Fig. 2. Effect of hypoxia on CD105 transcription. (A) Northern blot analysis. Total RNA was extracted from HDMECs and fractionated on a 1% denaturing agarose gel. After blotting onto a nitrocellulose membrane, the fractionated RNA was probed using ³²P-labelled cDNAs for CD105 or GAPDH, visualised on a phosphorimager and quantified using a densitometer. Maximal expression of CD105 mRNA was observed at 3 hours with a threefold increase over normoxic culture. The bar chart represents CD105 mRNA relative to GAPDH mRNA signal intensity collected from three experiments. (B) Hypoxia activates the CD105 promoter. HDMECs (the same batch of cells as for panel A) co-transfected with plasmid pXP2/pCD105/luc and CMVßgal were grown under hypoxic condition for up to 24 hours. Luciferase activity was determined and normalised to ß-galactosidase activity. CD105 promoter activity peaked between 3 hours and 6 hours of culture (*P<0.05 and **P<0.01 compared with 0 hours as analysed by one-way ANOVA followed by the Duncan test). Data represent six replicates at each time point collected from three separate experiments. Vertical bars indicate standard error of the means.