Inhibition of Eg5, but not loss of K fibers, prevents the formation of minor multipolar spindles. (A, B, and F) HeLaM cells were treated as indicated, labeled, and scored for mitotic phenotype (>100 cells in each of four independent experiments, means ± SEM). (A) Scrambled (SC) or LIC siRNA-treated HeLaM cells were incubated for 48 h and then transfected with scrambled or Nuf2 siRNAs and fixed after a further 24-h incubation. (B) Depleted cells were treated with DMSO or 100 nM CENP-E inhibitor (GSK-923295; CI) for 90 min before fixation. (C) Confocal images of mitotic spindles in control (Ctrl) or LIC1 and 2 Xenopus morphants treated with DMSO or STLC to inhibit Eg5. Spindles were stained with anti–α-tubulin, DAPI, and anti–γ-tubulin. Insets show zoom in of a monopolar spindle: two adjacent γ-tubulin foci indicate a single pole. Bar, 20 µm. (D) Quantitation of spindle morphology in control and LIC1 and 2 morphants treated with DMSO or STLC (means ± SEM, six independent experiments). (E) The mitotic index of control (C) and LIC morphants was determined ± STLC treatment (ns = not significant, Student’s t test, means ± SEM, n = 6 independent experiments). (F) Scrambled or LIC siRNA-treated HeLaM cells were treated with DMSO or 2 µM STLC for 2 h. See Tables S1 and S2 for two-way ANOVA results for A, B, D, and E.