Downregulation of IL-1β-induced MMP-1/MMP-13 expression by 13-HODE and 15-HETE does not require de novo protein synthesis. (a, b) Chondrocytes were treated with 100 pg/ml IL-1β in the presence of the control vehicle dimethyl sulfoxide or increasing concentrations of 13-HODE (panel a) or 15-HETE (panel b) for 8 hours. (c) Chondrocytes were pretreated with control vehicle dimethyl sulfoxide or cycloheximide (10 μg/ml) for 30 minutes before stimulation with 100 pg/ml IL-1β in the absence or presence of 50 μmol/l 13-HODE or 15-HETE for 8 hours. Total RNA was isolated, reverse transcribed into cDNA, and MMP-1 and MMP-13 mRNAs were quantified using real-time PCR. The housekeeping gene GAPDH was used for normalization. All experiments were performed in triplicate, and negative controls without template RNA were included in each experiment. Results are expressed as fold changes, considering 1 as the value of untreated cells, and are the mean ± standard deviation of three independent experiments. *P < 0.05 versus cells treated with IL-1β alone. CHX, cycloheximide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HETE, hydroxyeicosatetraenoic acid; HODE, hydroxy octadecadienoic acid; MMP, matrix metalloproteinase; TNF, tumor necrosis factor.