Quantification of γH2AX DNA Damage Foci

LC3 puncta analysis was performed as we previously reported [34 (link)]. The Cells were seeded on coverslips in 24-well plates at a density of 5 × 10² cells per well in 1 ml of medium. After 24 h, cells were cotreated with HCQ and BKM120. After 48 h, cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in PBS. Samples were then blocked in 5% donkey serum in the presence of 0.1% Triton X-100 and stained with the primary antibody γH2AX (Abcam, ab81299). After the cells were washed three times with PBS, the secondary antibody coupled to Alexa Fluor 488 was added and incubated for 1 h at room temperature. After being rinsed and washed three times with PBS, slides were mounted using VECTASHIELD mounting medium (Vector Laboratories) containing DAPI. Cells were then visualized with Zeiss Confocal microscope LSM880 for the presence of γH2AX puncta. The puncta was quantified using ImageJ.