Fig. 7. The effect of GS on mitochondrial function was abrogated by NAM, a SIRT1 inhibitor. (A-B) Basal OCR and MRC in H9c2 cells were measured, after the treatment by GS and/or NAM for 48 h.(C) H9c2 cells were treated with GS and/or NAM for 48 h to measure ATP level by a bioluminescence detection kit. (D) The expressions of CV-ATP5A, CIII-UQCRC2, CIV-MTCO1, CII-SDHB, and CI-NDUFB8 in H9c2 cells treated with GS and/or NAM for 48 h were detected by Western blot. (E) Relative levels of five mitochondrial complexes from (D) are shown, after the quantification by ImageJ.(F) The effect of GS combined with NAM on the NAD+ level was analyzed by a NAD/NADH-Glo assay kit.(G-H) Total proteins of H9c2 cells treated with GS and/or NAM for 48 h were prepared and followed by Western blot analysis using antibodies against SIRT1, PGC1α, Nrf1, Nrf1, and β-Actin. Relative expression of these proteins was quantified with ImageJ and shown on the right. β-Actin is loading control. Ctrl: control group, OCR: oxygen consumption rate, MRC: maximal respiration capacity. *P < 0.05 and***P < 0.001 versus Ctrl group; #P < 0.05 and##P < 0.01 versus GS+NAM group.