Fig. 5: Effects of SP2509 and Vorinostat on ARPE-19 cells.

A ARPE-19 cells were treated under hypoxia or hypoxidative stress conditions for 24 h with SP2509, Vorinostat, or vehicle (DMSO) and cell death was determined with LDH assays (N = 3–4). B Micrographs of ARPE-19 cells treated with 30 µM SP2509 and 10 µM Vorinostat confirmed that both inhibitors fully rescued cells from cell death. C, D Western blots of ARPE-19 cells treated under hypoxia for 6 h revealed that 30 µM SP2509 increased and 10 µM Vorinostat significantly decreased HIF-1α protein levels (N = 5–7). Consequently, the HIF-1 target gene CA9 was upregulated by SP2509 and downregulated by Vorinostat. E ARPE-19 cells treated with 10 µM Vorinostat for 24 h under hypoxidative stress exhibited decreased TFR1 and HO-1 protein levels. F ARPE-19 cells were treated under hypoxidative stress conditions for 18 h together with 10 µM Vorinostat or vehicle (DMSO). C11-BODIPY^581/591 treatment revealed that lipid peroxidation was significantly decreased by Vorinostat (N = 3). G To test metabolic effects of Vorinostat on metabolism of stressed ARPE-19 cells, cells were treated under hypoxia or hypoxidative stress conditions with 10 µM Vorinostat or vehicle (DMSO) for 24 h and metabolic flux analyses were conducted using a Seahorse Bioanalyzer. A Mitochondrial Stress Test (left) and a Glycolytic Rate Assay (right) were performed to assess mitochondrial function and glycolytic flux. H While basal respiration was significantly decreased by Vorinostat treatment, maximal respiration and spare respiratory capacity were significantly increased by Vorinostat. I Glycolytic Flux was not affected by the treatments. LDH and Seahorse assays were statistically analyzed with two-way ANOVA followed by Tukey’s multiple comparisons test, Western blots were analyzed with t test (C, D) or mixed-effects model followed by Šídák’s multiple comparisons test (E), and lipid peroxidation data were analyzed with t test. Data are expressed as mean ± SD (except for G: mean ± SEM). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, and ^****p < 0.0001. Scale bar: 50 µm.