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. 2010 May 12;285(29):22244–22253. doi: 10.1074/jbc.M110.121327

FIGURE 5.

FIGURE 5.

Enforced expression of xCT in Txnrd1−/− cells does not rescue cells from BSO-induced cell death. A and B, xCT overexpression in Txnrd1+/− and Txnrd1−/− cells was confirmed by quantitative RT-PCR (A) and uptake measurements of radiolabeled cystine (B). A 3-fold (2.9 ± 1.95-fold) increase in xCT mRNA levels in xCT-Txnrd1+/− cells resulted in a marginal increase in cystine uptake, whereas in xCT-Txnrd1−/− cells, there was an 18-fold (17.73 ± 6.73-fold) increase in xCT mRNA levels, resulting in a 7-fold higher uptake of radiolabeled (Cys)2 (0.208 ± 0.022 versus 1.385 ± 0.347 nmol of cystine/min/mg protein]). C, despite the marginal increase in cystine uptake in xCT-Txnrd1+/− cells, ectopic xCT expression was associated with increased resistance to BSO in xCT-Txnrd1+/− cells as compared with eGFP-transfected Txnrd1+/− cells (80% versus 30% viability at 10 μm BSO) (upper panel). In stark contrast, xCT overexpression not only failed to rescue Txnrd1−/− cells from BSO-induced cell death, it rendered the cells significantly more susceptible to BSO-induced cell death (23% viability of xCT-Txnrd1−/− cells versus 81.42% viability of eGFP-Txnrd1−/− control cells at 2.5 μm BSO) (lower panel). The data are representative of two separate sets of experiments (means ± S.D.). D, the increased susceptibility of xCT-transfected Txnrd1−/− cells to BSO-induced cell death as compared with mock transfected Txnrd1−/− cells was confirmed by annexin V and propidium iodide staining. The cells were treated with 10 μm BSO for 24 h and then stained with annexin V and PI. Upon BSO treatment, the percentage of annexin V-PI-positive Txnrd1−/− cells was 4-fold higher when xCT was overexpressed as compared with control cells (23.8 ± 5.4% in xCT-Txnrd1−/− versus 7.5 ± 0.8% in mock Txnrd1−/− cells). Pooled data from two separate experiments are presented as percentages with means ± S.D. E, proliferation and viability over time of Txnrd1+/− and Txnrd1−/− cells transfected with xCT or eGFP in the presence of 10 μm BSO. The BSO treatment accelerated cell death in xCT-transfected as compared with eGFP-transfected Txnrd1−/− cells (lower panel) but only marginally affected the viability and proliferation of Txnrd1+/− cells (upper panel). The data are representative of two independent sets of experiments (means ± S.D.).