FIGURE 2.

LTB₄-dependent stabilization of COX-2 mRNA, effects of chemical kinase inhibitors. A, quiescent HSF were treated with vehicle (Con) or with 100 pg/ml (5.7 pmol/liter) of rhIL-1β for 2 h (steady state), after which time cells were washed out (Wo) and treated with actinomycin D (1 μg/ml) for 30 min, and then fresh medium was added containing either vehicle (Wo) or LTB₄ (50 nmol/liter). After an additional 1, 2, 4, or 8 h of incubation, monolayers were extracted for RNA at each time point, and 5 μg of total RNA was analyzed for COX-2 mRNA and GAPDH mRNA by Northern hybridization using specific DIG-labeled cDNA probes. B, cells were treated with vehicle (Con) or with 100 pg/ml (5.7 pmol/liter) of rhIL-1β for 2 h, washed out (Wo), and treated with actinomycin D (1 μg/ml) for 30 min, and then fresh medium was added containing either vehicle (washed out 2 h) or LTB₄ (50 nmol/liter) alone or in the presence of l-Nil (1 μm), SB202190 (SB, 1 μmol/liter), U0126 (2 μmol/liter), Bay-11-7082 (BY, 5 μmol/liter), wortmannin (Wort, 200 nmol/liter), or staurosporine (ST, 10 nmol/liter) for 2 more h. 5 μg of total RNA was analyzed for COX-2 mRNA and GAPDH mRNA by Northern hybridization using specific DIG-labeled cDNA probes. ANOVA of the means for densitometric analysis is as follows: A, IL-1β versus IL-1β + washed out (Wo) + LTB₄ (1–4 h), not significant (NS), and ND, not determined; IL-1β versus washed out (1, 2 h), p < 0.001; B, Student's t test; IL-1β versus IL-1β + washed out 2 h, p < 0.003; IL-1β versus IL-1β + washed out 2 h + LTB₄, not significant; IL-1β versus IL-1β + washed out 2 h + LTB₄ + U0126, p < 0.006; IL-1β versus IL-1β + washed out 2 h + LTB₄ + other kinase inhibitors, not significant.