SCF, IL-3 and SDF-1α synergistically act to promote EC tube morphogenesis in the presence or absence of pericytes and function downstream of VEGF-A/FGF-2 EC priming events. (A) SCF, IL-3, and SDF-1α were each provided as morphogenic stimuli for ECs individually or in different combinations and compared with control conditions in 3D collagen matrices. Total EC tube area was quantitated for each condition after 72 hours of culture (n ≥ 10; P ≤ .01). (B) Representative images of control versus SCF/IL-3/SDF-1α–stimulated cultures are shown (Bar equals 100 μm, left) as well as an electron micrograph of EC tube assembly (10 μm, right; n ≥ 10; P ≤ .01). (C) EC-pericyte morphogenic coculture assays were established in the presence of the hematopoietic cytokines individually or in different combinations and compared with control conditions in 3D collagen matrices. EC tube areas were quantified after both 72 and 120 hours. (D) Representative images of the cocultures under control versus SCF/IL-3/SDF-1α–stimulated conditions are shown (bar equals 50 μm), along with an electron micrograph of these cultures demonstrating the relationship between the ECs and pericytes (bar equals 2 μm). (E) EC-only morphogenic assays were established in the presence of each hematopoietic cytokine alone or the 3 factors together (All) versus controls. Cell lysates were collected at day 3 and Western blot analysis performed to assess kinase signaling and protein expression changes that correlate with the ability of hematopoietic cytokines to stimulate tube morphogenesis, and determine synergistically acting promorphogenic signaling pathways. (F) A schematic depicting a 2-step process of EC priming versus morphogenic cues. (G) E6 Quail vitelline vein explants primed with VEGF-A/FGF-2 and placed into morphogenic assays containing either VEGF-A or the combined hematopoietic cytokines. *Significance over control. Fixed cultures were immunostained with QH-1 antibodies. Arrows indicate the vessel explant border (n ≥ 10; P ≤ .01).