Figure 4

HIMF increases the transcription activities, but not mRNA stability, of Flk-1 in SVEC 4–10 cells. (4A) SVEC 4–10, SVEC-zeo and SVEC-HIMF cells were co-transfected with pGL-Flk-1 (-258/+299) and pRL-TK. Twenty-four hours later, cells were lysed with passive lysis buffer, and luciferase activity was measured according to the dual-luciferase assay manual. The results indicated that SVEC-HIMF cells have higher Flk-1 transcription activities than those of their controls. (4B) SVEC 4–10 cells were co-transfected with pGL-Flk-1 (-258/+299) and pRL-TK. Twenty-four hours later, the cells were incubated with HIMF protein as indicated. Then, cells were lysed with passive lysis buffer, and luciferase activity was measured according to the dual-luciferase assay manual. The time-course study demonstrated that HIMF (40 nmol/L)-induced Flk-1 transcription is detectable at 6 h, and persisted for 24 h. After incubation with 10–80 nmol/L of HIMF, Flk-1 promoter activities in SVEC 4–10 were enhanced in a dose-dependent manner. (4C) SVEC 4–10 were treated with different concentrations of HIMF and incubated with 5 μg/ml of Actinomycin D for 6, 12 and 24 h. Real-time RT-PCR indicated that HIMF did not prevent Flk-1 degradation when treated with Actinomycin D in SVEC 4–10 cells. The symbol (*) indicates a significant increase from SVEC 4–10 controls without HIMF (P < 0.05). Triplicate experiments were performed with essentially identical results (n = 3).