Assays to measure apoptosis, caspase-Glo 3/7 activities, and annexin V level

The apoptotic cell population was measured using a DeadEnd Colorimetric TUNEL System (Promega) following the manufacturer’s protocol. Briefly, the cells were seeded at 5 × 10⁵ per well in six-well chamber slides and then fixed with 4% formaldehyde. The permeabilized cells were labelled with a terminal-deoxy-transferase reaction mixture for 1 h at 37 °C, the reaction was terminated by washing with 2× SSC buffer (0.3 M NaCl, 30 mM sodium citrate), and the cells were treated with streptavidin–HRP solution for 30 min at room temperature as indicated. The brown coloration developed by the diaminobenzene chromogen (Dako-Agilent, Santa Clara, CA, USA) was visualized. The apoptotic index was calculated as the percentage of positive cells in ≥500 cells at 400× magnification. Cell death was also determined by staining with Annexin V–fluorescein isothiocyanate and propidium iodide (PI) (Abcam, Cambridge, MA, USA; 14085). The cells were treated with the binding buffer with Annexin V and PI for 5 min in the dark and fixed in formaldehyde (4%) for 10 min, and the signals were measured on a FACSCalibur flow cytometer (BD Biosciences). The activities of caspase 3 and caspase 7 were measured using Caspase-Glo 3/7 Assay kits (Promega). The relative luminescence units were measured using a GloMax Luminometer (Promega).