Cells under dual ROCK and MLCK inhibition remain motile. (A) Cell tracking analysis of overall EPHB2 cell movement over 16 h. EPHB2 cells in segregation conditions migrate less than when mixed with other EPHB2-expressing cells; however, addition of Y27632 and ML7 does not diminish migration from what is seen in cell segregation conditions. Error bars represent mean ± SEM. (B) Representative images of WT HEK293 cells in regular media and in low-Ca²⁺ media. Cell contact is dramatically reduced in the absence of calcium. Scale bar, 20 μm. (C) Quantification of cell segregation in the presence of mitomycin C. Inhibition of cellular proliferation does not block cell segregation. Column heights represent means of the technical replicates, and error bars represent SEM. (D) Representative images of cell segregation in mixed population of HEK293 cells in the presence of mitomycin C inhibitor. Scale bar, 200 µm. ****, P < 0.0001.