Skip to main content
. 2010 Nov 30;124(1):68–81. doi: 10.1242/jcs.071340

Fig. 1.

Fig. 1.

Persistent DNA damage foci lack evidence of ssDNA and DNA synthesis. (A) HCA2 [population doubling 25 (PD25)] cells were irradiated with 0.5 or 10 Gy X-rays and followed for the indicated intervals before being fixed and stained for 53BP1. Scale bars: 10 μm. (B) Cells, either untreated or irradiated with 10 Gy, were pulsed with BrdU for the indicated intervals after irradiation before being fixed and stained for 53BP1 (red) and BrdU (green). Arrows indicate cells with BrdU-positive 53BP1 foci. (C) Cell populations in B were analyzed for the percentage of cells synthesizing DNA (BrdU positive throughout the nucleus) and the percentage that harbor BrdU-positive 53BP1 foci (BrdU in foci). Shown are the means ± s.d. from three or more independent measurements. (D) Cells were irradiated with 10 Gy and followed for the indicated intervals before being fixed and stained for 53BP1(red) and RPA70 (RPA, green). Note the RPA70 foci 24 hours after irradiation. (E) Cells treated and stained in D were analyzed for the percentage harboring three or more 53BP1 foci and the percentage with three or more foci positive for both RPA and 53BP1. Shown are the means ± s.d. from three or more independent measurements. (F) Cells were irradiated with 10 Gy. Three hours later, they were pulsed for 4 hours with BrdU, then fixed and stained for Rad51 (red), PML (green) and BrdU (blue). The arrow indicates a BrdU-Rad51-negative cell. (G) Single cells in the cell populations in F were analyzed simultaneously for the presence of nuclear RAD51 foci and nucleoplasmic BrdU. Shown are the means ± s.d. from three or more independent measurements. (H) Cells were given 10 μg/ml bleomycin (BLEO) for 30 minutes and processed 30 minutes later (1 hour BLEO), or induced to senesce by an exposure for 2 hours to 20 μg/ml BLEO and processed 12 days later (SEN-BLEO). Cells were either fixed (‘Non-permeabilized’) or permeabilized before fixation (‘Permeabilized’), then stained for RPA70 (green) and DNA (DAPI, blue) in the top panels or 53BP1 (red), γ-H2AX (green) and PML (blue) in the lower panels.