Figure 9. HKL directly binds to and activates SIRT3.

(A) Steady-state fluorescence anisotropy values are shown as a function of increased concentrations of HKL. The concentration of human SIRT3 was 3μM (a representative experiment). (B) In a deacetylase buffer 0.5ug of acetylated MnSOD was incubated with 0.5ug of SIRT3 in the presence or absence of HKL at the indicated concentrations of NAD. Samples were analyzed by immunoblotting with use of anti-MnSOD.AcK122 antibody. Blot was stripped and probed for MnSOD for equal loading. (C) Cardiomyocytes were treated with cycloheximide (10 μM) for 1hr and then with HKL (5 μM) for next 2 hrs. Mitochondrial lysate was prepared and analyzed by western blotting with use of indicated antibodies. (D) SIRT3 mRNA levels were measured after 6hrs of treatment of cardiomyocytes with 5 and 10 μM HKL, mean ± SE, values are average of three independent experiments; Students t test. (E) Cardiomyocytes were treated with 10μM HKL and PGC1α mRNA levels were measured 6 hrs after treatment. Values are average of three independent experiments (mean ± SE); Students t test (F) Wild-type or SIRT3KO fibroblasts were co-transfected with a PGC1α responsive promoter/ luciferase reporter plasmid. After 16 hours of transfection, cells were treated with 5 or 10μM of HKL for 8 hours. Cell lysates were prepared; luciferase activity was measured and normalized to protein content, mean ± SE, Values are average of four independent experiments; Students t test.