Actomyosin contractility drives increased cellular interfacial tension. (A) Representative images of cell doublets in agarose microwells. EPHRIN-B1-mCherry (magenta) and EPHB2-GFP (green) treated with vehicle (DMSO) or inhibitors. Scale bar, 20 µm. (B) Quantification of cell:cell contact angles 4 h after plating. In HEK293 media with DMSO, EPHB2:EPHRIN-B1 cell pairs show a decreased contact angle compared with EPHRIN-B1:EPHRIN-B1 homotypic cell pairs or EPHB2:EPHB2 homotypic cell pairs. Upon the addition of Y27632 and ML7 or the addition of blebbistatin, EPHB2:EPHRIN-B1 cell pairs no longer show diminished contact compared with EPHB2:EPHRIN-B1 cell pairs in media with DMSO control. Error bars represent mean ± SD. **, P < 0.01; ****, P < 0.0001 (ANOVA followed by Dunnett’s multiple comparison test). (C) Representative images of EPHRIN-B1-mCherry (magenta) cells and EPHB2-GFP (green) cells mixed and segregated after 24 h, when AFM was performed. White outline represents location of AFM machinery in each image. Scale bar, 200 µm. (D) Quantification of cellular elasticity (Pa) determined by AFM either when EPHRIN-B1 or EPHB2 cells were cultured alone or when these cells were mixed and allowed to segregate. Both cell types in sorted conditions show increased stiffness compared with when cultured alone. Error bars represent mean with 95% CI. ****, P < 0.0001 (ANOVA followed by Dunnett’s multiple comparison test).