Figure 4. Bub1b^ΔI MEFs have an increased time in mitosis and duration of mitotic arrest.

(A) Representative time-lapse images of live MEF cells of indicated genotypes progressing from prophase (t = 0) to anaphase (A). Time is indicated in min. WT, wild-type. P, prophase. M, metaphase. (B) Analysis of the time from nuclear envelope breakdown (NEBD) to anaphase onset in H2B-RFP MEFs of the indicated genotypes by live cell time-lapse imaging. n = 3 lines, ≥ 20 cells per line. Data are mean ± s.d. **p<0.01. FL, full-length. (C) In a nocodazole challenge, H2B-RFP MEFs of indicated genotypes were treated with 100 ng/ml of nocodazole (noc) and monitored by live cell time-lapse imaging. The point of time in which 50% of cells are arrested in mitosis is plotted. n ≥ 3 lines, ≥ 20 cells per line. Data are mean ± s.d. *p<0.05, **p<0.01. (D) H2B-RFP wild-type and mutant transgenic MEFs were treated concurrently with 100 ng/ml nocodazole and indicated concentrations of the Mps1 kinase inhibitor, AZ3146. The point of time in which 50% of cells are arrested in mitosis is plotted. n = 3 lines, ≥ 20 cells per line. Data are mean ± s.d. *p<0.05. Scale bar, 10 µm (See associated Figure 4—source data 1).

DOI: http://dx.doi.org/10.7554/eLife.16620.015

Figure 4—figure supplement 1. Bub1b^ΔI MEFs do not have persistent Mad2 signaling.

(A) MEFs of indicated genotypes and cell stage were stained for Mad2 (red), centromeres (cyan), and DNA (blue). White arrowhead depicts misaligned chromosome. WT, wild-type. FL, full-length (B) Table depicting the number of metaphases with at least one Mad2-positive kinetochore. n = 3 lines, 15 cells per line. (C) MEFs of indicated genotypes and cell stage were stained for Mad2 (red), centromeres (cyan), and DNA (blue). Scale bar 10 µm.

DOI: http://dx.doi.org/10.7554/eLife.16620.017