Fig. 3: Effects of HIF knockdown on regulators of the antioxidative response under hypoxia and hypoxidative stress.
A Expression of antioxidative response genes in ARPE-19 cells treated under hypoxia or hypoxidative stress conditions (N = 5–6). The fold change in HO1 expression under hypoxidative stress conditions exceeded the range observed for the other genes. Consequently, HO1 is presented separately using a distinct, higher range. B, C Representative images and quantification of Western blots of NRF2 and Tubulin as loading control from ARPE-19 cells with HIF-1α and HIF-2α knockdown treated under hypoxia or hypoxidative stress conditions for (B) 6 h and (C) 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.). The analysis revealed that NRF2 was significantly upregulated by HIF-1α knockdown under hypoxidative stress (N = 4). D, E Representative images and quantification of Western blots of HO-1 and Tubulin as loading control from ARPE-19 cells with HIF-1α and HIF-2α knockdown treated under hypoxia or hypoxidative stress conditions for (D) 6 h and (E) 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). The analysis revealed that HO-1 was significantly upregulated by hypoxidative stress (N = 3–4). F Knockdown efficiency of siRNA-mediated knockdown of HO-1 quantified by qRT-PCR (N = 4). G Quantification of cell death by LDH assay showed that HO-1 knockdown protected ARPE-19 cells from hypoxidative stress-induced cell death (N = 5). LDH assays were statistically analyzed with two-way ANOVA followed by Tukey’s multiple comparisons test, Western blot data were statistically analyzed with mixed-effect 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.
