Figure S4.
Spindle midzone and astral microtubules are not affected by NuMA depletion. (A) Schematic representation of quantification method used for analyzing midzone microtubule fluorescence intensity. (B and C) IF analysis of HeLa cells that were either transfected with control siRNA (B), or siRNA directed against NuMA (C) for 60 h, were fixed and stained with antibodies directed against ⍺-tubulin (in green). The scale bar in the panel and following panels represent 10 μm. More than 30 anaphase cells were analyzed from two independent experiments, and the representative cells are shown here. (D) Midzone microtubule fluorescence intensity quantification for control siRNA, or NuMA siRNA was performed as described in A. ns- P > 0.05 as determined by two-tailed unpaired Student’s t test. n = 15 cells, error bars: SD. (E) Schematic representation of quantification method used for analyzing midzone enrichment for Cyk4 (F and G) or Ect2 (I and J). (F–K) IF analysis of HeLa cells that were either transfected with control siRNA (F and I), or siRNA directed against NuMA (G and J) for 60 h, were fixed and stained with antibodies directed against Cyk4 or Ect2 (in green) as indicated. More than 30 anaphase cells were analyzed from two independent experiments, and the representative cells are shown here. Quantification on the right represent midzone enrichment for Cyk4 (H) or Ect2 (K) for control siRNA, or NuMA siRNA as described in E. ns, P > 0.05 as determined by two-tailed unpaired Student’s t test. n = 15 cells, error bars: SD. (L) A schematic representation of anaphase cell and highlighting the midzone microtubules bundles created by Prc1 (Protein Regulator of Cytokinesis 1) homodimer. (M and N) IF analysis of HeLa cells that were either transfected with control siRNA (M), or siRNA directed against Prc1 (N) for 36 h were fixed and stained with antibodies directed against ⍺-tubulin (in green). More than 30 anaphase cells were analyzed from two independent experiments, and the representative cells are shown here. (O and P) Schematic representation of quantification method (O) used to determine the RhoA zone (in gray) and outcome of such analysis in cells transfected with control siRNA (P). n = 25 cells; error bars: SD. (Q and R) Schematic representation of quantification method (Q) used to determine the cytoplasmic RhoA intensity in arbitrary unit (au), and outcome of such analysis in cells transfected with control siRNA, NuMA siRNA, Prc1 siRNA, or NuMA and Prc1 siRNA (R). ns, P > 0.05 as determined by two-tailed unpaired Student’s t test. n = 10 cells in each condition; error bars: SD. (S and T) Representative IF image of HeLa Kyoto cell in anaphase fixed and stained with α-tubulin antibodies (gray). The areas used to analyze relative astral microtubules intensity is shown (S). Quantification of relative astral microtubule intensity in cells transfected with control siRNA (control), Prc1 siRNA, NuMA siRNA or NuMA and Prc1 siRNA (T). The intensities of the spindle (Ispindle) and the total cell (Itotal) were determined with ImageJ software. Relative astral MT intensity was calculated by ([Itotal−Ispindle]/Ispindle). ns, P > 0.05 as determined by two-tailed unpaired Student’s t test. n = 11 cells in each condition; error bars: SD.

Spindle midzone and astral microtubules are not affected by NuMA depletion. (A) Schematic representation of quantification method used for analyzing midzone microtubule fluorescence intensity. (B and C) IF analysis of HeLa cells that were either transfected with control siRNA (B), or siRNA directed against NuMA (C) for 60 h, were fixed and stained with antibodies directed against ⍺-tubulin (in green). The scale bar in the panel and following panels represent 10 μm. More than 30 anaphase cells were analyzed from two independent experiments, and the representative cells are shown here. (D) Midzone microtubule fluorescence intensity quantification for control siRNA, or NuMA siRNA was performed as described in A. ns- P > 0.05 as determined by two-tailed unpaired Student’s t test. n = 15 cells, error bars: SD. (E) Schematic representation of quantification method used for analyzing midzone enrichment for Cyk4 (F and G) or Ect2 (I and J). (F–K) IF analysis of HeLa cells that were either transfected with control siRNA (F and I), or siRNA directed against NuMA (G and J) for 60 h, were fixed and stained with antibodies directed against Cyk4 or Ect2 (in green) as indicated. More than 30 anaphase cells were analyzed from two independent experiments, and the representative cells are shown here. Quantification on the right represent midzone enrichment for Cyk4 (H) or Ect2 (K) for control siRNA, or NuMA siRNA as described in E. ns, P > 0.05 as determined by two-tailed unpaired Student’s t test. n = 15 cells, error bars: SD. (L) A schematic representation of anaphase cell and highlighting the midzone microtubules bundles created by Prc1 (Protein Regulator of Cytokinesis 1) homodimer. (M and N) IF analysis of HeLa cells that were either transfected with control siRNA (M), or siRNA directed against Prc1 (N) for 36 h were fixed and stained with antibodies directed against ⍺-tubulin (in green). More than 30 anaphase cells were analyzed from two independent experiments, and the representative cells are shown here. (O and P) Schematic representation of quantification method (O) used to determine the RhoA zone (in gray) and outcome of such analysis in cells transfected with control siRNA (P). n = 25 cells; error bars: SD. (Q and R) Schematic representation of quantification method (Q) used to determine the cytoplasmic RhoA intensity in arbitrary unit (au), and outcome of such analysis in cells transfected with control siRNA, NuMA siRNA, Prc1 siRNA, or NuMA and Prc1 siRNA (R). ns, P > 0.05 as determined by two-tailed unpaired Student’s t test. n = 10 cells in each condition; error bars: SD. (S and T) Representative IF image of HeLa Kyoto cell in anaphase fixed and stained with α-tubulin antibodies (gray). The areas used to analyze relative astral microtubules intensity is shown (S). Quantification of relative astral microtubule intensity in cells transfected with control siRNA (control), Prc1 siRNA, NuMA siRNA or NuMA and Prc1 siRNA (T). The intensities of the spindle (Ispindle) and the total cell (Itotal) were determined with ImageJ software. Relative astral MT intensity was calculated by ([Itotal−Ispindle]/Ispindle). ns, P > 0.05 as determined by two-tailed unpaired Student’s t test. n = 11 cells in each condition; error bars: SD.

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