Figure 5. The RNF20/40 complex participates in spindle assembly through stabilizing Eg5.

(a) Depletion of Eg5, RNF20/40 and inhibition of Eg5 activity leads to monopolar spindle formation. Asynchronous MCF-7 cells treated with indicated siRNAs or 100 μM monastrol were grown on glass coverslips, stained with anti-α-tubulin. DAPI staining was included to visualize the chromosome. Scale bar, 5 μm. According to the morphology of chromosomes, 100 mitotic cells were found out, among which the percentage of monopolar spindles was calculated. Each bar represents the mean±s.d. for triplicate experiments and the P value were determined by Student's t-test. *P<0.001. Western blotting was performed using antibodies against the indicated proteins. (b) Depletion of Eg5, RNF20, or RNF40 leads to monopolar spindle formation in HeLa and PANC-1 cells. The percentage of monopolar spindles was examined by immunofluorescence microscopy. Each bar represents the mean±s.d. for triplicate experiments and the P value were determined by Student's t-test. *P<0.001. (c) Eg5 overexpression efficiently offsets monopolar spindle formation induced by RNF20/40 inhibition. MCF-7 cells with RNF20 or RNF40 depletion were transfected with the empty vector, FLAG-Eg5 or FLAG-Eg5K745A expression constructs, and the percentage of monopolar spindles was examined using immunofluorescence microscopy. Each bar represents the mean±s.d. for triplicate experiments and the P value between indicated treatments is examined. Western blotting was performed using antibodies against the indicated proteins.