XB-IMG-144777
Xenbase Image ID: 144777
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FIGURE 5. Myc deregulation induces replication-dependent DNA damage. a, Replication reactions in Xenopus extracts, assembled in the presence or absence of XMyc (75 nM) and geminin, were stopped at 120 min (top panel). Middle panel: western blot analysis of chromatin fractions. Chromatin-bound gamma-H2A.X was quantified by Scion software and normalized to histone H3 (graph). b, Immunodepletion of DNA polymerase alpha (Pol alpha) from Xenopus extracts (top panel). Replication reactions were then assembled with buffer or XMyc (30 nM) (middle panel) and quantified. The graph shows an average of three experiments (plusminuss.d.). c, Replication reactions assembled in Xenopus extracts in the presence or absence of XMyc. Samples taken at indicated time points were quantified (average of two experiments, plusminuss.d.; top graph). The bottom graph shows fold change between control (set at 1) and XMyc-treated extracts. d, Replication reactions in Xenopus extracts performed in the presence or absence of XMyc (wild type (WT) and mutants, 75 nM, lanes 1–4), or 5 mM caffeine (lanes 5–7). Reactions were analysed after 120 min. The graph shows the average of three experiments (plusminuss.d.). e, Western blot analysis of cytosolic fractions from replication reactions performed in the presence or absence of XMyc for 20 min, and probed with phospho-specific anti-ATM antibodies. f, Mammalian U2OS cells transfected with Myc and synchronized by nocodazole (G1, G2) or thymidine (S) blocks. Where indicated, aphidicolin (2.5 muM) was added to prevent S-phase entry. FACS profiles depict cell cycle status at collection. Bottom panel: western blot analysis of whole-cell lysates. g, Analysis of gamma-H2A.X distribution in splenic B cells from lambda-Myc mice and wild-type littermates. Top panel: immunofluorescence analysis for geminin and gamma-H2A.X distribution. Numbers identify cells within the images; arrows indicate cells with nuclear geminin (S/G2). The graph shows cell cycle distribution (based on geminin staining) and quantification of cells with nuclear gamma-H2A.X foci within these subpopulations (average of two experiments (plusminuss.d.), n > 1,000). A western blot analysis on whole-cell lysates from both B-cell populations is shown (bottom right panel). Image published in: Dominguez-Sola D et al. (2007) Copyright © 2007. Image reproduced with permission of the Publisher, Macmillan Publishers Ltd. Larger Image Printer Friendly View |