Extended Data Fig. 7: Lipophagy released FFAs in energy-stressed mTORC1 hyperactivated Tsc-deficient cells.

a,b, Mean ± s.e. of glycerol release from (a) and the number of LDs in (b) WT MEFs under normal and glucose-free media (without FBS) for 2 h supplemented with DMSO, orlistat, atglistat or JZL were shown. n = 3 (a) and 5 (b) independent experiments. c, Immunofluorescence of LAMP2, mTOR and DAPI in Orlistat-treated WT and Tsc2 KO MEFs under glucose-free medium (without FBS) for 2 h. Insets in details on right panels. Three independent experiments gave similar results. d, Lysates from WT and Tsc2 KO MEFs were examined by immunoblot of LAL and actin. Three independent experiments gave similar results. e, Lysates from scrambled shRNA and two individual LAL shRNAs infected WT MEF were examined by immunoblot of LAL and actin. Three independent experiments gave similar results. f,g, Mean ± s.e. of the glycerol release (f) and FFA content (g) from scrambled-shRNA- and LAL-shRNA-#1-nfected WT and Tsc2 KO MEF in normal and glucose-free media for 2 h were shown. n = 3 (f) and 6 (g) independent experiments. h,i, Mean ± s.e. of the number of LDs (h) and the content of TG (i) in scrambled-shRNA- and LAL-shRNA-#1-infected Tsc2 KO MEFs under normal medium and glucose-deprivation conditions. n = 5 independent experiments. j, Mean ± s.e. of ATP-related OCR of scrambled-shRNA- and LAL-shRNA-#1-infected WT and Tsc2 KO MEFs under glucose deprivation conditions. n = 4 independent experiments. k, Mean ± s.e. of the ATP content of scrambled-shRNA- and LAL-shRNA-#1-infected Tsc2 KO MEFs in normal, glucose-free and 2DG media with or without supplement of BSA–palmitate for 2 h were shown. n = 3 independent experiments. l, Lysates from scrambled-shRNA- and LAL-shRNA-#1-infected WT and Tsc2 KO MEFs in normal, glucose-free and 2DG-supplemented media for 2 hours. The levels of phosphorylated S6K, total S6K, phosphorylated S6RP, total S6RP and vinculin were examined by Western blot as indicated. Three independent experiments gave similar results. m–o, Immunofluorescence of pS6RP (m), pACC (n), pAMPK (o) and DAPI in SVZ of Tsc1^GFAPcKO mice treated with vehicle, 2DG, CQ and CQ + 2DG. Five independent experiments gave similar results. p, Immunofluorescence of pAKT and DAPI in SVZ of Tsc1^GFAPcKO mice treated with vehicle, 2DG, CQ and CQ + 2DG. Five independent experiments gave similar results. q, Mean ± s.e. of the percentage of pAKT⁺ cells in SVZ of Tsc1^GFAPcKO mice treated with vehicle, 2DG, CQ and CQ + 2DG. n = 5 independent experiments. r, Immunofluorescence of pERK and DAPI in SVZ of Tsc1^GFAPcKO mice treated with vehicle, 2DG, CQ and CQ + 2DG. Five independent experiments gave similar results. s, Mean ± s.e. of the percentage of pERK⁺ cells in SVZ of Tsc1^GFAPcKO mice treated with vehicle, 2DG, CQ and CQ + 2DG. n = 5 independent experiments. Dotted lines indicated the boundaries between SVZ and LV. E, ependymal layer; LV, lateral ventricle; ST, striatum; SVZ, subventricular zone. Scale bar, 10 μm (c); 100 μm (m–p,r). Data were analysed by two-tailed Student’s t test (a,b,f–k), one-way ANOVA with Tukey’s post-hoc test (q,s).