Figure 4.
Pericentrin and CDK5RAP2 are crucial for spindle elongation and spindle bipolarization of one-centrosome cells.(A) Mitotic spindle structures upon siRNA treatment with or without 500 nM centrinone B. Green, red, and blue represent GT335, α-tubulin, and DNA, respectively. Z-projections: 5 planes, 0.3 µm apart. Scale bar, 5 µm. (B) Frequency of mitotic spindle structures after siRNA treatment against the indicated proteins in A. Values are presented as mean percentages. n > 86, data from two independent experiments were pooled. (C) Time-lapse observation of the structure of NuMA and microtubules upon siRNA treatment against the indicated proteins. Centrinone-treated one-centrosome HeLa cells expressing EGFP-centrin1 and mCherry-NuMA were observed with a 40× objective. Red, green, and gray represent mCherry-NuMA, EGFP-centrin1, and SiR-tubulin, respectively. Z-projections: 10 planes, 2.2 µm apart. Scale bar, 10 µm. Time zero corresponds to NEBD. Arrowheads indicate the separated two NuMA foci. (D) The time required for the initial establishment of two poles of NuMA in C. Line and error bars represent the mean and SD (n ≥ 60 cells from three independent experiments). Kruskal–Wallis test was used to determine the significance of the difference. n.s., not significantly different (P > 0.05). (E) Mitotic duration, the time required from NEBD to cytokinesis, in C. Line and error bars represent the mean and SD (n ≥ 60 cells from three independent experiments). Kruskal–Wallis test was used to determine the significance of the difference. *, P < 0.01; **, P < 0.001. (F) Table of the times from NEBD to cytokinesis in E. (G) Distribution of CEP192 in centriolar and acentriolar spindle poles in proTAME-treated one-centrosome cells. Green, red, and blue represent GT335, CEP192, and DNA, respectively. Z-projections: 21 sections, 0.5 µm apart. Scale bar, 10 µm. (H) Quantification of pole patterns in G. Values are presented as mean percentages from triplicates (n = 3, triplicates, n ≥ 20 cells for each assay). Error bars represent SD. (I) The signal intensity of CEP192 on acentrosomal spindle poles in G. Line and error bars represent the mean and SD (n = 3, triplicates, n ≥ 20 cells for each assay). Kruskal–Wallis test was used to determine the significance of the difference. ***, P < 0.0001. A.U., arbitrary units.

Pericentrin and CDK5RAP2 are crucial for spindle elongation and spindle bipolarization of one-centrosome cells. (A) Mitotic spindle structures upon siRNA treatment with or without 500 nM centrinone B. Green, red, and blue represent GT335, α-tubulin, and DNA, respectively. Z-projections: 5 planes, 0.3 µm apart. Scale bar, 5 µm. (B) Frequency of mitotic spindle structures after siRNA treatment against the indicated proteins in A. Values are presented as mean percentages. n > 86, data from two independent experiments were pooled. (C) Time-lapse observation of the structure of NuMA and microtubules upon siRNA treatment against the indicated proteins. Centrinone-treated one-centrosome HeLa cells expressing EGFP-centrin1 and mCherry-NuMA were observed with a 40× objective. Red, green, and gray represent mCherry-NuMA, EGFP-centrin1, and SiR-tubulin, respectively. Z-projections: 10 planes, 2.2 µm apart. Scale bar, 10 µm. Time zero corresponds to NEBD. Arrowheads indicate the separated two NuMA foci. (D) The time required for the initial establishment of two poles of NuMA in C. Line and error bars represent the mean and SD (n ≥ 60 cells from three independent experiments). Kruskal–Wallis test was used to determine the significance of the difference. n.s., not significantly different (P > 0.05). (E) Mitotic duration, the time required from NEBD to cytokinesis, in C. Line and error bars represent the mean and SD (n ≥ 60 cells from three independent experiments). Kruskal–Wallis test was used to determine the significance of the difference. *, P < 0.01; **, P < 0.001. (F) Table of the times from NEBD to cytokinesis in E. (G) Distribution of CEP192 in centriolar and acentriolar spindle poles in proTAME-treated one-centrosome cells. Green, red, and blue represent GT335, CEP192, and DNA, respectively. Z-projections: 21 sections, 0.5 µm apart. Scale bar, 10 µm. (H) Quantification of pole patterns in G. Values are presented as mean percentages from triplicates (n = 3, triplicates, n ≥ 20 cells for each assay). Error bars represent SD. (I) The signal intensity of CEP192 on acentrosomal spindle poles in G. Line and error bars represent the mean and SD (n = 3, triplicates, n ≥ 20 cells for each assay). Kruskal–Wallis test was used to determine the significance of the difference. ***, P < 0.0001. A.U., arbitrary units.

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