Figure 4
Effect of PPARγ overexpression on the inhibition of IL-1β-induced responses by 15d-PGJ2. Chondrocytes in six-well plates were transfected with pcDNA3.1 peroxisome-proliferator-activated receptor γ (PPARγ) construct (500 ng) for 36 hours. Thereafter, cells were pretreated for 4 hours with 10 μM 15-deoxy-Δ12,14prostaglandin J2 (15d-PGJ2), then stimulated with 10 ng/ml IL-1β for 24 hours before extraction of total RNA and collection of culture supernatant. (a) PGE2 levels assayed by ELISA in culture supernatant; (b) relative abundance of microsomal prostaglandin E synthase-1 (mPGES-1) mRNAs analysed by real-time PCR and normalized to S29 mRNA; (c) western blot control experiment of PPARγ and β-actin expression; (d) modulation of adiponectin (a PPARγ target gene) mRNAs by PPARγ agonists and pcDNA3.1 PPARγ construct, analysed by real-time PCR and normalized to S29 mRNA. Results are expressed as means ± SD for at least three independent experiments. Statistically significant differences (P < 0.05): *, comparison with non-stimulated controls; #, comparison with IL-1β-stimulated cells; †, comparison with PPARγ agonists alone or in combination with PPARγ plasmid.