H2AX is required for the DNA damage response in human MCF10A cells. (A) H2AX promotes cellular resistance to DNA damage. Clonogenic survival assays of MCF10A H2AX+/+ and H2AX−/− cells were performed with various phleomycin concentrations as shown. n = 3 ± SEM. (B) DNA damage-induced MRE11 foci require H2AX. Cells were treated with phleomycin (60 μg/ml) for 1 h and analyzed by IF with the indicated antibodies. (C) DNA damage-dependent p53 activation requires H2AX. MCF10A H2AX+/+ and H2AX−/− cells were either untreated or were treated with phleomycin (30 μg/ml) for 2 h. After 2 h, fresh medium was used to wash out phleomycin for postrepair analysis. Samples were taken at the indicated times and analyzed by Western blotting with the antibodies as shown with both short and long exposures. Note that the long exposure of p53-S15p shows activation in MCF10A H2AX−/− cells under undamaged conditions. (D) p53 and p21 mRNA levels are increased in MCF10A H2AX−/− cells. RNA was isolated from both MCF10A H2AX+/+ and H2AX−/− cells and analyzed by RT-qPCR with primers specific for p53 and p21 and with control primers (GAPDH). Experiments were analyzed as in Fig. 5C. The graph represents data from 3 independent experiments, and error bars represent SEM. (E) DNA damage signaling is defective in MCF10A H2AX−/− cells. Experiments were performed and analyzed as in panel C with the indicated antibodies. (F) Summary of defects occurring in untreated and DNA damage-treated human MCF10A H2AX−/− cells.