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. 2012 Feb 15;23(4):567–576. doi: 10.1091/mbc.E11-09-0781

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© 2012 Orth et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 1: — Prolonged mitotic arrest results in two types of postslippage p53 induction dynamics. (A) Mock, untreated (M), G2-synchronized cells (synch; see Materials and Methods), and synchronized cells released into normal (n) or K5I-medium (K5I) were immunoblotted for p53, p21, and the mitotic marker p-H3. p53 and p21 were not persistently induced to high levels in normal medium. Release into K5I resulted in p53 induction beginning at 12–16 h and p21 induction after 16 h. All blots in each panel are from the same gels and/or samples. (B–D) Live MCF7 cells expressing p53-Venus were imaged for >60 h. Times are from start of time lapse. First frame is start of mitotic arrest. No p53 increase was noted during mitotic arrest. Sixty percent of arrested cells showed sustained, high-level, p53-induction (cell A), while 30% showed a pulsatile response with a large first pulse (cell B). (B and C) Scale bars: 10 μm. See Movies S1 and S2 and note additional cells in the same microscopic field. Vertical lines in representative traces (D) mark mitotic slippage. RFU, relative fluorescence units. (E) Box-and-whisker plots of peak p53 levels in individual cells with sustained and pulsatile dynamics; p53 induction is significantly higher in cells with sustained induction. For cells in each group, the peak p53 intensity is determined, and the resulting values are assorted in ascending order. The boxes represent the second and third quartile of the population, the red horizontal line represents the median. Upper and lower “whiskers” represent data points from the first and fourth quartile that are within 1.5-fold of the interquartile range. Outliers are marked by red +. The notches about the median represent the 95% confidence interval. As the notches between the two compared populations do not overlap, the median values are significantly different (p < 0.0001 for sustained vs. pulsatile). (F) Box-and-whisker plots of hours arrested for the types of p53 dynamics. The medians are not significantly different. Forty cells were tracked: 26 showed a sustained induction, and 14 were pulsatile.