Figure 2

Effects of TNF-α and atRA on Sox9 activity. Chondrocytes were transfected with (a) the type II collagen enhancer luciferase reporter and some cultures were also co-transfected with (b) a phosphorylation site-deficient inhibitor of nuclear factor-κB (IκB-2N). Cultures were then treated with or without tumour necrosis factor (TNF)-α (30 ng/ml) and/or all-trans retinoic acid (atRA; 1, 10, or 100 nmol/l) for 24 hours. Panels a: TNF-α and atRA separately decreased Sox9 activity. Greater decreases in Sox9 activity were induced by treatment with TNF-α and atRA (1 nmol/l). Panel b: IκB-2N did not significantly increase basal levels of Sox9 activity (1.3 ± 0.2 fold increase [mean ± standard deviation]) compared with untreated cells transfected with Sox9 reporter only (P > 0.05). IκB-2N attenuated the effect of TNF-α on Sox9 activity, but regulation of Sox9 activity by atRA was maintained. Data are ratios of Sox9-regulated firefly luciferase units to constitutive SV40-regulated renilla luciferase units, normalized as a fraction of the ratios in untreated cultures (first bar), and are expressed as means ± standard error (the number of independent experiments was six for panel a and four for panel b). Data were evaluated by repeated measures analysis of variance and Tukey's multiple comparisons test. Unlabelled bars or bars labelled with the same lower case letters are not significantly different (P > 0.05).