Malfunctioning of the Iron–Sulfur Cluster Assembly Machinery in Saccharomyces cerevisiae Produces Oxidative Stress via an Iron-Dependent Mechanism, Causing Dysfunction in Respiratory Complexes
Figure 3
Determination of Fe2+ release in S. cerevisiae ISC mutants.
Yeast cultures were grown in liquid YPD medium, harvested and suspended in YPD at 1×107 cells/mL and charged with the fluorescent probe PGFL and incubated for 2 h at 30°C with light shaking in darkness. Then, yeast suspensions were treated with and without a stressor and incubated at 30°C with light shaking. Samples (100 µL) were collected, suspended in PBS buffer, and the fluorescence intensity in the cells was evaluated by real-time flow cytometry within 6 h. Free Fe2+ determination in yeast suspensions without a stressor (dashed lines) and with stressor treatment (continuous lines). A) H2O2 12 mM, B) menadione 80 µM, C) ethanol 10% v/v, D) Free Fe2+ determination at 6 h of treatment with ethanol (10%). Results represent the fluorescence intensity of yeast cells. Values are the mean of three independent experiments with 20,000 cells counted by flow cytometry per each point. SEM values are indicated as bars (n = 3), one-way ANOVA with Bonferroni's post-hoc test was used to compare mutants to controls. Significant differences (p<0.05) are indicated with (*) for (A–C). Tukey's post-hoc test was used for (D), and significant differences (p<0.05) with respect to the WT control are indicated with different letters for the treatments; lowercase and uppercase letters indicate without and with ethanol treatment, respectively.