Figure 9. DUX4 expression induces DNA... | Rockefeller University Press

$DUX4 expression induces DNA damage but fail to activate DNA damage response pathways. (A) Cell cycle flow cytometry analysis in −dox cells (iDUX4 or MB135 parentals), cells briefly induced with 2 μg/ml doxycycline for 4-h (+dox) and fixed at 24 h (iDUX4 or MB135 parentals) or 48 h (iDUX4 only), or cells treated with 0.5 μg/ml nocodazole for 24-h (control, iDUX4 only). The cell cycle was determined by BrdU-APC and 7-AAD staining. G1 accumulation in −dox cells at 24 h compared with +dox cells at 24 or 48 h after induction. N = 3. (B) Representative immunofluorescence of HSATII RNA (green) and BrdU (magenta) in induced cells 12 h after a 4-h pulse of doxycycline and 12-h pulse of BrdU leading up to fixation. Arrows indicate HSATII+ nuclei. Scale bar = 20 μm. Images are representative from three independent experiments conducted on separate days. N ≥ 100 nuclei imaged. (C) Fraction of nuclei that contain BrdU staining in either uninduced and induced cells either with no HSATII RNA (HSATII−) or with HSATII RNA (HSATII+). Dots represent independent experiments. N ≥ 100 nuclei. (D) Representative immunofluorescence of HSATII RNA (green) and BrdU (magenta) in induced cells 12 h after a 4-h pulse of doxycycline and 30-min pulse of BrdU prior to fixation. Arrows indicate HSATII+ nuclei. Scale bar = 20 μm. Images are representative of two independent experiments conducted on separate days. N ≥ 100 nuclei imaged. (E) Percent of nuclei that contain BrdU staining in induced cells either with no HSATII RNA (HSATII−) or with HSATII RNA (HSATII+). Dots represent fields taken. N ≥ 100 nuclei. (F) Immunofluorescence of γH2A.X (green) or BrdU (red) signal in −dox cells or cells briefly induced with 2 μg/ml doxycycline for 4 h (+DOX) and fixed at 24, 48, or 72 h. BrdU pulse labeling was performed on cells 30 min prior to fixation for each indicated time point. Arrows indicate nuclei with γH2A.X foci. Scale bar = 20 μm. Images are representative of three independent experiments conducted on separate days. N ≥ 100 nuclei imaged. (G) Frequency of BrdU positive (BrdU+) cells for each condition. N = 3; data are mean ± SD of experimental replicates. (H) Fraction of nuclei containing γH2A.X foci that overlap with nuclei that also contain BrdU signal. The fraction calculated represents the mean taken from each independent experiment and N ≥ 50 nuclei. (I) Western blot was performed on whole cell lysate and probed for various NHEJ and HR DNA damage response factors. GAPDH was used as the loading control. iDUX4 cells were either untreated (no etoposide) and uninduced (−dox), briefly treated with 10 μM etoposide for 4 h and harvested immediately following treatment or at 24-h timepoint intervals (24, 48, or 72 h), or briefly induced with 2 μg/ml doxycycline for 4 h and harvested immediately following treatment or at 24-h timepoint intervals (24, 48, or 72 h). The numbers indicated above phosphor-blots are phosphorylation signals relative to total protein normalized to loading control. Shown is a representative blot of three biological replicates. (C, E, and G) 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 F9.$

Figure 9.

DUX4 expression induces DNA damage but fail to activate DNA damage response pathways. (A) Cell cycle flow cytometry analysis in −dox cells (iDUX4 or MB135 parentals), cells briefly induced with 2 μg/ml doxycycline for 4-h (+dox) and fixed at 24 h (iDUX4 or MB135 parentals) or 48 h (iDUX4 only), or cells treated with 0.5 μg/ml nocodazole for 24-h (control, iDUX4 only). The cell cycle was determined by BrdU-APC and 7-AAD staining. G1 accumulation in −dox cells at 24 h compared with +dox cells at 24 or 48 h after induction. N = 3. (B) Representative immunofluorescence of HSATII RNA (green) and BrdU (magenta) in induced cells 12 h after a 4-h pulse of doxycycline and 12-h pulse of BrdU leading up to fixation. Arrows indicate HSATII+ nuclei. Scale bar = 20 μm. Images are representative from three independent experiments conducted on separate days. N ≥ 100 nuclei imaged. (C) Fraction of nuclei that contain BrdU staining in either uninduced and induced cells either with no HSATII RNA (HSATII−) or with HSATII RNA (HSATII+). Dots represent independent experiments. N ≥ 100 nuclei. (D) Representative immunofluorescence of HSATII RNA (green) and BrdU (magenta) in induced cells 12 h after a 4-h pulse of doxycycline and 30-min pulse of BrdU prior to fixation. Arrows indicate HSATII+ nuclei. Scale bar = 20 μm. Images are representative of two independent experiments conducted on separate days. N ≥ 100 nuclei imaged. (E) Percent of nuclei that contain BrdU staining in induced cells either with no HSATII RNA (HSATII−) or with HSATII RNA (HSATII+). Dots represent fields taken. N ≥ 100 nuclei. (F) Immunofluorescence of γH2A.X (green) or BrdU (red) signal in −dox cells or cells briefly induced with 2 μg/ml doxycycline for 4 h (+DOX) and fixed at 24, 48, or 72 h. BrdU pulse labeling was performed on cells 30 min prior to fixation for each indicated time point. Arrows indicate nuclei with γH2A.X foci. Scale bar = 20 μm. Images are representative of three independent experiments conducted on separate days. N ≥ 100 nuclei imaged. (G) Frequency of BrdU positive (BrdU+) cells for each condition. N = 3; data are mean ± SD of experimental replicates. (H) Fraction of nuclei containing γH2A.X foci that overlap with nuclei that also contain BrdU signal. The fraction calculated represents the mean taken from each independent experiment and N ≥ 50 nuclei. (I) Western blot was performed on whole cell lysate and probed for various NHEJ and HR DNA damage response factors. GAPDH was used as the loading control. iDUX4 cells were either untreated (no etoposide) and uninduced (−dox), briefly treated with 10 μM etoposide for 4 h and harvested immediately following treatment or at 24-h timepoint intervals (24, 48, or 72 h), or briefly induced with 2 μg/ml doxycycline for 4 h and harvested immediately following treatment or at 24-h timepoint intervals (24, 48, or 72 h). The numbers indicated above phosphor-blots are phosphorylation signals relative to total protein normalized to loading control. Shown is a representative blot of three biological replicates. (C, E, and G) 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 F9.

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