To select ethanol concentrations that would not be toxic over the 24 h course of the experiment, the response of three LCLs to increasing concentrations of ethanol up to 100 mM were examined. The three LCLs differed in their rates of doubling in the absence of ethanol (22, 28 and 35 hours). Ethanol prolonged the lag phase before LCLs began logarithmic growth, but in the period from 22-70 hours after ethanol was added, LCLs treated with 0, 50, 75 or 100 mM ethanol were in log phase. A plot of log10 (cell number) vs. time during this period fit a linear regression with r2 ≥ 0.98 for all LCLs with all concentrations of ethanol. The average doubling time in the absence of ethanol was 27.4 h, and it was 27.7 h in 75 mM ethanol (Supplementary Figure 1). Thus at the time studied, the cells were growing exponentially. Based upon these data, we chose to examine gene expression with and without 24 h exposure to 75 mM ethanol.
