J Clin Invest.
Posttranscriptional control of COX-2 mRNA. (a) Steady-state levels of COX-2 and c-myc mRNA in HT29 and LoVo cells were detected by RNase protection assays. The protected mRNA for c-myc was detected as a doublet. GAPDH mRNA is shown as a control for RNA loading. The data shown represent three experiments. (b) Levels of constitutive COX-2 transcription in HT29 and LoVo cells. A luciferase reporter construct containing the COX-2 promoter (filled bars) or the pGL3 vector (open bars) were cotransfected with the pSV-β-galactosidase control vector. Promoter activity was assessed as luciferase activity normalized to β-galactosidase activity and is the average of three experiments. (c) mRNA half-life experiments were initiated by adding 5 μg/ml ActD to HT29 and LoVo cells for the indicated times and COX-2 mRNA decay was analyzed by Northern blot probing for COX-2 and the internal control GAPDH. Blot exposure times were adjusted to yield similar starting intensities of COX-2 mRNA. c-myc mRNA decay was analyzed similarly by RNase protection assay using riboprobes for c-myc and the internal control GAPDH. The data shown represent duplicate experiments.