Extended Data Figure 6.

a, Phosphorylation of S6K (T389) was decreased in U87 cells treated with octyl α-KG, but not in cells treated with octanol control. Same results were obtained using HEK-293 and MEF cells. b, Phosphorylation of AMPK (T172) is upregulated in WI-38 cells upon Complex V inhibition by α-KG, consistent with decreased ATP content in α-KG treated cells and animals. However, this activation of AMPK appears to require more severe Complex V inhibition than the inactivation of mTOR, as either oligomycin or a higher concentration of octyl α-KG was required for increasing P-AMPK whereas concentrations of octyl α-KG comparable to those that decreased cellular ATP content (Fig. 2d) or oxygen consumption (Fig. 2f) were also sufficient for decreasing P-S6K. Same results were obtained using U87 cells. Western blotted with specific antibodies against P-AMPK T172 (Cell Signaling, 2535S) and AMPK (Cell Signaling, 2603S). c, α-KG still induces autophagy in aak-2(RNAi) worms; **P < 0.01 (t-test, two-tailed, two-sample unequal variance). Number of GFP::LGG-1 containing puncta was quantitated using ImageJ. Bars indicate the mean. d-e, α-KG does not bind to TOR directly as determined by DARTS. HEK-293 (d) or HeLa (e) cells were lysed in M-PER buffer (Thermo Scientific, 78501) with the addition of protease inhibitors (Roche, 11836153001) and phosphatase inhibitors (50 mM NaF, 10 mM β-glycerophosphate, 5 mM sodium pyrophosphate, 2 mM Na₃VO₄). Protein concentration of the lysate was measured by BCA Protein Assay kit (Pierce, 23227). Chilled TNC buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl, 10 mM CaCl₂) was added to the protein lysate, and the protein lysate was then incubated with vehicle control (DMSO) or varying concentrations of α-KG for 1 h (for d) or 3 h (for e) at room temperature. Pronase (Roche, 10165921001) digestions were performed for 20 min at room temperature, and stopped by adding SDS loading buffer and immediately heating at 95 °C for 5 min (for d) or 70 °C for 10 min (for e). Samples were subjected to SDS-PAGE on 4–12% Bis-Tris gradient gel (Invitrogen, NP0322BOX) and Western blotted with specific antibodies against ATP5B (Santa Cruz, sc58618), mTOR (Cell Signaling, 2972), or GAPDH (Ambion, AM4300). ImageJ was used to quantify the mTOR/GAPDH and ATP5B/GAPDH ratios. Susceptibility of the mTOR protein to Pronase digestion is unchanged in the presence of α-KG, whereas, as expected, Pronase resistance in the presence of α-KG is increased for ATP5B, which we identified as a new binding target of α-KG. f, Increased autophagy in HEK-293 cells treated with octyl α-KG was confirmed by Western blot analysis of MAP1 LC3 (Novus, NB100-2220), consistent with decreased phosphorylation of the autophagy initiating kinase ULK1 (Fig. 4a).