Figure 5.

Effect of sildenafil and vincristine on BUBR1 expression and the distance between kinetochore pairs in PC-3 cells. The cells were incubated in the absence or presence of sildenafil (10 μM) and/or vincristine (10 nM) for 24 h. The confocal immunofluorescence examination was performed to detect BUBR1 (green) and chromosome (blue) using BUBR1 antibody and DAPI, respectively (A), or the cells were harvested and lysed for the detection of protein expression by Western blot analysis (B). Data are expressed as mean ± SEM of three independent determinations. Furthermore, BUBR1 phosphorylation was validated by the absence of phosphatase inhibitors or the presence of phosphatase. Both conditions significantly decreased the phosphorylation levels. (C) Confocal immunofluorescence microscopic examination was performed to detect kinetochore (green), microtubule (red), and chromosome (blue) using CENP-A antibody, β-tubulin antibody, and DAPI, respectively. (D) The distances between paired kinetochores (n = 100) were blindly measured at individual z planes, scored from 5 to 8 cells.