Figure 7. Mechanistic target of rapamycin complex 1 (mTORC1) regulates cellular glutathione levels through activating transcription factor 4 (ATF4) and SLC7A11-mediated cystine uptake.
(A) Total glutathione in serum-deprived Tsc2-/-mouse embryo fibroblasts (MEFs) treated with rapamycin (20 nM), Torin1 (250 nM), or buthionine-sulfoximine (BSO) (10 μM) for 16 hr is graphed as mean ± SEM relative to vehicle-treated cells from three independent experiments, each with three biological replicates (n = 9). Relative abundance of reduced (GSH) and oxidized (GSSG) glutathione from this experiment is shown in Figure 7—figure supplement 1A. (B) Relative total glutathione in serum-deprived Tsc2-/- MEFs with stable reconstitution of a cDNA encoding TSC2 or empty vector (EV) control is graphed as mean ± SD from a representative experiment with three biological replicates (n = 3). (C, D) Immunoblot (C) and relative glutathione levels measured by LC-MS/MS (D) from Tsc2-/- ELT3 xenograft tumors resected from mice treated for 5 days with vehicle or rapamycin (1 mg/kg on days 1, 3, and 5) (n = 3 mice/group). Relative glutathione levels from rapamycin-treated human TSC2-/- tumor cells are shown in Figure 7—figure supplement 1B. (E) Total glutathione in serum-deprived LNCaP (left) and PC3 (right) cells treated with vehicle, rapamycin (20 nM), Torin1 (250 nM), or BSO (50 μM) for 24 hr is graphed as mean ± SD relative to vehicle-treated cells from a representative experiment with three biological replicates (n = 3). (F) Total glutathione in serum-deprived Tsc2-/- MEFs (Atf4 wild-type [WT]) and Tsc2-/- Atf4-/- MEFs with stable expression of cDNAs encoding GFP (control), ATF4, ATF4DBD, or SLC7A11 grown in Dulbecco’s Modified Eagle’s Medium and supplemented, where indicated, with cysteine (1 mM, Cys) or cystine (0.5 mM, Cys2) is graphed as mean ± SD relative to WT cells from a representative experiment with three biological replicates (n = 3). Relative glutathione in these cells supplemented with nonessential amino acid with or without Cys, measured by LC-MS/MS, is shown in Figure 7—figure supplement 1C. (G) Total glutathione in serum-deprived Atf4+/+ and Atf4-/- MEFs pretreated 30 min with vehicle or rapamycin (20 nM) prior to insulin stimulation (500 nM, 24 hr) or treated with BSO (10 μM, 24 hr) is graphed as mean ± SEM relative to unstimulated Atf4+/+ cells from two independent experiments, with three biological replicates each (n = 6). (H) Total glutathione in serum-deprived Tsc2-/- MEFs (Atf4 WT) and Tsc2-/- Atf4-/- MEFs with stable expression of cDNAs encoding GFP, ATF4 lacking its 5′-UTR, or a DNABD mutant (DBD) of this ATF4 treated with vehicle or rapamycin (20 nM) for 16 hr is graphed as mean ± SD relative to vehicle-treated WT cells from a representative experiment with three biological replicates (n = 3). Effects of mTORC1 signaling and ATF4 on GCLC and GCLM transcript and protein levels are shown in Figure 7—figure supplement 1D–G. (B, E, F, H) are representative of at least two independent experiments. *p<0.05, **p<0.01, ***p<0.001, ns = not significant, calculated in (A, E, F, G, H) via one-way analysis of variance with Holm–Sidak method for multiple comparisons and in (B, D) via unpaired two-tailed t-test.
Figure 7—figure supplement 1. Supplemental data supporting Figure 7.
