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. 2022 Apr 28;221(6):e202109065. doi: 10.1083/jcb.202109065

Figure 7.

Figure 7.

Both GEF-H1 and Ect2 regulate ERM phosphorylation and cell rounding in metaphase. (A and B) Immunofluorescence of HEK293T cells transiently transfected with non-target siRNA (NT) or two independent siRNA targeting Ect2 (A). Metaphase and interphase cells were identified based on DNA staining (DAPI, blue; A; arrowheads indicate metaphase cells), and p-ERM (green) signal intensity at the plasma membrane was quantified and normalized to interphase cells transfected with non-target siRNA (B; n > 60 cells). (C and D) Sphericity and mitotic rounding defects were measured using immunofluorescence 3D reconstitution after confocal microscopy (see Fig. S5 B) in HEK293T cells transiently transfected with non-target siRNA (NT) or two independent siRNA targeting Ect2. Sphericity was measured as in Fig. 3 G (C). Mitotic rounding defects were assessed by measuring the mean sphericity ratio of metaphase to interphase cell populations (D). (n > 55 cells). (E and F) Immunofluorescence of HEK293T cells transiently transfected with non-target siRNA (NT), siRNA targeting GEF-H1 and/or siRNA targeting Ect2 (E). Metaphase and interphase cells were identified based on DNA staining (DAPI, blue; E; arrowheads indicate metaphase cells), and p-ERM signal intensity at the plasma membrane was quantified and normalized to interphase cells transfected with non-target siRNA (F). Quantifications of p-ERM signal intensity of cells transfected with non-target siRNA or siRNA #1 targeting Ect2 (Ect2 #1; F) were already shown in B (n > 45 cells). (G–K) Sphericity and mitotic rounding defects were measured using immunofluorescence 3D reconstitution after confocal microscopy in HEK293T cells transiently transfected with non-target siRNA (NT), siRNA targeting GEF-H1 and/or siRNA targeting Ect2 (G–I; n > 55 cells), or two independent siRNA targeting SLK and incubated with vehicle (DMSO) or 10 µM Y-27632 for 4 h (J and K; n > 55 cells). Sphericity was measured as in Fig. 3 G (H and K). Mitotic rounding defects were assessed by measuring the mean sphericity ratio of metaphase to interphase cell populations (I). Quantifications of sphericity of cells transiently transfected with non-target siRNA or siRNA #1 targeting Ect2 (Ect2 #1; H) were already shown in C. Quantifications of sphericity of metaphase cells transiently transfected with non-target siRNA or siRNA targeting SLK and treated with vehicle (K) were already shown in Fig. 5 B. (G and J) Top panels show confocal planes (Top view), and lower panels show orthogonal views (Ortho view). I, interphase; M, metaphase. Immunofluorescences (A, E, G, and J) are representative of three independent experiments. Quantifications of p-ERM, sphericity, and sphericity ratio of metaphase-to-interphase populations represent the mean ± SD of three independent experiments. Dots represent individual cells (B, C, F, H, and K) or independent experiments (D and I). P values were calculated using Holm-Sidak’s multiple comparisons test with a single pooled variance (B, C, F, H, and K) or using a two-tailed paired t test (D and I). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.