Figure 1.

The local impact of laser microirradiation. (A) U2OS cells were either sensitized with BrdU followed by laser microirradiation or cultured without any presensitization followed by exposure to IR. 1 h later, the cells were fixed, immunostained with an antibody to the p32 subunit of RPA, and subject to z stack recording. (left) Representative 3D projections of cells exposed to the microlaser and 3 Gy of IR, respectively, are shown. (right) Quantification of the RPA foci in the microirradiated tracks or in the whole cell nuclei exposed to IR was obtained from 10 independent cells for each treatment. (B) U2OS cells were treated by the microlaser or exposed to increasing doses of IR. 1 h later, the cells were fixed and processed for RPA immunodetection as in A. A region spanning the entire microirradiated nuclear track containing the RPA foci (left) was placed over the maximum nuclear diameter of the IR-treated cells (right). The graph summarizes quantification of the RPA foci in these regions from 10 independent cells for each treatment. All images in this section are 3D projections as in A. (C) U2OS cells were microirradiated as in A. 1 h later, the cells were fixed and coimmunostained with antibodies to γ-H2AX and phospho-serine 15 of p53 (S15-P). The total nuclear fluorescence associated with S15-P was determined and compared with that measured in cells exposed for 1 h to the indicated doses of IR. The blue line marks the nucleus of an unirradiated cell to illustrate the background fluorescence associated with the S15-P antibody. The graph represents quantification of the S15-P fluorescence intensities from at least 50 cells for each treatment. Error bars indicate standard deviation. Bars, 10 μm.