EPH/EPHRIN signaling increases heterotypic interfacial tension. (A) Schematic for cell:cell contact angle measurements. (B) Representative images of cell doublets in agarose microwells. EPHRIN-B1-mCherry (magenta) and EPHB2-GFP (green). Scale bars, 20 µm. (C) Quantification of cell:cell contact angles 4 h after plating. EPHB2:EPHRIN-B1 cell pairs show a decreased contact angle or increased interfacial tension. Upon the addition of unclustered-EPHRIN-B1-Fc, to block EPH/EPHRIN signaling, this relative increase in interfacial tension between heterotypic cell pairs is diminished. (D) Individual frames from live imaging experiments (Videos 1, 2, and 3) at 10 min, 4 h, 8 h, and 12 h of live imaging, showing that cell:cell contacts are dynamic over time. EPHRIN-B1-mCherry (magenta) and EPHB2-GFP-LifeAct-mCherry (green). Scale bars, 20 µm. (E) HEK293 3D cell aggregates in circular agarose microwells (180 µm). Scale bars, 50 µm. Error bars represent mean ± SD. *, P < 0.05; ****, P < 0.0001 (ANOVA followed by Dunnett’s multiple comparison test).