Extended Data Fig. 6: Regulation of activation of mTORC1, AMPK and ATP content by inhibitors for β-oxidation and AMPK in energy-stressed Tsc-deficient cells.

a–d, Lysates were extracted from WT MEFs and Tsc2 KO MEFs in normal medium, glucose-free medium and 2DG-supplemented medium (without FBS) for 2 h. Cells were preincubated with β-oxidation inhibitors of Rano (a,b) and TMZ (c,d) for 12 hours and followed by 2 hours treatment. In a and c, the levels of phosphorylated Raptor, total Raptor, phosphorylated ACC, total ACC and vinculin were examined. In b and d, the levels of phosphorylated S6K, total S6K, phosphorylated S6RP, total S6RP and vinculin were examined. Three independent experiments gave similar results. e, Immunofluorescence of mTOR, LAMP2 and DAPI in ETO and Rano treated Tsc2 KO MEFs under normal and glucose-free medium for 2 h. Five independent experiments for at least 200 cells gave similar results. f, Mean ± s.e. of the Pearson correlation coefficient of mTOR localization on LAMP2⁺ structure in Tsc2 KO MEF treated with ETO or Rano under glucose-free conditions. n = 5 independent experiments. g, Lysates from vehicle-, ETO- or Compound C (CC)-treated Tsc2 KO MEFs in normal, glucose-free and 2DG supplemented media (without FBS) for 2 h were examined by immunoblot using antibodies as indicated. Cells were preincubated with ETO with or without CC for 24 h. Three independent experiments gave similar results. h, Mean ± s.e. of the ATP content of β-oxidation inhibitors of ETO-, TMZ- and Rano-treated WT MEFs in normal, glucose-free and 2DG media (without FBS) for 2 h. The β-oxidation inhibitors were preincubated for 12 h. n = 3–6 independent experiments. i, Mean ± s.e. of ATP content in ETO-treated Ctrl and Tsc1 KD 293 cells under glucose deprivation conditions for 2 h. n = 5 independent experiments. Scale bar,10 μm. Data were analysed by two-tailed Student’s t test (h,i) or Pearson’s correlation coefficient (f).