Fig. 2. Localisation of pol and PCNA after UV irradiation. MRC5 cells were transfected with pEGFPpol and incubated for 20 hours. Cells were UV irradiated with 10 J/m² and incubated for 6 hours. Cells were then fixed and stained with anti-PCNA mouse monoclonal antibody and rhodamine-conjugated secondary antibody. Colocalisation of eGFPpol and PCNA foci was analysed. (A) eGFPpol-expressing cells were selected and sorted by the foci formation property of pol and PCNA. To check the colocalisation of foci, images of the cells that form both eGFPpol and PCNA foci were captured and then further analysed. Experiments were carried out in triplicate, and indicated numbers are the averages and the standard deviations of three independent experiments. More than 100 cells were analysed in each experiment. (B-E) Typical staining patterns of the auto-fluorescent signal of eGFPpol (green) and PCNA (red) in the same cell are shown. (B) Complete colocalisation of eGFPpol foci and PCNA foci was observed as shown by yellow staining. (C) Partial colocalisation of eGFPpol foci and PCNA foci. Most of the PCNA foci colocalised with eGFPpol foci, but significant numbers of eGFPpol foci were missing (white arrows). (D) eGFPpol foci were completely absent in cells with PCNA foci. (E) eGFPpol foci and PCNA foci were not colocalised at all.