Fig. 4: The VPS37A UEVL domain is indispensable for autophagosome closure.
c–i VPS37A KO U-2 OS cells were stably transduced with the indicated GFP-tagged VPS37A constructs. a Overlay of ten lowest-energy NMR structures of VPS37A21–148. The unstructured region of residues 133 to 148 is not shown. N and C indicate N- and C- terminals, respectively. b Diagram of VPS37A and mutants for in vivo study. c Immunoblot analysis of whole-cell lysates (input) and immunoprecipitates (GFP-Trap) from the indicated cells. d Confocal images of cells that were transfected with the indicated siRNAs for 45 h and starved for 3 h. Magnified images in the arrowhead-indicated areas are shown in the bottom panels. Scale bars represent 10 μm, and 1 μm in the magnified images. e Quantification of GFP-VPS37A-positive and CHMP4B-positive foci area per cell in d (n ≥ 60) with one-way ANOVA followed by Tukey’s multiple comparisons. f Confocal images of cells that were transduced with HT-LC3, starved for 3 h in the presence or absence of BafA1, and subjected to the HT-LC3 autophagosome completion assay using the indicated HaloTag ligands. Scale bars represent 10 μm. g Quantification of the MIL/MPL fluorescence intensity ratio for each cell in the starvation plus BafA1 treatment group (n = 50) with the Kruskal–Wallis test followed by Dunn’s multiple comparisons. The data shown are relative to the mean of GFP-WT-expressing cells. h Immunoblot analysis of cells that were starved for 3 h in the presence or absence of BafA1. i Dot plots of LC3-II and p62 degradation ratios relative to GFP-WT-expressing cells in h (n = 3) with one-way ANOVA followed by Tukey’s multiple comparisons. All values in the graphs are mean ± SD. ns, not significant.
