Figure 1.
Inhibiting p110 activates CMA in NIH3T3 cells. (A) Western blots showing effects of selected doses of buparlisib (2 µM), pictilisib (500 nM), and copanlisib (50 nM) on phosphorylation of Akt, MAPKAP1, and GFAP. Blots are representative of six experimental replicates. DMSO is the solvent control for buparlisib and pictilisib. TFA is the solvent control for copanlisib. (B) Quantification of GFAP phosphorylation change in response to p110 inhibitors, as shown in A, n = 6. For C–H, data points are pooled from at least three independent experiments. (C and D) 10 h buparlisib treatment induces the accumulation of Dendra2 CMA reporter puncta. DMSO = 92 cells; buparlisib = 83 cells. (E and F) 10 h pictilisib treatment induces the accumulation of Dendra2 CMA reporter puncta. DMSO = 71 cells; pictilisib = 86 cells. (G and H) 10 h copanlisib treatment induces the accumulation of Dendra2 CMA reporter puncta. TFA = 132 cells; copanlisib = 116 cells. (I–K) Effects of buparlisib, pictilisib, and copanlisib on macroautophagy, as measured by 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. **, P < 0.01 by unpaired t test. For charts in I–K, PINT is the interaction term P value from a two-way ANOVA. Error bars are SEM. Scale bars are 20 µm. ACTB, β-Actin; Bup, buparlisib; Cop, copanlisib; Pic, pictilisib; TFA, trifluoroacetic acid.