Figure 6

COE inhibits the binding activity of NF-κB in the MMP-9 promoter. FLSs were pretreated with the indicated amounts of COE for 1 h, and stimulated with IL-1β (10 ng/ml) and TNF-α (10 ng/ml) for 48 h. (A) The DNA binding activity of the nuclear extracts was examined by EMSA using a probe containing the NF-κB motif in the MMP-9 promoter. NF-κB DNA binding activity was analysed by EMSA and shown is a representative blot from three independent experiments. (B) The cross-linked chromatin was prepared and immunoprecipitated with antibody and IgG to NF-κB p65 before amplification of the MMP-9 gene region containing the NF-κB site. Immunoprecipitates were analysed by PCR for the presence of the MMP-9 gene promoter sequence using the primer pair described in material and methods. DNA purified from the sonicated chromatin was directly analysed by PCR using the same primer set, which served as an input control (Input). Similar results were obtained in three independent experiments. (C) FLSs were harvested and whole cell extracts were subjected to western blot analysis for the indicated proteins (IκBα, p-IκBα, p65 and p-p65). A representative protein blot of three independent experiments is shown. β-actin served as a control. The histogram shows the protein relative expression changes from three independent experiments. ^* P < 0.05, ^** P < 0.01 versus normal control group, ^◆ P < 0.05, ^◆◆ P < 0.01 versus IL-1β and TNF-α combination-treated group.