Figure 7

Supernumerary centrosomes in aphidicolin-treated U2OS cells. (A) Effect of siRNAs for ARHGEF10, RhoA and KIF3B on mRNA expression. Expression levels of ARHGEF10, RhoA, KIF3B and GAPDH (control) mRNAs in U2OS cells transfected with indicated siRNAs were measured by RT-PCR. (B) Effect of siRNAs for ARHGEF10, RhoA and KIF3B on protein expression. Expression levels of Myc-tagged ARHGEF10, HA × 3-tagged RhoA(wt), FLAG-tagged KIF3B and α-tubulin (control) in U2OS cells transfected with indicated siRNAs were measured by immunoblotting. M, molecular weight markers. (C) Immunostaining of centrin and γ-tubulin in ARHGEF10-, RhoA- and KIF3B-knockdown cells. U2OS cells were transfected with ARHGEF10 siRNA #1, RhoA siRNA #3 or KIF3B siRNA #2 and synchronized in S phase with aphidicolin. Indicated proteins were immunostained. High magnification images of boxed areas are shown in right panels. Scale bar, 2 μm (right panels) and 10 μm (other panels). (D) Immunostaining of pericentrin and γ-tubulin in ARHGEF10-, RhoA- and KIF3B-knockdown cells. Indicated proteins in U2OS cells treated as in (C) were immunostained. Scale bar, 10 μm. (E) Quantification of supernumerary centrosomes in ARHGEF10-, RhoA- and KIF3B-knockdown cells. U2OS cells were transfected with indicated siRNAs and synchronized in S phase with aphidicolin. The percentage of cells containing multiple centrosomes are shown as the means ± S.E. for three independent experiments. *, P < 0.01. In (C) and (D), nuclei were stained with DAPI, and arrowheads indicate centrioles (C) or centrosomes (D). Thin dotted lines in cell images in (C) and (D) indicate the contour of the cell.