Figure 4

The sensitizing effects of hPUMA are mediated by enhanced caspase activation and release of cytochrome c and Smac into the cytosol. (A) A2780s and SKOV3 cells were subjected to the indicated treatments as described in Materials and Methods. PUMA expression and caspase activation were analyzed by Western blotting. Arrows indicate active forms of caspases. β-Actin was used as a loading control. Densitometric analysis of the Western blots was performed and the amounts of hPUMA were compared with the protein loading control. Relative amount of hPUMA from the untreated cells was set as 1. (B) A2780s and SKOV3 cells were subjected to the indicated treatments as described in Materials and Methods. Release of cytochrome c and Smac into the cytosol were analyzed by Western blotting. Cox-IV blots indicate mitochondrial loading controls, whereas β-actin was used as a loading control for the cytosolic fraction. Densitometric analysis of the Western blots was performed, and the amounts of Smac and cytochrome c were compared with the protein loading control. Relative amount of cytosol Smac and cytochrome c from the cells treated with hPUMA plus Cis was set as 1, whereas a relative amount from untreated cells was set as 1 for mitochondria Smac and cytochrome c. Mito, mitochondria; Cyto, cytosol.