Pitstop 2 disrupts the mitotic spindle and impairs mitotic progression. A-B, Asynchronously growing HeLa cells were treated with the indicated concentration of pitstop 2, 0.1% DMSO vehicle, or 0.3 μM MLN8237 for 6 h (A). Asynchronously growing CHC-depleted cells were treated with pitstop 2 (30 μM), dynole 34-2 (30 μM), MLN8237 (0.3 μM) or DMSO vehicle (0.1%) for 6 h (B). Graphs show the percentage of cells in metaphase (mitotic index, mean ± S.E.M. from two independent experiments, n≥300 per sample). (C) Representative microscopy images of G2/M-synchronized HeLa cells after 30 min treatment with pitstop 2 (30 μM), dynole 34-2 (30 μM), MLN8237 (0.3 μM) or DMSO vehicle (0.1%). Images of untreated, CHC-depleted and epsin-depleted G2/M-synchronized cells are also shown. Alpha-tubulin (red); CHC (green); DNA (blue).D-E, Graphs (mean ± S.E.M. of two independent experiments; n≥30 per sample) show the percentage of metaphase cells with a narrow mitotic spindle in (D) cells described in C as well as in (E) untreated, CHC-depleted, epsin-depleted G2/M-synchronized and in CHC-depleted cells treated with pitstop 2 (30 μM), dynole 34-2 (30 μM), MLN8237 (0.3 μM), or DMSO vehicle (0.1%) for 30 min following G2/M synchronization. F-G, Dot blots show the ratio of the width of the mitotic spindle/total cell of metaphase cells quantified in (D) and (E). Pitstop 2 assessed at 30 μM. Each dot represents one cell. Solid black line represents median (n≥2, 30 cells per sample). Statistical significance determined by Student’s t-test (* p < 0.05, ** p < 0.01).