Cells expressing URA3 plasmids (pDRF1-GW, pYES2, or pYX212) were grown overnight in 1x YNB medium (Sigma–Aldrich, St. Louis, MO, USA), containing 1% ethanol (Boom BV, Meppel, The Netherlands), 20 mg/L adenine hemisulfate (Sigma–Aldrich), 20 mg/L L-tryptophan (Sigma–Aldrich), 20 mg/L L-histidine (Sigma–Aldrich), and 60 mg/L L-leucine (SERVA Electrophoresis GmbH, Heidelberg, Germany). For WT strains, uracil (Sigma–Aldrich) was added to a final concentration of 20 mg/L. The cells were subsequently diluted and grown overnight to an OD600 of 0.1–1.5, with at least 5 divisions.
For the experiments that involved S25-31C, cells were grown on 1x YNB medium containing 1% ethanol, 5 mM glucose (Boom BV, Meppel, The Netherlands), 20 mg/L adenine hemisulfate, 20 mg/L L-tryptophan, 20 mg/L L-histidine, and 60 mg/L L-leucine until glucose was exhausted. Next, cells were kept on this medium for 2 more days, after which they were visualized under the microscope.
For the experiments that involved S25-31C, cells were grown on 1x YNB medium containing 1% ethanol, 5 mM glucose (Boom BV, Meppel, The Netherlands), 20 mg/L adenine hemisulfate, 20 mg/L L-tryptophan, 20 mg/L L-histidine, and 60 mg/L L-leucine until glucose was exhausted. Next, cells were kept on this medium for 2 more days, after which they were visualized under the microscope.