Cell segregation is abolished by dual inhibition of ROCK and MLCK. (A) Cell segregation in mixed populations of HEK293 cells. In the far-left panels EPHB2-GFP (green) cells were mixed with EPHB2-GFP-LifeAct-mCherry (magenta) cells. In the rest of the panels, EPHB2-GFP-LifeAct-mCherry (green) cells were mixed with EPHRIN-B1-mCherry (magenta) cells and treated with vehicle (DMSO) or inhibitors or cultured in low-Ca²⁺ media to determine effect on cell segregation. For images at 48 h, some inhibitors were added or removed at 24 h. Scale bars, 200 µm. (B) Quantification of cell segregation for several of the conditions illustrated in (A). Dual inhibition of ROCK and MLCK abolished cell segregation. (C) Quantification of cell segregation in the absence of calcium. Cell segregation was undisrupted by the lack of calcium in the media. (D) Quantification of cell segregation upon the addition or removal of inhibitors. Cell segregation is still able to occur upon removal of ROCK and MLCK inhibitors after 24 h, and addition of these inhibitors to media at 24 h after sorting disrupts cell segregation. Cell segregation was quantified using the nearest neighbor method. Column heights represent means of the technical replicates, and error bars represent SEM. Results are representative of three experiments. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 versus (B) EPHB2 + EPHRIN-B1 + DMSO condition, (C) EPHB2 + EPHRIN-B1 regular media condition, and (D) EPHB2 + EPHRIN-B1 + DMSO condition at 48 h (ANOVA followed by Dunnett’s multiple comparison test).