PI4KIIIβ acts at initiation sites of autophagosomes in the absence of PI3P. (A) HEK293A-GFP-ATG13 were incubated in ES with or without VPS34 IN1 for 1 h, fixed, and labeled for WIPI2. Airyscan imaging. Scale bars, 5 µm; inset, 1 µm. (B) HEK293A cells stably expressing GFP-ATG13 were incubated in ES with or without VPS34 IN1 for 1 h, and the GFP-positive compartment was immunoisolated before immunoblot for GFP and PI4KIIIβ. (C) Quantification of the relative amount of PI4KIIIβ after normalization to immunoisolated GFP-ATG13. Statistical analysis using two-tailed unpaired Student’s t test, mean ± SEM, n = 3 experiments; **, P ≤ 0.01. (D) HEK293A-GFP-ATG13 cells were incubated in ES with or without VPS34 IN1 for 1 h, fixed, and labeled using antibodies to PI4P. Airyscan imaging. Scale bars, 5 µm; inset, 1 µm. Arrowheads indicate colocalized structures. (E) Quantification of GFP-ATG13 and PI4P colocalization in D; statistical analysis using one-way ANOVA with Tukey’s multiple comparisons test, mean ± SEM, n = 3 experiments, Pearson’s coefficient of 30 cells per condition per independent experiment were quantified; ***, P ≤ 0.001. (F) HEK293A-GFP-ATG13 cells were incubated in ES with or without VPS34 IN1 for 1 h, fixed, and labeled using antibodies to PI4KIIIβ. Airyscan imaging. Scale bars, 5 µm; inset, 1 µm. Arrowheads indicate colocalized structures. (G) Quantification of GFP-ATG13 and PI4KIIIβ colocalization in F; statistical analysis using one-way ANOVA with Tukey’s multiple comparisons test, mean ± SEM, n = 3 experiments, Pearson’s coefficient of 30 cells per condition per independent experiment were quantified; ***, P ≤ 0.001.