Quantifying Autophagy Flux via LC3 Puncta

LC3 puncta analysis was performed as we previously reported [32 (link)]. The GFP-LC3 plasmid or GFP-RFP-LC3 plasmid was transiently transfected into the cells using Lipofectamine 3000 according to the manufacturer’s instructions with slight modification. After incubation overnight, the medium was replaced with fresh growth medium. The cells were incubated with HCQ and then exposed to BKM120 for 48 h. Cells were collected, washed with PBS, and fixed in 4% paraformaldehyde for 30 min at room temperature. Then, the slides were processed and kept in the dark until analysis. Cells were then visualized with Zeiss Confocal microscope LSM880 using 488 nm and 643 nm channels for the presence of GFP-LC3 puncta and RFP-LC3 puncta.