Figure 5
Stability of Hif-1α mRNA is increased in VEGF-KO cells. (A, B) Expression levels of Hif-1α mRNA in parental and VEGF-KO cell lines under normoxia and hypoxia. Cells were exposed to hypoxia (~0.1% O2) for 16 h. Hif-1α mRNA levels were determined by RT-qPCR (n=5, means ± S.D.). *P < 0.01. (C-F) Hif-1α mRNA stability was increased by HuR in VEGF-KO cells. Stability of Hif-1α (C, E) and Gapdh (D, F) mRNA was determined in cells transfected with the indicated siRNA in the presence of actinomycin D (Act. D) under hypoxia for 8 h, (n=5, means ± S.D.). *P < 0.01. (G, H) Knockdown efficiency of HuR mRNA. Cells were untransfected (none) or transfected with siRNA targeting HuR mRNA or control siRNA for 8 h, then they were exposed to hypoxia or normoxia for 16 h. HuR mRNA levels were determined by RT-qPCR (n=5, means ± S.D.). (I, J) VEGF promoter activity. Cells were transfected with a GFP reporter construct containing VEGF promoter sequence for 12 h, then they were exposed to hypoxia for additional 16 h. VEGF promoter activity was assessed by quantification of GFP reporter mRNA levels by RT-qPCR and normalized to the levels of Renilla luciferase mRNA (n=5, means ± S.D.). *P < 0.01.