Figure 3

Effect of pargyline and tranylcypromine on interleukin 1β-induced histone H3 lysine 9 demethylation and microsomal prostaglandin E synthase 1 protein expression. Osteoarthritis (OA) chondrocytes were pretreated with control vehicle (dimethyl sulfoxide) or increasing concentrations of pargyline (A) through (C) and tranylcypromine (TCP) (D) through (F) for 1 hour prior to stimulation with 100 pg/ml interleukin 1β (IL-1β) for 8 hours (A, B, D and E) or 24 hours (C) and (F). (A), (B), (D) and (E) Chromatin immunoprecipitation (ChIP) assays, coupled with real-time PCR, were performed using antibodies specific to mono- and dimethylated histone H3 lysine 9 (H3K9). The results are expressed as the percentage of control values (that is, untreated cells) and represent the mean ± SD of four independent experiments. For each ChIP assay, the immunoprecipitated DNA was quantitated in triplicate on two separate occasions. *P < 0.05 compared with IL-1β-treated cells. TCP, tranylcypromine. (C) and (F) Cell lysates were prepared and analyzed for microsomal prostaglandin E synthase 1 (mPGES-1) protein expression by Western blotting. In the lower panels, the blots were stripped and reprobed with specific anti-β-actin antibody. The blots are representative of similar results obtained in four independent experiments using cells from four separate donors. cPGES, Cytosolic prostaglandin E synthase; me1, Monomethylation; me2, Dimethylation; me3, Trimethylation.