Figure 6. DDR factors have impaired... | Rockefeller University Press

$DDR factors have impaired recruitment to sites of DNA damage in DUX4-expressing cells. (A) Representative immunofluorescence images of γH2A.X signal (green) and 53BP1 signal (magenta) in uninduced (−dox), treated with 10 μM etoposide for 30 min and immediately fixed (+eto), briefly induced with 2 μg/ml doxycycline for 4 h and fixed 20 h after induction (+dox), or briefly induced with 2 μg/ml doxycycline for 4 h and at 20 h after induction treated with 10 μM etoposide for 30 min and then immediately fixed (+dox +eto), (scale bar = 20 μm). White arrows indicate nuclei with γH2A.X foci that lack 53BP1 foci. The asterisk indicates nuclei with γH2A.X foci that have 53BP1 foci colocalization. Images are representative of four independent experiments conducted on separate days. (B) Quantification of fraction nuclei with 53BP1 signal overlap with γH2A.X signal are indicated for two independent experiments and N ≥ 100 nuclei per condition within each experiment. (C) Quantification of total 53BP1 protein levels in +eto, −dox, pulse, or continuous +dox conditions. N = 3 per condition; blot shows two biological replicates for each condition. (D) Representative immunofluorescence images of γH2A.X signal (green) and RAD51 signal (magenta) in −dox, +eto, or +dox iDUX4 cells, (scale bar = 20 μm). The cell cycle state for each imaged nucleus was not determined. Images are representative of three independent experiments conducted on separate days. N ≥ 100 nuclei imaged. (E) Quantification of fraction RAD51 signal overlap with γH2A.X signal in nuclei are indicated for two independent experiments and N ≥ 100 nuclei per condition within each experiment. (F) Quantification of total RAD51 protein levels in +eto, −dox, pulse, or continuous +dox conditions. Three biological replicates were used; blot shows two biological replicates for each condition. Note that Fig. S3 F uses the same GAPDH control image because the same western membrane was used to probe for RAD51 and RNF168. (G) Representative immunofluorescence images of γH2A.X signal (green) and phosphor-p53 (Ser15) signal (magenta) in −dox, +eto, or +dox iDUX4 cells, (scale bar = 20 μm). White arrows indicate nuclei with γH2A.X foci that lack phosphor-p53 foci. Asterisk indicates nuclei with γH2A.X foci that have phosphor-p53 foci colocalization. Images are representative of two independent experiments conducted on separate days. N ≥ 50 nuclei imaged. (H) Quantification of the fraction of nuclei containing phosphor-p53 that colocalize with γH2A.X foci. Fraction calculated per field of images taken from each independent experiment and N ≥ 50 nuclei. (I) Quantification of total phosphor-p53 protein levels in +eto, −dox, pulse, or continuous +dox conditions. Relative levels are normalized to total p53 levels and then to loading control (GAPDH). N = 3; Blot shows two biological replicates for each condition. (B, C, E, F, H, and I). Data represent means ± SD. Statistical differences between groups were analyzed employing one-way ANOVA Tukey’s multiple comparison test between each group and a control. Source data are available for this figure: SourceData F6.$

Figure 6.

DDR factors have impaired recruitment to sites of DNA damage in DUX4-expressing cells. (A) Representative immunofluorescence images of γH2A.X signal (green) and 53BP1 signal (magenta) in uninduced (−dox), treated with 10 μM etoposide for 30 min and immediately fixed (+eto), briefly induced with 2 μg/ml doxycycline for 4 h and fixed 20 h after induction (+dox), or briefly induced with 2 μg/ml doxycycline for 4 h and at 20 h after induction treated with 10 μM etoposide for 30 min and then immediately fixed (+dox +eto), (scale bar = 20 μm). White arrows indicate nuclei with γH2A.X foci that lack 53BP1 foci. The asterisk indicates nuclei with γH2A.X foci that have 53BP1 foci colocalization. Images are representative of four independent experiments conducted on separate days. (B) Quantification of fraction nuclei with 53BP1 signal overlap with γH2A.X signal are indicated for two independent experiments and N ≥ 100 nuclei per condition within each experiment. (C) Quantification of total 53BP1 protein levels in +eto, −dox, pulse, or continuous +dox conditions. N = 3 per condition; blot shows two biological replicates for each condition. (D) Representative immunofluorescence images of γH2A.X signal (green) and RAD51 signal (magenta) in −dox, +eto, or +dox iDUX4 cells, (scale bar = 20 μm). The cell cycle state for each imaged nucleus was not determined. Images are representative of three independent experiments conducted on separate days. N ≥ 100 nuclei imaged. (E) Quantification of fraction RAD51 signal overlap with γH2A.X signal in nuclei are indicated for two independent experiments and N ≥ 100 nuclei per condition within each experiment. (F) Quantification of total RAD51 protein levels in +eto, −dox, pulse, or continuous +dox conditions. Three biological replicates were used; blot shows two biological replicates for each condition. Note that Fig. S3 F uses the same GAPDH control image because the same western membrane was used to probe for RAD51 and RNF168. (G) Representative immunofluorescence images of γH2A.X signal (green) and phosphor-p53 (Ser15) signal (magenta) in −dox, +eto, or +dox iDUX4 cells, (scale bar = 20 μm). White arrows indicate nuclei with γH2A.X foci that lack phosphor-p53 foci. Asterisk indicates nuclei with γH2A.X foci that have phosphor-p53 foci colocalization. Images are representative of two independent experiments conducted on separate days. N ≥ 50 nuclei imaged. (H) Quantification of the fraction of nuclei containing phosphor-p53 that colocalize with γH2A.X foci. Fraction calculated per field of images taken from each independent experiment and N ≥ 50 nuclei. (I) Quantification of total phosphor-p53 protein levels in +eto, −dox, pulse, or continuous +dox conditions. Relative levels are normalized to total p53 levels and then to loading control (GAPDH). N = 3; Blot shows two biological replicates for each condition. (B, C, E, F, H, and I). Data represent means ± SD. Statistical differences between groups were analyzed employing one-way ANOVA Tukey’s multiple comparison test between each group and a control. Source data are available for this figure: SourceData F6.

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