Nuclei with PRC1 aggregation, HSATII RNA accumulation, and H2AUb depletion have increased incidence of DNA damage. (A) Representative immunofluorescence images of γH2A.X signal (green) in uninduced (−dox; no DUX4 expression) or induced with 2 μg/ml doxycycline for 24-h (+dox; DUX4-expressing) iDUX4 cells (scale bar = 20 μm). Images are representative of four independent experiments conducted on separate days. (B) Percent γH2A.X positive (containing at least 5 γH2A.X foci) nuclei are indicated for three independent experiments. N ≥ 100 random nuclei per condition within each experiment. (C) Western blot was performed on whole-cell lysate and probed for phosphorylated H2A.X at Serine 139. GAPDH was used as the loading control. iDUX4 cells were either uninduced (−dox), briefly induced with 2 μg/ml doxycycline for 4-h (pulse) and harvested 20 h after induction or induced with 2 μg/ml doxycycline for 24 h (continuous, “cont.”). The blot shows two biological replicates. (D) Combined RNA-FISH and immunofluorescence of γH2A.X (red) and HSATII RNA (green) in −dox or +dox (4-h pulse and fixed/analyzed 20 h after induction) iDUX4 cells (scale bar = 20 μm). Images are representative of two independent experiments conducted on separate days. N ≥ 100 nuclei imaged. (E) Percentage of cells with HSATII RNA accumulation (pos) or no HSATII RNA (neg) that contain γH2A.X foci within +dox only condition. Dots represent the mean number of nuclei per field. (F) Representative immunofluorescence images of γH2A.X signal (green) and H2AK119Ub (H2AUb) signal (red) in iDUX4 cells. Cells were either uninduced (−dox), briefly induced with 10 μM etoposide for 30 min and immediately harvested (+eto), briefly induced with 2 μg/ml doxycycline for 4 h (pulse) and harvested 20 h after induction (+dox), or pulsed and treated with etoposide 30 min before harvest (+dox +eto) (scale bar = 10 μm). White arrows indicate nuclei that contain γH2A.X signal but low/negative H2AUb signal. Asterisks indicate nuclei that contain γH2A.X and H2AUb signal. Images are representative from two independent experiments conducted on separate days. (G) Nuclear H2AUb mean fluorescence intensity (MFI) was calculated for all γH2A.X positive nuclei. Nuclei are indicated for each independent experiment and N = 50–100 random nuclei per condition for the representative experiment. (H) Percentage of cells with no H2AUb signal (neg) or cells with H2AUb signal (pos) that contain γH2A.X foci within +dox only condition. Dots represent mean number of nuclei per field. (I) Representative immunofluorescence images of γH2A.X signal (green) and RNF2 signal (magenta) in −dox, or +dox (4 h pulse and fixed/analyzed 20 h after induction) iDUX4 cells, (scale bar = 20 μm). Images are representative of two independent experiments conducted on separate days. (J) Percentage of cells with RNF2 signal type (pan versus foci) that contain γH2A.X foci within +dox only condition. Dots represent the mean number of nuclei per field. (K) Phospho-H2A.X signal intensity measured within nuclei in −dox, +dox cells with RNF2 pan-nuclear signal (pan) or RNF2 foci signal, compared to γH2A.X signal intensity measured within RNF2 foci or randomly drawn ROI within the nucleoplasm in +dox RNF2 foci+ nuclei. Each dot represents either individual nuclei or individual foci, respectively. Nuclei are indicated for representative experiment and N = 10–50 nuclei per condition or N ≥ 40 ROI. (B, C, E, G, H, J, and K) Data represent means ± SD. Statistical differences between groups were analyzed employing either two-tailed paired t test or were assessed with one-way ANOVA Tukey’s multiple comparison test between each group and a control. Source data are available for this figure: SourceData F5.
Figure 5.

Nuclei with PRC1 aggregation, HSATII RNA accumulation, and H2AUb depletion have increased incidence of DNA damage. (A) Representative immunofluorescence images of γH2A.X signal (green) in uninduced (−dox; no DUX4 expression) or induced with 2 μg/ml doxycycline for 24-h (+dox; DUX4-expressing) iDUX4 cells (scale bar = 20 μm). Images are representative of four independent experiments conducted on separate days. (B) Percent γH2A.X positive (containing at least 5 γH2A.X foci) nuclei are indicated for three independent experiments. N ≥ 100 random nuclei per condition within each experiment. (C) Western blot was performed on whole-cell lysate and probed for phosphorylated H2A.X at Serine 139. GAPDH was used as the loading control. iDUX4 cells were either uninduced (−dox), briefly induced with 2 μg/ml doxycycline for 4-h (pulse) and harvested 20 h after induction or induced with 2 μg/ml doxycycline for 24 h (continuous, “cont.”). The blot shows two biological replicates. (D) Combined RNA-FISH and immunofluorescence of γH2A.X (red) and HSATII RNA (green) in −dox or +dox (4-h pulse and fixed/analyzed 20 h after induction) iDUX4 cells (scale bar = 20 μm). Images are representative of two independent experiments conducted on separate days. N ≥ 100 nuclei imaged. (E) Percentage of cells with HSATII RNA accumulation (pos) or no HSATII RNA (neg) that contain γH2A.X foci within +dox only condition. Dots represent the mean number of nuclei per field. (F) Representative immunofluorescence images of γH2A.X signal (green) and H2AK119Ub (H2AUb) signal (red) in iDUX4 cells. Cells were either uninduced (−dox), briefly induced with 10 μM etoposide for 30 min and immediately harvested (+eto), briefly induced with 2 μg/ml doxycycline for 4 h (pulse) and harvested 20 h after induction (+dox), or pulsed and treated with etoposide 30 min before harvest (+dox +eto) (scale bar = 10 μm). White arrows indicate nuclei that contain γH2A.X signal but low/negative H2AUb signal. Asterisks indicate nuclei that contain γH2A.X and H2AUb signal. Images are representative from two independent experiments conducted on separate days. (G) Nuclear H2AUb mean fluorescence intensity (MFI) was calculated for all γH2A.X positive nuclei. Nuclei are indicated for each independent experiment and N = 50–100 random nuclei per condition for the representative experiment. (H) Percentage of cells with no H2AUb signal (neg) or cells with H2AUb signal (pos) that contain γH2A.X foci within +dox only condition. Dots represent mean number of nuclei per field. (I) Representative immunofluorescence images of γH2A.X signal (green) and RNF2 signal (magenta) in −dox, or +dox (4 h pulse and fixed/analyzed 20 h after induction) iDUX4 cells, (scale bar = 20 μm). Images are representative of two independent experiments conducted on separate days. (J) Percentage of cells with RNF2 signal type (pan versus foci) that contain γH2A.X foci within +dox only condition. Dots represent the mean number of nuclei per field. (K) Phospho-H2A.X signal intensity measured within nuclei in −dox, +dox cells with RNF2 pan-nuclear signal (pan) or RNF2 foci signal, compared to γH2A.X signal intensity measured within RNF2 foci or randomly drawn ROI within the nucleoplasm in +dox RNF2 foci+ nuclei. Each dot represents either individual nuclei or individual foci, respectively. Nuclei are indicated for representative experiment and N = 10–50 nuclei per condition or N ≥ 40 ROI. (B, C, E, G, H, J, and K) Data represent means ± SD. Statistical differences between groups were analyzed employing either two-tailed paired t test or were assessed with one-way ANOVA Tukey’s multiple comparison test between each group and a control. Source data are available for this figure: SourceData F5.

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