Fig. 5. Effects of Cox-2 inactivation on apoptosis after DNA damage in 16N hNMECs. 16N cells were exposed to either doxorubicin (0.5 µg/ml) and DMSO (a solvent for NS-398) or doxorubicin and NS-398 (20 µM), the Cox-2 selective inhibitor, for 48 h. Cells were (A) harvested at the indicated times for western blot analysis using antibodies against Cox-2, p53, phospho-ERK, Bcl-xL and p27, or (B) harvested after 48 h for Trypan Blue staining. The percentages of dead cells were compared. Error bars indicate means ± SD of three independent experiments with duplicate plates. (C) FACS analysis of drug-treated cells. Z-VAD-fmk (50 µM), the general caspase inhibitor, abolished the effect of NS-398 (upper panels). The activation of caspase 3 induced by doxorubicin was increased further by NS-398 (lower panel). Cells were pre-treated with DMSO, NS-398 or Z-VAD-fmk for 1 h and then co-treated with doxorubicin for 48 h.