( B ) Fold - change ± standard error in baseline IL - 6 plasma at week 12 from RA treated with placebo ( n = 47 ) , baricitinib 1 - mg ( n = 23 ) , 2 - mg ( n = 24 ) , 4 - mg ( n = 23 ) , 8 - mg ( n = 23 ) in phase 2b randomized , placebo - controlled study NCT01469013 . p values are for comparisons of baricitinib 1 - mg , 2 - mg , 4 - mg , 8 - mg change from baseline compared to placebo change from baseline . Treatment effects were estimated using a mixed effects repeated measures model ( SAS Proc Mixed ) using log10 transformed IL - 6 with an unstructured covariance matrix .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O

(B) Fold-change ± standard error in baseline IL-6 plasma levels at week 12 from RA patients treated with placebo (n=47), baricitinib 1-mg (n=23), 2-mg (n=24), 4-mg (n=23), and 8-mg (n=23) in the phase 2b randomized, placebo-controlled study NCT01469013. p values are for comparisons of baricitinib 1-mg, 2-mg, 4-mg, and 8-mg change from baseline compared to the placebo change from baseline. Treatment effects were estimated using a mixed effects repeated measures model (SAS Proc Mixed) using log10 transformed IL-6 levels with an unstructured covariance matrix.


( A B ) Binding affinities in Kd are shown for baricitinib ( A B ) , tofacitinib , upadacitinib , AZD7762 ( positive control ) , broad - spectrum tyrosine kinase inhibitors sunitinib erlotinib ( B ) . Kd values were estimated from an 11 - point response curve with fixed top parameters a four - parameter logistic model . Kd , equilibrium affinity constant ( n = 3 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A and B) Binding affinities in Kd are shown for baricitinib (A and B), tofacitinib, upadacitinib, AZD7762 (positive control), and broad-spectrum tyrosine kinase inhibitors sunitinib and erlotinib (B). Kd values were estimated from an 11-point concentration response curve with fixed top parameters and a four-parameter logistic model. Kd, equilibrium affinity constant (n=3)


( C ) mRNA expression of relevant NAKs in human liver organoids ( n = 3 ) ( p < 0 . 05 ) . immunofluorescence shows juxtaposition of 1A9 ( signal from SARS - CoV - 2 ) in red ACE2 in green along with spheroid nuclei in blue as a DAPI stain ( n = 1 ) . Colocalization of 1A9 ACE2 is depicted with an arrow .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O

(C) Protein and mRNA expression of relevant NAKs in primary human liver organoids (n=3) (p<0.05). The immunofluorescence shows the juxtaposition of 1A9 (signal from the SARS-CoV-2) in red and ACE2 in green along with the spheroid nuclei in blue as a DAPI stain (n=1). Colocalization of 1A9 and ACE2 is depicted with an arrow.


( D , E ) Time course of days ( one , three , six days ) post infection ( dpi ) using liver spheroids SARS - CoV - 2 at 0 . 1 1 . 0 multiplicity of infection ( MOI ) ( n = 1 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D, E) Time course of days (one, three, and six days) post infection (dpi) using liver spheroids and SARS-CoV-2 at 0.1 and 1.0 multiplicity of infection (MOI) (n=1).


( F ) Viability of spheroids was quantified by measuring intracellular ATP concentrations . Note that no significant ( n . s . ; Student ' s t - ) decrease in viability was observed for any tested ( n = 10 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Viability of spheroids was quantified by measuring intracellular ATP concentrations. Note that no significant (n.s.; Student's t-test) decrease in cell viability was observed for any concentration tested (n=10).


( H ) Viral load in infected ( MOI 0 . 1 ) spheroids as measured by qPCR after treatment with 400 nM 800 nM of baricitinib 48 hours after infection ( * p < 0 . 05 ; Student ' s t - ) . Data are mean ± standard error of mean ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Viral load in infected (MOI 0.1) spheroids as measured by qPCR after treatment with 400 nM and 800 nM of baricitinib 48 hours after infection (*p<0.05; Student's t-test). Data are mean ± standard error of mean (n=3).


( A ) Detection of SARS - CoV - 2 in nasopharyngeal swabs peripheral blood using GeneFinderTM COVID - 19 Plus RealAmp Kit ( ELITechGroup S . p . A . , Turin , Italy ) . Three viral target genes , RdRp , N , E , together with housekeeping gene GAPDH were simultaneously amplified . Here most sensitive target gene , N , is shown . Samples with Ct values > 40 were defined as negative . A dashed horizontal indicates cut - off . IL - 6 values are shown in same graph . Time of baricitinib treatment is highlighted ( A , B , D for 10 days , C for 12 days ) with total time of evaluation on X - axis . Fever cough symptoms are indicated . bars in grey indicate that cough has improved but has not resolved .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Detection of SARS-CoV-2 in nasopharyngeal swabs and peripheral blood using the GeneFinderTM COVID-19 Plus RealAmp Kit assay (ELITechGroup S.p.A., Turin, Italy). Three viral target genes, RdRp, N, and E, together with the housekeeping gene GAPDH were simultaneously amplified. Here the most sensitive target gene, N, is shown. Samples with Ct values > 40 were defined as negative. A dashed horizontal line indicates the cut-off. IL-6 values are shown in the same graph. Time of baricitinib treatment is highlighted (Patients A, B, and D for 10 days, Patient C for 12 days) with total time of evaluation on the X-axis. Fever and cough symptoms are indicated. The bars in grey indicate that cough has improved but has not resolved.


( B ) of CD3 + , CD4 + , CD8 + , B ( CD19 + ) NK ( CD3 - , CD16 + , CD56 + ) were determined using AQUIOS CL Flow Cytometry System ( Beckman Coulter ) .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O

(B) Levels of CD3+, CD4+, CD8+, B cells (CD19+) and NK cells (CD3-, CD16+, CD56+) were determined using the AQUIOS CL Flow Cytometry System (Beckman Coulter).


( C ) Total serum IgG , IgA , IgM are shown for .

(C) Total serum IgG, IgA, and IgM levels are shown for all patients.


Detection of IgG antibodies against S1 / S2 antigens of SARS - CoV - 2 . Time of baricitinib treatment is indicated for ( A , B , D for 10 days , C for 12 days ) .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Detection of IgG antibodies against the S1/S2 antigens of SARS-CoV-2. Time of baricitinib treatment is indicated for all patients (patients A, B, and D for 10 days, patient C for 12 days).


( A ) of D - dimer , CRP , ferritin , blood , neutrophils are shown for .

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O

(A) Levels of D-dimer, CRP, ferritin, white blood cells, and neutrophils are shown for all patients.


( B ) of aspartate aminotransferase , alanine aminotransferase , bilirubin are shown for . ALT , alanine aminotransferase ; AST , aspartate

(B) Levels of aspartate aminotransferase, alanine aminotransferase, and bilirubin are shown for all patients. ALT, alanine aminotransferase; AST, aspartate aminotransferase


( C ) Chest CT scan for C on day one 19 from symptom onset showing clinical improvement over time . Day one CT scan shows ground glass opacity ( arrows ) sub - pleurally in lower lobes bilaterally ( early stage one according to Pan et al ( Pan , Ye et al . , 2020 ) . Day 19 CT scan shows that consolidation was gradually absorbed with evident residual fibrosis emphysema bubbles ( arrows ) in site of early lesions ( absorption stage four according to Pan et al ( Pan et al . , 2020 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Chest CT scan for Patient C on day one and 19 from symptom onset showing clinical improvement over time. Day one CT scan shows ground glass opacity (arrows) sub-pleurally in the lower lobes bilaterally (early stage one according to Pan et al (Pan, Ye et al., 2020). Day 19 CT scan shows that consolidation was gradually absorbed with evident residual fibrosis and emphysema bubbles (arrows) in the site of the early lesions (absorption stage four according to Pan et al (Pan et al., 2020).


B . 5 ' UTR of rne is bound by RNase E non - genomically encoded A - tails are maximally recovered - 9nt from rne start codon . Known loop strucutres ( HP1 - 3 ) ribosomal binding site ( RBS ) are shaded grey .

O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

B. The 5'UTR of rne is bound by RNase E and non-genomically encoded A-tails are maximally recovered -9nt from the rne start codon. Known stem loop strucutres (HP1-3) and the ribosomal binding site (RBS) are shaded grey.


C . RNase E binding oligoadenylation of pdlB - yigL dicistronic transcript . reported RNase E cleavage site ( red dashed ) SgrS binding site ( grey shading ) are indicated .

O O B-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O

C. RNase E binding and oligoadenylation of the pdlB-yigL dicistronic transcript. The reported RNase E cleavage site (red dashed line) and SgrS binding site (grey shading) are indicated.


D . of non - genomically encoded oligo ( A ) tails recovered from RNase E bound reads .

O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O

D. Length of non-genomically encoded oligo(A) tails recovered from RNase E bound reads.


E . Position of Hfq binding oliogadenylation relative to RNase E binding peaks . cumulative position of Hfq oligoadenylation peaks were determined relative to RNase E binding peaks for 672 RNase E binding sites that were within 1kb of an Hfq binding peak . A detailed description of data processing is presented in Appended Supplementary Methods .

O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

E. Position of Hfq binding and oliogadenylation relative to RNase E binding peaks. The cumulative position of Hfq and oligoadenylation peaks were determined relative to RNase E binding peaks for 672 RNase E binding sites that were within 1kb of an Hfq binding peak. A detailed description of the data processing is presented in the Appended Supplementary Methods.


B . Hfq binding ( blue ) , RNase E binding ( grey ) , oligoadenylation of MicA binding site in ompA mRNA . MicA seed is shaded blue .

O O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O

B. Hfq binding (blue), RNase E binding (grey), and oligoadenylation of the MicA binding site in the ompA mRNA. The MicA seed sequence is shaded blue.


C . Interaction strength ( Gibbs free energy ) of - interactions recovered by RNase E - CLASH ( solid ) for randomly paired hybrid read halves ( dashed ) .

O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O

C. Interaction strength (Gibbs free energy) of all RNA-RNA interactions recovered by RNase E-CLASH (solid line) and for randomly paired hybrid read halves (dashed line).


D . Interaction strength of sRNA - mRNA hybrid read halves ( solid ) randomly paired sRNA mRNA read halves ( dashed ) .

O O O O O O O O O O O O O O O O O O O O O O O O

D. Interaction strength of sRNA-mRNA hybrid read halves (solid line) and randomly paired sRNA and mRNA read halves (dashed line).


E . Position of sRNA - mRNA interactions relative to mRNA start codon .

E. Position of sRNA-mRNA interactions relative to the mRNA start codon.


classes are labeled on outer ring ( rRNA has been omitted to clarify interactions between other classes ) . - interactions with more than two unique hybrid sequences are presented . thickness of link represents number of unique hybrid sequences recovered ( up to a maximum of 50 sequences ) . - interactions where both hybrid halves overlap an Hfq binding site are colored blue non - overlapping interactions are colored green .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RNA classes are labeled on the outer ring (rRNA has been omitted to clarify interactions between other RNA classes). RNA-RNA interactions with more than two unique hybrid sequences are presented. The thickness of the link represents the number of unique hybrid sequences recovered (up to a maximum of 50 sequences). RNA-RNA interactions where both hybrid halves overlap an Hfq binding site are colored blue and non-overlapping interactions are colored green.


A B ( top ) . Sequences interacting with ChiX ( A ) or ompA ( B ) were analyzed for conserved motifs using MEME . Motifs that were enriched in target RNAs are shown above with proportion of target RNAs carrying motif ( N ) expected value for motif ( e - val ) . complementary motifs within ChiX or ompA are shown below logo . ( Middle ) Heatmaps showing position of predicted basepairing for each interaction within ChiX or ompA . Grey bars boxed below indicate position of basepairing with experimentally verified mRNAs ( known interactions ) . ( Bottom ) Hfq ( blue ) RNase E ( grey ) binding sites within ChiX ompA . Hfq RNase E - bound , non - genomically encoded oligo ( A ) tails within ChiX ompA are shown as plots where x - axis position correlates with heatmaps above .

O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O

A B (top). Sequences interacting with ChiX (A) or ompA (B) were analyzed for conserved motifs using MEME. Motifs that were enriched in the target RNAs and are shown above the heatmap with proportion of target RNAs carrying the motif (N) and the expected value for the motif (e-val). The complementary sequence motifs within ChiX or ompA are shown below the logo. (Middle) Heatmaps showing the position of predicted basepairing for each interaction within ChiX or ompA. Grey bars boxed below indicate the position of basepairing with experimentally verified mRNAs (known interactions). (Bottom) Hfq (blue) and RNase E (grey) binding sites within ChiX and ompA. Hfq and RNase E-bound sequence, and non-genomically encoded oligo(A) tails within ChiX and ompA are shown as line plots where the x-axis position correlates with heatmaps above.


C , D E . Cumulative plots of oligoadenylation ( C ) , Hfq ( D ) RNase E ( E ) binding at predicted seed motifs .

O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O

C, D E. Cumulative plots of oligoadenylation (C), Hfq (D) and RNase E (E) binding at predicted seed motifs.


A - H . Base pairing patterns for sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) are shown ( left ) for frdA - RyhB ( A ) . Point mutations predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - t - was used to calculate significance from biological triplicate cultures . * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for frdA-RyhB (A). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.


A - H . Base pairing patterns for sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) are shown ( left ) for zapB - RyhB ( B ) . Point mutations predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - t - was used to calculate significance from biological triplicate cultures . * p < 0 . 05 .

A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for zapB-RyhB (B). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.


A - H . Base pairing patterns for sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) are shown ( left ) for hdeA - RyhB ( C ) . Point mutations predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - t - was used to calculate significance from biological triplicate cultures . * p < 0 . 05 .

A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for hdeA-RyhB (C). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.


A - H . Base pairing patterns for sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) are shown ( left ) hdeA - GadY ( D ) . Point mutations predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - t - was used to calculate significance from biological triplicate cultures . * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) hdeA-GadY (D). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.


A - H . Base pairing patterns for sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) are shown ( left ) for rssA - RyeB ( E ) . Point mutations predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - t - was used to calculate significance from biological triplicate cultures . * p < 0 . 05 .

A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for rssA-RyeB (E). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.


A - H . Base pairing patterns for sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) are shown ( left ) for chuA - Esr41 ( F ) . Point mutations predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - t - was used to calculate significance from biological triplicate cultures . * p < 0 . 05 .

A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for chuA-Esr41 (F). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.


A - H . Base pairing patterns for sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) are shown ( left ) for cirA - Esr41 ( G ) . Point mutations predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - t - was used to calculate significance from biological triplicate cultures . * p < 0 . 05 .

A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for cirA-Esr41 (G). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.


A - H . Base pairing patterns for sRNA - mRNA pairs identified by CLASH were calculated using IntaRNA software ( Busch et al . 2008 ) are shown ( left ) for bfr - Esr41 ( H ) . Point mutations predicted sRNA seed sequences ( blue shading ) are indicated . ( Histograms , right ) Median ( MFI ) was assessed by FACS for mRNA - sfGFP fusions with compensatory base changes . In each case , introduction of a point mutation into an sRNA or mRNA construct ( M1 ) is expected to reduce sRNA repression , which should be restored by combining complementary point mutants ( last bar , M1 - M1 ) . A two - t - was used to calculate significance from biological triplicate cultures . * p < 0 . 05 .

A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for bfr-Esr41 (H). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.


A - C . FACS of constitutively expressed sfGFP fusions to chuA ( A ) , cirA ( B ) , or bfr ( C ) in presence of Esr41 ( orange ) , RyhB ( blue ) , or a scrambled control ( red ) . histogram shows GFP for each sRNA - mRNA fusion .

O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

A-C. FACS analysis of constitutively expressed sfGFP fusions to chuA (A), cirA (B), or bfr (C) in the presence of Esr41 (orange), RyhB (blue), or a scrambled RNA control (red). The histogram shows GFP fluorescence for each sRNA-mRNA fusion.


D . Esr41 confers resistance to Colicin 1A 1B in sensitive background , E . coli DH5alpha . Top agar lawns of DH5alpha expressing a control scrambled ( pJV300 ) , Esr41 ( pZE12 : : esr41 ) , or RyhB ( pZE12 : : ryhB ) were spotted with colicins indicated ( top ) . Zones of clearing indicate sensitivity to tested colicin .

O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

D. Esr41 confers resistance to Colicin 1A and 1B in the sensitive background, E. coli DH5alpha. Top agar lawns of DH5alpha expressing a control scrambled RNA (pJV300), Esr41 (pZE12::esr41), or RyhB (pZE12::ryhB) were spotted with colicins indicated (top). Zones of clearing indicate sensitivity to the tested colicin.


E . Esr41 confers a competitive disadvantage on EHEC under iron - limiting conditions . Wild type EHEC isogenic ∆ esr41 ( black ) , or EHEC ∆ esr41and chromosomally repaired EHEC ∆ esr41 : : esr41 were inoculated at equal densities cultured in MEM - HEPES media for 3 days . proportion of each was determined at each time point ( days ) is expressed as a relative to starting inoculum where 1 is an equal fitness ( see Appended Supplementary Methods ) .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Esr41 confers a competitive disadvantage on EHEC under iron-limiting conditions. Wild type EHEC and isogenic ∆esr41 strain (black), or EHEC ∆esr41and the chromosomally repaired strain EHEC ∆esr41::esr41 were inoculated at equal densities and cultured in MEM-HEPES media for 3 days. The proportion of each strain was determined at each time point (days) and is expressed as a ratio relative to the starting inoculum where 1 is an equal fitness (see Appended Supplementary Methods).


A - D of uninjected , FLT3 / WT mRNA FLT3 / ITD mRNA injected ( 150 ng per ) on day 2 post fertilization ( dpf ) . Data information : ov : otic vesicles Scale bar = 500 μm .

O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-D The morphology of uninjected, FLT3/WT mRNA and FLT3/ITD mRNA injected (150 ng per embryo) embryos on day 2 post fertilization (dpf). Data information: ov: otic vesicles Scale bar = 500 μm.


E - H Whole mount in situ hybridization ( WISH ) of notochord - specific marker col9a2 in uninjected , FLT3 / WT mRNA FLT3 / ITD mRNA injected on 2 dpf . Data information : ov : otic vesicles ; cm : cephalic mesoderm ; nc : notochord . Scale bar = 500 μm .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E-H Whole mount in situ hybridization (WISH) of notochord-specific marker col9a2 in uninjected, FLT3/WT mRNA and FLT3/ITD mRNA injected embryos on 2 dpf. Data information: ov: otic vesicles; cm: cephalic mesoderm; nc: notochord. Scale bar = 500 μm.


FLT3 signaling was detected by Western blotting in 293FT - transfected with FLT3 / ITD mRNA ( I )

B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O

FLT3 signaling was detected by Western blotting in 293FT cells-transfected with FLT3/ITD mRNA (I)


FLT3 signaling was detected by Western blotting in zebrafish - injected with FLT3 / ITD mRNA ( J ) .

B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

FLT3 signaling was detected by Western blotting in zebrafish embryos-injected with FLT3/ITD mRNA (J).


K effect of FLT3 inhibitor quizartinib ( Qui ) on dorsalization axis - duplication phenotype - induced by FLT3 / ITD mRNA injection in zebrafish . Data information : experiments were performed in triplicates data are presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) . NS , not significant .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K The effect of FLT3 inhibitor quizartinib (Qui) on the dorsalization and axis-duplication phenotype-induced by FLT3/ITD mRNA injection in zebrafish. Data information: the experiments were performed in triplicates and the data are presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test). NS, not significant.


of fst expression by RT - qPCR ( L ) after FLT3 / ITD overexpression in zebrafish at 6 hpf . Data information : experiments were performed in triplicates data are presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) . NS , not significant .

O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of fst expression by RT-qPCR (L) after FLT3/ITD overexpression in zebrafish embryos at 6 hpf. Data information: the experiments were performed in triplicates and the data are presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test). NS, not significant.


of fst expression by Western Blotting ( M ) , after FLT3 / ITD overexpression in zebrafish at 6 hpf .

O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

Quantification of fst expression by Western Blotting (M), after FLT3/ITD overexpression in zebrafish embryos at 6 hpf.


of fst expression by WISH ( N ) after FLT3 / ITD overexpression in zebrafish at 6 hpf .

O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

Quantification of fst expression by WISH (N) after FLT3/ITD overexpression in zebrafish embryos at 6 hpf.


A - C WISH of fst in FLT3 / WT - ( A ) , FLT3 / ITD - plasmid injected zebrafish without ( B ) or with ( C ) quizartinib treatment ( 2 . 5 μM ) from 6 to 36 hpf . fst expression was expanded by FLT3 / ITD in 86 % of ( B , arrow , 32 / 37 ) which could be effectively blocked by treating with FLT3 inhibitor quizartinib in 83 % of ( C , 29 / 35 ) . Data information : Scale bar = 500 μm .

O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

A-C WISH of fst in FLT3/WT- (A), FLT3/ITD- plasmid DNA injected zebrafish embryos without (B) or with (C) quizartinib treatment (2.5 μM) from 6 to 36 hpf. fst expression was expanded by FLT3/ITD DNA in 86% of embryos (B, arrow, 32/37) which could be effectively blocked by treating with FLT3 inhibitor quizartinib in 83% of embryos (C, 29/35). Data information: Scale bar = 500 μm.


Generation characterization of FLT3 / ITD - transgenic zebrafish . GFP expression was detected by florescent microscopy ( F - H ) in blood circulation thymus by WISH ( I J , blue arrow ) in WT sibling Runx1 - FLT3 / ITD transgenic zebrafish ( F1 ) at 4 dpf . FLT3 / ITD - positive zebrafish ( F1 ) were confirmed by PCR genotyping of GFP FLT3 / ITD using genomic from fin clip of WT siblings Runx1 - FLT3 / ITD transgenic zebrafish ( F1 ) at two months old . Fish 4 , 5 6 showed germline transmission of FLT3 / ITD transgene ( K ) . Data information : Scale bar = 500 μm .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

Generation and characterization of FLT3/ITD-transgenic zebrafish. GFP expression was detected by florescent microscopy (F-H) and in blood circulation and thymus by WISH (I and J, blue arrow) in WT sibling and Runx1-FLT3/ITD transgenic zebrafish (F1) embryos at 4 dpf. FLT3/ITD-positive zebrafish (F1) were confirmed by PCR genotyping of GFP and FLT3/ITD using genomic DNA from fin clip of WT siblings and Runx1-FLT3/ITD transgenic zebrafish (F1) at two months old. Fish 4, 5 and 6 showed germline transmission of FLT3/ITD transgene (K). Data information: Scale bar = 500 μm.


Kidney marrow ( KM ) was collected from Runx1 - FLT3 / ITD transgenic zebrafish ( F1 ) at 18 months old . hematopoietic composition of KM from WT siblings ( n = 6 ) Runx1 - FLT3 / ITD transgenic ( n = 6 ) zebrafish were examined by Giemsa ( L ) flow cytometry ( abbreviation for panel M : M , Myeloid ; P , Progenitor ; L , Lymphoid ; E , Erythroid ) . Data are presented in box plot . * * P < 0 . 01 ( Student ' s t - ) .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kidney marrow (KM) was collected from Runx1-FLT3/ITD transgenic zebrafish (F1) at 18 months old. The morphology and hematopoietic composition of KM from WT siblings (n = 6) and Runx1-FLT3/ITD transgenic (n = 6) zebrafish were examined by Giemsa staining (L) and flow cytometry (abbreviation for panel M: M, Myeloid cells; P, Progenitor cells; L, Lymphoid cells; E, Erythroid cells). Data are presented in box plot. **P<0.01 (Student's t-test).


Kidney marrow ( KM ) was collected from Runx1 - FLT3 / ITD transgenic zebrafish ( F1 ) at 18 months old . hematopoietic composition of KM from WT siblings ( n = 6 ) Runx1 - FLT3 / ITD transgenic ( n = 6 ) zebrafish were examined by flow cytometry Data are presented in box plot . whiskers , boxes , central lines in panel N represented minimum - to - maximum values , 25th - to - 75th percentile , 50th percentile ( median ) , respectively . * * P < 0 . 01 ( Student ' s t - ) .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kidney marrow (KM) was collected from Runx1-FLT3/ITD transgenic zebrafish (F1) at 18 months old. The morphology and hematopoietic composition of KM from WT siblings (n = 6) and Runx1-FLT3/ITD transgenic (n = 6) zebrafish were examined by flow cytometry Data are presented in box plot. The whiskers, boxes, and central lines in panel N represented the minimum-to-maximum values, 25th-to-75th percentile, and the 50th percentile (median), respectively. **P<0.01 (Student's t-test).


O Expression of fst was detected by RT - qPCR in KM from WT sibling Runx1 - FLT3 / ITD transgenic zebrafish at 18 months old . RT - qPCR experiments were performed in triplicates data were presented as mean ± . * * P < 0 . 01 .

O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

O Expression of fst was detected by RT-qPCR in KM from WT sibling and Runx1-FLT3/ITD transgenic zebrafish at 18 months old. The RT-qPCR experiments were performed in triplicates and data were presented as mean ± SEM. **P<0.01.


P Detection of FST expression , p - ERK1 / 2 , p - CREB in mononuclear from normal peripheral blood ( PBSC ) FLT3 / ITD AML ( diagnostic samples with leukemia blasts > 80 % ) by Western blotting . ^ : non - specific of p - ATF1 due to conserved motif .

O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O

P Detection of FST expression, p-ERK1/2, and p-CREB in mononuclear cells from normal peripheral blood stem cell (PBSC) and FLT3/ITD AML patients (diagnostic samples with leukemia blasts > 80%) by Western blotting. ^: non-specific staining of p-ATF1 protein due to the conserved motif.


B - C direct binding of p - CREB to human FST promoter was detected by ChIP - PCR ( B ) ChIP - qPCR ( C ) . c - Fos was used as positive control of p - CREB target gene . Normal IgG was used as negative control of ChIP . Data information experiments were performed in triplicates , data were presented as mean ± . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B-C The direct binding of p-CREB to human FST promoter was detected by ChIP-PCR (B) and ChIP-qPCR (C). c-Fos was used as positive control of p-CREB target gene. Normal IgG was used as negative control of ChIP. Data information the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).


D Dual luciferase demonstrating direct binding of pCREB on human FST promoter . pRL - CMV , Renilla luciferase vector ; pGL - CRE - pGL - CRE + , firefly luciferase expression driven by human FST promoter with deleted CRE site ( CRE - ) or wildtype ( CRE + ) ; p - GFPSpark , GFP expressing vector ; p - CREBY134F , CREBY134F - GFP expressing vector . Data information : experiments were performed in triplicates , data were presented as mean ± . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Dual luciferase assay demonstrating the direct binding of pCREB on human FST promoter. pRL-CMV, Renilla luciferase vector; pGL-CRE- and pGL-CRE+, firefly luciferase expression driven by human FST promoter with deleted CRE site (CRE-) or wildtype (CRE+); p-GFPSpark, GFP expressing vector; p-CREBY134F, CREBY134F-GFP expressing vector. Data information: the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).


E FST expression FLT3 / ITD signaling were detected by Western blotting in Ba / F3 - parental ( P in short ) Ba / F3 - FLT3 / ITD ( ITD in short ) .

O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

E FST expression and FLT3/ITD signaling were detected by Western blotting in Ba/F3-parental (P in short) and Ba/F3-FLT3/ITD (ITD in short) cells.


F - H Phosphor - flow of p - CREB in Ba / F3 - parental , Ba / F3 - FLT3 / ITD , Ba / F3 - FLT3 / ITD - treated with FLT3 inhibitor Quizartinib ( Qui in short ) . Isotype antibody were used as control to calculate mean ( MFI ) ( F - G ) . transcription expression of Fst was detected by RT - qPCR after Quizartinib treatment ( 10 nM ) in Ba / F3 - FLT3 / ITD for one day ( H ) . Data information : experiments were performed in triplicates , data were presented as mean ± . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F-H Phosphor-flow analysis of p-CREB in Ba/F3-parental, Ba/F3-FLT3/ITD, and Ba/F3-FLT3/ITD cells-treated with FLT3 inhibitor Quizartinib (Qui in short). Isotype antibody were used as control to calculate the mean fluorescence intensity (MFI) ratio (F-G). The transcription and expression of Fst was detected by RT-qPCR after Quizartinib treatment (10 nM) in Ba/F3-FLT3/ITD cells for one day (H). Data information: the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).


I - K expression of FST phosphorylation of CREB were detected by Western Blotting ( I K ) phospho - flow ( J ) in MOLM - 13 ( I ) Ba / F3 - FLT3 / ITD ( K ) treated with Quizartinib BRD7389 for one day , respectively . Data information : experiments were performed in triplicates , data were presented as mean ± . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I-K The expression of FST and phosphorylation of CREB were detected by Western Blotting (I and K) and phospho-flow analysis (J) in MOLM-13 (I) and Ba/F3-FLT3/ITD (K) cells treated with Quizartinib and BRD7389 for one day, respectively. Data information: the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).


L RSK FST expression were detected by Western Blotting after p90RSK knockout by CRISPR / Cas9 in MOLM - 13 .

O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

L RSK and FST expression were detected by Western Blotting after p90RSK knockout by CRISPR/Cas9 in MOLM-13 cells.


M phosphorylation of CREB FST expression were detected by Western Blotting in Ba / F3 - FLT3 / ITD treated with CREB inhibitor 666 - 15 for one day . ^ : non - specific of p - ATF1 due to conserved motif .

O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O

M The phosphorylation of CREB and FST expression were detected by Western Blotting in Ba/F3-FLT3/ITD cells treated with CREB inhibitor 666-15 for one day. ^: non-specific staining of p-ATF1 protein due to the conserved motif.


N CREB FST expression were detected by Western Blotting after CREB knockout by CRISPR / Cas9 in MOLM - 13 .

N CREB and FST expression were detected by Western Blotting after CREB knockout by CRISPR/Cas9 in MOLM-13 cells.


O growth of Ba / F3 - parental ( with IL - 3 ) , Ba / F3 - FLT3 / ITD ( without IL - 3 ) Ba / F3 - FLT3 / ITD ( with IL - 3 ) was measured after three days treatment of CREB inhibitor 666 - 15 in vitro . Data information : experiments were performed in triplicates , data were presented as mean ± . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

O The growth of Ba/F3-parental (with IL-3), Ba/F3-FLT3/ITD (without IL-3) and Ba/F3-FLT3/ITD (with IL-3) cells was measured after three days treatment of CREB inhibitor 666-15 in vitro. Data information: the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).


P rescue effect of CREB inhibitor 666 - 15 on FLT3 / ITD - induced dorsalization axis duplication in zebrafish at 1 dpf .

O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

P The rescue effect of CREB inhibitor 666-15 on FLT3/ITD-induced dorsalization and axis duplication in zebrafish embryos at 1 dpf.


A FST expression in different AML lines was detected by Western Blotting .

A FST expression in different AML cell lines was detected by Western Blotting.


FST317 FST344 overexpression resulted in significant increases of FST transcription by RT - qPCR by Western Blot ( B ) Green , ML - 2 - GFP ; Blue , ML - 2 - FST317 ; Red , ML - 2 - FST344 . RT - qPCR experiments were performed in triplicates ( B ) . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) . ns : Not significant .

B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

FST317 and FST344 overexpression resulted in significant increases of FST transcription by RT-qPCR and protein by Western Blot (B) Green, ML-2-GFP; Blue, ML-2-FST317; Red, ML-2-FST344. The RT-qPCR experiments were performed in triplicates (B). Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). ns: Not significant.


FST317 FST344 overexpression promoted ML - 2 growth in vitro ( C ) . Green , ML - 2 - GFP ; Blue , ML - 2 - FST317 ; Red , ML - 2 - FST344 . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) . ns : Not significant .

B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

FST317 and FST344 overexpression promoted ML-2 cell growth in vitro (C). Green, ML-2-GFP; Blue, ML-2-FST317; Red, ML-2-FST344. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). ns: Not significant.


D - E clonogenicity of ML - 2 overexpressing GFP , FST317 , FST344 in vitro for 14 days . CFU experiments were performed in triplicates ( E ) . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) . ns : Not significant .

O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D-E The clonogenicity of ML-2 overexpressing GFP, FST317, and FST344 in vitro for 14 days. The CFU experiments were performed in triplicates (E). Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). ns: Not significant.


engraftment of ML - 2 ( with luciferase gene ) overexpressing GFP , FST317 , FST344 was quantified by bioluminescence imaging ( F G ) Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) . ns : Not significant .

O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The engraftment of ML-2 (with luciferase gene) overexpressing GFP, FST317, and FST344 was quantified by bioluminescence imaging (F and G) Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). ns: Not significant.


engraftment of ML - 2 ( with luciferase gene ) overexpressing GFP , FST317 , FST344 survival of ML - 2 - engrafted NSG mice in vivo was recorded ( H ) . Survival curve in panel H was analyzed by Log - rank . * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The engraftment of ML-2 (with luciferase gene) overexpressing GFP, FST317, and FST344 and the survival of ML-2-engrafted NSG mice in vivo was recorded (H). Survival curve in panel H was analyzed by Log-rank test. *P<0.05, **P<0.01.


- seq of upregulation of RET , IL2RA CCL5 after FST344 overexpression in ML - 2 .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O

RNA-seq of upregulation of RET, IL2RA and CCL5 after FST344 overexpression in ML-2 cells.


RT - qPCR validation of upregulation of RET , IL2RA CCL5 after FST344 overexpression in ML - 2 . RT - qPCR experiments were performed in triplicates ( J ) . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) . ns : Not significant .

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RT-qPCR validation of upregulation of RET, IL2RA and CCL5 after FST344 overexpression in ML-2 cells. RT-qPCR experiments were performed in triplicates (J). Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). ns: Not significant.


Overall survival of from TCGA - AML on differential expression of

Overall survival analysis of patients from TCGA-AML based on the differential expression of IL2RA


Overall survival of from TCGA - AML on differential expression of CCL5 .

Overall survival analysis of patients from TCGA-AML based on the differential expression of CCL5.


M Activation of MAPK / ERK pathway in ML - 2 by FST344 overexpression were validated by Western Blotting . Scale bar = 1 mm .

O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

M Activation of MAPK/ERK pathway in ML-2 by FST344 overexpression were validated by Western Blotting. Scale bar = 1 mm.


A FST knockdown in MOLM - 13 by shRNA effectively reduced FST expression .

O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O

A FST knockdown in MOLM-13 by shRNA effectively reduced FST expression.


were measured after FST knockdown in vitro . Scale bar = 10 μm .

O O O B-CONTROLLED_VAR O O O O O O O O O O

The morphology were measured after FST knockdown in vitro. Scale bar = 10 μm.


apoptosis ( C ) measured after FST knockdown in vitro . Scale bar = 10 μm . apoptosis assays ( C ) were performed in triplicates . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The apoptosis (C) measured after FST knockdown in vitro. Scale bar = 10 μm. The apoptosis assays (C) were performed in triplicates. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


clonogenicity of MOLM - 13 ( D E ) were measured after FST knockdown in vitro . Scale bar = 10 μm . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The clonogenicity of MOLM-13 (D and E) were measured after FST knockdown in vitro. Scale bar = 10 μm. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


engraftment of MOLM - 13 after FST knockdown was detected by flow cytometry of human CD45 mouse CD45 . 1 positive in recipient mouse BM aspiration at week 2 post transplantation .

O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O

The engraftment of MOLM-13 after FST knockdown was detected by flow cytometry of human CD45 and mouse CD45.1 positive cells in recipient mouse BM aspiration at week 2 post transplantation.


engraftment of MOLM - 13 after FST knockdown was detected by flow cytometry of human CD45 mouse CD45 . 1 positive in recipient mouse BM aspiration at week 2 post transplantation . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The engraftment of MOLM-13 after FST knockdown was detected by flow cytometry of human CD45 and mouse CD45.1 positive cells in recipient mouse BM aspiration at week 2 post transplantation. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


effect of FST knockdown on survival of NSG mice - engrafted with MOLM - 13 . scr : scrambled control ( 7 mice ) ; sh : short hairpin ( 8 mice for sh1 sh2 respectively ) . survival curve was analyzed by Log - rank . * * P < 0 . 01 .

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The effect of FST knockdown on the survival of NSG mice-engrafted with MOLM-13. scr: scrambled sequence control (7 mice); sh: short hairpin RNA (8 mice for sh1 and sh2 respectively). The survival curve was analyzed by Log-rank test. **P<0.01.


of MOLM - 13 after FST knockout by CRISPR / Cas9 in vitro . Scale bar = 10 μm .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

The morphology of MOLM-13 after FST knockout by CRISPR/Cas9 in vitro. Scale bar = 10 μm.


clonogenicity of MOLM - 13 after FST knockout by CRISPR / Cas9 in vitro . Scale bar = 10 μm . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The clonogenicity of MOLM-13 after FST knockout by CRISPR/Cas9 in vitro. Scale bar = 10 μm. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


engraftment of MOLM - 13 after FST knockout was detected by flow cytometry of human CD45 mouse CD45 . 1 positive in recipient mouse BM aspiration at week 2 post transplantation .

The engraftment of MOLM-13 after FST knockout was detected by flow cytometry of human CD45 and mouse CD45.1 positive cells in recipient mouse BM aspiration at week 2 post transplantation.


engraftment of MOLM - 13 after FST knockout was detected by flow cytometry of human CD45 mouse CD45 . 1 positive in recipient mouse BM aspiration at week 2 post transplantation . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

The engraftment of MOLM-13 after FST knockout was detected by flow cytometry of human CD45 and mouse CD45.1 positive cells in recipient mouse BM aspiration at week 2 post transplantation. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


E effect of FST knockout on survival of NSG mice - engrafted with MOLM - 13 . Cas 9 : Cas 9 only ( 10 mice ) ; sgRNA # 3 : Cas 9 + sgRNA # 3 ( 10 mice ) ; sgRNA # 4 : Cas 9 + sgRNA # 4 ( 8 mice ) . Data information : survival curves were analyzed by Log - rank . * P < 0 . 05 .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E The effect of FST knockout on the survival of NSG mice-engrafted with MOLM-13 cells. Cas 9: Cas 9 only (10 mice); sgRNA#3: Cas 9 + sgRNA#3 (10 mice); sgRNA#4: Cas 9 + sgRNA#4 (8 mice). Data information: survival curves were analyzed by Log-rank test. *P<0.05.


F knockdown efficiency of different FST - specific antisense oligoes ( ASOs ) in MOLM - 13 was detected by RT - qPCR after three days treatment in vitro . knockdown RT - qPCR experiments were performed in triplicates . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F The knockdown efficiency of different FST-specific antisense oligoes (ASOs) in MOLM-13 cells was detected by RT-qPCR after three days treatment in vitro. The knockdown and RT-qPCR experiments were performed in triplicates. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


G MOLM - 13 growth was measured after 3 days treatment of FST - ASO in vitro . ASO treatment experiments were performed in triplicates . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G MOLM-13 cell growth was measured after 3 days treatment of FST-ASO in vitro. The ASO treatment experiments were performed in triplicates. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


H Intraperitoneal injection of FST - ASO ( 10mg / kg weekly , 6 mice ) significantly prolonged survival of MOLM - 13 - engrafted NSG mice . random was used for negative control ( Neg - ASO , 6 mice ) . Data information : survival curves were analyzed by Log - rank . * P < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H Intraperitoneal injection of FST-ASO (10mg/kg weekly, 6 mice) significantly prolonged the survival of MOLM-13-engrafted NSG mice. The random sequence was used for negative control (Neg-ASO, 6 mice). Data information: survival curves were analyzed by Log-rank test. *P<0.05.


Flt3 / ITD knock - in mouse were genotyped ( A ) .

B-CONTROLLED_VAR O O O O O O O O O O O O

Flt3/ITD knock-in mouse were genotyped (A).


spleen ( B C , 4 mice each ) were measured in WT siblings Flt3 / ITD - knock - in mice ( 8 mice each ) . data were presented in box plot . whiskers , boxes , central lines represented minimum - to - maximum values , 25th - to - 75th percentile , 50th percentile ( median ) , respectively . * * P < 0 . 01 ( Student ' s t - ) .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The spleen weight (B and C, 4 mice each) were measured in WT siblings and Flt3/ITD-knock-in mice (8 mice each). data were presented in box plot. The whiskers, boxes, and central lines represented the minimum-to-maximum values, 25th-to-75th percentile, and the 50th percentile (median), respectively. **P<0.01 (Student's t-test).


serum Fst level ( D ) were measured in WT siblings Flt3 / ITD - knock - in mice ( 8 mice each ) . data were presented in box plot . whiskers , boxes , central lines represented minimum - to - maximum values , 25th - to - 75th percentile , 50th percentile ( median ) , respectively . * * P < 0 . 01 ( Student ' s t - ) .

O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The serum Fst level (D) were measured in WT siblings and Flt3/ITD-knock-in mice (8 mice each). data were presented in box plot. The whiskers, boxes, and central lines represented the minimum-to-maximum values, 25th-to-75th percentile, and the 50th percentile (median), respectively. **P<0.01 (Student's t-test).


engraftment of MOLM - 13 in NSG mice was confirmed at week 1 post injection ( E - F ) .

O O O O O O O O O O O O O O O O O O O O O

The engraftment of MOLM-13 in NSG mice was confirmed at week 1 post injection (E-F).


Serum FST level was measured in MOLM - 13 - engrafted NSG mice at pre - injection week 2 post injection ( G , PBS group , 3 mice ; MOLM - 13 group , 4 mice ) . Data information : data were presented as scatter dot plot . n . s : not significant , * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Serum FST level was measured in MOLM-13-engrafted NSG mice at pre-injection and week 2 post injection (G, PBS group, 3 mice; MOLM-13 group, 4 mice). Data information: data were presented as scatter dot plot. n.s: not significant, *P<0.05, **P<0.01 (Student's t-test). In


After FST knockdown , serum FST level was also measured in MOLM - 13 - engrafted NSG mice at week 2 post injection ( H , 3 mice for each group ) . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

After FST knockdown, serum FST level was also measured in MOLM-13-engrafted NSG mice at week 2 post injection (H, 3 mice for each group). Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


Serum FST level was significantly increased in AML - derived xenografted mouse at week 6 post injection . Human leukemic engraftments were confirmed by flow cytometry of human CD45 mouse CD45 . 1 in recipient mouse BM aspiration ( J ) .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O

Serum FST level was significantly increased in primary AML-derived xenografted mouse at week 6 post injection. Human leukemic engraftments were confirmed by flow cytometry of human CD45 and mouse CD45.1 cells in recipient mouse BM aspiration (J).


Serum FST level was significantly increased in AML - derived xenografted mouse at week 6 post injection . Serum FST from pre - injection post - engraftment mouse were measured ( K , one mouse for each AML sample ) . Data information : data were presented as scatter dot plot . n . s : not significant , * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Serum FST level was significantly increased in primary AML-derived xenografted mouse at week 6 post injection. Serum FST from pre-injection and post-engraftment mouse were measured (K, one mouse for each primary AML sample). Data information: data were presented as scatter dot plot. n.s: not significant, *P<0.05, **P<0.01 (Student's t-test).


L Correlation between serum FST leukemia blast from FLT3 / ITD - mutated AML at diagnosis . Correlation ( Pearson correlation coefficient ) was performed by Graphpad Prism 6 .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

L Correlation between serum FST levels and leukemia blast percentage from FLT3/ITD-mutated AML at diagnosis. Correlation analysis (Pearson correlation coefficient) was performed by Graphpad Prism 6.


Serum FST decreased in CR increased after relapse in 4 AML receiving Quizartinib monotherapy . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Serum FST decreased in CR and increased after relapse in 4 AML patients receiving Quizartinib monotherapy. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


N Serum FST continued to rise during disease progression from a who did not respond to Quizartinib . in N were recruited in QUANTUM - R accrual has been completed . data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) .

O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

N Serum FST continued to rise during disease progression from a patient who did not respond to Quizartinib. Patients in N were recruited in the QUANTUM-R and patient accrual has been completed. data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).


A . SF - 1 , FATE1 β - tubulin shown in basal condition after Dox treatment in H295R / TR , H295R / TRSF - 1 , H295R / TRFATE1 H295R / TR N - FlagFATE1cells .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O

A. SF-1, FATE1 and β-tubulin protein levels shown in basal condition and after Dox treatment in H295R/TR, H295R/TRSF-1, H295R/TRFATE1 and H295R/TR N-FlagFATE1cells.


B . FATE1 mRNA expression is increased in H295R / TRSF - 1 by Dox treatment . efficiency of FATE1 knockdown by specific ( siFATE1 ) vs . control ( siC ) siRNA nucleofection is shown ( mean ± ; n = 4 with 3 replicates / experiment ) .

O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. FATE1 mRNA expression is increased in H295R/TRSF-1 cells by Dox treatment. The efficiency of FATE1 knockdown by specific (siFATE1) vs. control (siC) siRNA nucleofection is shown (mean ± SEM; n=4 with 3 replicates/experiment).


C . Immunofluorescence microscopy showing endogenous FATE1 ( red ) induction of expression by Dox - treatment of H295R / TRSF - 1 . SF - 1 ( green ) , DAPI ( blue ) . Scale bars , 10 μm .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O

C. Immunofluorescence microscopy showing endogenous FATE1 protein (red) induction of expression by Dox-treatment of H295R/TRSF-1 cells. SF-1 (green), DAPI (blue). Scale bars, 10 μm.


D . Subcellular localization of endogenous FATE1 ( red ) transfected markers for Golgi , ER mitochondria , respectively ( green ) in Dox - treated H295R / TRSF - 1 . DAPI ( blue ) . Scale bars , 10 μm .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Subcellular localization of endogenous FATE1 (red) and transfected fluorescent markers for Golgi, ER and mitochondria, respectively (green) in Dox-treated H295R/TRSF-1 cells. DAPI (blue). Scale bars, 10 μm.


E . Subcellular localization of endogenous FATE1 ( green ) in Dox - treated H295R / TRSF - 1 costained with an antibody against mitochondrial marker HSP60 ( red ) . SF - 1 ( blue ) . Scale bar , 10 μm .

O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O

E. Subcellular localization of the endogenous FATE1 protein (green) in Dox-treated H295R/TRSF-1 cells costained with an antibody against the mitochondrial marker HSP60 (red). SF-1 (blue). Scale bar, 10 μm.


F . Immunogold electron microscopy showing association of FATE1 with mitochondrial outer surface in Dox - treated H295R / TR N - FlagFATE1 . Scale bar , 500 nm .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

F. Immunogold electron microscopy showing association of FATE1 with the mitochondrial outer surface in Dox-treated H295R/TR N-FlagFATE1 cells. Scale bar, 500 nm.


G . Dox - treated H295R / TR N - FlagFATE1 were fractioned into ( N ) , heavy membranes ( HM ) , membranes ( LM ) ( C ) fractions localization of SF - 1 , FATE1 , ribosomal RPL7 , VDAC1 GAPDH was revealed by immunoblot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O

G. Dox-treated H295R/TR N-FlagFATE1 cells were fractioned into nuclear (N), heavy membranes (HM), light membranes (LM) and cytosolic (C) fractions and localization of SF-1, FATE1, ribosomal protein RPL7, VDAC1 and GAPDH was revealed by immunoblot.


H . Effect of increasing concentrations ( 0 , 1 , 10 100 μg / ml ) of proteinase K 0 . 1 % Triton X - 100 ( TX - 100 ) treatment of mitochondrial fraction from Dox - treated H295R / TR N - FlagFATE1 on VDAC1 , FATE1 , TOM20 cytochrome c .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O

H. Effect of increasing concentrations (0, 1, 10 and 100 μg/ml) of proteinase K and 0.1% Triton X-100 (TX-100) treatment of the mitochondrial fraction from Dox-treated H295R/TR N-FlagFATE1 cells on VDAC1, FATE1, TOM20 and cytochrome c.


I . FATE1 VDAC1 are associated with pellet ( ; P ) fraction after high - speed centrifugation of alkaline - extracted mitochondrial fraction of Dox - treated H295R / TR SF - 1 , while cytochrome c is found in supernatant ( S ) .

O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O

I. FATE1 and VDAC1 are associated with the pellet (membrane; P) fraction after high-speed centrifugation of the alkaline-extracted mitochondrial fraction of Dox-treated H295R/TR SF-1 cells, while cytochrome c is found in the supernatant (S).


B . Subcellular localization of EGFP - fusions of full - ( FL ) FATE1 its mutants : N - terminal ( aa . 1 - 124 ) , C - terminal ( aa . 125 - 183 ) , 1 - 162 , transmembrane domain ( aa . 155 - 183 ) L151D ( green ) transfected in H295R / TRSF - 1 . Mitochondria were stained by anti - TOM20 antibody ( red ) by DAPI ( blue ) . Scale bars , 10 μm .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

B. Subcellular localization of EGFP-fusions of full-length (FL) FATE1 and its mutants: N-terminal (aa. 1-124), C-terminal (aa.125-183), 1-162, transmembrane domain (aa. 155-183) and L151D (green) transfected in H295R/TRSF-1 cells. Mitochondria were stained by anti-TOM20 antibody (red) and DNA by DAPI (blue). Scale bars, 10 μm.


C . EMD Flag - tagged FATE1 , but not other OMM TOM20 , were co - immunoprecipitated from Dox - treated H295R / TR N - FlagFATE1 . Control immunoprecipitations were performed with anti - myc ( α - myc ) non - immune rabbit IgG ( IgG - R ) antibodies .

O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. EMD and Flag-tagged FATE1, but not the other OMM protein TOM20, were co-immunoprecipitated from Dox-treated H295R/TR N-FlagFATE1 cells. Control immunoprecipitations were performed with anti-myc (α-myc) and non-immune rabbit IgG (IgG-R) antibodies.


D . Confocal microscopy showed colocalization of EMD , as revealed by immunofluorescence microscopy ( red ) , with ER , stained by ER - GFP BacMam ( green ) . Scale bars , 10 μm .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Confocal fluorescence microscopy showed colocalization of cytoplasmic EMD, as revealed by immunofluorescence microscopy (red), with ER, stained by ER-GFP BacMam (green). Scale bars, 10 μm.


E . Mic60 / mitofilin Flag - tagged FATE1 were coimmunoprecipitated from Dox - treated H295R / TR N - FlagFATE1 . Control immunoprecipitations were performed with anti - myc ( α - myc ) antibody .

O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Mic60/mitofilin and Flag-tagged FATE1 were coimmunoprecipitated from Dox-treated H295R/TR N-FlagFATE1 cells. Control immunoprecipitations were performed with anti-myc (α-myc) antibody.


F . Mitochondrial localization of Mic60 / mitofilin ( green ) FATE1 ( red ) in Dox - treated H295R / TRSF - 1 . Scale bar , 10 μm .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

F. Mitochondrial localization of Mic60/mitofilin (green) and FATE1 (red) in Dox-treated H295R/TRSF-1 cells. Scale bar, 10 μm.


G . Left , triple immunofluorescence microscopy labelling of endogenous FATE1 ( green ) , ER labelled by calreticulin ( red ) mitochondria labelled by HSP60 ( blue ) in Dox - treated H295R / TRSF - 1 . One showing close apposition of red , green blue ( signals ) is shown at higher magnification . Scale bar , 10 μm . Right , graph showing of FATE1 signal in total mitochondria ( histogram ) vs . ER - mitochondria contact sites ( red histogram ) ( mean ± ; n = 13 ) . * * p < 0 . 01 .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Left, triple immunofluorescence microscopy labelling of endogenous FATE1 (green), ER labelled by calreticulin (red) and mitochondria labelled by HSP60 staining (blue) in Dox-treated H295R/TRSF-1 cells. One area showing close apposition of red, green and blue staining (white signals) is shown at higher magnification. Scale bar, 10 μm. Right, graph showing quantification of FATE1 signal in total mitochondria (white histogram) vs. ER-mitochondria contact sites (red histogram) (mean ± SEM; n=13). **p<0.01.


H . H295R / TRSF - 1 were fractioned into crude mitochondria ( CM ) , endoplasmic reticulum ( ER ) , purified mitochondria ( PM ) mitochondria - associated membranes ( MAM ) fractions . H , total homogenate . localization of SERCA2 , SOAT1 , VDAC1 , GRP75 , S1R , EMD , Mic60 / mitofilin , FATE1 TOM20 in different fractions was revealed by immunoblot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O

H. H295R/TRSF-1 cells were fractioned into crude mitochondria (CM), endoplasmic reticulum (ER), purified mitochondria (PM) and mitochondria-associated membranes (MAM) fractions. H, total homogenate. The localization of SERCA2, SOAT1, VDAC1, GRP75, S1R, EMD, Mic60/mitofilin, FATE1 and TOM20 proteins in the different fractions was revealed by immunoblot.


A . H295R / TR N - FlagFATE1 were transfected with D1 ER marker for ER ( green ) mtRFP marker for mitochondria ( red ) . Manders ' coefficient for green - red signals colocalization in basal ( histograms ) Dox - treated ( black histograms ) is shown on right ( mean ± ; n = 12 ) . * * * p < 0 . 001 . Scale bars , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. H295R/TR N-FlagFATE1 cells were transfected with D1 ER marker for ER (green) and mtRFP marker for mitochondria (red). Manders' coefficient for green-red signals colocalization in basal (white histograms) and Dox-treated (black histograms) cells is shown on the right (mean ± SEM; n=12). ***p<0.001. Scale bars, 10 μm.


B . Effect of FATE1 expression on ER - mitochondria in H295R / TR N - FlagFATE1 measured using a split - GFP probe [ 29 ] . is shown in graph . histogram , number of objects / in basal conditions ; black histogram , number of objects / in Dox - treated ( mean ± ; n = 6 with 90 analyzed in total ) . * * p < 0 . 01 . Scale bars , 5 μm .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Effect of FATE1 expression on ER-mitochondria distance in H295R/TR N-FlagFATE1 cells measured using a split-GFP probe [29]. Quantification is shown in the graph. White histogram, number of fluorescent objects/cell in basal conditions; black histogram, number of fluorescent objects/cell in Dox-treated cells (mean ± SEM; n=6 with 90 cells analyzed in total). **p<0.01. Scale bars, 5 μm.


C . Transmission electron microscopy of H295R / TR N - FlagFATE1 cultured in basal conditions or treated with Dox . ER - mitochondria contacts are indicated by arrowheads . Scale bars , 200 nm . Right , of number of contacts normalized by number of mitochondria of mitochondria with ER contact sites is shown in basal conditions ( histograms ) after Dox treatment ( black histograms ) of ( mean ± ; n = 38 for basal 47 for Dox - treated ) . * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Transmission electron microscopy images of H295R/TR N-FlagFATE1 cells cultured in basal conditions or treated with Dox. ER-mitochondria contacts are indicated by white arrowheads. Scale bars, 200 nm. Right, quantification of the number of contacts normalized by the number of mitochondria and the percentage of mitochondria with ER contact sites is shown in basal conditions (white histograms) and after Dox treatment (black histograms) of cells (mean ± SEM; n=38 for basal and 47 for Dox-treated cells). ***p<0.001.


D . Transmission electron microscopy of H295R / TRSF - 1 cultured in basal conditions or treated with Dox . ER - mitochondria contacts are indicated by arrowheads . Scale bars , 200 nm . Right , of number of contacts normalized by number of mitochondria of mitochondria with ER contact sites is shown in basal conditions ( histograms ) after Dox treatment ( black histograms ) of ( mean ± ; n = 41 for basal 42 for Dox - treated ) . * * * p < 0 . 001 .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Transmission electron microscopy images of H295R/TRSF-1 cells cultured in basal conditions or treated with Dox. ER-mitochondria contacts are indicated by white arrowheads. Scale bars, 200 nm. Right, quantification of the number of contacts normalized by the number of mitochondria and the percentage of mitochondria with ER contact sites is shown in basal conditions (white histograms) and after Dox treatment (black histograms) of cells (mean ± SEM; n=41 for basal and 42 for Dox-treated cells). ***p<0.001.


E . Mitochondrial shape ( BacMam Mitochondria - RFP ; in red ) in H295R / TR N - FlagFATE1 shown by confocal microscopy in basal conditions after Dox treatment . FATE1 ( green ) , DAPI ( blue ) . A higher magnification of merged green red signal is shown in insets . Scale bars , 10 μm . F . Mitochondrial fragmentation index in H295R / TR N - FlagFATE1 after Dox treatment ( red histogram ) compared to cultured in basal conditions ( histogram ) ( mean ± ; n = 111 for basal 101 for Dox - treated ) . * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Mitochondrial shape (BacMam Mitochondria-RFP; in red) in H295R/TR N-FlagFATE1 cells shown by fluorescence confocal microscopy in basal conditions and after Dox treatment. FATE1 (green), DAPI (blue). A higher magnification of merged green and red signal is shown in the insets. Scale bars, 10 μm.F. Mitochondrial fragmentation index in H295R/TR N-FlagFATE1 cells after Dox treatment (red histogram) compared to cells cultured in basal conditions (white histogram) (mean ± SEM; n=111 for basal and 101 for Dox-treated cells). ***p<0.001.


G . Mitochondrial fragmentation index in H295R / TRSF - 1 after Dox treatment ( green histogram ) compared to cultured in basal conditions ( histogram ) . ( mean ± ; n = 37 for basal 49 for Dox - treated ) . * * * p < 0 . 001 .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Mitochondrial fragmentation index in H295R/TRSF-1 cells after Dox treatment (green histogram) compared to cells cultured in basal conditions (white histogram). (mean ± SEM; n=37 for basal and 49 for Dox-treated cells). ***p<0.001.


H . Mitochondrial potential ( ∆ Ψ ) measured by TMRM after Dox treatment of H295R / TR N - FlagFATE1 ( red histogram ) compared to cultured in basal conditions ( histogram ) . Mean ± is shown . ( mean ± ; n = 3 with 12 replicates / experiment ) . ns , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Mitochondrial membrane potential (∆Ψ) measured by TMRM fluorescence after Dox treatment of H295R/TR N-FlagFATE1 cells (red histogram) compared to cells cultured in basal conditions (white histogram). Mean ± SEM is shown. (mean ± SEM; n=3 with 12 replicates/experiment). ns, not significant.


A . MitochondrialCa2 + uptake . H295R / TR parental clone ( / grey histograms ) H295R / TRSF - 1 ( / green histograms ) , H295R / TR N - FlagFATE1 ( / red histograms ) H295R / TRFATE1 ( / violet histograms ) ( mean ± ; n = 3 - 5 with 12 replicates / condition ) . * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not significant . B . CytosolicCa2 + . Histograms as in A ( mean ± ; n = 3 - 5 with 12 replicates / condition ) . ns , not significant .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. MitochondrialCa2+ uptake. H295R/TR parental clone (white/grey histograms) H295R/TRSF-1 (white/green histograms), H295R/TR N-FlagFATE1 (white/red histograms) and H295R/TRFATE1 cells (white/violet histograms) (mean ± SEM; n=3-5 with 12 replicates/condition). **p<0.01; ***p<0.001; ns, not significant.B. CytosolicCa2+ concentration. Histograms as in A (mean ± SEM; n=3-5 with 12 replicates/condition). ns, not significant.


C . Mitochondrial ( left ) ( right ) Ca2 + concentrations in Dox - treated H295R / TRSF - 1 transfected with control ( siC ; histograms ) or FATE1 - specific ( siFATE1 ; green histograms ) siRNAs ( mean ± ; n = 3 - 5 with 12 replicates / condition ) . * * * p < 0 . 001 ; ns , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Mitochondrial (left) and cytosolic (right) Ca2+ concentrations in Dox-treated H295R/TRSF-1 cells transfected with control (siC; white histograms) or FATE1-specific (siFATE1; green histograms) siRNAs (mean ± SEM; n=3-5 with 12 replicates/condition). ***p<0.001; ns, not significant.


D . Caspase 3 / 7 activity in H295R / TR N - FlagFATE1 cultured in basal conditions ( histograms ) or in presence of Dox ( red histograms ) treated with H2O2 ( 500 μM ) C2 - ceramide ( 50 μM ) or staurosporine ( STS ) ( 1 μM ) . ( mean ± ; n = 5 - 8 with 3 replicates / condition ) . * p < 0 . 05 ; * * p < 0 . 01 ; ns , not significant .

O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Caspase 3/7 activity in H295R/TR N-FlagFATE1 cells cultured in basal conditions (white histograms) or in the presence of Dox (red histograms) treated with H2O2 (500 μM) C2-ceramide (50 μM) or staurosporine (STS) (1 μM). (mean ± SEM; n=5-8 with 3 replicates/condition). *p<0.05; **p<0.01; ns, not significant.


E . Caspase 3 / 7 activity in Dox - treated H295R / TRSF - 1 transfected with control ( siC ; histograms ) or FATE1 - specific ( siFATE1 ; green histograms ) siRNAs treated with H2O2 or STS ( mean ± ; n = 5 - 8 with 3 replicates / condition ) . * p < 0 . 05 ; ns , not significant .

O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Caspase 3/7 activity in Dox-treated H295R/TRSF-1 cells transfected with control (siC; white histograms) or FATE1-specific (siFATE1; green histograms) siRNAs and treated with H2O2 or STS (mean ± SEM; n=5-8 with 3 replicates/condition). *p<0.05; ns, not significant.


F . Flow cytometry of TUNEL in H295R / TR N - Flag FATE1 cultured in basal conditions or in presence of Dox treated with vehicle ( control ) H2O2 ( 500 μM ) , C2 - ceramide ( 50 μM ) or staurosporine ( STS ) ( 1 μM ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Flow cytometry analysis of TUNEL staining in H295R/TR N-Flag FATE1 cells cultured in basal conditions or in the presence of Dox treated with vehicle (control) H2O2 (500 μM), C2-ceramide (50 μM) or staurosporine (STS) (1 μM).


G . Caspase 3 / 7 activity in H295R / TR N - FlagFATE1 cultured in basal conditions ( histograms ) or in presence of Dox ( red histograms ) treated with mitotane ( 50 μM ) ( mean ± ; n = 5 - 8 with 3 replicates / condition ) . * * p < 0 . 01 .

O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Caspase 3/7 activity in H295R/TR N-FlagFATE1 cells cultured in basal conditions (white histograms) or in the presence of Dox (red histograms) and treated with mitotane (50 μM) (mean ± SEM; n=5-8 with 3 replicates/condition). **p<0.01.


H . Caspase 3 / 7 activity in Dox - treated H295R / TRSF - 1 transfected with control ( siC ; histograms ) or FATE1 - specific ( siFATE1 ; green histograms ) siRNAs treated with mitotane ( mean ± ; n = 5 - 8 with 3 replicates / condition ) . * p < 0 . 05 .

O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Caspase 3/7 activity in Dox-treated H295R/TRSF-1 cells transfected with control (siC; white histograms) or FATE1-specific (siFATE1; green histograms) siRNAs and treated with mitotane (mean ± SEM; n=5-8 with 3 replicates/condition). *p<0.05.


A . Immunohistochemistry of FATE1 expression in normal adrenal cortex adrenal hyperplasia , adenoma carcinoma ( ACC ) . Testis tissue was used as positive control . Scale bars , 200 µm ( low magnification ) ; 50 μ m ( high magnification ) . B . Table summarizing relative of FATE1IHC ( H - score ) in groups of normal , benign malignant tissues . highest FATE1 expression were found in ACC . n , number of samples studied for each category of tissues . NBST , normal benign steroidogenic tissues .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

A. Immunohistochemistry analysis of FATE1 expression in normal adrenal cortex and adrenal hyperplasia, adenoma and carcinoma (ACC). Testis tissue was used as positive control. Scale bars, 200 µm (low magnification); 50 μ m (high magnification).B. Table summarizing the relative intensity of FATE1IHC staining (H-score) in groups of normal, benign and malignant tissues. The highest FATE1 expression levels were found in ACC. n, number of samples studied for each category of tissues. NBST, normal and benign steroidogenic tissues.


C . Prognostic parameters in ACC . Results are presented as hazard ratios ( HR ) including 95 % confidence interval ( CI ) . significance level was set at α = 5 % for comparisons . results for SF - 1 , FATE1 combined SF - 1 / FATE1 expression are shown both in univariate mutivariate ( adjusted for , sex ENSAT tumour stage ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

C. Prognostic parameters in ACC patients. Results are presented as hazard ratios (HR) including 95% confidence interval (CI). The significance level was set at α=5% for all comparisons. The results for SF-1, FATE1 and combined SF-1/FATE1 expression levels are shown both in univariate and mutivariate (adjusted for age, sex and ENSAT tumour stage) analysis.


D . Overall survival of ACC according to FATE1 expression in their tumours , as measured by quantitative immunohistochemistry ( low , green curve ; high , red curve ) in mutivariate . p = 0 . 008 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Overall survival of ACC patients according to FATE1 expression in their tumours, as measured by quantitative immunohistochemistry (low, green curve; high, red curve) in mutivariate analysis. p=0.008.


A ) of effect of BCL - 2 - family on HCT AKO , measured as of showing released cyt c or pyknotic nuclei . Data correspond to at least three independent experiments , n > 30 per condition per experiment . * * * p < 0 , 001 * p < 0 , 05 with respect to GFP condition .

O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) Quantification of the effect of individual BCL-2-family proteins on HCT AKO cell death, measured as percentage of cells showing released cyt c or pyknotic nuclei. Data correspond to at least three independent experiments, n>30 cells per condition per experiment. *** p<0,001 and * p<0,05 with respect to GFP condition.


C ) Western blot of SMAC cyt c release in GFP - BAX , tBID - GFP GFP - BCLXL expressing at 12 h , 16 h 24 h after transfection in HCT AKO . SN ( supernatant , fraction ) , P ( pellet , fraction ) PS ( Ponceau ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) Western blot analysis of SMAC and cyt c release in GFP-BAX, tBID-GFP and GFP-BCLXL expressing cells at 12 h, 16 h and 24 h after transfection in HCT AKO. SN (supernatant, cytosolic fraction), P (pellet, membrane fraction) and PS (Ponceau Staining).


F ) Kinetics of expression of tBID - GFP ( blue ) PI intake upon tBID expression ( purple ) unstransfected HCT116 AKO ( in grey ) . Plots show average ( lines ) its standard deviation ( shaded areas ) from 3 technical replicates 3 independent experiments . was obtained by total per well using incucyte system every 60 min .

O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F) Kinetics of the expression of tBID-GFP (blue) and PI intake upon tBID expression (purple) and unstransfected HCT116 AKO cells (in grey). Plots show the average fluorescence intensity (dark lines) and its standard deviation (shaded areas) from 3 technical replicates and 3 independent experiments. Fluorescence intensity was obtained by analysis the total fluorescence per well using incucyte system every 60 min.


G ) of PI + from total transfected with GFP - BAX , tBID - GFP , GFP - BCLXL GFP . n = 3 independent experiments with > 10000 per condition per experiment . * * * p < 0 , 001 with respect to untransfected condition .

O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G) Quantification of PI+ cells from total transfected cells with GFP-BAX, tBID-GFP, GFP-BCLXL and GFP. n=3 independent experiments with >10000 cells per condition per experiment. *** p<0,001 with respect to untransfected condition.


H ) induced by tBID - GFP in HCT AKO in absence presence of ZVAD , NSA Ferrostatin - 1 . Data correspond to three independent experiments , n > 30 per condition per experiment . * * * p < 0 , 001 with respect to MOCK condition .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H) Cell death induced by tBID-GFP in HCT AKO cells in the absence and presence of ZVAD, NSA and Ferrostatin-1. Data correspond to three independent experiments, n>30 cells per condition per experiment. *** p<0,001 with respect to MOCK condition.


A ) Representative confocal immunofluorescence bright field ( BF ) of HCT DKO expressing tBID - GFP ( in green ) treated or not with COMBO ( ABT737 + S63586 ) . Cyt c shown in magenta nuclei in cyan . Scale bar 10 µm . B ) Effect of tBID - GFP mCherry - BCLXL on , measured as of showing released cyt c or pyknotic nuclei . Data correspond to at least three independent experiments , n > 30 per condition per experiment . * * * p < 0 , 001 , * * p < 0 , 025 * p < 0 , 05 with respect to tBID - GFP in HCT AKO condition .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

A) Representative confocal immunofluorescence and bright field (BF) images of HCT DKO cells expressing tBID-GFP (in green) treated or not with COMBO (ABT737 + S63586). Cyt c shown in magenta and nuclei in cyan. Scale bar 10 µm. B) Effect of tBID-GFP and mCherry-BCLXL on cell death, measured as percentage of cells showing released cyt c or pyknotic nuclei. Data correspond to at least three independent experiments, n> 30 cells per condition per experiment. *** p<0,001, ** p<0,025 and * p<0,05 with respect to tBID-GFP in HCT AKO condition.


C ) Representative confocal immunofluorescence of HCT DKO upon indicated treatments . Nuclei ( cyan ) cyt c ( magenta ) are shown . Scale bar 10µm . Arrowheads represent showing released cytochrome c pyknotic nuclei .

O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O

C) Representative confocal immunofluorescence images of HCT DKO cells upon indicated treatments. Nuclei (cyan) and cyt c (magenta) are shown. Scale bar 10µm. Arrowheads represent cells showing released cytochrome c and pyknotic nuclei.


E ) % of induced by TRAIL ( 1µg / ml ) , COMBO their combination at different treatment durations on HCT DKO measured as in panel B . Paired Student ' s t - * * p < 0 , 025 * p < 0 , 05 with respect to MOCK condition .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) % of cell death induced by TRAIL (1µg/ml), COMBO and their combination at different treatment durations on HCT DKO measured as in panel B. Paired Student's t-test ** p<0,025 and * p<0,05 with respect to MOCK condition.


I ) Effect of TRAIL ( 1µg / ml ) , COMBO their combination on HCT DKO TKO at different time points normalized to control . Each dot corresponds to average of 3 technical replicates from 4 independent experiments . * * * p < 0 , 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I) Effect of TRAIL (1µg/ml), COMBO and their combination on HCT DKO and TKO cell death at different time points normalized to control cells. Each dot corresponds to the average of 3 technical replicates from 4 independent experiments. *** p<0,001.