SIRT1 activity assay

SIRT1 deacetylase activity was evaluated in crude nuclear extracts from heart samples. Trichostain A (0.2 mM; Sigma-Aldrich, St. Louis, MO, USA), components of Fluor de Lys SIRT1 Fluorescent Activity Assay/Drug Discovery Kit (Enzo Life Sciences, Farmingdale, NY, USA), and 170 μM NAD⁺ were mixed at 37°C for 1 h, followed by incubation in developer for 15 min at room temperature according to the manufacturer's instructions. Fluorescence intensity was measured using a Fluoroskan Ascent® microplate fluorometer (Thermo Electron Corp., Milford, MA, USA). No-enzyme and time 0 negative controls were generated by incubating developer II solution with 2 mM NAM before mixing the substrates with or without samples. SIRT1 activity was calculated with the corrected arbitrary fluorescence units of the tested samples and expressed as fluorescence units relative to the control ⁴⁸.