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. 2015 Aug 19;4:e07909. doi: 10.7554/eLife.07909

Figure 2. The asymmetric distribution of unaligned chromosomes depends on CENP-E.

(A) An illustrative example of a HeLa-eGFP-centrin1/mCherry-CENP-A cell where the distances from centrosomes to kinetochores were measured 30 s before nuclear envelope breakdown (left top), assay to calculate the distances between kinetochores and centrosomes (left bottom), and distribution (right) of the measured distances. Values are determined from 24 cells and 1434 kinetochores in 6 independent experiments. White arrowheads indicate old centrosomes in all panels. Scale bars in all panels = 5 μm. (B) Proportion of unaligned chromosomes at the old centrosomes in hTert-RPE1-eGFP-centrin1 cells after indicated treatments. Error bars indicate s.e.m. *** indicates p ≤ 0.01 in Binomial test. (C) hTert-RPE1-eGFP-centrin1 cells stained for CENP-A and DAPI after indicated treatments. (D) Differences in the intensity of the microtubule asters in a re-nucleation assay at old and young centrosomes as shown in E, calculated in 32–49 cells in 3 independent experiments. Columns indicate the median, errors bars the 99% CI. Precise methodology is shown in Figure 2—figure supplement 2. (E) HeLa-eGFP-centrin1 or hTert-RPE1-eGFP-centrin1 cells stained for α-tubulin after a microtubule re-nucleation assay. (F) hTert-RPE1-eGFP-centrin1 cells treated with 10 ng/ml nocodazole and/or CENP-E inhibitor, and stained for CENP-A. Yellow arrowheads indicate unaligned kinetochores in the proximity of the young centrosome (G) Proportion of unaligned chromosomes at old centrosomes in hTert-RPE1-eGFP-centrin1 cells treated with 10 ng/ml nocodazole and/or CENP-E inhibitor. Error bars indicate s.e.m. *** indicates p ≤ 0.01 in Binomial test. For results of all individual experiments see Figure 2—source data 1.

DOI: http://dx.doi.org/10.7554/eLife.07909.006

Figure 2—source data 1. Values of individual experiments of graphs shown in Figure 2.
DOI: http://dx.doi.org/10.7554/eLife.07909.007
elife07909s002.xlsx (54.6KB, xlsx)
DOI: 10.7554/eLife.07909.007

Figure 2.

Figure 2—figure supplement 1. Monastrol wash-out does not change the alignment bias in HeLa cells.

Figure 2—figure supplement 1.

Proportion of unaligned chromosomes at the old centrosome in HeLa-eGFP-centrin1 cells after monastrol washout. Errors bars indicate s.e.m. *** indicates p ≤ 0.01 in Binomial test.
DOI: http://dx.doi.org/10.7554/eLife.07909.008

Figure 2—figure supplement 2. Methodology to compare microtubule re-nucleation at old and new pole.

Figure 2—figure supplement 2.

To calculate in hTert-RPE1-eGFP-centrin1 cells the relative differences in microtubule nucleation, cells were stained after a release from an ice-cold treatment with a-tubulin antibodies (microtubules) and DAPI (chromosomes). The intensity of the centrosomal aster (minus the background) at old and young centrosomes was measured and the relative differences calculated with the indicated formula. Scale bar = 5 μm.
DOI: http://dx.doi.org/10.7554/eLife.07909.009