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. 2006 Jul 17;174(2):195–206. doi: 10.1083/jcb.200604022

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Copyright © 2006, The Rockefeller University Press

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Figure 5. — Biochemical analysis of premature mitosis versus G2/M arrest and normal mitotic events. (A) Condensin recruitment and UCC develop in DNA-damaged HeLa but not in U2OS cells. Cells were treated with NCS (200 ng/ml for 24 h), immunostained with anti-SMC2 antibody (red), and counterstained with DAPI (blue). Bars, 20 μm. (B) Western blotting analyses of chromatin fractions from NCS-treated (200 ng/ml for 24 h) HeLa and U2OS cells. Note that in HeLa cells, recruitment of SMC2 to damaged chromatin coincides with the phosphorylation of histone 3 on Ser10 (p-H3). (C) Biparametric FACS analysis demonstrating that DNA damage drives HeLa cells into mitosis and arrests U2OS cells in G2/M. The figure represents one of three independent experiments. (D) Levels of expression of p53 and its inducible regulator p21^waf1 in DNA-damaged U2OS cells. U₂₄ and U₇₂ represent two untreated controls obtained at 24 and 72 h, respectively. (E) Western blotting analysis of G2→M transition regulators in DNA-damaged cells. Total cell lysates were obtained from NCS-treated U2OS and HeLa cells 24–72 h after 200 ng/ml NCS administration. U₂₄ and U₇₂ represent two untreated controls obtained at 24 and 72 h, respectively. Hsc70 was used as a loading control. (F) Comparative analysis of Cdk1 phosphorylation pattern in DNA-damaged and mitotic-arrested HeLa cells. Cells were either treated with 200 ng/ml NCS (for 24 h) or arrested in metaphase using colcemid. Total cell lysates were analyzed by Western blotting. (G) DNA damage leads to the appearance of two Cdk1 pools marked with the activating phosphorylation on Thr161 or the inhibitory phosphorylation on Tyr15, each with a distinct subcellular localization. Cytosolic and nuclear fractions were prepared from untreated and NCS-treated HeLa cells 24–72 h after the treatment and subjected to Western blotting analysis. The degree of fractionation is demonstrated using antibodies against α-tubulin and PARP-1, reflecting cytosolic and nuclear protein fractions, respectively. Asterisks in the cytosol fraction of the PARP-1 immunoblot denote the cross-reactive bands.