Fig. 3.

Intracellular GSH depletion aberrantly activates mTORC1 in both serum-free and serum-containing media. Primary neuronal-glial cerebrocortical cultures were treated with increasing concentrations of l-buthionine-(S,R)-sulfoximine (BSO) in MEM media and GSH, GSSG levels were measured by HPLC. 20 nM rapamycin was used as an additional negative control for this experiment. (A) GSH levels and (B) Total glutathione levels. Cultures were treated with increasing BSO concentrations for 24 h in MEM (serum-fee) (C and D) or GMEM (serum-containing) (E and F) media and mTORC1 activity represented by p-S6/t-S6 was assessed by immunoblotting. (C) Representative mTORC1 immunoblot (MEM media), and (D) Blot quantification. (E) Representative mTORC1 immunoblot (GMEM media), and (F) Blot quantification. 20 nM rapamycin was used as procedural/positive control for the immunoblotting experiments. Data are represented as mean ± SEM (error bars). **p < 0.01, ****p < 0.0001, ns: not significant versus vehicle control by one-way ANOVA with Dunnett's post-hoc test (A, B, D and F). n = 3/grp. N = 3 experimental replicates. Mean ± SD of raw data values of GSH and total glutathione normalized to total protein (nmol/mg protein) are stated in parentheses (GSH; total glutathione) for each treatment group: a) Veh ctrl (6.3 ± 4.9; 9.5 ± 2.0); b) 10 μM BSO (3.0 ± 2.3; 4.6 ± 0.6); c) 30 μM BSO (3.0 ± 1.9; 4 ± 0.3); d) 100 μM BSO (2.0 ± 1.5; 3.0 ± 0.3); e) 300 μM BSO (1.7 ± 1.3; 2.6 ± 0.2); and f) 20 nM Rapamycin (10.4 ± 1.1; 10.5 ± 1.1) (3A and 3B).