Fig. 4. Localization of Wipi3 and GS15 on isolated autophagic membranes.

a Successful fractionation of trans-Golgi membranes. Atg5^KO MEFs were treated with etoposide (10 µM), lysed in isotonic buffer, and fractionated by sucrose-gradient centrifugation. The expression of each molecules were analyzed by western blotting. Detailed data are shown in Supplementary Fig. 12. b, c Representative electron micrographs of fraction 5 from untreated and etoposide-treated Atg5^KO MEFs. Arrowheads indicate autophagic vacuoles. Representative isolation membrane (IM)-like and autophagic structures are shown in the right panels. Bars = 0.2 µm. d The populations of IM-like and autophagic structures per total structures were calculated. Data are shown as the mean ± SEM (NT: n = 25 structures; Etoposide: n = 122 structures). e Immunoelectron micrographs of fraction 5 from etoposide-treated Atg5^KO MEFs. HA-GS15 and Flag-Wipi3 were recognized by 15-nm gold-conjugated anti-HA antibodies and 10-nm gold-conjugated anti-Flag antibodies, respectively. Bars = 0.2 µm. f Association of Wipi3 with PI3P in etoposide-treated Atg5^KO MEFs. Atg5^KO/Wipi3^Cr MEFs expressing Flag-Wipi3 and its mutants together with mCherry-FYVE were left untreated or were treated with etoposide (10 µM). Green and red signals indicate Flag-Wipi3 derivatives and PI3P, respectively. Bars = 10 µm. Magnified images are shown in the right panels. Bars = 2 µm. g, h The indicated MEFs were treated with etoposide (10 µM; 18 h), and were stained with an anti-Lamp2 antibody. In (g), Bars = 10 µm. Magnified images are shown in the inset. Bars = 1 µm. Ring-like Lamp2 puncta were autolysosomes. h The populations of MEFs with ring-like Lamp2 puncta were calculated. Data are shown as the mean ± SD (n > 200 cells examined over 3 independent experiments). In (d, h), comparisons were performed using one-way ANOVA followed by the Tukey post hoc test. In (d), *p < 0.01 vs the value of untreated MEFs (IM-like: exact p value is too small [p < 0.0001], autophagic vacuole: p = 0.0083). In (h), *p < 0.01 vs the value of etoposide-treated Atg5^KO MEFs (exact p value is too small [p < 0.0001]). Source data are provided as a Source Data file.