FIGURE 5: Dram1 is sufficient to induce conventional autophagy, but is not involved in etoposide-induced conventional autophagy. (A-C) Dram1 is sufficient to induce conven-tional autophagy. (A) WT MEFs were transfected with dram1-flag or a control vector for the indicated times in the presence or absence of bafilomycin A1 (BafA; 10 nM), and the expression of each protein was analyzed by western blotting. GAPDH was included as a loading control. (B, C) GFP-LC3-expressing WT MEFs were transfected with dram1-flag or control vector for the indicated times, and then immunostained with an anti-Flag antibody (red). GFP-LC3 signals are shown in green. Representative images at 24 h are shown in (B). (C) The proportion of cells with LC3 puncta was calculated (n > 100 cells in each experiment). Data are shown as the mean  +  SD (n = 3 experiments). *p < 0.05 vs the value of “vector 24 h”. (D, E) Dram1 is not involved in etoposide-induced conventional autophagy. (D) shDram1-transfected and control WT MEFs were incubated with 10 µM etoposide for 8 h, and Dram1 expression was assessed by qPCR using mRNA. *p < 0.05. (E) The indicated WT MEFs were treated with etoposide (10 µM) for the indicated times in the presence or absence of bafilomycin A1 (10 nM), and the expression of each protein was analyzed by western blotting. GAPDH was included as a loading control.