Chromokinesin knockdown results in prometaphase arrest. (A) Total cell extracts prepared from HeLa cells 36 h post-transfection (pt) with siRNAs were analyzed by immunoblotting for KIF4A, hKID, and α-tubulin. (B) HeLa cells transfected with 50 nM of siRNAs targeting hKID, KIF4A, or both (KK) were methanol-fixed 40 h pt, DNA stained, and the mitotic index and (C) the mitotic phases determined (mean of three independent experiments + SEM). (D) Maximum intensity projections (MIP) of deconvolved z-stacks showing representative mitotic spindles of different siRNA treatments. Arrows indicate the misoriented chromosome arms and uncongressed chromosomes in hKID/KIF4A RNAi. Arrowheads mark stabilized, aberrant nonkinetochore MTs in KIF4A knockdown cells. Note the increased interkinetochore distance (sister separation) in KIF4A and KK knockdown cells (arrowhead with asterisk). Bar, 5 µm. (E) Quantification of chromosome congression failures from mitotic cells of three independent experiments. (F) HeLa cells were transfected with different siRNAs and treated with 15 nM nocodazole or solvent 24 h later. After 12 h treatment, cells were imaged and analyzed for their mitotic index. Mean ± SEM of three independent experiments. Student’s t test significance values: *, P < 0.05; **, P < 0.01; ***, P < 0.001.