Smurf2 silencing inhibits mitotic progression and cytokinesis. (a) Smurf2 levels determined by immunoblotting in HeLa cells (top) and U2OS cells (bottom) 48 h after transfection with anti–Smurf2 siRNA (siSm2) or nonspecific dsRNA (siNS). UN, untransfected control. (b) Morphology of multinucleated HeLa cells 48 h after transfection with siRNA #1. Hoechst was used for DNA staining. (c) Percentages of multinucleated cells in HeLa cultures at the indicated hours after transfection with siNS (open bars) or Smurf2 siRNA #1 (shaded bars). At least 300 cells were counted per time point per cohort. Mean of data from two independent experiments are shown. See Fig. S2 a for raw data from the individual experiments. (d) Percentages of cells that failed cytokinesis in siNS- or siSm2-transfected HeLa cultures quantified from the time-lapse microscopy. At least 65 mitotic cells per cohort (siNS vs. siSmurf2) were analyzed for the progression of cytokinesis. (e) Representative time-lapse pictures of siSm2-transfected HeLa cells that showed impaired mitotic progression and failed cytokinesis. Bars: (b) 50 μm; (e) 10 μm.