Fig. 6

ERO1α upregulates SLC7A11 via activation of the IL-6/STAT3 pathway. A Schematic diagram of the screening of co-differentially expressed cytokines upon knockout or knockdown of ERO1α using a cytokines array assay. B and C Cell supernatants from the indicated cells were collected, and IL-6, MIP-1α, and MIG levels were determined using an ELISA. D sgERO1α NTC/T1 null cells were treated with IL-6 (20 ng/ml), MIP-1α (100 ng/ml), or MIG (100 ng/ml) for 24 h. E ERO1α-overexpressing Tsc2 + / + MEFs were transfected with IL-6 siRNAs or control siRNA (siNC) for 48 h. D and E Cell lysates were subjected to immunoblotting with the indicated antibodies (left panels), the expression of SLC7A11 mRNA was detected by qRT–PCR (right panels). F Representative IF showing the localizations of STAT3 in the indicated cells. Scale bar, 20 μm. G IL-6 (20 ng/ml, 12 h) pre-treated sgERO1α NTC/T1 null cells were transfected with STAT3 siRNAs or control siRNA (siNC) for 48 h. H IL-6 (20 ng/ml, 12 h) pre-treated sgERO1α NTC/T1 null cells were treated with different concentrations of S3I-201 for 24 h. I IL-6 siRNA-transduced ERO1α-overexpressing Tsc2 + / + MEFs were transfected with a constitutively activated STAT3 (STAT3C) or its control vector pBabe-puro (pBabe). G–I The expression of SLC7A11 was examined by western blotting (left panels) and qRT–PCR (right panels). J Schematic representation of the putative STAT3-binding sites in the promoter of mouse SLC7A11 gene. K HEK 293 T cells were co-transfected with the indicated promoter constructs plus pBabe-STAT3C or empty vector pBabe and the internal control plasmid pRL-TK. The relative luciferase activity was determined 24 h after transfection. L The enrichment of STAT3 in the promoter of SLC7A11 was analyzed by ChIP-PCR assay. M sgERO1α and sgCtrl NTC/T1 null cells were subjected to ChIP analysis with antibodies to p-STAT3 or control rabbit IgG. qRT–PCR was performed to amplify regions surrounding the putative STAT3 binding Site 2 (PBR) and a nonspecific STAT3 binding region (NBR). The data were plotted as the ratio of immunoprecipitated DNA to total input DNA. Error bars indicate mean ± SD of triplicate samples. **P < 0.01; ****P < 0.0001. n.s: no significance