Figure 1
Partial inhibition of Cdk1 results in a spindle checkpoint-dependent mitotic arrest.
(A) Flow cytometry of RPE1 and U2OS cells treated with increasing concentrations of RO-3306. Cells were treated for 16 hours (Mean ± s.e.m, n = 3). (B) Cell death correlates to entering mitosis, whereas a full blockade of Cdk1 activity prevents cell death. Cell death was determined by flow cytometry analysis. (Mean ± s.e.m, n = 3). (C) Partial Cdk1 inhibition triggers PARP cleavage. Lysates of U2OS cells are shown. (D) Cyclin B1 degradation in metaphase is delayed in cells with impaired Cdk1 activity. U2OS-CCNB1-EYFP cells were followed by differential interference contrast (DIC) and fluorescence microscopy. NEB, nuclear envelope breakdown; nt, nuclear translocation; ana, anaphase. Scale bar, 10 μm. (E) Quantification of the cyclin B1-EYFP levels in cells treated with or without 3 μM RO-3306. Fluorescence intensity was plotted against time after NEB. (Mean ± s.e.m, n = 3, untreated = 20 cells analysed, 3 μM RO-3306 treated = 13 cells analysed). (F) Unaligned sister chromatids are detected by the mitotic checkpoint. U2OS cells, treated with or without 3 μM RO-3306 were fixed after 16 hours. Mad2-positive kinetochores are indicative of a functional spindle checkpoint. Insets: overlay of Mad2 and CREST stainings. Scale bar, 10 μm. (G) Partial Cdk1 inhibition results in sister chromatid attachment defects to the mitotic spindle. Treatments are performed as in (F). Sister chromatid pairs that are not aligned are negative for kinetochore-associated astrin. Insets: overlay of astrin and CREST stainings. Scale bar, 10 μm. (H) Quantification of the mitotic duration after partial Cdk1 inhibition in presence or absence of a functional checkpoint. Mitosis is plotted from NEB till anaphase. (Mean ± s.e.m, n = 3, 50 cells/experiment; ****p < 0.0001, Student’s t test). (I) Cdk1 inhibition results in the formation of micronuclei during mitotic exit. Analysis is performed using the cells from panel (H). (Mean ± s.e.m, n = 3, 50 cells/experiment; ***p < 0.001, Student’s t test). Western blots in panel (C) have been cropped and full-length gels can be viewed in Supplementary Fig. 6.
