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. 2022 Jul 16;13:4146. doi: 10.1038/s41467-022-31905-0

Fig. 3. Autophagy induction by SMER28 depends on VCP and PI3K activity.

Fig. 3

a–c PI(3)P puncta in HeLa cells in basal (DMEM), starvation (EBSS 1 h; a, b) and in basal conditions treated with 20 μM SMER28 (a, c) or with or without 1 μM VPS34 inhibitor (IN1) or 10 μM DBeQ for indicated time points (b, c); PI(3)P puncta per cell normalized to control; n = 4 representative images in a; quantification in b (one-way ANOVA: P = 0.004 with post hoc Tukey test; EBSS P = 0.0255, EBSS + IN1 P = 0.0039) and in c (for SMER28 treatment alone: one-way ANOVA: P = 0.0079 with post hoc Tukey test, 4 h SMER28 P = 0.0131, 6 h SMER28 P = 0.0126; for SMER28 6 h treatment with or without inhibitors: one-way ANOVA: P = 0.0153 with post hoc Tukey test, SMER28 + IN1 P = 0.0284, SMER28 + DBeQ P = 0.028). d PI(3)P puncta in cells treated with 20 μM SMER28 for 6 h with or without 5 μM CB-5083, 10 μM NMS873; n = 4; two-tailed paired Student’s t test; DMSO vs. SMER28 P = 0.0444. e, f PI(3)P puncta in control and VCP knockdown HeLa cells in basal (DMEM), starvation (EBSS 2 h) and in basal conditions treated with 20 μM SMER28 for 4 h; n = 4; representative images in f; quantification in e (for control siRNA cells: one-way ANOVA P = 0.0022 with post hoc Tukey test; for VCP knockdown cells: one-way ANOVA P = 0.0294 with post hoc Tukey test). g Purified VPS34 was incubated in the presence of phosphatidylinositols (PtdIns) and ATP with 20 μM SMER28 for 20 min. The levels of inorganic phosphate were measured; n = 3; paired two-tailed Student’s t test. h SRAI-LC3B cells were treated with 20 μM SMER28 in the presence or absence of 0.3 μM CB-5083 for 24 h, followed by FACS analysis, n = 5; one-way ANOVA: P = 0.01 with post hoc Tukey test (DMSO vs. SMER28 P = 0.0008, CB-5083 P = 0.0213). Data presented as normalized mean ± SEM, *P < 0.05, **P < 0.001, ***P < 0.0001; scale bar = 10 μm; ns not significant. Source data are provided as a Source Data file.