Fig. 1: Effects of HIF-1α and HIF-2α knockdown on hypoxidative stress-induced cell death in ARPE-19 cells.
A Hypoxidative stress is induced by the combination of hypoxia treatment in a hypoxia chamber at 1% O2 (7.5 mmHg O2) and oxidative stress induced by NaIO3. Created in BioRender.com. B Hypoxidative stress resulted in substantial cell death. Ferroptosis inhibitors protected cells from hypoxidative stress-induced cell death (N = 3). C Knockdown efficiency of siRNA-mediated silencing of HIF-1α (left panel) and HIF-2α (N = 3). D HIF-1α knockdown protected ARPE-19 cells from hypoxidative stress-induced cell death (N = 4). E Micrographs of knockdown cells treated either under hypoxia alone or hypoxidative stress conditions showed many regions with intact cells in the HIF-1α knockdown group. F Representative plots of flow cytometry analyses using Zombie Aqua dye to quantify cell death. G Analysis of Zombie Aqua-positive cells confirmed that cell death was significantly reduced by HIF-1α knockdown (N = 3). H The protective effect of HIF-1α knockdown was further confirmed by MTT assay (N = 3). I C11-BODIPY581/591 staining revealed that lipid peroxidation is downregulated by HIF-1α knockdown pointing towards ferroptosis inhibition (left panel, N = 4). RSL3, a ferroptosis inducer, was used in parallel as positive control (right panel, N = 3). LDH, flow cytometry and MTT assays were statistically analyzed with two-way ANOVA followed by Tukey’s multiple comparisons test, knockdown efficiencies were analyzed with t test, and lipid peroxidation assays were analyzed with one-way ANOVA followed by Tukey’s multiple comparison’s test. All data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Scale bar = 50 μm.
