Adhesion-dependent nuclear movement is differentially regulated by the actin and MT cytoskeletons. (A) Diagram illustrating treatment of WT MKCs with the indicated drug before Ca²⁺ addition in B–D. (B) E-cadherin (E-cad; green), β-tubulin (magenta), actin (phalloidin, red), and nuclei (Hoechst) localization are shown. Diagrams illustrate the effect of drug treatment on nuclear position. (C and D) Plots of the nuclear centroid to cell centroid distance normalized to cell radius. n > 50 cells per condition (representative of three experiments). Nuclei in blebbistatin-treated cells remained located at the cell center. Nuclei in nocodazole-treated cells moved closer to adhesions than in DMSO-treated cells, which was rescued by blebbistatin treatment. (E) Diagram illustrating treatment of WT MKCs with the indicated drug after Ca²⁺ addition in F–H. (F) Staining as in B. (G and H) Quantification as in C and D. n > 50 cells per condition (representative of three experiments). Nuclear position was unaltered in blebbistatin-treated cells, whereas latrunculin A reduced nuclear movement. Nuclei in nocodazole-treated cells moved closer to adhesions than in control cells, which were partially rescued by blebbistatin. Asterisks denote indicated significance (**, P ≤ 0.01; ****, P ≤ 0.0001) as assessed by unpaired, two-tailed t test (C), ANOVA with Tukey’s posttest (D and H), or ANOVA with Dunnett’s posttest (G). All data plotted as box and whiskers plots; the bottom and top of the box display the 25th and 75th percentiles, whereas the central band represents the median. The whiskers indicate the minimum and maximum values, and the plus signs indicate the means. AU, arbitrary unit; Noco, nocodazole; Blebb, blebbistatin; Lat A, Latrunculin A.