ARFIP2 controls starvation-induced autophagy and the trafficking of ATG9A. (A) HEK293A CTRL or ARFIP2 KO cells (CrARFIP2; clone 1 or clone 2) were incubated in FM or ES without or with Bafilomycin A1 (EB) for 2 h before immunoblotting for ARFIP2, Actin, and LC3B. (B) Quantification of A. Statistical analysis using one-way ANOVA with Tukey’s multiple comparisons test, mean ± SEM, n = 3 experiments; **, P ≤ 0.01; ***, P ≤ 0.001. (C) CTRL or CrARFIP2 KO (clone 1) cells were incubated in ES for 2 h, fixed, and labeled using antibodies to ULK1, WIPI2, or LC3B. Scale bars, 10 µm. (D) ULK1, WIPI2, and LC3B puncta in C were counted. Statistical analysis using two-tailed unpaired Student’s t test, mean ± SEM, n = 2 experiments for ULK1 puncta and n = 3 experiments for WIPI2 and LC3B puncta, 100 cells per independent experiment; ***, P ≤ 0.001. (E) HEK293A stably expressing mRFP-ATG9A and GFP-ATG13 were transfected with iRFP-ARFIP2, incubated in ES, and imaged live by Airyscan microscopy. Scale bars, 5 µm; inset, 1 µm. (F) CTRL or CrARFIP2 KO (clone 1) cells were incubated in FM or ES for 2 h before immunostaining for ATG9A and GM130. Scale bars, 10 µm. (G) Quantification of ATG9A and GM130 colocalization in F. Mean ± SEM, n = 3 experiments, Pearson’s coefficient was measured in 30 cells per condition per independent experiment, and statistical analysis was done using one-way ANOVA with Tukey’s multiple comparisons test; ***, P ≤ 0.001.