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Fig. 3. Condensin II is required for chromosome condensation in prophase. (A) Analysis of chromosome condensation in fixed prophase-cells depleted of condensin I or II. Logarithmically proliferating immortalized RPE cells were transfected with CAP-D2 or CAP-D3 siRNAs. Sixty hours after the transfection cells were fixed and co-incubated with antibodies against histone H3-phospho-Ser10 and either CAP-D2 or CAP-D3, and probed with Texas Red and Alexa 488, respectively. DNA was counterstained with DAPI. Cells were scored as being in prophase when the shape of their nucleus indicated that they possessed an intact nuclear envelope, and when they were positive for histone H3-phospho-Ser10. Based on DAPI staining, the extent of chromosome condensation in these cells was classified into `none', `mild', `moderate' or `strong' as exemplified in the top panels. Cells were also grouped into three different classes according to their CAP-D2 or CAP-D3 staining intensity as `no reduction', `reduced but still detectable' or `undetectable' (these different signal intensities are graphically represented by black triangles on the left). Each dot represents one prophase cell. (B) Analysis of chromosome condensation during prophase in live HeLa cells depleted of condensin I or II. To visualize chromatin, we analyzed HeLa cells that stably express H2B-EGFP. Prophase image-sequences were extracted from long-term imaging experiments and aligned on the time axis according to NEBD, as determined from the loss of a defined nuclear boundary. Imaging was 56-70 hours after siRNA transfection. Bar, 10 µm.
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