Fig. 4. Survivin RNAi causes accumulation in prometaphase, failure in cytokinesis and multinucleation. (A,B) Time course of mitotic staging of cells transfected with Survivin siRNA. Survivin-positive cells were scored in A and Survivin-depleted cells were scored in B. From 36 hours post transfection, the percentage of Survivin-depleted cells in prometaphase increases and the percentage of cells in later mitotic stages decreases. (C,D) Examples of Survivin-positive and Survivin-depleted cells in mitosis. Centromeres (red); tubulin (green); DNA (blue). Inset panels show the Survivin staining in the region indicated by the box. (C) Survivin-positive cell with all chromosomes aligned. (D) Survivin-depleted cells have misaligned chromosomes (arrows). (C',D') Chromosomes in C and D in greyscale. Bars, 5 µm. (E) Representative graph showing the percentage of multinucleated cells in cultures following transfection with control (red) versus Survivin (blue) siRNA. Multinucleation increases gradually in cultures transfected with Survivin siRNA. (F) Phase-contrast images of cells from cultures transfected with control or Survivin siRNAs, blocked in S-phase, released in fresh medium and followed by time-lapse since entering mitosis (which begins approximately 10 hours post-release from S-phase). Numbers in images indicate time (in hours) since entering mitosis. The two Survivin siRNA transfected cells shown spent >1 hour in mitosis and failed cytokinesis. (G) Histogram showing the percentages of cells from cultures transfected with control (n=73) or Survivin siRNAs (n=101) that spent up to 1 hour or more than 1 hour in mitosis before completing (yellow) or failing (green) cytokinesis.

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