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

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© 2022 Leguay et al.

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Figure S4. — Microtubule dissociation at mitotic entry regulates cell rounding depending on GEF-H1 and RhoA. (A and B) Cell height and cell shape within the x/y plane were measured in HEK293T cells incubated with vehicle (DMSO) or taxol for 90 min (A and B; n > 60 cells). (C–F) Sphericity (D), cell height (E), and cell shape within the x/y plane (F) were measured using immunofluorescence 3D reconstitution after confocal microscopy (C) in HEK293T metaphase cells treated with vehicle (DMSO) or 10 nM taxol for 15 min. Sphericity was measured as in Fig. 3 G (D). (C) Top panels show confocal planes (Top view), and lower panels show orthogonal views (Ortho view; n > 45 cells). (G and H) Cell height (G) and cell shape within the x/y plane (H) were measured in HEK293T cells transiently transfected with non-target siRNA (NT) or two independent siRNA targeting GEF-H1 (n > 50 cells). (I–M) Sphericity (J), cell height (K), cell shape within the x/y plane (L), and mitotic rounding defects (M) were measured using immunofluorescence 3D reconstitution after confocal microscopy (I) in HEK293T cells stably expressing non-target shRNA (NT) or two independent shRNA targeting GEF-H1. Sphericity was measured as in Fig. 3 G (F). Mitotic rounding defects were assessed by measuring the mean sphericity ratio of metaphase-to-interphase cell populations (M). (I) Top panels show confocal planes (Top view), and lower panels show orthogonal views (Ortho view; n > 70 cells). (N–Q) Cell height and cell shape within the x/y plane were measured in HEK293T cells incubated with vehicle (water) or 1 µg/ml C3 transferase for 6 h (N and O; n > 50 cells), or incubated with vehicle (DMSO), 10 nM taxol for 90 min and/or 1 µg/ml Rho activator II (CN03) for 1 h (P and Q; n > 50 cells). I, interphase; M, metaphase. Immunofluorescences (C and I) are representative of at least two independent experiments. All quantifications represent the mean ± SD of at least two independent experiments. Dots represent individual cells (A, B, D–H, J–L, and N–Q) or independent experiments (M). P values were calculated using Holm-Sidak’s multiple comparisons test with a single pooled variance (A, B, G, H, J–L, and N–Q), two-tailed unpaired t test (D–F) or two-tailed paired t test (M). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.