Fig. 5.

Calorie restriction extends the chronological lifespan of the yeast. The prototrophic yeast strain (CEN.PK113-7D) was grown in the synthetic defined medium containing 2%, 0.5%, and 0.25% glucose 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 glucose concentrations. B The chronological lifespan (CLS) of the aged cells grown under different glucose concentrations 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 grown under different glucose concentrations 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 graph 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 < 0.0001 based on two-way ANOVA followed by Dunnett’s multiple comparisons test (A, B, and D). n.s, non-significant