Fig. 1. Growth characteristics and phenotypic changes. (A) Time course of growth with HUVEC and GFP-U87 in physical contact in co-culture. Cells were plated at a 1:5 U87:HUVEC ratio in complete media. , HUVEC monoculture; , HUVEC co-culture; , U87 monoculture; , U87 co-culture. (B) Growth after 48 hours with cells physically separated on Transwell inserts (0.4 µm pores). HUVECs established on inserts with either HUVECs (M) or GFP-U87 cells (C) added to wells in starvation medium. (C) Development of angiogenic phenotype in HUVECs cocultured with GFP-U87 cells. 0 hours is HUVEC monoculture at the start of the experiment, with 5 hour, 12 hour and 24 hour time points showing elongation (insert, 5 hours) and migration of cells to form a morphologically distinct phenotype.