Caspase-3 activity assay

Caspase-3 activity assay was performed as described previously (23). Briefly, 4 × 10⁵ cells/well were seeded in 6-well plates and incubated overnight. The next day, cells were treated with appropriated drugs and incubated for 30 hours. Cells were then collected using a cell lifter and lysed in caspase buffer [pH 7.2, 20 mmol/L PIPES, 100 mmol/L NaCl, 1 mmol/L EDTA (pH 8.0), 0.1% (w/v) CHAPS, 10% sucrose and 10 mmol/L DTT] and quantified with BCA assay. Twenty micrograms of total protein were used to combine with 2 μL of 2 mmol/L DEVD-Afc (Millipore) in 96-well flat-bottom plates and added 200 μL/well caspase buffer. The plate was covered and incubated war 37°C for 2 hours before measuring fluorescence at excitation of 400 nm and emission of 510 nm. Measurements were analyzed using Graph-Pad Prism 6.