Pim-1 is required for vasculogenesis and angiogenesis in vitro. (A) Western blot analysis of Pim-1 silencing obtained by infecting ES cells with a lentivirus vector expressing Pim-1 siRNA. (B) FACS analysis of differentiated ES cells at 5.5 and 8.5 days using specific antibodies for each marker as indicated. Green lines indicate the positive cell population, and numbers above each curve indicate the percentage of positive cells compared with red lines, which represent negative controls. ES cells were infected with a lentivirus vector generating, under the control of the U6 promoter, siRNA for renilla luciferase gene (control) or the murine Pim-1. (C) HUVECs were infected with the lentiviral vector expressing siRNA to silence the renilla luciferase (control) or human PIM-1 under the control of the U6 polymerase promoter. Pim-1 expression in HUVECs was valued by Western blot at 2 hours after VEGF-A induction. (D) PIM-1 silencing affects both proliferation and migration of HUVECs in vitro. Vertical bars represent the fold induction as indicated. Proliferation and migration tests were performed as described.²⁴  The data are the results of 3 independent experiments. (E) Pim-1 silencing inhibited the angiogenesis assay in vitro. A representative experiment is shown. The assay was performed by plating HUVECs either infected with the mock vector or infected with the siRNA lentiviral vector on Matrigel, and HUVECs were induced with VEGF-A as described.²⁴  (F) PIM-1 silencing inhibited sprouting of ECs. A representative experiment is shown. The sprouting assay was performed as described.³⁵  Original magnification × 40.