Figure 8. Depletion of DAXX rescues reduced interkinetochore distance in CENP-A overexpressing cells.

A. DAXX is efficiently depleted in DAXX siRNA-treated cells. Immunoblot shows DAXX levels in cell lysates treated with siRNAs as indicated. HeLa ^YFP-CENP-A cells were treated with siRNAs as indicated for 72 hours. Cell lysates were collected using 2x laemmli buffer for western blot analysis. Blots were probed with anti-DAXX Ab to assess depletion efficiency of DAXX following treatment with DAXX siRNA oligo. Anti-tubulin Ab was used as a loading control. B. DAXX depletion rescues reduced interkinetochore distance in CENP-A overexpressing cells. Representative immunostained images of HeLa ^{YFP CENP-A} cells treated with negative or DAXX siRNA oligos. Prior to immunostaining, cells were treated with 10 μM MG132 for 3 hours. Cells were then immunostained with antibodies against CENP-A, CREST antisera for centromeres and stained with DAPI for DNA. Insets correspond to red boxed areas in main images. White boxed areas in insets show examples of kinetochore pairs included in analysis. C. Prism graph shows interkinetochore distance ascertained by distance between two CENP-A signals within aligned chromosomes in MG132-arrested HeLa ^{YFP CENP-A} cells treated with negative or DAXX siRNA oliogs. Each circle represents a kinetochore pair and ‘Kts’ denotes number of kinetochore pairs analyzed in certain number of cells as denoted by ‘cells’. Red horizontal lines represent mean interkinetochore distance as indicated. Error bars represent standard error of mean (SEM) across kinetochores measured from at least two independent experiments. P-values calculated using the Mann-Whitney U test are indicated.