Figure 3

Heavy water extension of yeast chronological lifespan mediated by calorie restriction and mitochondria. See also Supplementary Figure S3. (a) CLS extension by D₂O (50%) is abrogated by mild calorie restriction (n=3). Two independent experiments were performed, and one is shown here. The P values (log-rank test) for comparison with respective control (no D₂O) are indicated in the legends. The P values (log-rank test) for comparison with 2% glucose are<1.0E−5 for all other five conditions. NS, not significant. Error bars=s.e.m. (b) CLS extension by D₂O (50%) is attenuated in yeast cells lacking mitochondria. Mitochondria-deficient petite strains (rho⁻) were generated through EtBr treatment (see Materials and Methods). The deletion strain mip1Δ (BY4741 background) lacks the gene encoding the single subunit of the mitochondrial DNA polymerase in S. cerevisiae. Lifespans upon treatment with 50% D2O is shown. The mean lifespans and P values (log-rank test, versus BY4741 (rho⁺, 0%) are indicated with colors respective to legends. n=3. Error bars=s.e.m. (c) Measurement of in vivo generation of reactive oxygen species (ROS). The ROS generation was continuously monitored by fluorescent dyes CellROX (total ROS) and MitoSOX (mitochondrial ROS) on a plate reader for three strains treated with D₂O (50%) (n=3). The ROS generation, as calculated by area under curve calculation in Supplementary Figure S3, was normalized to no D₂O controls for each strain (control as 100%). The P values (two-tailed t-test, unequal variance) are indicated on graph. Error bars=s.e.m.