Figure 2
Knockdown of HIF-2α suppressed Oct4 expression as well as Oct4- and hypoxia-dependent transactivation in bladder cancer cells. TCC-SUP and MBT-2 cultured in six-well plates were transfected with 5 μg of shRNA constructs specific to HIF-2α or GFP. After being cultured for 3 days, the cells were exposed to hypoxia or normoxia for 48 h, followed by immunoblot analysis. Expression of HIF-2α and Oct4 in HIF-2α knockdown TCC-SUP (a, left) and MBT-2 (b, left) cells was detected. The expression of β-actin served as the loading control. In another set of experiments, cells transfected with shRNA constructed as described above for 24 h were re-seeded in 24-well plates and cultured overnight, followed by transfection with 1 μg of pFRL2-9 × ORE-CMVmini, which drives firefly luciferase by the CMVmini promoter ligated with 9 × ORE and drives Renilla luciferase by the CMV promoter contained in the plasmid backbone. After 24 h, the cells were exposed to hypoxia or normoxia for 48 h. Transcriptional activities of the CMVmini promoter ligated with 9 × ORE in TCC-SUP (a, right) and MBT-2 (b, right) cells under normoxia or hypoxia 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<0.05.
