FIG. 6.

Superoxide anion is generated downstream of PLA₂ and is essential for apoptosis. (A) Generation of superoxide anion. SK-N-SH cells were infected with DEN-2 PL046 virus at an MOI of 5. At the indicated times, culture supernatants were collected and the amount of superoxide anion was detected using a Lumimax Superoxide Anion Detection Kit as described in Materials and Methods. (B) DEN-2 virus-induced apoptosis is blocked by SOD. SK-N-SH cells were pretreated with SOD at 1,000, 500, and 0 U/ml for 1 h and then were infected with DEN-2 PL046 virus at an MOI of 5. After 60 h of infection, apoptotic cells were determined by PI staining and flow cytometry analysis. (C) A cPLA₂-specific inhibitor, AACOCF₃, delayed DEN-2 virus-induced superoxide anion generation. SK-N-SH cells were pretreated with AACOCF₃ (10 μM) for 1 h and then were infected with DEN-2 PL046 virus at an MOI of 5. At 24 and 48 h p.i., culture supernatants were collected and the amount of superoxide anion was measured. The values represent the means ± SD of triplicate measurements from one of two similar experiments. An asterisk indicates a significant difference between the absence and presence of SOD (B) and AACOCF₃ (C) (P < 0.01 by Student's t test). Ctl, control.