Extended Data Fig. 3: Autophagy sustained mTORC1 hyperactivation in Tsc-deficient MEFs under energy stresses.

a, Lysates from WT and Tsc2 KO MEFs treated with DMSO or BafA1 examined by immunoblots with antibodies for LC3 and actin. Three independent experiments gave similar results. b, Mean ± s.e. of the number of LC3 puncta of DMSO or Spautin1 treated WT and Tsc2 KO MEFs in normal medium and glucose-free medium (without FBS) with or without BafA1 for 2 hours. Spautin1 was preincubated for 12 h. n = 6–12 independent experiments for at least 200 cells. c, Lysates from DMSO- or Spautin1-treated WT MEFs and Tsc2 KO MEFs under normal, glucose-free and amino-acid-free conditions (without FBS) for 2 h were examined with indicated antibodies. Spautin1 was preincubated for 12 h. Three independent experiments gave similar results. d, Immunofluorescence of mTOR, LAMP2 and DAPI in WT MEF, Tsc2 KO MEF and Tsc2/Atg7 2KO MEF under normal medium (without FBS), glucose-free medium (without FBS) or glucose-free medium with 10 μM Spautin1 (without FBS) for 2 h. Spautin1 was preincubated for 12 h. Four independent experiments gave similar results. e, Mean ± s.e. of the Pearson correlation coefficient (PCC) of mTOR co-localization on LAMP2⁺ structure in WT MEF, Atg7 KO MEF, Tsc2 KO MEF and 2KO MEF under normal medium, glucose-free medium and glucose-free medium (without FBS) with Spautin1 for 2 h. n = 5 independent experiments for at least 200 cells. f, Lysates from WT MEFs and Tsc2 KO MEFs treated with DMSO or Spautin1 were examined by immunoblots with antibody for LC3 and vinculin. BafA1 was used to block autophagosome degradation, and Spautin1 was preincubated for 12 h before experiments. Three independent experiments gave similar results. Mean ± s.e. of the relative level of LC3II of DMSO- or Spautin1-treated WT MEFs and Tsc2 KO MEFs with or without BafA1 for 2 h on the right. n = 3 independent experiments. g, Lysates from WT MEFs, Atg7 KO MEFs (2 independent clones), Tsc2 KO MEFs and 2KO MEFs (2 independent clones) were examined by immunoblots with antibodies as indicated. Three independent experiments gave similar results. h, Lysates were extracted from WT MEF, Atg7 KO MEF (clone 1), Tsc2 KO MEF and 2KO MEF (clone 1) in normal medium, glucose-free medium and 2DG-supplemented medium (without FBS) for 2 h. The levels of phosphorylated S6K, total S6K, phosphorylated S6RP, total S6RP and vinculin were examined. Three independent experiments gave similar results. Scale bar, 10 μm. Data were analysed by two-tailed Student’s t test (b,f) or Pearson’s correlation coefficient (e).