Fig. 3. AcCoA is essential for Leu metabolism-mediated autophagy regulation.

a AcCoA levels were assessed using the PicoProbe AcCoA assay kit. HeLa cells were incubated in nutrient-depleted conditions for 4 h followed by lysis and measurement of AcCoA levels. N = 4. Two-tailed unpaired t-test. b Effect of DCA and Leu on mTOR signaling and autophagy modulation caused by MCCC1 knockdown. Control cells, and cells treated with MCCC1 siRNA were treated with 10 mM DCA or 10 µM Leu for 1 h. Cells were lysed and western blots for phosphorylated S6K1 (p-S6K1) and total S6K1, as well as LC3 and MCCC1 are shown. N = 3. Two-tailed unpaired t-test. c Rescue of activated autophagy in MCCC1-depleted cells by DCA. 10 mM DCA for 1 h treatment restored the increased LC3 dots in MCCC1-depleted cells. N = 3, 60–70 cells scored per condition per experiment. Two-tailed unpaired t-test. Scale bar, 10 μm. AcCoA-induced autophagy regulation in mTORC1-dependent manner. Treatment with 0.5 µM Torin1 for 4 h or RRAGA and RRAGB double-knockdown (RRAGA + B) blocked the rescue effect of Leu or DCA on autophagy activation (LC3-II level (d) and LC3 dots (e)) by Leu depletion. HeLa cells were treated with scrambled siRNA or MCCC1 siRNA then incubated in Leu-depleted media for 4 h or incubated in Leu-depleted media for 4 h, followed by the re-addition of 10 µM of Leu or 10 mM DCA to the media for 1 h. N = 3, 40–50 cells scored per condition per experiment. Two-way ANOVA with post-hoc Tukey’s multiple comparison test. Scale bar, 10 μm. Data are presented as mean values ± SEM. Source data are provided as a Source data file.