Figure 3.

VPS37A depletion accumulates ULK1, p62, and Atg5 on phagophores. (A) crNT and crVPS37A U-2 OS cells stably expressing GFP-ULK1 were incubated in complete medium or starvation medium for 2 h, stained for LC3 and p62, and analyzed by confocal microscopy. Representative images from three independent experiments are shown. Magnified images in the boxed areas are shown in the bottom panels. Arrowheads indicate GFP-ULK1 and LC3 double-positive immature autophagosomal membranes. (B) The areas of LC3-positive (top) and p62-positive (bottom) autophagic structures that were positive for GFP-ULK1 in each cell in A were quantified and shown (n = 30). Statistical significance was determined by Kruskal–Wallis one-way ANOVA on ranks followed by Dunn’s multiple comparison test. All values are mean ± SD. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001; ns, not significant. (C) GFP-ATG5–expressing crNT and crVPS37A U-2 OS cells were starved for 2 h, stained for LC3, and analyzed by confocal microscopy. Representative images from three independent experiments are shown. Magnified images in the boxed areas are shown in the right panels. Arrowheads indicate GFP-ATG5– and LC3 double-positive immature autophagosomal membranes. (D) The areas of GFP-ATG5 and LC3 double-positive autophagic structures in each cell in C are quantified and shown (n = 30). Statistical significance was determined by Student’s t test. All values are mean ± SD. ****, P ≤ 0.0001. The scale bars represent 10 µm in A and C and 1 µm in the magnified images.