Figure 6.
The mitotic kinase Plk1 drives the enrichment of PCM proteins at old centrosomes. (A) Immunofluorescence images of WT RPE1 GFP-centrin1 G2 and prophase cells stained with DAPI (blue), centrin1-GFP (green), cenexin (yellow), and pericentrin (magenta) antibodies. Scale bars = 5 μm. (B) Quantification of the relative pericentrin distribution at centrosomes versus centrosome age in G2 and prophase WT RPE1 GFP-centrin1 cells. Means of 9.2 ± 12.9%, n = 36 cells, P = 0.0001 in G2 and 9.5 ± 9.3%, n = 29 cells, P < 0.0001 in prophase. One-sample t tests were used for statistical analyses. (C) Immunofluorescence images of WT RPE1 GFP-centrin1 G2 and prophase cells stained with DAPI (blue), centrin1-GFP (green), cenexin (yellow), and γ-tubulin (magenta) antibodies. Scale bars = 5 μm. (D) Quantification of the relative γ-tubulin distribution at centrosomes versus centrosome age in G2 and prophase WT RPE1 GFP-centrin1 cells. Means of 3.6 ± 16.8%, n = 50 cells, P = 0.1375 in G2 and 7.3 ± 10.8%, n = 53 cells, P < 0.0001 in prophase. One-sample t tests were used for statistical analyses. (E) Immunofluorescence images of WT RPE1 GFP-centrin1 prophase and metaphase cells stained with DAPI (blue), GFP-centrin1 (green), centriolin (yellow), and Plk1 (magenta) antibodies. Scale bars = 10 μm. (F) Quantification of the relative Plk1 distribution at centrosomes versus centrosome age in prophase and metaphase cells. Means of 10.9 ± 20.8%, n = 47 cells, P = 0.0008 for G2 cells and 6.6 ± 10.8%, n = 35 cells, P = 0.0009 for prophase cells. One-sample t tests were used for statistical analyses. (G) Immunofluorescence images of 1:1 RPE1 Centrin1-GFP cells treated with DMSO (control condition) or 50 nM Bi2536 (Plk1 inhibitor). Cells are stained with DAPI (blue), centrin1-GFP (green), cenexin (yellow), and γ-tubulin (magenta) antibodies. Scale bar = 10 μm. (H) Quantification of spindle size measurements in DMSO- and BI2536-treated cells. Means of 11.2 ± 1.3 µm, n = 43 cells, and 7.9 ± 1.3 µm, n = 37 cells in DMSO- and BI2536-treated cells, respectively, P value < 0.0001 in unpaired t test. (I) Quantification of the relative γ-tubulin distribution at centrosomes versus centrosome age in DMSO- and BI2536-treated RPE1 cells. Means of 4.0 ± 5.5%, n = 43 cells, P <0.0001 for and −0.6 ± 4.8%, n = 37 cells, P = 0.45 in DMSO- and BI2536-treated cells, respectively. One-sample t tests were used for statistical analyses. (J) Quantification of the relative pericentrin distribution at centrosomes versus centrosome age in DMSO- and BI2536-treated RPE1 cells. Means of 4.5 ± 9.6%, n = 46 cells, P = 0.0025 and −3.3 ± 12.4%, n = 24 cells, P = 0.2102 in DMSO- and BI2536-treated cells, respectively. One-sample t tests were used for statistical analyses. (K) SAI quantification in DMSO- and BI2536treated 1:1 RPE1 metaphase cells. SAI means of 2.4 ± 4.6%, n = 43 cells, P = 0.0012 and 0.4 ± 10.0%, n = 37 cells, P = 0.79 in DMSO- and BI2536-treated cells, respectively. One-sample t tests were used for statistical analyses. ns = not significant; P ≤ 0.01 = **; P ≤ 0.001 = ***; P ≤ 0.0001 = ****.

The mitotic kinase Plk1 drives the enrichment of PCM proteins at old centrosomes. (A) Immunofluorescence images of WT RPE1 GFP-centrin1 G2 and prophase cells stained with DAPI (blue), centrin1-GFP (green), cenexin (yellow), and pericentrin (magenta) antibodies. Scale bars = 5 μm. (B) Quantification of the relative pericentrin distribution at centrosomes versus centrosome age in G2 and prophase WT RPE1 GFP-centrin1 cells. Means of 9.2 ± 12.9%, n = 36 cells, P = 0.0001 in G2 and 9.5 ± 9.3%, n = 29 cells, P < 0.0001 in prophase. One-sample t tests were used for statistical analyses. (C) Immunofluorescence images of WT RPE1 GFP-centrin1 G2 and prophase cells stained with DAPI (blue), centrin1-GFP (green), cenexin (yellow), and γ-tubulin (magenta) antibodies. Scale bars = 5 μm. (D) Quantification of the relative γ-tubulin distribution at centrosomes versus centrosome age in G2 and prophase WT RPE1 GFP-centrin1 cells. Means of 3.6 ± 16.8%, n = 50 cells, P = 0.1375 in G2 and 7.3 ± 10.8%, n = 53 cells, P < 0.0001 in prophase. One-sample t tests were used for statistical analyses. (E) Immunofluorescence images of WT RPE1 GFP-centrin1 prophase and metaphase cells stained with DAPI (blue), GFP-centrin1 (green), centriolin (yellow), and Plk1 (magenta) antibodies. Scale bars = 10 μm. (F) Quantification of the relative Plk1 distribution at centrosomes versus centrosome age in prophase and metaphase cells. Means of 10.9 ± 20.8%, n = 47 cells, P = 0.0008 for G2 cells and 6.6 ± 10.8%, n = 35 cells, P = 0.0009 for prophase cells. One-sample t tests were used for statistical analyses. (G) Immunofluorescence images of 1:1 RPE1 Centrin1-GFP cells treated with DMSO (control condition) or 50 nM Bi2536 (Plk1 inhibitor). Cells are stained with DAPI (blue), centrin1-GFP (green), cenexin (yellow), and γ-tubulin (magenta) antibodies. Scale bar = 10 μm. (H) Quantification of spindle size measurements in DMSO- and BI2536-treated cells. Means of 11.2 ± 1.3 µm, n = 43 cells, and 7.9 ± 1.3 µm, n = 37 cells in DMSO- and BI2536-treated cells, respectively, P value < 0.0001 in unpaired t test. (I) Quantification of the relative γ-tubulin distribution at centrosomes versus centrosome age in DMSO- and BI2536-treated RPE1 cells. Means of 4.0 ± 5.5%, n = 43 cells, P <0.0001 for and −0.6 ± 4.8%, n = 37 cells, P = 0.45 in DMSO- and BI2536-treated cells, respectively. One-sample t tests were used for statistical analyses. (J) Quantification of the relative pericentrin distribution at centrosomes versus centrosome age in DMSO- and BI2536-treated RPE1 cells. Means of 4.5 ± 9.6%, n = 46 cells, P = 0.0025 and −3.3 ± 12.4%, n = 24 cells, P = 0.2102 in DMSO- and BI2536-treated cells, respectively. One-sample t tests were used for statistical analyses. (K) SAI quantification in DMSO- and BI2536treated 1:1 RPE1 metaphase cells. SAI means of 2.4 ± 4.6%, n = 43 cells, P = 0.0012 and 0.4 ± 10.0%, n = 37 cells, P = 0.79 in DMSO- and BI2536-treated cells, respectively. One-sample t tests were used for statistical analyses. ns = not significant; P ≤ 0.01 = **; P ≤ 0.001 = ***; P ≤ 0.0001 = ****.

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