Fig. 6. VEGF reporter activity is mediated by the expression of HIF-1 and PI3K signaling. A, the VEGF reporter was cotransfected with pCMVßGal plasmids into PC-3 cells and incubated with MEM supplemented with 10% fetal bovine serum overnight. The cells were switched to serum-free medium in the presence or absence of 20 µM LY294002 and 200 nM insulin for 36 h. Relative Luc activity was determined by the ratio of Luc:ß-Gal activity and normalized to the value in the solvent DMSO control. B, VEGF reporter and pCMVßGal plasmids were cotransfected with vector or 0.6 or 1.2 µg of HIF-1NBAB plasmid, respectively, into PC-3 cells. After transfection, the cells were cultured and treated as indicated in A. C, VEGF reporter and pCMVßGal plasmids were cotransfected with pSG5 vector or vector carrying the PI3K dominant negative construct, p85iSH2N, or tumor suppressor PTEN. The PC-3 cells were maintained in growth medium after transfection. The relative Luc activity was determined 48 h after transfection as described above.