Figure 2

Multipolar spindle formation in ARHGEF10-knockdown HeLa cells. (A) Effect of ARHGEF10 siRNAs on mRNA expression. Expression levels of endogenous ARHGEF10 and GAPDH (control) mRNAs in HeLa cells transfected with indicated siRNAs were measured by RT-PCR. (B) Effect of ARHGEF10 siRNA #1 on protein expression. Expression levels of Myc-tagged ARHGEF10 and α-tubulin (control) in HeLa cells transfected with indicated siRNAs were measured by immunoblotting. M, molecular weight markers. (C) Subcellular localization of ARHGEF10 in M phase. HeLa cells were transfected with ARHGEF10 siRNA #1 or control siRNA and synchronized in M phase with nocodazole. Indicated proteins were immunostained. Scale bar, 10 μm. (D) Immunostaining of centrin and γ-tubulin in ARHGEF10-knockdown cells. HeLa cells were transfected with ARHGEF10 siRNA #1 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). (E) Immunostaining of α-tubulin and γ-tubulin in ARHGEF10-knockdown cells. Indicated proteins in HeLa cells treated as in (D) were immunostained. Scale bar, 10 μm. (F) Immunostaining of α-tubulin and pericentrin in ARHGEF10-knockdown cells. Indicated proteins in HeLa cells treated as in (D) were immunostained. Scale bar, 10 μm. (G) Immunostaining of α-tubulin and Aurora-A in ARHGEF10-knockdown cells. Indicated proteins in HeLa cells treated as in (D) were immunostained. Scale bar, 10 μm. (H) Quantification of multipolar spindle formation in ARHGEF10-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 (G), nuclei were stained with DAPI, and arrowheads indicate centrosomes (C, E, F and G) or centrioles (D). Thin dotted lines in cell images in (C) and (D) indicate the contour of the cell.