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. 2014 Oct 15;25(20):3105–3118. doi: 10.1091/mbc.E14-03-0844

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© 2014 Panopoulos 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 8: — Changes in protein expression in tetraploid arrest. (A) HFF cells were exposed to 10 μM DCB for 24 h, released for 6 h, and then exposed again to DCB for 24 h to maximize tetraploidy. Extracts were harvested for assay 2 d after DCB release. ERK1/2 is highly phosphorylated (p-ERK1/2), and cells have a high level of cyclin D expression. There is no increase in ERK1/2 expression. Results were compared with contact-inhibited (CI) and random cycling controls. Samples are from the same extracts, and tubulin is a loading control. (B) Immunofluorescence of cells treated as in A shows that phospho-ERK (p-ERK) is perinuclear and intranuclear in binucleate cells. Counterstains are propidium iodide for DNA and anti–α-tubulin antibody. Scale bar, 40 μm. (C) HFF cells, doubly exposed to DCB as in A, were assayed at the times indicated after release and compared with CI and random cycling controls (–). DCB-treated cells were positive for p21waf1 and p27kip1. Tubulin is a loading control. (D) Western blot showing that tetraploidy induces expression of p16INK4a at 3 d after transfection of HFF cells with PRC1 siRNA or 2 d after release from 10 μM DCB (DCB release). Controls are cells transfected with scrambled siRNA or cells not treated with DCB. The DCB lane indicates a sample taken after 24 h in 10 μM DCB. Tubulin is a loading control. Bottom, microscope images show HFF cells released from 10 μM DCB for 24 h and stained for p16INK4a (green) and DNA (blue, DAPI). Binucleate cells are positive for p16INK4a, and mononucleate cells are negative. Microscope settings were constant for all images. Scale bars, 40 μm.