Fig 2: The PLA fluorescent signals detected using the antibodies to CDC20 and MAD2 reflect the physical interaction between these two proteins in vivo.HeLa cells were treated with 0.5% DMSO (control in a) or 50 µM M2I-1 in 0.5% DMSO (b) for 16 hours. The PLA signals (TexasRed) of the CDC20-MAD2 were quantified from projected confocal images of relevant Z-scan stacks from cells at different cell cycle stages as indicated. Arrows indicate the cytoplasmic regions of the prophase cells. DNA morphologies (DAPI staining in grey in the top panel and blue in the bottom panel) and centrosome morphologies (Arrowheads, pericentrin staining in green) were used to determine the cell cycle stages. The white dash circle lines are used to highlight the boundaries of the cell or nucleus where appropriate. (c) Quantitative results comparing the PLA signals between DMSO (control) and 50 µM M2I-1 in 0.5% DMSO treated cells. A two-tailed unpaired t-test was used to assess the significance between the two groups as indicated. Statistical significance was assigned where ***P < 0.0008, ****P < 0.0001. Standard deviation bars are in red. (d) Western blot results showing that similar levels of CDC20 and MAD2 are found in the samples extracted from the cells with or without M2I-1 treatment. GAPDH is the loading control. (e) Western blot results showing cyclin B1 levels in the relevant samples. Actin is the loading control. (f) HeLa cells released after 7 hours of thymidine synchronization, were then treated with either 0.1% DMSO as the control or 0.5 µM reversine in 0.1% DMSO for 1.5 hours and then for another 3 hours with either 10 µM MG132 in 0.1% DMSO or 10 µM MG132 and 0.5 µM reversine in 0.1% DMSO. Cells were then fixed and PLA treated as described before. (g) The quantitative average PLA fluorescent intensities from individual arrested prometaphase and metaphase cells encircled with the dash lines in (f) as examples of the regions of interest. P value: ****p < 0.0001. n: The number of cells used for quantification. Scale bar = 5 µM.

Fig 3: BUB1 and SURVIVIN protein levels decrease in a similar pattern during a prolonged mitosis in HCT116 and HT29 cells. Western blot analysis of paclitaxel-treated HCT116 (a) and HT29 (b) cells after thymidine block. In both HCT116 and HT29 cell lines, BUB1 and SURVIVIN levels diminish over time, whereas the levels of the mitotic checkpoint protein MAD2 remain constant during mitotic arrest.

Fig 4: Mad2 phosphorylation impaired DSB repair. (A,B) Homology-directed repair assay in control shRNA or ATM shRNA cells subjected to vector, WT, S195A, or S195D transfection. (C,D) Nonhomologous end-joining assay in control shRNA or ATM shRNA cells subjected to vector, WT, S195A, or S195D transfection. (E) Colony formation assay of control shRNA or ATM shRNA cells transfected with empty vector only, WT, S195A, or S195D with or without interventional radiology treatment. Mean ± SD, Student's t-test, n = 3, *p < 0.05. (F) CCK-8 assay analysis of cell proliferation abilities in control shRNA or ATM shRNA cells subjected to vector, WT, S195A, or S195D transfection.

Fig 5: Inhibition of PLK1 in APC-?C-expressing HCT116 cells reduces the recruitment of BUBR1, MAD1 to kinetochore and decreases the mitotic checkpoint complex association. a Scheme of the experimental procedure. APC-?C-expressing and control HCT116 cells were seeded on day 1, treated on day 2 either with 2 µM STLC or 100 nM BI6727 for 16 h and harvested on day 3. The kinetochore recruitment of the checkpoint proteins (b) BUBR1 and (c) MAD1 was monitored. After treatment, cells were fixed and stained with the indicated antibodies. Scale bar, 5 µm. The kinetochore intensities of BUBR1 and MAD1 staining in the different treatment groups were quantified. The intensities were normalized to ACA. Values were calculated from at least 50 cells per treatment (means ± s.d., n = 3, for each treatment). ***P < 0.001, Student’s t-test, unpaired and two-tailed. d Lysates (left panel) and immunoprecipitation of CCD20 from asynchronous and BI6727 (100 nM)-treated HCT-116 and APC-?C-expressing HCT116 cells were analyzed (right panel). CCD20-interacting proteins from IP were analyzed using western blot for MAD2, BUB3, BUBR1, and p55-CDC20