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Figure 1


Fig. 1. UV-induced H2AX phosphorylation occurs independently in quiescent cells and cycling S-phase cells. (A) MSU-2 cells growing asynchronously (AS) or growth-arrested by contact inhibition and serum starvation (G0) were irradiated with 10 J/m2 UV, incubated for 4 hours and treated with 50 µM BrdU for 15 minutes before fixation. Cells were stained with anti-{gamma}-H2AX and anti-BrdU antibodies and DAPI was used for nuclear counterstaining. Note that the BrdU-labeling index was less than 1% in the growth-arrested population. (B) MSU-2 cells (AS or G0) were irradiated with UV (black line, 0 J/m2; red line, 10 J/m2), incubated for 1 hour and treated with 50 µM BrdU for 30 minutes before fixation. Cells were stained with anti-{gamma}-H2AX and anti-BrdU antibodies, and PI staining was performed to measure cellular DNA content. For the asynchronous population, cells were divided into three subpopulations (G1, 65%; S, 19%; G2/M, 14%) based on the staining with PI and anti-BrdU antibody, and histograms of {gamma}-H2AX intensity in each subpopulation are shown in the upper panels. For quiescent cells, a BrdU-negative 2N subpopulation (92%) was analyzed and shown as a histogram of {gamma}-H2AX in the lower panel. (C) Growth-arrested MSU-2 cells were released by subculturing at lower density under normal serum conditions and irradiated with 10 J/m2 UV at 12, 15 or 18 hours after the release. The cells were incubated for 30 minutes and processed for immunostaining as described in A.





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