Figure 3.

Inhibiting Vps34 does not activate CMA in NIH3T3 cells. (A and B) 10 h treatment with autophinib (10 µM) does not induce the formation of Dendra2 CMA reporter puncta. DMSO = 131 cells; autophinib = 126 cells. (C and D) 10 h treatment with VPS34-IN1 (4 µM) does not induce the formation of Dendra2 CMA reporter puncta. DMSO = 102 cells; VPS34-IN1 = 75 cells. For A–D, data were pooled from at least three independent experiments. (E) Autophinib and VPS34-IN1 do not inhibit Akt, MAPKPA1, or GFAP phosphorylation. DMSO is the solvent control, and buparlisib (2 µM) is the positive control for Akt inhibition. Blots are representative of six experimental replicates. (F and G) Autophinib and VPS34-IN1 completely block LC3-II flux, n = 6. For all experiments, cells were maintained in complete growth medium with 10% serum. P values written above brackets are derived from unpaired t tests. For F and G, the interaction P value to the right of each graph is derived by two-way ANOVA. Error bars are SEM. Scale bars are 20 µm. ACTB, β-Actin.