Fig. 2. Effect of cell-cell contact on proliferation when spreading is controlled. (A) Schematic outline of method used to pattern substrates to control cell spreading and cell-cell contact simultaneously. (B) Differential interference contrast images of single cells or pairs of cells in agarose wells of 750 µm²/half (left two images) and 1000 µm²/half (right two images). Bovine pulmonary artery and adrenal microvascular endothelial cells were G₀-synchronized and cultured on arrays of wells for 24 hours and fixed for analysis. Cells distributed randomly as single cells and pairs of cells in the wells. (C) Immunofluorescence images of pairs of cells in wells of 750 µm²/half (top images) or monolayers (bottom images) stained for VE-cadherin (VEcad) or ß-catenin (ßcat). Both VE-cadherin and ß-catenin specifically localized to the zone of contact. Broken lines (white) indicate the borders of the wells. (D) Graph of percentage of cells entering S phase (incorporating BrdU) for single cells and pairs of cells in both sizes of wells. (E) Graph of percentage of cells entering S phase for cells in wells of 750 µm²/half treated with VE-cadherin antibody. Similar results were seen with both types of endothelial cells analyzed. Error bars represent the s.d., with (*) P<0.05 relative to single cells (D) or controls (E) as calculated by Student's t-test. (F) Immunofluorescence images of pairs of cells in wells treated with control (top) and anti-VE-cadherin (bottom) antibodies and stained for VE-cadherin (VEcad) or ß-catenin (ßcat). (G) Phase contrast images of endothelial cells treated with control (left) or anti-VE-cadherin (right) antibodies. Scale bars: 25 µm.