Figure 4

Effect of lysine-specific demethylase 1 silencing on interleukin 1β–induced histone H3 lysine 9 demethylation at microsomal prostaglandin E synthase 1 promoter. Osteoarthritis (OA) chondrocytes were transfected with 100 nM control scrambled small interfering RNA (siRNA) or lysine-specific demethylase 1 (LSD1). At 48 hours posttransfection, cells were left untreated or treated with 100 pg/ml interleukin 1β (IL-1β) for 8 hours (A) or 24 hours (B). CTL, Control. (A) 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 percentages of control values (that is, untreated cells), and the data are 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 nontransfected cells stimulated with IL-1β. (B) Cell lysates were prepared and analyzed for microsomal prostaglandin E synthase 1 (mPGES-1) protein expression by Western blotting. The blots were stripped and reprobed with specific anti-β-actin antibody. The blots are representative of similar results obtained from four independent experiments using cells from four separate donors. Knockdown of LSD1 was confirmed by Western blotting using a specific anti-LSD1 antibody (lower panels).