Chromatin - enriched fractions of HeLa cells transfected with indicated siRNAs and subsequently exposed or not to 5 - azadC for 2 h were analyzed by immunoblotting with SUMO2 / 3 and Actin antibodies .

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Chromatin-enriched fractions of HeLa cells transfected with indicated siRNAs and subsequently exposed or not to 5-azadC for 2 h were analyzed by immunoblotting with SUMO2/3 and Actin antibodies.


HeLa cells stably expressing GFP - DNMT1 and transfected with indicated siRNAs were left untreated or exposed to 5 - azadC for 2 h . Cells were then preextracted in stringent preextraction buffer and fixed at the indicated time points after 5 - azadC removal . Mean detergent - resistant GFP signal was determined by quantitative image analysis ( > 6000 cells analyzed per condition ) . Data from a representative experiment are shown .

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HeLa cells stably expressing GFP-DNMT1 and transfected with indicated siRNAs were left untreated or exposed to 5-azadC for 2 h. Cells were then preextracted in stringent preextraction buffer and fixed at the indicated time points after 5-azadC removal. Mean detergent-resistant GFP signal was determined by quantitative image analysis (>6000 cells analyzed per condition). Data from a representative experiment are shown.


Proliferative capacity of HeLa / GFP - DNMT1 cells transfected with control or UBC9 siRNAs and exposed to the indicated 5 - azadC doses for 2 h was assayed by measuring cell proliferation with the SRB assay ( mean ± SEM ; n = 3 independent experiments ; * * * p < 0 . 001 , Student ' s t - test ) .

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Proliferative capacity of HeLa/GFP-DNMT1 cells transfected with control or UBC9 siRNAs and exposed to the indicated 5-azadC doses for 2 h was assayed by measuring cell proliferation with the SRB assay (mean±SEM; n=3 independent experiments; ***p<0.001, Student's t-test).


untransfected HeLa cells were exposed or not to 5 - azadC in the presence or absence of a small molecule SUMO E1 enzyme inhibitor ( SUMO - E1i ) .

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untransfected HeLa cells were exposed or not to 5-azadC in the presence or absence of a small molecule SUMO E1 enzyme inhibitor (SUMO-E1i).


HeLa cells treated with 5 - azadC for 2 h in the presence or absence of SUMO - E1i were preextracted in stringent preextraction buffer and fixed at the indicated time points after 5 - azadC removal , and immunostained with DNMT1 antibody . Mean detergent - resistant DNMT1 signal was determined by quantitative image analysis ( > 6000 cells analyzed per condition ) . Data from a representative experiment are shown .

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HeLa cells treated with 5-azadC for 2 h in the presence or absence of SUMO-E1i were preextracted in stringent preextraction buffer and fixed at the indicated time points after 5-azadC removal, and immunostained with DNMT1 antibody. Mean detergent-resistant DNMT1 signal was determined by quantitative image analysis (>6000 cells analyzed per condition). Data from a representative experiment are shown.


Proliferative capacity of HeLa cells transfected with SPRTN or ZNF451 siRNAs and exposed to indicated 5 - azadC doses for 2 h was

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Proliferative capacity of HeLa cells transfected with SPRTN or ZNF451 siRNAs and exposed to indicated 5-azadC doses for 2 h was assayed


iPOND analysis of 5 - azadC - induced DNMT1 trapping and SUMOylation . HeLa cells were mock - treated or pre - incubated with 5 - azadC for 5 min before addition of EdU for 10 min . Cells were then washed and incubated with thymidine for 0 ( pulse ) or 60 min ( chase ) before performing iPOND .

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iPOND analysis of 5-azadC-induced DNMT1 trapping and SUMOylation. HeLa cells were mock-treated or pre-incubated with 5-azadC for 5 min before addition of EdU for 10 min. Cells were then washed and incubated with thymidine for 0 (pulse) or 60 min (chase) before performing iPOND.


HeLa cells stably expressing GFP - DNMT1 were pre - incubated or not with SUMO - E1i for 30 min , and where indicated 5 - azadC was added to the medium for an additional 60 min . Cells were then processed for GFP immunoprecipitation ( IP ) followed by immunoblotting for the indicated proteins .

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HeLa cells stably expressing GFP-DNMT1 were pre-incubated or not with SUMO-E1i for 30 min, and where indicated 5-azadC was added to the medium for an additional 60 min. Cells were then processed for GFP immunoprecipitation (IP) followed by immunoblotting for the indicated proteins.


Immunoblot analysis of chromatin - enriched fractions of HeLa cells that were synchronized in early S phase by double thymidine block , pulse - labeled with 5 - azadC for 30 min and grown in the presence or absence of proteasome inhibitor ( MG132 ) for the indicated times .

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Immunoblot analysis of chromatin-enriched fractions of HeLa cells that were synchronized in early S phase by double thymidine block, pulse-labeled with 5-azadC for 30 min and grown in the presence or absence of proteasome inhibitor (MG132) for the indicated times.


cells were treated or not with aphidicolin following pulse - labeling with 5 - azadC .

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cells were treated or not with aphidicolin following pulse-labeling with 5-azadC.


Representative images of U2OS cells stably expressing GFP - SPRTN or GFP - ACRC that were transfected with control ( CTRL ) or UBC9 siRNAs , exposed to formaldehyde in the presence or absence of ubiquitin E1 enzyme ( UBA1 ) inhibitor TAK - 243 ( Ub - E1i ) as indicated and fixed one h later . Scale bar , 10 μm .

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Representative images of U2OS cells stably expressing GFP-SPRTN or GFP-ACRC that were transfected with control (CTRL) or UBC9 siRNAs, exposed to formaldehyde in the presence or absence of ubiquitin E1 enzyme (UBA1) inhibitor TAK-243 (Ub-E1i) as indicated and fixed one h later. Scale bar, 10 μm.


Quantification of data showing proportion of U2OS / GFP - SPRTN cells displaying nuclear GFP - SPRTN foci ( mean ± SEM ; at least 100 cells quantified per condition per experiment ; n = 3 independent experiments ) . As in ( C ) , but showing proportion of U2OS / GFP - ACRC cells displaying nuclear GFP - ACRC foci ( mean ± SEM ; at least 100 cells quantified per condition per experiment ; n = 3 independent experiments ) .

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Quantification of data showing proportion of U2OS/GFP-SPRTN cells displaying nuclear GFP-SPRTN foci (mean±SEM; at least 100 cells quantified per condition per experiment; n=3 independent experiments). As in (C), but showing proportion of U2OS/GFP-ACRC cells displaying nuclear GFP-ACRC foci (mean±SEM; at least 100 cells quantified per condition per experiment; n=3 independent experiments).


U2OS cells expressing GFP - ACRC were exposed to the indicated genotoxic agents ( formaldehyde : 600 μM , 1 h ; UV : 20 J / m2 , 6 h recovery ; hydroxyurea ( HU ) : 2mM , 24 h ; mitomycin C ( MMC ) : 40 ng / mL , 24 h ) , preextracted and fixed , and analyzed by microscopy . Representative images are shown . Scale bar , 10 μm .

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U2OS cells expressing GFP-ACRC were exposed to the indicated genotoxic agents (formaldehyde: 600 μM, 1 h; UV: 20 J/m2, 6 h recovery; hydroxyurea (HU): 2mM, 24 h; mitomycin C (MMC): 40 ng/mL, 24 h), preextracted and fixed, and analyzed by microscopy. Representative images are shown. Scale bar, 10 μm.


HeLa cells transfected with plasmids encoding GFP alone or indicated GFP - ACRC alleles were subjected to GFP IP under denaturing conditions . Beads were incubated with recombinant polySUMO22 - 8 chains , washed extensively and processed for immunoblotting with SUMO2 / 3 and GFP antibodies .

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HeLa cells transfected with plasmids encoding GFP alone or indicated GFP-ACRC alleles were subjected to GFP IP under denaturing conditions. Beads were incubated with recombinant polySUMO22-8 chains, washed extensively and processed for immunoblotting with SUMO2/3 and GFP antibodies.


Representative images of U2OS cells expressing indicated GFP - ACRC alleles that were treated with 5 - azadC in the presence or absence of SUMO inhibitor ( SUMO - E1i ) , fixed 2 h later and immunostained with DNMT1 antibody . Scale bar , 10 μm . Quantification of data in ( G ) , showing proportion of cells displaying GFP - ACRC co - localization with DNMT1 foci ( mean ± SEM ; at least 100 cells quantified per condition per experiment ; n = 3 independent experiments ) .

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Representative images of U2OS cells expressing indicated GFP-ACRC alleles that were treated with 5-azadC in the presence or absence of SUMO inhibitor (SUMO-E1i), fixed 2 h later and immunostained with DNMT1 antibody. Scale bar, 10 μm.   Quantification of data in (G), showing proportion of cells displaying GFP-ACRC co-localization with DNMT1 foci (mean±SEM; at least 100 cells quantified per condition per experiment; n=3 independent experiments).


HeLa cells transfected with plasmids encoding GFP alone , GFP - ACRC or GFP - tagged C . elegans GCNA - 1 were subjected to GFP IP under denaturing conditions . Beads were incubated with recombinant polySUMO23 - 8 chains , washed extensively and processed for immunoblotting with SUMO2 / 3 and GFP antibodies .

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HeLa cells transfected with plasmids encoding GFP alone, GFP-ACRC or GFP-tagged C. elegans GCNA-1 were subjected to GFP IP under denaturing conditions. Beads were incubated with recombinant polySUMO23-8 chains, washed extensively and processed for immunoblotting with SUMO2/3 and GFP antibodies.


GFP expression driven by the gcna - 1 promoter was observed in germ cells , proliferating embryos and young larvae but not in post - mitotic tissues in the head Scale bars , 50 µm .

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GFP expression driven by the gcna-1 promoter was observed in germ cells, proliferating embryos and young larvae but not in post-mitotic tissues in the head Scale bars, 50 µm.


Formaldehyde survival of wild type ( wt ) , dvc - 1 , gcna - 1 loss - of - function ( lof ) and gcna - 1 ; dvc - 1 double mutant C . elegans ( mean ± SEM ; n = 4 independent experiments ) .

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Formaldehyde survival of wild type (wt), dvc-1, gcna-1 loss-of-function (lof) and gcna-1; dvc-1 double mutant C. elegans (mean±SEM; n=4 independent experiments).


Cisplatin survival of wild type , dvc - 1 , gcna - 1 and gcna - 1 ; dvc - 1 double mutant C . elegans ( mean ± SEM ; n = 3 independent experiments ) .

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Cisplatin survival of wild type, dvc-1, gcna-1 and gcna-1; dvc-1 double mutant C. elegans (mean±SEM; n=3 independent experiments).


Formaldehyde survival of gcna - 1 deletion ( del ) and E364Q mutant C . elegans ( mean ± SEM ; n = 2 independent experiments ) .

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Formaldehyde survival of gcna-1 deletion (del) and E364Q mutant C. elegans (mean±SEM; n=2 independent experiments).


Formaldehyde survival of C . elegans grown on L4440 control ( CTRL ) or smo - 1 RNAi bacteria ( mean ± SEM ; n = 2 independent experiments ) .

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Formaldehyde survival of C. elegans grown on L4440 control (CTRL) or smo-1 RNAi bacteria (mean±SEM; n=2 independent experiments).


Formaldehyde survival of C . elegans grown on L4440 control ( CTRL ) or gei - 17 RNAi bacteria ( mean ± SEM ; n = 2 independent experiments ) .

Formaldehyde survival of C. elegans grown on L4440 control (CTRL) or gei-17 RNAi bacteria (mean±SEM; n=2 independent experiments).


Formaldehyde survival of wild type and gcna - 1 deletion ( del ) mutant C . elegans grown on L4440 control ( CTRL ) or smo - 1 RNAi bacteria ( mean ± SEM ; n = 2 independent experiments ) .

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Formaldehyde survival of wild type and gcna-1 deletion (del) mutant C. elegans grown on L4440 control (CTRL) or smo-1 RNAi bacteria (mean±SEM; n=2 independent experiments).


WB analysis of smc5 - AID clones in the absence or presence of Auxin using FLAG . Tubulin is used as loading control and wt cells is used as a negative control for the FLAG tag .

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WB analysis of smc5-AID clones in the absence or presence of Auxin using FLAG. Tubulin is used as loading control and wt cells is used as a negative control for the FLAG tag.


Proliferation curve of wt and smc5 - AID cells in the presence or absence of Auxin . The data represents means ± SD of 3 independent experiments .

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Proliferation curve of wt and smc5-AID cells in the presence or absence of Auxin. The data represents means ± SD of 3 independent experiments.


Proliferation curves of wt and 2 independent smc5 clones grown at 39 . 5ºC , with the estimated doubling time on the right . Data represents means ± SD of 4 experiments . Asterisks indicate p value ≤ 0 . 05 , as derived from unpaired t - test type 3 , two - sample unequal variance , carried out to check statistical significance between smc5 clones and wt . smc5 clone 1 versus wt , p = 0 . 016 , smc5 clone 2 versus wt , p = 0 . 04 .

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Proliferation curves of wt and 2 independent smc5 clones grown at 39.5ºC, with the estimated doubling time on the right. Data represents means ± SD of 4 experiments. Asterisks indicate p value ≤ 0.05, as derived from unpaired t-test type 3, two-sample unequal variance, carried out to check statistical significance between smc5 clones and wt. smc5 clone 1 versus wt, p=0.016, smc5 clone 2 versus wt, p=0.04.


Bidimensional FACS analysis shows significant accumulation in G2 / M and decrease in the S phase in smc5 mutants . The data represents means ± SD of 3 independent experiments for smc5 clones 2 and 3 , and 4 independent experiments for wt and smc5 clone 1 . Asterisks indicate p value ≤ 0 . 05 , detailed below , using paired t - test . S - phase ( smc5 clones 1 , 2 , 3 versus wt : p = 0 . 0031 ; p = 0 . 0002 ; p = 0 . 004 , respectively ) . G2 / M ( smc5 clones 1 , 2 , 3 versus wt : p = 0 . 029 ; p = 0 . 0014 ; p = 0 . 02 , respectively ) .

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Bidimensional FACS analysis shows significant accumulation in G2/M and decrease in the S phase in smc5 mutants. The data represents means ± SD of 3 independent experiments for smc5 clones 2 and 3, and 4 independent experiments for wt and smc5 clone 1. Asterisks indicate p value ≤ 0.05, detailed below, using paired t-test. S-phase (smc5 clones 1, 2, 3 versus wt: p= 0.0031; p=0.0002; p=0.004, respectively). G2/M (smc5 clones 1, 2, 3 versus wt: p= 0.029; p=0.0014; p=0.02, respectively).


Survival curve of smc5 cells treated with different concentrations of cisplatin ( CDDP ) . Data represents means ± SD of 3 experiments .

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Survival curve of smc5 cells treated with different concentrations of cisplatin (CDDP). Data represents means ± SD of 3 experiments.


Survival curve of smc5 cells complemented or not with chicken SMC5 cDNA and treated with different concentrations of cisplatin . The experiment is carried out at 39 . 5ºC . Data represents means ± SD of at least 3 experiments .

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Survival curve of smc5 cells complemented or not with chicken SMC5 cDNA and treated with different concentrations of cisplatin. The experiment is carried out at 39.5ºC. Data represents means ± SD of at least 3 experiments.


Survival curve of smc5 ku70 cells treated with different concentrations of cisplatin . Data represents means ± SD of the 3 experiments .

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Survival curve of smc5 ku70 cells treated with different concentrations of cisplatin. Data represents means ± SD of the 3 experiments.


Survival curve of smc5 fancc cells treated with different concentrations of cisplatin . Data represents means ± SD of 3 experiments .

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Survival curve of smc5 fancc cells treated with different concentrations of cisplatin. Data represents means ± SD of 3 experiments.


WB analysis of CHK1 - P levels at steady state or after treatment with 1 μM cisplatin for 3 hours . CHK1 total protein is used as loading control . Quantitative analysis of CHK1 - P levels at steady state versus total CHK1 is shown in the bottom panel . Data represents means ± SD of 4 experiments .

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WB analysis of CHK1-P levels at steady state or after treatment with 1 μM cisplatin for 3 hours. CHK1 total protein is used as loading control. Quantitative analysis of CHK1-P levels at steady state versus total CHK1 is shown in the bottom panel. Data represents means ± SD of 4 experiments.


WB analysis of FANCD2 ubiquitination levels at steady state after treatment with 1 μM cisplatin for 3 hours . Analysis of the ratio between FANCD2 ubiquitinated / non - ubiquitinated forms is shown on the bottom panel . Data represents means ± SD of 3 experiments .

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WB analysis of FANCD2 ubiquitination levels at steady state after treatment with 1 μM cisplatin for 3 hours. Analysis of the ratio between FANCD2 ubiquitinated/non-ubiquitinated forms is shown on the bottom panel. Data represents means ± SD of 3 experiments.


Left panel : Growth curve was carried out to test smc5 fancm cell proliferation . Data represents means ± SD of 3 experiments . Right panel : Survival curve of smc5 fancm cells treated with different concentrations of cisplatin . Data represents means ± SD of the 3 experiments .

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Left panel: Growth curve was carried out to test smc5 fancm cell proliferation. Data represents means ± SD of 3 experiments. Right panel: Survival curve of smc5 fancm cells treated with different concentrations of cisplatin. Data represents means ± SD of the 3 experiments.


Clonogenic assay to test cisplatin sensitivity of smc5 fancm cells in long term assays . Data represents means ± SD of 2 independent experiments with two independent clones analyzed for smc5 fancm .

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Clonogenic assay to test cisplatin sensitivity of smc5 fancm cells in long term assays. Data represents means ± SD of 2 independent experiments with two independent clones analyzed for smc5 fancm.


Left panel : Growth curve was carried out to test smc5 rad17 ( C ) cell proliferation . Data represents means ± SD of 3 experiments . Right panel : Survival curve of smc5 rad17 ( C ) cells treated with different concentrations of cisplatin . Data represents means ± SD of the 3 experiments .

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Left panel: Growth curve was carried out to test smc5 rad17 (C) cell proliferation. Data represents means ± SD of 3 experiments. Right panel: Survival curve of smc5 rad17 (C) cells treated with different concentrations of cisplatin. Data represents means ± SD of the 3 experiments.


Left panel : Growth curve was carried out to test smc5 ddx11 ( D ) cell proliferation . Data represents means ± SD of 3 experiments . Right panel : Survival curve of smc5 ddx11 ( D ) cells treated with different concentrations of cisplatin . Data represents means ± SD of the 3 experiments .

Left panel: Growth curve was carried out to test smc5 ddx11 (D) cell proliferation. Data represents means ± SD of 3 experiments. Right panel: Survival curve of smc5 ddx11 (D) cells treated with different concentrations of cisplatin. Data represents means ± SD of the 3 experiments.


Left panel : Representative Western blots illustrating the expression of SMC6 , SMC5 and FANCD2 in protein extracts from HeLa cells 48 hours after transfection with the indicated siRNAs . Right panel : Relative expression of SMC5 , SMC6 and FANCD2 . We performed individual experiments for each cell line with technical replicates . In each individual experiment , we first normalized SMC5 , SMC6 or FANCD2 expression to that of Vinculin ( internal control ) and calculated its relative level in each cell line in relation to the target protein / Vinculin ratio of the siLacZ transfected cells , which was set as one in each experiment . Data present the mean + / - S . E . M . and were analyzed by unpaired t - test . SMC5 in siSMC6 : n = 7 and p = 0 . 0002 ; SMC5 in siFANCD2 : n = 7 and p = 0 . 0016 ; SMC6 in siSMC5 : n = 8 and p < 0 . 0001 ; SMC6 in siFANCD2 : n = 24 and p < 0 . 0001 ; FANCD2 in siSMC6 : n = 30 and p < 0 . 0001 ; FANCD2 in siSMC5 : n = 10 and p = 0 . 0008 .

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Left panel: Representative Western blots illustrating the expression of SMC6, SMC5 and FANCD2 in protein extracts from HeLa cells 48 hours after transfection with the indicated siRNAs. Right panel: Relative expression of SMC5, SMC6 and FANCD2. We performed individual experiments for each cell line with technical replicates. In each individual experiment, we first normalized SMC5, SMC6 or FANCD2 expression to that of Vinculin (internal control) and calculated its relative level in each cell line in relation to the target protein/Vinculin ratio of the siLacZ transfected cells, which was set as one in each experiment. Data present the mean+/-S.E.M. and were analyzed by unpaired t-test. SMC5 in siSMC6: n=7 and p=0.0002; SMC5 in siFANCD2: n=7 and p=0.0016; SMC6 in siSMC5: n=8 and p<0.0001; SMC6 in siFANCD2: n=24 and p<0.0001; FANCD2 in siSMC6: n=30 and p<0.0001; FANCD2 in siSMC5: n=10 and p=0.0008.


Top . Representative Western blots illustrating the expression of SMC6 , FANCJ and FANCD2 in protein extracts from HeLa cells 48 hours after transfection with the indicated siRNAs . Bottom . Survival curves of cells transfected with indicated siRNA and treated with different concentrations of mitomycin C ( MMC ) . Data represents the means and SEM ( standard error of the mean ) of 3 independent experiments .

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Top. Representative Western blots illustrating the expression of SMC6, FANCJ and FANCD2 in protein extracts from HeLa cells 48 hours after transfection with the indicated siRNAs. Bottom. Survival curves of cells transfected with indicated siRNA and treated with different concentrations of mitomycin C (MMC). Data represents the means and SEM (standard error of the mean) of 3 independent experiments.


Representative Western blots illustrating the expression of SMC6 and of monoubiquitylated FANCD2 in protein extracts from HeLa cells transfected with the indicated siRNAs and treated 48 hours after transfection with HU ( 5mM , 18 hours ) or Mitomycin C ( 200 ng / ml , 18 hours ) . Vinculin is used as loading control .

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Representative Western blots illustrating the expression of SMC6 and of monoubiquitylated FANCD2 in protein extracts from HeLa cells transfected with the indicated siRNAs and treated 48 hours after transfection with HU (5mM, 18 hours) or Mitomycin C (200 ng/ml, 18 hours). Vinculin is used as loading control.


Top . Representative images of nuclei ( DAPI stained , blue ) with RAD51 ( green ) foci in HeLa cells transfected with the indicated siRNAs and treated with HU or MMC , as indicated in panel A . The scale bar represents 10 μm . Bottom . Histograms presenting the percentage of RAD51 foci - positive cells as evaluated in the different conditions of above . Data represent the means + / - S . E . M . and were analyzed by unpaired t - test . siSMC6 vs siLacZ : n = 3 , NT , MMC or HU p = n . s . ; siFANCD2 vs siLacZ : n = 4 , NT p = n . s . , MMC p = 0 . 0003 , HU p = 0 . 0479 ; siSMC6 + siFANCD2 vs siLacZ : n = 3 , NT p = n . s . , MMC p = 0 . 0439 , HU p = 0 . 0458 .

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Top. Representative images of nuclei (DAPI stained, blue) with RAD51 (green) foci in HeLa cells transfected with the indicated siRNAs and treated with HU or MMC, as indicated in panel A. The scale bar represents 10 μm. Bottom. Histograms presenting the percentage of RAD51 foci-positive cells as evaluated in the different conditions of above. Data represent the means+/-S.E.M. and were analyzed by unpaired t-test. siSMC6 vs siLacZ: n=3, NT, MMC or HU p=n.s.; siFANCD2 vs siLacZ: n=4, NT p=n.s., MMC p=0.0003, HU p=0.0479; siSMC6+siFANCD2 vs siLacZ: n=3, NT p=n.s., MMC p=0.0439, HU p=0.0458.


Left panel : Representative images of anaphase HeLa cells with micronuclei ( white arrows ) . 48 hours after transfection with indicated siRNAs cells were incubated 18 hours with 2 μg / ml cytochalasyn B to block cytokinesis . The scale bar represents 20 μm . Right panel : Histograms presenting the percentage of binucleate cells with micronuclei . Data represent the mean + / - SEM of 3 independent experiments , t - test , unpaired . siSMC6 vs siLacZ , p = 0 . 0085 ; siFANCD2 vs siLacZ , p = 0 . 0057 ; siSMC6 + siFANCD2 vs siLacZ , p = 0 . 0019 .

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Left panel: Representative images of anaphase HeLa cells with micronuclei (white arrows). 48 hours after transfection with indicated siRNAs cells were incubated 18 hours with 2 μg/ml cytochalasyn B to block cytokinesis. The scale bar represents 20 μm. Right panel: Histograms presenting the percentage of binucleate cells with micronuclei. Data represent the mean +/- SEM of 3 independent experiments, t-test, unpaired. siSMC6 vs siLacZ, p=0.0085; siFANCD2 vs siLacZ, p=0.0057; siSMC6+siFANCD2 vs siLacZ, p=0.0019.


Top : Examples of HeLa mitotic catastrophes and anaphase bridges as observed 48 hours after siRNA transfection . The scale bar represents 20 μm . Bottom : Histograms presenting the percentage of cells with mitotic abnormalities . Data represent the mean + / - S . E . M . of 3 independent experiments . t - test , unpaired . siSMC6 vs siLacZ : Aberrant mitosis p = 0 . 0125 , Nuclear bridges p = 0 . 0198 , Mitotic catastrophes p = 0 . 0145 ; siFANCD2 vs siLacZ : Aberrant mitosis p = 0 . 0005 , Nuclear bridges p = 0 . 0002 , Mitotic catastrophes p = 0 . 0011 ; siSMC6 + siFANCD2 vs siLacZ : Aberrant mitosis p = 0 . 0012 , Nuclear bridges p = 0 . 0116 , Mitotic catastrophes p = 0 . 0033 .

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Top: Examples of HeLa mitotic catastrophes and anaphase bridges as observed 48 hours after siRNA transfection. The scale bar represents 20 μm. Bottom: Histograms presenting the percentage of cells with mitotic abnormalities. Data represent the mean +/- S.E.M. of 3 independent experiments. t-test, unpaired. siSMC6 vs siLacZ: Aberrant mitosis p=0.0125, Nuclear bridges p=0.0198, Mitotic catastrophes p=0.0145; siFANCD2 vs siLacZ: Aberrant mitosis p=0.0005, Nuclear bridges p=0.0002, Mitotic catastrophes p=0.0011; siSMC6+siFANCD2 vs siLacZ: Aberrant mitosis p=0.0012, Nuclear bridges p=0.0116, Mitotic catastrophes p=0.0033.


Immunoprecipitation of FLAG - SMC6 and FLAG - NSMCE2 with FANCI in HEK293T cells overexpressing the SMC6 or NSMCE2 , respectively , in the presence or absence of Ethidium bromide ( EtBr ) . The experiment was performed 3 times , and the blot shows a representative experiment . The band under the one indicated as FANCI may represent a degradation product .

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Immunoprecipitation of FLAG-SMC6 and FLAG-NSMCE2 with FANCI in HEK293T cells overexpressing the SMC6 or NSMCE2, respectively, in the presence or absence of Ethidium bromide (EtBr). The experiment was performed 3 times, and the blot shows a representative experiment. The band under the one indicated as FANCI may represent a degradation product.


Co - immunoprecipitation of endogenous SMC5 - mAID - EGFP and FANCD2 in TK6 cells in the presence of benzonase . Both experiments were performed twice independently , with the note that the cisplatin condition was performed only in one of the experiments , and the FANCD2 pull down in the experiment not shown was performed without benzonase .

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Co-immunoprecipitation of endogenous SMC5-mAID-EGFP and FANCD2 in TK6 cells in the presence of benzonase. Both experiments were performed twice independently, with the note that the cisplatin condition was performed only in one of the experiments, and the FANCD2 pull down in the experiment not shown was performed without benzonase.


A Neurosphere assays were performed using mouse E14 . 5 primary neural progenitors treated with CRH with or without antalarmin ( Ant ) ( n = 3 biological replicates ) .

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A Neurosphere assays were performed using mouse E14.5 primary neural progenitors treated with CRH with or without antalarmin (Ant) (n = 3 biological replicates).


B Effects of CRH treatment on the stemness of neural progenitor cells at 2 days post - treatment were analyzed by Western blotting using an anti - SOX2 antibody ( n = 3 biological replicates ) .

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B Effects of CRH treatment on the stemness of neural progenitor cells at 2 days post-treatment were analyzed by Western blotting using an anti-SOX2 antibody (n = 3 biological replicates).


Effects of CRHR1 overexpression on the ( C ) neurosphere

Effects of CRHR1 overexpression on the (C) neurosphere formation


Effects of CRHR1 overexpression on the ( D ) SOX2 expression ( n = 3 biological replicates ) .

Effects of CRHR1 overexpression on the (D) SOX2 expression (n = 3 biological replicates).


E CRHR1 knockdown efficiency of shRNAs used in this study ( n = 4 biological replicates ) .

E CRHR1 knockdown efficiency of shRNAs used in this study (n = 4 biological replicates).


Effects of CRHR1 knockdown on the ( F ) neurosphere formation ,

Effects of CRHR1 knockdown on the (F) neurosphere formation,


Effects of CRHR1 knockdown on the ( G ) SOX2 expression n = 3 biological replicates for each experiment except for ( G , n = 5 biological replicates ) .

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Effects of CRHR1 knockdown on the (G) SOX2 expression n = 3 biological replicates for each experiment except for (G, n = 5 biological replicates).


Effects of CRHR1 knockdown on the ( H ) neurosphere morphology , n = 3 biological replicates for each experiment except for ( G , n = 5 biological replicates ) .

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Effects of CRHR1 knockdown on the (H) neurosphere morphology, n = 3 biological replicates for each experiment except for (G, n = 5 biological replicates).


Effects of CRHR1 knockdown on the ( I ) βIII - tubulin expression . n = 3 biological replicates for each experiment except for ( G , n = 5 biological replicates ) .

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Effects of CRHR1 knockdown on the (I) βIII-tubulin expression. n = 3 biological replicates for each experiment except for (G, n = 5 biological replicates).


J , K Immunostaining using anti - GFP ( green ) together with ( J ) anti - βIII - tubulin ( clone TuJ1 ; red ) ( n = 5 biological replicates ) or ( K ) anti - GFAP ( red ) antibodies ( n = 3 biological replicates ) after in vitro differentiation of E14 . 5 neural progenitor cells transduced with a lentiviral vector expressing shCRHR1 .

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J, K Immunostaining using anti-GFP (green) together with (J) anti-βIII-tubulin (clone TuJ1; red) (n = 5 biological replicates) or (K) anti-GFAP (red) antibodies (n = 3 biological replicates) after in vitro differentiation of E14.5 neural progenitor cells transduced with a lentiviral vector expressing shCRHR1.


A Embryonic brains that were intraventricularly electroporated CRHR1 - expressing plasmids at E13 . 5 were harvested at E15 . 5 and immunostained using an anti - GFP ( green ) and SOX2 ( red ) antibodies . B Quantification of the cell fraction in each laminar layer in ( A ) ( n = 3 biological replicates ) . C Percentage of SOX2 + cells among GFP + cells in ( A ) ( n = 4 biological replicates ) .

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A Embryonic brains that were intraventricularly electroporated CRHR1-expressing plasmids at E13.5 were harvested at E15.5 and immunostained using an anti-GFP (green) and SOX2 (red) antibodies. B Quantification of the cell fraction in each laminar layer in (A) (n = 3 biological replicates). C Percentage of SOX2+ cells among GFP+ cells in (A) (n = 4 biological replicates).


D Embryonic brains were electroporated with shCRHR1 plasmids with or without shCRHR1 - resistant CRHR1 expression vectors at E14 . 5 , and samples were harvested at E17 . 5 for anti - GFP immunolabeling . GFP + cell localization was quantified ( bottom panel ) ( n = 5 biological replicates ) .

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D Embryonic brains were electroporated with shCRHR1 plasmids with or without shCRHR1-resistant CRHR1 expression vectors at E14.5, and samples were harvested at E17.5 for anti-GFP immunolabeling. GFP+ cell localization was quantified (bottom panel) (n = 5 biological replicates).


E , F Brains electroporated with CRHR1 expression vectors at E13 . 5 were harvested at E15 . 5 and co - immunostained using anti - GFP ( green ) together with ( E ) anti - TBR2 ( red ) or ( F ) anti - CTIP2 ( red ) antibodies . n = 3 biological replicates .

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E, F Brains electroporated with CRHR1 expression vectors at E13.5 were harvested at E15.5 and co-immunostained using anti-GFP (green) together with (E) anti-TBR2 (red) or (F) anti-CTIP2 (red) antibodies. n = 3 biological replicates.


G E14 . 5 Embryonic brains that were electroporated with CRHR1 - expression vectors at E13 . 5 were pulse labeled with EdU and harvested at E15 . 5 . Samples were then visualized for GFP ( green ) , EdU ( blue ) and Ki67 ( red ) ( n = 4 biological replicates ) .

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G E14.5 Embryonic brains that were electroporated with CRHR1-expression vectors at E13.5 were pulse labeled with EdU and harvested at E15.5. Samples were then visualized for GFP (green), EdU (blue) and Ki67 (red) (n = 4 biological replicates).


Analysis of the effects of CRHR1 knockdown on neural stemness - and cell - fate - regulating transcription factors in neural progenitor cells by ( A ) qPCR at 2 days post - transduction .

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Analysis of the effects of CRHR1 knockdown on neural stemness- and cell-fate-regulating transcription factors in neural progenitor cells by (A) qPCR at 2 days post-transduction.


Analysis of the effects of CRHR1 knockdown on neural stemness - and cell - fate - regulating transcription factors in neural progenitor cells by ( B ) Western blotting ( n = 3 biological replicates ) at 2 days post - transduction .

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Analysis of the effects of CRHR1 knockdown on neural stemness- and cell-fate-regulating transcription factors in neural progenitor cells by (B) Western blotting (n = 3 biological replicates) at 2 days post-transduction.


The effects of CRHR1 overexpression on REST expression in neural progenitor cells analyzed by qPCR


The effects of CRHR1 overexpression on REST expression in neural progenitor cells analyzed by ( B ) Western blotting ( n = 6 biological replicates ) .

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The effects of CRHR1 overexpression on REST expression in neural progenitor cells analyzed by (B) Western blotting (n = 6 biological replicates).


E Double - immunolabeling of E15 . 5 brain sections electroporated in utero with CRHR1 - expressing plasmid with or without shREST vectors at E13 . 5 using anti - GFP ( green ) and SOX2 ( red ) antibodies .

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E Double-immunolabeling of E15.5 brain sections electroporated in utero with CRHR1-expressing plasmid with or without shREST vectors at E13.5 using anti-GFP (green) and SOX2 (red) antibodies.


F , G Quantification of ( F ) GFP + cell distribution ( n ≥ 4 biological replicates ) and ( G ) percentage of SOX2 + cells ( n ≥ 3 biological replicates ) in ( E ) .

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F, G Quantification of (F) GFP+ cell distribution (n ≥ 4 biological replicates) and (G) percentage of SOX2+ cells (n ≥ 3 biological replicates) in (E).


H Embryonic brains were electroporated with shCRHR1 plasmids with or without REST expression vectors at E14 . 5 , and brain samples were harvested at E17 . 5 for anti - GFP immunolabeling . I Quantification of GFP + cell localization in ( H )

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H Embryonic brains were electroporated with shCRHR1 plasmids with or without REST expression vectors at E14.5, and brain samples were harvested at E17.5 for anti-GFP immunolabeling. I Quantification of GFP+ cell localization in (H)


A , B Western blot analysis of E14 . 5 neural progenitor cells transduced with a lentiviral vector expressing ( A ) CRHR1 or ( B ) shCRHR1 using an anti - CREB and anti - phospho - CREB ( pCREB ) ( S133 ) antibodies . n = 5 biological replicates .

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A, B Western blot analysis of E14.5 neural progenitor cells transduced with a lentiviral vector expressing (A) CRHR1 or (B) shCRHR1 using an anti-CREB and anti-phospho-CREB (pCREB) (S133) antibodies. n = 5 biological replicates.


C E14 . 5 neural progenitor cells were transduced with CRHR1 - containing lentiviral vectors . At 2 days post - transduction , cells were treated with 10 µM H89 ( PKA inhibitor ) or 1 µM KN93 ( CaM kinase inhibitor ) for 8 h and harvested for Western blot analysis for CREB ( top panel ) . Band intensities were quantified ( bottom panel ) ( n = 3 biological replicates ) .

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C E14.5 neural progenitor cells were transduced with CRHR1-containing lentiviral vectors. At 2 days post-transduction, cells were treated with 10 µM H89 (PKA inhibitor) or 1 µM KN93 (CaM kinase inhibitor) for 8 h and harvested for Western blot analysis for CREB (top panel). Band intensities were quantified (bottom panel) (n = 3 biological replicates).


D Double - immunolabeling of E15 . 5 brain sections electroporated in utero with CRHR1 - expressing plasmid at E13 . 5 using anti - GFP ( green ) and pCREB ( S133 ) ( red ) as primary antibodies . Immunostaining was quantified ( right panel ) ( n = 5 biological replicates ) . White inset boxes were magnified and shown in the panels D ' and D ' ' .

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D Double-immunolabeling of E15.5 brain sections electroporated in utero with CRHR1-expressing plasmid at E13.5 using anti-GFP (green) and pCREB (S133) (red) as primary antibodies. Immunostaining was quantified (right panel) (n = 5 biological replicates). White inset boxes were magnified and shown in the panels D' and D''.


E Double - immunolabeling of E15 . 5 brain sections electroporated in utero with CRHR1 - expressing plasmid with or without a vector expressing CREB S133A , a dominant negative mutant form of CREB , at E13 . 5 using anti - GFP ( green ) and SOX2 ( red ) antibodies .

E Double-immunolabeling of E15.5 brain sections electroporated in utero with CRHR1-expressing plasmid with or without a vector expressing CREB S133A, a dominant negative mutant form of CREB, at E13.5 using anti-GFP (green) and SOX2 (red) antibodies.


F , G Quantification of ( F ) GFP + cell distribution ( n = 4 biological replicates ) and ( G ) percentage of SOX2 + cells ( n ≥ 3 biological replicates ) in ( E ) .

F, G Quantification of (F) GFP+ cell distribution (n = 4 biological replicates) and (G) percentage of SOX2+ cells (n ≥ 3 biological replicates) in (E).


H Embryonic brains were electroporated with shCRHR1 plasmids with or without CREB expression vectors at E14 . 5 , and then brain samples were harvested at E17 . 5 for anti - GFP immunolabeling . I Quantification of GFP + cell localization in ( L ) ( n ≥ 4 biological replicates ) .

H Embryonic brains were electroporated with shCRHR1 plasmids with or without CREB expression vectors at E14.5, and then brain samples were harvested at E17.5 for anti-GFP immunolabeling. I Quantification of GFP+ cell localization in (L) (n ≥ 4 biological replicates).


( A ) qPCR of E14 . 5 neural progenitor cells transduced with CREB - expressing lentiviral vectors , measuring REST

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(A) qPCR of E14.5 neural progenitor cells transduced with CREB-expressing lentiviral vectors, measuring REST mRNA


( B ) Western blot analyses ( n = 3 biological replicates ) of E14 . 5 neural progenitor cells transduced with CREB - expressing lentiviral vectors , measuring REST protein levels , respectively .

(B) Western blot analyses (n = 3 biological replicates) of E14.5 neural progenitor cells transduced with CREB-expressing lentiviral vectors, measuring REST protein levels, respectively.


Dose - dependent inhibition of endogenous REST expression by a CREB inhibitor , 666 - 15 . E14 . 5 neural progenitor cells were treated with the indicated concentrations of 666 - 15 for 48 h and subjected to ( C )

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Dose-dependent inhibition of endogenous REST expression by a CREB inhibitor, 666-15. E14.5 neural progenitor cells were treated with the indicated concentrations of 666-15 for 48 h and subjected to (C) qPCR


Dose - dependent inhibition of endogenous REST expression by a CREB inhibitor , 666 - 15 . E14 . 5 neural progenitor cells were treated with the indicated concentrations of 666 - 15 for 48 h and subjected to ( D ) Western blot

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Dose-dependent inhibition of endogenous REST expression by a CREB inhibitor, 666-15. E14.5 neural progenitor cells were treated with the indicated concentrations of 666-15 for 48 h and subjected to (D) Western blot analyses


Inhibition of CRHR1 - induced REST expression by 666 - 15 . E14 . 5 neural progenitor cells transduced with a lentiviral vector expressing CRHR1 were treated with 20 nM of 666 - 15 , and after 48 h , cells were lysed and subjected to ( E )

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Inhibition of CRHR1-induced REST expression by 666-15. E14.5 neural progenitor cells transduced with a lentiviral vector expressing CRHR1 were treated with 20 nM of 666-15, and after 48 h, cells were lysed and subjected to (E) qPCR


Inhibition of CRHR1 - induced REST expression by 666 - 15 . E14 . 5 neural progenitor cells transduced with a lentiviral vector expressing CRHR1 were treated with 20 nM of 666 - 15 , and after 48 h , cells were lysed and subjected to ( F ) Western blot analyses ( n = 4 biological replicates ) .

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Inhibition of CRHR1-induced REST expression by 666-15. E14.5 neural progenitor cells transduced with a lentiviral vector expressing CRHR1 were treated with 20 nM of 666-15, and after 48 h, cells were lysed and subjected to (F) Western blot analyses (n = 4 biological replicates).


H Luciferase activity was measured using E14 . 5 neural progenitor cells transfected with CREB expression vectors and pGL3 - Basic luciferase plasmids harboring the wild - type or mutated REST promoter at 2 days post - transfection ( n = 3 biological replicates ) .

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H Luciferase activity was measured using E14.5 neural progenitor cells transfected with CREB expression vectors and pGL3-Basic luciferase plasmids harboring the wild-type or mutated REST promoter at 2 days post-transfection (n = 3 biological replicates).


I A representative gel image of ChIP analysis showing association of CREB proteins with regions of the REST promoter by PCR amplification of putative CRE sequences in the REST promoter ( n = 3 biological replicates ) .

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I A representative gel image of ChIP analysis showing association of CREB proteins with regions of the REST promoter by PCR amplification of putative CRE sequences in the REST promoter (n = 3 biological replicates).


J Immunolabeling of E15 . 5 brain sections electroporated in utero with CREB - expressing plasmids with or without shREST vectors at E13 . 5 using anti - GFP antibody . GFP + cell localization was quantified ( right panel ) ( n ≥ 5 biological replicates ) . The upper and lower dashed lines indicate the boundaries between the CP and IZ , and IZ and VZ / SVZ , respectively .

J Immunolabeling of E15.5 brain sections electroporated in utero with CREB-expressing plasmids with or without shREST vectors at E13.5 using anti-GFP antibody. GFP+ cell localization was quantified (right panel) (n ≥ 5 biological replicates). The upper and lower dashed lines indicate the boundaries between the CP and IZ, and IZ and VZ/SVZ, respectively.


A Skeletal muscles in the lower legs were dissected and RNA was extracted on postnatal day 0 , 7 , and 21 . The expression of R3hdml was examined by RT - PCR . GAPDH was used as an internal control . MM , 100 bp DNA marker . B Quantification of RT - PCR results . Data are expressed as the means ± standard error of the mean ( SEM ) . The experiments were repeated at least three times . * * * * p < 0 . 0001 vs . day 0 group ; one - way ANOVA followed by Bonferroni post - hoc test .

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A Skeletal muscles in the lower legs were dissected and RNA was extracted on postnatal day 0, 7, and 21. The expression of R3hdml was examined by RT-PCR. GAPDH was used as an internal control. MM, 100 bp DNA marker. B Quantification of RT-PCR results. Data are expressed as the means ± standard error of the mean (SEM). The experiments were repeated at least three times. **** p < 0.0001 vs. day 0 group; one-way ANOVA followed by Bonferroni post-hoc test.


Satellite cells were isolated from skeletal muscles of adult mice by enzymatic digestion and placed in culture medium . Then , differentiation was induced in satellite cells ( C ) .

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Satellite cells were isolated from skeletal muscles of adult mice by enzymatic digestion and placed in culture medium. Then, differentiation was induced in satellite cells (C).


The proteins were extracted at the indicated time points . R3hdml expression was examined by western blotting using an anti - R3hdml antibody ( D ) . β - actin was used as internal control . Quantification of western blotting results ( E ) . Data are expressed as the means ± standard error of the mean ( SEM ) . The experiments were repeated at least three times . Representative data are shown . * p < 0 . 05 ; Student ' s t - test . Scale bar = 500 µm .

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The proteins were extracted at the indicated time points. R3hdml expression was examined by western blotting using an anti-R3hdml antibody (D). β-actin was used as internal control. Quantification of western blotting results (E). Data are expressed as the means ± standard error of the mean (SEM). The experiments were repeated at least three times. Representative data are shown. * p < 0.05; Student's t-test. Scale bar = 500 µm.


A Cultured C2C12 cells were transfected with the firefly luciferase construct containing the 781 bp fragment upstream of the translational start codon of the R3hdml coding sequence ( R3hdml - luc ) , together with Renilla luciferase vectors as controls ; the cells were then differentiated in the presence of 2 % horse serum . Cells were harvested at the indicated time points , after which luciferase activity of the cell lysate was measured .

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A Cultured C2C12 cells were transfected with the firefly luciferase construct containing the 781 bp fragment upstream of the translational start codon of the R3hdml coding sequence (R3hdml-luc), together with Renilla luciferase vectors as controls; the cells were then differentiated in the presence of 2% horse serum. Cells were harvested at the indicated time points, after which luciferase activity of the cell lysate was measured.


C Plasmids containing MyoD cDNA or empty vector were transfected into C2C12 cells . One day and 3 days after transfection , cells were lysed and subjected to SDS - PAGE and western blotting using anti - MyoD antibodies .

C Plasmids containing MyoD cDNA or empty vector were transfected into C2C12 cells. One day and 3 days after transfection, cells were lysed and subjected to SDS-PAGE and western blotting using anti-MyoD antibodies.


D Plasmids containing MyoD cDNA or empty vector together with R3hdml - luc and Renilla luciferase vectors were transfected into C2C12 cells . One day and 3 days after transfection , the luciferase activity of the cell lysate with or without MyoD overexpression was measured . Relative luciferase activity is expressed as the fold activity above the background conferred by the empty vector together with R3hdml - luc .

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D Plasmids containing MyoD cDNA or empty vector together with R3hdml-luc and Renilla luciferase vectors were transfected into C2C12 cells. One day and 3 days after transfection, the luciferase activity of the cell lysate with or without MyoD overexpression was measured. Relative luciferase activity is expressed as the fold activity above the background conferred by the empty vector together with R3hdml-luc.


A R3hdml siRNA or non - targeting siRNA was transfected into C2C12 cells . Then , C2C12 cells were differentiated into myotubes . RNA was extracted from the cells after induction of differentiation at the indicated time points . Closed bars , control ; open bars , silenced R3hdml gene ; * * p < 0 . 01 ; * p < 0 . 05 ; Student ' s t - test .

A R3hdml siRNA or non-targeting siRNA was transfected into C2C12 cells. Then, C2C12 cells were differentiated into myotubes. RNA was extracted from the cells after induction of differentiation at the indicated time points. Closed bars, control; open bars, silenced R3hdml gene; ** p < 0.01; * p < 0.05; Student's t-test.


B Immunofluorescence after C2C12 differentiation . MyHC is shown in green and the nuclei in blue ( counterstained with Hoechst ) . Representative images of MyHC immunofluorescence of C2C12 myocytes transfected with the siRNA or after addition of R3hdml protein . Scale bar = 100 µm .

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B Immunofluorescence after C2C12 differentiation. MyHC is shown in green and the nuclei in blue (counterstained with Hoechst). Representative images of MyHC immunofluorescence of C2C12 myocytes transfected with the siRNA or after addition of R3hdml protein. Scale bar = 100 µm.


C The number of nuclei per MyHC + cell were calculated on day 5 after differentiation . * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni post - hoc test .

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C The number of nuclei per MyHC+ cell were calculated on day 5 after differentiation. ** p < 0.01; one-way ANOVA followed by Bonferroni post-hoc test.


D The distribution of nuclei per myotube were calculated on day 5 after differentiation . * * p < 0 . 01 ; * p < 0 . 05 ; one - way ANOVA followed by Bonferroni post - hoc test .

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D The distribution of nuclei per myotube were calculated on day 5 after differentiation. ** p < 0.01; * p < 0.05; one-way ANOVA followed by Bonferroni post-hoc test.


A Satellite cells were isolated by enzymatic digestion from R3hdml KO ( n = 6 ) and wild type control ( n = 6 ) mice and placed in culture medium . Then , cell proliferation was evaluated by Ki67 staining . Cell nuclei were stained with DAPI . B The ratio of Ki67 positive cells to total cells ( DAPI - positive ) was evaluated . The number of cells was counted under approximately 60 fields of view for each well . Closed columns , wild type controls ; open columns , R3hdml KO mice . * p < 0 . 05 ; Student ' s t - test .

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A Satellite cells were isolated by enzymatic digestion from R3hdml KO (n = 6) and wild type control (n = 6) mice and placed in culture medium. Then, cell proliferation was evaluated by Ki67 staining. Cell nuclei were stained with DAPI. B The ratio of Ki67 positive cells to total cells (DAPI-positive) was evaluated. The number of cells was counted under approximately 60 fields of view for each well. Closed columns, wild type controls; open columns, R3hdml KO mice. * p < 0.05; Student's t-test.


Thirty freshly isolated myofibers from 8 - 12 - week - old R3hdml KO ( n = 7 ) and wild type mice ( n = 7 ) were cultured for 72 h . Then , the isolated myofibers were fixed with 4 % PFA followed by immunostaining using anti Pax - 7 , anti MyoD , and anti Myog antibodies . ( C , D ) Co - immunostaining parallel cultures for Pax7 and MyoD showed that there were fewer cells with the self - renewing Pax7 + / MyoD − phenotype in muscles from R3hdml KO mice compared with control ; Pax7 + / MyoD − cells ( arrowheads ) , Pax7 − / MyoD + cells ( yellow arrows ) and Pax7 + / MyoD + cells ( white arrows ) . * p < 0 . 05 ; Student ' s t - test .

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Thirty freshly isolated myofibers from 8-12-week-old R3hdml KO (n = 7) and wild type mice (n = 7) were cultured for 72 h. Then, the isolated myofibers were fixed with 4% PFA followed by immunostaining using anti Pax-7, anti MyoD, and anti Myog antibodies. (C, D) Co-immunostaining parallel cultures for Pax7 and MyoD showed that there were fewer cells with the self-renewing Pax7+/MyoD− phenotype in muscles from R3hdml KO mice compared with control; Pax7+/MyoD− cells (arrowheads), Pax7−/MyoD+ cells (yellow arrows) and Pax7+/MyoD+ cells (white arrows). * p < 0.05; Student's t-test.


( E , F ) Co - immunostaining parallel cultures for MyoD and Myog showed that there were fewer cells with the differentiating MyoD − / Myog + phenotype in muscles from R3hdml KO mice compared to control ; MyoD − / Myog + cells ( arrowheads ) , MyoD - / Myog + cells ( yellow arrows ) , and MyoD + / Myog + cells ( white arrows ) . * p < 0 . 05 ; Student ' s t - test .

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(E, F) Co-immunostaining parallel cultures for MyoD and Myog showed that there were fewer cells with the differentiating MyoD−/Myog+ phenotype in muscles from R3hdml KO mice compared to control; MyoD−/Myog+ cells (arrowheads), MyoD-/Myog+ cells (yellow arrows), and MyoD+/Myog+ cells (white arrows). * p < 0.05; Student's t-test.


A Seven days after CTX injection , skeletal muscles were dissected , fixed , and embedded in paraffin . Two consecutive sections were utilized , and the first section was subjected to in situ hybridization using an R3hdml sequence - specific RNA probe to detect Rh3dml , while the second section was used for IHC using the anti - MyoD antibody . MyoD protein was visualized with DAB stain ( brown ) . R3hdml expression is highly detected in MyoD - positive cells . Scale bar in low magnification images = 100 µm ; in high magnification images = 25µm .

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A Seven days after CTX injection, skeletal muscles were dissected, fixed, and embedded in paraffin. Two consecutive sections were utilized, and the first section was subjected to in situ hybridization using an R3hdml sequence-specific RNA probe to detect Rh3dml, while the second section was used for IHC using the anti-MyoD antibody. MyoD protein was visualized with DAB stain (brown). R3hdml expression is highly detected in MyoD-positive cells. Scale bar in low magnification images = 100 µm; in high magnification images = 25µm.


B One hundred microliters of 10 µM CTX was intramuscularly injected into the forearm muscle of anesthetized R3hdml KO ( [ b ] , n = 10 ) and wild type control ( [ a ] , n = 10 ) mice . Subsequently , hand grip was evaluated 0 , 3 , 6 , and 14 days after CTX injection . R3hdml genes were overexpressed in the forearm muscle of anesthetized R3hdml KO mice ( [ c ] , n = 10 ) and hand grip was also evaluated 0 , 3 , 6 , and 14 days after CTX injection . Data are expressed as the means ± standard error of the mean ( SEM ) . * * p < 0 . 01 ; * * * * p < 0 . 0001 vs . wild type control ; # p < 0 . 05 ; # # p < 0 . 01 vs . R3hdml KO mice ; two - way ANOVA followed by a Bonferroni post hoc comparison of the individual time points .

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 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 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

B One hundred microliters of 10 µM CTX was intramuscularly injected into the forearm muscle of anesthetized R3hdml KO ([b], n = 10) and wild type control ([a], n = 10) mice. Subsequently, hand grip was evaluated 0, 3, 6, and 14 days after CTX injection. R3hdml genes were overexpressed in the forearm muscle of anesthetized R3hdml KO mice ([c], n = 10) and hand grip was also evaluated 0, 3, 6, and 14 days after CTX injection. Data are expressed as the means ± standard error of the mean (SEM). ** p < 0.01; **** p < 0.0001 vs. wild type control; # p < 0.05; ## p < 0.01 vs. R3hdml KO mice; two-way ANOVA followed by a Bonferroni post hoc comparison of the individual time points.


C Myofibers were freshly dissected from 8 - 12 - week - old R3hdml KO ( n = 4 ) and control mice ( n = 4 ) 35 days after CTX injection , and the number of satellite cells was evaluated by Pax - 7 staining . Arrowheads indicate Pax - 7 - positive cells . The number of Pax - 7 - positive cells per myofiber was counted . At least 50 myofibers in each animal were evaluated . Closed columns , wild type controls ; open columns , R3hdml KO mice . Data are expressed as the means ± standard error of the mean ( SEM ) . * p < 0 . 05 ; Student ' s t - test .

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C Myofibers were freshly dissected from 8-12-week-old R3hdml KO (n = 4) and control mice (n = 4) 35 days after CTX injection, and the number of satellite cells was evaluated by Pax-7 staining. Arrowheads indicate Pax-7-positive cells. The number of Pax-7-positive cells per myofiber was counted. At least 50 myofibers in each animal were evaluated. Closed columns, wild type controls; open columns, R3hdml KO mice. Data are expressed as the means ± standard error of the mean (SEM). * p < 0.05; Student's t-test.


Western blot of the indicated proteins in T - inducible PSCs stimulated as indicated for 24 hr . GAPDH was used as a loading control .

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Western blot of the indicated proteins in T-inducible PSCs stimulated as indicated for 24 hr. GAPDH was used as a loading control.


Heatmap derived from mean - centered expression values ( log2 scale ) of selected key pluripotency genes . See also Fig EV2C , Dataset EV1 for all the clustered DEGs .

Heatmap derived from mean-centered expression values (log2 scale) of selected key pluripotency genes. See also Fig EV2C, Dataset EV1 for all the clustered DEGs.


ChIP - seq tracks of T ( in red ; input is in blue ) around the Oct4 ( 7 . 5 kb ) . DE , distal enhancer ; PE , proximal enhancer . The T - binding peak around the Oct4 - DE is indicated ( arrowhead ) . Right : ChIP - qPCR for the enrichment of T on the DE and the regions indicated in Fig EV3E . NC , negative control ( the locus without T binding ) . The enrichment is indicated by the percent input with SDs ( three independent experiments with two technical replicates ) .

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ChIP-seq tracks of T (in red; input is in blue) around the Oct4 (7.5 kb). DE, distal enhancer; PE, proximal enhancer. The T-binding peak around the Oct4-DE is indicated (arrowhead). Right: ChIP-qPCR for the enrichment of T on the DE and the regions indicated in Fig EV3E. NC, negative control (the locus without T binding). The enrichment is indicated by the percent input with SDs (three independent experiments with two technical replicates).


Luciferase assay with T - binding elements ( TBEs ) around the indicated loci as shown in ( A ) and Fig EV3E . Relative luciferase activities of sorted cell fractions are shown with SDs . ( three independent experiments with two technical replicates ) .

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Luciferase assay with T-binding elements (TBEs) around the indicated loci as shown in (A) and Fig EV3E. Relative luciferase activities of sorted cell fractions are shown with SDs. (three independent experiments with two technical replicates).


Left : A representative confocal image of a mouse embryo at E7 . 0 bearing T - GFP and Blimp1 - RFP transgenic reporters . Scale bar : 50um . Arrowheads indicate Blimp1 - positive nascent germ cells in the posterior proximal epiblast . Right : FACS analysis of dissociated mouse embryos without visceral endoderm .

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Left: A representative confocal image of a mouse embryo at E7.0 bearing T-GFP and Blimp1-RFP transgenic reporters. Scale bar: 50um. Arrowheads indicate Blimp1-positive nascent germ cells in the posterior proximal epiblast. Right: FACS analysis of dissociated mouse embryos without visceral endoderm.


Expression of the indicated genes measured by qPCR in FACS - sorted cells ( E ) derived from embryos at different developmental stages ( E7 . 0 - 7 . 5 ) . Ten FACS - sorted cells in different cell fractions ( E ) were used for each individual qPCR analysis with two technical replicates . The ∆ Ct values from the average Ct values of Arbp and Ppia are shown ( n ≥ 3 , log2 scale ) . P values were calculated by two - tailed unpaired t - test .

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Expression of the indicated genes measured by qPCR in FACS-sorted cells (E) derived from embryos at different developmental stages (E7.0-7.5). Ten FACS-sorted cells in different cell fractions (E) were used for each individual qPCR analysis with two technical replicates. The ∆Ct values from the average Ct values of Arbp and Ppia are shown (n≥3, log2 scale). P values were calculated by two-tailed unpaired t-test.


ChIP - seq tracks of T ( in red ; input is in blue ) and core pluripotency factors ( in green ) around the Blimp1 ( 100 kb ) and Prdm14 ( 100 kb ) . The co - bound locus ( L # 1 - 4 ) and the locus exclusively bound by core pluripotency factors but not T ( L # 5 ) are indicated by arrow . NC , negative control ( the locus without T binding ) .

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ChIP-seq tracks of T (in red; input is in blue) and core pluripotency factors (in green) around the Blimp1 (100 kb) and Prdm14 (100 kb). The co-bound locus (L#1-4) and the locus exclusively bound by core pluripotency factors but not T (L#5) are indicated by arrow. NC, negative control (the locus without T binding).


ChIP - qPCR for the enrichment of the indicated factors on the loci ( L # 1 - 5 ) indicated in ( A ) . The enrichment is indicated by the percent input with SDs ( three independent experiments with two technical replicates ) .

ChIP-qPCR for the enrichment of the indicated factors on the loci (L#1-5) indicated in (A). The enrichment is indicated by the percent input with SDs (three independent experiments with two technical replicates).


Luciferase assay with the specific sequence of the locus bound by T around L # 1 and L # 3 as shown in ( A ) . Relative luciferase activities of low T - inducible ZHBTc4 ESCs stimulated as indicated are shown with SDs . ( three independent experiments with two technical replicates ) .

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Luciferase assay with the specific sequence of the locus bound by T around L#1 and L#3 as shown in (A). Relative luciferase activities of low T-inducible ZHBTc4 ESCs stimulated as indicated are shown with SDs. (three independent experiments with two technical replicates).


Expression of Blimp1 and Prdm14 measured by qPCR in PGCLCs derived from indicated ESC lines . The ∆ Ct values from the average Ct values of Arbp and Ppia are shown ( log2 scale , the mean value of two independent experiments with two technical replicates ) .

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Expression of Blimp1 and Prdm14 measured by qPCR in PGCLCs derived from indicated ESC lines. The ∆Ct values from the average Ct values of Arbp and Ppia are shown (log2 scale, the mean value of two independent experiments with two technical replicates).


Top : Oct4 - GFP ( green ) reporter expression ( left ) , and Blimp1 - RFP ( red ) and Oct4 - GFP ( BROG ) reporter expression ( right ) in the gonads of E13 . 5 embryos generated by 4N complementation with the indicated ESC lines . Scale bar : 100um . Bottom : percentage of Oct4 - GFP reporter - positive PGCs ( left ) and BROG reporter - positive PGCs ( right ) measured by FACS analysis . Blue circles , red squares , and green triangles represent the percentage of gonadal PGCs in the first , second , and third independent mutant line , respectively , of the examined locus ( L # 1 - 5 ) .

Top: Oct4-GFP (green) reporter expression (left), and Blimp1-RFP (red) and Oct4-GFP (BROG) reporter expression (right) in the gonads of E13.5 embryos generated by 4N complementation with the indicated ESC lines. Scale bar: 100um. Bottom: percentage of Oct4-GFP reporter-positive PGCs (left) and BROG reporter-positive PGCs (right) measured by FACS analysis. Blue circles, red squares, and green triangles represent the percentage of gonadal PGCs in the first, second, and third independent mutant line, respectively, of the examined locus (L#1-5).


A MMS - induced checkpoint activation is not altered in irc5 - Δ1 cells . The level of Rad53 phosphorylation was analyzed in wild type ( W303 - 1a ) and irc5 - Δ1 ( IL012 ) cells by Western blot with an anti - Rad53 antibody

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A MMS-induced checkpoint activation is not altered in irc5-Δ1 cells. The level of Rad53 phosphorylation was analyzed in wild type (W303-1a) and irc5-Δ1 (IL012) cells by Western blot with an anti-Rad53 antibody


B Prolonged checkpoint activation in irc5 - Δ1 cells during recovery from MMS treatment . Rad53 phosphorylation status was analyzed in wild type ( W303 - 1a ) and irc5 - Δ1 ( IL012 ) cells by Western blot with an anti - Rad53 antibody ; M , MMS

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B Prolonged checkpoint activation in irc5-Δ1 cells during recovery from MMS treatment. Rad53 phosphorylation status was analyzed in wild type (W303-1a) and irc5-Δ1 (IL012) cells by Western blot with an anti-Rad53 antibody; M, MMS


C Lack of Irc5 results in an elevated number of cells with Rad52 - YFP foci . At indicated time points samples of wild type ( W3749 - 14C ) and irc5 - Δ1 ( TB039 ) cells were collected , washed and processed for microscopic analysis . Error bars represent mean value ± standard deviations of mean ( n = 5 ) . Unpaired t - test was used to calculate the P - value . Representative microscopic photos of cells are shown . Scale bars : 5 μm

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C Lack of Irc5 results in an elevated number of cells with Rad52-YFP foci. At indicated time points samples of wild type (W3749-14C) and irc5-Δ1 (TB039) cells were collected, washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=5). Unpaired t-test was used to calculate the P-value. Representative microscopic photos of cells are shown. Scale bars: 5 μm


D Disruption of IRC5 leads to accumulation of ssDNA - containing lesions . ChEC analysis of wild type ( BYR52MN ) and irc5 - Δ1 ( TB040 ) cells cultured in the presence or absence of 0 . 03 % MMS for 2 hours ( n = 3 )

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D Disruption of IRC5 leads to accumulation of ssDNA-containing lesions. ChEC analysis of wild type (BYR52MN) and irc5-Δ1 (TB040) cells cultured in the presence or absence of 0.03% MMS for 2 hours (n=3)


F Increased levels of bright Rfa1 - YFP foci in cells lacking Irc5 . At indicated time points culture samples of wild type ( W3775 - 12C ) and irc5 - Δ1 ( TB041 ) cells were collected , washed and processed for microscopic analysis . Error bars represent mean value ± standard deviations of mean ( n = 5 ) . Unpaired t - test was used to calculate the P - value . Representative microscopic photos of cells are shown . Scale bars : 5

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F Increased levels of bright Rfa1-YFP foci in cells lacking Irc5. At indicated time points culture samples of wild type (W3775-12C) and irc5-Δ1 (TB041) cells were collected, washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=5). Unpaired t-test was used to calculate the P-value. Representative microscopic photos of cells are shown. Scale bars: 5 μm


A The irc5 - Δ1 mutant exhibits S - phase delay in the presence of MMS . DNA content of wild type ( W303 - 1a ) and irc5 - Δ1 ( IL012 ) cells was measured by FACS and plotted as histograms

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A The irc5-Δ1 mutant exhibits S-phase delay in the presence of MMS. DNA content of wild type (W303-1a) and irc5-Δ1 (IL012) cells was measured by FACS and plotted as histograms


B Completion of DNA replication is delayed in the irc5 - Δ1 mutant after MMS treatment . Intact chromosomes were isolated from wild type ( W303 - 1a ) and irc5 - Δ1 ( IL012 ) cells and processed for PFGE . M , 0 . 03 % MMS

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B Completion of DNA replication is delayed in the irc5-Δ1 mutant after MMS treatment. Intact chromosomes were isolated from wild type (W303-1a) and irc5-Δ1 (IL012) cells and processed for PFGE. M, 0.03% MMS


D Samples used for PFGE in B were processed for FACS analysis


A Lack of Irc5 translocase activity results in elevated number of cells with Rfa1 - YFP foci . At indicated time points samples of wild type ( TB042 ) and irc5DAEA ( TB043 ) cells were collected , washed and processed for microscopic analysis . Error bars represent mean value ± standard deviations of mean ( n = 5 ) . Unpaired t - test was used to calculate the P -

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A Lack of Irc5 translocase activity results in elevated number of cells with Rfa1-YFP foci. At indicated time points samples of wild type (TB042) and irc5DAEA (TB043) cells were collected, washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=5). Unpaired t-test was used to calculate the P-value


B Increased levels of bright Rad52 - YFP foci in cells expressing Irc5DAEA lacking ATPase activity . At indicated time points samples of wild type ( TB044 ) and irc5DAEA ( TB045 ) cells were collected , washed and processed for microscopic analysis . Error bars represent mean value ± standard deviations of mean ( n = 5 ) . Unpaired t - test was used to calculate the P -

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B Increased levels of bright Rad52-YFP foci in cells expressing Irc5DAEA lacking ATPase activity. At indicated time points samples of wild type (TB044) and irc5DAEA (TB045) cells were collected, washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=5). Unpaired t-test was used to calculate the P-value


C Translocase activity of Irc5 is important for completion of DNA replication after MMS treatment . PFGE was performed as in Fig 2B using wild type ( EMD09 ) and irc5DAEA ( EMD10 )

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C Translocase activity of Irc5 is important for completion of DNA replication after MMS treatment. PFGE was performed as in Fig 2B using wild type (EMD09) and irc5DAEA (EMD10) strains


E Samples used for PFGE in C were processed for FACS analysis


A , B ChIP - qPCR analysis of Scc1 association with early replication origin ARS305 or ARS607 . PK - tagged Scc1 interaction with chromatin was monitored in wild type ( JC1513 ) and irc5Δ - 1 ( TB064 ) cells . Error bars represent mean value ± standard deviations of mean ( n = 3 ) . ARS305 + 7 kb locus was used as a control for cohesin - free chromosomal region . POA1 was used as a control locus for the chromosomal region that is bound by cohesin but is located ~ 30 . 000 bp from the nearest replication origin

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A, B ChIP-qPCR analysis of Scc1 association with early replication origin ARS305 or ARS607. PK-tagged Scc1 interaction with chromatin was monitored in wild type (JC1513) and irc5Δ-1 (TB064) cells. Error bars represent mean value ± standard deviations of mean (n=3). ARS305 + 7 kb locus was used as a control for cohesin-free chromosomal region. POA1 was used as a control locus for the chromosomal region that is bound by cohesin but is located ~ 30.000 bp from the nearest replication origin


C , D ChIP - qPCR analysis of Pol2 association with early replication origin ARS305 or ARS607 as well as late replication origin ARS501 . HA - tagged Pol2 interaction with chromatin was monitored in wild type ( TB065 ) and irc5Δ - 1 ( TB066 ) cells . Error bars represent mean value ± standard deviations of mean ( n = 3 )

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C, D ChIP-qPCR analysis of Pol2 association with early replication origin ARS305 or ARS607 as well as late replication origin ARS501. HA-tagged Pol2 interaction with chromatin was monitored in wild type (TB065) and irc5Δ-1 (TB066) cells. Error bars represent mean value ± standard deviations of mean (n=3)


A , B ChIP - qPCR analysis of Scc1 association with early replication origin ARS305 or ARS607 . HA - tagged Scc1 interaction with chromatin was monitored in wild type ( Y39 ) and scc2 - 4 ( Y4366 ) cells . Error bars represent mean value ± standard deviations of mean ( n = 3 )

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A, B ChIP-qPCR analysis of Scc1 association with early replication origin ARS305 or ARS607. HA-tagged Scc1 interaction with chromatin was monitored in wild type (Y39) and scc2-4 (Y4366) cells. Error bars represent mean value ± standard deviations of mean (n=3)


C , D ChIP - qPCR analysis of Pol2 association with early replication origin ARS305 or ARS607 . HA - tagged Pol2 interaction with chromatin was monitored in wild type ( TB067 ) and scc2 - 4 ( TB068 ) cells . Error bars represent mean value ± standard deviations of mean ( n = 3 )

C, D ChIP-qPCR analysis of Pol2 association with early replication origin ARS305 or ARS607. HA-tagged Pol2 interaction with chromatin was monitored in wild type (TB067) and scc2-4 (TB068) cells. Error bars represent mean value ± standard deviations of mean (n=3)


E Cohesin loading complex is required for completion of chromosome replication after MMS treatment . Intact chromosomes were isolated from wild type ( Y39 ) and scc2 - 4 ( Y4366 ) cells cultivated at permissive temperature ( 25 ° C ) and processed for PFGE . M ,

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E Cohesin loading complex is required for completion of chromosome replication after MMS treatment. Intact chromosomes were isolated from wild type (Y39) and scc2-4 (Y4366) cells cultivated at permissive temperature (25°C) and processed for PFGE. M, MMS


A Sensitivity of scc2 - 4 and scc2 - 4 irc5 - Δ1 mutants to DNA damaging agents at permissive temperature 25 ° C . Logarithmically growing cultures of wild type ( W303 - 1a ) , irc5 - Δ1 ( IL012 ) , scc2 - 4 ( TB069 ) and irc5 - Δ1 scc2 - 4 ( TB070 ) strains were 10 - fold serially diluted and plated onto solid YPD containing indicated concentrations of DNA damaging

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A Sensitivity of scc2-4 and scc2-4 irc5-Δ1 mutants to DNA damaging agents at permissive temperature 25°C. Logarithmically growing cultures of wild type (W303-1a), irc5-Δ1 (IL012), scc2-4 (TB069) and irc5-Δ1 scc2-4 (TB070) strains were 10-fold serially diluted and plated onto solid YPD containing indicated concentrations of DNA damaging agents


B , C Levels of DNA repair foci in scc2 - 4 and scc2 - 4 irc5 - Δ1 mutants . Logarithmically growing cultures of scc2 - 4 ( TB071 or TB073 ) and scc2 - 4 irc5 - Δ1 ( TB072 or TB074 ) were treated with MMS for 1 hour at 25 ° C . Next , cells were washed and processed for microscopic analysis . Error bars represent mean value ± standard deviations of mean ( n = 3 )

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B, C Levels of DNA repair foci in scc2-4 and scc2-4 irc5-Δ1 mutants. Logarithmically growing cultures of scc2-4 (TB071 or TB073) and scc2-4 irc5-Δ1 (TB072 or TB074) were treated with MMS for 1 hour at 25°C. Next, cells were washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=3)


D Sensitivity of scc1 - 73 and scc1 - 73 irc5 - Δ1 mutants to DNA damaging agents at permissive temperature 25 ° C or semi - permissive temperature 30 ° C . Logarithmically growing cultures of wild type ( W303 - 1a ) , irc5 - Δ1 ( IL012 ) , scc1 - 73 ( JC1339 ) and irc5 - Δ1 scc1 - 73 ( TB075 ) strains were 10 - fold serially diluted and plated onto solid YPD containing indicated concentrations of DNA damaging

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D Sensitivity of scc1-73 and scc1-73 irc5-Δ1 mutants to DNA damaging agents at permissive temperature 25°C or semi-permissive temperature 30°C. Logarithmically growing cultures of wild type (W303-1a), irc5-Δ1 (IL012), scc1-73 (JC1339) and irc5-Δ1 scc1-73 (TB075) strains were 10-fold serially diluted and plated onto solid YPD containing indicated concentrations of DNA damaging agents


E , F Levels of Rfa1 - YFP and Rad52 - YFP foci in scc1 - 73 and irc5 - Δ1 scc1 - 73 mutants . Logarithmically growing cultures of scc1 - 73 ( TB076 or TB078 ) and irc5 - Δ1 scc1 - 73 ( TB077 or TB079 ) were treated with MMS for 1 hour at 25 ° C . Next , cells were washed and processed for microscopic analysis . Error bars represent mean value ± standard deviations of mean ( n = 3 )

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E, F Levels of Rfa1-YFP and Rad52-YFP foci in scc1-73 and irc5-Δ1 scc1-73 mutants. Logarithmically growing cultures of scc1-73 (TB076 or TB078) and irc5-Δ1 scc1-73 (TB077 or TB079) were treated with MMS for 1 hour at 25°C. Next, cells were washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=3)


G Replication completion defect of scc1 - 73 is not exacerbated by IRC5 disruption . Intact chromosomes were isolated from scc1 - 73 ( JC1339 ) and irc5 - Δ1 scc1 - 73 cells ( TB075 ) , cultured at 25 ° C , and processed for PFGE . M , 0 . 03 %

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G Replication completion defect of scc1-73 is not exacerbated by IRC5 disruption. Intact chromosomes were isolated from scc1-73 (JC1339) and irc5-Δ1 scc1-73 cells (TB075), cultured at 25°C, and processed for PFGE. M, 0.03% MMS


Western blot analysis of nephrin expression in immortalized podocytes cultured in normal ( 5 mM ) or high glucose ( HG ; 30 mM ) or high mannitol ( Mann ; 5 mM glucose plus 25 mM mannitol ) for 24 , 48 and 72 hours . * P < 0 . 05 versus normal controls for the indicated time points ( Wilcoxon two - sample test ; n = 3 ) .

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Western blot analysis of nephrin expression in immortalized podocytes cultured in normal (5 mM) or high glucose (HG; 30 mM) or high mannitol (Mann; 5 mM glucose plus 25 mM mannitol) for 24, 48 and 72 hours. *P < 0.05 versus normal controls for the indicated time points (Wilcoxon two-sample test; n = 3).


B Analysis of the methylation status at nephrin gene promoter in podocytes cultured in normal , high glucose or high mannitol at the indicated time points . U , unmethylated - specific primers ; M , methylated - specific primers .

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B Analysis of the methylation status at nephrin gene promoter in podocytes cultured in normal, high glucose or high mannitol at the indicated time points. U, unmethylated-specific primers; M, methylated-specific primers.


C Effect of pargyline hydrochloride , a pan - KDM inhibitor , on high glucose - mediated nephrin expression . Immortalized mouse podocytes were cultured in the indicated cultured media in the presence or absence of pargyline hydrochloride ( 1 μM ) for 48 hours . Protein lysates from the treated podocytes were analyzed for nephrin expression by immunoblotting . * P < 0 . 05 versus normal controls , # P < 0 . 05 versus untreated HG - incubated cells ( Wilcoxon two - sample test ; n = 3 ) .

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C Effect of pargyline hydrochloride, a pan-KDM inhibitor, on high glucose-mediated nephrin expression. Immortalized mouse podocytes were cultured in the indicated cultured media in the presence or absence of pargyline hydrochloride (1 μM) for 48 hours. Protein lysates from the treated podocytes were analyzed for nephrin expression by immunoblotting. *P < 0.05 versus normal controls, #P < 0.05 versus untreated HG-incubated cells (Wilcoxon two-sample test; n = 3).


D Effect of pargyline hydrochloride on high glucose - mediated DNA methylation at nephrin gene promoter . Genomic DNA samples from the treated podocytes were analyzed for methylation status of nephrin gene promoter by methylation - specific PCR .

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D Effect of pargyline hydrochloride on high glucose-mediated DNA methylation at nephrin gene promoter. Genomic DNA samples from the treated podocytes were analyzed for methylation status of nephrin gene promoter by methylation-specific PCR.


E Quantitative RT - PCR analyses of different KDMs in immortalized podocytes exposed to high glucose or high mannitol for 48 hours . * P < 0 . 05 versus normal controls ( Wilcoxon two - sample test ; n = 3 ) .

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E Quantitative RT-PCR analyses of different KDMs in immortalized podocytes exposed to high glucose or high mannitol for 48 hours. *P < 0.05 versus normal controls (Wilcoxon two-sample test; n = 3).


F Western blot analysis of KDM6A expression in podocytes cultured in normal , high glucose or high mannitol conditions for 24 , 48 ad 72 hours . * P < 0 . 05 versus normal controls for the indicated time points ( Wilcoxon two - sample test ; n = 3 ) .

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F Western blot analysis of KDM6A expression in podocytes cultured in normal, high glucose or high mannitol conditions for 24, 48 ad 72 hours. *P < 0.05 versus normal controls for the indicated time points (Wilcoxon two-sample test; n = 3).


G Western blot analysis of H3K27me1 , H3K27me2 , H3K27me3 and pan - methyl H3K9 in podocytes cultured in normal , high glucose or high mannitol conditions for 9 and 24 hours . * P < 0 . 05 versus normal controls ( Wilcoxon two - sample test ; n = 3 ) .

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G Western blot analysis of H3K27me1, H3K27me2, H3K27me3 and pan-methyl H3K9 in podocytes cultured in normal, high glucose or high mannitol conditions for 9 and 24 hours. *P < 0.05 versus normal controls (Wilcoxon two-sample test; n = 3).


A Effect of KDM6A knockdown on the expression of nephrin and WT - 1 in immortalized mouse podocytes cultured under high glucose conditions . Western blot analyses were performed using protein lysates of podocytes that were transfected with scrambled siRNAs or KDM6A - specific siRNAs , and cultured in high glucose ( HG ) or high mannitol ( Mann ) for 48 hours . * P < 0 . 05 versus normal controls , # P < 0 . 05 versus control siRNA with HG incubation ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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A Effect of KDM6A knockdown on the expression of nephrin and WT-1 in immortalized mouse podocytes cultured under high glucose conditions. Western blot analyses were performed using protein lysates of podocytes that were transfected with scrambled siRNAs or KDM6A-specific siRNAs, and cultured in high glucose (HG) or high mannitol (Mann) for 48 hours. *P < 0.05 versus normal controls, #P < 0.05 versus control siRNA with HG incubation (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


B Effect of KDM6A overexpression on expression levels of nephrin and WT - 1 . Immortalized podocytes were transfected with the KDM6A expression plasmid or the empty vector control for 48 hours , and the expression levels of KDM6A , nephrin and WT - 1 in these transfected podoytes were analyzed by immunoblotting . * P < 0 . 05 versus vector controls ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

B Effect of KDM6A overexpression on expression levels of nephrin and WT-1. Immortalized podocytes were transfected with the KDM6A expression plasmid or the empty vector control for 48 hours, and the expression levels of KDM6A, nephrin and WT-1 in these transfected podoytes were analyzed by immunoblotting. *P < 0.05 versus vector controls (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


C Changes in H3K27me3 levels in podocytes after treatment with high glucose , the combination of high glucose and KDM6A knockdown , or KDM6A overexpression for 48 hours . * P < 0 . 05 versus normal controls , # P < 0 . 05 versus control siRNA with HG incubation ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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C Changes in H3K27me3 levels in podocytes after treatment with high glucose, the combination of high glucose and KDM6A knockdown, or KDM6A overexpression for 48 hours. *P < 0.05 versus normal controls, #P < 0.05 versus control siRNA with HG incubation (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


D Changes in DNA methylation at nephrin gene promoter by high glucose , by the combination of high glucose plus KDM6A knockdown , or by KDM6A overexpression in immortalized podocytes . U , unmethylated - specific primers ; M , methylated - specific primers . Experiments were repeated three times and a representative gel from one experiment is shown .

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D Changes in DNA methylation at nephrin gene promoter by high glucose, by the combination of high glucose plus KDM6A knockdown, or by KDM6A overexpression in immortalized podocytes. U, unmethylated-specific primers; M, methylated-specific primers. Experiments were repeated three times and a representative gel from one experiment is shown.


E Effect of GSK - J4 ( 40 μM ) , a specific KDM6A inhibitor , on high glucose - mediated changes in levels of nephrin , WT - 1 and H3K27me3 . * P < 0 . 05 versus normal controls , # P < 0 . 05 versus untreated HG - incubated cells ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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E Effect of GSK-J4 (40 μM), a specific KDM6A inhibitor, on high glucose-mediated changes in levels of nephrin, WT-1 and H3K27me3. *P < 0.05 versus normal controls, #P < 0.05 versus untreated HG-incubated cells (Parametric ANOVA and a Bonferroni post hoc test; n = 3).