Fig. 1: SLC7A11-mediated cystine uptake promotes GPX4 protein synthesis.

a UMRC6 cells were cultured in control (200 μM) or treatment (1 μM cystine) media for 30 h followed by MS analysis for regulated proteins. Volcano plot shows the differentially expressed proteins in treatment vs control cells. Each filled circle represents a protein; significantly upregulated and downregulated proteins are highlighted with blue and red, respectively. The curve is derived at FDR = 0.05 and S0 = 0.1 as described in the Methods. b UMRC6 cells were cultured in media with indicated concentrations of cystine for 24 h. Protein levels were evaluated by Western blotting. c GPX4 and SLC7A11 mRNA levels were measured by RT-PCR in UMRC6 cells cultured in media with indicated concentrations of cystine for 24 h. n = 3. d UMRC6 cells were cultured in media with indicated concentrations of erastin for 24 h. GPX4 and SLC7A11 protein levels were evaluated by Western blotting. e GPX4 and SLC7A11 mRNA levels were measured by RT-PCR in UMRC6 cells treated with 5 μM erastin for 24 h. n = 3. f GPX4 and SLC7A11 protein levels were evaluated by Western blotting in control (sgCon) and SLC7A11-knockout (sgSLC-1 and sgSLC-2) UMRC6 cells. g GPX4 mRNA level was determined by RT-PCR in SLC7A11-knockout UMRC6 cells. n = 3. h GPX4 and SLC7A11 protein levels were evaluated by Western blotting in indicated SLC7A11-overexpressing cell lines. i GPX4 mRNA level was determined by RT-PCR in SLC7A11-overexpressing cell lines. n = 3. j Empty vector (EV) and SLC7A11-overexpressing (SLC) 786-O cells were cultured in complete (Con) or cystine-free (-cystine) media for 20 h followed by Western blotting to monitor GPX4 protein levels. k 786-O-EV and -SLC cell lines were subjected to polyribosome fractionation followed by RT-PCR to analyze GPX4 mRNA distribution profiles during protein translation. n = 3. l UMRC6-sgCon, -sgSLC-1, and -sgSLC-2 cells were subjected to polyribosome fractionation followed by RT-PCR to analyze GPX4 mRNA distribution profiles during protein translation. n = 3. m UMRC6 cells were cultured in control media, cystine-free media, or treated with erastin (10 μM) for 24 h followed by polyribosome fractionation and RT-PCR to analyze GPX4 mRNA distribution profiles during protein translation. n = 3. n Luciferase reporter activity for 3′-UTR of GPX4 gene was measured in UMRC6 cells treated with indicated concentrations of cystine for 24 h. n = 4. o RT-PCR analysis of 3′-UTR or exon region of GPX4 gene in UMRC6 cells treated with indicated concentrations of cystine for 24 h. n = 3. For all panels, error bars are mean ± SD. n indicates biologically independent repeats. P value was determined by two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.