IL-3 induces angiogenic cell expansion. (A) The number of colonies obtained by culturing angiogenic cells in EGM-2 or in EBM-2, with or without IL-3, for 4 or 12 days is reported. Data are the mean of 10 fields (± SD; *P < .05, EBM-2 + IL-3 vs EBM-2–IL-3 or vs EGM-2). Magnification, 10×. (B) Representative photomicrographs of colonies obtained by culturing angiogenic cells for 4 days in EBM-2 + IL-3 (panels i,ii), in EGM-2 (panel iii), or in EBM-2–IL-3 (panel iv). Magnification, 10× (panels i,iii,iv) and 40× (panel ii). (C) The percentage of angiogenic cells in the S phase was evaluated by FACS analysis in different culture conditions. (D) IL-3-cultured angiogenic cells were lysed and cell extracts were subjected to sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE). The filter was immunoblotted (IB) with anti–cyclin D1 and anti–β-actin antibodies, as indicated. (E) FACS analysis was performed after 4 days of IL-3 culture. Black lines, preimmune IgG used as negative control; gray lines, CD45, CD14, CD146, and CD105 markers. Three different experiments were performed with comparable results. Photomicrographs of cells were acquired with a Leica DM IL inverted contrasting microscope (Leica Microsystems, Wetzlar, Germany) equipped with 10×/20×/40× hiPlan objective lenses. Images were analyzed using Leica QWin software, version 3.