Extended Data Fig. 7. Lipophagy released FFAs in energy stressed mTORC1 hyperactivated Tsc-deficient cells.
(A, B) Mean ± SE of glycerol release from (A) and the number of LDs in (B) WT MEF under normal media and glucose-free media (without FBS) for 2 hours 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 MEF under glucose-free media (without FBS) for 2 hours. Insets in details on right panels. Three independent experiments gave similar results.
(D) Lysates from WT and Tsc2 KO MEF 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 and G) Mean ± SE of the glycerol release (F) and FFA content (G) from scrambled shRNA and LAL shRNA #1 infected WT and Tsc2 KO MEF in normal media and glucose-free media for 2 hours were shown. n = 3 (F) and 6 (G) independent experiments.
(H and I) Mean ± SE of the number of LDs (H) and the content of triglyceride (I) in scrambled shRNA and LAL shRNA #1 infected Tsc2 KO MEF under normal media and glucose deprivation conditions. n = 5 independent experiments.
(J) Mean ± SE of ATP related OCR of scrambled shRNA and LAL shRNA #1 infected WT and Tsc2 KO MEF under glucose deprivation conditions. n = 4 independent experiments.
(K) Mean ± SE of the ATP content of scrambled shRNA and LAL shRNA #1 infected Tsc2 KO MEF in normal media, glucose-free media and 2DG media with or without supplement of BSA-Palmitate for 2 hours were shown. n = 3 independent experiments.
(L) Lysates from scrambled shRNA and LAL shRNA #1 infected WT and Tsc2 KO MEF in normal media, glucose-free media, 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 Tsc1GFAP cKO mice treated with vehicle, 2DG, CQ, and CQ+2DG. Five independent experiments gave similar results.
(P) Immunofluorescence of pAKT and DAPI in SVZ of Tsc1GFAP cKO mice treated with vehicle, 2DG, CQ, and CQ+2DG. Five independent experiments gave similar results.
(Q) Mean ± SE of the percentage of pAKT+ cells in SVZ of Tsc1GFAP cKO mice treated with vehicle, 2DG, CQ, and CQ+2DG. n = 5 independent experiments.
(R) Immunofluorescence of pERK and DAPI in SVZ of Tsc1GFAP cKO mice treated with vehicle, 2DG, CQ, and CQ+2DG. Five independent experiments gave similar results.
(S) Mean ± SE of the percentage of pERK+ cells in SVZ of Tsc1GFAP cKO 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.
Bar = 10 μm (C); Bar = 100μm (M-P, R). Data were analyzed by two-tailed Student’s t test (A, B, F-K), one-way ANOVA with Tukey’s post-hoc test (Q, S).