Fig. 2

KO MEFs accumulate elevated levels of DNA damage in response to IR and ETO treatment. A Representative images of γH2AX foci staining. Elp1^flox/flox MEFs were infected with Ctrl or Cre-expressing lentivirus. Cells were either non-irradiated (0 h) or treated with 4 Gy of IR at D5, before being returned to normal culture conditions for a further 1, 4 or 24 h. Cells were then fixed and an immunofluorescence assay was performed using antibodies against γH2AX (green). Nuclei were stained with DAPI (blue). Scale bars, 25 µm. B Quantification of the number of cells with more than 10 γH2AX-positive foci per cell. For each time-point, at least 200 cells per group were counted. Results represent the average of three independent experiments. C Representative images of cells analyzed by comet assay. Ctrl and KO MEFs were either non-irradiated (0 h) or exposed to 4 Gy of IR, and then 24 h later they were subjected to comet assay. Arrows indicate genomic DNA migrating away from its original location. Scale bars, 50 µm. D Quantification of DNA damage was performed by comet assay. We measured the parameters of % tail DNA (left panel) and tail moment (right panel). Data shown are from three independent experiments, and at least 50 comets per group were measured per experiment. E, F Detection of DNA damage by staining for γH2AX foci. Ctrl and KO MEFs were either untreated (no treatment) or treated with 40 μM of ETO for 2, 16 or 24 h, and then subjected to γH2AX immunofluorescence staining. Representative images are shown in (E), and γH2AX intensity per cell is summarized in (F). γH2AX intensity per nucleus was analyzed using Image J software. The images of at least 200 cells per group were taken and used to quantify γH2AX intensity. Scale bars, 10 µm. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 compared to Ctrl cells using Student’s t-test