Functional analysis of COX-2 promoter in MG-63 osteoblast-like line cells. The -415 constructs of the COX-2 promoter fused to a Luciferase (Luc) reporter gene, its mutated ATF/CRE derivative (muATF/CRE), and mutated NF-κB derivative (muNF-κB) are shown in schematic representation. The constructs were co-transfected in MG-63 osteoblast-like line cells with pCMV-β-galactosidase (pCMV-β-gal). Six hours after transfection, fresh 0.5% foetal bovine serum/Dulbecco's modified Eagle's medium was added in the absence or presence of 20 μM HNE, 1 ng/ml TNF-α, and 20 μM HNE + 1 ng/ml TNF-α for another 24 hours. The β-gal and Luc levels were then measured in cellular extracts using specific commercial kits, and data were normalised for Luc and β-gal activities. Values are mean ± standard error of the mean of three experiments. Statistics: p values determined by Student unpaired t test: ***p < 0.001. P values are versus autologous untreated cells (control). COX-2, cyclooxygenase-2; HNE, 4-hydroxynonenal; NF-κB, nuclear factor-κB; TNF-α, tumour necrosis factor-α.