Effect of HNE on IL-6 protein production in osteoblasts. Osteoblasts were treated with HNE (0 to 20 μM) for 48 or 4 hours for IL-6 protein (a) (n = 7) and mRNA (b) (n = 3) determination, respectively. The effect of HNE combined with TNF-α was evaluated by incubating osteoblasts with HNE (20 μM) for 30 minutes and subsequently stimulating them with TNF-α (1 ng/ml) for 48 or 4 hours for IL-6 protein (c) (n = 7) and mRNA (d) (n = 3) determination, respectively. mRNA levels of each gene were quantified by real-time polymerase chain reaction as described in Materials and methods and normalised to those of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA. Data are means ± standard error of the mean and expressed as a percentage of untreated cells. Statistics: Student unpaired t test; *p < 0.05, **p < 0.01, ***p < 0.001. HNE, 4-hydroxynonenal; IL-6, interleukin-6; TNF-α, tumour necrosis factor-α.