Quantifying UPR under Ethanol Stress

To measure UPR activity under ethanol stress we used a modified plasmid reporter called UPR-mCherry that encodes red fluorescent protein mCherry (Merksamer et al., 2008 (link)), driven by a minimal CYC1 promoter and four tandem unfolded protein response elements. Cells containing the plasmid were grown overnight in GPY with geneticine medium at 28°C and were allowed to reach the early exponential phase (an approximate OD600 value of 0.4) for the analysis. Then the culture was divided into sterile centrifuge tubes, pelleted and incubated with GPY media with geneticine, with or without 10% (v/v) ethanol. Cells were grown at 28°C, sampled every 2 h, pelleted and frozen in liquid nitrogen until use. GFP fluorescence was measured by flow cytometry in a LSR Fortessa flow cytometer (BD Biosciences) and analyzed with the FACS DIVA software to compile.fcs files. Files were analyzed using FloJo (Tree Star Ashland, OR). Median fluorescence intensities (MFI) were calculated for each channel and were normalized with time cero sample data. To quantify UPR induction, fractional area (fa) of 10% ethanol condition was normalized against control (see above). Biological triplicates were performed in all cases.