Figure 5

Promoter deletion assay for HIMF-induced Flk-1 expression in SVEC 4–10 cells. SVEC 4–10 cells were co-transfected with pRL-TK and each Flk-1 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) HIMF induced high Flk-1 promoter activities within cells transfected with pGL-Flk-1 (-258/+299), pGL-Flk-1 (-96/+299) or pGL-Flk-1 (-71/+299), which contain one NF-κB binding site within Flk-1 promoter. Deletion of binding sites for E-Box, Sp1 and AP-2 partially attenuated the transcription activity. In addition, deletion of NF-κB binding site completely abolished HIMF-induced Flk-1 promoter activity. (5C) Further mutation or deletion NF-κB binding site within pGL-Flk-1 (-71/+299) abolished HIMF-induced Flk-1 transcripts in SVEC 4–10 cells. The symbol (*) indicates a significant increase from SVEC 4–10 controls treated without HIMF (P < 0.05). The symbol (#) indicates a significant decrease from SVEC 4–10 transfected with pGL-Flk-1 (-258/+299) or pGL-Flk-1 (-71/+299) and treated with HIMF (P < 0.05). Triplicate experiments were performed with essentially identical results (n = 3).