Effect of caMEKK1 and ectopic p300 on Sox9 activity. (a) Chondrocytes were co-transfected with the Sox9 reporter and constitutively active mitogen-activated protein kinase kinase kinase (caMEKK)1 expression vector and treated for 24 hours with or without tumour necrosis factor (TNF)-α (30 ng/ml) and/or all-trans retinoic acid (atRA; 1, 10, or 100 nmol/l). TNF-α significantly increased Sox9 activity, whereas the highest concentration of atRA decreased Sox9 activity. Co-treatment with atRA and TNF-α resulted in Sox9 activity levels equivalent to that observed in untreated cultures containing caMEKK1 (first bar). Data are ratios of Sox9-regulated firefly luciferase units to renilla luciferase units normalized as a fraction of the ratio in untreated cultures (first bar), and are expressed as means ± standard error. Data were evaluated by repeated measures analysis of variance and Tukey's multiple comparisons test (three independent experiments). Bars labelled with the same lower case letters are not significantly different (P > 0.05). (b) Chondrocytes were transfected with Sox9 reporter alone (closed bars) or in combination with p300 expression vector (open bars) and then treated with TNF-α and/or atRA, as indicated. Co-transfection with the p300 expression vector significantly increased Sox9 activity in comparison with similarly treated normal cells (* significant effect of p300, P < 0.05). Under most conditions, over-expression of p300 also maintained Sox9 activity at a level comparable to that observed in normal, untreated chondrocytes (first closed bar). Data are Sox9-regulated luciferase units normalized to level in normal, untreated chondrocytes. #Significant difference compared with the first closed bar (P < 0.05). Data are means ± standard error (three independent experiments). Data were analyzed by paired t-tests.