Fig. 7.

Inhibition of PLK1 in APC-ΔC-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-ΔC-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-ΔC-expressing HCT116 cells were analyzed (right panel). CCD20-interacting proteins from IP were analyzed using western blot for MAD2, BUB3, BUBR1, and p55-CDC20