Caspase-3 activity assay

A Caspase-3 Activity Assay kit (BioVision, Mountain View, CA, USA) was used to measure caspase-3 activity according to the manufacturer's instructions. Cells were plated on 60 mm dishes at a density of 2 × 10⁶ cells/mL and treated with various concentrations of metformin for 24 or 48 h. Cells were washed with PBS and harvested with lysis buffer, which was included in the kit. Cells were incubated on ice for 10 min. Cell lysates were then centrifuged at 4°C and 12,000 × g, and the supernatant was then transferred to a new tube and stored on ice. Protein content was analyzed using the Bradford assay (Sigma, St Louis, MO, USA). Assays were performed in 96-well plates containing 90 μg of protein in 50 μL lysis buffer, followed by the addition of 5 µL 4 mM N-Acetyl-Asp-Glu-Val-Asp p-nitroanilide (DEVD-pNA). The samples were incubated at 37°C for 2 h. Absorbance was measured at 405 nm using a DTX 880 Multimode Detector.