Fig. 2: Effects of HIF knockdown on iron regulation under hypoxia and hypoxidative stress.

A Expression of HIF-associated and iron regulatory genes in ARPE-19 cells treated under hypoxia or hypoxidative stress conditions (N = 4–6). B Representative images of Western blots of HIF-1α, HIF-2α, and Tubulin as loading control and quantification of HIF-1α and HIF-2α protein levels (N = 6–7) in ARPE-19 cells with HIF-1α and HIF-2α knockdown treated under hypoxia or hypoxidative stress conditions for 24 h. C, D Representative images and quantification of TFR1 protein levels normalized to Tubulin revealed that HIF-1α knockdown resulted in a significant downregulation of TFR1 after (C) 6 h and (D) 24 h (24 h treatments consisted of 18 h preincubation at 1% O₂ followed by 6 h incubation with 10 mM NaIO₃ at 1% O₂ to avoid cell death by NaIO₃) (N = 4). E Quantification of intracellular Fe³⁺ by flow cytometry in ARPE-19 cells with HIF-1α and HIF-2α knockdown showed that intracellular Fe³⁺ was downregulated by HIF-1α knockdown under hypoxidative stress conditions. F Knockdown efficiency of siRNA-mediated knockdown of TFR1 quantified by qRT-PCR. G TFR1 knockdown under hypoxidative stress conditions protected cells from cell death quantified by LDH assay. LDH assays were statistically analyzed with two-way ANOVA followed by Tukey’s multiple comparisons test, Western blot data were statistically analyzed with mixed effects model followed by Šídák’s multiple comparisons test, and knockdown efficiencies were analyzed with t test. All data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.