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. 2019 Dec 30;295(5):1350–1365. doi: 10.1074/jbc.RA119.011471

Figure 5.

Figure 5.

l-Cystine import from system xc− is required for glucose deprivation–induced cell death. A, expression of the specific light chain of system xc−, xCT/SLC7A11, correlates with sensitivity to glucose deprivation in GBM cell lines. The resistant and sensitive (Sens.) GBM cell lines were starved of glucose for the indicated times and then lysed. Expression of xCT/SLC7A11 was assessed by Western blotting. Actin was used as an equal loading control. B and C, pharmacological inhibition of xCT-rescued sensitive cells from glucose deprivation–induced cell death. The indicated sensitive GBM cells were treated with either SASP (500 μm) or erastin (10 μm) in the presence or absence of glucose for 8 h, and viability was assessed by trypan blue exclusion. *** denotes p < 0.001 by Student's t test (n = 3). D, genetic knockdown of xCT promoted resistance to glucose deprivation in sensitive LN18 cells. Cells were first infected with CRISPRi machinery (dCas9-KRAB (66)) and then infected with a second vector carrying the indicated guide RNA. Cells were then cultured for 8 h in the presence or absence of glucose, and viability was measured by trypan blue exclusion. * denotes p < 0.05 by Student's t test (n = 3). Western blotting with antibodies against xCT and the equal loading control actin confirmed knockdown. NT, nontargeting control. E, l-cystine starvation rescues sensitive cells from glucose deprivation–induced death. The sensitive GBM cell lines were cultured in l-cystine-free DMEM in the presence and absence of glucose for 16 h. The media were supplemented with either l-cystine (200 μm) or l-glutamate (100 μm), and viability was measured by trypan blue exclusion. *** denotes p < 0.001 by Student's t test (n = 3). F, inhibition of the xCT cystine/glutamate antiporter prevents GSH depletion following glucose deprivation. The sensitive GBM cell line LN18 was cultured with and without glucose in the presence or absence of the xCT inhibitor SASP (500 μm) for 3 h, and then intracellular metabolite concentrations were measured using LC-MS metabolomics. xCT inhibition prevented the accumulation of l-cystine and l-cysteine and depletion of GSH following glucose deprivation. N.S. denotes p > 0.05; ** denotes p < 0.01, *** denotes p < 0.001 by Student's t test (n = 3). Error bars are standard deviation of the mean.