H2AX loss affects 53BP1 focus formation in unperturbed and DNA damage-treated MCF10A cells. (A) Untreated MCF10A H2AX−/− cells accumulate 53BP1 foci. Wild-type and H2AX-deleted MCF10A cells were cultured, fixed, and analyzed by immunofluorescence (IF) with the indicated antibodies. The percentages of total cells staining positive for 53BP1 foci are shown in white. Over 100 cells from each cell line were quantified for 53BP1 foci and graphed. The numbers shown represent the numbers of 53BP1 foci, and error bars represent SEM (n = 3). Note that large 53BP1 foci in MCF10A cells contain γH2AX. (B) 53BP1 foci in untreated cells are enriched in the G1 phase of the cell cycle. Asynchronous cells were analyzed by IF with the indicated antibodies. Cyclin A staining was performed to detect S/G2-phase cells. Over 100 cells were counted for 53BP1 foci and cyclin A staining in each cell line, and error bars represent SEM (n = 3). We note that MCF10A H2AX−/− cells exhibit reduced cyclin A-positive cells and an increase in 53BP1 foci relative to MCF10A cells. (C) Impaired recruitment of MDC1 to DNA damage in MCF10A H2AX−/− cells. Cells were treated with 3 Gy IR and analyzed 60 min post-IR with the indicated antibodies. (D) DNA-damage-induced 53BP1 foci are defective in MCF10A H2AX−/− cells. Cells were irradiated and analyzed with the indicated antibodies at 15, 30, and 60 min after 3 Gy IR. Scale bars, 10 μm.