Figure 4

Multinucleation in Y-27632-treated, but not ARHGEF10-knockdown, HeLa cells. (A) Immunostaining of centrin and γ-tubulin in Y-27632-treated cells. HeLa cells were treated with Y-27632 for 4 h 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). (B) Immunostaining of α-tubulin and γ-tubulin in Y-27632-treated cells. Indicated proteins in HeLa cells treated as in (A) were immunostained. Scale bar, 10 μm. (C) Quantification of multipolar spindle formation in Y-27632-treated cells. HeLa cells were treated with Y-27632 for 4 h 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. (D) Increase of multinucleated cells upon Y-27632 treatment, but not ARHGEF10 knockdown. HeLa cells were transfected with ARHGEF10 siRNA #1 or treated with Y-27632 for 48 h and synchronized in M phase with nocodazole. Multinucleated cells as shown in the right panel were counted after 4 h-incubation without nocodazole. The percentage of multinucleated cells are shown as the means ± S.E. for three independent experiments. *, P < 0.01. Scale bar, 10 μm. (E) Flow cytometric analysis of DNA content in ARHGEF10-knockdown and Y-27632-treated HeLa cells. Representative fluorescence-activated cell sorter histograms and the percentage of cells contained in each peak are shown. (F) Cytokinesis of ARHGEF10-knockdown cells. HeLa cells were transfected with ARHGEF10 siRNA #1 or control siRNA in combination with an expression plasmid for HA × 3-tagged RhoA(wt) and synchronized in M phase with nocodazole. Indicated proteins were immunostained after 3 h-incubation without nocodazole. Representative cells during cytokinesis are shown. Scale bar, 10 μm. In (A), (B), (D) and (F), nuclei were stained with DAPI, and arrowheads indicate centrioles (A) or centrosomes (B). Thin dotted lines in cell images in (A) and (F) indicate the contour of the cell.