Azaspirane induces caspase-dependent apoptotic cell death. (A) MM.1S and U266 cells were cultured with azaspirane (5 μM) for 48 and 72 hours. Azaspirane-induced apoptosis was confirmed by the TUNEL method, using flow cytometry. Percentage demonstrated is TUNEL-negative fraction gated by horizontal bars. (B) MM.1S cells were cultured with azaspirane (5 μM) for 4 and 8 hours. Whole-cell lysates were subjected to caspase-3 colorimetric assay. The absorbance was measured at 405 nm, using a spectrophotometer, and data represent mean (± SD) of triplicate cultures. (C) MM.1S cells were cultured with azaspirane (5 μM) for 4, 8, and 12 hours. Whole-cell lysates were subjected to Western blotting, using anti–caspase-8, anti–caspase-3, PARP Abs, as well as anti–caspase-9, BAX, Bcl-2, and Mcl-1 Abs. (D) MM.1S cells were preincubated with Z-VAD-FMK (20 μM) for 1 hour prior to treatment with azaspirane (5 μM) for 4, 8, and 12 hours. Whole-cell lysates were subjected to Western blotting using anti-PARP Ab.