Figure 5.
A pericentrin–TPX2 axis drives the formation of asymmetric spindles. (A) Immunofluorescence images of siCtrl and siTPX2-treated 1:1 RPE1 GFP-centrin1 cells stained with DAPI (blue), GFP-centrin1 (green), cenexin (magenta), and TPX2 (yellow) antibodies. (B) Quantification of the SAI of siCtrl and siTPX2-treated 1:1 RPE1 GFP-centrin1 cells. SAI means of 2.5 ± 5.9%, n = 74 cells, P = 0.0005, in siCtrl cells and 1.1 ± 9.2%, n = 102 cells, P = 0.2454, in siTPX2 cells. One-sample t tests were used for statistical analyses. (C) Quantification of the TPX2 asymmetry versus centrosome age in 2:2 RPE1 GFP-centrin1 WT and centriolin−/− cells. Relative TPX2 distribution means of 2.9 ± 6.5%, n = 36 cells, P = 0.0210, in WT cells and 0.1 ± 5%, n = 55 cells, P = 0.5917, in centriolin−/− cells. One-sample t tests were used for statistical analyses. (D) Left panels: immunofluorescence images of siCtrl, siPCNT, and siTPX2-treated 2:2 RPE1 GFP-centrin1 cells stained with DAPI (blue), GFP-centrin1 (green), TPX2 (yellow), and pericentrin (magenta) antibodies; right panels: immunofluorescence images of siCtrl, siPCNT, and sich-TOG–treated 2:2 RPE1 GFP-centrin1 cells stained with DAPI (blue), GFP-centrin1 (green), ch-TOG (yellow), and pericentrin (magenta) antibodies. Scale bars = 10 μm. (E) Quantification of TPX2 fluorescence intensity at spindle poles. Means of 100 ± 26.3%, n = 44 cells in siCtrl cells, 50.1 ± 32.7%, n = 45 cells, P < 0.0001 in siPCNT cells, and 27.6 ± 13.4%, n = 30 cells, P < 0.0001 in siTPX2-treated cells. One-way Kruskal–Wallis with Dunnett’s multiple comparisons tests. (F) Quantification of the relative protein distribution of ch-TOG in siCtrl-treated 1:1 RPE1 GFP-centrin1 mScarlet-cenexin cells. Mean of 2.9 ± 6.6%, n = 34 cells, P = 0.0249, one-sample Wilcoxon-test. (G) Quantification of the SAI of siCtrl and sich-TOG–treated 1:1 RPE1 Centrin1-GFP mScarlet-cenexin cells. SAI means of 2.8 ± 7.4%, n = 59 cells, P = 0.0057, in siCtrl cells and 1.5 ± 8.4%, n = 63 cells, P = 0.1587, in sich-TOG cells. One-sample t tests were used for statistical analyses. (H) Quantification of ch-TOG fluorescence intensity at spindle poles. Means of 100 ± 16.6%, n = 43 cells in siCtrl cells, 27.2 ± 8.3%, n = 41 cells, P < 0.0001 in siPCNT cells, and 44.2 ± 10%, n = 47 cells, P < 0.0001 in sich-TOG–treated cells. One-way Kruskal–Wallis with Dunnett’s multiple comparisons tests. ns = not significant; P ≤ 0.05 = *; P ≤ 0.01 = **; P ≤ 0.001 = ***; P ≤ 0.0001 = ****.

A pericentrin–TPX2 axis drives the formation of asymmetric spindles. (A) Immunofluorescence images of siCtrl and siTPX2-treated 1:1 RPE1 GFP-centrin1 cells stained with DAPI (blue), GFP-centrin1 (green), cenexin (magenta), and TPX2 (yellow) antibodies. (B) Quantification of the SAI of siCtrl and siTPX2-treated 1:1 RPE1 GFP-centrin1 cells. SAI means of 2.5 ± 5.9%, n = 74 cells, P = 0.0005, in siCtrl cells and 1.1 ± 9.2%, n = 102 cells, P = 0.2454, in siTPX2 cells. One-sample t tests were used for statistical analyses. (C) Quantification of the TPX2 asymmetry versus centrosome age in 2:2 RPE1 GFP-centrin1 WT and centriolin−/− cells. Relative TPX2 distribution means of 2.9 ± 6.5%, n = 36 cells, P = 0.0210, in WT cells and 0.1 ± 5%, n = 55 cells, P = 0.5917, in centriolin−/− cells. One-sample t tests were used for statistical analyses. (D) Left panels: immunofluorescence images of siCtrl, siPCNT, and siTPX2-treated 2:2 RPE1 GFP-centrin1 cells stained with DAPI (blue), GFP-centrin1 (green), TPX2 (yellow), and pericentrin (magenta) antibodies; right panels: immunofluorescence images of siCtrl, siPCNT, and sich-TOG–treated 2:2 RPE1 GFP-centrin1 cells stained with DAPI (blue), GFP-centrin1 (green), ch-TOG (yellow), and pericentrin (magenta) antibodies. Scale bars = 10 μm. (E) Quantification of TPX2 fluorescence intensity at spindle poles. Means of 100 ± 26.3%, n = 44 cells in siCtrl cells, 50.1 ± 32.7%, n = 45 cells, P < 0.0001 in siPCNT cells, and 27.6 ± 13.4%, n = 30 cells, P < 0.0001 in siTPX2-treated cells. One-way Kruskal–Wallis with Dunnett’s multiple comparisons tests. (F) Quantification of the relative protein distribution of ch-TOG in siCtrl-treated 1:1 RPE1 GFP-centrin1 mScarlet-cenexin cells. Mean of 2.9 ± 6.6%, n = 34 cells, P = 0.0249, one-sample Wilcoxon-test. (G) Quantification of the SAI of siCtrl and sich-TOG–treated 1:1 RPE1 Centrin1-GFP mScarlet-cenexin cells. SAI means of 2.8 ± 7.4%, n = 59 cells, P = 0.0057, in siCtrl cells and 1.5 ± 8.4%, n = 63 cells, P = 0.1587, in sich-TOG cells. One-sample t tests were used for statistical analyses. (H) Quantification of ch-TOG fluorescence intensity at spindle poles. Means of 100 ± 16.6%, n = 43 cells in siCtrl cells, 27.2 ± 8.3%, n = 41 cells, P < 0.0001 in siPCNT cells, and 44.2 ± 10%, n = 47 cells, P < 0.0001 in sich-TOG–treated cells. One-way Kruskal–Wallis with Dunnett’s multiple comparisons tests. ns = not significant; P ≤ 0.05 = *; P ≤ 0.01 = **; P ≤ 0.001 = ***; P ≤ 0.0001 = ****.

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