Figure 3.

Elevation of NAD⁺ enhances sirtuin activation and autophagy induction and extends the replicative lifespan. (a) Sirtuin activation of cell lysates was analyzed. (b) Autophagy induction: the degree of autophagy was measured using an autophagosome-staining dye. Scale bar (White) = 10 $\mathsf{\mu }$m. (c) RT-qPCR was performed to analyze autophagy-related gene expression. (d) Replicative lifespan of fibroblasts: cells were cultured for 4 weeks, and cell populations were measured using CCK-8. The absorbance at 450 nm proportional to the number of cells was measured. Visualization was achieved by calcein-AM staining. Scale bar (White) = 20 $\mathsf{\mu }$m. All experiments were performed in triplicate. One-way ANOVA (Dunnett’s test) was performed for comparison between control and experimental groups. * Significantly different results (p < 0.05).