FIG 5.

Both dsVEGF706 and NcVEGF positively modify VEGF promoter activity and VEGF expression. (A) PC3 cells were cotransfected, in the absence or presence of dsRNA (control [dsCT] or dsVEGF706 [dsVEGF]), with a β-galactosidase-expressing vector (CMV-βgal) and with either empty (pGL3B) or VEGF promoter (from position −990 to position +145) containing luciferase vector (VEGF promoter). After 72 h, the cells were lysed, and both the luciferase and the β-galactosidase activities were measured. The relative luciferase and β-galactosidase activities are reported on a histogram. Values are means ± the SD of three independent experiments, with each sample run in triplicate. (B) PC3 cells were cotransfected with a β-galactosidase-expressing vector (CMV-βgal), with either empty (pGL3B) or VEGF promoter containing (VEGF promoter) the luciferase vector and with either the expression vector empty (3.1) or encoding either sense (sense NcVEGF) or antisense (antisense NcVEGF) NcVEGF transcripts. After 72 h, the cells were lysed, and both the luciferase and the β-galactosidase activities were measured. The relative luciferase and β-galactosidase activities are reported on a histogram. Values are means ± the SD of three independent experiments, with each sample run in triplicate. (C) PC3 cells transfected for 72 h with an expression vector either empty (3.1) or in which the CMV promoter drives the expression of either sense (sense NcVEGF; −990/−145 or −990/+163) or antisense (antisense NcVEGF; −145/−990 or +168/−990) NcVEGF transcripts. cDNAs were used to analyze VEGF expression with qPCR, and results are reported as the means ± the SD of two independent experiments, with each sample run in triplicate. 36B4 and GAPDH were used for normalization in qPCR experiments. *, P ≤ 0.05; **, P ≤ 0.005.