Measurement of contractility markers in control and TEM4-depleted cells. (A) Circularity in control and TEM4-depleted metaphase cells, where 1 is considered a perfect circle. Data shown is mean ± SEM with ≥20 cells/condition, n = 3 independent experiments. A one-way ANOVA Kruskal–Wallis test followed by Dunn’s multiple comparison’s test was performed for analysis of significance. (B) Polarization microscopy of control and TEM4-depleted cells in interphase. Representative images and quantification optical retardance, n = 3. Scale bar = 10 μm. The graph illustrates retardance reading from individual cells (13–36 cells analyzed/condition/replicate acquired over 3 independent experiments). A one-way ANOVA test for significance was performed and the P values are shown from Holm-Šídák’s multiple comparisons test. (C) RhoA-GTP staining in control and TEM4-depleted cells. Fixed cells were stained with Phalloidin-Atto 565 (red), RhoA-GTP (green), and Hoechst (blue). Scale bar = 10 μm for the main image and 2 μm for the zoomed image. (D) pERM staining in control and TEM4 depleted cell. Representative IF images of cells stained with pERM (red) and Hoescht (blue) Data shown are mean ± SD, n = 2 independent experiments; N = between 18 and 22 cells/condition per experiment. Scale bar = 5 μm. Relative intensity of pERM signals is shown in the adjacent graph. A one-way ANOVA test for significance was performed and the P values shown from Tukey’s multiple comparisons test.