TEM4 depletion leads to excessive cortical actin and loss of retraction fibers in mitosis. (A) Control, TEM4-21, and TEM4-22 expressing cells before and after 72 h of induction were synchronized in mitosis after release from double thymidine block, then fixed before staining with Phalloidin-Atto 565 (magenta) and Hoechst (cyan). Scale bar = 5 μm in the main figure and 1 μm in the zoomed image. (B) The percentage of cells with or without retraction fibers was quantified in metaphase after 72 h of TEM4 depletion. Mean percentages ± SEM is shown, n = 3 independent experiments, N ≥ 20 cells/condition. Statistical significance of the differences in protrusion-positive cells between control and TEM4-depleted cells was determined by the Brown-Forsythe and Welch ANOVA tests. (C) Actin intensity at the cortex in control and TEM4-depleted metaphase cells, means ± SEM (n = 3 independent experiments, ≥20 cells/condition) are shown. P values were determined by ANOVA-Kruskal–Wallis followed by Dunn’s multiple comparisons test. (D) Optical retardance measurements of control, TEM4-21, and TEM4-22 cells. N ≥ 90 cells per condition from three independent experiments. Bars indicate mean ± 95% CI. P was determined by a one-way ANOVA test followed by Tukey’s multiple comparison test. (E) Immunofluorescence of Rho A-GTP (green) and F-actin (red) at the cell cortex of control and TEM4-depleted cells. Main image scale bar = 10 μM, inset scale bar = 2 μM. (F) Quantification of the experiment in D. Rho A-GTP at the cortex was measured in maximum projections of cells stained for active Rho A. Data shows mean ± SEM, n = 3 independent experiments, N ≥ 20 cells/condition. A one-way ANOVA-Kruskal–Wallis test for significance was performed followed by Dunn’s multiple comparisons test. (G) Percentage of control cells and TEM4-21 cells rescued with TEM4^WT, TEM4^ΔABD, TEM4^Y1216A-exhibiting retraction fibers. Data shown are means ± SEM, n = 3 independent experiments, N ≥ 100 cells per condition. Statistical significance of the differences in protrusion-positive cells between control and TEM4 depleted cells was determined by a one-way ANOVA and Tukey’s multiple comparison tests. (H) Percentage of control cells, and TEM4-22 cells rescued with TEM4^WT, TEM4^ΔABD, TEM4^Y1216A exhibiting retraction fibers. Data shown are means ± SEM, n = 3 independent experiments, N ≥ 100 cells per condition. Statistical differences were measured by a One-way ANOVA and Tukey’s multiple comparison test for protrusion-positive cells.