Figure 3

Examination of autophagy and inhibition of irradiation-induced autophagy in WT, Bax^−/− and PUMA^−/− HCT116 cells by 3-MA. WT, Bax^−/− and PUMA^−/− HCT116 cells were transfected with GFP-tagged LC3 plasmids. Twenty-four hours later, the transfected cells were subjected to irradiation of 8 Gy. The expression of GFP-LC3 was then examined by a fluorescence microscopy after 72 h incubation. Representative images are shown in (a) and the percentage of cells with punctate GFP-LC3 was calculated relative to all GFP-LC3 positive cells and shown in (b). GFP-LC3 puncta were pointed out by white arrows. WT, Bax^−/− and PUMA^−/− HCT116 cells that were transiently expressing GFP-LC3 were incubated in the presence or absence of 3-MA and then exposed to irradiation of 8 Gy. Seventy-two hours later, images were taken by a fluorescent microscope and the percentage of cells with punctate GFP were calculated (d and e). White arrow marks the GFP-LC3 puncta. WT, Bax^−/− and PUMA^−/− HCT116 cells were cultured with or without 3-MA and then subjected to irradiation of 8 Gy. Cell death (c) and apoptosis (f and g) were determined at 72 h after irradiation by CCK-8 kit and Annexin V-FITC staining with flow cytometry, respectively. Con, control, no irradiation. IR, irradiation. Statistical significant differences are marked as *P<0.05, **P<0.01, ***P<0.001 or ****P<0.0001.