Fig. 5. Mistargeted CENP-C does not recruit CENP-A to ectopic loci. Similar to CENP-A, CENP-C can be mistargeted to non-centromeric regions of chromatin by overexpression. To determine if mistargeted CENP-C is capable of recruiting CENP-A to non-centromeric chromatin, a tetracycline-regulated CENP-C expression vector was constructed and transfected into a stable HeLa cell line in which HA-tagged CENP-A is expressed at near-physiological levels and faithfully targeted to centromeres (Fig. 1A). CENP-C and CENP-A were coexpressed for 48 hours in the cells. Cells were grown and harvested on coverslips. Overexpressed CENP-C was observed to mistarget throughout interphase chromatin (cell on left), visualized using an anti-CENP-C antibody that stains exclusively centromeres in untransfected cells (cell on right). CENP-A was immunolocalized with anti-HA antibody and was found to be faithfully targeted to centromeres despite the overexpression and mistargeting of CENP-C. DNA was counterstained with DAPI. Bar, 10 µm.
