Figure 3

Role of RhoA downstream of ARHGEF10. (A) Effect of RhoA siRNAs on mRNA expression. Expression levels of endogenous RhoA and GAPDH (control) mRNAs in HeLa cells transfected with indicated siRNAs were measured by RT-PCR. (B) Effect of RhoA siRNA #3 on protein expression. Expression levels of HA × 3-tagged RhoA(wt) 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 RhoA-knockdown cells. HeLa cells were transfected with RhoA 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 RhoA-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 RhoA-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 RhoA-knockdown cells. Indicated proteins in HeLa cells treated as in (C) were immunostained. Scale bar, 10 μm. (G) Quantification of multipolar spindle formation in RhoA-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. (H) Suppression of multipolar spindle formation in ARHGEF10-knockdown cells by RhoA. HeLa cells transfected with ARHGEF10 siRNA #1 or control siRNA in combination with an expression plasmid for green fluorescent protein (GFP) (control), Myc-tagged ARHGEF10(376–665) or HA × 3-tagged constitutively activated GTPases were 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.