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Fig. 2. The DSB repair response to local pulsed 800 nm laser irradiation. (A) XPC-GFP-expressing cells were treated with pulsed 800 nm irradiation and presence of DSBs is shown by immunohistochemical staining with a PKcs antibody (lines in right panel indicated by arrowheads). The bright spots outside the damaged area in the right panel are nucleolar structures of unknown origin and it is unknown if they exist in a living cell as well. (B) XPC-GFP-expressing cells were treated as in panel A and stained for the presence of phosphorylated histone H2AX (H2AX). Accumulation of H2AX at areas irradiated by the pulsed 800 nm laser confirms the presence of DSBs (right panel, arrowheads). No accumulation of H2AX is found on UV-C irradiated spots (arrows). Earlier it was shown that phosphorylation of H2AX takes place after UV-C irradiation (Marti et al., 2006; O'Driscoll et al., 2003) and we have found this as well in other experiments (data not shown). It is possible that in this case the specific immunohistochemical staining of H2AX at UV-C damage was not strong enough to be detected over background signals. (C) Rad54-GFP expressing cells were irradiated in an area of approximately 5 µm² with pulsed 800 nm light and the redistribution of fluorescence was studied in time. The boxed area is two times enlarged in the left bottom of both panels. Rad54-GFP accumulates in small foci at the damaged area. (D) YFP-MDC1(BRCT) expressing cells were irradiated in a rectangular line through the nucleus and fluorescence redistribution was followed in time. YFP-MDC1(BRCT) accumulates in large foci at the damaged area (boxed area, left panel).

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