Figure 4.

VDU2 enhances HIF-1α-mediated HRE activity and VEGF expression. (A) Regulation of HIF-1α-mediated HRE luciferase reporter gene activity by VDU2. Various combinations of expression plasmids as indicated with 300 ng of pRE–tk–LUC and 10 ng of pRLSV40–LUC were transfected into HEK293 cells. At 24 h after transfection, cells were collected and measured in a dual-luciferase assay. (B) Regulation of HIF-1α-mediated VEGF protein production by VDU2. Various combinations of expression plasmids as indicated were transfected into HEK293 cells. At 36 h after transfection, aliquots of medium were collected for VEGF ELISA. Histograms represent the results of three independent experiments. (C) Effects of overexpression of VDU2 on VEGF mRNA levels. HEK293 cells stably transfected with GFP–VDU2 or GFP were cultured under normoxia or hypoxia (1% O₂). VEGF mRNA levels were determined by real-time PCR. (D) Downregulation of HRE luciferase reporter gene activity in HEK293 cells stably transfected with VDU2 by VDU2–RNAi-1406 under hypoxia. Various combinations of expression plasmids as indicated with the dual reporter system were transfected into HEK293 cells stably transfected with GFP–VDU2. At 24 h after transfection, cells were cultured under normoxia or hypoxia (1% O₂) overnight and collected for a dual-luciferase assay.