Fig. 4: ATP6AP1 regulates mTORC1 signaling.

a–c Serum-starved HeLa cells transiently expressing Flag-tagged Rheb, ATP6AP1 or ATP6AP1ΔC were cultured with 0.9 μM insulin for 15 min, and collected for western blot analysis using the indicated antibodies (a). Flag-GFP-expressing cells with insulin treatment served as negative controls. GAPDH was used as a loading control. Intensity values were obtained using ImageJ. p-S6K (b) and p-S6 (c) signals were normalized to total S6K/S6 as well as GAPDH signals and graphed as mean ± SEM (n = 3). Statistical significance was determined using the two-way ANOVA followed by Dunnett’s multiple comparisons test. **P < 0.01, ****P < 0.0001. ns, not significant. d–f HeLa cells stably expressing ATP6AP1-targeting shRNAs were serum starved for 16 h and then cultured with 0.9 μM insulin for 15 min, and cells were collected for western blotting (d). shNC, negative control shRNA. GAPDH served as a loading control. Intensity values for p-S6K (e) and p-S6 (f) were similarly processed to a–c and graphed as mean ± SEM (n = 3). Statistical significance was determined using the two-way ANOVA followed by Sidak’s multiple comparisons test. ****P < 0.0001. shNC with insulin treatment served as a negative control. g–i MDA-MB-231 cells stably expressing ATP6AP1-targeting shRNAs were treated and analyzed as described in d–f. Statistical significance was determined using the two-way ANOVA followed by Dunnett’s multiple comparisons test. *P < 0.05, ****P < 0.0001. j–m HeLa cells stably expressing ATP6AP1-targeting shRNAs were serum starved for 16 h and then treated with 200 ng/mL FGF plus 50 ng/mL heparin for 15 min, and cells were harvested for western blotting and data analysis as described in d–f. Statistical significance was determined using the two-way ANOVA followed by Dunnett’s multiple comparisons test. **P < 0.01, ***P < 0.001. ns, not significant. n Construct encoding shRNA-resistant SFB-tagged full-length ATP6AP1 or its C-tail alone mutant was transfected at different amount into HeLa cells stably expressing an ATP6AP1-targeting shRNA. The cells were serum starved and then cultured with 0.9 μM insulin for 15 min, and harvested for western blot analysis. The depth of the circle color represents different doses. Dark circles represent high doses (4 μg) of the indicated plasmids per well for transfection, while gray circles denote 2 μg of plasmids. The hollow white circles indicate no addition of relevant plasmid.