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. 2013 Jan;33(1):111–126. doi: 10.1128/MCB.01024-12

Fig 7.

Fig 7

Complementation of 53BP1 focus formation in MCF10A H2AX−/− cells. (A) Wild type, fH2AX E92A, and fH2AX-9K to R, but not fH2AX S139A, rescued 53BP1 IR-induced foci in MCF10A H2AX−/− cells. MCF10A H2AX−/− cells were transfected with either empty vector or vectors containing the indicated fH2AX constructs followed by 48 h of recovery. Cells were then treated and analyzed as in Fig. 6C at 15 min posttreatment with 3 Gy IR. Flag IF identified H2AX-expressing cells. (B) Quantification of panel A. More than 50 cells from both Flag-positive and Flag-negative cells were scored from wild-type, fH2AX E92A-, fH2AX-9K to R-, and fH2AX S139A-transfected MCF10A H2AX−/− cells for 53BP1 focus formation. Cells were scored as having either 0 to 10 53BP1 foci or more than 10 53BP1 foci. Cells staining negative for Flag from each of the 4 fH2AX samples were scored and combined to represent the Flag-negative sample. (C) Impaired BRCA1 recruitment to DNA damage sites in MCF10A H2AX−/− cells is rescued by H2AX-9K to R. Cells were treated and analyzed as in panel A, except using GFP-tagged H2AX and H2AX-9K. Dotted circles indicate cell nuclei. Scale bars, 10 μm.