Representative images of recombined IAHCs following IgG or αDll4 conditions . Kit is shown in white in the image , in black in the label . Multistack reconstruction of confocal images . Scale bar : 5μm .

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Representative images of recombined IAHCs following IgG or αDll4 conditions. Kit is shown in white in the image, in black in the label. Multistack reconstruction of confocal images. Scale bar: 5μm.


Quantification and classification of IAHC containing recombined cells in TAM - induced VeCadCreER : Confetti embryos after IgG or αDll4 treatment . Embryos treated with αDll4 contain more IAHC with colored cells . Quantification of confocal images ( n = 7 embryos ) .

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Quantification and classification of IAHC containing recombined cells in TAM-induced VeCadCreER:Confetti embryos after IgG or αDll4 treatment. Embryos treated with αDll4 contain more IAHC with colored cells. Quantification of confocal images (n=7 embryos).


Representative images of recombined endothelial cells in transversal sections of the aorta . The distribution and amount of recombined endothelial cells is similar in IgG and αDll4 conditions . CD31 is shown in white in the image , in black in the label . Multistack reconstruction of confocal images . Scale bar : 50μm . Quantification of recombined cells in the aortic endothelium using confocal images . Mean ± SE ( n = 3 ) .

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Representative images of recombined endothelial cells in transversal sections of the aorta. The distribution and amount of recombined endothelial cells is similar in IgG and αDll4 conditions. CD31 is shown in white in the image, in black in the label. Multistack reconstruction of confocal images. Scale bar: 50μm. Quantification of recombined cells in the aortic endothelium using confocal images. Mean±SE (n=3).


Snapshots of Movie EV5 . Time Lapse of embryonic organotypic slice from a CD41 : YFP : H2B - GFP reporter mouse showing the recruitment of CD41 : YFP negative cells ( magenta central point - tracking analyses ) migrating towards the IAHC ( CD41 : YFP + cells ) . Green central point and relative tracking identify cells with non - directional movement . Scale bar : 5μm . Time expressed in hh : mm : ss .

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Snapshots of Movie EV5. Time Lapse of embryonic organotypic slice from a CD41:YFP: H2B-GFP reporter mouse showing the recruitment of CD41:YFP negative cells (magenta central point-tracking analyses) migrating towards the IAHC (CD41:YFP+ cells). Green central point and relative tracking identify cells with non-directional movement. Scale bar: 5μm. Time expressed in hh:mm:ss.


Colony - forming assays using E10 . 5 AGMs derived from embryos treated with IgG or αDll4 .

Colony-forming assays using E10.5 AGMs derived from embryos treated with IgG or αDll4.


FACS analyses of Gfi1 + cells derived from explants treated with IgG or αDll4 for 5 hours ( C ) . Analyses of the hemogenic Gfi1 + Kit - ( D ) and the Gfi1 + Kit + ( E ) within the CD31 + CD45 - population .

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FACS analyses of Gfi1+ cells derived from explants treated with IgG or αDll4 for 5 hours (C). Analyses of the hemogenic Gfi1+Kit-(D) and the Gfi1+Kit+ (E) within the CD31+CD45- population.


Percentage of reconstitution ( chimerism determined by CD45 . 1 and CD45 . 2 expression ) in peripheral blood ( PB ) . Embryos treated with IgG or αDll4 were kept in explant culture for 48hours and then transplanted into irradiated recipients . Peripheral blood was analyzed at different time points . Each dot / square represents a single transplanted mouse .

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Percentage of reconstitution (chimerism determined by CD45.1 and CD45.2 expression) in peripheral blood (PB). Embryos treated with IgG or αDll4 were kept in explant culture for 48hours and then transplanted into irradiated recipients. Peripheral blood was analyzed at different time points. Each dot/square represents a single transplanted mouse.


Analysis of progenitors ( lin - sca1 + kit + ) and HSCs ( Lin - sca1 + kit + CD48 - CD150 + ) in the donor population in the bone marrow after 4 months of transplantation .

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Analysis of progenitors (lin-sca1+kit+) and HSCs (Lin-sca1+kit+CD48-CD150+) in the donor population in the bone marrow after 4 months of transplantation.


B . DIC microscopic image of WTSOD12SH diluted to 100 µM in HEPES buffer , pH 7 . 4 and 100 mM NaCl and incubated 37 ° C , 180 RPM for 30 min shows no droplet formation ; scale bar : 100 µm ; inset shows Alexa Fluor 488 maleimide labelled WTSOD12SH ;

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B. DIC microscopic image of WTSOD12SH diluted to 100 µM in HEPES buffer, pH 7.4 and 100 mM NaCl and incubated 37 °C, 180 RPM for 30 min shows no droplet formation; scale bar: 100 µm; inset shows Alexa Fluor 488 maleimide labelled WTSOD12SH;


C . Solution turbidity plot ( absorbance at 600 nm ) shows that WTSOD12SH ( at concentrations ranging from 25 to 200 µM does not undergo LLPS in presence or absence of heparin , LLPS inducer ; data is represented as mean ± SD ( from 3 biological replicates ) .

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C. Solution turbidity plot (absorbance at 600 nm) shows that WTSOD12SH (at concentrations ranging from 25 to 200 µM does not undergo LLPS in presence or absence of heparin, LLPS inducer; data is represented as mean ± SD (from 3 biological replicates).


D . DIC microscopic image of ApoSOD12SH under condensate inducing conditions ( 37  C , 180 RPM ) at a concentration of 100 µM incubated 100 mM NaCl ; scale bar : 100 µm ( inset on bottom left shows Alexa Fluor 488 maleimide labeled protein condensates ;

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D. DIC microscopic image of ApoSOD12SH under condensate inducing conditions (37 C, 180 RPM) at a concentration of 100 µM incubated 100 mM NaCl; scale bar:100 µm (inset on bottom left shows Alexa Fluor 488 maleimide labeled protein condensates;


E . Fluorescent microscopic images of Alexa Fluor 488 maleimide labeled ApoSOD12SH condensates diluted in HEPES buffer , pH 7 . 4 and 100 mM NaCl and incubated ( 180 RPM , 37  C ) in absence ( left ) and presence ( right ) of heparin at 30 min time -

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E. Fluorescent microscopic images of Alexa Fluor 488 maleimide labeled ApoSOD12SH condensates diluted in HEPES buffer, pH 7.4 and 100 mM NaCl and incubated (180 RPM, 37 C) in absence (left) and presence (right) of heparin at 30 min time-point


F . Solution turbidity measurements with ApoSOD12SH ( absorbance at 600 nm ) shows that while in the absence of heparin the turbidity increases with time , the presence of heparin results in faster LLPS ;

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F. Solution turbidity measurements with ApoSOD12SH (absorbance at 600 nm) shows that while in the absence of heparin the turbidity increases with time, the presence of heparin results in faster LLPS;


G . Comparison of ApoSOD12SH droplet numbers per microscopic field of view ( view area 0 . 001963 mm2 ) calculated from 5 different images of droplets incubated with and without heparin at 2 h timepoint .

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G. Comparison of ApoSOD12SH droplet numbers per microscopic field of view (view area 0.001963 mm2) calculated from 5 different images of droplets incubated with and without heparin at 2 h timepoint.


H . Liquid nature of ApoSOD12SH showing droplet fusion and surface

H. Liquid nature of ApoSOD12SH showing droplet fusion and surface wetting


I . Amplitude normalized FCS curves of ApoSOD12SH ( in blue ) of dilute phase ( DP ) and condensed phase ( CP ) . Intense color indicates CP and light variant indicates DP . The increase in τD ( as shown by the arrow ) translates to a decrease in

I. Amplitude normalized FCS curves of ApoSOD12SH (in blue) of dilute phase (DP) and condensed phase (CP). Intense color indicates CP and light variant indicates DP. The increase in τD (as shown by the arrow) translates to a decrease in D


J . Scheme depicting that presence of Zn and not Cu in pre - incubation mixture inhibits LLPS . K . DIC microscopic images of ApoSOD12SH in presence of Zn ( top ) showing no droplet formation and Cu ( below ) showing the presence of

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J. Scheme depicting that presence of Zn and not Cu in pre-incubation mixture inhibits LLPS. K. DIC microscopic images of ApoSOD12SH in presence of Zn (top) showing no droplet formation and Cu (below) showing the presence of droplets


L . Solution turbidity plot ( absorbance at 600 nm ) of ApoSOD12SH subjected to increasing concentrations of Cu and Zn ; turbidity decreases in a dose dependent manner for Zn while no significant change is observed in presence of Cu ;

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L. Solution turbidity plot (absorbance at 600 nm) of ApoSOD12SH subjected to increasing concentrations of Cu and Zn; turbidity decreases in a dose dependent manner for Zn while no significant change is observed in presence of Cu;


A . DIC microscopic images of I113T SOD12SH ( top ) and G85R SOD12SH ( bottom ) droplets incubated in the absence and presence of Zn and Cu respectively ( scale bar : 100 µm ) ; insets on bottom left show Alexa Fluor 488 maleimide labeled protein condensates for I113T SOD12SH and G85R SOD12SH respectively ; scale bar : 5 µm . Droplet dissolution was observed for I113T SOD12SH on addition of Zn while droplets persisted on addition of Cu . Red arrowhead indicates fusion of liquid droplets .

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A. DIC microscopic images of I113T SOD12SH (top) and G85R SOD12SH (bottom) droplets incubated in the absence and presence of Zn and Cu respectively (scale bar: 100 µm); insets on bottom left show Alexa Fluor 488 maleimide labeled protein condensates for I113T SOD12SH and G85R SOD12SH respectively; scale bar: 5 µm. Droplet dissolution was observed for I113T SOD12SH on addition of Zn while droplets persisted on addition of Cu. Red arrowhead indicates fusion of liquid droplets.


B . Amplitude normalized FCS curves of I113T SOD12SH ( in green ) and G85R SOD12SH ( in grey ) of dilute phase ( DP ) and condensed phase ( CP ) showing an increase in diffusion time from DP to CP . The intense color indicates CP and light variant indicates DP .

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B. Amplitude normalized FCS curves of I113T SOD12SH (in green) and G85R SOD12SH (in grey) of dilute phase (DP) and condensed phase (CP) showing an increase in diffusion time from DP to CP. The intense color indicates CP and light variant indicates DP.


C . On metal cofactor removal , ApoG37R SOD12SH formed condensates which ceased to exist when protein was incubated with Zn , while condensates persisted on Cu addition ( left to right ) ;

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C. On metal cofactor removal, ApoG37R SOD12SH formed condensates which ceased to exist when protein was incubated with Zn, while condensates persisted on Cu addition (left to right);


B . Plot of the loge of the hydrodynamic radius ( rH ) versus the log e of the number of residues in the polypeptide chain . The line fitted to these data for the native folded proteins ( yellow dashed ) has a slope of 0 . 29 ± 0 . 02 and a y - axis intercept of 1 . 56 ± 0 . 1 , while the other fitted to the chemically denatured protein ( grey dashed ) data has a slope of 0 . 57 ± 0 . 02 and a y - axis intercept of 0 . 79 ± 0 . 07 . Literature data have been used for folded and chemically denatured proteins while we employed FCS to calculate the rH of all SOD12SH variants with ( solid shapes ) and without Zn ( hollow shapes ) . The hollow grey circle of G85R SOD12SH is directly under hollow green I113T SOD12SH in the log - log plot .

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B. Plot of the loge of the hydrodynamic radius (rH) versus the log e of the number of residues in the polypeptide chain. The line fitted to these data for the native folded proteins (yellow dashed) has a slope of 0.29 ± 0.02 and a y-axis intercept of 1.56 ± 0.1, while the other fitted to the chemically denatured protein (grey dashed) data has a slope of 0.57 ± 0.02 and a y-axis intercept of 0.79 ± 0.07. Literature data have been used for folded and chemically denatured proteins while we employed FCS to calculate the rH of all SOD12SH variants with (solid shapes) and without Zn (hollow shapes). The hollow grey circle of G85R SOD12SH is directly under hollow green I113T SOD12SH in the log-log plot.


C . Variation of diffusion coefficients of de - metalated protein variants determined from FCS measurement with increasing Zn concentration ( inset shows a comparison of binding constants between mutants and Zn ; ApoSOD12SH and ApoG37R SOD12SH has a higher binding affinity to Zn than ApoI113T SOD12SH and ApoG85R SOD12SH ) .

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C. Variation of diffusion coefficients of de-metalated protein variants determined from FCS measurement with increasing Zn concentration (inset shows a comparison of binding constants between mutants and Zn; ApoSOD12SH and ApoG37R SOD12SH has a higher binding affinity to Zn than ApoI113T SOD12SH and ApoG85R SOD12SH).


D . Disordered / extended conformation content decreases in ApoSOD12SH and ApoI113T SOD12SH with the addition of Zn calculated from FTIR spectra ;

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D. Disordered/ extended conformation content decreases in ApoSOD12SH and ApoI113T SOD12SH with the addition of Zn calculated from FTIR spectra;


C . Salt - bridge analysis of ApoG85R SOD12SH ( dist . cutoff < 0 . 5 nm = salt - bridge ) .

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C. Salt-bridge analysis of ApoG85R SOD12SH (dist. cutoff <0.5 nm = salt-bridge).


C . Upper panel shows fluorescence images of the maturation of Alexa Fluor 488 labeled ApoSOD12SH droplets with time , samples were incubated at 37  C up to ~ 25 hours . Images are taken at indicated time point : 0 * hour ( the time after incubation required for droplet formation ) , 12 hours and > 24 hours ; images below show magnified scale bar : 5 µm . The middle panel shows magnified view of the marked region from corresponding images on the upper panel . Red arrowhead indicates matured ( solid ) portion inside the droplet . Lower panel shows coexistence of liquid and solid phase inside droplet ; scale bar : 10 µm . Intensity plot ( bottom right ) shows the distribution of high intensity region within matured droplet .

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C. Upper panel shows fluorescence images of the maturation of Alexa Fluor 488 labeled ApoSOD12SH droplets with time, samples were incubated at 37 C up to ~25 hours. Images are taken at indicated time point: 0* hour (the time after incubation required for droplet formation), 12 hours and >24 hours; images below show magnified scale bar: 5 µm. The middle panel shows magnified view of the marked region from corresponding images on the upper panel. Red arrowhead indicates matured (solid) portion inside the droplet. Lower panel shows coexistence of liquid and solid phase inside droplet; scale bar: 10 µm. Intensity plot (bottom right) shows the distribution of high intensity region within matured droplet.


D . Diffusion coefficient obtained from point FCS measurement at different regions of the droplet during maturation course ; 0 * ( x - axis label ) indicates the time after incubation required for droplet formation .

D. Diffusion coefficient obtained from point FCS measurement at different regions of the droplet during maturation course; 0* (x-axis label) indicates the time after incubation required for droplet formation.


E . ThT fluorescence assay plot showing aggregation kinetics of all SOD12SH variants . ApoSOD12SH , I113T SOD12SH and G85R SOD12SH show high ThT fluorescence .

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E. ThT fluorescence assay plot showing aggregation kinetics of all SOD12SH variants. ApoSOD12SH, I113T SOD12SH and G85R SOD12SH show high ThT fluorescence.


F . AFM micrographs of ApoSOD12SH , I113T SOD12SH and G85R SOD12SH show fibrillar aggregates after prolonged incubation at 37 ℃ , 180 RPM in absence of Zn ( left panel ) ; AFM micrographs of ApoSOD12SH , I113T SOD12SH and G85R SOD12SH incubated with Zn ( at 1 : 5 protein : Zn molar ratio ) show presence of oligomers for ApoSOD12SH , I113T SOD12SH and short fibrils for G85R SOD12SH ( right panel ) ;

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F. AFM micrographs of ApoSOD12SH, I113T SOD12SH and G85R SOD12SH show fibrillar aggregates after prolonged incubation at 37 ℃, 180 RPM in absence of Zn (left panel); AFM micrographs of ApoSOD12SH, I113T SOD12SH and G85R SOD12SH incubated with Zn (at 1:5 protein: Zn molar ratio) show presence of oligomers for ApoSOD12SH, I113T SOD12SH and short fibrils for G85R SOD12SH (right panel);


G . Cell proliferation of SHSY5Y human neuroblastoma cells assessed by MTT assay post 12 h treatment with 5 µM ApoSOD12SH , I113T SOD12SH and G85R SOD12SH condensates and aggregates ( fibrils ) . Cell culture medium ( DMEM ) was used as negative control and untreated SHSY5Y cells were used as positive control .

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G. Cell proliferation of SHSY5Y human neuroblastoma cells assessed by MTT assay post 12 h treatment with 5 µM ApoSOD12SH, I113T SOD12SH and G85R SOD12SH condensates and aggregates (fibrils). Cell culture medium (DMEM) was used as negative control and untreated SHSY5Y cells were used as positive control.


H . Dot plots showing flow cytometric analysis of annexin V and propidium iodide ( PI ) staining of apoptotic SHSY5Y neuroblastoma cells following a 12 h treatment with 5 µM ApoSOD12SH condensates , I113T SOD12SH condensates , G85R SOD12SH condensates , ApoSOD12SH fibrils , I113T SOD12SH fibrils and G85R SOD12SH fibrils respectively ( see Methods for details ) . High cytotoxicity was observed for SOD12SH condensates .

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H. Dot plots showing flow cytometric analysis of annexin V and propidium iodide (PI) staining of apoptotic SHSY5Y neuroblastoma cells following a 12 h treatment with 5 µM ApoSOD12SH condensates, I113T SOD12SH condensates, G85R SOD12SH condensates, ApoSOD12SH fibrils, I113T SOD12SH fibrils and G85R SOD12SH fibrils respectively (see Methods for details). High cytotoxicity was observed for SOD12SH condensates.


Coomassie Blue staining of recombinant human DDX5 purified in bacteria . M denotes the molecular mass markers in kDa .

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Coomassie Blue staining of recombinant human DDX5 purified in bacteria. M denotes the molecular mass markers in kDa.


R - loop unwinding assay in presence of increasing DDX5 . The top panel shows a typical image obtained after the assay . The bar graph ( bottom ) shows the quantification . The average is expressed as percentage unwinding , and standard error of the mean ( S . E . M . ) , n = 4 .

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R-loop unwinding assay in presence of increasing DDX5. The top panel shows a typical image obtained after the assay. The bar graph (bottom) shows the quantification. The average is expressed as percentage unwinding, and standard error of the mean (S.E.M.), n=4.


S9 . 6 signal in the nucleoplasm of U2OS cells . An average from 3 independent experiments performed in triplicate . In total , ~ 90 images for each condition ( 10 images / slide and 9 slides for each condition ) were taken and two cells per image were quantified . Statistical significance was assessed using one - way ANOVA t - test . * : p < 0 . 05 ; * * * * : p < 0 . 0001 .

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S9.6 signal in the nucleoplasm of U2OS cells. An average from 3 independent experiments performed in triplicate. In total, ~90 images for each condition (10 images/slide and 9 slides for each condition) were taken and two cells per image were quantified. Statistical significance was assessed using one-way ANOVA t-test. *: p < 0.05; ****: p < 0.0001.


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 .

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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 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 .

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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.


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

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


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 ) .

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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).


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


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 .

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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 and DDX5 knockdowns .

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Western blotting of PRMT5 and DDX5 knockdowns.


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 .

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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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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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 cell extracts from siCTL , siPRMT5 , siDDX5 or siDDX5 / siPRMT5 transfected cells .

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


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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


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 .

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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 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 .

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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 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 .

O O O O O O O O O O O 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 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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

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 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 .

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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 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 .

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 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

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 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 .

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 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 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 .

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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 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 .

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

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 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 .

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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 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 .

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(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 ) .

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

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 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 ) .

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

(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 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 .

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

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 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 ) .

O O O O O O O O O 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 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 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 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 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 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 ) .

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

(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 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 ) .

O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B-MEASURED_VAR O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O 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 ) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O 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

,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 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 ) .

O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O 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 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 .

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

(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 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 .

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

(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 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 .

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

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 B-MEASURED_VAR O O 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 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 )

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 B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O 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 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 O O O O O O O O O O O O O O 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 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 ) .

O O O 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 B-MEASURED_VAR 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 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

(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 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

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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR 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. TFEB knockdown efficiency by TFEB siRNAs and TFEB deficiency by knockout were checked by SDS-PAGE and immunoblot


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 .

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 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

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 ) 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

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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR 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. TFEB knockdown efficiency by TFEB siRNAs and TFEB deficiency by knockout were checked by SDS-PAGE and immunoblot


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 .

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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 ) 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 ) .

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 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

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 ) 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 ) .

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 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 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 ) .

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

(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 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 ) .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O 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 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 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 ) .

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

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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 ) .

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

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 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 ) .

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 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 .

O O 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 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 ) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . 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 .

O O O O O O O O O O O O O O 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 tumor infiltrating lymphocytes ( TIL ) , in particular FOXP3 + regulatory T cells ( Treg ) , CD8 + cytotoxic T lymphocytes and the ratio of CD8 + T cells 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 and 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 ) and 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 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 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 ) .

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 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 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 ) .

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

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

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 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 .

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 O O O O O O O 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 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 + 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 ) .

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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 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 ) .

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 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

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 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 ) .

O O 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 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

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 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 ) .

O O O O B-MEASURED_VAR O B-MEASURED_VAR 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

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 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 .

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 B-MEASURED_VAR 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

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 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 ) .

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 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

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 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 ) .

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

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 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 ) .

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 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 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

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 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 .

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 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 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 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 ) .

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

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).