Fig. 4: HIF-1α alleviated ferroptosis via targeting GPX4 transcription in inflammatory intestinal epithelial cells (IECs).
From: HIF-1α alleviates ferroptosis in ulcerative colitis by regulation of GPX4
A, B Fluorescence in situ hybridization (FISH) and subcellular fractionation assays showing the main localization of HIF-1α within NCM460 cell nucleus. 18S rRNA, GAPDH, and U6 served as cytoplasmic and nuclear controls, respectively (Scale: 25 μm). C JASPAR database predicted HIF-1α and GPX4 promoter binding site schematic. D RT-qPCR detected the mRNA level of GPX4 in inflammatory IECs with HIF-1α overexpression. E Co-IP indicated that HIF-1α might bind to GPX4 to form the HIF-1α/GPX4 complex in inflammatory IECs. F ChIP-qPCR confirmed HIF-1α binding to GPX4 promoter. G Dual-luciferase reporter assay was performed after transfection with GPX4 promoter wild-type (WT) or mutant luciferase reporter plasmids in HEK 293 T cells. H CCK-8 cell assay to assess cell survival under different conditions. I Fluorescent detection of cell death by PI staining of RSL3 (4 μM) for 24 h on colon epithelial cells. J Flow cytometric detection of PI-positive cells to analyze necrotic cell death after RSL3 and LPS intervention. K Western blot analysis of FTL, FTH, and HIF-1α with β-actin as a control. L, M Fluorescent histogram of lipid peroxidation level in colon epithelial cells detected by C11 BODIPY581/591 fluorescent probe (Scale: 100 μm). N MDA levels in human colon epithelial cells with and without RSL3 (4 μM) intervention. O Protein expression levels of ferroptosis essential genes (ACSL4, SLC7A11, GPX4, LPCAT3, and TXNRD1) and HIF-1α detected by Western blot in IECs at different RSL3 concentrations. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
