NAC treatment prevents loss of ability to regrow after starvation. (A) Wild-type, grh1Δ, and vps23Δ cells were grown overnight in logarithmic phase, washed twice, and cultured in 2% potassium acetate for 3.5–4 h in the absence or presence of 10 mM NAC. Cell density was normalized to 1 OD/ml during the time of starvation. Subsequently, cells were serially diluted to a final dilution of 1:5,000, and 100 µl was plated on rich medium. After 2 d of growth at 30°C, the number of CFU per milliliter was counted. For wild-type cells, n = 7; for grh1Δ cells, n = 4 of freshly generated deletion strains; and for vps23Δ cells, n = 3. All relevant differences were found to be statistically significant by unpaired Student’s t test. Wild type (±NAC), P = 0.0218; wild type – grh1Δ (−NAC), P = 0.009; wild type – vps23Δ (−NAC), P = 0.0322; grh1Δ – grh1Δ (±NAC), P = 0.0049; and vps23Δ – vps23Δ (±NAC), P < 0.0001. Error bars indicate SEM. (B) The starving cultures without NAC from (A) were incubated with 5 µg/ml PI and 1 µg/ml calcein AM (Thermo Fisher Scientific) for 30 min in the dark before standard flow cytometry analysis using a FACSCanto (BD Biosciences). “negative” indicates wild-type cells without PI or calcein AM labeling. Data were analyzed with FlowJo software, v10. Representative dot plots are shown, indicating all three strains were mostly viable after 3.5–4 h of starvation based on calcein AM fluorescence (y axis, Q3). PI fluorescence is in the y axis.