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. 2020 Jun 19;11:3148. doi: 10.1038/s41467-020-16886-2

Fig. 4. Raptor deacetylation by EP300 inactivation mediates autophagy activation after Leu deprivation.

Fig. 4

a HeLa cells were treated with control siRNA or MCCC1 siRNAs, and cytosolic EP300 activity was assessed. N = 4. Two-tailed unpaired t-test. b Reduced raptor acetylation in MCCC1 knockdown cells. HeLa cells were treated with control or MCCC1 siRNA and transfected with HA-raptor. Following incubation in Leu-depleted media for 4 h, cells were lysed and raptor was immunoprecipitated using an anti-HA antibody. N = 4. c HeLa cells were depleted of raptor with siRNA and transfected with cDNA constructs encoding either raptor WT or raptor K1097R mutant (KR) (both HA-tagged). N = 4. d mTORC1 activity (phosphorylated S6) and LC3 dot numbers in HeLa cells expressing raptor WT or raptor KR. *HA-raptor WT or KR-expressing cells, #nontransfected cells. Scale bar, 10 μm. N = 3, 20–30 cells scored per condition per experiment. Two-tailed unpaired t-test. Acetylated PIK3C3 (e) and BECN1 levels (f) in HeLa cells depleted of raptor then reconstituted with raptor WT or raptor KR. Two-tailed unpaired t-test. HC, heavy chain. N = 4. g Effects of EP300 activator on mTORC1 signaling and autophagy in response to Leu deprivation with or without DCA in HeLa cells depleted of raptor then reconstituted with raptor WT or raptor KR. HeLa cells were incubated in Leu-depleted media for 4 h or incubated in Leu-depleted media with Torin1 for 4 h, followed by re-addition of 10 mM DCA to the media for 1 h. 50 µM CTB for 12 h was used to activate EP300. Blots are representative of three biologically independent experiments (N = 3). h Leu abundance regulates autophagy in many cell types via its metabolite AcCoA. AcCoA activates EP300, which acetylates raptor, leading to mTORC1 activation, which inhibits autophagy. In Leu deprivation conditions, autophagy activation is mainly mediated by decreased raptor acetylation causing mTORC1 inhibition, rather than by altered acetylation of other autophagy regulators. Data are presented as mean values ± SEM. Source data are in Source data file.