FIGURE 2: Inhibition of alternative autophagy in etoposide-treated Atg5 KO MEFs by an shRNA for Dram1. (A, B) Dram1 expression was assessed by qPCR using mRNA. (A) Atg5 KO MEFs and Atg5/p53 DKO MEFs were incubated with 10 µM etoposide for the indicated times. *p < 0.05 vs the value of “Atg5 KO 0 h”. (B) shDram1-transfected and control Atg5 KO MEFs were incubated with 10 µM etoposide for the indicated times. *p < 0.05 vs the value of “shControl 0 h”. (C, D) Electron micrograph of shDram1-transfected Atg5 KO MEFs and control Atg5 KO MEFs treated with etoposide (10 µM) for 18 h. Blue, yellow, and red arrows indicate autoly-sosomes, autophagosomes, and isolation membranes, respectively. “G” indicates Golgi membranes. In (C), bar = 2 µm. (D) The number of each type of autophagic structure appearing in Atg5 KO MEFs treated with etoposide. shDram1-transfected and control Atg5 KO MEFs were incubated with 10 µM etoposide for 18 h, and the number of autophagic vacuoles per cell was counted on the EM photographs. White and black columns represent the number of autophagic structures in control and shDram1-transfected Atg5 KO MEFs, respectively. Data are the mean + SD obtained from 10 cells. *p < 0.05 vs the value of “shControl”. (E, F) Keima analysis indicated the requirement of Dram1 in etoposide-induced alternative autophagy. The indicated MEFs were treated with etoposide (10 µM) for 12 h. Alternative autophagy was then analyzed using Keima. (E) Keima signals (red) were merged with images obtained from phase-contrast microscopy at 12 h. (F) The extent of Keima fluorescence is shown as the mean + SD (n = 4). *p < 0.05 vs the value of “shControl 0 h”. (G) Electron micrograph of shDram1-transfected Atg5 KO MEFs treated with etoposide (10 µM) for 18 h. Red arrows indicate isolation membranes. “G” indicates Golgi membranes. Bar = 0.5 µm.