(A) A549 cells (6 × 106 cells/flask) were treated with IL-1β (1 ng/ml) for 24 hr, subjected to freeze-thaw (two cycles), exposed for 20 min to either vehicle (0.1% v/v EtOH), ASA, the cytochrome P450 inhibitor (17-ODYA, 5 µM) or the 5-LO inhibitor (Rev-5901 isomer, 5 µM) and incubated with arachidonic acid (20 µM) for 20 min at 37°C. In some experiments, cells were heat-denatured (100°C, 60 min) before incubation. Incubations were stopped with addition of methanol (2 v), and products were extracted for RP-HPLC. Data are mean ± SEM from four to six separate flasks. *p < 0.05 and **p < 0.01 for treatments versus control. (B) Time course of 15-HETE formation from endogenous sources. A549 cells (1.5 × 106 cells/ml) were grown for 48 hr in the absence or presence of IL-1β (1 ng/ml) and incubated (30 min at 37°C) in 4 ml of HBSS with or without A23187 (5 µ M). 15-HETE levels were determined by RIA. Results represent the mean ± SEM of three different experiments determined by duplicate. *p < 0.05 for treatments versus vehicle.