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. 2010 Oct 4;191(1):45–60. doi: 10.1083/jcb.201003034

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© 2010 Ismail et al.

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Figure 6. — BMI1 is required for efficient DDR. (A) BMI1 WT and BMI1 KO MEFs were irradiated (2 Gy) and allowed to recover for 60 min. Cells were stained with γ-H2AX and FK2 antibodies. The number of foci per cell was counted manually on the microscope using a 63× lens. The mean number of FK2 foci per cell is shown from 40 cells counted. The arrow points to micronuclei that are commonly observed in BMI1 KO MEFs. (B) BMI1 WT and BMI1KO MEFs expressing GFP RAP80 were irradiated (2 Gy, 120 min), and the number of GFP-RAP80 IRIF per cell was counted. (C) BMI1 WT, BMI1 KO, and RNF8 KO MEFs were microirradiated, allowed to recover for 5 or 30 min, and stained with the indicated antibodies. The relative intensity of 53BP1 recruited to the sites of DNA damage was quantified and shown as the mean of at least five cells. (D) BMI1 WT and BMI1KO MEFs expressing GFP BRCA1 were monitored after microirradiation using time-lapse microscopy. (D, left) Representative images of BRCA1 time lapse. (D, right) The integrated intensity in the microirradiated area is plotted as a percentage of maximum versus time. Error bars represent standard error from two independent experiments. Bars, 5 µm.