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. 2016 Dec 15;27(25):4011–4020. doi: 10.1091/mbc.E16-06-0428

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© 2016 Irianto, Pfeifer, 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 4: — FokI nuclease is confirmed to enact on/off-inducible damage at a specific locus on chromosome 1 and also to segregate/mislocalize during Transwell migration. (A) A lac operator transgene integrated into the p-end of chromosome 1 in the U2OS line. Expression of the integrated mCherry-Lac repressor-FokI construct can be stably induced with 4-OHT (tamoxifen) and Shield-1 to creates DNA breaks. FokI intensity correlates with γH2AX intensity, and the area of GFP-53BP1 at the damaged site is linearly correlated to γH2AX focus area. (B) A comet assay is sensitive enough to detect induced DNA breaks: the mean centroid distance is higher for FokI-activated cells (≥175 nuclei/group; at least three experiments). (C, D) FRAP shows that both nucleoplasmic and chromatin-bound mCherry-FokI are mobile, with half-lives of 6 s and >2 min, respectively. The former is thus much more mobile, and it indeed segregates outside a micropipette in D during aspiration (≥4 cells/group; at least three experiments). (E, F) Migration through 3-μm Transwell pores also causes FokI to segregate and mislocalize, as seen in migrated cells that have been fixed for confocal imaging. Intensity profiles are shown along the dashed arrows (>10 cells/group; at least three experiments).