Fig. 2. P53-induced cell cycle arrest occurs after 2 failed cytokinesis.

A RB+p53 wild type (wt) HCT116 and HT1080, and p53 deleted HCT116 cell lines were treated without (Control) or with 0.2 μM AZD2811 for 48 h then harvested for flow cytometry of DNA content. The data is representative of three independent experiments. B HCT116 and HT1080 cells either untreated (Control) or treated with AZD2811 for 24 or 48 h were labelled with EdU for 2 h then fixed and stained for EdU incorporation and p53, then >1000 cells/well in triplicate were analysed by high content image analysis. The data represents the pooled wells. The level of EdU and p53 staining in each cell is shown and percentage of cells in each quadrant shown. C, D HCT116 wild type cells were either untreated (Control) or treated with 0.2 μM AZD2811 then followed by time lapse microscopy, imaging every 30 min. A control mitosis and cytokinesis is shown (D white arrowheads) and two failed division in AZD2811-treated cells (D white arrow heads). E HCT116 and HT1080 cells were treated with 0.2 μM AZD2811 and followed by time lapse microscopy. Timelines for 50 cells in each cell line are shown, with the time in mitosis shown. All cells failed cytokinesis. F Analysis of time lapse imaging of AZD2811 treated HCT116 and HT1080 cells to quantify the number of mitoses each cell underwent over 80 h of treatment. Data are mean and SD for 100 cells were followed for each cell line from three independent experiments. G Cells from the control and cultures treated with AZD2811 for 1 day then drug washed out, were allowed to proliferate for a week. The resultant cultures were pulsed with EdU for 2 h to identify the proliferating population, and then fixed, stained and >1000 cells/well in triplicate were analysed by high content image analysis. The data represents the pooled wells. The EdU incorporation and DNA content for the control and AZD2811-treated cultures was overlaid.