Figure 3

Generation of HIMF overexpressing endothelial cells. SVEC 4–10 cells were transfected with HIMF cDNA or control vector. Stable cell lines, SVEC-HIMF, along with their transfection control cells SVEC-Zeo, were screened based on resistance to Zeocin (400 μg/ml). Western blots with cell culture medium for HIMF and protein from cell lysate for VEGF (3A), immunofluorescence staining for Flk-1 (3B) and real-time RT-PCR with cell total RNA (3C) demonstrated that SVEC-HIMF cells have higher HIMF protein and mRNA levels than their parent (SVEC 4–10) and vector-transfection (SVEC-Zeo) counterparts. The levels of Flk-1, but not VEGF, in SVEC-HIMF were also increased significantly compared with those of their controls. The symbol (*) indicates a significant increase from parent controls (P < 0.05). Triplicate experiments were performed with essentially identical results (n = 3).