Figure 2.
Defective loading of Condensin II complexes on RB1ΔL/ΔL chromosomes. (A) Chromatin fractions were prepared from MEFs that were either proliferating asynchronously or enriched for M-phase cells. Protein content in these fractions was analyzed by SDS-PAGE, followed by Coomassie staining for histone proteins, or Western blotting for the indicated components of the Cohesin and Condensin complexes. (B) Wild-type MEFs were transduced with retroviruses expressing the indicated shRNAs and H2B-GFP. Cell extracts were analyzed by Western blotting for CAP-D3 and Actin. (UT) Untransduced cells. (C) Video microscopy was performed on cells expressing either a control luciferase shRNA, or shRNAs directed against CAP-D3 (sh63 and sh64). Phase-contrast and GFP images were taken every 3 min for 15 h. Representative pictures include the onset of prophase in the left-most panel, and the last view of the metaphase plate before anaphase along with the time elapsed from prophase. The last frame on the right shows the cells after either cytokinesis (shLuc), or failure to resolve as two daughter cells (resulting in binucleation [sh63]) or as persistent anaphase bridges (sh64). The numbers in the left-most image correspond to references in the Supplemental Material and Supplemental Movies. Bars, 50 μm. (D) Extracts were prepared from MEFs of the indicated genotypes. Chromatin fractions from these cells were then subjected to immunoprecipitation with anti-pRB antibodies. The relative amount of CAP-D3, precipitated with wild-type and mutant pRB, was detected by Western blotting. Input levels of relevant proteins from chromatin fractions are shown, and the CAP-D3 blot is overexposed to demonstrate that Condensin II complexes are present in the Rb1 mutant input.