Measurement of intracellular ROS.

Yeast cells were incubated in 1.5 ml of YPD medium at 30°C for 24 h. An aliquot of the culture (OD₆₀₀, 3) was sampled, washed twice with sterile water. The cells were dissolved in 3 ml of CSM medium (0.67% [wt/vol] yeast nitrogen base without amino acid, 0.077% [wt/vol] complete supplement mixture, 10% [wt/vol] glucose) and incubated at 30°C with shaking for 24 h. An aliquot of the culture (OD₆₀₀, 1) was centrifuged at 800 × g for 1 min and washed with sterile water once. The cells were dissolved in 987 μl of 10 mM potassium phosphate buffer (pH 6.42) and 13 μl of 10-mg ml⁻¹ 2′,7′-dichlorofluorescein diacetate (DCF-DA; final concentration, 10 μM) and incubated with gentle shaking at 30°C for 20 min. The cells were washed twice with sterile water and dissolved in 100 μl of sterile water, and 100 cells were observed under a fluorescence microscope. All procedures were performed within 90 min.