Figure 4
TNFα-induced changes to Sox9 and NFκB functional activity are independent of MEK1/2 activity. Chondrocytes transfected with (a) Sox9 or (b) NFκB reporters were pretreated with dimethyl sulfoxide (DMSO), U0124 (10 μM) or U0126 (10 μM) for 30 minutes followed by treatment with TNFα (30 ng/ml) for 24 hours. Data are ratios of (a) Sox9-regulated or (b) NFκB-regulated firefly luciferase units to constitutive cytomegalovirus-regulated renilla luciferase units in TNFα-treated cultures normalized to their respective DMSO-treated, U0124-treated or U0126 control-treated cultures. Data were log-transformed prior to analysis by paired t tests to determine significant reporter regulation by TNFα, followed by one-way analysis of variance to determine significant differences between the effects of DMSO, U0124 or U0126 pretreatment on TNFα-regulated reporter activity. Data are expressed as the mean ± standard error of four independent experiments. (c) Cells were pretreated with vehicle, DMSO, U0124 (10 μM) or U0126 (10 μM), or PD153035 (1 μM) for 30 minutes followed by treatment with TNFα (30 ng/ml) for 24 hours. Nuclear extracts (10 μg) were incubated with 32P-radiolabelled κB-consensus DNA. Resulting protein-DNA complexes were resolved on 4% polyacrylamide gels and exposed by autoradiography. Arrow, NFκB p65-containing protein-DNA complexes, as previously described [12]. The autoradiograph displayed is representative of three independent experiments.