SLK and ERMs promote cell rounding in metaphase. (A–J) Sphericity and mitotic rounding defects were measured using immunofluorescence 3D reconstitution after confocal microscopy in HEK293T cells transiently transfected with non-target siRNA (NT) or two independent siRNA targeting SLK (A–C; n > 55 cells) or a combination of three siRNA targeting each ERM (D–G; n > 55 cells), or transiently transfected with non-target siRNA (NT) or two independent siRNA targeting SLK and co-transfected with ezrinT567D-dsRed or ezrinT567A-dsRed constructs (H–J; n > 48 cells). (A, E, and I) Top panels show confocal planes (Top view), and lower panels show orthogonal views (Ortho view). Sphericity was measured as in Fig. 3 G (B, F, and J). Mitotic rounding defects were assessed by measuring the mean sphericity ratio of metaphase to interphase cell populations (C and G). Knock down efficiency and overexpression were assessed using immunoblots (D and H). Immunofluorescences (A, E, and I) and immunoblots (D and H) are representative of three independent experiments. Quantifications of sphericity and sphericity ratio of metaphase to interphase populations represent the mean ± SD of three independent experiments. Dots represent individual cells (B, F, and J) or independent experiments (C and G). P values were calculated using Holm-Sidak’s multiple comparisons test with a single pooled variance (B, F, and J) or using a two-tailed paired t test (C and G). *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Numbers associated with Western blots indicate molecular weight in kD. Source data are available for this figure: SourceData F5.