Caspase activity assay

Cells were plated into 96-well plates at a density of 2 × 10⁴ cells per well and incubated overnight. Small molecules were then added and incubated at 37 °C for 4 h. TRAIL (100 ng/ml), cisplatin (50 μM), and doxorubicin (2 μM) were then added. Cycloheximide (5 μg/ml) was added along with TRAIL at the indicated time points. Caspase 3/7 activity was measured using the flourogenic substrate CellTitier-Glo for caspase 3/7 activity (Promega, Fitchburg, WI, USA) following the manufacturer's instructions. Briefly, cells were lysed by the addition of 20 μl of 5 × passive lysis buffer (Promega). The plate was put on an orbital shaker and incubated for 10 min at room temperature. In all, 20 μl of cell lysates were transferred to white plates, and either substrate alone or substrate plus the caspase 3/7 inhibitor was added to the appropriate wells. After a 10-min incubation, the released fluorophore was measured using a plate-reading fluorimeter (Flx800, Bio-Tek Instrument Co., Winooski, VT, USA). The activity in wells treated with inhibitor was subtracted from the activity in wells lacking inhibitor. The resulting difference was expressed as a percentage of the caspase activity of the untreated cells.