Immunofluorescence Staining of γH2AX

The treated cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.2% Triton-X 100 for 10 min at 4°C, blocked with 3% bovine serum albumin for 30 min at room temperature, and incubated overnight at 4°C with γH2AX antibody (diluted at 1:1000). Cells were washed 3 times for 5 min in PBS and then incubated for 2 h with AlexaFluor secondary antibody (diluted 1:1000 in blocking buffer). DAPI was used to stain nuclei. γH2AX foci were visualized with a laser scanning confocal microscope (LSM 510 Meta), and the number of H2AX foci per nucleus was quantified.