Figure 6

Multipolar spindle formation in KIF3B-knockdown HeLa cells. (A) Effect of KIF3B siRNAs on mRNA expression. Expression levels of endogenous KIF3B and GAPDH (control) mRNAs in HeLa cells transfected with indicated siRNAs were measured by RT-PCR. (B) Effect of KIF3B siRNA #2 on protein expression. Expression levels of FLAG-tagged KIF3B and α-tubulin (control) in HeLa cells transfected with indicated siRNAs were measured by immunoblotting. M, molecular weight markers. (C) Immunostaining of centrin and γ-tubulin in KIF3B-knockdown cells. HeLa cells were transfected with KIF3B siRNA #2 and synchronized in M phase with nocodazole. Indicated proteins were immunostained. High magnification images of boxed areas are shown in lower panels. Scale bar, 10 μm (upper panels) and 1 μm (lower panels). (D) Immunostaining of α-tubulin and γ-tubulin in KIF3B-knockdown cells. Indicated proteins in HeLa cells treated as in (C) were immunostained. Scale bar, 10 μm. (E) Immunostaining of α-tubulin and pericentrin in KIF3B-knockdown cells. Indicated proteins in HeLa cells treated as in (C) were immunostained. Scale bar, 10 μm. (F) Immunostaining of α-tubulin and Aurora-A in KIF3B-knockdown cells. Indicated proteins in HeLa cells treated as in (C) were immunostained. Scale bar, 10 μm. (G) Quantification of multipolar spindle formation in KIF3B-knockdown cells. HeLa cells were transfected with indicated siRNAs and synchronized in M phase with nocodazole. The percentage of cells containing multiple centrosomes are shown as the means ± S.E. for three independent experiments. *, P < 0.01. In (C) to (F), nuclei were stained with DAPI, and arrowheads indicate centrioles (C) or centrosomes (D, E and F). Thin dotted lines in cell images in (C) indicate the contour of the cell.