Figure 6.

Centromere-tethered HP1 and Wapl depletion bypass the requirement for INCENP–HP1 interaction in centromeric cohesion protection. A–C, the indicated stable cell lines were transfected with CB-Myc or CB-CSD-Myc and treated with nocodazole for 3 h. Mitotic chromosome spreads were stained with DAPI and antibodies for Myc and CENP-C. Example images are shown (A). The inter-KT distance was measured on over 616 chromosomes in 20 cells (B). The asynchronous cell lysates were immunoblotted (C). D–G, HeLa cells and the indicated stable cell lines transfected with control or Wapl siRNA were immunoblotted (D); exposed to MG132, fixed at the indicated time points for DNA staining, and quantified in around 200 cells (n = 2) (E); used to prepare chromosome spreads after 7-h treatment with MG132 followed by determining the percentage of cells with cohesion loss in around 60 cells (F); or treated with nocodazole for 3 h to make chromosome spreads and stained with ACA and DAPI followed by measuring the inter-KT distance on over 749 chromosomes in over 30 cells (G). Means and error bars representing S.D. are shown (unpaired t test). Scale bars, 10 μm. See also Fig. S5. ns, not significant.