SIRT1 Deacetylase Activity Assay

A commercial assay kit of Sigma Inc (St. Louis, MO, USA) was utilized. Collected cells were re-suspended in a lysate, centrifuged at 1300 × g for 10 min at 4°C and supernatant was removed. The nuclei were rinsed, sonicated for 30 s and placed on ice for later usage. After 10-min centrifugation at 20,000 × g, the supernatant was harvested and stored at −80°C or immediately utilized. Assay working buffer was added into 20 μl sample and agitated thoroughly to ensure sufficient reaction at room temperature. Fluorescent intensity was measured by a Full-Wavelength Microplate Reader at an emission wavelength of 460 nm and an excitation wavelength of 355 nm (measured every 2 min for 60 min). The activity of SIRT1 was expressed as protein concentration integrated by the change rate of fluorescent intensity.