Fig. 4: GRAMD1C regulates autophagy initiation during amino acid starvation.

a U2OS cells were transfected with siRNA against control (siNC) or GRAMD1C for 72 h prior to starvation in EBSS for 1 h or incubation in DMEM (control). The cells were then fixed, immunostained with antibodies against ATG16L1, ATG13 or WIPI2 and subjected to wide-field microscopy. Scale bar = 20 µm. The number of b ATG16L1, c ATG13, and d WIPI2 puncta per cell were quantified and normalized to siNC control from n = 3 independent experiments, >500 cells per condition. Significance was determined using two-way ANOVA followed by Tukey’s comparison test. Error bar = SEM. P value (ATG16L1) = 0.0441, 0.0042, and 0.008, P value (ATG13) = 0.0013 and <0.0001, P value (WIPI2) = 0.0014 and <0.0001. e Wild-type (Wt) or GRAMD1C knockout (GKO) U2OS cells were starved or not in EBSS for 1 h before immunostaining for ATG16L1 or ATG13. Scale bar = 20 µm. f, g The number of ATG13 (f) and ATG16L1 (g) puncta per cell were quantified and normalized to Wt control from n = 3 independent experiments, >500 cells per condition. Significance was determined using two-way ANOVA followed by Sidak’s comparison test. P value (ATG13) = 0.0012 and <0.0001, and P value (ATG16L1) = 0.0008 and 0.0003. h U2OS cells expressing EGFP-BATS were transfected with siRNA against control or GRAMD1C for 72 h before starvation in EBSS for 1 h. Scale bar = 20 µm. i The number of EGFP-BATS puncta per cell was quantified and normalized to siNC control from n = 3 independent experiments, >500 cells per condition. Significance was determined using two-way ANOVA followed by Tukey’s comparison test. Error bar = SEM. P value = 0.0003 and 0.0069. Source data are provided as a Source Data file.