Figure 6. SIRT1 is required for SPD-induced PGC-1α activation in aging cardiomyocytes. (A) Western blot detection of SIRT1, PGC-1α, NRF1, NRF2, and TFAM in nuclear fractions isolated from cardiac tissue. (B) Quantification of protein expression based on L-lamin as a nuclear loading control (n = 4). (C) Quantification of NAD+ levels by fluorimetry (n = 8). # P < 0.05 vs. young, ## P < 0.01 vs. young, * P < 0.05 vs. old, ** P < 0.01 vs. old, $ P < 0.05 vs. SPD, $$ P < 0.01 vs. SPD. (D) Co-localization of SIRT1 (red) and PGC-1α (green) by immunofluorescence in H9C2 cells. Nuclei were stained with DAPI (blue) (n = 8); scale bars: 20 μm. (E, F) Western blot and immunoprecipitation (IP) analysis of PGC-1α acetylation status in NRCMs. Quantification is shown on the right-hand side of the graphs. GAPDH was used as loading control (n = 4). # P < 0.05 vs. Control, ## P < 0.01 vs. Control, * P < 0.05 vs. H2O2, ** P < 0.01 vs. H2O2, $ P < 0.05 vs. H2O2 + SPD, $$ P < 0.01 vs. H2O2 + SPD.