Stained cells were subjected selleck chemical to confocal microscopy. The fluorescence ratio of the two dyes was determined for quantitative Inhibitors,Modulators,Libraries analysis of MOMP. The Leica confocal software, a MetaMorph ver. 7. 8 was used for the analysis. Determination of cytochrome c release The release of cytochrome c was determined using an ApoAlert cell fractionation kit. The cells were processed according to the manufac turers instructions and the concentration of released cyto chrome c in the cytosolic fractions was determined by immunoblotting with anti cytochrome c antibody. used for ATO/BSO treatment 48 h after transfection with siRNA. Statistical analysis Experimental values are represented as the mea standard deviation in triplicate. The experiments were carried out at least 3 times.

The significance of differ ence between experimental and control groups was de termined by the Students t test. A value of p 0. 05 was considered statistically significant. Results BSO augments ATO induced cell death via intracellular ROS generation The effect of BSO on ATO induced cell death HL60 cells was examined by determining cell viability. BSO significantly augmented ATO Inhibitors,Modulators,Libraries induced cell death. Approximately 80% of HL60 cells died when exposed to ATO in the presence of BSO, whereas ATO alone killed approximately 30% of the cells. Since ATO induced cell death is associated with generation of intracellular ROS, the effect of the antioxidants, NAC and DTT, on ATO/BSO induced cell death was examined. Antioxidants prevented ATO/BSO induced cell death, suggesting that ROS play an important role in BSO mediated augmentation of ATO induced cell death.

To confirm the ROS generation directly in ATO/BSO treated cells, intracellular ROS generation was determined using a fluorescent probe, CM H2DCFDA dye. ATO/BSO treatment markedly increased the ROS gener ation, although ATO alone treatment did it only slightly. BSO Inhibitors,Modulators,Libraries augments ATO induced cell death via ROS mediated mitochondrial injury In the preceding section, BSO augmented ATO induced cell death via intracellular ROS generation. To clarify in volvement of ROS mediated mitochondrial injury in BSO mediated augmentation, the effect of BSO on the release of cytochrome c, a marker of mitochondrial in jury, in ATO treated cells was examined by immuno blotting. BSO significantly augmented ATO induced cytochrome c release whereas ATO alone induced slight Inhibitors,Modulators,Libraries release of cytochrome c.

The cytochrome c release was abolished by antioxidants. Fur ther, Inhibitors,Modulators,Libraries BSO markedly augmented the activation of caspase 9, which is triggered by released cytochrome c and is in volved in an early stage of mitochondrial apoptosis. On the other hand, the caspase 9 activation was hardly de tected in ATO alone treatment. To confirm BSO mediated mitochondrial selleckchem 17-AAG injury, the effect of ATO/BSO treatment on MOMP was examined with a confocal microscope.