FIGURE 2: Expression of Coq7 phosphomutant versions increases both the endogenous generation and the sensitivity to oxidative stress.

(A) Quantification of hydrogen peroxide generation was performed in presence of NADH and succinate as electron donors and 50 μg of purified mitochondria from the indicated strains. Data are mean ± SD, N ≥ 3 independent assays.

(B) Superoxide anion generation was measured using acetylated cytochrome c as electron acceptor. Quantification was produced in presence of reduced decylubiquinone as electron donor and 50 μg of purified mitochondria from the indicated strains. Data are mean ± SD, N ≥ 3 independent assays. ** P ≤ 0.001 compared to positive control (coq7∆/pNMQ7).

(C) Oxidative stress sensitivity was measured in the indicated strains. Cells were subjected to oxidative stress treatment by H₂O₂ (0.75 mM) or linolenic acid (1 mM) in 0.1 M sodium phosphate buffer plus 0.2 % glucose buffer at 150 x 10⁶ cells/ml. At the indicated times cells were harvested, diluted in sterile water and seeded in triplicate in YPD plates to calculate the number of CFU. The experiment is representative of a set of three.

(D) Cell viability was monitored under induced oxidative stress. Yeast cells were grown in SDc –ura glucose. After two days, cells were spotted by serial dilutions (1/10) from 0.5 OD_660nm/ml onto YPD plates containing the indicated concentrations of oxidative stress agents. Plates were incubated at 30°C for 3 days and imaged.