Figure S1.
Formin, Rock, and Myosin II are not critical for NuMA exclusion from equatorial membrane. (A–D) Assessing the depletion efficiency of siRNA against Cyk4, Mklp1, Ect2, or Anillin by immunoblot analysis. Mitotically synchronized protein extracts made from HeLa Kyoto cells were either transfected with control siRNA or siRNA-against above-mentioned genes for 60 h. The resulting blot was probed with antibodies directed against Cyk4 (A), Mklp1 (B), Ect2 (C), or Anillin (D). Antibodies against β-actin were used for loading control. In this and other Figure panels for immunoblot analysis, the molecular mass is indicated in kilodaltons (kD) and is shown on the left. (E–H) Immunofluorescence (IF) analysis of HeLa cells that were either transfected with control siRNA (E) siRNA-against mDia (Formin; F) for 60 h, treated with DMSO (Control; G), or with Y-27632 (H) for 12 h to inactivate Rho-associated protein kinase (Rock). Cells were fixed thereafter and stained with anti-p150Glued antibodies (green). In this, and other IF-analysis panels DNA is shown in blue. Multinucleation percentage (%) is shown in the figure panel. (n > 500 cells were analyzed from two independent experiments). The scale bar in this panel and the following panels represent 10 μm. (I–L) HeLa cells transfected with control siRNA (I), siRNA against mDia (J) for 60 h, treated with DMSO (Control; K), or treated with Y-27632 (L) for 1 h to inactivate Rock in anaphase. Cells were fixed and thereafter costained with anti-NuMA (green) and anti-p150Glued (red) antibodies. More than 50 cells from two independent experiments were analyzed, and the representative cells are shown here. (M) Synchronization scheme for myosin II inactivation using PNBB to analyze cells in anaphase for NuMA and p150Glued localization, and after 12 h for assessing cytokinesis failure. (N and O) IF analysis of mitotically synchronized populations of HeLa cells that were either treated with DMSO (Control; N) or PNBB (O) for 12 h. Thereafter, cells were fixed and stained with anti-p150Glued antibodies (green). Multinucleation percentage (%) is shown in the figure panel. (n > 500 cells were analyzed from two independent experiments). (P–S) HeLa cells in telophase (P and Q) or during early anaphase (R and S) that were either treated with DMSO (Control) or PNBB for 30 min. Cells were fixed and costained with anti-NuMA (green) and anti-p150Glued (red) antibodies. Please note that during telophase (P and Q), NuMA has localized to the nucleus, but the cleavage furrow has not formed because of myosin II inactivation. n > 50 cells, from two independent experiments, and the representative cells are shown here. (T and U) Confocal live-imaging analysis of HeLa Kyoto cells stably expressing GFP-DHC1 that were transfected with control siRNA (T) or siRNA against Ect2 (U). The recording was started 26 h post-transfection for control and Ect2 siRNA. “0 min” time-point represents metaphase to anaphase transition. Yellow arrowheads depict GFP-DHC1 localization at the equatorial membrane. Representative images from the time-lapse recording are shown here (n > 10 cells). Source data are available for this figure: SourceData FS1.

Formin, Rock, and Myosin II are not critical for NuMA exclusion from equatorial membrane. (A–D) Assessing the depletion efficiency of siRNA against Cyk4, Mklp1, Ect2, or Anillin by immunoblot analysis. Mitotically synchronized protein extracts made from HeLa Kyoto cells were either transfected with control siRNA or siRNA-against above-mentioned genes for 60 h. The resulting blot was probed with antibodies directed against Cyk4 (A), Mklp1 (B), Ect2 (C), or Anillin (D). Antibodies against β-actin were used for loading control. In this and other Figure panels for immunoblot analysis, the molecular mass is indicated in kilodaltons (kD) and is shown on the left. (E–H) Immunofluorescence (IF) analysis of HeLa cells that were either transfected with control siRNA (E) siRNA-against mDia (Formin; F) for 60 h, treated with DMSO (Control; G), or with Y-27632 (H) for 12 h to inactivate Rho-associated protein kinase (Rock). Cells were fixed thereafter and stained with anti-p150Glued antibodies (green). In this, and other IF-analysis panels DNA is shown in blue. Multinucleation percentage (%) is shown in the figure panel. (n > 500 cells were analyzed from two independent experiments). The scale bar in this panel and the following panels represent 10 μm. (I–L) HeLa cells transfected with control siRNA (I), siRNA against mDia (J) for 60 h, treated with DMSO (Control; K), or treated with Y-27632 (L) for 1 h to inactivate Rock in anaphase. Cells were fixed and thereafter costained with anti-NuMA (green) and anti-p150Glued (red) antibodies. More than 50 cells from two independent experiments were analyzed, and the representative cells are shown here. (M) Synchronization scheme for myosin II inactivation using PNBB to analyze cells in anaphase for NuMA and p150Glued localization, and after 12 h for assessing cytokinesis failure. (N and O) IF analysis of mitotically synchronized populations of HeLa cells that were either treated with DMSO (Control; N) or PNBB (O) for 12 h. Thereafter, cells were fixed and stained with anti-p150Glued antibodies (green). Multinucleation percentage (%) is shown in the figure panel. (n > 500 cells were analyzed from two independent experiments). (P–S) HeLa cells in telophase (P and Q) or during early anaphase (R and S) that were either treated with DMSO (Control) or PNBB for 30 min. Cells were fixed and costained with anti-NuMA (green) and anti-p150Glued (red) antibodies. Please note that during telophase (P and Q), NuMA has localized to the nucleus, but the cleavage furrow has not formed because of myosin II inactivation. n > 50 cells, from two independent experiments, and the representative cells are shown here. (T and U) Confocal live-imaging analysis of HeLa Kyoto cells stably expressing GFP-DHC1 that were transfected with control siRNA (T) or siRNA against Ect2 (U). The recording was started 26 h post-transfection for control and Ect2 siRNA. “0 min” time-point represents metaphase to anaphase transition. Yellow arrowheads depict GFP-DHC1 localization at the equatorial membrane. Representative images from the time-lapse recording are shown here (n > 10 cells). Source data are available for this figure: SourceData FS1.

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