Figure 7. Depletion of DAXX prevents mislocalization of CENP-A and rescues chromosome segregation defects 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 prevents CENP-A mislocalization. Representative images of chromosomes show expression and localization of YFP-CENP-A in HeLa ^YFP-CENP-A cells treated with siRNAs as indicated. Chromosome spreads were prepared and immunostained with antibody against GFP to visualize CENP-A and stained with DAPI for DNA. Cells were then imaged and analyzed for YFP-CENP-A localization. Scale bar: 1 μm. C. CENP-A levels are reduced in non-centromeric regions following DAXX depletion. Prism graphs show CENP-C signal intensities at non-centromeric regions (left) and at centromeres (right) in chromosome spreads of HeLa ^YFP-CENP-A cells treated with siRNAs as indicated. Each circle represents a spot and ‘n’ denotes number of spots analyzed. Red horizontal lines represent mean signal intensities as indicated. Error bars represent standard error of mean (SEM) across the areas measured. P-values calculated using the Mann-Whitney U test are indicated. # represents statistically insignificant values. D. DAXX depletion suppresses chromosome segregation defects. Bar chart graph shows proportion of HeLa ^YFP-CENP-A cells with defective chromosome segregation in different conditions. Error bar represents standard error of mean across three independent experiments. ‘n’ denotes number of cells analyzed. P value calculated using proportion test is indicated.