Measurement of SIRT1 activity

SIRT1 activity was measured using the SIRT1 Activity Assay Kit (Abcam). Cells were seeded into 6 well plates (2 × 10⁵ cells/well). The day after, the medium was removed and cells were treated with 1 or 2 μM KL1333. After 1 h, cells were harvested and lysed in lysis buffer. Reactions were initiated by adding cell lysates to the reaction mixture containing SIRT1 assay buffer, fluoro-substrate peptides (100 mM), and NAD⁺ (100 mM). Fluorescence intensity was measured for 30 min at 2–3 min intervals on a microplate fluorometer (excitation, 350 nm; emission, 460 nm). SIRT1 activity was calculated within the linear range of reaction velocity, and normalized against the protein concentration in WT control cells.