Fig. 4: Atg7-PDCD4 pathway saves energy to reduce apoptosis.

a HCT116 cells were transfected with Flag-Atg7, Flag-Atg7^D476AQ398A or control vector. Intracellular ATP levels were detected after 2DG (5 mM) and oligomycin (2.5 μM) treatment for four hours. Data represent the mean ± SD of four independent experiments (n = 4). P values were calculated by t test. *P < 0.05. b HCT116 cells or shPDCD4 cells were transfected with si-Atg7 or NC control. Intracellular ATP levels were detected after 2DG (5 mM) and oligomycin (2.5 μM) treatment for four hours. Data represent the mean ± SD of three independent experiments (n = 3). P values were calculated by t test. *P < 0.05. c HEK293 cells were transfected with Flag-Atg7, Flag-Atg7^D476AQ398A or control vector. Cells were labeled with fluorescent substrates for Caspase 3 (green) and mounted with DAPI. Then, they were visualized under a microscope. Data represent the mean ± SD of six independent experiments (n = 100 cells). P values were calculated by t test. *p < 0.05. Scale bar, 5 μm. d HCT116 cells or shPDCD4 cells were transfected with si-Atg7 or NC control. Cells were labeled with fluorescent substrates for Caspase 3 (green) and mounted with DAPI. Then, they were visualized under a microscope. Data represent the mean ± SD of five independent experiments (n = 100 cells). P values were calculated by t test. ****p < 0.0001. Scale bar, 5 μm. e HEK293 cells were transfected with Flag-Atg7, Flag-Atg7^D476AQ398A or control vector. After 2DG (5 mM) and oligomycin (2.5 μM) treatment for 24 h, a western blot was performed to detect PDCD4, Atg7, p-AMPKα Thr172, AMPKα, Caspase-3, and cleaved-PARP. The band intensities were quantified by gray values (n = 3). P values were calculated by t test. *P < 0.05. f HEK293 cells were transfected with Flag-Atg7, Flag-Atg7^D476AQ398A or control vector. After 2DG (5 mM) and oligomycin (2.5 μM) treatment for 72 h, the apoptosis was analyzed with FACS. Data represent the mean ± SD of three independent experiments (n = 3). P values were calculated by t test. *P < 0.05, **p < 0.005. g HCT116 cells were transfected with Flag-Atg7 or control. shPDCD4 cells were transfected with si-Atg7 or NC control. After 2DG (5 mM) and oligomycin (2.5 μM) treatment for 12 h, a western blot was performed to detect PDCD4, Atg7, p-AMPKα Thr172, AMPKα, Caspase-3, and cleaved-PARP. The band intensities were quantified by gray values (n = 3). P values were calculated by t test. *P < 0.05, **p < 0.005. h HCT116 cells were transfected with Flag-Atg7 or control. shPDCD4 cells were transfected with si-Atg7 or NC control. After 2DG (5 mM) and oligomycin (2.5 μM) treatment for 48 h, the apoptosis was analyzed with FACS. Data represent the mean ± SD of four independent experiments (n = 4). P values were calculated by t test. *P < 0.05, **p < 0.005.