γh2ax

Two thousand cells were seeded on a 10-hole slide and incubated for 24 h. The cells were treated with radiation (4 Gy) and allowed to react for 1, 3, or 6 h. Cells were fixed with 4% paraformaldehyde and blocked with 5% donkey serum (Sigma, St. Louis, MO, USA) at room temperature for 1 h. The antibody for the phosphorylated form of H2A histone family member X (γH2AX (Cell signaling Technology, Danvers, MA, USA)), was incubated overnight at 4 °C. Cells were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (Life Technologies Corporation, Carlsbad, CA, USA) and 4′,6-diamidino-2-phenylindole (DAPI). After mounting with Vectashield H-1200 (Vector Laboratories, Burlingame, CA, USA), the number of γH2AX foci was measured using a confocal microscope (LSM 900, Carl Zeiss, Oberkochen, Germany).

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.