EPHB2 cells increase homotypic contacts due to high cell:medium cortical tension. (A) Cell segregation in mixed populations of HEK293 cells. In the left panels, EPHB2-GFP (green) cells were mixed EPHB2-GFP-LifeAct-mCherry (magenta) cells in either regular or low-Ca²⁺ media. In the right panels, EPHB2-GFP-LifeAct-mCherry (green) cells were mixed with EPHRIN-B1-LifeAct-mCherry (magenta) cells 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) Representative images of cell doublets in agarose microwells. EPHB2-GFP (green). Scale bar, 20 µm. (D) Quantification of cell:cell contact angles in the absence of calcium. Cell:cell contacts diminish in the absence of calcium; however, EPHB2:EPHB2 homotypic contacts are somewhat retained. In low-Ca²⁺ media with the addition of blebbistatin, EPHB2:EPHB2 contacts are diminished. Dashed lines indicate average cell:cell contact angles in regular media conditions. Error bars represent mean ± SD. ****, P < 0.0001.