Figure 5

Autophagic cell death in Bid^−/− MEFs in response to irradiation. (a) WT and Bid^−/− MEFs were subjected to irradiation of the indicated doses. Seventy-two hours later, cell death was measured by CCK-8 kit and shown as means±S.D. (b) WT and Bid^−/− MEFs were exposed to 10 Gy of irradiation, and at 72 h after irradiation, apoptosis was assessed by Annexin-V and PI staining with flow cytometry. Representative flow cytometric plots are shown. (c) Quantification of apoptosis was shown as means±S.D. (d and e) WT and Bid^−/− MEFs were transfected with GFP-LC3 plasmids, and 24 h later, the transfected cells were subjected to irradiation of 10 Gy. After 72 h incubation, the expression of GFP-LC3 was examined by a fluorescent microscopy and the percentage of cells with punctate GFP was calculated. White arrow marks the GFP-LC3 puncta. (f) WT and Bid^−/− MEFs were incubated with or without 3-MA and then subjected to irradiation of 10 Gy, and cell death was determined 72 h later by CCK-8 kit. (h and j) Apoptosis was also measured by Annexin V-FITC staining with flow cytometry. (g and i) WT and Bid^−/− MEFs that were transiently expressing GFP-LC3 were cultured in the presence or absence of 3-MA and then exposed to irradiation of 10 Gy. Seventy-two hours after irradiation, images were taken and the percentage of cells with punctate GFP was calculated. White arrow marks the GFP-LC3 puncta. IR, irradiation; NT, non-treatment. Statistical significant differences are marked as *P<0.05 or **P<0.01.