Figure 4. β-oxidation of FFAs to sustain hyperactivation of mTORC1 in energy stressed Tsc-deficient cells.
(A and B) Lysates from WT and Tsc2 KO MEF in FBS-free normal, glucose-free, and 2DG media for 2 hrs. ETO was pre-incubated for 12 hrs followed by 2 hrs energy stress. (A) Phosphorylated and total Raptor, phosphorylated and total ACC, and vinculin. (B) Phosphorylated and total S6K, phosphorylated and S6RP, and vinculin.
(C) Lysates from Ctrl and Tsc1 cKO neurosphere in FBS-free normal, glucose-free, and 2DG media for 2 hrs. ETO was pre-incubated for 12 hrs and followed by 2 hrs energy stress. Phosphorylated and total S6K, phosphorylated and total S6RP are shown. Three independent experiments gave similar results. (A-C).
(D) Mean ± SE of ATP content of Tsc2 KO MEF treated with ETO (200 μM), TMZ (1 mM), and Rano (200 μM) in FBS-free normal, glucose-free, and 2DG media for 2 hrs. Inhibitors were pre-incubated for 12 hrs. n = 3 independent experiments.
(E) Mean ± SE of ATP of Tsc1GFAP cKO primary neurospheres treated with ETO (200 μM) or Rano (200 μM) in normal or glucose-free media. Inhibitors were pre-incubated for 12 hrs. n = 5 independent experiments.
(F and G) Mean ± SE of maximum OCR of ETO, TMZ, and Rano treated WT and Tsc2 KO MEF under glucose-free (F) or 2DG treatment (G). n = 4 independent experiments.
(H) Immunofluorescence of LDs, LAMP2 and DAPI in ETO treated WT and Tsc2 KO MEF under FBS-free normal and glucose-free media for 2 hrs. Insets in details on right panels.
(I and J) Mean ± SE of percentage of LDs on LAMP2+ structure (I) and on LC3+ puncta (J) of ETO, TMZ, and Rano treated Tsc2 KO MEF in FBS-free normal and glucose-free media for 2 hrs. n = 5–10 (I) and 10 (J) independent experiments.
(K) Mean ± SE of FFA content of ETO, TMZ, and Rano treated Tsc2 KO MEF in FBS-free normal, glucose-free, and 2DG media for 2 hrs. n = 5 independent experiments.
Bar = 10 μm. Data were analyzed by two-tailed Student’s t test, (D-G, I-K).