Figure 5

Promoter deletion assay for HIMF-induced VEGF expression in MLE-12 cells. MLE-12 cells were co-transfected with pRL-TK and each VEGF luciferase reporter construct (5A) for 24 h, then cells were incubated with HIMF protein (40 nmol/L) for another 24 h. Luciferase activity was measured and the firefly luciferase signal was normalized to the renilla luciferase signal for each individual well. (5B) Deletion of NF-κB binding site, but not HRE or binding sites for AP-1 and AP-2, completely abolished HIMF-induced VEGF promoter activity. Deletion of all cis-acting elements resulted in complete loss of induction of VEGF promoter activity. The symbol (*) indicates a significant increase from MLE-12 controls untreated with HIMF (P < 0.05). The symbol (#) indicates a significant decrease from MLE-12 transfected with pGL-VEGF and treated with HIMF (P < 0.05). Triplicate experiments were performed with essentially identical results.