Lymphatic endothelial cells expressing Dll4 show an advantage in competition for the tip cell position. (A) Schematic representation of the mouse Dll4-EGFP fusion protein. (B-G) LECs transduced with mDll4-ECTM-EGFP or empty retrovirus were prelabeled with Green (green) or Orange (red) CellTracker (CT), respectively, and mixed in a 1:1 ratio to form the spheroids, which were treated with VEGF (100 ng/mL) or VEGF-C (100 ng/mL) for 48 hours. The spheroids were subsequently stained for mDll4 (white) and DAPI (blue). The arrows indicate Dll4+ tip cells. (D,G) Low-power magnification of the spheroids in panels B,C,E and F (□). Scale bars, 25 μm in panels B,C,E and F and 100 μm in panels D and G. (H) The percentage of mDll4-positive tip cells (white cells in panels A-D) out of the total tip cells was quantified and plotted against their expected values. The figure summarizes data from 2-3 independent experiments with a total of n = 122 for VEGF and n = 229 for VEGF-C treatments. A 2-tailed binomial test was used for statistical analysis. **P < .01; ***P < .001.