ATLs and VAPs act partially redundantly in mediating the formation of FIP200/ULK1 puncta. (A and B) Compared with ATL2/3 DKO COS-7 cells (A), the number of LC3 puncta is further decreased by simultaneous KD of VAPA/B (B). Scale bars, 5 µm. (C) Immunoblotting assays showing LC3 levels in control and ATL2/3 DKO COS-7 cells with or without KD of VAPA/B. Quantifications of LC3-I and LC3-II/LC3-I ratios are also shown (normalized by actin level). The level of LC3-I and the ratio of LC3-II/LC3-I in control cells is set to 1.00. (D and E) Compared with ATL2/3 DKO COS-7 cells (D), the number of ULK1-GFP puncta is much lower after simultaneous KD of VAPA/B (E). Scale bars, 5 µm. (F–K) Compared with ATL2/3 DKO COS-7 cells (F, H, and J), the numbers of ATG101 puncta, FIP200 puncta, and ATG13 puncta are dramatically decreased by simultaneous KD of VAPA/B (G, I, and K). Scale bars, 5 µm. (L) Quantitative data for A and B and D–K are shown in L as mean ± SEM (n = 32 cells in A and B, n = 26 cells in D, n = 21 cells in E, n = 20 cells in F–K). ***, P < 0.001. (M–Q) EM micrographs showing autophagic structures in ATL2/3 DKO COS-7 cells (M and O) and ATL2/3 DKO cells with simultaneous depletion of VAPA/B (N and P) after 1 h of starvation (M and N) or after Bfa.A1 treatment (O and P). Arrowheads indicate autophagosomes. Quantifications of the numbers of autophagosomes are shown in Q as mean ± SEM (n = 50 sections in each group, one section for each cell). *, P < 0.05; **, P < 0.01. Scale bars, 0.5 µm. ctrl, control; NC, negative control; Strv, starved.