Acquired stress resistance experiments

An overnight BY4741 preculture grown in SC medium at 28°C was split into three cultures: two received a single dose of mild stress (0.1 and 0.2 μg/mL tunicamycin), and the other served as a control to which no stress was added. Each culture was grown in a total volume of 30 mL of SC. Once in the exponential phase [an optical density (OD₆₀₀)~0.4], cells were collected, supernatant was removed and cells were resuspended to OD₆₀₀ of 0.1 in 96-well plates that contained SC or SC plus 6, 8, and 10% ethanol. Yeast growth curves were monitored in a SPECTROstar Omega instrument (BMG Labtech, Offenburg, Germany) until yeast cells reached the stationary phase. Six replicates were used per condition. The areas under the OD₆₀₀-time curves, linearly related to both the maximum population level and maximum specific growth rate, were calculated by integration of the curves using OriginPro 7.5 software (OriginLab Corporation, Northampton, USA) (Arroyo-López et al., 2010).