TI - Results DSB-repair measurements during the mammalian cell cycle . AB - To investigate the contribution of NHEJ and HR in different cell -cycle phases , we analysed asynchronously growing cells to avoid potential introduction of DSBs during synchronization . For human fibroblasts , we used pan-nuclear CENP-F staining to identify G2 cells ( Liao et al , 1995 ; Kao et al , 2001 ) and added aphidicolin to prevent irradiated S-phase cells progressing into G2 during analysis ( Figure 1A ) . Aphidicolin caused pronounced pan-nuclear H2AX PHOSphorylation in S-phase cells , likely due to DSBs resulting from collapsed replication forks ( Figure 1A ) . These S-phase cells , as well as mitotic cells , identified by their condensed chromatin and CENP-F staining were excluded from analysis . Flow cytometry ( FACs ) analysis demonstrated that up to 8 h post-IR the majority of irradiated G2 cells remain in G2 and that S-phase cells do not progress into G2 ( Supplementary Figure 1A ) , providing sufficient time to detect the repair defect in ATM - and Artemis-deficient cells , which is measurable at >4 h post-IR ( Riballo et al , 2004 ) . To identify cell -cycle phases in mouse embryonic fibroblasts ( MEFs ) , phosphoH3 was used instead of CENP-F ( Supplementary Figure 1B ) . We used gammaH2AX foci analysis to monitor DSB repair . Enumeration of gammaH2AX foci in CENP-F-positive primary human fibroblasts following 2-Gy X-irradiation provided DSB-repair kinetics in G2 (Figure 1B) . Enumeration of gammaH2AX foci in CENP-F-negative cells , which were also negative for the pan-nuclear aphidicolin-induced gammaH2AX signal , allowed the analysis of repair in G1 (Figure 1B) . We observed similar kinetics and magnitude of repair in G1 and G2 , which was also similar to that previously observed in G0 cells ( Riballo et al , 2004 ) . Aphidicolin treatment did not affect the repair capacity and did not form gammaH2AX foci in G1 or G2 ( Figure 1B ) . Initial gammaH2AX foci increased linearly with dose up to 80-90 foci per cell ( Figure 1C and Supplementary Figure 1C ) . Foci numbers correlated with DNA content , being twice as high in G2 compared with G1 (Figure 1C) . Since gammaH2AX foci in G1 are lost in a manner entirely dependent on NHEJ factors and represent DSBs ( Riballo et al , 2004 ) , the two-fold higher numbers in G2 strongly suggest that gammaH2AX foci in G2 also represent DSBs . To further substantiate this contention , we treated control and ATR-deficient cells with the specific ATM and DNA-PK inhibitors , KU55933 and NU7026 . IR -induced gammaH2AX foci formation was identical between control and ATR-deficient cells and was abolished by joint KU55933/NU7026 treatment , demonstrating that ATM and DNA-PKcs contribute to foci formation in G1 and G2 ( Supplementary Figure 1D ) .