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. 2016 Feb 25;7:10818. doi: 10.1038/ncomms10818

Figure 1. Bub1 depletion and Plk1 inhibition synergize to inactivate the spindle checkpoint.

Figure 1

(a) Domains and motifs of Bub1. GLEBS, Gle2-binding sequence; TPR, tetratricopeptide repeat. (b) Quantification of the mitotic index (defined as the percentage of MPM2+, 4N cells) of HeLa Tet-On cells treated with 5 μM nocodazole and the indicated siRNAs and kinase inhibitors. Mean±s.d. for columns 1, 2, 5, 6 and 10 (3 or more independent experiments); mean±range for other columns (2 independent experiments). (c) Quantification of the mitotic index of HeLa Tet-On cells treated with 500 nM nocodazole and the indicated siRNA and Plk1 inhibitors (BI, BI 2536; GSK, GSK461364). Mean±s.d. (n=3 independent experiments). (d) Differential interference contrast (DIC) images of representative cells with the indicated siRNA and inhibitor treatments at different time points in 200 nM taxol. Time 0 marks the start of imaging and is ∼40 min after the addition of BI 2536. Dashed lines demarcate nucleus. Arrowheads indicate nucleoli. Scale bar, 5 μm. (e) Quantification of the percentage of cells in d remaining in mitosis at different time points (n=39 cells for each group). (f) Immunoblots of lysates of parental HeLa Tet-On cells and cells stably expressing GFP-Bub1 WT or kinase-dead mutant (KD) treated with doxycycline in the presence or absence of Bub1 siRNA and BI 2536. (g) Quantification of the mitotic index of HeLa Tet-On parental cells and cells expressing GFP-Bub1 WT and KD treated with 200 nM taxol in the presence or absence of doxycycline (Dox), Bub1 siRNA and BI 2536. Mean±s.d. (n=3 independent experiments).