Persistent DNA damage induces replication stress and genomic instability DUX4-expressing myoblast cells. (A) Flow cytometry analysis of TUNEL experiments in untreated (no etoposide or dox), briefly treated with 10 μM etoposide for 4 h only or for 4 h and assayed at 24-h timepoint intervals (24, 48, or 72 h), or briefly induced with 2 μg/ml doxycycline for 4 h and assayed at 24-h timepoint intervals (24, 48, or 72 h). Intracellular BrdU fluorescence (BrdU-APC) was measured indicating DNA breaks. N = 3. (B) Paired protein samples from A. Western blot was performed on whole cell lysate and probed for activated (p-RPA) and total RPA. GAPDH was used as the loading control. The numbers indicated above phosphor-RPA blot is a phosphorylation signal relative to total RPA normalized to loading control. Shown is a representative blot of three biological replicates. (C) Proximity-ligation assay (PLA) performed using antibodies from two different species targeting PCNA or γH2A.X (green signal indicates proximity = PLA foci) in −dox or +dox iDUX4 cells, (scale bar = 10 μm). Images are representative from two independent experiments conducted on separate days. (D) Number of PLA foci per nucleus in either −dox or +dox iDUX4 cells. Nuclei are indicated for each independent experiment and N ≥ 60 nuclei per condition. (E and F) Representative images and ratio data from DNA fiber assay. DNA was labeled 24 or 48 h after a 4-h pulse of doxycycline, and the lengths of red and green segments were measured to calculate the ratio of each DNA fiber. N > 300 fibers. (G) Representative images of micronuclei in +dox cells compared to uninduced (−dox), (scale bar = 5 μm in −dox or 10 μm in +dox). Images are representative of two independent experiments conducted on separate days. (H) Percent cells with micronuclei in −dox cells or briefly induced with 2 μg/ml doxycycline for 4 h and fixed at 24-h time-point intervals (24, 48, or 72 h). Percent calculated represents the mean taken from each independent experiment and N ≥ 100 nuclei. (D–F and H) Data represent means ± SD. Statistical differences between groups were analyzed employing nonparametric Mann–Whitney test in the absence of normal distribution or one-way ANOVA Dunnett’s multiple comparison test between each group and a control. Source data are available for this figure: SourceData F10.
Figure 10.

Persistent DNA damage induces replication stress and genomic instability DUX4-expressing myoblast cells. (A) Flow cytometry analysis of TUNEL experiments in untreated (no etoposide or dox), briefly treated with 10 μM etoposide for 4 h only or for 4 h and assayed at 24-h timepoint intervals (24, 48, or 72 h), or briefly induced with 2 μg/ml doxycycline for 4 h and assayed at 24-h timepoint intervals (24, 48, or 72 h). Intracellular BrdU fluorescence (BrdU-APC) was measured indicating DNA breaks. N = 3. (B) Paired protein samples from A. Western blot was performed on whole cell lysate and probed for activated (p-RPA) and total RPA. GAPDH was used as the loading control. The numbers indicated above phosphor-RPA blot is a phosphorylation signal relative to total RPA normalized to loading control. Shown is a representative blot of three biological replicates. (C) Proximity-ligation assay (PLA) performed using antibodies from two different species targeting PCNA or γH2A.X (green signal indicates proximity = PLA foci) in −dox or +dox iDUX4 cells, (scale bar = 10 μm). Images are representative from two independent experiments conducted on separate days. (D) Number of PLA foci per nucleus in either −dox or +dox iDUX4 cells. Nuclei are indicated for each independent experiment and N ≥ 60 nuclei per condition. (E and F) Representative images and ratio data from DNA fiber assay. DNA was labeled 24 or 48 h after a 4-h pulse of doxycycline, and the lengths of red and green segments were measured to calculate the ratio of each DNA fiber. N > 300 fibers. (G) Representative images of micronuclei in +dox cells compared to uninduced (−dox), (scale bar = 5 μm in −dox or 10 μm in +dox). Images are representative of two independent experiments conducted on separate days. (H) Percent cells with micronuclei in −dox cells or briefly induced with 2 μg/ml doxycycline for 4 h and fixed at 24-h time-point intervals (24, 48, or 72 h). Percent calculated represents the mean taken from each independent experiment and N ≥ 100 nuclei. (D–F and H) Data represent means ± SD. Statistical differences between groups were analyzed employing nonparametric Mann–Whitney test in the absence of normal distribution or one-way ANOVA Dunnett’s multiple comparison test between each group and a control. Source data are available for this figure: SourceData F10.

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