Figure 7.

p53 protects cells from inhibition of PLK1 by maintaining centrosome separation. (A) Schematic showing the protein kinase cascade responsible for regulating centrosome uncoupling and separation. (B) HCT116-p53+/+ and -p53−/− cells were treated with 20 nM GSK461364 or DMSO for 8 h, and subsequently stained for γ-tubulin antibody (a centrosome marker). The number of mitotic cells showing a normal bipolar spindle (separated centrosomes) or monopolar spindle (non-separated centrosomes) was determined using fluorescence microscopy. Over 100 cells were counted for each condition (scored by two independent individuals) and plotted as percentages of separated v non-separated centrosomes (bar graph). (Counting was based on the detection of mitotic cells by use of DAPI, and then switching channels to look at gamma tubulin to assess separation of centrosomes in the mitotic cells). Data are representative of two independent experiments and error bars represent the standard deviation of the mean. (C) HCT116-p53+/+ and -p53−/− cells were treated with DMSO, 250 or 500 nM STLC. Time-lapse analysis was then used to determine the duration of mitosis. The graph represents the cumulative data from 50 cells for each treatment and are representative of three replicates. (D) HCT116 p53+/+ and p53−/− cells were treated for 72 h with increasing concentrations of STLC. Cell viability was measured using an MTS assay. Data are representative of three independent experiments each conducted in triplicate. Error bars represent the standard deviation of the mean.