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Fig. 1. Preservation of chromosome arm cohesion/cohesin in MG132-arrested cells. (A) Schematic overview of the experimental procedure. Synchronized HeLa cells were released from early S phase and treated with 100 ng/ml nocodazole, 25 µM MG132 or the solvent DMSO at G2/M transition. Mitotic cells were collected at the indicated times (hours) after the treatment and chromosome morphology was analyzed by Giemsa staining. (B) Representative pictures of chromosomes in the absence (left panel; `open') or presence (right panel; `closed') of arm cohesion. Insets show magnified images of the single chromosomes in the boxed region. (C) Four-hundred prometaphase/metaphase cells were scored for arm status at each time-point; the results are summarized in the histogram. The dark-grey and white populations represents cells with open and closed arms, respectively, as exemplified in B. An unclassified population is shown in light grey. (D) HeLa cells that had been induced to express Scc1-Myc for 2 days were treated with nocodazole or MG132 for 2 hours to enrich mitotic cells, which were then collected and further incubated in a new dish for 2 hours in the presence of the respective drugs. For a control experiment, cells were treated with an equivalent amount of DMSO for 4 hours. Mitotic cells were shaken off, cytospun, fixed and stained with Myc antibodies. Representative Myc-staining patterns including `throughout chromosomes', `centromere enrichment' and `centromere predominant' are shown. (E) One-hundred prometaphase/metaphase cells, indicated by condensin-I-specific subunit CAP-G staining (not shown), were classified into three categories based on Myc-staining pattern as exemplified and colour-coded in D. An unclassified population is shown in grey. Bar, 10 µm.
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