Figure 1.
Centrosome age breaks spindle size symmetry in RPE1 cells. (A) Immunofluorescence images of 2:2, 1:1, 1:0, and 0:0 RPE1 GFP-centrin1 metaphase cells. Cells are stained with cenexin (yellow) and γ-tubulin (magenta) antibodies, DAPI (blue), and GFP-centrin1 (green). Note that these images are also shown in Fig. S2 B. (B) Quantification of the SAI of 2:2, 1:1, 1:0, and 1:0 cells. The dashed line and full lines of each condition represent the mean and SD, respectively. Each condition was compared with the null hypothesis, a symmetric spindle centered at 0 (dashed line). For 2:2 cells, mean SAI = 1.4 ± 6.2%, n = 133 cells, P = 0.0104; for 1:1 cells = 4.1 ± 7.3%, n = 96 cells, P < 0.0001; for 1:0 cells = 19.3 ± 11.2%, n = 111 cells, P < 0.0001; and for 0:0 cells = 0.9 ± 13.3%, n = 77 cells, P = 0.54 in one-sample t tests. (C) Immunofluorescence images of 2:2 and 1:1 centriolin−/− RPE1 metaphase cells, stained with DAPI (blue), GFP-centrin1 (green), and cenexin antibody (yellow). (D) SAI quantification of 2:2 and 1:1 WT RPE1 cells (light blue) versus centriolin−/− RPE1 cells (light grey). For 2:2 centriolin−/− RPE1 cells, mean SAI = 0.7 ± 4.7%, n = 56 cells, P = 0.3573; and for 1:1 centriolin−/− RPE1 cells, mean SAI = 0.4 ± 4.7%, n = 65 cells, P = 0.4229 in one-sample t tests. (E) Immunofluorescence images of 2:2 WT GFP-centrin1 and 2:2 centriolin−/− GFP-centrin1 RPE1 metaphase cells treated with nocodazole and stained with cenexin (yellow) and CENP-A (green) antibodies, DAPI (blue), and GFP-centrin1 (green). (F) Quantification of the percentage of polar chromosome distribution versus centrosome age. The dashed line represents a symmetric distribution (50%) of the polar chromosomes between the two poles. Means of 69.2 ± 11.6% (from 223 polar chromosomes in 86 cells) in WT 2:2 cells, and 67.2 ± 7.8% (from 203 polar chromosomes in 169 cells) in centriolin−/− 2:2 cells. P values of < 0.0001 for both conditions using binomial tests to compare individual conditions to a random distribution of 50%. P value of 0.8902 using a Fisher’s exact test to compare the two conditions. All scale bars = 10 μm. ns = not significant; P ≤ 0.05 = *; P ≤ 0.01 = **; P ≤ 0.0001 = ****.

Centrosome age breaks spindle size symmetry in RPE1 cells. (A) Immunofluorescence images of 2:2, 1:1, 1:0, and 0:0 RPE1 GFP-centrin1 metaphase cells. Cells are stained with cenexin (yellow) and γ-tubulin (magenta) antibodies, DAPI (blue), and GFP-centrin1 (green). Note that these images are also shown in Fig. S2 B. (B) Quantification of the SAI of 2:2, 1:1, 1:0, and 1:0 cells. The dashed line and full lines of each condition represent the mean and SD, respectively. Each condition was compared with the null hypothesis, a symmetric spindle centered at 0 (dashed line). For 2:2 cells, mean SAI = 1.4 ± 6.2%, n = 133 cells, P = 0.0104; for 1:1 cells = 4.1 ± 7.3%, n = 96 cells, P < 0.0001; for 1:0 cells = 19.3 ± 11.2%, n = 111 cells, P < 0.0001; and for 0:0 cells = 0.9 ± 13.3%, n = 77 cells, P = 0.54 in one-sample t tests. (C) Immunofluorescence images of 2:2 and 1:1 centriolin−/− RPE1 metaphase cells, stained with DAPI (blue), GFP-centrin1 (green), and cenexin antibody (yellow). (D) SAI quantification of 2:2 and 1:1 WT RPE1 cells (light blue) versus centriolin−/− RPE1 cells (light grey). For 2:2 centriolin−/− RPE1 cells, mean SAI = 0.7 ± 4.7%, n = 56 cells, P = 0.3573; and for 1:1 centriolin−/− RPE1 cells, mean SAI = 0.4 ± 4.7%, n = 65 cells, P = 0.4229 in one-sample t tests. (E) Immunofluorescence images of 2:2 WT GFP-centrin1 and 2:2 centriolin−/− GFP-centrin1 RPE1 metaphase cells treated with nocodazole and stained with cenexin (yellow) and CENP-A (green) antibodies, DAPI (blue), and GFP-centrin1 (green). (F) Quantification of the percentage of polar chromosome distribution versus centrosome age. The dashed line represents a symmetric distribution (50%) of the polar chromosomes between the two poles. Means of 69.2 ± 11.6% (from 223 polar chromosomes in 86 cells) in WT 2:2 cells, and 67.2 ± 7.8% (from 203 polar chromosomes in 169 cells) in centriolin−/− 2:2 cells. P values of < 0.0001 for both conditions using binomial tests to compare individual conditions to a random distribution of 50%. P value of 0.8902 using a Fisher’s exact test to compare the two conditions. All scale bars = 10 μm. ns = not significant; P ≤ 0.05 = *; P ≤ 0.01 = **; P ≤ 0.0001 = ****.

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