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. Author manuscript; available in PMC: 2020 Sep 30.
Published in final edited form as: Nat Cell Biol. 2020 Mar 30;22(4):476–486. doi: 10.1038/s41556-020-0496-x

Extended Data Fig. 3. High expression of SLC7A11 promote disulfide stress, deplete NADPH and causes redox system collapse under glucose deprivation.

Extended Data Fig. 3

a, Simplified schematic of how SLC7A11 can be linked to NADPH and the PPP. b, c, Measurement of intracellular GSSG (b) and GSH (c) concentrations in EV and SLC7A11-overexpressing 786-O cells cultured with (+Glc) or without glucose (-Glc). d, Diagrams illustrating the structures of γ-glutamylcysteine, γ-glutamyl-cystine, GSH, and glutathionyl-cysteine. Glu: glutamate; Gly: glycine; Cys: cysteine. e, f, The relative levels of intracellular γ-glutamyl-cystine (e) and glutathionyl-cysteine (f) in EV and SLC7A11-overexpressing 786-O cells cultured with (+Glc) or without glucose (-Glc). g, Representative phase-contrast images of indicated cells cultured with or without glucose. h, Western blotting analysis of SLC7A11 protein levels in the control (sgCtrl) and SLC7A11 knockout (sgSLC-1/2) UMRC6 cells. i-l, Measurement of intracellular GSSG (i) and GSH (j) concentrations and the relative levels of intracellular γ-glutamyl-cystine (k) and glutathionyl-cysteine (l) in control (sgCtrl) and SLC7A11 knockout (sgSLC-1/2) UMRC6 cells cultured with (+Glc) or without glucose (-Glc). m, Representative phase-contrast images of indicated cells cultured with (+Glc) or without glucose (-Glc). n, o, Cystine uptake levels in EV and SLC7A11- overexpressing 786-O cells (n) or UMRC6 cells (o) upon treatment with 1 mM sulfasalazine (SAS). p-u, Cell death with or without representative phase-contrast images (p, s), NADP+/NADPH ratios (q, t), and ROS levels (r, u) of EV and SLC7A11- overexpressing 786-O or UMRC6 cells cultured in glucose-containing or glucose free medium with or without treatment of 1 mM SAS. Error bars are mean ± s.d, all bar graphs have 3 independent repeats. All scale bars=100 μm. The experiment (g, h, m, p) was repeated twice, independently, with similar results. All p values were calculated using two-tailed unpaired Student’s t-test. Scanned images of unprocessed blots are shown in Source Data Extended Data Fig. 3. Numeral data are provided in Statistics Source Data Extended Data Fig. 3.