Quantification as percentage of phosphoprotein level normalized to total VEGFR2 . each dot ( B ) represent one individual mouse .

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Quantification as percentage of phosphoprotein level normalized to total VEGFR2. each dot (B) represent one individual mouse.


Summary plot of 51 proteins enriched in the immobilized pY1212 peptide precipitate . 10 proteins ( red oval ) were selected for further analysis . PSM : peptide - spectrum matches . Area : average area of spectra for the three most abundant peptides in arbitrary units .

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Summary plot of 51 proteins enriched in the immobilized pY1212 peptide precipitate. 10 proteins (red oval) were selected for further analysis. PSM: peptide-spectrum matches. Area: average area of spectra for the three most abundant peptides in arbitrary units.


Blot for peptide - enriched GRB2 and PI3Kp85 . HDMEC total lysate incubated with indicated immobilized peptides followed by immunoblotting for GRB2 and PI3Kp85 .

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Blot for peptide-enriched GRB2 and PI3Kp85. HDMEC total lysate incubated with indicated immobilized peptides followed by immunoblotting for GRB2 and PI3Kp85.


VEGFR2 / GRB2 and VEGFR2 / PI3Kp85 complexes in isolated ( i ) ECs . E . PLA for VEGFR2 / GRB2 and VEGFR2 / PI3Kp85 complexes ( black puncta ) in iECs from FVB wildtype ( WT ) and Vegfr2Y1212F / Y1212F lungs . Hoechst 33342 show nuclei ( grey ) . Scale bar , 20 µm . F , G . Fold - increase over PBS - treated sample ; each dot represents the mean of 6 fields . 2 - way ANOVA p = 0 . 0206 ( F ) , p = 0 . 0368 ( G ) ; Sidak ' s multiple comparison test , * p < 0 . 05 . n = 3 experiments .

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VEGFR2/GRB2 and VEGFR2/PI3Kp85 complexes in isolated (i)ECs. E. PLA for VEGFR2/GRB2 and VEGFR2/PI3Kp85 complexes (black puncta) in iECs from FVB wildtype (WT) and Vegfr2Y1212F/Y1212F lungs. Hoechst 33342 show nuclei (grey). Scale bar, 20 µm. F, G. Fold-increase over PBS-treated sample; each dot represents the mean of 6 fields. 2-way ANOVA p=0.0206 (F), p=0.0368 (G); Sidak's multiple comparison test, * p <0.05. n=3 experiments.


phosphoERK1 / 2 and phosphoAkt immunostaining ( green ) of iECs from FVB wildtype ( WT ) and Vegfr2Y1212F / Y1212F lungs . Hoechst 33342 ( blue ) shows nuclei . Arrows indicate nuclear accumulation of phosphoERK1 / 2 and phosphoAkt , respectively . Scale bar , 20 µm .

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phosphoERK1/2 and phosphoAkt immunostaining (green) of iECs from FVB wildtype (WT) and Vegfr2Y1212F/Y1212F lungs. Hoechst 33342 (blue) shows nuclei. Arrows indicate nuclear accumulation of phosphoERK1/2 and phosphoAkt, respectively. Scale bar, 20 µm.


Nuclear accumulation expressed as phoshoERK1 / 2 - positive nuclear area normalized to the total phosphoERK1 / 2 area by field . Each dot represents 1 field . Min , minutes of stimulation . 2 - way ANOVA p = 0 . 0248 ; Sidak ' s multiple comparison test , * p < 0 . 05 , * * p < 0 . 01 . n = 3 experiments .

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Nuclear accumulation expressed as phoshoERK1/2-positive nuclear area normalized to the total phosphoERK1/2 area by field. Each dot represents 1 field. Min, minutes of stimulation. 2-way ANOVA p=0.0248; Sidak's multiple comparison test, * p <0.05, ** p <0.01. n=3 experiments.


Nuclear accumulation expressed as fold - increase of nuclear phosphoAkt area in Vegfr2Y1212F / Y1212F over PBS - treated wildtype ( WT ) iECs , normalized to the number of nuclei . Each dot represents 1 field . Min , minutes of stimulation . Unpaired T test , * p < 0 . 05 . n = 3 experiments .

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Nuclear accumulation expressed as fold-increase of nuclear phosphoAkt area in Vegfr2Y1212F/Y1212F over PBS-treated wildtype (WT) iECs, normalized to the number of nuclei. Each dot represents 1 field. Min, minutes of stimulation. Unpaired T test, *p < 0.05. n=3 experiments.


Immunoblot for phosphoERK1 / 2 , total ERK1 / 2 , phosphoAkt and total Akt on lung lysates from wiltype ( WT ) and Vegfr2Y1212F / Y1212F FVB mice tail - vein injected with PBS or VEGFA followed by circulation for time points indicated . Each lane represents one individual mouse . Quantification of phosphoERK1 / 2 / total ERK1 / 2 expressed as Fold change to PBS . Min , minutes of stimulation . Error bars : SD ; 2 - way ANOVA p = 0 . 0025 ; unpaired T test , * p < 0 . 05 . n = 3 experiments . Quantification of phosphoAkt / total Akt expressed as Fold change to PBS . Min , minutes of stimulation . Error bars : SD ; 2 - way ANOVA p = 0 . 0003 ; unpaired T test , * p < 0 . 05 . n = 3 experiments .

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Immunoblot for phosphoERK1/2, total ERK1/2, phosphoAkt and total Akt on lung lysates from wiltype (WT) and Vegfr2Y1212F /Y1212F FVB mice tail-vein injected with PBS or VEGFA followed by circulation for time points indicated. Each lane represents one individual mouse. Quantification of phosphoERK1/2 /total ERK1/2 expressed as Fold change to PBS. Min, minutes of stimulation. Error bars: SD; 2-way ANOVA p=0.0025; unpaired T test, *p < 0.05. n=3 experiments. Quantification of phosphoAkt/total Akt expressed as Fold change to PBS. Min, minutes of stimulation. Error bars: SD; 2-way ANOVA p=0.0003; unpaired T test, *p < 0.05. n=3 experiments.


Immunoblot for Myc on lung lysates from wiltype ( WT ) and Vegfr2Y1212F / Y1212F FVB mice tail - vein injected with PBS or VEGFA followed by circulation for time points indicated . Each lane represents one individual mouse . Quantification of Myc / Actin expressed as Fold change to PBS . Min , minutes of stimulation . Error bars : SD ; 2 - way ANOVA p = 0 . 0058 ; unpaired T test , * p < 0 . 05 , * * p < 0 . 01 . n = 3 experiments .

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Immunoblot for Myc on lung lysates from wiltype (WT) and Vegfr2Y1212F /Y1212F FVB mice tail-vein injected with PBS or VEGFA followed by circulation for time points indicated. Each lane represents one individual mouse. Quantification of Myc/Actin expressed as Fold change to PBS. Min, minutes of stimulation. Error bars: SD; 2-way ANOVA p=0.0058; unpaired T test, *p < 0.05, ** p <0.01. n=3 experiments.


Myc protein expression . C . Myc and Actin immunoblotting on lysates of lung isolated ECs from untreated wildtype ( WT ) and mutant ( VEGFR2 Y1212F ) C57Bl / 6 and FVB mice . D . Quantification of Myc / Actin expressed as Arbitrary unit . Each lane ( C ) and each dot ( D ) represent one individual mouse . 2 - way ANOVA p < 0 . 0001 ; unpaired T test , * * p < 0 . 01 . n = 5 - 7 .

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Myc protein expression. C. Myc and Actin immunoblotting on lysates of lung isolated ECs from untreated wildtype (WT) and mutant (VEGFR2 Y1212F) C57Bl/6 and FVB mice. D. Quantification of Myc/Actin expressed as Arbitrary unit. Each lane (C) and each dot (D) represent one individual mouse. 2-way ANOVA p <0.0001; unpaired T test, ** p <0.01. n=5-7.


Baseline transcriptional level of Myc from lung isolated ECs from untreated wildtype ( WT ) and mutant ( VEGFR2 Y1212F ) C57Bl / 6 and FVB mice . Each dot represents one individual mouse . 2 - way ANOVA p = 0 . 0041 ; Sidak ' s multiple comparison test , * p < 0 . 05 . n = 4 - 5 .

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Baseline transcriptional level of Myc from lung isolated ECs from untreated wildtype (WT) and mutant (VEGFR2 Y1212F) C57Bl/6 and FVB mice. Each dot represents one individual mouse. 2-way ANOVA p=0.0041; Sidak's multiple comparison test, * p <0.05. n=4-5.


Proliferation of C57Bl / 6 and FVB iECs . CD31 ( white ) , ERG ( red ) , EdU ( green ) and double - positive cells ( yellow ) in C57Bl / 6 ( F ) and FVB ( G ) iECs . EdU signal was masked with ERG signal .

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Proliferation of C57Bl/6 and FVB iECs. CD31 (white), ERG (red), EdU (green) and double-positive cells (yellow) in C57Bl/6 (F) and FVB (G) iECs. EdU signal was masked with ERG signal.


Proliferation of C57Bl / 6 and FVB iECs . EdU incorporation in ERG - positive ECs normalized to total ERG - positive ECs . Each dot represents 2 fields / well , one well / mouse . Scale bar , 100 µm . Error bars : SD ; 2 - way ANOVA p = 0 . 0843 ( H ) , p = 0 . 0474 ( I ) ; unpaired T test , * p < 0 . 05 ; * * p < 0 . 01 . C57Bl / 6 n = 3 - 6 mice , FVB n = 3 - 5mice .

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Proliferation of C57Bl/6 and FVB iECs. EdU incorporation in ERG-positive ECs normalized to total ERG-positive ECs. Each dot represents 2 fields/well, one well/mouse. Scale bar, 100 µm. Error bars: SD; 2-way ANOVA p =0.0843 (H), p=0.0474 (I); unpaired T test, *p < 0.05; **p < 0.01. C57Bl/6 n=3-6 mice, FVB n=3-5mice.


Rescue of angiogenic sprouting by Myc overexpression in E11 . 5 C57Bl / 6 Vegfr2Y1212F / Y1212F explant tissue . Representative images of E11 . 5 explants from C57Bl / 6 embryos taken from wildtype ( WT ) and Vegfr2Y1212F / Y1212F littermates with ( MycOE / Cre + ) and without ( MycOE / Cre - ) endothelial - specific overexpression of Myc . Endothelial cell marker ( CD31 ) immunostaining highlights the vasculature in white . Embryonic tissue blacked out to highlight sprouting area .

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Rescue of angiogenic sprouting by Myc overexpression in E11.5 C57Bl/6 Vegfr2Y1212F /Y1212F explant tissue. Representative images of E11.5 explants from C57Bl/6 embryos taken from wildtype (WT) and Vegfr2Y1212F /Y1212F littermates with (MycOE/Cre +) and without (MycOE/Cre-) endothelial-specific overexpression of Myc. Endothelial cell marker (CD31) immunostaining highlights the vasculature in white. Embryonic tissue blacked out to highlight sprouting area.


Rescue of angiogenic sprouting by Myc overexpression in E11 . 5 C57Bl / 6 Vegfr2Y1212F / Y1212F explant tissue . Quantification as fold - increase of vessel sprouting area compared to WT MycOE / Cre - . Scale bar , 250 µm . Error bars : SD ; unpaired T test , * * p < 0 . 01 , * * * * p < 0 . 0001 . n = 4 - 11 explants , one explant / embryo .

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Rescue of angiogenic sprouting by Myc overexpression in E11.5 C57Bl/6 Vegfr2Y1212F /Y1212F explant tissue. Quantification as fold-increase of vessel sprouting area compared to WT MycOE/Cre -. Scale bar, 250 µm. Error bars: SD; unpaired T test, ** p <0.01, **** p <0.0001. n=4-11 explants, one explant/embryo.


Rescue of Ec proliferation . Myc overexpression in E11 . 5 C57Bl / 6 Vegfr2Y1212F / Y1212F explant tissue . ERG ( red ) , EdU ( green ) and double - positive cells ( yellow ; arrows ) .

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Rescue of Ec proliferation. Myc overexpression in E11.5 C57Bl/6 Vegfr2Y1212F /Y1212F explant tissue. ERG (red), EdU (green) and double-positive cells (yellow; arrows).


Rescue of Ec proliferation . Quantification of EdU / ERG double positive ECs normalized to total ECs . Scale bar , 50 µm . Error bars : SD ; unpaired T test , * * * p < 0 . 001 , * * * * p < 0 . 0001 . n = 3 - 7 explants , one explant / embryo .

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Rescue of Ec proliferation. Quantification of EdU/ERG double positive ECs normalized to total ECs. Scale bar, 50 µm. Error bars: SD; unpaired T test, *** p <0.001, **** p <0.0001. n=3-7 explants, one explant/embryo.


GSEAs showing upregulation of genes belonging to the K - Ras , Angiogenesis and mTORC1 gene sets in ( WT ) compared to VEGFR2 Y1212F isolated ECs . K - Ras and Angiogenesis gene sets derived from C57Bl / 6 iECs kept in basal condition ; mTORC1 gene set derived from C57Bl / 6 iECs treated with VEGFA for 3h . See Supplementary Table 2 . Enrichment score ( ES ) , normalized ES ( NES ) are shown . P - values < 0 . 05 were regarded as significant . Heat maps displaying gene regulation in gene sets shown in A . The color in the heatmap shows the gene expression value , expressed as a z - score across animals . Red color represents relative higher expression and blue color represents relative lower expression value . The z - score is calculated by ( x - x ' ) / s where x is individual gene expression , x ' is the mean of the gene expression across samples and s is standard deviation of the gene expression across samples .

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GSEAs showing upregulation of genes belonging to the K-Ras, Angiogenesis and mTORC1 gene sets in (WT) compared to VEGFR2 Y1212F isolated ECs. K-Ras and Angiogenesis gene sets derived from C57Bl/6 iECs kept in basal condition; mTORC1 gene set derived from C57Bl/6 iECs treated with VEGFA for 3h. See Supplementary Table 2. Enrichment score (ES), normalized ES (NES) are shown. P-values <0.05 were regarded as significant. Heat maps displaying gene regulation in gene sets shown in A. The color in the heatmap shows the gene expression value, expressed as a z-score across animals. Red color represents relative higher expression and blue color represents relative lower expression value. The z-score is calculated by (x-x')/s where x is individual gene expression, x' is the mean of the gene expression across samples and s is standard deviation of the gene expression across samples.


Myc - regulated gene expression downstream of VEGFR2 pY1212 . Quantitative PCR analysis of a set of known Myc - regulated transcripts in C57Bl / 6 ( C , D ) and FVB ( E , F ) adult lungs at basal ( C , E ) and at 1 hour after tail - vein administration of VEGFA ( D , F ) . FASN , fatty acid synthase ; Ldha and - b , Lactate dehydrogenase A and B ; Acod1 , Aconitate Decarboxylase 1 ; Ppat , Amidophosphoribosyltransferase ; Pde4d , cAMP - specific 3 ' , 5 ' - cyclic phosphodiesterase 4D ; Prkaca , cAMP - dependent protein kinase catalytic subunit alpha . Error bars : SD ; 2 - way ANOVA between WT and Vegfr2Y1212F / Y1212F p = 0 . 0453 ( C ) , p < 0 . 0001 ( D ) , p = 0 . 0018 ( E ) , p = 0 . 09 ( F ) ; unpaired T test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . C57Bl / 6 n = 8 - 11 mice , FVB n = 9 - 14 mice .

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Myc-regulated gene expression downstream of VEGFR2 pY1212. Quantitative PCR analysis of a set of known Myc-regulated transcripts in C57Bl/6 (C, D) and FVB (E, F) adult lungs at basal (C, E) and at 1 hour after tail-vein administration of VEGFA (D, F). FASN, fatty acid synthase; Ldha and -b, Lactate dehydrogenase A and B; Acod1, Aconitate Decarboxylase 1; Ppat, Amidophosphoribosyltransferase; Pde4d, cAMP-specific 3',5'-cyclic phosphodiesterase 4D; Prkaca, cAMP-dependent protein kinase catalytic subunit alpha. Error bars: SD; 2-way ANOVA between WT and Vegfr2Y1212F /Y1212F p=0.0453 (C), p < 0.0001 (D), p=0.0018 (E), p=0.09 (F); unpaired T test, *p < 0.05, **p < 0.01, ***p < 0.001. C57Bl/6 n=8-11 mice, FVB n=9-14 mice.


A . FGF21SS and FGF21 improve insulin resistance by countering the inflammation in 3T3 - L1 adipocytes . Cells were cultured in RAW - CM to induce inflammation and insulin resistance , while vehicle , FGF21 or FGF21SS were added simultaneously . The insulin sensitivity was indicated by insulin - induced AKT phosphorylation using western blotting analysis . Error bars show the SEM of four independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( Student ' s t - test ) vs RAW - CM treated only group .

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A. FGF21SS and FGF21 improve insulin resistance by countering the inflammation in 3T3-L1 adipocytes. Cells were cultured in RAW-CM to induce inflammation and insulin resistance, while vehicle, FGF21 or FGF21SS were added simultaneously. The insulin sensitivity was indicated by insulin-induced AKT phosphorylation using western blotting analysis. Error bars show the SEM of four independent experiments. *p < 0.05, **p < 0.01 (Student's t-test) vs RAW-CM treated only group.


Anti - diabetic effect evaluation in ob / ob mice . Mice were injected subcutaneously with vehicle , FGF21 or FGF21SS at indicated doses every day . Body weight ( B ) was measured Error bars show the SEM of ten independent experiments . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ( Student ' s t - test ) vs vehicle .

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Anti-diabetic effect evaluation in ob/ob mice. Mice were injected subcutaneously with vehicle, FGF21 or FGF21SS at indicated doses every day. Body weight (B) was measured Error bars show the SEM of ten independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 (Student's t-test) vs vehicle.


Anti - diabetic effect evaluation in ob / ob mice . Mice were injected subcutaneously with vehicle , FGF21 or FGF21SS at indicated doses every day . plasma glucose ( D ) was measured every day . Plasma insulin ( C ) was analyzed at day 7 using insulin ELISA kit . Error bars show the SEM of ten independent experiments . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ( Student ' s t - test ) vs vehicle .

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Anti-diabetic effect evaluation in ob/ob mice. Mice were injected subcutaneously with vehicle, FGF21 or FGF21SS at indicated doses every day. plasma glucose (D) was measured every day. Plasma insulin (C) was analyzed at day 7 using insulin ELISA kit. Error bars show the SEM of ten independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 (Student's t-test) vs vehicle.


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).


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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 .

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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 .

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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 .

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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 .

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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). 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 .

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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 .

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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 .

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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 .

B-CELL_LINE O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


HEK293 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 .

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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.


In vitro niche dependency of HGSOC tumor cells . Phase - contrast pictures illustrate that isolated ovarian cancer cells rely on EGF supplementation for growth , while they do not grow at all in Wnt3a supplemented medium . Also , inhibition of BMP signaling through Noggin has strong negative effect on the initial growth . Scale bar : 500 µm . E - EGF , F - FGF10 , N - Noggin , R - R - spondin1 , B - Basic medium , P -

O O O O O B-DISEASE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-DISEASE I-DISEASE O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

In vitro niche dependency of HGSOC tumor cells. Phase-contrast pictures illustrate that isolated ovarian cancer cells rely on EGF supplementation for growth, while they do not grow at all in Wnt3a supplemented medium. Also, inhibition of BMP signaling through Noggin has strong negative effect on the initial growth. Scale bar: 500 µm. E - EGF, F - FGF10, N -Noggin, R - R-spondin1, B - Basic medium, P - Passage


Cancer organoids express HGSOC markers Pax8 and EpCAM and have lost the cystic phenotype suggesting complete breakdown of epithelial polarity as seen on confocal images from two representative organoid lines . Scale bar : 20 µm .

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Cancer organoids express HGSOC markers Pax8 and EpCAM and have lost the cystic phenotype suggesting complete breakdown of epithelial polarity as seen on confocal images from two representative organoid lines. Scale bar: 20 µm.


HE staining of organoids and respective tissue confirm high similarity in cellular structure and tissue organization . Scale bar : 100 µm .

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HE staining of organoids and respective tissue confirm high similarity in cellular structure and tissue organization. Scale bar: 100 µm.


HGSOC organoids show differential response to carboplatin treatment , confirming patient - specific sensitivity of the cultures . Cell viability assay was performed after 5 days of treatment with different concentrations of carboplatin on mature organoids from three different donors . Data represent mean ± SD of technical triplicates .

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HGSOC organoids show differential response to carboplatin treatment, confirming patient-specific sensitivity of the cultures. Cell viability assay was performed after 5 days of treatment with different concentrations of carboplatin on mature organoids from three different donors. Data represent mean ±SD of technical triplicates.


Overview of mutations found in organoid cultures and matching tumor tissue obtained by targeted sequencing of 121 HGSOC related genes confirms almost identical profile between parental tissue and in vitro long - term organoid culture . Color code indicates type of mutations which were detected .

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Overview of mutations found in organoid cultures and matching tumor tissue obtained by targeted sequencing of 121 HGSOC related genes confirms almost identical profile between parental tissue and in vitro long-term organoid culture. Color code indicates type of mutations which were detected.


Representative western blot showing loss or overexpression of p53 in individual organoid lines .

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Representative western blot showing loss or overexpression of p53 in individual organoid lines.


Strong nuclear p53 signal in immunostainings of HGSOC organoids is indicative of a gain - of - function TP53 mutation . Scale bars : 100 µm ( Tissue ) and 50 µm ( Organoid ) .

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Strong nuclear p53 signal in immunostainings of HGSOC organoids is indicative of a gain-of-function TP53 mutation. Scale bars: 100 µm (Tissue) and 50 µm (Organoid).


Box plots depicting overall constant level in normalized expression of major HGSOC marker genes between organoids and parental tissue . Data represent the median , quartiles , maximum , and minimum of the normalized expression from 8 different donors .

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Box plots depicting overall constant level in normalized expression of major HGSOC marker genes between organoids and parental tissue. Data represent the median, quartiles, maximum, and minimum of the normalized expression from 8 different donors.


Heat map of differentially expressed genes between cancer and healthy tissue / organoids reveals upregulation of several HGSOC biomarkers and reduction of FT differentiation markers in the cancer samples . Differential expression determined by single - color microarray for 8 different patient samples was significant for all genes with p < 0 . 05 except for OVGP1 and TOP2A in organoids .

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Heat map of differentially expressed genes between cancer and healthy tissue/organoids reveals upregulation of several HGSOC biomarkers and reduction of FT differentiation markers in the cancer samples. Differential expression determined by single-color microarray for 8 different patient samples was significant for all genes with p < 0.05 except for OVGP1 and TOP2A in organoids.


Experimental approach for genetic manipulation of FT epithelial cells ( FTECs ) . FTECs were sequentially transduced in 2D culture with replication - deficient viruses containing specific shRNAs and different selection markers . After sorting cells were seeded in Matrigel for organoid formation . Scale bars : 2 cm ( tissue ) , 200 µm ( 2D culture ) and 500 µm ( 3D culture ) . Confirmation of robust knockdowns of p53 , PTEN and RB in FT organoids on RNA and protein level . Relative mRNA levels were normalized to the WT mRNA level and are given as the mean ± SEM from 3 different donors . A representative western blot from one donor is depicted . RNA as well as protein samples were taken at passage 1 or 2 of each organoid line . KD - Knockdown , Vec Ctrl - Vector Control , WT - Wild

O O O O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O B-GENEPROD O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Experimental approach for genetic manipulation of FT epithelial cells (FTECs). FTECs were sequentially transduced in 2D culture with replication-deficient viruses containing specific shRNAs and different selection markers. After sorting cells were seeded in Matrigel for organoid formation. Scale bars: 2 cm (tissue), 200 µm (2D culture) and 500 µm (3D culture). Confirmation of robust knockdowns of p53, PTEN and RB in FT organoids on RNA and protein level. Relative mRNA levels were normalized to the WT mRNA level and are given as the mean ±SEM from 3 different donors. A representative western blot from one donor is depicted. RNA as well as protein samples were taken at passage 1 or 2 of each organoid line. KD - Knockdown, Vec Ctrl - Vector Control, WT- Wild type


Cyclin E1 overexpression as a downstream effect of RB knockdown was confirmed by western blot analysis .

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Cyclin E1 overexpression as a downstream effect of RB knockdown was confirmed by western blot analysis.


Confocal images of triple KD organoids reveal increased DNA damage ( yH2AX , marked by asterisks ) , atypic nuclei ( DRAQ5 ) and loss of apicobasal polarity . Scale bar : 20µm .

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Confocal images of triple KD organoids reveal increased DNA damage (yH2AX, marked by asterisks), atypic nuclei (DRAQ5) and loss of apicobasal polarity. Scale bar: 20µm.


Confirmation of robust knockdowns of p53 , PTEN and RB in FT organoids on RNA and protein level . Relative mRNA levels were normalized to the WT mRNA level and are given as the mean ± SEM from 3 different donors . A representative western blot from one donor is depicted . RNA as well as protein samples were taken at passage 1 or 2 of each organoid line . KD - Knockdown , Vec Ctrl - Vector Control , WT - Wild type Cyclin E1 overexpression as a downstream effect of RB knockdown was confirmed by western blot analysis . Presence of ciliated cells ( arrowheads ) , as revealed by immunostaining against detyrosinated Tubulin ( dtyrTub ) in triple KD organoids , proves capacity for terminal differentiation . Scale bar : 50µm .

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O O O O O O

Confirmation of robust knockdowns of p53, PTEN and RB in FT organoids on RNA and protein level. Relative mRNA levels were normalized to the WT mRNA level and are given as the mean ±SEM from 3 different donors. A representative western blot from one donor is depicted. RNA as well as protein samples were taken at passage 1 or 2 of each organoid line. KD - Knockdown, Vec Ctrl - Vector Control, WT- Wild type Cyclin E1 overexpression as a downstream effect of RB knockdown was confirmed by western blot analysis. Presence of ciliated cells (arrowheads), as revealed by immunostaining against detyrosinated Tubulin (dtyrTub) in triple KD organoids, proves capacity for terminal differentiation. Scale bar: 50µm.


In long - term 3D culture triple KD organoids show loss of growth capacity and premature growth arrest compared to the controls as indicated by the representative growth curves of FT234 . Scale bar : 500 µm .

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In long-term 3D culture triple KD organoids show loss of growth capacity and premature growth arrest compared to the controls as indicated by the representative growth curves of FT234. Scale bar: 500 µm.


Growth curves and phase - contrast images of triple KD organoids grown in OCM compared to FTM reveal successful rescue of long - term growth capacity . Scale bar : 500 µm .

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Growth curves and phase-contrast images of triple KD organoids grown in OCM compared to FTM reveal successful rescue of long-term growth capacity. Scale bar: 500 µm.


Levels of DNA damage ( yH2AX ) and apoptosis ( cleaved caspase - 3 , PARP1 ) do not differ among the different media as shown by representative western blots comparing triple KD organoids and their controls .

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Levels of DNA damage (yH2AX) and apoptosis (cleaved caspase-3, PARP1) do not differ among the different media as shown by representative western blots comparing triple KD organoids and their controls.


Triple KD ( p53 / PTEN / RB ) organoids are characterized by a HGSOC gene expression signature as revealed by microarray analysis comparing WT and KD organoids grown in either OCM or FTM . Differential expression determined by dual - color microarray for 2 biological replicates was significant for all genes with p < 0 . 00005 .

O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-DISEASE B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O

Triple KD (p53/PTEN/RB) organoids are characterized by a HGSOC gene expression signature as revealed by microarray analysis comparing WT and KD organoids grown in either OCM or FTM. Differential expression determined by dual-color microarray for 2 biological replicates was significant for all genes with p < 0.00005.


Differential expression of stemness - and differentiation - related genes of triple KD organoids grown in OCM vs . FTM . Fold - changes were derived from microarray data and indicate and an increase in stemness ( CD133 , SOX2 , MYCN ) and a drop in differentiation ( OVGP1 , PGR , FOXJ1 ) under OCM conditions . Differential expression determined by dual - color microarray for 2 biological replicates was significant for all genes with p < 0 . 05 .

O B-EXP_ASSAY O 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-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O

Differential expression of stemness- and differentiation-related genes of triple KD organoids grown in OCM vs. FTM. Fold-changes were derived from microarray data and indicate and an increase in stemness (CD133, SOX2, MYCN) and a drop in differentiation (OVGP1, PGR, FOXJ1) under OCM conditions. Differential expression determined by dual-color microarray for 2 biological replicates was significant for all genes with p < 0.05.


FOXJ1 expression determined by qPCR was significantly diminished in triple KD organoids grown in OCM compared to FTM , suggesting a decrease in differentiation capacity . Data represent the mean ± SEM from technical triplicates for two independent knockdown cultures .

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FOXJ1 expression determined by qPCR was significantly diminished in triple KD organoids grown in OCM compared to FTM, suggesting a decrease in differentiation capacity. Data represent the mean ±SEM from technical triplicates for two independent knockdown cultures.


Proportion of ciliated cells ( detyrosinated Tubulin ; dtyrTub ) is significantly reduced in KD organoids under OCM growth conditions , confirming inhibition of differentiation as illustrated by confocal images ( Scale bar : 50 µm ) . The quantification plot depicts the mean ± SEM from 3 independent biological replicates , based on quantification of the average number of ciliated cells per counted nuclei . 1000 cells were counted per individual experiment . * p < 0 . 05 , two - sided Student ' s t - test .

O O B-SUBCELLULAR O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O 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-SUBCELLULAR O O B-EXP_ASSAY B-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

Proportion of ciliated cells (detyrosinated Tubulin; dtyrTub) is significantly reduced in KD organoids under OCM growth conditions, confirming inhibition of differentiation as illustrated by confocal images (Scale bar: 50 µm). The quantification plot depicts the mean ±SEM from 3 independent biological replicates, based on quantification of the average number of ciliated cells per counted nuclei. 1000 cells were counted per individual experiment. *p < 0.05, two-sided Student's t-test.


The proportion of CD133 + cells was determined by FACS analysis after growing triple KD and control cells in FTM vs . OCM for two passages . The graph is representative of two biological replicates .

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The proportion of CD133+ cells was determined by FACS analysis after growing triple KD and control cells in FTM vs. OCM for two passages. The graph is representative of two biological replicates.


Growth capacity increases when KD organoids are grown in OCM medium as shown by difference in total viable cell number ( representative graph of 3 independent experiments ) . The luminescent cell viability assay was performed in triplicate and the bar plot depicts the mean ± SEM . * p < 0 . 05 , two - sided Student ` s t - test ; KD - Knockdown , Vec Ctrl - Vector Control , WT - Wild

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Growth capacity increases when KD organoids are grown in OCM medium as shown by difference in total viable cell number (representative graph of 3 independent experiments). The luminescent cell viability assay was performed in triplicate and the bar plot depicts the mean ±SEM. * p < 0.05, two-sided Student`s t-test; KD - Knockdown, Vec Ctrl - Vector Control, WT - Wild type


Phase - contrast images of cancer organoids which entered growth arrest upon treatment with Wnt agonists ( Wnt3a / RSPO1 ) over 2 passages ( Scale bar : 500µm ) . Differences in cell number were confirmed by the respective cell viability assays ( performed in technical triplicates ) . Data represent the mean ± SEM for 5 different OC organoid lines ( n = 5 ) . * * * p < 0 . 001 , two - sided Student ` s t - test .

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Phase-contrast images of cancer organoids which entered growth arrest upon treatment with Wnt agonists (Wnt3a/RSPO1) over 2 passages (Scale bar: 500µm). Differences in cell number were confirmed by the respective cell viability assays (performed in technical triplicates). Data represent the mean ±SEM for 5 different OC organoid lines (n=5). *** p < 0.001, two-sided Student`s t-test.


The number of CD133 + cells determined by FACS for OC7 and OC4 organoids grown in different media with or without Wnt3a / RSPO1 and with or without Noggin over 2 passages , confirms a sharp drop in stemness in the presence of Wnt agonists .

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The number of CD133+ cells determined by FACS for OC7 and OC4 organoids grown in different media with or without Wnt3a/RSPO1 and with or without Noggin over 2 passages, confirms a sharp drop in stemness in the presence of Wnt agonists.


Addition of conditioned medium collected from the parental Wnt3a cell line did not suppress growth of cancer organoids , proving that the growth inhibitory effect is due to the presence of Wnt agonists . The bar plot represents the mean ± SEM of triplicates performed with a luminescent cell viability assay . * p < 0 . 05 , * * * p < 0 . 001 , two - sided Student ` s t - test .

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Addition of conditioned medium collected from the parental Wnt3a cell line did not suppress growth of cancer organoids, proving that the growth inhibitory effect is due to the presence of Wnt agonists. The bar plot represents the mean ±SEM of triplicates performed with a luminescent cell viability assay. * p < 0.05, *** p < 0.001, two-sided Student`s t-test.


Comparative analysis of microarray data revealed consistent up - regulation of MYCN , the canonical Wnt inhibitors KREMEN2 and DKK3 as well as differentiation marker FOXJ1 , AR and PGR in HGSOC cancer organoids as well as triple KD FT organoids . Differential expression was determined either by single - color microarray for the cancer samples ( 8 replicates ) or dual - color microarray for the knockdowns ( 2 replicates ) and is significant for all genes with p < 0 . 05 .

O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-DISEASE I-DISEASE O O O O O O B-TISSUE O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O B-DISEASE O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

Comparative analysis of microarray data revealed consistent up-regulation of MYCN, the canonical Wnt inhibitors KREMEN2 and DKK3 as well as differentiation marker FOXJ1, AR and PGR in HGSOC cancer organoids as well as triple KD FT organoids. Differential expression was determined either by single-color microarray for the cancer samples (8 replicates) or dual-color microarray for the knockdowns (2 replicates) and is significant for all genes with p < 0.05.


Transcriptional induction of MYCN is confirmed by qPCR for three patients between cancer organoids and normal FT organoids as well as triple KD organoids grown in OCM vs . FTM conditions . Depicted is the mean ± SEM of the normalized MYCN mRNA expression level of three biological replicates ( n = 3 ) . * * p < 0 . 01 , * * * p < 0 . 001 , two - sided Student ` s t - test .

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Transcriptional induction of MYCN is confirmed by qPCR for three patients between cancer organoids and normal FT organoids as well as triple KD organoids grown in OCM vs. FTM conditions. Depicted is the mean ±SEM of the normalized MYCN mRNA expression level of three biological replicates (n=3). ** p < 0.01, *** p < 0.001, two-sided Student`s t-test.


Microarray data revealed that expression levels of Wnt target genes are downregulated in triple KD compared to WT control FT organoids . Differential expression determined by dual - color microarray for 2 biological replicates was significant for all genes with p ≤ 0 . 005 .

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Microarray data revealed that expression levels of Wnt target genes are downregulated in triple KD compared to WT control FT organoids. Differential expression determined by dual-color microarray for 2 biological replicates was significant for all genes with p ≤ 0.005.


FOXJ1 expression was upregulated in HGSOC organoids in FTM compared to OCM as shown by qPCR analysis of 3 different patient samples . Data represent the mean ± SEM of technical triplicates .

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FOXJ1 expression was upregulated in HGSOC organoids in FTM compared to OCM as shown by qPCR analysis of 3 different patient samples. Data represent the mean ±SEM of technical triplicates.


A ) Perturbation in the free energy of PKD2L1 opening ( ∆ G ° ) and difference in closure rates ( ∆ τclose ) inducted by substitutions within the pore module . Error bars = S . D as estimated by the propagation of error equation , as descried in statistics subsection .

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A) Perturbation in the free energy of PKD2L1 opening (∆G°) and difference in closure rates (∆τclose) inducted by substitutions within the pore module. Error bars = S.D as estimated by the propagation of error equation, as descried in statistics subsection.


B ) Exemplar tail currents from mutant channels where the energetics of opening are disassociated with the rate of channel closure - mutations which cause slower closure ( + ∆ τclose ) while also increasing the free energy opening ( + ∆ G ° ) .

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B) Exemplar tail currents from mutant channels where the energetics of opening are disassociated with the rate of channel closure- mutations which cause slower closure (+∆τclose) while also increasing the free energy opening (+∆G°).


D ) Comparing voltage - dependent opening based on normalized tail currents measured from WT , single and double mutant channels . Top , single and double mutant channels including mutations to the outer and inner gate . Middle and bottom , control groups comparing tail currents from non - gate S4 - S5 linker mutations ( D476A and G479A ) and those from the outer and inner gates . Error bars indicate standard deviation for as estimated by the propagation of error equation , as descried in statistics subsection . Number cells tested ( N ) are indicated in parentheses for each group .

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D) Comparing voltage-dependent opening based on normalized tail currents measured from WT, single and double mutant channels. Top, single and double mutant channels including mutations to the outer and inner gate. Middle and bottom, control groups comparing tail currents from non-gate S4-S5 linker mutations (D476A and G479A) and those from the outer and inner gates. Error bars indicate standard deviation for as estimated by the propagation of error equation, as descried in statistics subsection. Number cells tested (N) are indicated in parentheses for each group.


E ) The coupling energy ( ∆ ∆ G ° ) resulting from the mutant cycle analysis resulting from the data shown in D . Error bars indicate standard deviation for as estimated by the propagation of error equation , as descried in statistics subsection . Number cells tested ( N ) are indicated in parentheses for each group .

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E) The coupling energy (∆∆G°) resulting from the mutant cycle analysis resulting from the data shown in D. Error bars indicate standard deviation for as estimated by the propagation of error equation, as descried in statistics subsection. Number cells tested (N) are indicated in parentheses for each group.


B ) Summary of the change in voltage - dependent inactivation ( ∆ V1 / 2 Inact . ) of pore mutant channels . ∆ V1 / 2 Inact . , are plotted relative to the pore module topology of the channel .

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B) Summary of the change in voltage-dependent inactivation (∆V1/2 Inact.) of pore mutant channels. ∆V1/2 Inact., are plotted relative to the pore module topology of the channel.


C ) Voltage - dependent inactivation of test pulse tail currents recorded from WT , single and double mutant channels .

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C) Voltage-dependent inactivation of test pulse tail currents recorded from WT, single and double mutant channels.


C ) Exemplar whole cell currents ( Top ) at the zero , three and seven minute time points . Current records ( Bottom ) from cells expressing the F514C : P538C and treated with extracellular 100 μM 1 , 2 - methanediyl bismethanethiosulfonate ( M2M ) .

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C) Exemplar whole cell currents (Top) at the zero, three and seven minute time points. Current records (Bottom) from cells expressing the F514C:P538C and treated with extracellular 100 μM 1,2-methanediyl bismethanethiosulfonate (M2M).


D ) Box ( S . E . M . ) and whisker ( S . D . ) plots comparing tail currents normalized to their initial amplitude at 3 minute ( Left ) and 7 - minute time points ( Right ) . N = 6 cells per group . Results from each individual cells are indicated by grey open circles and the average for the channel is indicated by a horizontal line . P - values results from a one - way ANOVA statistical analysis is shown above the plots . Dashed lines the level normalcy , no change in Itail relative to time point zero .

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D) Box (S.E.M.) and whisker (S.D.) plots comparing tail currents normalized to their initial amplitude at 3 minute (Left) and 7-minute time points (Right). N = 6 cells per group. Results from each individual cells are indicated by grey open circles and the average for the channel is indicated by a horizontal line. P-values results from a one-way ANOVA statistical analysis is shown above the plots. Dashed lines the level normalcy, no change in Itail relative to time point zero.


Left : Fluorescence changes of IRDye700 labelled dsDNA ( at 125 nM ) when incubated at increasing ID2 ( I + D2 , IUb + D2Ub , I + D2Ub ) or ubiquitinated FANCD2 ( D2Ub ) concentrations ( ranging from 1 . 3 nM to 5 . 9 µM ) . Measurement of fluorescence enhancement for each ID2 complex was conducted for two separately prepared complexes ( two technical repeats ) and all data points for each protein - combination were used in fitting of a one - site binding model . Right : Bar graph showing mean apparent Kd values calculated from model fitting . Error bars : Asymmetric 95 % confidence intervals from non - linear regression ( 23 - 24 data points each ) .

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Left: Fluorescence changes of IRDye700 labelled dsDNA (at 125 nM) when incubated at increasing ID2 (I+D2, IUb+D2Ub, I+D2Ub) or ubiquitinated FANCD2 (D2Ub) concentrations (ranging from 1.3 nM to 5.9 µM). Measurement of fluorescence enhancement for each ID2 complex was conducted for two separately prepared complexes (two technical repeats) and all data points for each protein-combination were used in fitting of a one-site binding model. Right: Bar graph showing mean apparent Kd values calculated from model fitting. Error bars: Asymmetric 95% confidence intervals from non-linear regression (23-24 data points each).


Assessment of protein - DNA interactions using Electro - mobility shift assays ( EMSA ) . IRDye700 labelled dsDNA ( at 2 nM ) was incubated with indicated amounts of non / single / double - ubiquitinated ID2 ( His6 - TEV - V5 - FANCI and FLAG - FANCD2 ) protein complexes ( I + D2 , IUb + D2Ub , I + D2Ub ) or ubiquitinated FLAG - FANCD2 ( D2Ub ) . Mixes were ran on non - denaturing gels , and the resolved free - and protein - bound DNA bands were visualized using an infrared scanner . EMSA gels of ID2 complexes are representative of 3 replicate experiments .

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Assessment of protein-DNA interactions using Electro-mobility shift assays (EMSA). IRDye700 labelled dsDNA (at 2 nM) was incubated with indicated amounts of non/single/double-ubiquitinated ID2 (His6-TEV-V5-FANCI and FLAG-FANCD2) protein complexes (I+D2, IUb+D2Ub, I+D2Ub) or ubiquitinated FLAG-FANCD2 (D2Ub). Mixes were ran on non-denaturing gels, and the resolved free- and protein-bound DNA bands were visualized using an infrared scanner. EMSA gels of ID2 complexes are representative of 3 replicate experiments.


Left : CryoEM 2D classes of ID2Ub and IUbD2Ub - dsDNA . Right : 3D reconstructions of ID2Ub ( EMD - 10843 ) and IUbD2Ub - DNA ( EMD - 10844 ) . Both structures exhibit a torus - like shape .

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Left: CryoEM 2D classes of ID2Ub and IUbD2Ub-dsDNA. Right: 3D reconstructions of ID2Ub (EMD-10843) and IUbD2Ub-DNA (EMD-10844). Both structures exhibit a torus-like shape.


Both wild - type ( IWT ) and R1285Q mutant ( IR1285Q ) FANCI can be efficiently ubiquitinated in the presence of DNA . Reactions were performed at 2 μM FANCI substrate for 60 min in the absence or presence of 4 µM ssDNA .

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Both wild-type (IWT) and R1285Q mutant (IR1285Q) FANCI can be efficiently ubiquitinated in the presence of DNA. Reactions were performed at 2 μM FANCI substrate for 60 min in the absence or presence of 4 µM ssDNA.


Both wild - type ( IWT ) and R1285Q mutant ( IR1285Q ) FANCI efficiently associate with FANCD2 . Fluorescence changes occurring when RED - tris - NTA - labelled FANCI ( IWT or IR1285Q ; both at 60 nM ) is incubated at increasing concentrations of FANCD2 ( ranging from 2 . 48 nM to 5 . 08 µM ) . Titrations were conducted twice ( two technical replicates ) and all data points for each protein - combination were used in fitting of a one - site binding model . Apparent Kd values ( mean ± SEM ) derived from fitting of a one - site binding model to the 24 data points of the two technical replicates are shown .

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Both wild-type (IWT) and R1285Q mutant (IR1285Q) FANCI efficiently associate with FANCD2. Fluorescence changes occurring when RED-tris-NTA-labelled FANCI (IWT or IR1285Q; both at 60 nM) is incubated at increasing concentrations of FANCD2 (ranging from 2.48 nM to 5.08 µM). Titrations were conducted twice (two technical replicates) and all data points for each protein-combination were used in fitting of a one-site binding model. Apparent Kd values (mean ± SEM) derived from fitting of a one-site binding model to the 24 data points of the two technical replicates are shown.


FANCD2 within an IR1285QD2 complex is resistant to ubiquitination . Reactions were performed at 4 μM ID2 substrate and 16 μM dsDNA . Progress of FANCD2 and FANCI ubiquitination was monitored by western blotting following SDS - PAGE .

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FANCD2 within an IR1285QD2 complex is resistant to ubiquitination. Reactions were performed at 4 μM ID2 substrate and 16 μM dsDNA. Progress of FANCD2 and FANCI ubiquitination was monitored by western blotting following SDS-PAGE.


FANCD2 ubiquitination cannot robustly enhance ID2 - DNA binding when complexed with IR1285Q , as determined by PIFE . Left : Fluorescence changes when IRD700 - labelled dsDNA ( at 125 nM ) is incubated at increasing concentrations of IR1285Q + D2 or IR1285Q + D2Ub ( complex concentrations ranging from 1 . 54 nM to 3 . 8 µM ) . Measurement of fluorescence enhancement for each protein combination was conducted for two separately prepared complexes ( two technical repeats ) and all data points for each protein - combination were used in fitting of a one - site binding model . Right : Bar graph showing mean apparent Kd values calculated from the one - site binding model . Error bars : Asymmetric 95 % confidence intervals from non - linear regression ( 22 data points each ) . IWT + D2 and IWT + D2Ub previously calculated curves and corresponding Kd values ( from data points shown in Figure 1A ) are also shown for comparison .

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FANCD2 ubiquitination cannot robustly enhance ID2-DNA binding when complexed with IR1285Q, as determined by PIFE. Left: Fluorescence changes when IRD700-labelled dsDNA (at 125 nM) is incubated at increasing concentrations of IR1285Q +D2 or IR1285Q +D2Ub (complex concentrations ranging from 1.54 nM to 3.8 µM). Measurement of fluorescence enhancement for each protein combination was conducted for two separately prepared complexes (two technical repeats) and all data points for each protein-combination were used in fitting of a one-site binding model. Right: Bar graph showing mean apparent Kd values calculated from the one-site binding model. Error bars: Asymmetric 95% confidence intervals from non-linear regression (22 data points each). IWT + D2 and IWT + D2Ub previously calculated curves and corresponding Kd values (from data points shown in Figure 1A) are also shown for comparison.


FANCD2 ubiquitination cannot robustly enhance ID2 - DNA binding when complexed with IR1285Q , as determined by EMSAs . IRDye700 - labelled dsDNA ( at 2 nM ) was incubated with indicated amounts of ID2 ( His6 - FANCI or His6 - FANCIR1285Q mixed with non - ubiquitinated or ubiquitinated FLAG - FANCD2 . Mixes were ran on non - denaturing gels , and the resolved free - and protein - bound DNA bands were visualized using an infrared scanner . Gels shown are representative of two replicate experiments .

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FANCD2 ubiquitination cannot robustly enhance ID2-DNA binding when complexed with IR1285Q, as determined by EMSAs. IRDye700-labelled dsDNA (at 2 nM) was incubated with indicated amounts of ID2 (His6-FANCI or His6-FANCIR1285Q mixed with non-ubiquitinated or ubiquitinated FLAG-FANCD2. Mixes were ran on non-denaturing gels, and the resolved free- and protein-bound DNA bands were visualized using an infrared scanner. Gels shown are representative of two replicate experiments.


USP1 - UAF1 can efficiently deubiquitinate D2Ub in the presence or absence of FANCI ( I ) , but not in the presence of ubiquitinated FANCI ( IUb ) . Ubiquitinated FANCD2 ( D2Ub ) was mixed with a 50 base pair dsDNA and either His6 - TEV - V5 - FANCI ( I ) , ubiquitinated His6 - TEV - V5 - FANCI ( IUb ) or no protein ; protein - DNA mixes were subsequently incubated with USP1 - UAF1 ( at 25 nM or 100 nM ) for indicated time periods . Deubiquitination of D2Ub and IUb was assessed , following SDS - PAGE , by Coomassie staining of the gels , as well as by western blotting of transferred blots with a specific FANCD2 antibody .

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USP1-UAF1 can efficiently deubiquitinate D2Ub in the presence or absence of FANCI (I), but not in the presence of ubiquitinated FANCI (IUb). Ubiquitinated FANCD2 (D2Ub) was mixed with a 50 base pair dsDNA and either His6-TEV-V5-FANCI (I), ubiquitinated His6-TEV-V5-FANCI (IUb) or no protein; protein-DNA mixes were subsequently incubated with USP1-UAF1 (at 25 nM or 100 nM) for indicated time periods. Deubiquitination of D2Ub and IUb was assessed, following SDS-PAGE, by Coomassie staining of the gels, as well as by western blotting of transferred blots with a specific FANCD2 antibody.


USP7 and USP2 can deubiquitinate D2Ub , but their activity towards D2Ub is greatly reduced in the presence of FANCI ( I ) or ubiquitinated FANCI ( IUb ) . Reactions were set - up as in A , but with 100 nM USP7 or USP2 . Deubiquitination was assessed , following SDS - PAGE , by Coomassie staining .

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USP7 and USP2 can deubiquitinate D2Ub, but their activity towards D2Ub is greatly reduced in the presence of FANCI (I) or ubiquitinated FANCI (IUb). Reactions were set-up as in A, but with 100 nM USP7 or USP2. Deubiquitination was assessed, following SDS-PAGE, by Coomassie staining.


Time course of FANCI ubiquitination with various ubiquitin mutants and corresponding sensitivity / resistance to USP1 - UAF1 mediated deubiquitination of resulting products . Deubiquitination reactions in the presence or absence of USP1 - UAF1 ( 100 nM ) incubated for 30 minutes .

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Time course of FANCI ubiquitination with various ubiquitin mutants and corresponding sensitivity/resistance to USP1-UAF1 mediated deubiquitination of resulting products. Deubiquitination reactions in the presence or absence of USP1-UAF1 (100 nM) incubated for 30 minutes.


Time course of USP1 - UAF1 mediated deubiquitination of IUbD2Ub - DNA complexes consisting of D2Ub a 50 base pair dsDNA and IUb produced with various ubiquitin mutants or wild - type ( WT ) ubiquitin . Progress of deubiquitination reaction was assessed following SDS - PAGE , by both Coomassie staining of the gels , as well as by western blotting of transferred blots with a specific FANCD2 antibody . FANCI ubiquitination with indicated ubiquitin - mutants ( or WT ) and subsequent deubiquitination of resulting IUbD2Ub - DNA complexes were conducted twice ; the residual FANCD2 ubiquitination , calculated from the FANCD2 blots for each time - point , was plotted for each type of ubiquitin in the protein complex , and corresponding deubiquitination progress curves were fitted using a one - phase decay model .

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Time course of USP1-UAF1 mediated deubiquitination of IUbD2Ub-DNA complexes consisting of D2Ub a 50 base pair dsDNA and IUb produced with various ubiquitin mutants or wild-type (WT) ubiquitin. Progress of deubiquitination reaction was assessed following SDS-PAGE, by both Coomassie staining of the gels, as well as by western blotting of transferred blots with a specific FANCD2 antibody. FANCI ubiquitination with indicated ubiquitin-mutants (or WT) and subsequent deubiquitination of resulting IUbD2Ub-DNA complexes were conducted twice; the residual FANCD2 ubiquitination, calculated from the FANCD2 blots for each time-point, was plotted for each type of ubiquitin in the protein complex, and corresponding deubiquitination progress curves were fitted using a one-phase decay model.


( B ) Confocal fluorescence microscopy of 293T cells transfected with 0 . 5 μg pQCXIP encoding IFITM3 variants containing N - terminal FLAG ( pQCXIP - FLAG - IFITM3 ) and immunostained with anti - FLAG M2 antibody . Scale bar , 5 μm .

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(B) Confocal fluorescence microscopy of 293T cells transfected with 0.5 μg pQCXIP encoding IFITM3 variants containing N-terminal FLAG (pQCXIP-FLAG-IFITM3) and immunostained with anti-FLAG M2 antibody. Scale bar, 5 μm.


( C ) Flow cytometry analysis of transfected cells in ( B ) immunostained with anti - FLAG M2 antibody . Corresponding mean flourescence intensity values are shown .

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(C) Flow cytometry analysis of transfected cells in (B) immunostained with anti-FLAG M2 antibody. Corresponding mean flourescence intensity values are shown.


( D ) SDS - PAGE of cell lysates derived from 293T cells transfected in ( B ) followed by immunoblotting with anti - FLAG M2 and anti - actin antibodies .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_LINE O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O

(D) SDS-PAGE of cell lysates derived from 293T cells transfected in (B) followed by immunoblotting with anti-FLAG M2 and anti-actin antibodies.


( E ) SDS - PAGE of Opti - prep purified supernatants ( 25 ng p24 equivalent ) derived from 293T cells transfected with pQCXIP - FLAG - IFITM3 variants , HIV - 1 pNL4 - 3 , and Vpr - Blam plasmid followed by immunoblotting with anti - FLAG M2 and anti - p24 Gag ( 183 - H12 - 5C ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O

(E) SDS-PAGE of Opti-prep purified supernatants (25 ng p24 equivalent) derived from 293T cells transfected with pQCXIP-FLAG-IFITM3 variants, HIV-1 pNL4-3, and Vpr-Blam plasmid followed by immunoblotting with anti-FLAG M2 and anti-p24 Gag (183-H12-5C).