Spindle checkpoint activation and aberrant chromosome segregation in ATRX-depleted cells. (A) The spindle checkpoint protein BubR1 was detected in ATRX-depleted metaphase cells at both aligned and misaligned chromosomes (top). Another checkpoint protein, Bub1, was also found to be adjacent to the centromeres (CREST staining) in ATRX-depleted metaphase cells. (B) Mitotic index of control and ATRX-depleted cells after 16 h of nocodazole treatment. Control and ATRX-depleted cells were cultured with or without nocodazole for 16 h and live cells were photographed and scored for the percentage of mitotic rounded cells by phase imaging (n > 1,000). (C) Live cell imaging of control and ATRX-depleted mitotic cells (n = 100 for each cell line) revealed an increased number of cells that undergo repeated failed attempts at initiating anaphase, an increased number of cells that initiate anaphase without full chromosome alignment, and an increased incidence of segregation defects as indicated by the formation of chromosome DNA bridges and micronuclei. (D) The number of internuclear bridges and micronuclei indicative of chromosome missegregation were quantified at 2–4 d after transfection of the siATRX1 duplex and the fold difference between mock and depleted cells was calculated. (E) Fixed mitotic cells stained with DAPI (blue) and a phosphohistone H3S10 antibody (red) showing intranuclear DNA bridges and micronuclei in transiently depleted cells. Bars, 5 μm.