Figure 1.

SLC7A11 mRNA is regulated by cellular stress and NMD. A. SLC7A11 mRNA was assessed by real-time PCR in U2OS, PC3, and HCT116 control, UPF1 depleted, UPF2 depleted, hypoxic (8 hours) or tunicamycin (Tm) (2.5 μg/ml 8 hours) cells. Experiments were repeated in triplicate, and the data reflect average ± standard error. * = p<0.05 by Students T test. B. Control and UPF1 over-expressing U2OS, PC3, and HCT116 cells were treated with tunicamycin (8 hrs) or rendered hypoxic (8 hrs), and expression of SLC7A11 mRNA was quantitated by real-time PCR. Experiments were repeated in triplicate, and the data reflect average ± standard error. * = p<0.05 by Students T test. C. (left panel) eIF2α wild type MEFs, either control or over-expressing UPF1, and eIF2α S51A cells were treated with cellular stress and SLC7A11 mRNA was assessed. Experiments were repeated in triplicate, and the data reflect average ± standard error. * = p<0.05 by Students T test. (right panel) Representative immunoblots of ATF4, UPF1, phosphorylated eIF2α, and (as loading control) total eIF2α in stressed eIF2α wild-type MEFs, wild-type MEFs over-expressing UPF1/RRent1, and eIF2aα S51A/S51A MEFs. D. ATF4 wild-type and ATF4 −/− MEFs were treated with cellular stress and SLC7A11 mRNA was assessed. Experiments were repeated in triplicate, and the data reflect average ± standard error. * = p<0.05 by Students T test. E. Control (sh Scr) and ATF4 depleted (sh ATF4) U2OS, PC3 and HCT116 cells were rendered hypoxic or treated with tunicamycin and SLC7A11 mRNA was assessed by quantitative PCR (left panel). Immunoblots of ATF4 expression in control and ATF4 depleted cells (right panel). F. U2OS cells were depleted of UPF1, UPF2, or treated with cellular stress for three hours, and then DRB was added to cease new RNA synthesis. SLC7A11 mRNA was serially assessed by quantitative PCR. Experiments were repeated in triplicate.