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Fig. 6. Decrease in securin levels in nocodazole-arrested cells. (A) Schematic overview of the experimental settings. Asynchronously grown HeLa cells were transfected with Cdc20 siRNA for 22 hours. To obtain a synchronous culture of mitotic cells, we first shook-off mitotic cells to remove cells that had already spent some time in mitosis, and then collected cells that entered and stayed in mitosis within the next 2 hours. Subsequently, cells were transferred to a growing medium containing 100 ng/ml nocodazole, incubated for another 1.5 hours, and analyzed by chromosome spreading and Giemsa staining. (B) Depletion of Cdc20 by RNAi. Total cell extracts were prepared from mitotic cells that were used in the experiment. (C) One-hundred prometaphase/metaphase cells were classified based on chromosome-arm status. The dark-grey and white populations represent cells with open and closed arms, respectively. An unclassified population is shown in light grey. (D) HeLa cells were synchronized at early S phase by the double-thymidine-block regimen and, at 6.5 hours after the release, cells were treated either with 100 ng/ml nocodazole or 25 µM MG132, or with the solvent DMSO. Total cell extracts were analyzed by immunoblotting with the indicated antibodies. In nocodazole-arrest experiments, we noticed a slight reduction in the protein levels of securin. Note that phospho-H3 first appears at 6 hours after the release in both nocodazole- and MG132-treated cells, as in DMSO-treated cells, suggesting that the timing of mitotic entry is not largely affected by these treatments.
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-galactosidase (lacZ) at MOI 200. After 2 days, mitotic cells were collected following 1 hour of treatment with 100 ng/ml nocodazole (or with 25 µM MG132, as a control; data not shown) and incubated in the presence of nocodazole (or MG132) for another 0.5, 1 or 3 hours, when cells were fixed and chromosome spreads were stained with Giemsa. (B) Representative chromosome configurations with a normal number of chromatids (two chromatids; left panels) and diplochromosomes (four chromatids; right panels) are shown. Note that chromosomes appear V-shaped when arm cohesion is dissolved, because mouse chromosomes are acrocentric. Insets show magnification images of the chromosomes in the boxed regions. (C) Approximately 200 cells were assessed for arm cohesion and summarized. Note that diplochromosomes (four chromatids) appeared only in Cre-introduced separase-depleted cells, which were scored separately from chromosomes with two chromatids. (D) Experiment designed to enrich the first mitosis after separase depletion. At 10 hours after adenovirus infection, cells were treated with 1 µM aphidicholin for 24 hours to arrest cells at early S phase. At 7 hours after the release from aphidicholin, when many cells were in G2 phase, either 100 ng/ml nocodazole (Noc) or 25 µM MG132 were added and cells were incubated for 2-4 hours. Mitotic cells were collected and analyzed by chromosome spreading. (E) Quantification data of arm cohesion from the experiment in D. Approximately 200 cells with two chromatids were assessed for each time-point.
100 prometaphase cells for each experiment are summarized in the histogram. The cell population for the open and closed arms is shown in dark grey and white, respectively, and an unclassified population is in light grey. (E) The Scc1-Myc-positive prometaphase and metaphase cells were further classified accordingly to the distribution pattern of Scc1-Myc on chromosomes, as colour-coded. (F) One-hundred prometaphase/metaphase cells were classified into three categories based on Myc-staining pattern, as exemplified in E. An unclassified population is shown in grey. Bar, 10 µm.