Smurf2 silencing leads to chromosomal misalignment at metaphase and premature onset of anaphase with defective chromosome segregation. HeLa cells stably expressing GFP-H2B were transfected with Smurf2 siRNA #1, and chromosomal movement was monitored 24–25 h after transfection by time-lapse fluorescence microscopy. Quantified data are representative of at least two independent experiments. (a) Defects in chromosomal alignment in Smurf2-depleted cells at metaphase. Metaphase defects in cells transfected with Smurf2 siRNA (shaded bars) or nonspecific dsRNA (siNS, open bars) were categorized into the two indicated groups. (b) Unaligned chromosomes (*) and lack of metaphase plates (#) during mitosis with Smurf2 depletion. Arrowheads denote lagging chromosomes. (c) Chromosomal segregation defects in GFP-H2B HeLa cells transfected with Smurf2 siRNA (shaded bars) or nonspecific dsRNA (open bars) at anaphase. (a and c) Data are means ± SEM (error bars) from three independent experiments. (d) Lagging chromosomes (arrowheads, top row), major segregation defects (arrowheads, middle row), and lack of segregation in GFP-H2B HeLa cells with Smurf2 depletion. (e) Premature anaphase onset in Smurf2-depleted cells. The metaphase–anaphase transition of GFP-H2B HeLa cells was monitored by time-lapse microscopy, and the time from nuclear envelope breakdown (NEBD) until anaphase onset was determined. (f) Representative pictures of the metaphase–anaphase transition. AO, anaphase onset. Bars, 10 μm.