Fig. 6: BH3 mimetics suppress oxidative damage by intrinsic antioxidant activities.

A Voltammetry measurements for ferrostatin-1 (FER) and BH3-mimetics. Red and black curves correspond to the first oxidation or reduction measurement, respectively for each substrate. Peak undefined due to substrate deterioration (undef.). B FENIX assay to assess antioxidant activities of FER and BH3 mimetics by measuring STY-BODIPY (1 mM) co-autoxidation. All compounds were tested at 2 μM. Data were normalised to the DMSO control that lacked STY-BODIPY and are presented as the mean ± SD of three technical replicates from one out of three independent experiments. C Pfa1 cells were stimulated with 50 nM RSL3, 10 µM WEHI-539 (W),10 µM A-1331852 (A) and 2 µM ferrostatin-1 (F) for the indicated times before they were analysed for lipid peroxidation (C11 BODIPY 581/591 conversion) via flow cytometry. Data shown are mean ± SD of three independent experiments. One-way ANOVA with Tukey’s multiple comparisons test *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns = not significant. Overlay graphs are from one representative experiment. DMSO control (0 h) is the same in all bar graphs and plots. D Pfa1 cells were stimulated with 100 nM RSL3, 10 μM WEHI-539 (W) and 2 μM FER (F) for the indicated times before they were analysed for lipid peroxidation (C11 BODIPY 581/591 conversion) via flow cytometry. Data shown are mean ± SD of three independent experiments. One-way ANOVA with Tukey‘s multiple comparisons test *p ≤ 0.05, ****p ≤ 0.0001, ns not significant. Overlay graphs are from one representative experiment. DMSO control (0h) is the same in all bar graphs and plots. E Oxidised lipids species measured by LC-MS/MS. Pfa1 cells were treated with 10 µM WEHI-539 and 100 nM RSL3 alone or in combination for 4 h. The heat map shows relative amounts of significantly altered oxidized PEs (ANOVA, adjusted p value [FDR] cutoff: 0.05). The color scheme corresponds to auto-scaled log fold change relative to the mean log value within the samples. PE, phosphatidylethanolamines, i indicates isomeric lipids. Samples were clustered by average linkage weighted pair group method with arithmetic mean (WPGMA) agglomeration rule. Data are from one experiment with three technical replicates.