Fig. 5. DMOG + SI co-treatment is likely to promote the Fenton reaction in DMOG + SI co-treated ARPE-19 cells.

A–C The Fe²⁺-specific fluorescence sensor RPA revealed significantly lower Fe²⁺ levels in SI-treated cells compared to controls after 8 h at 3% O₂ while no difference between controls and DMOG + SI co-treated cells were detected. In contrast, Fe³⁺ levels were significantly increased by DMOG + SI co-treatment compared to the other treatment groups after 8 h, which was assessed with the Fe³⁺-specific fluorescence sensor NBD-DFO. After 16 h, iron levels were similar in all treatment groups. While chelation of Fe³⁺ by Deferoxamine Mesylate (D) did not improve cell viability, Fe²⁺ chelation by 2’2-Bipyridyl E resulted in full rescue of cells suggesting that Fe²⁺ plays a detrimental role in DMOG + SI-induced ferroptosis. Data are presented as means + SD. RPA and NBD-DFO assays (3 independent groups) were statistically analyzed with one-way ANOVA and LDH assays were analyzed with two-way ANOVA. SOD2 knockdown (6 independent groups) were analyzed with two-way ANOVA. ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.