Pitstop 2 disrupts chromosome congression and K-fibre organization during metaphase. A-B, Metaphase synchronized HeLa cells were treated with the indicated siRNA either alone or in combination with pitstop 2 (30 μM), dynole 34-2 (30 μM), MLN8237 (0.3 μM), or DMSO (0.1%) (A) or with the indicated drug alone (B). The graph shows the width of the metaphase plate in each experimental condition, where each dot represents a cell (n≥2 30 cells were scored per sample). Median is indicated by the solid black line. Statistical significance was determined by a Student’s t-test (* p < 0.05, ** p < 0.01). C-E, Metaphase synchronized HeLa cells treated with the indicated siRNA or drugs were stained for α-tubulin (red), HURP (green), and DNA (DAPI, blue). Representative immunofluorescence microscopy images are shown in (C). The graphs (mean ± S.E.M.; n≥30 per sample) show the percentage of siRNA-treated (D) and drug-treated (E) cells with abnormal HURP localisation. Statistical significance was determined using a Student’s t-test (* p < 0.05, ** p < 0.01).