Figure 2. DEK controls VEGF expression in HIF-1α-dependent and -independent manners.

A. Luciferase reporter assays in MCF-7 breast cancer cells cotransfected with the VEGF-Luc reporter and DEK shRNA1 or control shRNA. At 24 h post-transfection, cells were exposed to either normoxic (20% O₂) or hypoxic (1% O₂) conditions for another 24 h. Cells were harvested for determination of VEGF-Luc reporter activity. Representative immunoblot shows the expression of DEK. B. Real-time RT-PCR analysis of VEGF₁₆₅ expression in MCF-7 cells stably infected with lentivirus carrying DEK shRNA1 or control shRNA. Cells were exposed to either normoxic (20% O₂) or hypoxic (1% O₂) conditions for 24 h before collected for immunoblot. Representative immunoblot indicates the expression of DEK. C. ELISA analyses of the VEGF concentration in cell supernatants from MCF-7 cells stably infected and treated as in B. D. Luciferase reporter assays in MCF-7 cells cotransfected with the VEGF-Luc reporter, DEK and HIF-1α shRNA as indicated. Cells were treated and analyzed as in A. Representative immunoblot shows the expression of DEK and HIF-1α. E. Real-time RT-PCR was performed to analyze VEGF₁₆₅ expression in MCF-7 cells stably infected with lentivirus carrying DEK and HIF-1α shRNA as indicated. Cells were treated and analyzed as in B. Representative immunoblot shows the expression of DEK and HIF-1α. F. ELISA assay was performed to analyze VEGF concentration in cell supernatants from MCF-7 cells stably infected and treated as in E. All values shown are mean ± SD of triplicate measurements and have been repeated 3 times with similar results. The average of triplicate measurements of the control under normoxia condition was set as 1, and all other groups were compared with the control under normoxia condition. *P < 0.05, **P < 0.01 versus control shRNA or control shRNA plus empty vector.