EPHB2 cells increase homotypic contacts in response to EPH/EPHRIN signaling. (A) Cell segregation in mixed populations of HEK293 cells at 48 h. In the left panels, EPHB2-GFP (green) cells were mixed with EPHB2-GFP-LifeAct-mCherry (magenta) cells in either regular or low-Ca²⁺ media. In the right panels, cells EPHB2-GFP-LifeAct-mCherry were mixed with cells overexpressing EPHRIN-B1-LifeAct-mCherry (magenta) in regular HEK293 or low-Ca²⁺ media. Hoescht images shown to visualize nuclei. Yellow dashed lines outline EPHB2 cell patches. Scale bar, 200 µm. (B) Quantification of nuclear density for the conditions illustrated in (A). In both regular and low-Ca²⁺ media, EPHB2 cells have a significantly increased density. Column heights represent means of the technical replicates, and error bars represent SEM. **, P < 0.01; ****, P < 0.0001. (C) Cell segregation in mixed populations of HEK293 cells. EPHB2-GFP (green) cells were mixed with EPHRIN-B1-LifeAct-mCherry (magenta) cells and WT-LifeAct-BFP (blue) cells. Cell segregation robustly occurs, and WT-LifeAct-BFP cells do not intermix with EPHB2 cells. Scale bar, 200 µm.