Figure 6.

Mutations in the putative catalytic domain of CENP-V abolish the chromatin hypercondensation phenotype of the wild-type protein but do not affect levels of histone modification H3K9me3. (A) Diagram depicting the cysteine residues mutated to alanine in CENP-V^C174A (red) and CENP-V^CC172/177AA (blue) mutants. (B) Chromatin morphology of non-transfected cells (NT) and cells transiently over expressing CENP-V–GFP (CENP-V) and the C-A mutants (C174A–GFP and CC172/177AA–GFP). The panel shows deconvolved images of DAPI staining of the DNA. (C) Dendrogram based on image similarity of the overexpression phenotype of non-transfected cells and cells overexpressing wild-type or mutant CENP-V–GFP. (D) Quantification of percentages of HeLa cells that undergo mitosis or enter apoptosis in non-transfected cells and in cells overexpressing CENP-V–GFP. (E) Immunoblots reveal that the levels of H3K9me3 are not affected when CENP-V–GFP, CENP-V^C174A–GFP or CENP-V^CC172/177AA–GFP are transiently overexpressed in HeLa cells. To enrich for transfected cells expressing the fusion proteins, at 24 h after transfection, cells were incubated 10 h with puromycin and extracts were prepared 14 h thereafter (nt, non-transfected cells). Bar, 5 μm.