DNA damage signal γH2AX in human keratinocytes peaks at metaphase, where it depends on both ATM and ATR. (a) Expression of γH2AX (red) is highest in metaphasic exponentially proliferating keratinocytes treated with DMSO vehicle only (CT, arrowheads). This localization is present upon a single 24-h treatment with ATRi, ATMi, or combined ATMi/DNA-PKi, but is inhibited by combined ATRi/ATMi or by ATRi/ATMi/DNA-PKi. (b) FC analyses of DNA content (PI) and DNA damage (γH2AX) in primary keratinocytes treated with the genotoxic agent DOXO (right) for 24 h. γH2AX-positive cells according to negative isotype antibody (red). DMSO vehicle as CT (left). (c and d) Double IF for γH2AX (red) and 53BP1 (green) in keratinocytes. In c, the three patterns of 53BP1: (i) diffuse in absence of significant DNA damage (arrowhead), (ii) granulated when acute DNA damage (arrow), and (iii) large spots corresponding to unrepaired NBs (thin arrowheads). Note that 53BP1 NBs colocalize with γH2AX spots (arrows in d). Scale bar, 50 µm. Nuclear DNA by DAPI. This figure complements Fig. 1 and Fig. 2.