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Figure 3


Fig. 3. Nucleotide excision repair is required for the UV-induced H2AX phosphorylation in quiescent cells. (A) MSU-2 or XP2BI cells (AS or G0) were irradiated with 10 J/m2 UV and incubated for 4 hours. 50 µM BrdU was included in the asynchronous population for 15 minutes before fixation and cells were costained with anti-{gamma}-H2AX and anti-BrdU antibodies. (B) MSU-2 or XP2BI cells under growth-arrested conditions were locally irradiated with 40 J/m2 UV and incubated for 4 hours. Cells were fixed and double-stained with anti-{gamma}-H2AX and anti-CPD antibodies. (C) MSU-2, TIG-120, XP12BE, XP1CTA and XP2BI cells growth-arrested by contact inhibition and serum starvation were irradiated with no UV (black lines) or 10 J/m2 UV (red lines) and incubated for 1 hour before fixation. Cells were stained with anti-{gamma}-H2AX and PI and analyzed by flow cytometry. (D) XP3OS/T-n cells were transfected with pCMV-Myc-XPA plasmid using TransFectin lipid reagent (Bio-Rad) and incubated for 2 days. The cells were growth-arrested by serum starvation for 4 days and exposed to 20 J/m2 UV. After 3 hours of incubation, cells were fixed and double-stained with anit-{gamma}-H2AX and anti-6-4PP antibodies. (E) MSU-2 or XP2BI cells (AS or G0) were treated with 2 µM NA-AAF for 30 minutes and incubated for another 30 minutes after medium change. BrdU was used for labeling S-phase cells in the AS population, and {gamma}-H2AX and BrdU were detected as described in A.





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