FIG. 2.
Time course of caspase-3 activation and protection by inhibitors of caspase and endonuclease. (A) Caspase-3 activation. SK-N-SH cells were infected with DEN-2 PL046 virus at an MOI of 5. At the indicated times, cells were lysed and lysates were detected for cleavage of the caspase-3 by Western immunoblotting analysis using monoclonal antibody specific to caspase-3 p20 subunit (Santa Cruz). (B) Effects of a pan-caspase inhibitor, Z-VADfmk, and an endonuclease inhibitor, ZnSO4, on DEN-2 virus-induced apoptosis. SK-N-SH cells were pretreated or not pretreated with Z-VADfmk (100 μM) or ZnSO4 (20 mM) for 3 h and infected with DEN-2 virus (MOI of 5). Cells were then cultured in the presence of either inhibitor. After 60 h of infection, apoptotic cells were determined by PI staining and flow cytometry analysis as described in Materials and Methods. The values represent the means ± SD of triplicate determinations from one of two similar experiments, and the asterisks indicate a significant difference between the absence and presence of inhibitor (P < 0.01 by Student's t test).