Apoptosis assay

Transfected cells were treated with or without 100 nM vincristine for 48 h. Apoptosis was analyzed using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) assay (BD Biosciences, San Jose, CA, USA), according to the manufacturer's protocol. The amount of phosphatidylserine on the outer surface of the plasma membrane (a biochemical alteration unique to membranes of apoptotic cells) and the amount of PI, a dye that easily enters dead cells or cells in the late stages of apoptosis and binds DNA, but does not bind the plasma membrane of viable cells, were detected. Fluorescence was detected using a FACSCalibur flow cytometer (BD Biosciences) by fluorescence activated cell sorter analysis, and data were analyzed using CellQuest software (BD Biosciences). Cells with phosphatidylserine on their surface were considered to be apoptotic. To verify the effect on apoptosis, caspase-3 activation was also determined using the human active caspase-3 ELISA kit (cat. no. ABIN1568799; R&D Systems, Inc., Minneapolis, MN, USA). Survivin siRNA-transfected or control siRNA-transfected cells were treated with or without 100 nM vincristine. Following a 24-h incubation, the cells were lysed and assayed as described by the manufacturer.