Figure 1
The CMVmini-6 × HRE-9 × ORE promoter was highly responsive to endogenous Oct4 and HIFs in hypoxic human and murine bladder cancer cells. (a) Expression of Oct4 mRNA in bladder cancer cells under normoxic and hypoxic conditions for 48 h, as determined by quantitative real-time RT-PCR. (b) Detection of HIF-1α, HIF-2α and Oct4 expression in bladder cancer cells after exposure to normoxia (N) or hypoxia (H) for 48 h. The expression of β-actin served as the loading control. (c) Determination of promoter activities. TCC-SUP and MBT-2 cells were transfected with single dual-luciferase reporter constructs, which contained the CMVmini promoter ligated with either 6 × HRE or 9 × ORE, or both to drive firefly luciferase, as well as the CMV promoter to drive Renilla luciferase, and then exposed to normoxia or hypoxia for 48 h. Promoter activities were determined by a dual-luciferase reporter assay. The ratio of firefly luciferase activity to Renilla luciferase activity was expressed as relative light units (RLU) (n=4). Values are the mean±s.e.m. of the mean. ***P<0.001; **P<0.01; *P<0.05.
