Cat D overexpression leads to apoptosis. (A) Hoechst staining was performed on BE-2 neuroblastoma cells transfected with or without Cat D. Cat D overexpression clearly indicates fragmentation of nucleus in Cat-D GFP cells (see white arrow). The experiment is representative of n = 4. Bar = 5 μm (B) In situ measurement of apoptosis in Cat D transfected BE-2 neuroblastoma cells. As seen, the CY-5 staining in nucleus can be strongly visualized in Cat D transfected cells but not in control cells indicating the presence of fragmented DNA in Cat D. The experiment is representative of n = 4. Bar = 5 μm (C) Protein extracts from mitochondrial and cytosolic fractions, isolated from Cat D transfected and control cells, were blotted for Cyto-C, GAPDH, Tom20 and β-actin. As can be seen from western blot, there is clear increase in cytosolic presence of Cyto-C in Cat D transfected cells compared to controls, whereas another mitochondrial marker (Tom20) as well as a cytoplasmic marker (GAPDH) remain similar, indicating an activation of apoptotic pathway.