Figure 4

Autophagic flux in WT and PUMA^−/− HCT116 cells. (a) WT and PUMA^−/− HCT116 cells were subjected to irradiation of 8 Gy and proteins were extracted 72 h later. The protein level of LC3-I/II and p62 were determined by western blotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. (b) WT and PUMA^−/− HCT116 cells were exposed to irradiation of 8 Gy in the presence of 3-MA, and proteins were collected 72 h later for the determination of LC3-I/II and p62 protein level. GAPDH was used as internal control. (c) mCherry-GFP-LC3 plasmids were transfected into WT and PUMA^−/− HCT116 cells before the treatment by 3-MA, and then the cells were subjected to irradiation of 8 Gy. After 72 h incubation, images were taken by fluorescent microscope and mCherry and/or GFP puncta are pointed by white arrows.