Figure 2

Endothelial cells induce various branching phenotypes in a time-dependent fashion through soluble factors. A. Time series of VA10/HUVEC co-culture in Matrigel. Days after seeding are indicated. A colony undergoing branching morphogenesis is shown during days 8-17 after seeding. The colonies can be categorized as follows: Day 8 spherical; day 9 budding; days 10-12 early branching; days 13-17 complex branching. Scale bars 100 μm. B. The number of colonies per well over time in A549 rBM culture (crosses), VA10 culture (triangles), and co-culture of HUVEC cells with A549 (empty boxes) or VA10 cell (solid circles). C. The number of branching colonies over time in co-culture of VA10 and HUVEC and VA10 cells cultured alone. The percentage of total colonies is shown above each point. Error bars indicate standard deviation. D. The proportion of the different colony forms at several time-points. Irregular colonies do not show any sign of regular branching but cannot be categorized as spherical. The total number of colonies is indicated at the base of each column. E. Culture of endothelial cells on transwell (TW) filters suspended above rBM gels containing VA10 cells. Total and branching epithelial colonies are indicated. Error bars indicate standard deviation. TW: HUVEC kept on filters throughout experiment. TW off day 6: Transwells were removed on day 6. Control: Negative control without HUVEC in TW.