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. 2010 Jul 26;190(2):197–207. doi: 10.1083/jcb.200911156

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© 2009 Giunta et al.

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Figure 2. — 53BP1 is excluded from IRIF during mitosis. White arrows point to mitotic cells. (A) Costaining of either mock- or IR-treated asynchronously growing U2OS cells with 53BP1 and γH2AX antibodies. Phleomycin-treated U2OS cells costained for 53BP1 and MDC1. (B) Enlarged images of mitotic cells show exclusion of 53BP1 from chromatin and a lack of colocalization between the punctate 53BP1 staining and γH2AX foci. The histogram represents a quantification of γH2AX, MDC1, and 53BP1 focus-positive mitotic cells after IR treatment from three independent experiments (n > 200). Error bars indicate standard deviation. (C) Active exclusion of 53BP1 from mitotic IRIF. After a 3-h incubation with 50 nM of Chk1 inhibitor (Chk1i), cells were treated with phleomycin for 2 h, fixed and costained for 53BP1 and γH2AX, or collected for FACS analyses to determine mitotic indices by H3 pS10 immunofluorescence (purple boxes). (D) Time-lapse frames of U2OS cells stably expressing EGFP-53BP1. After irradiation with 0.5 Gy, two mitotic cells marked with arrows were imaged for up to 3 h.