Figure 5
Contribution of NF-κB pathway to IL-1β-induced responses and 15d-PGJ2 inhibitory effects. In one set of experiments (a, b), chondrocytes cultured in six-well plates were transfected with 500 ng of IKBα dominant-negative (IκBαΔN) vector for 24 hours, then stimulated for 24 hours with 10 ng/ml IL-1β. (a) PGE2 levels in culture supernatant assayed by ELISA; (b) Relative abundance of microsomal prostaglandin E synthase-1 (mPGES-1) mRNAs analysed by real-time PCR and normalized to S29 mRNA. Results are expressed as means ± SD for at least three independent experiments. In another set of experiments (c, d), chondrocytes cultured in six-well plates were exposed to 10 ng/ml IL-1β for 15 min in the presence or absence of 10 μM 15-deoxy-Δ12,14prostaglandin J2 (15d-PGJ2) before extraction of nuclear proteins. Activation of NF-κB was determined by EMSA (c) and by ELISA with the TransAm® technology (d). Results in (d) are expressed as relative arbitrary units with IL-1β treatment set at 100, and are representative of three different experiments. Statistically significant differences (P < 0.05): *, comparison with non-stimulated controls; #, comparison with IL-1β-stimulated cells.