Increased interkinetochore distances and reduced cohesion in ATRX-depleted cells. (A) ATRX-depleted metaphase cells were stained for the kinetochore proteins CENP-F or CENP-E (green) in combination with CREST (red) and imaged at different z planes. Images represent an extended focus rendering of deconvoluted z stacks. (B) Control and ATRX-depleted cells were stained with anti-CREST antibody to stain kinetochores. Distances between paired kinetochores (n = 100) were measured at individual z planes and were significantly increased in shATRX1 and shATRX2 cells compared with controls (P < 0.05 by ANOVA). (C) Mitotic HeLa cells were transiently transfected with either siATRX-Scr control or siATRX1 and siATRX2 duplexes. After mitotic shake-off to remove any cells in mitosis, cells were treated with colcemid and chromosome spreads were stained with DAPI and scored for the percentage of chromatids displaying cohesion defects (n = 50 per treatment). Chromosomes from ATRX-depleted cells showed reduced cohesion compared with control-treated cells. (D) Microscopic images of representative chromosome spreads scored as normal (<10% cohesion defect), moderate (10–90% cohesion defect), or severe (>90% cohesion defect). Insets show representative chromosomes from each spread with normal (left), moderate (middle), and severe (right) cohesion defects. Bars: (A and B) 5 μm; (C) 20 μm.