Fig. 4.

Effect of CCN2 on the activation of HRE at −1006/−954 of the human VEGF promoter. (A) HCS-2/8 cells pre-treated with 100 ng/ml rCCN2 (closed column) or the same volume of PBS (open column) were transiently transfected either with the firefly luciferase reporter construct, 1HRE/WT-LUC containing the HRE at −1006/−954 of VEGF promoter, or its mutant, 1HRE/HM-LUC and thymidine kinase promoter-Renilla luciferase reporter plasmid (pRL-TK, internal control). After 24 h, cells were exposed to hypoxia (5% oxygen), and the cells were then assayed for luciferase activities. The ordinate shows the mean and standard error of relative value standardized against control culture of the measured luminescence of firefly versus Renilla luciferase of duplicate cultures. Relative luciferase activity of control culture is represented as 1.0. (B) HCS-2/8 cells pre-treated with 50 or 100 ng/ml rCCN2 (closed column) or same volume of PBS (open column) were transiently transfected with the reporter construct, 2HRE-LUC. After 24 h, the cells were exposed to normoxia or hypoxia (5% oxygen) for 24 h, and the cells were then assayed for luciferase activities. The ordinate shows the mean and standard error of relative value of the measured luminescence of firefly versus Renilla luciferase of duplicate cultures.