Induction of caspase-independent cell death by reversine. (A) Two hours before reversine addition, OSCC cells were pre-treated with or without 20 μM Z-VAD-fmk, followed by treated with or without 5 μM reversine, and harvested at 12, 24 and 48 h later. Cell numbers were counted as mentioned. (B) OSCC cells in (A) panel at 24th hour were used to monitor the cleaved caspase 3 protein as a marker of caspase-dependent apoptosis. (C) Similar to (B) panel, OC2 cells were used for DNA fragmentation analysis. (D) OC2 cells were treated with 5 μM reversine, stained with anti-AIF antibody and DAPI and visualized with a fluorescence microscope (magnification, ×200). AIF was retained in the cytoplasm in the absence of reversine and translocated into the nucleus after reversine treatment.