Figure 8.

Mutation of HIF-1 binding site in VEGF promoter does not prevent its regulation by Akt. (A) Schematic of VEGF promoter constructs. The mut1 construct contains a mutation in the HIF-1 binding site of CGT to AAA as indicated (first box in solid black). The mut2 construct contains mutations in Sp1/AP-2 binding sites in the proximal promoter (second box in solid black). The mut3 construct contains mutations in both the HIF-1 binding site and Sp1/AP-2 region (fist and second boxes in solid black). (B) SF188 cells were cotransfected with (a) 0.2 μg of pCMV/myrAkt or pCMV6 (empty vector), (b) 2 μg of 1.5-kB wt VEGF, mut1, mut2, or mut3 vector, and (c) pSV-β-galactosidase (0.1 μg). Forty-eight hours later, dishes were harvested for both luciferase and β-galactosidase determination. (C) U87MG cells were cotransfected with (i) 2 μg of mut2 or mut3 vector and (ii) pSV-β-galactosidase (0.1 μg). Twenty-four hours later they were subjected to 1% O₂. Samples were harvested after 8 h in hypoxia. (D) U87MG cells were cotransfected with (a) pCMV6/Akt K179M (0.2 μg), which is a plasmid expressing dominant negative Akt, (b) 2 μg of either a 1.4-kB wt VEGF reporter or mut1, and (c) pSV-β-galactosidase (0.1 μg). Forty-eight hours later, dishes were harvested.