Rho kinase inhibition restores retraction fibers and reduces accumulation of f-actin at the cortex. (A) Control and TEM4-depleted cells were synchronized in mitosis after release from a double thymidine block and treated for 30 min with DMSO or the ROCK inhibitor, Y27632 (10 μM) prior to fixation. Cells were stained with Phalloidin-Atto 565 (magenta) and Hoechst (cyan). Scale bar = 5 μm. Orthogonal views are shown in the rightmost panel. (B) The percentage of cells from A with or without retraction fibers was quantified in metaphase cells in the presence of DMSO or Y27632. Data is mean ± SEM, n = 3 independent experiments, N ≥ 50 cells/condition. Statistical differences were measured by a One-way ANOVA and pairwise comparisons of protrusion-positive significance were determined by Tukey’s multiple comparison test. (C) Quantification of cortical actin intensity of cells from A. Data shown is mean ± SEM, n = 3 independent experiments, N ≥ 20 cells/condition. A one-way ANOVA test followed by Kruskal–Wallis for significance was performed followed by Dunn’s multiple comparisons test to determine P values. (D) Mitotic index in cells treated as in A. Data is shown from two representative experiments with ≥100 cells/condition. Individual datapoints represent individual fields of imaging within an experiment. A one-way ANOVA-Kruskal–Wallis for significance was performed followed by Dunn’s multiple comparisons test to determine P values.