U2OS transfected with siCTL , or siDDX5 were subjected to DRIP - qPCR with anti - IgG , anti - S9 . 6 antibodies with or without RNaseH treatment . gene location genomic qPCR amplification region are shown at top of each panel . B , E , H , S X denote location of BsrGI , EcoRI , HindIII , SspI , XbaI . identified R - loop peaks were extracted from R - loop database ( R - loop DB ) for each region . bar graphs are average S . E . M . from 3 independent experiments . Statistical significance was assessed using t - . * : 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 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

U2OS cells transfected with siCTL, or siDDX5 were subjected to DRIP-qPCR analysis with anti-IgG, and anti-S9.6 antibodies with or without RNaseH treatment. The gene location and genomic qPCR amplification region are shown at the top of each panel. B, E, H, S and X denote the location of the BsrGI, EcoRI, HindIII, SspI, and XbaI. The identified R-loop peaks were extracted from the R-loop database (R-loop DB) for each region. The bar graphs are the average and S.E.M. from 3 independent experiments. Statistical significance was assessed using t-test. *: p < 0.05.


U2OS were lysed lysates were incubated with anti - PRMT5 antibody or immunoglobulin G ( IgG ) as control followed by A Sepharose bead addition . immunoprecipitated ( IP ) or whole extract ( WCE ) were separated by SDS - PAGE Western blotted ( WB ) with indicated antibodies . Same as panel ( A ) except anti - DDX5 antibody was used instead of anti - PRMT5 antibody for immunoprecipitation .

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

U2OS cells were lysed and the lysates were incubated with anti-PRMT5 antibody or immunoglobulin G (IgG) as control followed by protein A Sepharose bead addition. The immunoprecipitated (IP) proteins or whole cell extract (WCE) were separated by SDS-PAGE and Western blotted (WB) with the indicated antibodies. Same as panel (A) except anti-DDX5 antibody was used instead of anti-PRMT5 antibody for immunoprecipitation.


mass spectrum of R502 monomethylation from Flag - DDX5 expressed in U2OS .

The mass spectrum of R502 monomethylation from Flag-DDX5 expressed in U2OS.


HEK293 transfected with Flag - DDX5 were lysed immunoprecipitated with anti - Flag antibodies . bound were along with WCE from untransfected ( - ) Flag - DDX5 ( + ) transfected were Western blotted with anti - FLAG , anti - methylarginine antibodies ( MeR ) .

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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

HEK293 cells transfected with Flag-DDX5 were lysed and immunoprecipitated with anti-Flag antibodies. The bound proteins were along with WCE from untransfected (-) and Flag-DDX5 (+) transfected cells were Western blotted with anti-FLAG, anti-methylarginine antibodies (MeR).


knockdown using siRNAs of indicated gene in U2OS was confirmed by Western

The knockdown using siRNAs of the indicated gene in U2OS cells was confirmed by Western blotting


knockdown using siRNAs of indicated gene in U2OS were analyzed by immunofluorescence with S9 . 6 anti - nucleolin antibodies . A typical image is shown .

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

The knockdown using siRNAs of the indicated gene in U2OS cells were analyzed by immunofluorescence with S9.6 and anti-nucleolin antibodies. A typical image is shown.


Western blotting of PRMT5 DDX5 knockdowns .

Western blotting of PRMT5 and DDX5 knockdowns.


Representative of S9 . 6 in siCTL , siDDX5 , or siPRMT5 knockdown at transcription on ( TA - KR ) or off ( TetR - KR ) genomic loci in U2OS TRE . nuclei are visualized by DAPI .

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

Representative images of S9.6 staining in siCTL, siDDX5, or siPRMT5 knockdown at transcription on (TA-KR) or off (TetR-KR) genomic loci in U2OS TRE cells. The nuclei are visualized by DAPI.


of S9 . 6 foci in indicated conditions . Three independent experiments were performed 50 were analyzed in each experiment . Error bars represent mean of S9 . 6 foci with s . e . m . statistical was performed by two - Student ' s t - ( Mann - Whitney U - ) . * * * * , p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 the S9.6 foci intensity in the indicated conditions. Three independent experiments were performed and 50 cells were analyzed in each experiment. Error bars represent mean of S9.6 foci intensity quantification with s.e.m. The statistical analysis was performed by two-tailed Student's t-test (Mann-Whitney test U-test). ****, p < 0.0001.


U2OS transfected with siCTL or siPRMT5 were subjected to DRIP - qPCR . average S . E . M . from 3 independent experiments is shown . Statistical significance was assessed using Student ' s t - . * : p < 0 . 05 ; * * : p < 0 . 01 .

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

U2OS cells transfected with siCTL or siPRMT5 were subjected to DRIP-qPCR analysis. The average and S.E.M. from 3 independent experiments is shown. Statistical significance was assessed using Student's t-test. *: p < 0.05; and **: p < 0.01.


Western blotting of whole extracts from siCTL , siPRMT5 , siDDX5 or siDDX5 / siPRMT5 transfected .

Western blotting of whole cell extracts from siCTL, siPRMT5, siDDX5 or siDDX5/ siPRMT5 transfected cells.


U2OS transfected with siCTL , siPRMT5 , siDDX5 or siDDX5 / siPRMT5 were subjected to DRIP - qPCR . average S . E . M . from 3 independent experiments is shown . Statistical significance was assessed using Student ' s t - . * : p < 0 . 05 ; * * : p < 0 . 01 .

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

U2OS cells transfected with siCTL, siPRMT5, siDDX5 or siDDX5/ siPRMT5 were subjected to DRIP-qPCR analysis. The average and S.E.M. from 3 independent experiments is shown. Statistical significance was assessed using Student's t-test. *: p < 0.05; and **: p < 0.01.


U2OS were transfected with a Flag - DDX5 expression vector in presence of siCTL or siPRMT5 . Lysates were immunoprecipitated with IgG or anti - Flag antibodies as indicated . WCE bound were Western blotted with anti - symmetrical dimethylarginine antibody or anti - Flag antibodies .

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

U2OS cells were transfected with a Flag-DDX5 expression vector in the presence of siCTL or siPRMT5. Lysates were immunoprecipitated with IgG or anti-Flag antibodies as indicated. The WCE and the bound proteins were Western blotted with anti-symmetrical dimethylarginine antibody or anti-Flag antibodies.


Coomassie blue ( left panel ) in vitro methylation ( right panel ) of indicated GST - DDX5 fragments GST - DDX5 - RK mutant .

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 O O O O

Coomassie blue (left panel) and in vitro methylation assay (right panel) of indicated GST-DDX5 fragments and the GST-DDX5-RK mutant.


Immunofluorescence with S9 . 6 anti - Flag antibodies of U2OS transfected with Flag - tagged DDX5 , DDX5 - RK or DDX5 - XD ( helicase dead ) . S9 . 6 signal was counted in both Flag - negative Flag - positive . Flag - negative were considered as untransfected . graphs shown represent with S . E . M from 3 independent experiments . Statistical significance was assessed using one - way ANOVA t - . * * * * , p < 0 . 0001 .

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 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

Immunofluorescence analysis with S9.6 and anti-Flag antibodies of U2OS cells transfected with Flag-tagged DDX5, DDX5-RK or DDX5-XD (helicase dead). Nuclear S9.6 signal was counted in both Flag-negative and Flag-positive cells. The Flag-negative cells were considered as untransfected cells. The graphs shown represent the quantification with the S.E.M from 3 independent experiments. Statistical significance was assessed using one-way ANOVA t-test. ****, p < 0.0001.


Immunofluorescence with S9 . 6 anti - Flag antibodies of U2OS transfected with siCTL or siDDX5 - 1 Flag - tagged DDX5 ( + WT ) or DDX5 - RK ( + RK ) as indicated . graphs show average S . E . M . from three independent experiments . Statistical significance was assessed using one - way ANOVA t - . * * * * , p < 0 . 0001 .

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 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

Immunofluorescence analysis with S9.6 and anti-Flag antibodies of U2OS cells transfected with siCTL or siDDX5-1 and Flag-tagged DDX5 (+WT) or DDX5-RK (+RK) as indicated. The graphs show the average and S.E.M. from three independent experiments. Statistical significance was assessed using one-way ANOVA t-test. ****, p < 0.0001.


HEK293 were transfected with siCTL , or siDDX5 . next day , that received siDDX5 were subsequently transfected with empty pcDNA3 vector ( - ) , expression vectors encoding Flag - DDX5 ( WT ) , or Flag - DDX5 - RK ( RK ) . Forty eight hours later , were subjected to DRIP - qPCR . average with S . E . M . from 3 independent experiments is shown . Statistical significance was assessed using Student ' s t - . * : p < 0 . 05 ; * * : p < 0 . 01 n . s . : 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 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 O O O O O O O O O

HEK293 cells were transfected with siCTL, or siDDX5. The next day, the cells that received siDDX5 were subsequently transfected with empty pcDNA3 vector (-), expression vectors encoding Flag-DDX5 (WT), or Flag-DDX5-RK (RK). Forty eight hours later, all the cells were subjected to DRIP-qPCR analysis. The average with the S.E.M. from 3 independent experiments is shown. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and n.s.: not significant.


HEK293 were transfected with empty pcDNA3 vector ( - ) or Flag - DDX5 ( + ) . Whole lysates ( WCE ) anti - Flag immunoprecipitations ( IP ) were immunoblotted with anti - Flag anti - XRN2 antibodies .

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 B-MEASURED_VAR O O

HEK293 cells were transfected with empty pcDNA3 vector (-) or Flag-DDX5 (+). Whole cell lysates (WCE) and anti-Flag immunoprecipitations (IP) were immunoblotted with anti-Flag and anti-XRN2 antibodies.


Untransfected U2OS were lyzed subjected to immunoprecipitation with control immunoglobulin G ( IgG ) or anti - XRN2 antibodies . bound were separated by SDS - PAGE followed by Western blotting with anti - DDX5 or anti - XRN2 antibodies . migration of DDX5 XRN2 is depicted with an arrow .

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

Untransfected U2OS cells were lyzed and subjected to immunoprecipitation with control immunoglobulin G (IgG) or anti-XRN2 antibodies. The bound proteins were separated by SDS-PAGE followed by Western blotting with anti-DDX5 or anti-XRN2 antibodies. The migration of DDX5 and XRN2 is depicted with an arrow.


U2OS were transfected with empty pcDNA3 vector ( pcDNA3 ) or expression vectors encoding Flag - DDX5 WT ( 1 - 614 ) or truncated Flag - DDX5 ( 60 - 614 ; 1 - 554 ; 1 - 435 ) . WCE of transfected ( left panel ) were Western blotted with anti - Flag anti - XRN2 antibodies to confirm equivalent expression . transfected were lysed anti - Flag immunoprecipitations ( right panel ) performed for presence of co - immunoprecipitating endogenous XRN2 by Western blotting with anti - XRN2 antibodies . M denotes molecular mass markers in kDa .

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

U2OS cells were transfected with empty pcDNA3 vector (pcDNA3) or expression vectors encoding Flag-DDX5 WT (1-614) or truncated Flag-DDX5 proteins (60-614; 1-554; 1-435). WCE of the transfected cells (left panel) were Western blotted with anti-Flag and anti-XRN2 antibodies to confirm equivalent expression. The transfected cells were lysed and anti-Flag immunoprecipitations (right panel) performed for the presence of co-immunoprecipitating endogenous XRN2 by Western blotting with anti-XRN2 antibodies. M denotes molecular mass markers in kDa.


Untransfected U2OS stably expressing Flag - DDX5 WT or Flag - DDX5 - RK were subjected to immunoprecipitation with anti - Flag antibody . WCE anti - Flag immunoprecipitated were Western blotted with anti - monomethylarginine ( MeR ) , anti - Flag anti - XRN2 antibodies , respectively . M denotes molecular mass markers in kDa .

O O O 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 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

Untransfected and U2OS stably expressing Flag-DDX5 WT or Flag-DDX5-RK were subjected to immunoprecipitation with anti-Flag antibody. The WCE and the anti-Flag immunoprecipitated proteins were Western blotted with anti-monomethylarginine (MeR), anti-Flag and anti-XRN2 antibodies, respectively. M denotes molecular mass markers in kDa.


U2OS transfected with siCTL , siDDX5 , or siXRN2 were subjected to DRIP - qPCR with anti - IgG , anti - S9 . 6 antibodies with or without RNaseH treatment . average S . E . M . from 3 independent experiments is shown . Statistical significance was assessed using Student ' s t - . * : p < 0 . 05 ; * * : p < 0 . 01 .

O O O O O 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

U2OS cells transfected with siCTL, siDDX5, or siXRN2 were subjected to DRIP-qPCR analysis with anti-IgG, and anti-S9.6 antibodies with or without RNaseH treatment. The average and S.E.M. from 3 independent experiments is shown. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01.


HEK293 were transfected with siCTL , siDDX5 , or siXRN2 . gene location qPCR amplification region are shown at top of each panel . identified R - loop peaks were extracted from R - loop database ( R - loop DB ) for each gene region . R - loop : DRIP denotes anti - S9 . 6 DRIP - qPCR Pol II ChIP denotes pol II ChIP . bar graphs show average S . E . M . from at least three independent experiments . Statistical significance was assessed using Student ' s t - . * : p < 0 . 05 ; * * : p < 0 . 01 * * * * : p < 0 . 0001 . B , E , H , S X denote location of BsrGI , EcoRI , HindIII , SspI , XbaI .

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

HEK293 cells were transfected with siCTL, siDDX5, or siXRN2. The gene location and qPCR amplification region are shown at the top of each panel. The identified R-loop peaks were extracted from R-loop database (R-loop DB) for each gene region. R-loop: DRIP denotes anti-S9.6 DRIP-qPCR and Pol II ChIP denotes RNA pol II ChIP. The bar graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001. B, E, H, S and X denote the location of the BsrGI, EcoRI, HindIII, SspI, and XbaI.


Schematic representation of β - actin locus location of genomic PCR primers used for ChIP - qPCR DRIP - qPCR . Prom ( promoter ) , RB ( region B ) , poly ( A ) RD ( region D ) indicate regions analyzed by qPCR .

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

Schematic representation of the β-actin locus and the location of the genomic PCR primers used for ChIP-qPCR and DRIP-qPCR. Prom (promoter), RB (region B), poly(A) and RD (region D) indicate the regions analyzed by qPCR.


HEK293 were transfected with siCTL or siDDX5 XRN2 ChIP assays were performed . XRN2 ChIP signal was normalized to Pol II ChIP signal . WCE were immunoblotted with anti - DDX5 α - tubulin antibodies , latter was used as a loading control . graphs show average S . E . M . from at least three independent experiments . Statistical significance was assessed using Student ' s t - . * : p < 0 . 05 ; * * : p < 0 . 01 .

O O O O 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 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

HEK293 cells were transfected with siCTL or siDDX5 and XRN2 ChIP assays were performed. The XRN2 ChIP signal was normalized to the Pol II ChIP signal. WCE were immunoblotted with anti-DDX5 and α-tubulin antibodies, the latter was used as a loading control. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01.


HEK293 were transfected with siCTL , siDDX5 or siXRN2 . were subjected to Western blotting , Pol II ChIP DRIP of β - actin gene . graphs show average S . E . M . from at least three independent experiments . Statistical significance was assessed using Student ' s t - . * : p < 0 . 05 ; * * : p < 0 . 01 * * * * : p < 0 . 0001 .

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 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

HEK293 cells were transfected with siCTL, siDDX5 or siXRN2. The cells were subjected to Western blotting, RNA Pol II ChIP and DRIP analysis of β-actin gene. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001.


HEK293 were transfected with siCTL or siDDX5 . next day , that received siDDX5 were subsequently transfected with empty pcDNA3 vector ( - ) , expression vectors encoding Flag - DDX5 ( WT ) , or Flag - DDX5 - RK ( RK ) . were subjected to Western blotting to visualize Flag - DDX WT RK expression . Pol II ChIP DRIP of β - actin gene were performed . For ChIP , Y - axis shows signal - to - noise of Pol II IP relative to control IgG IP . graphs show average S . E . M . from at least three independent experiments . Statistical significance was assessed using Student ' s t - . * : p < 0 . 05 ; * * : p < 0 . 01 * * * * : p < 0 . 0001 .

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

HEK293 cells were transfected with siCTL or siDDX5. The next day, the cells that received siDDX5 were subsequently transfected with empty pcDNA3 vector (-), expression vectors encoding Flag-DDX5 (WT), or Flag-DDX5-RK (RK). The cells were subjected to Western blotting to visualize Flag-DDX WT and RK expression. Pol II ChIP and DRIP analysis of β-actin gene were performed. For the ChIP analysis, the Y-axis shows the signal-to-noise ratio of RNA Pol II IP relative to control IgG IP. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001.


HEK293 were transfected with siCTL or siPRMT5 . were subjected to Western blotting , Pol II ChIP DRIP of β - actin gene . graphs show average S . E . M . from at least three independent experiments . Statistical significance was assessed using Student ' s t - . * : p < 0 . 05 ; * * : p < 0 . 01 .

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

HEK293 cells were transfected with siCTL or siPRMT5. The cells were subjected to Western blotting, RNA Pol II ChIP and DRIP analysis of β-actin gene. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01.


HEK293 were transfected with empty plasmid ( - ) , wild type Flag - DDX5 ( WT ) or its RK mutant ( RK ) subjected to western blot Flag - DDX5 ChIP - qPCR of β - actin gene . graphs show average S . E . M . from at least three independent experiments . Statistical significance was assessed using Student ' s t - . * : p < 0 . 05 ; * * : p < 0 . 01 * * * * : p < 0 . 0001 ; n . s . not significant .

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 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

HEK293 cells were transfected with empty plasmid (-), wild type Flag-DDX5 (WT) or its RK mutant (RK) and subjected to western blot and Flag-DDX5 ChIP-qPCR analysis of β-actin gene. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001; n.s. not significant.


( A ) Human osteosarcoma U2OS GFP - LC3 were treated with a library of polyphenols polyamines for 6 h then GFP - LC3 dots were counted to measure autophagy activity . Top 20 hits were shown in right frame .

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

(A) Human osteosarcoma U2OS GFP-LC3 cells were treated with a library of polyphenols and polyamines for 6 h And then GFP-LC3 dots were counted to measure autophagy activity. Top 20 hits were shown in the right frame.


Human osteosarcoma U2OS ( B or neuroglioma H4 ( D stably expressing GFP - LC3 were treated with a library of chalcones ( 30 µM ) as indicated , or were left untreated for 6 h . were then fixed GFP - LC3 dots were counted as an indicator for autophagy . Data are means ± SD ( * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ) . Representative are shown in ( B , D ) . Scale bar equals 10 µm . ( C , E ) Acetylation : U2OS H4 were treated as described above , followed by incubation with specific antibodies to block acetylated tubulin ; Thereafter immunofluorescence was conducted with antibodies against acetylated lysine residues appropriate AlexaFluor conjugated secondary antibodies before assessment of intensities . Representative of acetylation are shown in ( C , E ) . Scale bar equals 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Human osteosarcoma U2OS cells (B or neuroglioma H4 cells (D stably expressing GFP-LC3 were treated with a library of chalcones (30 µM) as indicated , or were left untreated for 6 h. The cells were then fixed and GFP-LC3 dots were counted as an indicator for autophagy. Data are means ± SD (* = p < 0.05; ** = p < 0.01;*** = p < 0.001). Representative images are shown in (B,D). Scale bar equals 10 µm. (C,E) Acetylation: U2OS and H4 cells were treated as described above, followed by incubation with specific antibodies to block acetylated tubulin; Thereafter immunofluorescence was conducted with antibodies against acetylated lysine residues and appropriate AlexaFluor conjugated secondary antibodies before the assessment of cytoplasmic fluorescence intensities. Representative images of acetylation are shown in (C,E). Scale bar equals 10 µm.


Human osteosarcoma U2OS F ) or neuroglioma H4 H ) stably expressing GFP - LC3 were treated with a library of chalcones ( 30 µM ) as indicated or rapamycin ( 10 µM ) , or were left untreated for 6 h . were then fixed GFP - LC3 dots were counted as an indicator for autophagy . Data are means ± SD ( * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ) . Viability : U2OS H4 were treated with 30 µM of indicated chalcones for 24 h then nuclei were stained with Hoechst 33342 number of harboring normal nuclei ( i . e . non pyknotic , " healthy " ) was determined . chalcones were hierarchically clustered upon z - scoring following : autophagy ( number of GFP - LC3 dots ) , viability ( number of healthy ) acetylation ( of acetylated lysine residues ) in U2OS H4 . Results are reported as a ( G , I ) .

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

Human osteosarcoma U2OS cells F) or neuroglioma H4 cells H) stably expressing GFP-LC3 were treated with a library of chalcones (30 µM) as indicated or rapamycin (10 µM), or were left untreated for 6 h. The cells were then fixed and GFP-LC3 dots were counted as an indicator for autophagy. Data are means ± SD (* = p < 0.05; ** = p < 0.01;*** = p < 0.001). Viability: U2OS and H4 cells were treated with 30 µM of the indicated chalcones for 24 h and then nuclei were stained with Hoechst 33342 and the number of cells harboring normal nuclei (i.e. non pyknotic, "healthy cells") was determined. All chalcones were hierarchically clustered upon z-scoring the following phenotypes: autophagy (number of GFP-LC3 dots), viability (number of healthy cells) and acetylation (cytoplasmic fluorescence intensity of acetylated lysine residues) in U2OS and H4 cells. Results are reported as a heatmap (G, I).


( J , K ) U2OS were treated with 3 , 4 - DC in presence or absence of dichloroacetate ( DCA ) , α - ketoisocaproic acid ( KIC ) , or L - leucine ( Leu ) . GFP - LC3 dots were quantified in ( J ) . Acetylation was performed as previously described , acetylation in cytoplasm was measured in ( K ) . Data are means ± SD ( * * * = p < 0 . 001 versus DMSO / Ctr ; # # = p < 0 . 01 , # # # = p < 0 . 001 versus 3 , 4 - DC / Ctr ) .

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

(J, K) U2OS cells were treated with 3,4-DC in the presence or absence of dichloroacetate (DCA), α-ketoisocaproic acid (KIC), or L-leucine (Leu). GFP-LC3 dots were quantified in (J). Acetylation staining was performed as previously described, and acetylation intensity in the cytoplasm was measured in (K). Data are means ± SD (*** = p < 0.001 versus DMSO/Ctr; ##=p<0.01, ###=p<0.001 versus 3,4-DC/Ctr).


Hepatoma HepG2 were treated with indicated concentrations of 3 , 4 - DC for 8 h ( A , B ) GAPDH was measured as a loading control . Band intensities of p62 , GAPDH , LC3 - I LC3 - II were measured ratios of p62 or LC3 - II versus GAPDH ( LC3 - II / GAPDH , p62 / GAPDH ) LC3 - II versus LC3 - I ( LC3 - II / LC3 - I ) were calculated in ( B Data are means ± of at least three independent experiments ( LC3 - II / GAPDH : * = p < 0 . 05 , * * = p < 0 . 01 ; p62 / GAPDH : # = p < 0 . 05 , # # # = p < 0 . 001 ; LC3 - II / LC3 - I : $ = p < 0 .

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

Hepatoma HepG2 cells were treated with the indicated concentrations of 3,4-DC for 8 h (A,B) GAPDH was measured as a loading control. Band intensities of p62, GAPDH, LC3-I and LC3-II were measured and ratios of p62 or LC3-II versus GAPDH (LC3-II/GAPDH, p62/GAPDH) and LC3-II versus LC3-I (LC3-II/LC3-I) were calculated in (B Data are means ± SEM of at least three independent experiments (LC3-II/GAPDH: * = p < 0.05, ** = p < 0.01; p62/GAPDH: # = p < 0.05, ### = p < 0.001; LC3-II/LC3-I: $=p<0.05


Hepatoma HepG2 were treated with 30 µM 3 , 4 - DC for indicated time points ( C , D ) . Then , were processed to measure LC3 p62 by SDS - PAGE immunoblot GAPDH was measured as a loading control . Band intensities of p62 , GAPDH , LC3 - I LC3 - II were measured ratios of p62 or LC3 - II versus GAPDH ( LC3 - II / GAPDH , p62 / GAPDH ) LC3 - II versus LC3 - I ( LC3 - II / LC3 - I ) were calculated in D ) . Data are means ± of at least three independent experiments ( LC3 - II / GAPDH : * = p < 0 . 05 , * * = p < 0 . 01 ; p62 / GAPDH : # = p < 0 . 05 , # # # = p < 0 . 001 ; LC3 - II / LC3 - I : $ = 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 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-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 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 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 I-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

Hepatoma HepG2 cells were treated with 30 µM 3,4-DC for the indicated time points (C,D). Then, cells were processed to measure LC3 and p62 protein levels by SDS-PAGE and immunoblot GAPDH was measured as a loading control. Band intensities of p62, GAPDH, LC3-I and LC3-II were measured and ratios of p62 or LC3-II versus GAPDH (LC3-II/GAPDH, p62/GAPDH) and LC3-II versus LC3-I (LC3-II/LC3-I) were calculated in D). Data are means ± SEM of at least three independent experiments (LC3-II/GAPDH: * = p < 0.05, ** = p < 0.01; p62/GAPDH: # = p < 0.05, ### = p < 0.001; LC3-II/LC3-I: $=p<0.05).


( E , F ) HepG2 were treated with 30 µM 3 , 4 - DC for 8 h in presence or absence of chloroquine ( CQ , 50µM ) for 4 h . SDS - PAGE immunoblot were performed Band intensities of LC3 - II GAPDH were assessed their ( LC3 - II / GAPDH ) was calculated in ( F ) . Data are means ± of at least three independent experiments ( * = p < 0 . 05 , * * = p < 0 . 01 versus untreated control ; # # = p < 0 . 01 versus CQ ) .

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

(E,F) HepG2 were treated with 30 µM 3,4-DC for 8 h in the presence or absence of chloroquine (CQ, 50µM) for 4 h. SDS-PAGE and immunoblot were performed Band intensities of LC3-II and GAPDH were assessed and their ratio (LC3-II/GAPDH) was calculated in (F). Data are means ± SEM of at least three independent experiments (* = p < 0.05, ** = p < 0.01 versus untreated control; ##=p<0.01 versus CQ).


( G , H ) U2OS - GFP - LC3 were treated with 30 µM 3 , 4 - DC , 10 µM rapamycin , or left untreated for 16 h in presence or absence of CQ for 4 h . GFP - LC3 dots were measured . Data are means ± SD ( * * * = p < 0 . 001 versus untreated ; # # # = p < 0 . 001 versus CQ ) .

O O O O O O O O O O O O O O O O O O O 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,H) U2OS-GFP-LC3 cells were treated with 30 µM 3,4-DC, 10 µM rapamycin, or left untreated for 16 h in the presence or absence of CQ for 4 h. GFP-LC3 dots were measured. Data are means ± SD (*** = p < 0.001 versus untreated; ###=p<0.001 versus CQ).


( I , J ) U2OS stably expressing LC3 fused with tandem GFP - RFP ( GFP - RFP - LC3 ) were treated with indicated raising doses of 3 , 4 - DC or 50 µM chloroquine ( CQ ) for 16 h . After fixation , GFP - LC3 RFP - LC3 dots were measured by automated image acquisition . GFP RFP double positive LC3 dots indicating autophagosomes ( GFP + ) , GFP negative but RFP positive LC3 dots indicating autolysosomes ( GFP - ) were counted in ( J ) . Data are means ± SD ( * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ) . Representative are shown in I . Scale bar equals 10 µm .

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 B-MEASURED_VAR O O B-MEASURED_VAR 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 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

(I,J) U2OS cells stably expressing LC3 fused with tandem fluorescent GFP-RFP proteins (GFP-RFP-LC3) were treated with the indicated raising doses of 3,4-DC or 50 µM chloroquine (CQ) for 16 h. After fixation, GFP-LC3 and RFP-LC3 dots were measured by automated image acquisition and analysis. GFP and RFP double positive LC3 dots indicating autophagosomes (GFP+), and GFP negative but RFP positive LC3 dots indicating autolysosomes (GFP-) were counted in (J). Data are means ± SD (* = p < 0.05;** = p < 0.01;*** = p < 0.001). Representative images are shown in I. Scale bar equals 10 µm.


A , B ) HepG2 were treated for indicated time with 30 µM 3 , 4 - DC ( A ) or indicated dose of 3 , 4 - DC for 8 h ( B ) . Thereafter , were collected SDS - PAGE immunoblots were performed as described before . Total p70S6K , phosphorylated p70S6K at T389 ( P - p70S6K ) , Atg14 , Lamp1 , LC3 , p62 , GAPDH were measured with specific antibodies . Samples for immunoblots in A B were run in parallel instances , then cut into horizontal stripes probed separately . Blots probed for P - p70S6K were stripped reprobed to detect p70 independently from its phosphorylation status . same applies for LAMP1 GAPDH .

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

A, B) HepG2 cells were treated for the indicated time with 30 µM 3,4-DC (A) or indicated dose of 3,4-DC for 8 h (B). Thereafter, cells were collected and SDS-PAGE and immunoblots were performed as described before. Total p70S6K, phosphorylated p70S6K at T389 (P-p70S6K), Atg14, Lamp1, LC3, p62, and GAPDH protein levels were measured with specific antibodies. Samples for immunoblots in A and B were run in parallel instances, then cut into horizontal stripes and probed separately. Blots probed for P-p70S6K were stripped and reprobed to detect p70 independently from its phosphorylation status. The same applies for LAMP1 and GAPDH.


( C , D ) H4 - GFP - LC3 were treated with 30 µM 3 , 4 - DC in presence or absence of CHX or AMD or with CQ for 16 h as controls , as indicated . GFP - LC3 dots were quantified in ( D ) . Data are means ± SD ( * = p < 0 . 05 , * * = p < 0 . 01 versus untreated control ; # = p < 0 . 05 versus untreated with CQ ) .

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

(C, D) H4-GFP-LC3 cells were treated with 30 µM 3,4-DC in the presence or absence of CHX or AMD or with CQ for 16 h as controls, as indicated. GFP-LC3 dots were quantified in (D). Data are means ± SD (* = p < 0.05, ** = p < 0.01 versus untreated control; # = p < 0.05 versus untreated with CQ).


( E ) expression 16 h after exposure to 3 , 4 - DC of indicated genes in HepG2 were measured by RT - PCR normalized to expression of housekeeping gene ( GAPDH ) . Data are means ± SD ( * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = 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

(E) RNA expression levels 16 h after exposure to 3,4-DC of the indicated genes in HepG2 cells were measured by RT-PCR and normalized to the expression of the housekeeping gene (GAPDH). Data are means ± SD (* = p < 0.05;** = p < 0.01;*** = p < 0.001).


, G ) U2OS GFP - LC3 were enucleated to obtain cytoplasts , which were further treated with 3 , 4 - DC torin1 . GFP - LC3 dots in cytoplasts were counted as shown in ( G ) . Data are means ± SD ( * * = p < 0 . 01 ) . Representative with cytoplast marked by a square are shown in ( F ) . Scale bar equals 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 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

,G) U2OS GFP-LC3 cells were enucleated to obtain cytoplasts, which were further treated with 3,4-DC and torin1. GFP-LC3 dots in cytoplasts were counted as shown in (G). Data are means ± SD (** = p < 0.01). Representative images with cytoplast marked by a white square are shown in (F). Scale bar equals 10 µm.


U2OS stably expressing GFP - TFEB fusion were treated with indicated chalcones at 30 µM for 6 h . GFP intensities in nuclei cytoplasm were measured of GFP intensities in nuclei cytoplasm were calculated to indicate TFEB translocation to nuclei ( A ) . Data are means ± SD ( * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = 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 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

U2OS cells stably expressing GFP-TFEB fusion protein were treated with indicated chalcones at 30 µM for 6 h. GFP intensities in nuclei and cytoplasm were measured and the ratio of GFP intensities in nuclei and cytoplasm were calculated to indicate TFEB translocation to nuclei (A). Data are means ± SD (* = p < 0.05;** = p < 0.01;*** = p < 0.001).


U2OS stably expressing GFP - TFEB fusion were treated with indicated chalcones at 30 µM for 6 h . Representative are shown in ( B ) . Scale bar equals 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

U2OS cells stably expressing GFP-TFEB fusion protein were treated with indicated chalcones at 30 µM for 6 h. Representative images are shown in (B). Scale bar equals 10 µm.


( C , D ) U2OS were treated with 30 µM 3 , 4 - DC for 6 h , then endogenous TFEB translocation was assessed by immunostaining ( C ) . TFEB intensities in nuclei cytoplasm were measured of TFEB intensities in nuclei cytoplasm were calculated to indicate TFEB translocation to nuclei ( D ) . Data are means ± SD ( * * * = p < 0 . 001 ) . Representative are shown in ( C ) . Scale bar equals 10 µm .

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 B-MEASURED_VAR 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 O O O O 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, D) U2OS cells were treated with 30 µM 3,4-DC for 6 h, and then endogenous TFEB translocation was assessed by immunostaining (C). TFEB intensities in nuclei and cytoplasm were measured and the ratio of TFEB intensities in nuclei and cytoplasm were calculated to indicate TFEB translocation to nuclei (D). Data are means ± SD (*** = p < 0.001). Representative images are shown in (C). Scale bar equals 10 µm.


( E , F ) U2OS were treated with 30 µM 3 , 4 - DC for 6 h or were left untreated . fractions were assessed for translocation of transcription factors TFEB TFE3 by SDS page .

O O O O O O O O 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

(E, F) U2OS cells were treated with 30 µM 3,4-DC for 6 h or were left untreated. Cytoplasmic and nuclear fractions were assessed for nuclear translocation of the transcription factors TFEB and TFE3 by SDS page.


MEF wild type TSC2 knockout ( TSC2 KO ) were treated with 3 , 4 - DC for 6 h . were fixed immunofluorescence was performed to detect endogenous TFEB . of TFEB intensities between nuclei cytoplasm was calculated in ( H ) . Data are means ± SD ( * * * = p < 0 . 001 versus Ctr ; # # # = p < 0 . 001 versus WT / 3 , 4 - DC ) . Representative are shown in ( G ) . Scale bar equals 10 µm .

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

MEF wild type and TSC2 knockout (TSC2 KO) cells were treated with 3,4-DC for 6 h. The cells were fixed and immunofluorescence was performed to detect endogenous TFEB. The ratio of TFEB intensities between nuclei and cytoplasm was calculated in (H). Data are means ± SD (*** = p < 0.001 versus Ctr; ###=p<0.001 versus WT/3,4-DC). Representative images are shown in (G). Scale bar equals 10 µm.


MEF wild type TSC2 knockout ( TSC2 KO ) were treated with 3 , 4 - DC for 6 h . ( I ) After treatment , nuclei were isolated western blot was performed to detect cytosol TFEB .

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 B-MEASURED_VAR O

MEF wild type and TSC2 knockout (TSC2 KO) cells were treated with 3,4-DC for 6 h. (I) After the treatment, nuclei were isolated and western blot was performed to detect nuclear and cytosol TFEB protein levels.


( J - L ) MEF wild type TSC2 KO were collected lysed for western blot after treatment with 3 , 4 - DC . Antibodies against LC3 , p62 , TSC2 , or GAPDH were administrated to detect , phosphorylation of mTOR substrate p70 S6K at Threonine 389 ( T389 ) was measured with phosphorylation specific antibody ( P - p70 ( T389 ) ) anti - p70 antibody . bands intensities of LC3 - II , p62 , GAPDH were measured with Image J , ratios of LC3 - II / GAPDH p62 / GAPDH were calculated in ( K ) ( L ) . Data are means ± of at least three independent experiments ( * * = p < 0 . 01 , * * * = p < 0 . 001 vs Ctr ; # = p < 0 . 05 , # # = p < 0 . 01 vs WT / 3 , 4 - DC )

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

(J-L) MEF wild type and TSC2 KO cells were collected and lysed for western blot after the treatment with 3,4-DC. Antibodies against LC3, p62, TSC2, or GAPDH were administrated to detect protein levels, and phosphorylation of mTOR substrate p70 S6K at Threonine 389 (T389) was measured with the phosphorylation specific antibody (P-p70(T389)) and anti-p70 antibody. The bands intensities of LC3-II, p62, and GAPDH were measured with Image J, and the ratios of LC3-II/GAPDH and p62/GAPDH were calculated in (K) and (L). Data are means ± SEM of at least three independent experiments (** = p < 0.01, *** = p < 0.001 vs Ctr; #=p<0.05, ##=p<0.01 vs WT/3,4-DC)


( M , N ) U2OS treated with 3 , 4 - DC or Torin for 6 h were collected processed for western blot . TFEB phosphorylation at Serine 211 was checked with phosphorylation specific antibody ( P - TFEB ( S211 ) ) total TFEB level was also detected with anti - TFEB . bands intensities of P - TFEB total TFEB were measured with Image J , their was calculated in ( N ) . Data are means ± of at least three independent experiments ( * = p < 0 . 05 vs Ctr ) .

O 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 B-MEASURED_VAR 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 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

(M,N) U2OS cells treated with 3,4-DC or Torin for 6 h were collected and processed for western blot. TFEB phosphorylation at Serine 211 was checked with the phosphorylation specific antibody (P-TFEB(S211)) and total TFEB protein level was also detected with anti-TFEB. The bands intensities of P-TFEB and total TFEB were measured with Image J, and their ratio was calculated in (N). Data are means ± SEM of at least three independent experiments (* = p < 0.05 vs Ctr).


U2OS - GFP - LC3 transfected with 3 siRNAs specifically targeting TFEB ( siTFEB - # 1 , siTFEB - # 2 , siTFEB - # 3 ) or scrambled siRNA ( siCtr ) were treated with 3 , 4 - DC as indicated for 16 h . TFEB knockdown efficiency by TFEB siRNAs TFEB deficiency by knockout were checked by SDS - PAGE

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

U2OS-GFP-LC3 cells transfected with 3 individual siRNAs specifically targeting TFEB (siTFEB-#1, siTFEB-#2, siTFEB-#3) or scrambled siRNA (siCtr) were treated with 3,4-DC as indicated for 16 h. TFEB knockdown efficiency by TFEB siRNAs and TFEB deficiency by knockout were checked by SDS-PAGE and immunoblot


U2OS - GFP - LC3 transfected with 3 siRNAs specifically targeting TFEB ( siTFEB - # 1 , siTFEB - # 2 , siTFEB - # 3 ) or scrambled siRNA ( siCtr ) were treated with 3 , 4 - DC as indicated for 16 h . GFP - LC3 dots were quantified as indicator of autophagy Data are means ± SD ( * = p < 0 . 05 , * * = p < 0 . 01 versus siCtr or WT treated with 3 , 4 - DC ) . Representative are shown Scale bar equals 10 µm .

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 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

U2OS-GFP-LC3 cells transfected with 3 individual siRNAs specifically targeting TFEB (siTFEB-#1, siTFEB-#2, siTFEB-#3) or scrambled siRNA (siCtr) were treated with 3,4-DC as indicated for 16 h. GFP-LC3 dots were quantified as indicator of autophagy Data are means ± SD (* = p < 0.05, ** = p < 0.01 versus siCtr or WT cells treated with 3,4-DC). Representative images are shown Scale bar equals 10 µm.


U2OS wild type ( wt ) TFEB knockout ( TFEB KO ) GFP - LC3 were treated with 3 , 4 - DC as indicated for 16 h . TFEB knockdown efficiency by TFEB siRNAs TFEB deficiency by knockout were checked by SDS - PAGE

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

U2OS wild type (wt) and TFEB knockout (TFEB KO) GFP-LC3 cells were treated with 3,4-DC as indicated for 16 h. TFEB knockdown efficiency by TFEB siRNAs and TFEB deficiency by knockout were checked by SDS-PAGE and immunoblot


U2OS wild type ( wt ) TFEB knockout ( TFEB KO ) GFP - LC3 were treated with 3 , 4 - DC as indicated for 16 h . GFP - LC3 dots were quantified as indicator of autophagy ata are means ± SD ( * = p < 0 . 05 , * * = p < 0 . 01 versus siCtr or WT treated with 3 , 4 - DC ) . Representative are shown cale bar equals 10 µm .

O O 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 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

U2OS wild type (wt) and TFEB knockout (TFEB KO) GFP-LC3 cells were treated with 3,4-DC as indicated for 16 h. GFP-LC3 dots were quantified as indicator of autophagy ata are means ± SD (* = p < 0.05, ** = p < 0.01 versus siCtr or WT cells treated with 3,4-DC). Representative images are shown cale bar equals 10 µm.


U2OS wild type ( WT ) , TFEB or TFE3 knockout , or double knockout ( TF DKO ) were treated with 30 µM 3 , 4 - DC for 16 h . Following , were collected SDS - PAGE immunoblots were performed as described before . TFEB , TFE3 , LC3 , p62 , GAPDH were measured with specific antibodies ( G ) .

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

U2OS wild type (WT), TFEB or TFE3 knockout, or double knockout (TF DKO) cells were treated with 30 µM 3,4-DC for 16 h. Following, cells were collected and SDS-PAGE and immunoblots were performed as described before. TFEB, TFE3, LC3, p62, and GAPDH protein levels were measured with specific antibodies (G).


U2OS wild type ( WT ) , TFEB or TFE3 knockout , or double knockout ( TF DKO ) were treated with 30 µM 3 , 4 - DC for 16 h . GFP - LC3 dots were quantified as indicator of autophagy ( H ) . Data are means ± SD ( * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 versus Ctr , respectively ) .

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 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

U2OS wild type (WT), TFEB or TFE3 knockout, or double knockout (TF DKO) cells were treated with 30 µM 3,4-DC for 16 h. GFP-LC3 dots were quantified as indicator of autophagy (H). Data are means ± SD (* = p < 0.05, ** = p < 0.01, *** = p < 0.001 versus Ctr, respectively).


( I ) U2OS WT , knockout for TFEB ( TFEB - / - ) or TFE3 ( TFE3 - / - ) , double knockout ( DKO ) were treated with 3 , 4 - DC for 24 h stained with LysoTracker Red for 30 min . Thereafter , red ( positive ) dots were measured . Data are means ± SD ( * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ) .

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(I) U2OS WT, knockout for TFEB (TFEB-/-) or TFE3 (TFE3-/-), double knockout (DKO) were treated with 3,4-DC for 24 h and stained with LysoTracker Red for 30 min. Thereafter, the red (positive) dots were measured. Data are means ± SD (* = p < 0.05; ** = p < 0.01; *** = p < 0.001).


( J ) Relative mRNA expression of key autophagy genes such as Lamp1 , Lc3b p62 were detected in controls 3 , 4 - DC treated U2OS . Data is depicted as a showing means of at least three independent experiments ( * * * = p < 0 . 001 versus WT / Ctr # = p < 0 . 05 , # # = p < 0 . 01 , as compared to WT treated with 3 , 4 - DC ) .

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

(J) Relative mRNA expression levels of key autophagy genes such as Lamp1, Lc3b and p62 were detected in controls and 3,4-DC treated U2OS cells. Data is depicted as a heatmap showing means of at least three independent experiments (*** = p < 0.001 versus WT/Ctr and # = p < 0.05, ## = p < 0.01, as compared to WT treated with 3,4-DC).


( A - D ) C57 / BL6 were treated with 3 , 4 - DC for 24 h . Following , liver heart tissue was excised were subjected to subcellular fractionation . Thereafter were separated by SDS PAGE immunoblots were performed to detect translocation of TFEB TFE3 ( A , C ) . GAPDH H3 were used as controls for fractions , respectively . Band intensities of TFEB , TFE3 H3 were measured ratios of TFs versus H3 ( TFsnuc / H3 ) were calculated in ( B , D ) . Data are means ± of at least three mice ( * * * = p < 0 . 001 versus Ctr )

O O O O O O O O O O O O O O O O O O O O O O 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 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 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) Female C57/BL6 animals were treated with 3,4-DC for 24 h. Following, liver and heart tissue was excised and cells were subjected to subcellular fractionation. Thereafter proteins were separated by SDS PAGE and immunoblots were performed to detect the nuclear translocation of TFEB and TFE3 (A, C). GAPDH and H3 were used as controls for cytosolic and nuclear fractions, respectively. Band intensities of nuclear TFEB, TFE3 and H3 were measured and ratios of nuclear TFs versus H3 (TFsnuc/H3) were calculated in (B, D). Data are means ± SEM of at least three mice (*** = p < 0.001 versus Ctr)


GFP - LC3 expressing mice were i . p . injected with 3 , 4 - DC for 24 h . Leupeptin ( Leu ) was used to autophagic flux in vivo GFP - LC3 dots were measured in liver tissue . Data are means ± of at least three mice ( * = p < 0 . 05 versus ctr without Leu ; # = p < 0 . 05 versus ctr with Leu ) .

O O O O O O O O O O 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

GFP-LC3 expressing mice were i.p. injected with 3,4-DC for 24 h. Leupeptin (Leu) was used to test autophagic flux in vivo and GFP-LC3 dots were measured in liver tissue. Data are means ± SEM of at least three mice (*= p < 0.05 versus ctr without Leu; #= p < 0.05 versus ctr with Leu).


C57 / BL6 mice were i . p . injected with 3 , 4 - DC or were starved for 24 hours . Following liver tissue was excised subjected to SDS PAGE immunoblot GAPDH was measured as a loading control . Band intensities of GAPDH LC3 - II were measured ratios LC3 - II versus GAPDH ( LC3 - II / GAPDH ) were calculated Data are means ± of at least three mice ( * = p < 0 . 05 , * * = p < 0 . 01 ) .

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

C57/BL6 mice were i.p. injected with 3,4-DC or were starved for 24 hours. Following liver tissue was excised and subjected to SDS PAGE and immunoblot GAPDH was measured as a loading control. Band intensities of GAPDH and LC3-II were measured and ratios LC3-II versus GAPDH (LC3-II/GAPDH) were calculated Data are means ± SEM of at least three mice (* = p < 0.05, ** = p < 0.01).


GFP - LC3 expressing mice were i . p . injected with 3 , 4 - DC for 24 h . Leupeptin ( Leu ) was used to autophagic flux in vivo GFP - LC3 dots were measured in heart ( I , J ) tissue . Data are means ± of at least three mice ( * = p < 0 . 05 versus ctr without Leu ; # = p < 0 . 05 versus ctr with Leu ) .

O O O O O O O O O O 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

GFP-LC3 expressing mice were i.p. injected with 3,4-DC for 24 h. Leupeptin (Leu) was used to test autophagic flux in vivo and GFP-LC3 dots were measured in heart (I, J) tissue. Data are means ± SEM of at least three mice (*= p < 0.05 versus ctr without Leu; #= p < 0.05 versus ctr with Leu).


C57 / BL6 mice were i . p . injected with 3 , 4 - DC or were starved for 24 hours . Following heart tissue was excised subjected to SDS PAGE immunoblot GAPDH was measured as a loading control . Band intensities of GAPDH LC3 - II were measured ratios LC3 - II versus GAPDH ( LC3 - II / GAPDH ) were calculated Data are means ± of at least three mice ( * = p < 0 . 05 , * * = p < 0 . 01 ) .

C57/BL6 mice were i.p. injected with 3,4-DC or were starved for 24 hours. Following heart tissue was excised and subjected to SDS PAGE and immunoblot GAPDH was measured as a loading control. Band intensities of GAPDH and LC3-II were measured and ratios LC3-II versus GAPDH (LC3-II/GAPDH) were calculated Data are means ± SEM of at least three mice (* = p < 0.05, ** = p < 0.01).


( A ) Twelve - old wild - type cardiac specific Atg7 knockout ( Atg7cKO ) mice were injected with corn oil ( vehicle control , Ctr ) or 3 , 4 - DC 24 h before surgery subjected to 3 h prolonged ischemia . Representative of left ventricular ( LV ) myocardial sections after alcian blue triphenyltetrazolium chloride ( TTC ) are depicted . Scale bar equals 1 mm .

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

(A) Twelve weeks-old wild-type and cardiac specific Atg7 knockout (Atg7cKO) mice were injected with corn oil (vehicle control, Ctr) or 3,4-DC 24 h before surgery and subjected to 3 h prolonged ischemia. Representative images of left ventricular (LV) myocardial sections after alcian blue and triphenyltetrazolium chloride (TTC) staining are depicted. Scale bar equals 1 mm.


( B , C ) of infarction ( indicated by pale color ) per at risk ( AAR ) ( B ) global AAR per LV ( C ) were measured ( mean value ± , * * p < 0 . 01 , # p < 0 . 05 vs WT / Ctr ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 size of the infarction area (indicated by pale color) per area at risk (AAR) (B) and the global AAR per LV (C) were measured (mean value ±SEM, **p<0.01, #p<0.05 vs WT/Ctr).


A . Induction of autophagy in murine MCA205 fibrosarcomas . were treated with 3 , 4 DC alone or in combination with chloroquine , were harvested 6 hours later for immunoblot detection of LC3 lipidation .

O O O O O O O O O O O O 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. Induction of autophagy in murine MCA205 fibrosarcomas. Cells were treated with 3,4 DC alone or in combination with chloroquine, and were harvested 6 hours later for immunoblot detection of LC3 lipidation.


( C - E ) . Cytofluorometric characterization of infiltrating lymphocytes ( TIL ) , in particular FOXP3 + regulatory T ( Treg ) , CD8 + cytotoxic T lymphocytes of CD8 + T over Treg .

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(C-E). Cytofluorometric characterization of tumor infiltrating lymphocytes (TIL), in particular FOXP3+ regulatory T cells (Treg), CD8+ cytotoxic T lymphocytes and the ratio of CD8+ T cells over Treg.


Growth kinetic of MCA205 fibrosarcomas that were wild type were evolving in immunocompetent C57Bl / 6 mice treated as

Growth kinetic of MCA205 fibrosarcomas that were wild type and were evolving in immunocompetent C57Bl/6 mice treated as indicated


Growth kinetic of MCA205 fibrosarcomas that were either wild type or Atg5KD ( H - I ) were evolving in immunocompetent C57Bl / 6 mice or immunodeficient nu / nu mice ( G ) , treated as

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

Growth kinetic of MCA205 fibrosarcomas that were either wild type or Atg5KD (H-I) and were evolving in immunocompetent C57Bl/6 mice or immunodeficient nu/nu mice (G), treated as indicated


( J - L ) Immunocompetent C57Bl / 6 mice were subcutaneously inoculated with TFEB / TFE3 double knockdown MCA205 or its scramble control ( K ) . When tumors became palpable , mice were treated as indicated growth curves from mice subjected to 3 , 4 - DC administration alone or in combination with MTX are shown ( J , L ) . Asterisks indicate significant effect of MTX with respect to untreated controls ( mean value ± , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) , while hash symbols refer to comparison of effects of MTX plus 3 , 4 - DC to MTX alone ( # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 ) ( n = 6 - 14 ) .

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

(J-L) Immunocompetent C57Bl/6 mice were subcutaneously inoculated with TFEB/TFE3 double knockdown MCA205 cells or its scramble control cells (K). When tumors became palpable, mice were treated as indicated Tumor growth curves from mice subjected to 3,4-DC administration alone or in combination with MTX are shown (J,L). Asterisks indicate significant effect of MTX with respect to untreated controls (mean value ±SEM, *p<0.05, **p<0.01, ***p<0.001), while hash symbols refer to the comparison of the effects of MTX plus 3,4-DC to MTX alone (#p<0.05, ##p<0.01, ###p<0.001)(n=6-14).


B Expression of RBM15 AS - RBM15 in different blood lineages by meta - of - seq data [ 38 ] : HSC ( hematopoietic ) , MPP ( multipotent progenitor ) , CMP ( common myeloid progenitor ) , CLP ( common lymphoid progenitor ) , GMP ( Granulocyte - macrophage progenitor ) , MEP ( megakaryocyte - erythrocyte progenitor ) , MK ( megakaryocyte ) , EB ( erythroid ) .

O O O B-MEASURED_VAR I-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

B Expression levels of RBM15 and AS-RBM15 in different blood lineages by meta-analysis of RNA-seq data [38]: HSC (hematopoietic stem cells), MPP (multipotent progenitor cells), CMP (common myeloid progenitor cells), CLP (common lymphoid progenitor cells), GMP (Granulocyte-macrophage progenitor cells), MEP (megakaryocyte- erythrocyte progenitor cells), MK (megakaryocyte), and EB (erythroid body).


C Relative expression of AS - RBM15 ( left ) RBM15 ( right ) in MEG - 01 treated with PMA were measured by real - time PCR assays ( n = 4 , mean ± SD ) . SD : standard deviation .

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

C Relative expression levels of AS-RBM15 (left) and RBM15 (right) in MEG-01 cells treated with PMA were measured by real-time PCR assays (n=4, mean ± SD). SD: standard deviation.


D Relative expression of AS - RBM15 RBM15 in CD34 + grown in pro - MK medium were measured by real - time PCR . GAPDH was used as an internal control ( n = 3 , mean ± SD ) . Also see Fig EV 1 .

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D Relative expression levels of AS-RBM15 and RBM15 in CD34+ cells grown in pro-MK medium were measured by real-time PCR. GAPDH was used as an internal control (n=3, mean ± SD). Also see Fig EV 1.


A CFU - MK assays with CD34 + expressing AS - RBM15 . MK colonies for acetylcholinesterase activity were counted ( right ) . P value was calculated using Student ' s t ( n = 3 , mean ± SD ) .

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

A CFU-MK assays with CD34+ cells expressing AS-RBM15. MK colonies staining for acetylcholinesterase activity were counted (right). P value was calculated using Student's t test (n=3, mean ± SD).


B number of CD41 + CD42 + mature MK was determined by FACS . CD34 + expressing sh - AS - RBM15 were grown in pro - MK medium containing thrombopoietin factor ( TPO / SCF ) for five days ( n = 4 , mean ± SD ) .

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

B The number of CD41+CD42+ mature MK cells was determined by FACS analysis. CD34+ cells expressing sh-AS-RBM15 were grown in pro-MK medium containing thrombopoietin and stem cell factor (TPO/SCF) for five days (n=4, mean ± SD).


D FACS of CD34 + grown in pro - MK medium expressing AS - RBM15 full or Δ5 or Δ3 . CD41 + CD42 + percentages were analyzed using one way ANOVA , * * P < 0 . 01 , ( n = 4 , mean ± SD ) .

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D FACS analysis of CD34+ cells grown in pro-MK medium and expressing AS-RBM15 full length or Δ5 or Δ3. The CD41+CD42+ cell percentages were analyzed using one way ANOVA, ** P<0.01, (n=4, mean ± SD).


E of CD41 + CD42 + in CD34 + expressing AS - RBM15 Exon 1 were determined by FACS after these were grown in pro - MK medium for five days . P value was calculated by t , p < 0 . 01 , ( n = 3 , mean ± SD ) .

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

E The percentage of CD41+CD42+ cells in CD34+ cells expressing AS-RBM15 Exon 1 were determined by FACS analysis after these cells were grown in pro-MK medium for five days. P value was calculated by t test, p<0.01, (n=3, mean ± SD).


A RBM15 in leukemia lines ( MEG - 01 , K562 , HEL ) were assessed by Western blotting ( WB ) ( left ) ( n = 3 ) . mRNA of RBM15 in overexpressing AS - RBM15 were measured by real - time PCR ( right , n = 3 , mean ± SD ) . GAPDH was used as an internal control for WB PCR .

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 B-MEASURED_VAR O O B-CONTROLLED_VAR I-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

A RBM15 protein levels in leukemia cell lines (MEG-01, K562, HEL) were assessed by Western blotting (WB) (left) (n=3). The mRNA levels of RBM15 in cells overexpressing AS-RBM15 were measured by real-time PCR (right, n=3, mean ± SD). GAPDH was used as an internal control for WB and PCR.


B ( left ) mRNA ( right ) of RBM15 in MEG - 01 expressing two different shRNAs against AS - RBM15 respectively were measured using WBs real - time PCR assays , respectively ( n = 3 , mean ± SD ) .

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B Protein levels (left) and mRNA levels (right) of RBM15 in MEG-01 cells expressing two different shRNAs against AS-RBM15 respectively were measured using WBs and real-time PCR assays, respectively (n=3, mean ± SD).


C ( left ) mRNA ( right ) of RBM15 in MEG - 01 expressing truncated AS - RBM15 RNAs ( schematic as shown on Fig 2C ) were measured using WB real - time PCR respectively ( n = 3 , mean ± SD ) .

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

C Protein levels (left) and mRNA levels (right) of RBM15 in MEG-01 cells expressing truncated AS-RBM15 RNAs (the schematic as shown on Fig 2C) were measured using WB and real-time PCR respectively (n=3, mean ± SD).


D role of AS - RBM15 in translation was assessed by reporter assays . reporter under control of CMV promoter contains 5 ' UTR of RBM15 mRNA ligated to firefly luciferase followed by an IRES element controlling Renilla luciferase as an internal control . reporter plasmid was cotransfected with plasmids expressing full - , exon 1 Δ5 AS - RBM15 respectively . vector reporter without 5 ' UTR is a negative control . ( n = 3 , mean ± SD ) . P values were calculated by one - way ANOVA ( * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ) .

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

D The role of AS-RBM15 in protein translation was assessed by reporter assays. The reporter under control of the CMV promoter contains the 5'UTR of RBM15 mRNA ligated to firefly luciferase followed by an IRES element controlling Renilla luciferase as an internal control. The reporter plasmid was cotransfected with plasmids expressing full-length, exon 1 and Δ5 AS-RBM15 respectively. The vector reporter without 5'UTR is a negative control. (n=3, mean ± SD). P values were calculated by one-way ANOVA test (*p<0.05, **p<0.01 and ***p<0.001).


A extracts from MEG - 01 lines stably expressing AS - RBM15 , Exon 1 , or shRNA - 02 against AS - RBM15 were fractionated through sucrose gradients ( 15 % - 60 % ) ( n = 4 ) . Global polyribosome profiles of these lines were generated by UV absorbance at 254 nm ( A254 ) . arrow indicates increasing polyribosome weights . Low molecular , LMW ; high molecular , HMW .

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

A Cytoplasmic extracts from MEG-01 cell lines stably expressing AS-RBM15, Exon 1, or shRNA-02 against AS-RBM15 were fractionated through sucrose gradients (15%-60%) (n=4). Global polyribosome profiles of these cell lines were generated by UV absorbance at 254 nm (A254). The arrow indicates increasing polyribosome weights. Low molecular weight, LMW; high molecular weight, HMW.


B distributions of RBM15 mRNA ( top ) , GAPDH mRNA ( middle ) , AS - RBM15 ( bottom ) were measured by real - time PCR represented as percentages of total in fractions ( n = 3 ) .

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B The distributions of RBM15 mRNA (top), GAPDH mRNA (middle), and AS-RBM15 (bottom) were measured by real-time PCR and represented as percentages of total RNA in the fractions (n=3).


C MS2 pull - down assays : AS - RBM15 full - ( FL ) exon 1 were fused to MS2RNA as baits . Plasmids expressing MS2 binding ( HA - tagged ) fusion baits were co - transfected into 293T . HA antibody was used to immunoprecipitateHA - MS2 binding as shown by a WB on bottom panel . RBM15 mRNA MS2 fusions were assessed by real - time PCR ( n = 3 , mean ± SD ) . Fold enrichment was calculated as 2 to power of [ ( CI . R - Cb . R ) - ( CI . MS2 - Cb . MS2 ) ] . I : input i . e . in solution before immunoprecipitation , R : RBM15 mRNA , b : bead - bound mRNA , MS2 : of MS2 fusions , C : number of cycles in real - time PCR . One - way ANONA for p value : p < 0 . 05 .

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C MS2 pull-down assays: AS-RBM15 full-length (FL) and exon 1 were fused to MS2RNA as baits. Plasmids expressing MS2 binding protein ( HA-tagged) and fusion baits were co-transfected into 293T cells. HA antibody was used to immunoprecipitateHA-MS2 binding protein as shown by a WB on the bottom panel. RBM15 mRNA and MS2 fusions were assessed by real-time PCR (n=3, mean ± SD). Fold enrichment was calculated as 2 to the power of [(CI.R-Cb.R)-(CI.MS2-Cb.MS2)]. I: input i.e. the RNA in solution before immunoprecipitation, R: RBM15 mRNA, b: bead-bound mRNA, MS2: RNA levels of MS2 fusions, C: number of cycles in real-time PCR. One-way ANONA for p value: p<0.05.


D WB of endogenous RBM15 in 293T overexpressing two different doses of AS - RBM15 - MS2 MS2 ( n = 3 ) .

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D WB analysis of the endogenous RBM15 protein levels in 293T cells overexpressing two different doses of AS-RBM15-MS2 and MS2 (n=3).


E RBM15 mRNA interacts with AS - RBM15 via - interaction . AS - RBM15 - MS2 - associated RNAs were purified by phenol extraction measured by reverse transcription PCR for detection of RBM15 , AS - RBM15 MS2 under denaturing ( heat ) non - denaturing conditions ( n = 3 ) .

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E RBM15 mRNA interacts with AS-RBM15 via RNA-RNA interaction. AS-RBM15-MS2-associated RNAs were purified by phenol extraction and measured by reverse transcription and PCR for detection of RBM15, AS-RBM15 and MS2 under denaturing (heat) and non-denaturing conditions (n =3).


F RNase A protection assays for detecting endogenous association of AS - RBM15 RBM15 mRNA ( n = 4 ) . was extracted from MEG - 01 with phenol digested with RNase A . cDNA was synthesized after RNase A treatment detected using AS - RBM15 exon - specific primers as shown in schematic diagram on top . RT : reverse transcriptase .

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F RNase A protection assays for detecting endogenous association of AS-RBM15 and RBM15 mRNA (n=4). RNA was extracted from MEG-01 cells with phenol and digested with RNase A. cDNA was synthesized after RNase A treatment and detected using AS-RBM15 exon-specific primers as shown in the schematic diagram on top. RT: reverse transcriptase.


G MEG - 01 expressing AS - RBM15 or vector control were treated with DMSO or rapamycin for 45 minutes . were harvested for WB using antibodies against GAPDH RBM15 ( n = 2 ) .

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G MEG-01 cells expressing AS-RBM15 or vector control were treated with DMSO or rapamycin for 45 minutes. Cells were harvested for WB using antibodies against GAPDH and RBM15 (n=2).


H RBM15 5 ' UTR reporter remains sensitive to rapamycin treatment with or without overexpression of antisense AS - RBM15 ( n = 4 , mean ± SD ) . P values were calculated by one - way ANOVA ( * * p < 0 . 01 * * * p < 0 . 001 ) .

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H The RBM15 5'UTR reporter remains sensitive to rapamycin treatment with or without overexpression of antisense AS-RBM15 (n=4, mean ± SD). P values were calculated by one-way ANOVA test (**p<0.01 and ***p<0.001).


subcellular distribution of RBM15 AS - RBM15 in CD34 + grown in TPO - containing medium for 5 days . of AS - RBM15 RBM15 were normalized to GAPDH ( control for fraction ) MALAT1 ( control for fraction ) , respectively , using ΔΔCt method ( n = 4 , mean ± SD ) .

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The subcellular distribution of RBM15 and AS-RBM15 in CD34+ cells grown in TPO-containing medium for 5 days. Cytoplasmic and nuclear RNA levels of AS-RBM15 and RBM15 were normalized to GAPDH (control for cytoplasmic fraction) and MALAT1 (control for nuclear fraction), respectively, using the ΔΔCt method (n=4, mean ± SD).


A RUNX1 expression in human CD34 + grown in pro - MK medium in a time course were measured by real - time PCR ( n = 4 , mean ± SD ) .

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A RUNX1 expression levels in human CD34+ cells grown in pro-MK medium in a time course were measured by real-time PCR (n=4, mean ± SD).


B CD34 + were grown in pro - MK medium for 5 days stained with antibodies against CD41 CD42 for flow cytometry . CD41 + CD42 + percentages were analyzed using one way ANOVA , * * P < 0 . 01 , ( n = 4 , mean ± SD ) .

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B CD34+ cells were grown in pro-MK medium for 5 days and stained with antibodies against CD41 and CD42 for flow cytometry analysis. The CD41+CD42+ cell percentages were analyzed using one way ANOVA, ** P<0.01, (n=4, mean ± SD).


C mRNA expression of RUNX1 , RBM15 , AS - RBM15 in RUNX1 knockdown MEG - 01 were measured by real - time PCR ( n = 4 , mean ± SD ) .

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C The mRNA expression levels of RUNX1, RBM15, and AS-RBM15 in RUNX1 knockdown MEG-01 cells were measured by real-time PCR (n=4, mean ± SD).


D immunoprecipitation using anti - RUNX1 antibody . Immunoprecipitated fragments were measured by real - time PCR using HEL expressing shRUNX1 control ( n = 4 , means ± SD ) .

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D Chromatin immunoprecipitation using anti-RUNX1 antibody. Immunoprecipitated DNA fragments were measured by real-time PCR using HEL cells expressing shRUNX1 and control (n=4, means ± SD).


E CD34 + human cord blood were transduced with MIG retroviruses co - expressing A / E ( AML1 - ETO , aka RUNX1 - ETO ) GFP . Around 5 days after transduction , GFP + CD11b - were sorted for real - time PCR ( n = 4 ) . Also see Fig EV6 for RUNX1 - ETO binding profile on RBM15 locus .

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E CD34+ human cord blood cells were transduced with MIG retroviruses co-expressing A/E (AML1-ETO, aka RUNX1-ETO) and GFP. Around 5 days after transduction, the GFP+CD11b- cells were sorted for real-time PCR (n=4). Also see Fig EV6 for RUNX1-ETO binding profile on RBM15 locus.


A ) Representative specimen of colorectal ( CRC ) tumour stained with haematoxylin / eosin / saffron ( HES ) , or antibodies against E - cadherin or Vimentin . i ) blue , orange pink dotted lines highlight normal mucosa , submucosa muscularis propria respectively . Red dotted highlights neoplastic tissue . Black arrowheads indicate direction of invasion . Boxed regions ii iii show high magnification of normal colonic ( ii ) CRC invasive front ( iii ) . Scale bar : 2 mm 500 μm .

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A) Representative specimen of colorectal (CRC) primary tumour stained with haematoxylin/eosin/saffron (HES), or antibodies against E-cadherin or Vimentin. i) The blue, orange and pink dotted lines highlight the normal mucosa, the submucosa and the muscularis propria respectively. Red dotted line highlights the neoplastic tissue. Black arrowheads indicate the direction of invasion. Boxed regions ii and iii show high magnification of normal colonic glands (ii) and the CRC invasive front (iii). Scale bar: 2 mm and 500 μm.


B ) Representative of histological sections of normal colon CRC stained for EpCam Villin . Boxed regions i , ii iii are high magnifications of luminal cavity of normal colonic ( i ) colorectal carcinoma ( ii iii ) . Aarrowheads point to apical pole enriched in villin . Scale bars : 50 μm .

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B) Representative images of histological sections of normal colon and primary CRC stained for EpCam and Villin. Boxed regions i, ii and iii are high magnifications of the luminal cavity of normal colonic gland (i) and colorectal carcinoma glands (ii and iii). Aarrowheads point to the apical pole enriched in villin. Scale bars: 50 μm.


( C ) Graph presenting of tumour displaying a glandular architecture from a cohort of 16

(C) Graph presenting the percentage of the tumour area displaying a glandular architecture from a cohort of 16 patients


A ) Representative confocal of non - invasive or invasive human colorectal cancer explants collected from 8 with NOS adenocarcinoma explants were fixed 4 days after recovery stained for lumen ( Ezrin ) , F - actin ( Phalloidin ) nuclei ( DAPI ) . Boxed regions i , ii , iii iv are displayed at high magnification . Arrowheads point to non - protruding , arrows point to protruding stars show off - centered nuclei . Scale bars : 20 μm .

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A) Representative confocal images of non-invasive or invasive human colorectal cancer explants collected from 8 patients with NOS adenocarcinoma The explants were fixed 4 days after recovery and stained for the lumen (Ezrin), F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i, ii, iii and iv are displayed at high magnification. Arrowheads point to non-protruding cells, arrows point to protruding cells and white stars show off-centered nuclei. Scale bars: 20 μm.


Graphs representing of adenocarcinoma explants displaying lumens ( B ) from 8 . Evaluation performed from DIC observation of live tissues , at least 50 explants were analysed per . error bar represents Standard Error of Mean ( Means ± ) . P values were calculated using unpaired t - ( ns : non significant ) .

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Graphs representing the percentage of adenocarcinoma explants displaying lumens (B) from 8 patients. Evaluation performed from DIC observation of live tissues, at least 50 explants were analysed per patients. The error bar represents the Standard Error of the Mean (Means ± SEM). P values were calculated using unpaired t-test (ns: non significant).


Graphs representing of adenocarcinoma explants displaying protrusions ( C ) from 8 . Evaluation performed from DIC observation of live tissues , at least 50 explants were analysed per . error bar represents Standard Error of Mean ( Means ± ) . P values were calculated using unpaired t - ( ns : non significant ) .

Graphs representing the percentage of adenocarcinoma explants displaying protrusions (C) from 8 patients. Evaluation performed from DIC observation of live tissues, at least 50 explants were analysed per patients. The error bar represents the Standard Error of the Mean (Means ± SEM). P values were calculated using unpaired t-test (ns: non significant).


D ) Time - lapse sequences of an adenocarcinoma explant undergoing collective invasion into collagen - I gel monitored by DIC microscopy over 2 days Arrowheads point to non - protruding , arrows point to protruding stars point to lumens . Scale bars : 50 μm .

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D) Time-lapse sequences of an adenocarcinoma explant undergoing collective invasion into collagen-I gel monitored by DIC microscopy over 2 days Arrowheads point to non-protruding cells, arrows point to protruding cells and white stars point to lumens. Scale bars: 50 μm.


E ) of an invading of a colorectal NOS adenocarcinoma , at microscopic level ( i , semithin section , 100x objective ) , as seen by electron microscopy ( ii , 1000x magnification ; iii , 4000x magnification ) . Note that bulk of invading neoplastic is in a state of glandular organization ( regions denoted by arrowheads in left panel ) : epithelia ( E , middle panel ) are polarized to form a lumen ( L ) , are attached to a basal ( asterisk ) by hemidesmosomes ; is surrounded by fibroblasts ( F ) . However , this organization is focally disrupted at tubular invasion pole ( arrows in left panel ) where neoplastic epithelial have lost polarization directly contact surrounding stroma as basal is missing ( right panel ) .

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E) Images of an invading gland of a colorectal NOS adenocarcinoma, at the light microscopic level (i, semithin section, 100x objective), and as seen by electron microscopy (ii, 1000x magnification; and iii, 4000x magnification). Note that the bulk of the invading neoplastic gland is in a state of glandular organization (regions denoted by arrowheads in the left panel): epithelia (E, middle panel) are polarized to form a lumen (L), and are attached to a basal membrane (asterisk) by hemidesmosomes; the gland is surrounded by fibroblasts (F). However, this organization is focally disrupted at the tubular invasion pole (arrows in the left panel) where neoplastic epithelial cells have lost polarization and directly contact the surrounding stroma as the basal membrane is missing (right panel).


A ) Representative of Caco - 2 cysts transfected with control siRNA ( siGlo ) or siRNAs targeting ROCK1 ROCK2 , fixed stained for prominin - 1 , F - actin ( Phalloidin ) Nuclei ( Hoechst ) . Confocal Z - sections of cysts are displayed . Arrowheads show non - protruding . Arrows point to protruding . stars mark nuclei that are off - centered relative to cyst ' s monolayer . Scale bars : 20 μm .

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A) Representative images of Caco-2 cysts transfected with control siRNA (siGlo) or siRNAs targeting ROCK1 and ROCK2, fixed and stained for prominin-1, F-actin (Phalloidin) and Nuclei (Hoechst). Confocal Z-sections of cysts are displayed. Arrowheads show non-protruding cells. Arrows point to protruding cells. White stars mark nuclei that are off-centered relative to the cyst's monolayer. Scale bars: 20 μm.


B ) Representative confocal of Caco - 2 cysts treated with ROCK inhibitors Y27632 H1152 , non - treated ( NT ) or transduced with ROCK1 ROCK2 shRNAs ( shROCK1 + 2 ) . cysts were fixed 2 days after invasion stained for E - cadherin , F - actin ( Phalloidin ) nuclei ( DAPI ) . Boxed regions i ii are displayed at high magnification . arrowheads point to non - protruding , green arrowheads point to apical pole , arrows point to protruding stars show off - centered nuclei . Scale bar : 20 μm .

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B) Representative confocal images of Caco-2 cysts treated with ROCK inhibitors Y27632 and H1152, non-treated (NT) or transduced with ROCK1 and ROCK2 shRNAs (shROCK1+2). The cysts were fixed 2 days after invasion and stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i and ii are displayed at high magnification. White arrowheads point to non-protruding cells, green arrowheads point to the apical pole, arrows point to protruding cells and white stars show off-centered nuclei. Scale bar: 20 μm.


C ) Bar graph representing of protrusive Caco - 2 cysts from three independent experiments . Caco - 2 cysts non - treated ( NT ) , incubated with ROCK inhibitors Y27632 H1152 or infected with lentiviruses encoding shRNA control targeting ROCK1 ROCK2 . cysts were fixed 2 days after invasion , over 100 cysts were counted for each condition . Error bar represents Standard Error of Mean ( Means ± ) . P values were calculated using unpaired t - ( * * * p < 0 . 001 , * * p < 0 . 01 ) .

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C) Bar graph representing the percentage of protrusive Caco-2 cysts from three independent experiments. Caco-2 cysts non-treated (NT), incubated with ROCK inhibitors Y27632 and H1152 or infected with lentiviruses encoding shRNA control and targeting ROCK1 and ROCK2. The cysts were fixed 2 days after invasion, over 100 cysts were counted for each condition. Error bar represents the Standard Error of the Mean (Means ± SEM). P values were calculated using unpaired t-test (***p<0.001, **p<0.01).


E ) number of that have invaded from top to bottom chamber of Boyden chamber was quantified by automated segmentation in non - treated ( NT ) , in ROCK inhibited conditions ( Y27632 , H1152 ) or in transfected with siRNA against ROCK1 ROCK2 ( siROCK1 + 2 ) . number of that have invaded was normalized to control conditions reported as average invasion rate from at least three independent experiments ( Means ± ) . P values were calculated using unpaired t - ( * * * * p < 0 . 0001 , * * p < 0 . 01 ) .

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E) The number of cells that have invaded from the top to the bottom chamber of the Boyden chamber was quantified by automated segmentation in non-treated (NT), in ROCK inhibited conditions (Y27632, H1152) or in cells transfected with siRNA against ROCK1 and ROCK2 (siROCK1+2). The number of cells that have invaded was normalized to control conditions and reported as the average invasion rate from at least three independent experiments (Means ± SEM). P values were calculated using unpaired t-test (****p<0.0001, **p<0.01).


F ) High magnification of boxed region from ( D ) . top ( i ) bottom ( ii ) side of Boyden chamber were stained for F - actin , E - cadherin , ZO - 1 , Ezrin Hoechst . Arrow points to protrusive of an invasive cyst stars show nuclei that engage in protrusion . Scale bars : 40 μm .

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F) High magnification of boxed region from (D). The top (i) and the bottom (ii) side of the Boyden chamber membrane were stained for F-actin, E-cadherin, ZO-1, Ezrin and Hoechst. Arrow points to the protrusive cells of an invasive cyst and the white stars show the nuclei that engage in the protrusion. Scale bars: 40 μm.


A ) Representative of organoids formed from derived xenografts ( PDXs ) that have been embedded in collagen - I gels during 3 days , in non - treated ( NT ) or ROCK inhibitors treated - conditions ( + Y27632 ) ( + H1152 ) . At end - point , organoids were stained for E - cadherin , F - actin ( Phalloidin ) nuclei ( DAPI ) . i ii are high magnification of boxed regions of + Y27632 + H1152 organoids respectively . Arrows point to protruding . stars show nuclei that engage in protrusions . Scale bars : 20μm . B ) bar graph represents of protrusive organoids resulting from three independent experiments . Over 50 organoids were counted per condition ( Means ± ) . P values were calculated using paired t - ( * * * p < 0 . 001 ) .

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A) Representative images of organoids formed from patient derived xenografts (PDXs) that have been embedded in collagen-I gels during 3 days, in non-treated (NT) or ROCK inhibitors treated-conditions (+Y27632) and (+H1152). At end-point, organoids were stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI). i and ii are high magnification of boxed regions of +Y27632 and +H1152 organoids respectively. Arrows point to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. B) The bar graph represents the percentage of protrusive organoids resulting from three independent experiments. Over 50 organoids were counted per condition (Means ± SEM). P values were calculated using paired t-test (***p<0.001).


C ) Representative of human adenocarcinoma explants after 2 days in 3D collagen - I gel , in non - treated ( NT ) or ROCK inhibitors treated - conditions ( + Y27632 ) ( + H1152 ) stained for apical pole ( Ezrin ) , F - actin ( Phalloidin ) nuclei ( DAPI ) . Boxed regions of NT , + Y27632 , + H1152 explants are shown at high magnification . Arrowheads point to non - protruding . Arrows point to protruding . stars show nuclei that engage in protrusions . Scale bars : 20μm . D Explants displaying ( D ) lumens from 10 were quantified results presented as of total explants . Over 50 explants were counted per condition ( duplicate ) per ( Means ± ) . P values were calculated using unpaired t - ( * * * * p < 0 . 0001 , n . s : non significant ) .

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C) Representative images of human adenocarcinoma explants after 2 days in 3D collagen-I gel, in non-treated (NT) or ROCK inhibitors treated-conditions (+Y27632) and (+H1152) and stained for the apical pole (Ezrin), F-actin (Phalloidin) and nuclei (DAPI). Boxed regions of NT, +Y27632, and +H1152 explants are shown at high magnification. Arrowheads point to non-protruding cells. Arrows point to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. D Explants displaying (D) lumens from 10 patients were quantified and results presented as percentage of total explants. Over 50 explants were counted per condition (duplicate) and per patient(Means ± SEM). P values were calculated using unpaired t-test (****p<0.0001 , n.s : non significant).


Explants displaying ( E ) protrusive from 10 were quantified results presented as of total explants . Over 50 explants were counted per condition ( duplicate ) per ( Means ± ) . P values were calculated using unpaired t - ( * * * * p < 0 . 0001 , n . s : non significant ) .

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Explants displaying (E) protrusive cells from 10 patients were quantified and results presented as percentage of total explants. Over 50 explants were counted per condition (duplicate) and per patient(Means ± SEM). P values were calculated using unpaired t-test (****p<0.0001 , n.s : non significant).


F ) Time - lapse sequences of an adenocarcinoma explant undergoing collective invasion into collagen - I gel in presence of Y27632 monitored by DIC microscopy for 2 days Arrowheads point to non - protruding , arrows point to protruding stars point to lumens . Scale bars : 50 μm .

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F) Time-lapse sequences of an adenocarcinoma explant undergoing collective invasion into collagen-I gel in the presence of Y27632 monitored by DIC microscopy for 2 days Arrowheads point to non-protruding cells, arrows point to protruding cells and white stars point to lumens. Scale bars: 50 μm.