Figure 4.

Spindly RNAi effects depend on ZW10. (A) CenpB-YFP–expressing cells were either transfected with 50 nM control or Spindly, ZW10-specific siRNAs or a combination of both. Thirty-six hours after transfection mitotic cells were monitored using live cell fluorescence imaging, taking images every 30 s. Arrows, slowly congressing; arrowheads, fast congressing chromosomes. Scale bar, 10 μm.(B) HeLa cells transfected with siRNAs for Spindly, ZW10, or DHC, alone or in combination were fixed 48 h after transfection, stained, and imaged as in A. Shown are maximum intensity projections (MIPs) of representative ZW10, ZW10+Spindly, DHC, and DHC+Spindly siRNA-transfected arrested cells stained for tubulin (green), kinetochores (red), pericentrin (yellow), and DNA (blue). (C) Mitotic index and phases were determined by visual inspection, and classification of images of fixed cells stained as in (B) taken on an a Axiovert200M widefield microscope with a 40× Plan Neofluar0.75 NA objective in three independent experiments with total cell numbers indicated in parentheses. (D) The same images were analyzed for spindle morphology according to the defined main phenotypes (see text), and the frequency of multipolar and long or twisted spindles shown in relation to the total number of mitotic cells was analyzed.