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. 2016 Jan 13;6:19332. doi: 10.1038/srep19332

Figure 2. Mitochondria are the major source of ROS production in PC(O-16:0/2:0) treated cells.

Figure 2

(A) H2-DCFDA fluorescence is increased in PC(O-16:0/2:0) treated cells. Wild type (BY4741) cells were grown to mid log in YPD and subsequently treated with Vehicle (0.2% Ethanol), PC(O-16:0/2:0) (20 μM) or H2O2 (0.8 mM) for t = 15 or 60 min prior to labelling with H2-DCFDA (10 μM). Representative brightfield (BF) and H2-DCFDA (DCF) images from t = 15 min are shown. (B) Quantifications of H2-DCFDA positive cells at the indicated time points from at least three independent experiments where a minimum of 150 cells were counted. Error bars = SD, (*p < 0.01, Kruskal-Wallis test). (C) Antioxidants reduce H2-DCFDA fluorescence. Wild type (BY4741) cells were grown to mid log in YPD with or without n-acetyl cysteine (NAC 20 mM, pH 7.5) or quercetin (300 μM) and subsequently treated with PC(O-16:0/2:0) (20 μM, 15 min) and labeled with H2-DCFDA (10 μM) and positive cells were quantified. A minimum of 150 cells from three independent experiments were counted. Error bars = SEM, (*p < 0.01, Kruskal-Wallis test). (D) Inhibition of ROS production reduces PC(O-16:0/2:0) toxicity. Sensitivity of wild type (WT, BY4741) and rrg1Δ (YKB3911) strains were assessed by examining growth on plates containing vehicle (Ethanol, EtOH) or PC(O-16:0/2:0) (7 μg/mL or 13.4 μM) at 30 °C for 2 days. Representative image from 3 experiments is shown. (E) Mitochondrial respiration is required for ROS production in PC(O-16:0/2:0) treated cells. Wild type (BY4741), rrg1Δ (YKB3911), rho° (YKB3925) cells were grown to mid log, treated with PC(O-16:0/2:0) (20 μM, 15 min) and labeled with H2-DCFDA (10 μM). A minimum of 150 cells from three independent experiments were counted. Error bars = SEM, (*p < 0.05, Kruskal-Wallis test). ND – not detected.