Knockdown of Eg5 causes an increase in monopolar spindles. Either PtK2 (A, B, C, D) or HeLa (E, F) cells were transfected with the identical 21 bp siRNA to knockdown Eg5 levels. (A, B, E) Control cells stained to visualize microtubules (green), Eg5 (red) and DNA (blue). Eg5 knockdown cells (C, F) or cells that were treated with the Eg5 inhibitor monastrol (D) were stained to visualize microtubules (green), Eg5 (red) and DNA (blue). Knockdown of Eg5 levels resulted in cells with monopolar spindles and a mitotic delay as reported previously. (G) Quantification of the percentage of RNAi cells with bipolar versus monopolar spindles in either buffer, negative control siRNA, Eg5 siRNA, or monastrol treated cells for PtK2, HeLa and PtK1 cell lines. (H) Quantification of the mitotic index in PtK2, HeLa, and PtK1 cells transfected with buffer, control siRNA, or Eg5 siRNA, or treated with monastrol. All data represent mean +/- SEM for 3+ experiments. N = 600 cells for mitotic index and N = 100 cells for mitotic phenotype counts for each experiment. Scale bar, 10 μm.