γ-H2AX focus formation assay

The induced DSBs were quantitatively detected by immunostaining with an antibody against γ-H2AX. After exposure, the cells were fixed in 4% paraformaldehyde for 10 min on ice. The cells rinsed with phosphate buffered saline (PBS) were permeabilized in 0.2% Triton X-100 in PBS for 5 min, and blocked in 1% bovine serum albumin (BSA) in PBS for 30 min. The cells were incubated at 4°C overnight with a primary antibody against γ-H2AX (ab26350, Abcam) diluted 1:400 by a 1% BSA in PBS, and rinsed with a 1% BSA in PBS three times. Subsequently, the cells were incubated for 2 h in the dark at room temperature with Alexa Fluor 594-conjugated goat-anti-mouse IgG H&L (ab150116, Abcam) diluted 1:250 by a solution of 1% BSA in PBS, as a secondary antibody. After rinsed with a 1% BSA in PBS three times, the cells were incubated with 1 μg/ml DAPI solution (62248, Thermo Fisher Scientific) for 15 min. After rinsed with methanol, γ-H2AX foci were detected by using a High Standard all-in-one fluorescent microscope (model BZ-9000; Keyence, Osaka, Japan). The number of nuclear foci (≥80 cells) was counted by using the Image J software^32,33, as reported previously¹⁵.