Figure 3. Autophagy degraded lipid storage of LDs in energy stressed Tsc-deficient cells.
(A) Mean ± SE of glycerol release from DMSO or Spautin1 treated WT and Tsc2 KO MEF in FBS-free normal and glucose-free media for 2 hrs. Spautin1 was pre-incubated for 12 hrs. n = 3 independent experiments.
(B, C) Mean ± SE of FFAs (B) and triglyceride (C) content of DMSO or Spautin1 treated Tsc2 KO MEF in FBS-free normal, glucose-free, and 2DG media for 2 hrs. n = 3 independent experiments.
(D) Fluorescence for lipid droplets and DAPI in WT, DMSO or Spautin1 treated Tsc2 KO and Tsc2/Atg7 2KO MEF under FBS-free normal or glucose-free conditions for 2 hrs.
(E) Mean ± SE of LDs of DMSO or Spautin1 treated WT, Tsc2 KO, and Tsc2/Atg7 2KO MEF in FBS-free normal and glucose-free media for 2 hrs. n = 5 independent experiments.
(F) Fluorescence for lipid droplets and DAPI in isolated primary neurospheres from Ctrl, Tsc1GFAP cKO, and 2cKO mice under FBS-free normal and glucose-free media for 2 hr.
(G) Mean ± SE of LDs in isolated primary neurospheric cells from Ctrl, Tsc1GFAP cKO, and 2cKO mice under FBS-free normal and glucose-free media for 2 hrs. n = 5 independent experiments.
(H) Immunofluorescence for lipid droplets, LC3 and DAPI in isolated primary neurospheres from Ctrl, Tsc1GFAP cKO, and 2cKO mice under FBS-free normal or glucose-free media for 2 hrs. Arrows: LDs/LC3 double positive; arrowheads: LD3 alone.
(I) Mean ± SE of percentage of LDs on LC3+ puncta of isolated primary neurospheres from Ctrl, Tsc1GFAP cKO, and 2cKO mice in FBS-free normal and glucose-free media for 2 hrs. n = 5 independent experiments.
(J) Mean ± SE of the percentage of LDs on LC3+ puncta of DMSO, BafA1 (100 nM), Spautin1 treated WT MEF and Tsc2 KO MEF in FBS-free normal and glucose-free media for 2 hrs. n = 4–9 independent experiments.
(K) Mean ± SE of the percentage of LDs on LAMP2+ lysosome of DMSO or Spautin1 treated WT MEF and Tsc2 KO MEF in FBS-free normal and glucose-free media for 2 hrs. n = 6–10 independent experiments.
(L) Mean ± SE of the β-oxidation activity of DMSO or Spautin1 treated WT MEF and Tsc2 KO MEF in FBS-free glucose-free media for 2 hrs. Etomoxir (ETO) inhibited β-oxidation. n = 6 independent experiments.
(M) Mean ± SE of ATP in Spautin1-treated Tsc2 KO MEF, re-supplemented with BSA or 1 mM BSA-FFAs in FBS-free glucose-free, and 2DG media for 2 hrs are shown. BSA-FFAs were pre-incubated for 2 hrs. n = 3–6 independent experiments.
(N) Mean ± SE of ATP related and maximum OCR of Spautin1 treated WT and Tsc2 KO MEF re-supplemented with BSA or 1 mM BSA-Palmitate and 2DG. n = 3–4 independent experiments.
(O) Lysates from DMSO and Spautin1 treated WT and Tsc2 KO MEF in normal and glucose-free media for 2 hrs probed by immunoblot. BSA or BSA-palmitate were pre-incubated for 2 hrs. Three independent experiments gave similar results.
Bar = 10 μm for D and 5 μm for F and H. Data were analyzed by two-tailed Student’s t test (A-C, M, N), one-way ANOVA with Tukey’s post-hoc test (E, G, I-L).