C Extracellular matrix following trichrome in sg - CTRL sg - HMGCL # 2 pancreatic tumors sections ( n = 8 9 mice / group respectively , left panel ) . Data are expressed as mean of of total tissue ± . Significance was defined by Mann - Whitney . * * : p < 0 . 01 . Representative of trichrome in sg - CTRL sg - HMGCL pancreatic tumors . Scale bar : 100 µm ( right panel ) .

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C Extracellular matrix quantification following trichrome staining in sg-CTRL and sg-HMGCL #2 pancreatic tumors sections (n=8 and 9 mice/group respectively, left panel). Data are expressed as mean of percentage of total tissue area ± SEM. Significance was defined by Mann-Whitney test. **: p<0.01. Representative images of trichrome staining in sg-CTRL and sg-HMGCL pancreatic tumors. Scale bar: 100 µm (right panel).


D , E of volume ( D ) ( E ) of sg - CTRL pancreatic tumors treated with 0 . 9 % NaCl ( i . p . ) ( n = 4 ) , pancreatic tumors from two different clones of sg - HMGCL treated with 0 . 9 % NaCl ( i . p . ) or βOHB ( 100mg / kg / bi - weekly , i . p . ) ( n = 10 / group ) . Data are expressed as mean of volume or ± . Significance was defined by Mann - Whitney . ns : not significant , * * p < 0 . 01 .

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D, E Quantification of volume (D) and weight (E) of sg-CTRL pancreatic tumors treated with 0.9% NaCl (i.p.) (n=4), and pancreatic tumors from two different clones of sg-HMGCL treated with 0.9% NaCl (i.p.) or βOHB (100mg/kg/bi-weekly, i.p.) (n=10/group). Data are expressed as mean of tumor volume or weight ± SEM. Significance was defined by Mann-Whitney test. ns: not significant, **p<0.01.


H [ U - 13C ] βOHB tracing into TCA intermediate : citrate in sg - CTRL sg - HMGCL # 2 # 3 PANC - 1 cultured in indicated glucose concentrations . Data are expressed as mean ± . ( n = 2 independent experiments ) . Significance was defined by one - way ANOVA followed by a Bonferroni ' s multiple comparisons , only significances between sg - HMGCL # 2 , # 3 PANC - 1 sg - CTRL PANC - 1 under same culture condition are mentioned . * * * p < 0 . 001 .

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H [U-13C]βOHB tracing into TCA intermediate: citrate in sg-CTRL and sg-HMGCL #2 and #3 PANC-1 cells cultured in indicated glucose concentrations. Data are expressed as mean ± SEM. (n=2 independent experiments). Significance was defined by one-way ANOVA followed by a Bonferroni's multiple comparisons test, only significances between sg-HMGCL #2, #3 PANC-1 cells and sg-CTRL PANC-1 cells under the same culture condition are mentioned. ***p<0.001.


( B ) Box - - whisker plots of ketone bodies ( KB ) metagene score defined as first component of PCA of genes in tumors ( n = 728 ) , in metastases ( n = 76 ) , in liver metastases specifically ( n = 35 ) . Box - - whisker plot were defined with default parameters by median value ( central band at 50th percentile ) , interquartile ranges ( IQR , box limited by 25th 75th percentile ) whisker boundaries defined at 1 . 5x IQR . Significance was defined by Student ' s t - .

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(B) Box-and-whisker plots of ketone bodies (KB) metagene score defined as first component of PCA of genes in primary tumors (n=728), in all metastases (n=76), and in liver metastases specifically (n=35). Box-and-whisker plot were defined with default parameters by median value (central band at the 50th percentile), interquartile ranges (IQR, box limited by 25th and 75th percentile) and whisker boundaries defined at 1.5x IQR. Significance was defined by Student's t-test.


D - F Effect of βOHB treatment on liver metastatic incidence , status . Representative HPS of liver lobes from metastatic mice treated with βOHB ( 100mg / kg / day , i . p . ) or 0 . 9 % NaCl ( i . p . ) ( n = 8 mice / group ) . Metastatic areas are separated from liver by yellow dotted lines . Scale bar : 1mm 500 µm for insets ( D ) . Number of metastasis per lobe classified in small , medium large ( E ) . Pathological status of metastasis from mice treated with βOHB or NaCl ( n = 13 or 5 lobes / group , respectively ) . Data are expressed as of total metastasis in liver lobes presenting metastasis ( F ) .

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D-F Effect of βOHB treatment on liver metastatic incidence, size and status. Representative HPS staining of liver lobes from metastatic mice treated with βOHB (100mg/kg/day, i.p.) or 0.9% NaCl (i.p.) (n=8 mice/group). Metastatic areas are separated from liver by yellow dotted lines. Scale bar: 1mm and 500 µm for insets (D). Number and size of metastasis per lobe and classified in small, medium and large size (E). Pathological status of metastasis from mice treated with βOHB or NaCl (n=13 or 5 lobes/group, respectively). Data are expressed as percentage of total metastasis in all liver lobes presenting metastasis (F).


( A ) Mouse were immunostained with anti - neurofilament antibody ( blue ) , showing mesencephalic projection . Dispersed axon projections ( arrows point to of superior colliculus ) decreased axon ( arrowheads point to of pretectal commissure ) were observed in Plcg1f / f ; Nes - Cre mice at E11 . 5 ( n = 20 ) . areas of superior colliculus is shown magnified in each inset ( scale bar = 200 μm )

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(A) Mouse embryos were immunostained with anti-neurofilament antibody (blue), showing mesencephalic projection. Dispersed axon projections (arrows point to the area of the superior colliculus) and decreased axon fibers (arrowheads point to the area of the pretectal commissure) were observed in Plcg1f/f;Nes-Cre mice at E11.5 (n = 20). The areas of the superior colliculus is shown magnified in each inset (scale bar = 200 μm)


( B ) Representative between posterior anterior borderlines of superior colliculus ( a b ) . red boxes were drawn as rostrocaudal region of interest ( ROI ) on upper part of pretectal commissure in embryonic mesencephalon ( scale bar = 100 μm )

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(B) Representative images between the posterior and anterior borderlines of the superior colliculus (a and b). The red boxes were drawn as rostrocaudal region of interest (ROI) based on the upper part of the pretectal commissure in the embryonic mesencephalon (scale bar = 100 μm)


( C D ) axons passing through ROI was quantified by measuring in ROI , distribution of axons was statistically analyzed ( n = 5 ; t - ; * * P < 0 . 005 )

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(C and D) The axons passing through the ROI was quantified by measuring the fluorescence intensity in the ROI, and the distribution of axons was statistically analyzed (n = 5; t-test; **P < 0.005)


( E ) Whole - mount immunostaining show neurofilament ( red ) , netrin - 1 ( purple ) , DCC ( green ) , respectively . An E11 . 5 Plcg1f / f ; Nes - Cre showed shortened dorsal ramus opthalmic nerves ( yellow boxes ) . Scale bar : 200

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(E) Whole-mount immunostaining images show neurofilament (red), netrin-1 (purple), and DCC (green), respectively. An E11.5 Plcg1f/f;Nes-Cre embryo showed shortened dorsal ramus and opthalmic nerves (white and yellow boxes). Scale bar: 200 μm


( F - G ) Enlarged views of opthalmic nerve ( yellow arrow heads ) dorsal ramus ( arrow heads ) ( scale bar : 50 μm )

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(F - G) Enlarged views of the opthalmic nerve (yellow arrow heads) and dorsal ramus (white arrow heads) (scale bar: 50 μm)


( H ) Nerve measurements ; Op : ophthalmic nerve ; DR : dorsal ramus ( n = 3 ; t - ; * * P < 0 . 005 ) . Data are presented as mean ± s . e .

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(H) Nerve length measurements; Op: ophthalmic nerve; DR: dorsal ramus (n = 3; t-test; **P < 0.005). Data are presented as the mean ± s.e.m


( I ) Double immunostaining . pattern of expression colocalization of DCC ( green ) - TH ( red ) - positive axon bundles extending toward alar thalamus . Scale bar : 100

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(I) Double immunostaining images. The pattern of expression and colocalization of DCC (green)- and TH (red)-positive axon bundles extending toward the alar thalamus. Scale bar: 100 μm


( A ) In mesencephalic neurons , phosphorylation of PLCγ1 on Y783 was increased proportionally to netrin - 1 . lower graph presents of band intensities in ( A ) ( n = 3 )

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(A) In primary mesencephalic neurons, the phosphorylation of PLCγ1 on Y783 was increased proportionally to the netrin-1 concentration. The lower graph presents quantification of the band intensities in (A) (n = 3)


( B ) Phosphorylation of PLCγ1 on Y783 was decreased after knockdown of DCC receptor by siRNA ( siDcc ) ( N = 3 )

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(B) Phosphorylation of PLCγ1 on Y783 was decreased after knockdown of the DCC receptor by siRNA (siDcc) (N = 3)


( C ) Western blot for NTN1 secretion . Lane # 1 : transfection - free lysate , # 2 : transfection - free supernatant , # 3 : NTN1 - transfected lyasate , # 3 # 4 : NTN - 1 - transfected

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(C) Western blot for the NTN1 secretion test. Lane #1: transfection-free lysate, #2: transfection-free supernatant, #3: NTN1-transfected cell lyasate, #3 and #4: NTN-1- transfected supernatant


( D ) Ventral mesencephalic ( A9 ) cultures ( daggers ) derived from control or Plcg1f / f ; Nes - Cre mice , were co - cultured with netrin - 1 - releasing 293T ( asterisks ; scale bar = 100 μm ) . areas delineated by boxes between mesencephalon tissue 293T are magnified in respective insets ( n = 4 )

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(D) Ventral mesencephalic (A9) cultures (daggers) derived from control or Plcg1f/f;Nes-Cre mice, were co-cultured with netrin-1-releasing 293T cell aggregates (asterisks; scale bar = 100 μm). The areas delineated by the white boxes between the mesencephalon tissue and 293T cell aggregates are magnified in the respective insets (n = 4)


(F) Mean angle of the axon extension


( G ) of ventral mesencephalic axons . bar graphs indicate percentages of proper targeting of ventral mesencephalic axons correctly targeting netrin - 1 - releasing ( n = 200 ) . Each experimental set was performed in

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(G) Quantification of the ventral mesencephalic axons. The bar graphs indicate the percentages of proper targeting of ventral mesencephalic axons correctly targeting the netrin-1-releasing cell aggregates (n = 200). Each experimental set was performed in quadruplicate


( H ) Anti - tyrosine hydroxylase ( TH ) immunostaining in ventral mesencephalic ( VM ) tissue . ( scale bar = 200 μm )

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(H) Anti-tyrosine hydroxylase (TH) immunostaining in ventral mesencephalic (VM) tissue. (scale bar = 200 μm)


( I ) mesencephalic neuron culture in absence or presence of NTN1 - conditioned media ( n = 100 ; scale bar : 500μm )

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(I) Primary mesencephalic neuron culture in the absence or presence of NTN1-conditioned media (n = 100; scale bar: 500μm)


( K ) Mean of neurites ( F - value : 1 . 82 , t - , P * * < 0 . 005 )

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(K) Mean length of neurites (F-value:1.82, t-test, P**<0.005)


( A ) Co - immunoprecipitation data showed that PLCγ1 did not interact with DCC receptor ( n = 2 , N = 3 ) .

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(A) Co-immunoprecipitation data showed that PLCγ1 did not interact with the DCC receptor (n = 2, N = 3).


( B ) Immunoprecipitation showed that Src kinase interacted with PLCγ1 . Rac1 is a positive interacting control for PLCγ1 ( N = 3 ) .

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(B) Immunoprecipitation assay showed that Src kinase interacted with PLCγ1. Rac1 is a positive interacting control for PLCγ1 (N = 3).


( C ) In vitro kinase confirmed that Src kinase directly phosphorylated Y783 of PLCγ1 . ( D ) of band intensities in ( C ) ( N = 3 ) .

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(C) In vitro kinase assay confirmed that Src kinase directly phosphorylated Y783 of PLCγ1. (D) Quantification of the band intensities in (C) (N = 3).


( E ) Src kinase inhibitor reduced specific phosphorylation of PLCγ1 on Y783 ( arrow ; Src I IC50 conc = 44 nM ; N = 3 ) .

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(E) Src kinase inhibitor reduced the specific phosphorylation of PLCγ1 on Y783 (arrow; Src I IC50 conc = 44 nM; N = 3).


( F ) Rac1 activity ( N = 3 ) . ( G ) of Rac1 activity in ( F ) .

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(F) Rac1 activity assay (N = 3). (G) Quantification of Rac1 activity levels in (F).


( H ) Representative immunoblot of cultured Plcg1f / f ( n = 2 , N = 3 ) Plcg1f / f ; Nes - Cre ( n = 2 , N = 3 ) mesencephalic neurons stimulated with conditioned media containing Netrin - 1 .

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(H) Representative immunoblot of cultured Plcg1f/f (n = 2, N = 3) and Plcg1f/f;Nes-Cre (n = 2, N = 3) mesencephalic neurons stimulated with conditioned media containing Netrin-1.


( B ) Immunohistochemical of control Plcg1 conditional knockout ( cKO ) sagittal brain sections . deletion of Plcg1 caused structural changes in SI OT projections in Plcg1f / f ; Nes - Cre ( d ) mice compared to controls ( a ) ( n = 4 ; scale bar = 1 mm ) . areas delineated by boxes ( insets ) are magnified of OT ( b e ) , yellow boxes are magnified of SI ( c f ) . ( C ) of axon bundle distribution . Green boxes in ( b ) ( e ) were selected as tegions of interest ( ROIs ) using ZEN software ( n = 4 ) ( one - way ANOVA , F - value : 118 . 5745 , P * * < 0 . 005 ) .

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(B) Immunohistochemical staining of control and Plcg1 conditional knockout (cKO) sagittal brain sections. The deletion of Plcg1 caused structural changes in SI and OT projections in the Plcg1f/f;Nes-Cre (d) mice compared to the controls (a) (n = 4; scale bar = 1 mm). The areas delineated by the white boxes (insets) are magnified images of the OT (b and e), and the yellow boxes are magnified images of the SI (c and f). (C) Quantification of the axon bundle distribution. Green boxes in (b) and (e) were selected as tegions of interest (ROIs) using the ZEN software (n = 4) (one-way ANOVA, F-value: 118.5745, P**<0.005).


( D ) Representative of tyrosine hydroxylase ( TH ) - positive neurons ( scale bar = 1 mm ) . areas delineated by boxes are magnified in insets . yellow arrow indicated misoriented TH - positive axons ( magenta : TH ) .

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(D) Representative images of tyrosine hydroxylase (TH)- positive neurons (scale bar = 1 mm). The areas delineated by the white boxes are magnified in the insets. The yellow arrow indicated misoriented TH-positive axons (magenta: TH).


( E ) intensities in ROIs were measured according to ( n = 4 ) ( one - way ANOVA , F - value : 271 . 8 , * * P < 0 . 005 ) . Cumulative values of intensities per section are shown in lower graph ( S : section ) .

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(E) Fluorescence intensities in the ROIs were measured according to distance (n = 4) (one-way ANOVA, F-value: 271.8, **P<0.005). Cumulative values of fluorescence intensities per section are shown in the lower graph (S: section).


( F ) In situ hybridization of PLCγ1 in mDA neurons ( scale bar = 50 μm ) ( green : TH , violet : DIG - labeled probe ) .

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(F) In situ hybridization of PLCγ1 in mDA neurons (scale bar = 50 μm) (green: TH, violet: DIG-labeled probe).


( G ) Representative of CC axon bundles in control ( left ) a Plcg1f / f ; Nes - Cre ( right ) mice ( n = 4 ) . tip of CC was triangular in control mouse , but oval in Plcg1f / f ; Nes - Cre mouse . areas delineated by boxes are magnified in insets ( red : neurofilament , green : glial fibrillary acidic , blue : Hoechst ; scale bar = 1 mm ) .

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(G) Representative images of the CC axon bundles in control (left) and a Plcg1f/f;Nes-Cre (right) mice (n = 4). The tip of the CC was triangular in the control mouse, but oval in the Plcg1f/f;Nes-Cre mouse. The areas delineated by the white boxes are magnified in the insets (red: neurofilament, green: glial fibrillary acidic protein, blue: Hoechst; scale bar = 1 mm).


( H ) of CC in Plcg1f / f ; Nes - Cre mouse was 36 % smaller than that in control mouse ( scale bar = 500 μm ) ( I ) Quantitative comparison of CC areas in control Plcg1f / f ; Nes - Cre mice ( n = 4 ; t - ; * * p < 0 . 005 ) . Data are presented as mean ± s . e . m .

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(H) The area of the CC in the Plcg1f/f;Nes-Cre mouse was 36% smaller than that in the control mouse (scale bar = 500 μm) (I) Quantitative comparison of the CC areas in the control and Plcg1f/f;Nes-Cre mice (n = 4; t-test; **p < 0.005). Data are presented as the mean ± s.e.m.


( A ) Cumulative histograms on fractional anisotropy ( FA ) values . ( B ) , show CC volume relative to FA values . As FA values increased , structural ( CC volume differences increased between control Plcg1f / f ; Nes - Cre mice . ( n = 4 ; Shapiro - Wilk t - ; * p < 0 . 05 ) . Data are presented as mean ± s . e . m .

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(A) Cumulative histograms based on fractional anisotropy (FA) values. (B), show the CC volume relative to FA values. As FA values increased, the structural (CC volume differences increased between the control and Plcg1f/f;Nes-Cre mice. (n = 4; Shapiro-Wilk and t-test; *p < 0.05). Data are presented as the mean ± s.e.m.


( C ) , Cumulative histograms on fractional anisotropy ( FA ) values . ( D ) , show number of tensors relative to FA values . As FA values increased , structural number of tensors ) differences increased between control Plcg1f / f ; Nes - Cre mice . ( n = 4 ; Shapiro - Wilk t - ; * p < 0 . 05 ) . Data are presented as mean ± s . e . m .

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(C), Cumulative histograms based on fractional anisotropy (FA) values. (D), show the number of tensors relative to FA values. As FA values increased, the structural number of tensors) differences increased between the control and Plcg1f/f;Nes-Cre mice. (n = 4; Shapiro-Wilk and t-test; *p < 0.05). Data are presented as the mean ± s.e.m.


( E ) Cumulative histograms on fractional anisotropy ( FA ) values . ( F ) show CC volume number of tensors relative to FA values . As FA values increased , structural ( CC volume number of tensors ) differences increased between control Plcg1f / f ; Nes - Cre mice . Depending on number of tensors , in CC appeared to be shortened ( E , F ) . ( n = 4 ; Shapiro - Wilk t - ; * p < 0 . 05 ) . Data are presented as mean ± s . e . m .

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(E) Cumulative histograms based on fractional anisotropy (FA) values. (F) show the CC volume and the number of tensors relative to FA values. As FA values increased, the structural (CC volume and the number of tensors) differences increased between the control and Plcg1f/f;Nes-Cre mice. Depending on the number of tensors, the length in the CC appeared to be shortened (E, F). (n = 4; Shapiro-Wilk and t-test; *p < 0.05). Data are presented as the mean ± s.e.m.


( A ) ( B ) CPT sensitivity assayed by 10 - fold serial dilutions of different mutant combinations on YPAD plates . Four biological replicates have been performed .

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(A) (B) CPT sensitivity assayed by 10-fold serial dilutions of different mutant combinations on YPAD plates. Four biological replicates have been performed.


( C ) Colony - forming unit assays to increasing CPT concentrations of same mutant combinations as in ( B ) . Mean values ± SD are plotted . Four biological replicates have been performed .

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(C) Colony-forming unit assays to increasing CPT concentrations of the same mutant combinations as in (B). Mean values ± SD are plotted. Four biological replicates have been performed.


( D ) UV sensitivity assayed by 10 - fold serial dilutions of different mutant combinations with rad3 - 102 allele on YPAD plates after exposure to indicated UV - C doses . Five biological replicates have been performed .

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(D) UV sensitivity assayed by 10-fold serial dilutions of different mutant combinations with rad3-102 allele on YPAD plates after exposure to the indicated UV-C doses. Five biological replicates have been performed.


( F ) BIR frequencies ( Lys + survivors among total ) for WT indicated mutant strains are plotted on a logarithmic scale . Data represent mean ± SD from 3 to 6 independent experiments . ns , non - significant difference , * P - value = 0 . 0134 , Mann - Whitney unpaired t .

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(F) BIR frequencies (Lys+ survivors among total cells) for the WT and indicated mutant strains are plotted on a logarithmic scale. Data represent the mean ± SD from 3 to 6 independent experiments. ns, non-significant difference, * P-value = 0.0134, Mann-Whitney unpaired t test.


( C ) UV sensitivity assayed by 10 - fold serial dilutions of different mutant combinations with rad3 - 102 allele on YPAD plates after exposure to indicated UV - C doses . Four biological replicates have been performed .

(C) UV sensitivity assayed by 10-fold serial dilutions of different mutant combinations with rad3-102 allele on YPAD plates after exposure to the indicated UV-C doses. Four biological replicates have been performed.


( D ) Synthetic combinations of rad3 - 102 mus81 ∆ with rad51 ∆ pol32 ∆ . Tetrads dissected on YPAD medium are shown . Triangles indicate either a severe growth defect or lethality .

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(D) Synthetic combinations of rad3-102 mus81∆ with rad51∆ and pol32∆. Tetrads dissected on YPAD medium are shown. Triangles indicate either a severe growth defect or lethality.


( A ) of content by flow cytometry of G1 phase synchronized wild type , rad52 ∆ , rad51 ∆ , mus81 ∆ pol32 ∆ further released into S phase . were synchronized in G1 with α - factor , treated with DMSO or 100 µM CPT , let in G1 for 1 h , released into S phase . Three biological replicates have been performed .

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(A) Analysis of DNA content by flow cytometry of G1 phase synchronized wild type, rad52∆, rad51∆, mus81∆ and pol32∆ cells and further released into S phase. Cells were synchronized in G1 with α -factor, treated with DMSO or 100 µM CPT, let in G1 for 1 h, and released into S phase. Three biological replicates have been performed.


( B ) ( C ) of content by flow cytometry of G1 phase synchronized wild type , rad3 - 102 , rad3 - 102 rad51 ∆ , rad3 - 102 mus81 rad3 - 102 mus81 - dd further released into S phase . were synchronized in G1 with α - factor , untreated or irradiated with 20 J / m2 UV - C , let in G1 for 2h , released into S phase . Three biological replicates have been performed .

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(B) (C) Analysis of DNA content by flow cytometry of G1 phase synchronized wild type, rad3-102, rad3-102 rad51∆, rad3-102 mus81 and rad3-102 mus81-dd cells and further released into S phase. Cells were synchronized in G1 with α-factor, untreated or irradiated with 20 J/m2 UV-C, let in G1 for 2h, and released into S phase. Three biological replicates have been performed.


( A ) of replicated tracks by single - molecule combing in WT , rad52 ∆ , rad51 ∆ , mus81 ∆ pol32 ∆ exposed to CPT . Exponentially growing were treated with DMSO or 50 µM CPT for 2h then - labeled with 50 µM EdU for 20min . were combed on silanized coverslips EdU - labeled was detected by Click chemistry . Graph depicts distribution of EdU tracks in kb . Box whiskers indicate 25 - 75 10 - 90 percentiles , respectively . Median EdU tracks is indicated in kb ( values from two biological replicates were pooled ) . Asterisks indicate P - value of Mann - Whitney unpaired t , * * : p = 0 . 0015 . of EdU track decrease between DMSO CPT conditions is indicated between parentheses for each . Representative of are shown . Red : EdU , green : .

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(A) Analysis of replicated DNA tracks length by single-molecule DNA combing in WT, rad52∆, rad51∆, mus81∆ and pol32∆ cells exposed to CPT. Exponentially growing cells were treated with DMSO or 50 µM CPT for 2h and then pulse-labeled with 50 µM EdU for 20min. DNA fibers were combed on silanized coverslips and EdU-labeled DNA was detected by Click chemistry. Graph depicts the distribution of EdU tracks length in kb. Box and whiskers indicate 25-75 and 10-90 percentiles, respectively. Median EdU tracks length is indicated in kb (values from two biological replicates were pooled). Asterisks indicate the P-value of the Mann-Whitney unpaired t test, **: p=0.0015. The percentage of EdU track length decrease between the DMSO and CPT conditions is indicated between parentheses for each strain. Representative images of DNA fibers are shown. Red and white: EdU, green: DNA.


( C ) of replication intermediates by 2D gel electrophoresis . Replication intermediates were monitored at early origin ARS305 region C in WT , rad3 - 102 , mus81 ∆ rad3 - 102 mus81 ∆ . were synchronized in G1 with α - factor collected at indicated time points after release into S phase . A scheme of studied chromosomal region is shown ( drawn to scale ) . Relevant probes are indicated by gray bars , coordinates of ARS restriction sites are indicated in bp . Two biological replicates have been performed .

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(C) Analysis of replication intermediates by 2D gel electrophoresis. Replication intermediates were monitored at early origin ARS305 and region C in WT, rad3-102, mus81∆ and rad3-102 mus81∆ cells. Cells were synchronized in G1 with α-factor and collected at the indicated time points after release into S phase. A scheme of the studied chromosomal region is shown (drawn to scale). Relevant probes are indicated by gray bars, and coordinates of ARS and restriction sites are indicated in bp. Two biological replicates have been performed.


( A ) of Rad52 - GFP foci formation . An illustrative image of experimental setup is shown . DIC , differential interference contrast ; yellow , Rad52 - GFP ; red , mCHERRY - Pus1 ( compartment marker ) . Scale bar = 5 µm .

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(A) Analysis of nuclear Rad52-GFP foci formation. An illustrative image of the experimental setup is shown. DIC, differential interference contrast; yellow, Rad52-GFP; red, mCHERRY-Pus1 (nuclear compartment marker). Scale bar = 5 µm.


( B ) ( C ) Kinetic of Rad52 foci formation . Wild - type mus81 ∆ containing Rad52 - YFP mCHERRY - Pus1 were synchronized in G1 with α - factor , treated with DMSO or 100 µM CPT , let in G1 for 30 min , released into S phase . were collected at indicated time points visualized by microscopy . Mean ± of with Rad52 foci are shown for each time point . Flow cytometry profiles corresponding experimental setup are shown . Four biological replicates have been performed .

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(B) (C) Kinetic analysis of Rad52 foci formation. Wild-type and mus81∆ cells containing Rad52-YFP and mCHERRY-Pus1 were synchronized in G1 with α-factor, treated with DMSO or 100 µM CPT, let in G1 for 30 min, and released into S phase. Cells were collected at the indicated time points and visualized by fluorescence microscopy. Mean ± SEM of cells with Rad52 foci are shown for each time point. Flow cytometry profiles corresponding the experimental setup are shown. Four biological replicates have been performed.


( D ) Mms4 - Flag10 phosphorylation analyzed by immunoblot in wild type exposed to CPT . Wild - type were synchronized in G1 with α - factor , treated with DMSO or 50 µM CPT , let in G1 for 1 h , released into S phase . were collected at indicated time points Mms4 was immunodetected with anti - Flag antibodies . Clb2 immunodetection serves as a marker for G2 phase entry . FACS profiles corresponding experimental setup are also shown . Two biological replicates have been performed .

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(D) Mms4-Flag10 phosphorylation analyzed by immunoblot in wild type cells exposed to CPT. Wild-type cells were synchronized in G1 with α-factor, treated with DMSO or 50 µM CPT, let in G1 for 1 h, and released into S phase. Cells were collected at the indicated time points and Mms4 was immunodetected with anti-Flag antibodies. Clb2 immunodetection serves as a marker for G2 phase entry. FACS profiles corresponding the experimental setup are also shown. Two biological replicates have been performed.


( E ) CPT sensitivity assayed by 10 - fold serial dilutions of S - MUS81 G2 - MUS81 alleles compared to wild type , mus81 , mms4 ∆ cdc5 - 2 mutants . Three biological replicates have been performed .

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(E) CPT sensitivity assayed by 10-fold serial dilutions of S-MUS81 and G2-MUS81 alleles compared to the wild type, mus81, mms4∆ and cdc5-2 mutants. Three biological replicates have been performed.


( F ) UV sensitivity assayed by 10 - fold serial dilutions of S - MUS81 G2 - MUS81 alleles in combination with rad3 - 102 on YPAD plates after exposure to indicated UV - C doses . Three biological replicates have been performed .

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(F) UV sensitivity assayed by 10-fold serial dilutions of S-MUS81 and G2-MUS81 alleles in combination with rad3-102 on YPAD plates after exposure to the indicated UV-C doses. Three biological replicates have been performed.


( A ) ( B ) Pulsed - field gel electrophoresis ( PFGE ) of wild type , mus81 ∆ , cdc5 - 2 G2 - MUS81 in response to CPT . were synchronized in G1 with α - factor , treated with DMSO or 100 µM CPT , let in G1 for 1 h , released into S phase . were collected at indicated time points . contents was analyzed by flow cytometry extracted in agarose plugs was analyzed by PFGE . Upper panel : agarose gel stained with ethidium bromide ; lower panel : Southern blot using a chromosome IV specific probe . JMs , joint molecules accumulated in gel wells . mean value of JMs relative to total amount of is indicated for each time point . Two to three biological replicates have been performed .

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(A) (B) Pulsed-field gel electrophoresis (PFGE) analysis of wild type, mus81∆, cdc5-2 and G2-MUS81 cells in response to CPT. Cells were synchronized in G1 with α-factor, treated with DMSO or 100 µM CPT, let in G1 for 1 h, and released into S phase. Cells were collected at the indicated time points. DNA contents was analyzed by flow cytometry and the DNA extracted in agarose plugs was analyzed by PFGE. Upper panel: agarose gel stained with ethidium bromide; lower panel: Southern blot using a chromosome IV specific probe. JMs, joint molecules accumulated in the gel wells. The mean value of JMs relative to the total amount of DNA is indicated for each time point. Two to three biological replicates have been performed.


( C ) ( D ) PFGE of rad52 ∆ rad52 ∆ mus81 ∆ in response to CPT performed as in ( A ) ( B ) . gel has been stained with Ethidium bromide densitometry profiles corresponding to + CPT 140 min time points in rad52 ∆ rad52 ∆ mus81 ∆ are shown . Two biological replicates have been performed .

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(C) (D) PFGE analysis of rad52∆ and rad52∆ mus81∆ cells in response to CPT performed as in (A) (B). The gel has been stained with Ethidium bromide and densitometry profiles corresponding to the +CPT 140 min time points in rad52∆ and rad52∆ mus81∆ cells are shown. Two biological replicates have been performed.


Cancer Genome Atlas ( TCGA RNAseq dataset ) was used on GlioVis platform ( Bowman et al , 2007 ) to analyze probability of survival ( log - rank p - value ) of 155 GBM , for each gene encoding for mediators of NF - κB pathway .

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The Cancer Genome Atlas (TCGA RNAseq dataset) was used on the GlioVis platform (Bowman et al, 2007) to analyze the probability of survival (log-rank p-value) of 155 GBM patients, for each gene encoding for the mediators of the NF-κB pathway.


Kaplan - Meier curve of probability of survival for 155 GBM with low or high MALT1 level , using median cut - off , on TCGA RNAseq dataset .

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Kaplan-Meier curve of the probability of survival for 155 GBM patients with low or high MALT1 RNA level, using median cut-off, based on the TCGA RNAseq dataset.


Box whisker plot of MALT1 mRNA expression in low - grade glioma ( LGG , grades II III ) or in GBM ( grade IV ) ( TCGA GBMLGG , RNAseq dataset ) ( D ) .

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Box and whisker plot of MALT1 mRNA expression in low-grade glioma (LGG, grades II and III) or in GBM (grade IV) (TCGA GBMLGG, RNAseq dataset) (D).


Alternatively , MALT1 mRNA expression was plotted in non - samples versus GBM samples ( TCGA RNAseq dataset ) ( E ) . Each dot represents one clinical sample .

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Alternatively, MALT1 mRNA expression was plotted in non-tumor samples versus GBM samples (TCGA RNAseq dataset) (E). Each dot represents one clinical sample.


Fraction of surviving over time in GSC # 1 GSC # 9 , transduced with control ( shc ) or bi - cistronic GFP plasmids using two different short hairpin ( shMALT1 sequences , seq # 1 # 2 ) . Data are plotted as of GFP - positive at day of ( Dx ) , normalized to starting point ( Day 4 post - infection , D4 ) .

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Fraction of surviving cells over time in GSC#1 and GSC#9, transduced with control (shc) or bi-cistronic GFP plasmids using two different short hairpin RNA (shMALT1 sequences, seq #1 and #2). Data are plotted as the percentage of GFP-positive cells at the day of the analysis (Dx), normalized to the starting point (Day 4 post-infection, D4).


EdU incorporation ( green , 2 hours ) was visualized by confocal imagery in GSC # 1 or by FACS in GSC # 9 transfected with sic or siMALT1 . In GSC # 1 of EdU - positive was quantified . Nuclei ( DAPI ) are shown in blue . n > 240 per replicate . Scale bar : 10 μm . Data are presented as mean + s . e . m . on 3 independent experiments .

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EdU incorporation (green, 2 hours) was visualized by confocal imagery in GSC#1 or by FACS in GSC#9 transfected with sic or siMALT1. In GSC#1 the percentage of EdU-positive cells was quantified. Nuclei (DAPI) are shown in blue. n> 240 cells per replicate. Scale bar: 10 μm. Data are presented as the mean + s.e.m. on 3 independent experiments.


FACS of propidium iodide ( PI ) in GSC # 1 # 9 transfected with non - silencing duplexes ( sic ) or MALT1 siRNA duplexes ( siMALT1 ) analyzed 72 hours later .

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FACS analysis of propidium iodide (PI) staining in GSC #1 and #9 transfected with non-silencing duplexes (sic) or MALT1 siRNA duplexes (siMALT1) and analyzed 72 hours later.


Linear regression plot of in vitro limiting dilution ( LDA ) for control ( shc ) or shMALT1 seq # 1 seq # 2 transduced GSC # 9 . Data are representative of n = 2 . Knockdown efficiency was verified at day 3 by western - blot using anti - MALT1 antibodies . GAPDH served as a loading control .

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Linear regression plot of in vitro limiting dilution assay (LDA) for control (shc) or shMALT1 seq#1 and seq#2 transduced GSC#9. Data are representative of n=2. Knockdown efficiency was verified at day 3 by western-blot using anti-MALT1 antibodies. GAPDH served as a loading control.


Tumorspheres per field of view ( fov ) were manually counted in sic or siMALT1 transfected GSC # 1 , # 4 # 9 . Data are presented as mean + s . e . m . on 3 independent experiments .

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Tumorspheres per field of view (fov) were manually counted in sic or siMALT1 transfected GSC#1, #4 and #9. Data are presented as the mean + s.e.m. on 3 independent experiments.


Linear regression plot of in vitro limiting dilution ( LDA ) for GSC # 9 treated with MALT1 inhibitor , mepazine ( MPZ , 20 μM , 14 days ) . DMSO vehicle was used as a control . Data are representative of n = 2 .

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Linear regression plot of in vitro limiting dilution assay (LDA) for GSC#9 treated with MALT1 inhibitor, mepazine (MPZ, 20 μM, 14 days). DMSO vehicle was used as a control. Data are representative of n=2.


frequency was calculated from LDA in GSCs # 1 , # 4 , # 12 treated with MPZ treatment ( 20 μM , 14 days ) . Data are presented as mean + s . e . m . on 2 independent experiments .

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Stem cell frequency was calculated from LDA in GSCs #1, #4, and #12 treated with MPZ treatment (20 μM, 14 days). Data are presented as the mean + s.e.m. on 2 independent experiments.


Tumorspheres per field of view ( fov ) were manually counted in GSCs # 1 , # 4 , # 9 , # 12 in response to MPZ ( 20 μM ) vehicle ( DMSO ) , in GSC # 9 treated with Z - VRPR - FMK ( 75 µM ) vehicle ( H2O ) for 4 days . Data are presented as mean + s . e . m . on 4 independent experiments for MPZ 3 independent experiments for Z - VRPR - FMK .

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Tumorspheres per field of view (fov) were manually counted in GSCs #1, #4, #9, and #12 in response to MPZ (20 μM) and vehicle (DMSO), and in GSC#9 treated with Z-VRPR-FMK (75 µM) and vehicle (H2O) for 4 days. Data are presented as the mean + s.e.m. on 4 independent experiments for MPZ and 3 independent experiments for Z-VRPR-FMK.


expression of stemness markers SOX2 NESTIN was evaluated by western - blot immunofluorescence ( SOX2 in red NESTIN in green ) in MPZ ( + , 20 μM , 16 hours ) vehicle ( - , DMSO , 16 hours ) treated GSC # 9 . GAPDH served as a loading control . Scale bar : 10 μm .

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The expression of the stemness markers SOX2 and NESTIN was evaluated by western-blot and immunofluorescence (SOX2 in red NESTIN in green) in MPZ (+, 20 μM, 16 hours) and vehicle (-, DMSO, 16 hours) treated GSC#9. GAPDH served as a loading control. Scale bar: 10 μm.


viability was measured using TiterGlo luminescent in GSCs # 1 , # 4 , # 9 , # 12 , human brain endothelial ( endo ) , human astrocytes ( astro ) , human neuron - like ( neuron ) treated for 48 hours with DMSO or MPZ ( 20 μM ) . Data were normalized to their respective DMSO - treated controls are presented as mean + s . e . m of 3 independent experiments in triplicate .

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Cell viability was measured using Cell TiterGlo luminescent assay in GSCs #1, #4, #9, and #12, human brain endothelial cells (endo), human astrocytes (astro), and human neuron-like cells (neuron) treated for 48 hours with DMSO or MPZ (20 μM). Data were normalized to their respective DMSO-treated controls and are presented as the mean + s.e.m of 3 independent experiments in triplicate.


FACS of EdU was performed on GSC # 1 treated overnight with MPZ ( 10 μM ) . Data are presented as mean + s . e . m . on 3 independent experiments .

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FACS analysis of EdU staining was performed on GSC#1 treated overnight with MPZ (10 μM). Data are presented as the mean + s.e.m. on 3 independent experiments.


FACS of propidium iodide ( PI ) in GSC # 9 treated for 48 hours with vehicle ( DMSO ) or MPZ ( 20 μM ) .

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FACS analysis of propidium iodide (PI) staining in GSC#9 treated for 48 hours with vehicle (DMSO) or MPZ (20 μM).


viability was measured using TiterGlo luminescent in differentiated GSC # 1 # 4 , # 9 ( DGCs ) treated for 48 hours with vehicle ( DMSO ) or MPZ ( 20 μM ) . Data were normalized to their respective DMSO - treated controls are presented as mean + s . e . m of 3 independent experiments . of GSCs # 1 , # 4 , # 9 DGCs # 1 , # 4 , # 9 was shown using brightfield .

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Cell viability was measured using Cell TiterGlo luminescent assay in differentiated GSC#1 #4, and #9 (DGCs) treated for 48 hours with vehicle (DMSO) or MPZ (20 μM). Data were normalized to their respective DMSO-treated controls and are presented as the mean + s.e.m of 3 independent experiments. Morphology of GSCs #1, #4, #9 and DGCs #1, #4, #9 was shown using brightfield images.


of viability of GSC # 9 using increasing doses ( 0 , 5 , 10 , 20 , 40 μM ) of phenothiazines : mepazine ( MPZ ) , fluphenazine ( FLU ) , cyamemazine ( CYAM ) , chlorpromazine ( CHLO ) , pipotiazine ( PIPO ) , alimemazine ( ALI ) , promethazine ( PRO ) , doxylamine ( DOXY ) . Data were normalized to their respective DMSO - treated controls .

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Heatmap of cell viability of GSC#9 using increasing doses (0, 5, 10, 20, 40 μM) of phenothiazines: mepazine (MPZ), fluphenazine (FLU), cyamemazine (CYAM), chlorpromazine (CHLO), pipotiazine (PIPO), alimemazine (ALI), promethazine (PRO), and doxylamine (DOXY). Data were normalized to their respective DMSO-treated controls.


Nude mice were implanted with GSC # 9 ( 106 ) in each flank randomized cages were treated with either vehicle ( DMSO ) or MPZ ( 8 mg / kg ) daily i . p . , for 14 consecutive days , once tumors were palpable . volume was measured from start of treatment until 1 week after treatment was removed . Graph of volume on day 21 post - treatment is presented . Data are presented as mean + s . e . m . n = 10 / group .

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Nude mice were implanted with GSC#9 (106 cells) in each flank and randomized cages were treated with either vehicle (DMSO) or MPZ (8 mg/kg) daily i.p., for 14 consecutive days, once tumors were palpable. Tumor volume was measured from the start of treatment until 1 week after treatment was removed. Graph of tumor volume on day 21 post-treatment is presented. Data are presented as the mean + s.e.m. n=10/group.


Cryosections from GSC - xenografted tumors were stained for endothelial marker PECAM1 ( red ) marker NESTIN ( green ) . Nuclei ( DAPI ) are shown in blue . Scale bar : 20 μm .

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Cryosections from GSC-xenografted tumors were stained for the endothelial marker PECAM1 (red) and tumor marker NESTIN (green). Nuclei (DAPI) are shown in blue. Scale bar: 20 μm.


Total lysates from GSCs # 1 # 9 challenged with TNFα ( 10 ng / mL , for indicated times ) were analyzed by western - blot for p - IκBα , IκBα p - JNK . Total JNK GAPDH served as loading controls .

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Total protein lysates from GSCs #1 and #9 challenged with TNFα (10 ng/mL, for the indicated times) were analyzed by western-blot for p-IκBα, IκBα and p-JNK. Total JNK and GAPDH served as loading controls.


Western - blot of p65 , cREL , RELB in ( cyt ) ( nuc ) fractionation from GSC # 1 GSC # 9 stimulated with TNFα ( 10 ng / mL , for indicated times ) . TUBULIN PARP served as controls for each fraction .

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Western-blot analysis of p65, cREL, and RELB in cytosolic (cyt) and nuclear (nuc) cell fractionation from GSC#1 and GSC#9 stimulated with TNFα (10 ng/mL, for the indicated times). TUBULIN and PARP served as controls for each fraction.


Jurkat T - , GSC # 1 GSC # 9 were stimulated with PMA ( 20 ng / mL ) Ionomycin ( Iono , 300 ng / mL ) for 30 minutes . Total lysates were analyzed by western - blot for CYLD ( full FL c ' d ) , HOIL1 ( full FL c ' d ) , p - IκBα IκBα . MALT1 BCL10 served as loading controls .

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

Jurkat T-cells, GSC#1 and GSC#9 were stimulated with PMA (20 ng/mL) and Ionomycin (Iono, 300 ng/mL) for 30 minutes. Total protein lysates were analyzed by western-blot for CYLD (full length FL and cleaved c'd), HOIL1 (full length FL and cleaved c'd), p-IκBα and IκBα. MALT1 and BCL10 served as loading controls.


Jurkat T - , GSC # 1 GSC # 9 were treated with vehicle ( DMSO ) mepazine ( MPZ , 20 μM ) for 4 hours . PMA / Ionomycin mixture was also administered to Jurkat for last 30 minutes . Total lysates were analyzed by western - blot for CYLD ( full FL c ' d ) . MALT1 served as a loading control .

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Jurkat T-cells, GSC#1 and GSC#9 were treated with vehicle (DMSO) and mepazine (MPZ, 20 μM) for 4 hours. PMA/Ionomycin mixture was also administered to Jurkat cells for the last 30 minutes. Total protein lysates were analyzed by western-blot for CYLD (full length FL and cleaved c'd). MALT1 served as a loading control.


Western - blot of CYLD ( full , FL , c ' d ) MALT1 in total lysates from GSC # 9 transfected with non - silencing duplexes ( sic ) or MALT1 targeting duplexes ( siMALT1 ) . GAPDH served as a loading control . Densitometric of c ' d CYLD / FL CYLD was performed ( right ) . Data are presented as mean + s . e . m . on 5 independent experiments .

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Western-blot analysis of CYLD (full length, FL and cleaved, c'd) and MALT1 in total protein lysates from GSC#9 transfected with non-silencing RNA duplexes (sic) or MALT1 targeting duplexes (siMALT1). GAPDH served as a loading control. Densitometric analysis of c'd CYLD/FL CYLD was performed (right). Data are presented as the mean + s.e.m. on 5 independent experiments.


( Left ) Western - blot of CYLD , ROQUIN1 / 2 , MALT1 , BCL10 in total lysates from GSC # 9 treated for 4 hours with vehicle ( H2O ) or Z - VRPR - FMK ( 75 μM ) . GAPDH served as a loading control . ( Right ) Densitometric of c ' d / FL was performed for ROQUIN1 / 2 CYLD . Data are presented as mean + s . e . m . on 3 independent experiments . Schematic drawing of MALT1 structures highlighting C464A substitution in protease - dead version . DD : Domain , C - like D : Caspase - like Domain , Ig : Immunoglobulin domain .

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(Left) Western-blot analysis of CYLD, ROQUIN1/2, MALT1, and BCL10 in total protein lysates from GSC#9 treated for 4 hours with vehicle (H2O) or Z-VRPR-FMK (75 μM). GAPDH served as a loading control. (Right) Densitometric analysis of c'd/FL was performed for ROQUIN1/2 and CYLD. Data are presented as the mean + s.e.m. on 3 independent experiments. Schematic drawing of MALT1 structures highlighting the C464A substitution in the protease-dead version. DD: Death Domain, C-like D: Caspase-like Domain, Ig: Immunoglobulin domain.


Western - blot of CYLD FLAG in total lysates from GSC # 9 transfected with WT or C464A MALT1 - FLAG . GAPDH served as a loading control .

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Western-blot analysis of CYLD and FLAG in total protein lysates from GSC#9 transfected with WT or C464A MALT1-FLAG. GAPDH served as a loading control.


Western - blot of CYLD ( full FL c ' d ) in total lysates from GSC # 9 after refreshing medium ( + ) , as compared to 3 days - old culture ( - ) . GAPDH served as a loading control . Densitometric of c ' d / FL CYLD was performed . Data are presented as mean + s . e . m . on 5 independent experiments .

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Western-blot of CYLD (full length FL and cleaved c'd) in total protein lysates from GSC#9 after refreshing the medium (+), as compared to 3 days-old culture (-). GAPDH served as a loading control. Densitometric analysis of c'd/FL CYLD was performed. Data are presented as the mean + s.e.m. on 5 independent experiments.


Western - blot of CYLD ( full FL c ' d ) in total lysates from GSC # 9 transfected with non - silencing duplexes ( sic ) or CARD10 targeting duplexes ( siCARD10 seq # 1 , seq # 2 , seq # 3 ) . GAPDH served as a loading control . qPCR confirmed knock - down of CARD10 in GSC # 9 . Data are presented as mean + s . e . m . on 3 independent experiments .

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Western-blot analysis of CYLD (full length FL and cleaved c'd) in total protein lysates from GSC#9 transfected with non-silencing RNA duplexes (sic) or CARD10 targeting duplexes (siCARD10 seq#1, seq#2, and seq#3). GAPDH served as a loading control. qPCR analysis confirmed the knock-down of CARD10 in GSC#9. Data are presented as the mean + s.e.m. on 3 independent experiments.


Western - blot of CYLD BCL10 in total lysates from GSC # 9 transfected with non - silencing duplexes ( sic ) or BCL10 targeting duplexes ( siBCL10 , seq # 1 seq # 3 ) . GAPDH served as a loading control . viability was measured using TiterGlo luminescent in sic seq # 1 siBCL10 - transfected . Data were normalized to their respective sic - treated controls are presented as mean + s . e . m of 3 independent experiments , in triplicate .

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Western-blot analysis of CYLD and BCL10 in total protein lysates from GSC#9 transfected with non-silencing RNA duplexes (sic) or BCL10 targeting duplexes (siBCL10, seq#1 and seq#3). GAPDH served as a loading control. Cell viability was measured using Cell TiterGlo luminescent assay in sic and seq#1 siBCL10-transfected cells. Data were normalized to their respective sic-treated controls and are presented as the mean + s.e.m of 3 independent experiments, in triplicate.


Transmission electron microscopy of GSC # 9 treated with vehicle ( DMSO ) or MPZ ( 20 μM ) for 16 hours . ER : endoplasmic reticulum ; MVB : multivesicular bodies ; lys : lysosome ; mit : mitochondria ; nuc : nucleus . Red stars denote lysosomes , blue stars vacuoles .

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Transmission electron microscopy of GSC#9 treated with vehicle (DMSO) or MPZ (20 μM) for 16 hours. ER: endoplasmic reticulum; MVB: multivesicular bodies; lys: lysosome; mit: mitochondria; nuc: nucleus. Red stars denote lysosomes, blue stars vacuoles.


Confocal of LAMP2 ( red ) at 0 , 1 , 2 , 4 , 6 hours post - MPZ ( 20 μM ) treatment . Nuclei ( DAPI ) are shown in blue . Scale bar : 10 μm .

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Confocal analysis of LAMP2 staining (red) at 0, 1, 2, 4, and 6 hours post-MPZ (20 μM) treatment. Nuclei (DAPI) are shown in blue. Scale bar: 10 μm.


Western - blot was performed in total lysates from GSC # 9 transfected with non - silencing duplexes ( sic ) or MALT1 targeting siRNA duplexes ( siMALT1 ) . Alternatively , western - blot of LAMP2 , CTSD , MALT1 was done in total lysates from GSC # 9 treated for 16 hours with MPZ ( 20 μM ) or Z - VRPR - FMK ( 75 µM ) . DMSO was used as vehicle . GAPDH served as a loading control .

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Western-blot analysis was performed in total protein lysates from GSC#9 transfected with non-silencing duplexes (sic) or MALT1 targeting siRNA duplexes (siMALT1). Alternatively, western-blot analysis of LAMP2, CTSD, and MALT1 was done in total protein lysates from GSC#9 treated for 16 hours with MPZ (20 μM) or Z-VRPR-FMK (75 µM). DMSO was used as vehicle. GAPDH served as a loading control.


Confocal of LAMP2 ( red ) in GSCs # 1 , # 4 , # 12 treated for 16 hours with vehicle ( DMSO ) or MPZ ( 20 μM ) . Alternatively , GSC # 9 were either treated for 16 hours with H2O or Z - VRPR - FMK ( 75 µM ) . Additionally , were transfected with non - silecing duplexes ( sic ) or MALT1 BCL10 targeting siRNA duplexes ( siMALT1 siBCL10 ) . Alternatively , lysotracker ( red ) was used to track for lysosomes in either GSC # 9 expressing either wild - type ( WT ) or C464A FLAG - MALT1 ( green ) . Scale bar : 10 μm .

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Confocal analysis of LAMP2 staining (red) in GSCs #1, #4, #12 treated for 16 hours with vehicle (DMSO) or MPZ (20 μM). Alternatively, GSC#9 were either treated for 16 hours with H2O or Z-VRPR-FMK (75 µM). Additionally, cells were transfected with non-silecing duplexes (sic) or MALT1 and BCL10 targeting siRNA duplexes (siMALT1 and siBCL10). Alternatively, lysotracker staining (red) was used to track for lysosomes in either GSC#9 expressing either wild-type (WT) or C464A FLAG-MALT1 (green). Scale bar: 10 μm.


of LAMP2 pixel on GSC # 9 treated as described in panel ( D ) . Data are presented as mean + s . e . m . on 3 independent experiments . Each dot represents one . n > 30 .

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Quantification of LAMP2 staining pixel intensity on GSC#9 treated as described in panel (D). Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>30.


Cryosections from GSC # 9 - xenografted tumors in vehicle MPZ - challenged ( as described in Fig 2J ) assessed for LAMP2 ( green ) . Nuclei ( DAPI ) are shown in blue . Scale bar : 10 μm . Western - blot of LAMP2 was performed in lysates . GAPDH served as a loading control .

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Cryosections from GSC#9-xenografted tumors in vehicle and MPZ-challenged animals (as described in Fig 2J) and assessed for LAMP2 staining (green). Nuclei (DAPI) are shown in blue. Scale bar: 10 μm. Western-blot analysis of LAMP2 was performed in tumor lysates. GAPDH served as a loading control.


Confocal of lysotracker ( red ) in GSC # 9 treated for 16 hours with vehicle ( DMSO ) or MPZ ( 20 μM ) . Alternatively , GSC # 9 were either treated for 16 hours with H2O or Z - VRPR - FMK ( 75 µM ) ( upper panel ) or transfected with sic siMALT1 ( bottom panel ) . As indicated , number of lysotracker - positive lysotracker pixel ( arbitrary unit , AU ) were quantified per . Data are presented as mean + s . e . m . on 3 independent experiments . Each dot represents one . n > 30 . Nuclei ( DAPI ) are shown in blue . Scale bars : 10 μm .

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Confocal analysis of lysotracker staining (red) in GSC#9 treated for 16 hours with vehicle (DMSO) or MPZ (20 μM). Alternatively, GSC#9 were either treated for 16 hours with H2O or Z-VRPR-FMK (75 µM) (upper panel) or transfected with sic and siMALT1 (bottom panel). As indicated, number of lysotracker-positive puncta and lysotracker pixel intensity (arbitrary unit, AU) were quantified per cell. Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>30. Nuclei (DAPI) are shown in blue. Scale bars: 10 μm.


( Left ) Confocal of LAMP2 ( red ) LC3B ( green ) in GSC # 9 treated for 4 16 hours with vehicle ( DMSO ) MPZ ( 20 µM ) . Nuclei ( DAPI ) are shown in blue . Scale bars : 10 μm . ( Right ) Super - resolution imaging ( SIM , Structured Illumination Microscopy ) of LAMP2 ( red ) LC3B ( green ) in GSC # 9 treated for 16 hours with vehicle ( DMSO ) or MPZ ( 20 µM ) .

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(Left) Confocal analysis of LAMP2 (red) and LC3B (green) in GSC#9 treated for 4 and 16 hours with vehicle (DMSO) and MPZ (20 µM). Nuclei (DAPI) are shown in blue. Scale bars: 10 μm. (Right) Super-resolution imaging (SIM, Structured Illumination Microscopy) of LAMP2 (red) and LC3B (green) staining in GSC#9 treated for 16 hours with vehicle (DMSO) or MPZ (20 µM).


Western - blot of LC3B P62 in total lysates from GSC # 9 at 0 , 2 , 4 , 6 , 16 hours post - MPZ treatment ( 20 µM ) . GAPDH served as a loading control .

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Western-blot analysis of LC3B and P62 in total protein lysates from GSC#9 at 0, 2, 4, 6, and 16 hours post-MPZ treatment (20 µM). GAPDH served as a loading control.


Western - blot of LC3B in total lysates from GSCs # 1 # 9 at 72 hours post - transfection with sic or siMALT1 , subsequently treated 4 hours with vehicle ( DMSO ) or chloroquine ( CQ , 20 µM ) . Knockdown was verified by MALT1 blotting GAPDH served as a loading control .

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Western-blot analysis of LC3B in total protein lysates from GSCs #1 and #9 at 72 hours post-transfection with sic or siMALT1, and subsequently treated 4 hours with vehicle (DMSO) or chloroquine (CQ, 20 µM). Knockdown was verified by MALT1 blotting and GAPDH served as a loading control.


Confocal of LC3B ( green ) in GSC # 9 treated for16 hours with vehicle ( DMSO ) MPZ ( 20 µM ) with or without chloroquine ( CQ , 20 µM ) . Nuclei ( DAPI ) are shown in blue . Scale bars : 10 μm .

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Confocal analysis of LC3B (green) in GSC#9 treated for16 hours with vehicle (DMSO) and MPZ (20 µM) with or without chloroquine (CQ, 20 µM). Nuclei (DAPI) are shown in blue. Scale bars: 10 μm.


GSC # 9 were transfected with LC3B reporters ( wild type WT or G120A mutant , which cannot be lipidated ) , treated 24 hours later with vehicle ( DMSO ) or MPZ ( 20 µM ) for 6 more hours . Ratios of WT / mutant luciferase signals are presented as mean + s . e . m of 3 independent experiments .

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GSC#9 were transfected with LC3B reporters (wild type WT or G120A mutant, which cannot be lipidated), treated 24 hours later with vehicle (DMSO) or MPZ (20 µM) for 6 more hours. Ratios of WT/mutant luciferase signals are presented as the mean + s.e.m of 3 independent experiments.


Confocal of P62 ( red ) in GSC # 9 treated for 16 hours with vehicle ( DMSO ) or MPZ ( 20 μM ) . Alternatively , GSC # 9 were either transfected with sic or siMALT1 ( middle ) or treated for 16 hours with H2O or Z - VRPR - FMK ( 75 µM ) ( bottom ) . of P62 pixel on GSC # 9 treated for 16 hours with vehicle ( DMSO or H2O ) , MPZ ( 20 µM ) or Z - VRPR - FMK ( 75 µM ) or sic siMALT1 . Data are presented as mean + s . e . m . on 3 independent experiments . Each dot represents one . n > 30 .

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Confocal analysis of P62 staining (red) in GSC#9 treated for 16 hours with vehicle (DMSO) or MPZ (20 μM). Alternatively, GSC#9 were either transfected with sic or siMALT1 (middle) or treated for 16 hours with H2O or Z-VRPR-FMK (75 µM) (bottom). Quantification of P62 staining pixel intensity on GSC#9 treated for 16 hours with vehicle (DMSO or H2O), MPZ (20 µM) or Z-VRPR-FMK (75 µM) or sic and siMALT1. Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>30.


viability was measuring using TiterGlo in GSCs # 1 # 9 pre - treated for 1 hour with vehicle ( DMSO ) or QVD ( 20 μM ) , treated for 72 hours more with indicated doses of MPZ . Data were normalized to vehicle - treated controls are presented as mean + s . e . m of 4 independent experiments .

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Cell viability was measuring using Cell TiterGlo in GSCs #1 and #9 pre-treated for 1 hour with vehicle (DMSO) or QVD (20 μM), and treated for 72 hours more with the indicated doses of MPZ. Data were normalized to the vehicle-treated controls and are presented as the mean + s.e.m of 4 independent experiments.


FACS of propidium iodide ( PI ) in GSC # 9 treated for 48 hours with vehicle ( DMSO ) or MPZ ( 15 μM ) in combination with QVD ( 20 μM ) . ( Left ) of PI positive , normalized to vehicle - treated controls are presented as mean + s . e . m . on 3 independent experiments . ( Right ) Histogram plots for representative experiment ( DMSO in red MPZ in blue ) .

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FACS analysis of propidium iodide (PI) staining in GSC#9 treated for 48 hours with vehicle (DMSO) or MPZ (15 μM) in combination with QVD (20 μM). (Left) Percentage of PI positive cells, normalized to vehicle-treated controls are presented as the mean + s.e.m. on 3 independent experiments. (Right) Histogram plots for representative experiment (DMSO in red and MPZ in blue).


CSTD ELISA was performed on culture media from GSC # 9 treated for 8 hours with vehicle ( DMSO ) or MPZ ( 20 µM ) . Alternatively , were transfected with sic or siMALT1 analyzed 72 hours later . Data are presented as mean + s . e . m of 3 independent experiments .

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CSTD ELISA was performed on culture media from GSC#9 treated for 8 hours with vehicle (DMSO) or MPZ (20 µM). Alternatively, cells were transfected with sic or siMALT1 and analyzed 72 hours later. Data are presented as the mean + s.e.m of 3 independent experiments.


( Left ) viability was measured using TiterGlo luminescent in GSC # 9 treated for 48 hours with vehicle ( DMSO ) or MPZ ( 10 μM ) , following a 30 minute - pre - treatment with following drugs : Bafilomycin A1 ( Baf , 100 nM ) , Pepstatin A ( Pep , 1 μg / mL ) , or CTS inhibitor 1 ( Ctsi , 1 μM ) . Data were normalized to vehicle - treated controls are presented as mean + s . e . m of 3 independent experiments in triplicate , stars refer to comparison to Vehicle + MPZ group ( blue squares ) . ( Right ) FACS of propidium iodide ( PI ) in GSC # 9 treated for 48 hours with vehicle ( DMSO ) or MPZ ( 15 μM ) in combination with Baf , Pep Ctsi . of PI positive normalized to vehicle - treated controls are presented as mean + s . e . m . on 3 independent experiments .

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

(Left) Cell viability was measured using Cell TiterGlo luminescent assay in GSC#9 treated for 48 hours with vehicle (DMSO) or MPZ (10 μM), following a 30 minute-pre-treatment with the following drugs: Bafilomycin A1 (Baf, 100 nM), Pepstatin A (Pep, 1 μg/mL), or CTS inhibitor 1 (Ctsi, 1 μM). Data were normalized to the vehicle-treated controls and are presented as the mean + s.e.m of 3 independent experiments in triplicate, stars refer to comparison to Vehicle + MPZ group (blue squares). (Right) FACS analysis of propidium iodide (PI) staining in GSC#9 treated for 48 hours with vehicle (DMSO) or MPZ (15 μM) in combination with Baf, Pep and Ctsi. Percentage of PI positive cells normalized to vehicle-treated controls are presented as the mean + s.e.m. on 3 independent experiments.


of differentially expressed genes obtained from RNAseq of GSC # 9 treated for 4 hours with vehicle ( DMSO ) or MPZ ( 20 μM ) , from three biological replicates .

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Heatmap of differentially expressed genes obtained from RNAseq analysis of GSC#9 treated for 4 hours with vehicle (DMSO) or MPZ (20 μM), from three biological replicates.


Volcano plot of differentially expressed genes in RNAseq of GSC # 9 , expressed as fold changes between vehicle ( DMSO ) MPZ - treated .

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Volcano plot of differentially expressed genes in RNAseq analysis of GSC#9, expressed as fold changes between vehicle (DMSO) and MPZ-treated cells.


GSEA ( gene set enrichment ) plot showing enrichment of " Bilanges serum rapamycin sensitive genes " signature in vehicle ( DMSO ) versus MPZ - treated triplicates .

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GSEA (gene set enrichment analysis) plot showing enrichment of "Bilanges serum and rapamycin sensitive genes" signature in vehicle (DMSO) versus MPZ-treated triplicates.


qRT - PCR was performed on total from GSC # 9 treated for 4 hours with vehicle ( DMSO ) or MPZ ( 20 μM ) . Histograms showed changes in expression of indicated targets . Data were normalized to two housekeeping genes ( ACTB , HPRT1 ) are presented as mean + s . e . m of technical triplicates .

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qRT-PCR was performed on total RNA from GSC#9 treated for 4 hours with vehicle (DMSO) or MPZ (20 μM). Histograms showed changes in RNA expression of indicated targets. Data were normalized to two housekeeping genes (ACTB, HPRT1) and are presented as the mean + s.e.m of technical triplicates.


Western - blot of LC3B , CSTD TFEB in total lysates from GSC # 9 transfected with non - silencing duplexes ( sic ) or siRNA duplexes targeting TFEB ( siTFEB ) treated with vehicle ( DMSO ) or MPZ ( 20 μM ) for 16 hours . GAPDH served as a loading control .

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Western-blot analysis of LC3B, CSTD and TFEB in total protein lysates from GSC#9 transfected with non-silencing duplexes (sic) or siRNA duplexes targeting TFEB (siTFEB) and treated with vehicle (DMSO) or MPZ (20 μM) for 16 hours. GAPDH served as a loading control.


Western - blot of p - ULK1 , p - AKT , p - S6 , p - p70S6K in GSC # 9 treated for 1 hour with MPZ ( 20 μM ) or rapamycin ( RAPA , 50 nM ) . Total ULK , AKT , S6 , p70S6K served as loading controls . DMSO was used as a vehicle .

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Western-blot analysis of p-ULK1, p-AKT, p-S6, and p-p70S6K in GSC#9 treated for 1 hour with MPZ (20 μM) or rapamycin (RAPA, 50 nM). Total ULK, AKT, S6, and p70S6K served as loading controls. DMSO was used as a vehicle.


Western - blot of MALT1 , p - AKT , p - S6 in total lysates from GSC # 9 transfected with non - silencing duplexes ( sic ) or MALT1 targeting siRNA duplexes ( siMALT1 ) . Total AKT S6 , as well as GAPDH served as loading controls .

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Western-blot analysis of MALT1, p-AKT, and p-S6 in total protein lysates from GSC#9 transfected with non-silencing duplexes (sic) or MALT1 targeting siRNA duplexes (siMALT1). Total AKT and S6, as well as GAPDH served as loading controls.


Western - blot of p - AKT , p - S6 , p - p70S6K in total lysates from GSC # 9 treated for 1 hour with vehicle ( DMSO ) or 20 μM of phenothiazine compounds ( MPZ , FLU , CHLO , CYA ) . Total AKT , total S6 total p70S6K served as loading controls .

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Western-blot analysis of p-AKT, p-S6, and p-p70S6K in total protein lysates from GSC#9 treated for 1 hour with vehicle (DMSO) or 20 μM of phenothiazine compounds (MPZ, FLU, CHLO, and CYA). Total AKT, total S6 and total p70S6K served as loading controls.


Western - blot of p - S6 FLAG in GSC # 9 expressing WT or C464A MALT - FLAG . Total S6 GAPDH served as loading controls .

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Western-blot analysis of p-S6 and FLAG in GSC#9 expressing WT or C464A MALT-FLAG. Total S6 and GAPDH served as loading controls.


Confocal of LAMP2 ( red ) mTOR ( green ) in GSC # 9 treated with vehicle ( DMSO ) or MPZ ( 20 μM ) , Z - VRPR - FMK ( 75 μM ) , FLU ( 20 μM ) , CHLO ( 20 μM ) , CYA ( 20 μM ) . Alternatively , were transfected with sic or siMALT1 . Nuclei ( DAPI ) are shown in blue . Arrows point to LAMP2 - positive . Scale bars : 10 μm . of mTOR colocalization score with LAMP2 is shown . Coloc2 plug - in from ImageJ was used to measure Mander ' s tM1 correlation factor in LAMP2 - positive ROI , using Costes threshold regression . Data are presented as mean + s . e . m . on 3 independent experiments . Each dot represents one . n > 10 .

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Confocal analysis of LAMP2 (red) and mTOR (green) staining in GSC#9 treated with vehicle (DMSO) or MPZ (20 μM), Z-VRPR-FMK (75 μM), FLU (20 μM), CHLO (20 μM), and CYA (20 μM). Alternatively, cells were transfected with sic or siMALT1. Nuclei (DAPI) are shown in blue. Arrows point to LAMP2-positive area. Scale bars: 10 μm. Quantification of mTOR colocalization score with LAMP2 is shown. The Coloc2 plug-in from ImageJ was used to measure Mander's tM1 correlation factor in LAMP2-positive ROI, using Costes threshold regression. Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>10.


( Left ) Kaplan - Meier curve of probability of survival for 155 GBM with low or high QKI level , using median cut - off , on TCGA RNAseq dataset . ( Right ) Differential expression related to either MALT1 or QKI expression highlighted a lysosomal lumen GO function . Venn diagram of overlapping lysosomal enriched encoding genes from this comparison showed 7 shared genes , together with 9 10 specific genes for MALT1 QKI expression , respectively . ( Bottom ) Correlation between MALT1 QKI expression was analyzed using Cancer Genome Atlas ( TCGA , HG - U133A dataset ) on GlioVis platform ( Bowman et al , 2007 ) . Pearson correlation factor = - 0 . 21 , p - value = 0 . 03 .

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(Left) Kaplan-Meier curve of the probability of survival for 155 GBM patients with low or high QKI RNA level, using median cut-off, based on the TCGA RNAseq dataset. (Right) Differential expression analysis related to either MALT1 or QKI expression highlighted a lysosomal lumen GO function. Venn diagram of overlapping lysosomal enriched protein encoding genes from this comparison showed 7 shared genes, together with 9 and 10 specific genes for MALT1 and QKI expression, respectively. (Bottom) Correlation between MALT1 and QKI expression was analyzed using The Cancer Genome Atlas (TCGA, HG-U133A dataset) on the GlioVis platform (Bowman et al, 2007). Pearson correlation factor=-0.21, p-value=0.03.


GSCs # 1 # 9 lysates ( input ) were processed for immunoprecipitation ( IP ) using control immunoglobulins ( Ig ) , anti - QKI or anti - BCL10 antibodies . Input IP fractions were separated on SDS - PAGE western - blots for MALT1 , QKI BCL10 antibodies were performed as specified .

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GSCs #1 and #9 protein lysates (input) were processed for immunoprecipitation (IP) using control immunoglobulins (Ig), anti-QKI or anti-BCL10 antibodies. Input and IP fractions were separated on SDS-PAGE and western-blots for MALT1, QKI and BCL10 antibodies were performed as specified.


Total lysates ( input ) from GSC # 9 treated with vehicle ( - , DMSO ) or MPZ ( + , 20 μM , 1 hour ) or with vehicle ( - , H2O ) or Z - VRPR - FMK ( + , 75 μM , 4 hours ) , were processed for control immunoglobulins ( Ig ) or anti - QKI antibodies immunoprecipitation ( IP ) . Western - blots were performed with indicated antibodies . Western - blots were performed with indicated antibodies .

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Total protein lysates (input) from GSC#9 treated with vehicle (-, DMSO) or MPZ (+, 20 μM, 1 hour) or with vehicle (-, H2O) or Z-VRPR-FMK (+, 75 μM, 4 hours), were processed for control immunoglobulins (Ig) or anti-QKI antibodies immunoprecipitation (IP). Western-blots were performed with indicated antibodies. Western-blots were performed with indicated antibodies.


Confocal of Lysotracker ( green ) or FLAG ( red ) in GSC # 9 overexpressing either empty vector ( mock ) or FLAG - QKI . Scale bars : 10 μm . Nuclei ( DAPI ) are shown in blue .

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Confocal analysis of Lysotracker (green) or FLAG (red) in GSC#9 overexpressing either empty vector (mock) or FLAG-QKI. Scale bars: 10 μm. Nuclei (DAPI) are shown in blue.


Confocal of LAMP2 ( green ) or FLAG ( red ) in GSC # 9 transfected with either empty vector ( mock ) or FLAG - QKI . Scale bars : 10 μm . Nuclei ( DAPI ) are shown in blue . of LAMP2 pixel on GSC # 9 transfected with mock FLAG - QKI . Data are presented as mean + s . e . m . on 3 independent experiments . Each dot represents one . n > 15 .

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Confocal analysis of LAMP2 (green) or FLAG (red) in GSC#9 transfected with either empty vector (mock) or FLAG-QKI. Scale bars: 10 μm. Nuclei (DAPI) are shown in blue. Quantification of LAMP2 staining pixel intensity on GSC#9 transfected with mock and FLAG-QKI. Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>15.


Western - blot of QKI , LAMP2 LC3B in GSC # 9 overexpressing either empty vector ( mock ) or FLAG - QKI . GAPDH served as a loading control .

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Western-blot analysis of QKI, LAMP2 and LC3B in GSC#9 overexpressing either empty vector (mock) or FLAG-QKI. GAPDH served as a loading control.


Confocal of mTOR ( green ) or FLAG ( red ) in GSC # 9 transfected with either empty vector ( mock ) or Flag - QKI . Nuclei ( DAPI ) are shown in blue . Scale bars : 10 μm .

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Confocal analysis of mTOR (green) or FLAG (red) in GSC#9 transfected with either empty vector (mock) or Flag-QKI. Nuclei (DAPI) are shown in blue. Scale bars: 10 μm.


GSC # 1 were transfected with either empty vector ( mock ) or FLAG - QKI . Total lysates were processed for western - blots against p - S6 FLAG . Total S6 served as a loading control .

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GSC#1 were transfected with either empty vector (mock) or FLAG-QKI. Total protein lysates were processed for western-blots against p-S6 and FLAG. Total S6 served as a loading control.


Fraction of surviving over time in GSCs # 1 # 9 , transduced with empty vector ( mock ) or FLAG - QKI bi - cistronic GFP plasmids . Data are plotted as of GFP - positive at day of ( Dx ) , normalized to starting point ( Day 4 post - infection , D4 ) . Data are representative of n = 3 .

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Fraction of surviving cells over time in GSCs #1 and #9, transduced with empty vector (mock) or FLAG-QKI bi-cistronic GFP plasmids. Data are plotted as the percentage of GFP-positive cells at the day of the analysis (Dx), normalized to the starting point (Day 4 post-infection, D4). Data are representative of n=3.


GSC # 9 transfected with non - silencing duplexes ( sic ) or QKI targeting siRNA duplexes ( siQKI ) were treated for 16 hours with vehicle ( DMSO ) or MPZ ( 10 μM ) . Total lysates were processed for western - blots against LAMP2 , CTSD , QKI , LC3B expression , as indicated . GAPDH served as a loading control .

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GSC#9 transfected with non-silencing RNA duplexes (sic) or QKI targeting siRNA duplexes (siQKI) were treated for 16 hours with vehicle (DMSO) or MPZ (10 μM). Total protein lysates were processed for western-blots against LAMP2, CTSD, QKI, and LC3B expression, as indicated. GAPDH served as a loading control.


Confocal of mTOR ( green ) LAMP2 ( red ) in GSC # 9 transfected with sic or siQKI , treated for 16 hours with vehicle ( DMSO ) or MPZ ( 20 μM ) . Nuclei ( DAPI ) are shown in blue . Scale bars : 10 μm . of mTOR colocalization score with LAMP2 is shown . Coloc2 plug - in from ImageJ was used to measure Mander ' s tM1 correlation factor in LAMP2 - positive ROI , using Costes threshold regression . Data are presented as mean + s . e . m . on 3 independent experiments . Each dot represents one . n > 10 .

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Confocal analysis of mTOR (green) and LAMP2 (red) in GSC#9 transfected with sic or siQKI, and treated for 16 hours with vehicle (DMSO) or MPZ (20 μM). Nuclei (DAPI) are shown in blue. Scale bars: 10 μm. Quantification of mTOR colocalization score with LAMP2 is shown. The Coloc2 plug-in from ImageJ was used to measure Mander's tM1 correlation factor in LAMP2-positive ROI, using Costes threshold regression. Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>10.


GSC # 9 transfected with non - silencing duplexes ( sic ) or QKI targeting siRNA duplexes ( siQKI ) were treated for 1 hour with vehicle ( DMSO ) or MPZ ( 20 μM ) . Total lysates were processed for western - blots against QKI p - S6 . TUBULIN Total S6 served as loading controls .

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GSC#9 transfected with non-silencing RNA duplexes (sic) or QKI targeting siRNA duplexes (siQKI) were treated for 1 hour with vehicle (DMSO) or MPZ (20 μM). Total protein lysates were processed for western-blots against QKI and p-S6. TUBULIN and Total S6 served as loading controls.


FACS of EdU was performed on GSC # 9 transfected with non - silencing duplexes ( sic , pink ) , QKI targeting siRNA duplexes ( siQKI , purple ) , MALT1 targeting siRNA duplexes ( siMALT1 , blue ) or double transfected with siQKI siMALT1 ( purple ) .

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FACS analysis of EdU staining was performed on GSC#9 cells transfected with non-silencing RNA duplexes (sic, pink), QKI targeting siRNA duplexes (siQKI, light purple), MALT1 targeting siRNA duplexes (siMALT1, blue) or double transfected with siQKI and siMALT1 (purple).


FACS of propidium iodide ( PI ) in GSC # 9 transfected with non - silencing duplexes ( sic ) , QKI targeting siRNA duplexes ( siQKI ) , MALT1 targeting siRNA duplexes ( siMALT1 ) or double transfected with siQKI siMALT1 analyzed 72 hours later . of PI - positive normalized to vehicle treated controls are presented as mean + s . e . m . on 3 independent experiments .

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FACS analysis of propidium iodide (PI) staining in GSC#9 transfected with non-silencing RNA duplexes (sic), QKI targeting siRNA duplexes (siQKI), MALT1 targeting siRNA duplexes (siMALT1) or double transfected with siQKI and siMALT1 and analyzed 72 hours later. Percentage of PI-positive cells normalized to vehicle treated controls are presented as the mean + s.e.m. on 3 independent experiments.


( A ) qRT - PCR for Islr , Chd1 Vim in total intestinal tissues , intestinal epithelium lamina propria . n = 4 .

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(A) qRT-PCR analysis for Islr, Chd1 and Vim in total intestinal tissues, intestinal epithelium and lamina propria. n = 4.


( B ) Western blotting for Islr E - cadherin in total intestinal tissues , intestinal epithelium lamina propria . α - Tubulin was used as a loading control .

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(B) Western blotting for Islr and E-cadherin in total intestinal tissues, intestinal epithelium and lamina propria. α-Tubulin was used as a loading control.


( C ) qRT - PCR for Islr in CD45 + , CD45 - Epcam + , CD45 - Epcam - CD90 + CD45 - Epcam - CD90 - sorted from intestinal tissues . n = 3 technical replicates .

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(C) qRT-PCR analysis for Islr in CD45+, CD45-Epcam+, CD45-Epcam-CD90+ and CD45-Epcam-CD90- cells sorted from intestinal tissues. n = 3 technical replicates.


( D ) In situ hybridization for Islr with RNAscope probe in intestine colon from 8 - week - old mice , showing that Islr is primarily expressed in stromal . Negative positive controls were shown in Appendix Figure S1A . Scale bar : 25 μm .

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(D) In situ hybridization for Islr with RNAscope probe in intestine and colon from 8-week-old mice, showing that Islr is primarily expressed in stromal cells. Negative and positive controls were shown in Appendix Figure S1A. Scale bar: 25 μm.


( E ) In situ hybridization for ISLR with RNAscope probe in human mucosa , inflamed mucosa from CD UC . Scale bar : 25 μm .

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(E) In situ hybridization for ISLR with RNAscope probe in human mucosa, inflamed mucosa from CD and UC patients. Scale bar: 25 μm.


( F ) qRT - PCR showing dynamic changes of Islr in colonic tissues from mice upon DSS treatment after DSS removal . n = 4 at each time points .

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(F) qRT-PCR analysis showing the dynamic changes of Islr in colonic tissues from mice upon DSS treatment and after DSS removal. n = 4 at each time points.


( G ) In situ hybridization for Islr with RNAscope probe in mouse colons without or with 3 - day or 5 - day DSS treatments . Scale bar : 50 μm .

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(G) In situ hybridization for Islr with RNAscope probe in mouse colons without or with 3-day or 5-day DSS treatments. Scale bar: 50 μm.


( H ) cancer genome atlas ( TCGA ) - seq showing that ISLR is upregulated in human rectal adenocarcinoma relative to normal rectal tissues . horizontal lines between box limits represent median , box limits indicate interquartile ranges whiskers indicate limit superior limit inferior respectively . n = 65 .

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(H) The cancer genome atlas (TCGA) RNA-seq analysis showing that ISLR is upregulated in human rectal adenocarcinoma relative to normal rectal tissues. The horizontal lines between the box limits represent the median, the box limits indicate the interquartile ranges and the whiskers indicate limit superior and limit inferior respectively. n = 65.


( I ) Kaplan - Meier survival curve of 270 CRC cases . P = 0 . 02 ( log - rank ) .

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(I) Kaplan-Meier survival curve of 270 CRC cases. P = 0.02 (log-rank test).


( J ) ISLR level increased with nodal metastasis in colorectal cancer . N0 , no regional lymph node metastasis ; N1 , metastases in 1 to 3 axillary lymph nodes ; N2 , metastases in 4 to 9 axillary lymph nodes ; N3 , metastases in 10 or more axillary lymph nodes . solid horizontal lines represent median , box limits indicate interquartile ranges whiskers indicate limit superior limit inferior respectively .

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(J) ISLR level increased with nodal metastasis in colorectal cancer patients. N0, no regional lymph node metastasis; N1, metastases in 1 to 3 axillary lymph nodes; N2, metastases in 4 to 9 axillary lymph nodes; N3, metastases in 10 or more axillary lymph nodes. The solid horizontal lines represent the median, the box limits indicate the interquartile ranges and the whiskers indicate limit superior and limit inferior respectively.


( K In situ hybridization for ISLR / Islr with RNAscope probes in normal human colon , human malignant adenocarcinoma , metastatic adenocarcinoma ( K ) , t indicating ; a indicating adjacent tissues of . Scale bar : 50 μm .

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(K In situ hybridization for ISLR/Islr with RNAscope probes in normal human colon, human malignant adenocarcinoma, metastatic adenocarcinoma (K), t indicating tumor; a indicating adjacent tissues of tumor. Scale bar: 50 μm.


L ) In situ hybridization for ISLR / Islr with RNAscope probes in mouse colon tumors from AOM - DSS model ( L ) . t indicating ; a indicating adjacent tissues of . Scale bar : 50 μm .

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L) In situ hybridization for ISLR/Islr with RNAscope probes in mouse colon tumors from AOM-DSS model (L). t indicating tumor; a indicating adjacent tissues of tumor. Scale bar: 50 μm.


( B ) qRT - PCR for Ets1 in colonic epithelium lamina propria . n = 3 .

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(B) qRT-PCR analysis for Ets1 in colonic epithelium and lamina propria. n = 3.


( C ) Double immunofluorescence for ETS1 β - catenin in intestinal biopsies of healthy controls inflamed mucosa from UC . Scale bar : 50 μm .

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(C) Double immunofluorescence for ETS1 and β-catenin in intestinal biopsies of healthy controls and inflamed mucosa from UC patients. Scale bar: 50 μm.


( D ) qRT - PCR for Ets1 in colons with or without 5 - day DSS treatment . n = 4 .

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(D) qRT-PCR for Ets1 in colons with or without 5-day DSS treatment. n = 4.


( E ) Double immunofluorescence for Ets1 β - catenin in normal mouse colons without DSS treatment , mouse colons with 5 - day DSS treatments , AOM - DSS mouse colon tumors . Scale bar : 50 μm .

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(E) Double immunofluorescence for Ets1 and β-catenin in normal mouse colons without DSS treatment, mouse colons with 5-day DSS treatments, and AOM-DSS mouse colon tumors. Scale bar: 50 μm.


( F ) Pearson correlation on ETS1 ISLR in colorectal adenocarcinoma TCGA - seq ( P < 0 . 001 ; R = 0 . 68 ) .

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(F) Pearson correlation analysis on ETS1 and ISLR in colorectal adenocarcinoma TCGA RNA-seq (P < 0.001; R = 0.68).


( G ) Western blotting for ISLR HA in lysates of UC - MSCs transfected with pCMV5 - HA or pCMV5 - ETS1 . α - Tubulin was used as a loading control .

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(G) Western blotting for ISLR and HA in lysates of UC-MSCs transfected with pCMV5-HA or pCMV5-ETS1. α-Tubulin was used as a loading control.


( H ) Luciferase activity in lysates of UC - MSCs transfected with luciferase reporter plasmids of pGL3 - basic empty vector ( basic ) , wild type ISLR promoter or mutant promoter with mutation of ETS1 binding sites under normal ETS1 overexpression conditions . n = 3 .

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(H) Luciferase activity in lysates of UC-MSCs transfected with luciferase reporter plasmids of pGL3-basic empty vector (basic), wild type ISLR promoter or mutant promoter with mutation of ETS1 binding sites under normal and ETS1 overexpression conditions. n = 3.