Cells were grown on coverslips at a density of 1×105 cells per well. After treatment with TMZ (100 μM) or additional KU0060648 (1 μM and 10 μM) for distinct time periods, cells were fixed in 4% paraformaldehyde for 10 min at room temperature and incubated in 0.1% Triton X-100 for 20 min, then blocked with 5% normal goat serum (Sigma-Aldrich). The cells were reacted with anti-γH2AX (1:500) at 4°C overnight. After that, cells were incubated with FITC- (fluorescein isothiocyanate) conjugated secondary antibodies for 1 h at 37°C. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) at a final concentration of 0.1 μg/mL. Images were visualized and recorded with a Zeiss LSM780 confocal microscope (Carl Zeiss Inc., Oberkochen, Germany).