Figure 6: Dram1 functions downstream of Ulk1 in alternative autophagy. (A) An electron micrograph of etoposide-treated Atg5 KO MEFs treated with shUlk1 for 18 h. None of the autophagic structures were observed. Bar = 5 µm. (B) Suppression of Ulk1-enhanced alternative autophagy by shDram1. shDram1-transfected and control Atg5 KO MEFs were transfected with ulk1 (1 µg) or a control vector for 24 h. The cells were then treated with etoposide (10 µM) for 8 h and analyzed for immunofluorescence of Lamp2. Percentages of cells with punctate Lamp2 immunofluorescence are shown as the mean + SD (n = 4). *p < 0.05 vs the value of “shControl (−)”. (C) Normal activation of Ulk1 in ShDram1-transfected Atg5 KO MEFs. shDram1-transfected and control Atg5 KO MEFs were treated with etoposide (10 µM) for the indicated times. The expression level of each protein was analyzed by western blotting. GAPDH was included as a loading control. Ulk1 upregulation was equivalently observed irrespective of the Dram1 silencing.