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Fig. 5. Cohesin dissociation from chromosome arms in prometaphase requires condensin I. (A) Quantitative analysis of sister chromatid cohesion in condensin RNAi cells. Chromosome spreads from HeLa cells transfected with either CAP-D2 or CAP-D3 siRNAs were prepared as described in Fig. 4B, using hypotonic conditions. For each chromosome, 20 line scans were randomly selected on chromosome arms excluding regions where chromatids converge into centromeres (exemplified in the upper left panel). The signal intensities over the defined lines were averaged, and the distance between the peak intensities were scored (upper right panel). The resulting distance values were plotted against chromosome number in histograms (bottom panel). Data are from cells that were not treated (black bars) or treated with nocodazole for 4 hours (purple bars). (B) Analysis of cohesin in condensin depleted cells. HeLa cells, stably expressing Scc1-myc, were transfected with control or condensin siRNAs and processed for IF microscopy following a 50 ng/ml nocodazole treatment for 4 hours. Cells were incubated with anti-myc antibodies (green) and either antibodies against kinetochores (CREST sera, red) or Smc2 antibodies (red, bottom panel). DNA was counterstained with DAPI. (C) Quantitative analysis of Scc1-myc signal-intensity on chromosomes. Chromosome regions were defined by DAPI staining, and the ratio of fluorescence intensities of myc signals to CREST signals was calculated. A Student's t-test demonstrated that the difference between CAP-D2 RNAi cells and control or CAP-D3 RNAi cells was significant (P<0.01).
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