Caspase activity assay

Total colorimetric caspase 3, 8, 9 assay were performed using commercial kit according to the manufacturer’s instructions. Briefly, cell were cultured O/N in 60 mm dishes, and then treated with various concentrations of DR (0, 20, 40, 60 and 80 μM) for 24 h. Cell were lysed with lysis buffer on ice for 30 min and then centrifuged at 13,200 rpm for 15 min. Equal amount of protein (50 μg) from each sample was added to the reaction mixture containing each substrate (DEVE-pNA, IETD-AFC or LEHD-pNA) and incubation for 120 min at 37°C. The absorbance was read at 405 nm in a micro plate reader. Each sample for the assay was taken in duplicates.