cytochrome c release is prevented by zVAD fmk in ceramide treated HL 60 cells To elucidate the position of caspases in ceramide induced apoptosis, HL 60 cells were treated with caspase inhibitor, and we investigate the efiects of apoptotic signaling events, including cytochrome c release throughout ceramide induced apoptosis. Induction of apoptosis by ceramide was conffrmed by detecting DNA fragmentation in HL 60 cells. In parallel, caspase activation and cytochrome c release were determined. In agreement with other cell lines, ceramide induced internucleosomal Crizotinib ic50 DNA fragmentation, cytochrome c release from mitochondria and subsequent activation of caspase 3. Nevertheless, activation of caspase 8 occurred in a time after ceramide treatment. While caspase 8 was triggered at 24 h after ceramide treatment cells treated with ceramide demonstrated activation of caspase 3 after 12 h. This observation implies that both caspases act as downstream caspases. DNA fragmentation induced by ceramide was inhibited by the vast caspase inhibitor benzoyloxycar bonyl VAD ffuoromethylketone, while cytochrome c release wasn’t affected by zVADfmk. Current results demonstrate that zVAD fmk has no effect upstream of cytochrome c release but blocks cell death, indicating downstream caspases are required for ceramidemediated cell death in HL 60 cells. 3. 2. Bax is required for cytochrome c release in ceramide induced apoptosis The tests described above show that ceramide induced cytochrome Organism c release in HL 60 cells is caspase independent. Recent studies show that Bax can directly induce cytochrome c release from mitochondria without requirement for caspases. Induction of cytochrome c release from mitochondria happens via caspase 8 mediated cleavage of Bax and Bid mitochondrial translocation. Because ceramide induced caspase 8 activation was seen after cytochrome c release, we examined whether Bax is involved with cytochrome c release. GW0742 To find out if Bax is essential for cytochrome c release in ceramide mediated apoptosis in HL 60 cells, we used Bax antisense oligodeoxynucleotides to speciffcally lower intracellular Bax levels. Cells subjected to 1 WM of Bax antisense oligodeoxynucleotides for 24 h indicated considerably paid off Bax protein levels. Bax antisense stopped nuclear DNA fragmentation and inhibited cell death, indicating that Bax is needed for mediating apoptosis induced by ceramide, as assessed by trypan blue o-r Hoechst dye discoloration. Furthermore, Bax antisense avoided ceramide induced cytochrome c release and PARP cleavage. These results show that Bax promotes apoptotic cell death and induces cytochrome c release downstream of ceramide.