A23240

Cells were seeded into a 24‐well plate containing coverslips before receiving IR. Twelve hours after 5 Gy radiotherapy, the cells were harvested, fixed with 1% paraformaldehyde, permeabilized with 0.5% Triton, blocked with 5% BSA and then incubated with primary antibodies at 4°C overnight. The primary antibodies were anti‐double stranded DNA (dsDNA; 1:200, ab27156, Abcam), anti‐cGAS (1:200, #79978, Cell Singling Technology) and anti‐NF‐κB P65 (1:200, A2547, ABclonal, Wuhan, Hubei, China). The next day, the cells were incubated with secondary antibodies for 1 h at room temperature followed by 2‐(4‐Amidinophenyl)‐6‐indolecarbamidine dihydrochloride (DAPI; Abbkine, Beijing, China) staining for 5 min. The secondary antibodies were Cy3, goat anti‐rabbit IgG (1:500, A22220, Abbkine, Beijing, China), DyeLight 488 goat anti‐mouse IgG (1:500, A23240, Abbkine), and DyeLight 488 goat anti‐rabbit IgG (1:500, A23220, Abbkine). Cover slides were then mounted for microscopy. For terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, sections were stained using the TUNEL staining kit (11684817910, Roche, Germany) according to the manufacturer's protocols. For the micronuclei assay, cells were stained with DAPI with antifade reagent for microscopy and scoring. At least 150 nuclei were counted in 10 fields.