Fig. 4.

Rapamycin drug extends the chronological lifespan of the yeast. The prototrophic yeast strain (CEN.PK113-7D) was grown in the synthetic defined medium with different concentrations of rapamycin in 96-well plates at 30 °C. A Cell growth OD600nm was measured at time points 24 h, 48 h, and 72 h using a microplate reader and graph plotted against different concentrations of rapamycin. B The chronological lifespan (CLS) of different concentrations of rapamycin-incubated cells was determined using the propidium iodide fluorescence–based method. Cell survival at different chronological age points was quantified and the growth time point 72 h was considered as day 1. C The CLS of the aged cells was determined by the outgrowth method in YPD liquid medium. The growth time point 72 h was considered as day 1. At various chronological age points, a 3-μL culture was transferred to a second 96-well plate containing 200μL YPD medium. Outgrowth in 96-well plate was photographed after incubation for 24 h at 30 °C. D Outgrowth OD600nm in YPD liquid medium was measured using a microplate reader. The outgrowth of different chronological age points is plotted relative to day 1. E At various chronological age points, 3-μL cultures were spotted onto the YPD agar plate. Outgrowth was photographed after incubation for 48 h at 30 °C. All data represent as means ± SD.; *P < 0.05, **P < 0.01, and ****P < 0.0001 based on two-way ANOVA followed by Dunnett’s multiple comparisons test (A, B, and D). n.s, non-significant