Actomyosin contractility increases cortical tension during cell segregation at early time points. (A) Quantification of cellular elasticity (Pa) determined by AFM in mixed EPHB2 cells cultured alone and EPHRIN-B1:EPHB2 mixed cultures at 2 and 4 h after plating. By 4 h after plating, EPHB2:EPHRIN-B1 mixed cultures show increased stiffness compared with EPHB2 cells cultured alone. *, P < 0.05. (B) Representative images of EPHB2-GFP-LifeAct-mCherry (magenta) cells mixed with EPHB2-GFP (green) cells mixed or EPHRIN-B1- LifeAct-mCherry (magenta) cells at 2 h and 4 h when AFM was performed. Cells in EPH:EPHRIN mixed cultures are not yet segregated. Scale bars, 200 µm. (C) Quantification of cellular elasticity (Pa) determined by AFM in mixed EPHRIN-B1:EPHB2 cultures in the presence of DMSO or actomyosin contractility inhibitors Y27632 and ML7 after 24 h. In the presence of contractility inhibitors, cells show a decrease in stiffness compared with DMSO controls. Error bars represent mean with 95% CI. ****, P < 0.0001 (ANOVA followed by Dunnett’s multiple comparison test).