Effects of TNF-α and atRA on matrix gene expression. Chondrocytes were 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. Total RNA was evaluated for aggrecan core protein (ACP), type II collagen (Col2a1) and 18S rRNA by (a) Northern blot analysis and by (b, c) quantitative real-time PCR, whereas link protein mRNA was evaluated only by (d) quantitative real-time PCR. TNF-α and atRA alone decreased aggrecan, type II collagen and link protein transcript levels. Co-treatment with TNF-α and atRA further decreased mRNA transcript levels of aggrecan, type II collagen and link protein. Panel a: Northern blots are representative of three independent experiments; 18S rRNA levels were used to verify equal loading. Panels b to d: data are ratios of matrix gene: Gapdh transcript levels 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 seven for panel b, five for panel c, and five for panel d). Data were evaluated by one-way 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).