Fixed analysis of P-MCAK knockdown by RNAi shows defects in spindle assembly and a mitotic delay in prometaphase. PtK2 cells were transfected with a 21 bp siRNA specific to P-MCAK and then processed after 72 hrs for immunofluorescence and for immunoblotting. (A) Immunoblot of serial dilutions of either control or MCAK RNAi cell extracts. K indicates the number of cells in thousands loaded in that lane. (B, C, D, E) Immunofluorescence images of control or MCAK RNAi cells show loss of MCAK staining at centromeres. (F-G) Immunofluorescence images of control or MCAK RNAi cells that show an increase in microtubule (green) density during prometaphase as well as an increase in chromosomes (blue) lingering near the poles (arrows) when MCAK levels are knocked down. (H, I) Quantification of mitotic defects caused by MCAK RNAi. At 72 hrs post-transfection, cells were quantified to determine the specificity of defects seen. (H) Knockdown of MCAK causes a prometaphase delay in mitosis. (I) Knockdown of MCAK causes an increase in cells with increased microtubule density. All data represent mean +/- SEM for 6 experiments. N = 600 cells for mitotic index and N = 100 cells for mitotic phenotype counts for each experiment. Scale bar, 10 μm.