Transient ATRX depletion in HeLa cells induces abnormal nuclear morphology. (A) Immunofluorescence detection of ATRX (green) and the kinetochore marker CREST (red) on prometaphase and metaphase chromosomes. (B) Western blot analysis of HeLa cells transiently transfected with siATRX 19-mer duplexes demonstrate ATRX protein depletion starting at 48 h and up to 96 h after transfection. ATRX protein levels remained constant upon mock transfection and in cells transfected with a control siATRX scrambled sequence duplex (left). Both ATRX isoforms were effectively silenced by 48 h after transfection (right). α-Tubulin protein expression (αTub) was used as a control in all experiments. Numbers on top of the blot indicate hours after transfection and numbers in parenthesis indicate the molecular mass in kD. (C) Costaining of kinetochores and ATRX with the CREST and 39f antibodies, respectively, reveals loss of ATRX protein at PCH in siRNA-treated cells. (D) Cells stained with DAPI show abnormal nuclear morphology in ATRX-depleted cells compared with control cells. Common defects include lobulated nuclei and intranuclear DNA bridges (right). (E) The number of nuclei displaying abnormalities was quantified at 48, 72, and 96 h after siRNA treatment (n > 1,000 nuclei at each time point). Bars: (A and C) 5 μm; (D) 20 μm.