Formation of multipolar spindles induced by Aki1 depletion is dependent on separase. (A) HeLa cells were transfected with control or Aki1-2 siRNA and costained for α-tubulin (α-tub; green) and centrin2 (red). Centrosomes are shown enlarged on the left. (B) Quantitative analysis of centriole number at each spindle pole in control or Aki1-2 siRNA–treated cells (mean of three experiments; 100 total cells were scored per condition). (C) HeLa cells were transfected with the indicated siRNA. Cell lysates were immunoblotted with the indicated antibodies. (D) Percentage of multipolar cells of mitotic cells in indicated siRNA-treated cells. Error bars represent SD of triplicate experiments (150–200 total cells were scored per condition). (E) HeLa cells were transfected with control or Aki1-2 siRNA and harvested asynchronously (Asyn) or 0 or 2 h after release (Rel) from nocodazole (Noc) arrest. Cell lysates were immunoblotted with the indicated antibodies. Bars, 5 µm.