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Fig. 5. UV-induced ssDNA focus formation is stimulated during S-phase. (A) GM-847 cells treated with nocodazole for 14 hours were subjected to mitotic shake-off and subsequently seeded on coverslips. At the indicated time-points, cells were pulsed with BrdU, and the number of cells in S-phase were determined by immunofluorescence labeling using an anti-BrdU antibody. For each sample more than 400 cells were scored. The data represent the average of two independent experiments ± s.d. (B) GM-847 cells were treated with BrdU for 24 hours and subsequently subjected to nocodazole treatment and mitotic shake-off in parallel with the experiment described in A. At the indicated time-points following mitotic shake-off, cells were UV-irradiated with 10 J/m2, and subsequently incubated in the presence of 4 mM caffeine for 2 hours. The number of cells containing PML-associated ssDNA foci were determined by immunofluorescence labeling. For each sample more than 400 cells were scored. The data represent the average of two independent experiments ± s.d. (C) Representative images of UV-induced PML-associated ssDNA foci in G1 and S-phase cells formed by UV-irradation at 5 and 12 hours post mitotic shake-off, respectively.
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