Figure 3.

xCT-dependent ROS clearance mediates starvation resistance. Both mRNA (a) and protein (b) expression of the glutamate/cystine antiporter xCT were elevated in GSCs with high Gln/Glu ratios (JHH520 and 407) (loading control = β actin) (n = 3, significance was calculated with a one-way ANOVA, * significantly decreased (p < 0.05) compared to JHH520, # significantly decreased (p < 0.05) compared to 407). (c) Relative GSH concentrations of L-Gln-starved cells (compared to controls in full medium) assessed with ¹H-NMR spectroscopy of metabolic extracts (n = 3, significance calculated with one-way ANOVA). (d) ROS accumulation in L-Gln-starved and control cells measured by means of DCF fluorescence intensity (n ≥ 3). (e) Immunoblotting verification of shRNA-based xCT knockdown and ZEB1 expression in shxCT and pLKO.1 (control) cells (loading control = β actin). (f) Soft agar colony formation in shxCT and pLKO.1 (control) cells (n = 3). (g) Soft agar colony formation in JHH shxCT cells cultured in the presence or absence of L-Gln (n = 3). (h) Soft agar colony formation in GBM1 cells starved for L-Gln in either the presence or absence of 3 mM NAC. For all soft agar assays, representative pictures of NBT-stained colonies and quantifications of three assays are shown (n = 3, scale bar equals 1 cm). For all assays, the data are presented as mean ± SD. Significance was calculated with a one-sided t-test if not specified elsewhere, * p < 0.05. Abbreviations: ctrl, control; CFU, colony-forming units; Gln, glutamine; GSH, glutathione; NAC, N-acetylcysteine; n.s., not significant; ROS, reactive oxygen species. Supplementary Figure 2 (S2) corresponds to Figure 3b, Supplementary Figure 3 (S3) corresponds to Figure 3e.