KT-NB induces chromosomal misalignment in cells with bipolar spindles. Cells were treated with DMSO as control (A) or 6.75 μg/ml of KT-NB (B, C) for 24 h. After incubation, cells were fixed and processed for immunofluorescence analysis. (A) The control cell in metaphase showed bipolar spindle with chromosome alignment to the metaphase plate. (B) The KT-NB treated cell showed bipolar spindle containing extra centrosome (arrowhead). (C) The KT-NB treated cells exhibited bipolar spindle with the normal two centrosomes. Arrows points to the misalignment chromosomes. DAPI was used for DNA staining, while anti-tubulin shows the mitotic spindle. Merged images: (DAPI is blue, tubulin is red). Scale bar: 5 μm. (D) Only the metaphase cells were analyzed. The percentage of metaphase cells with misaligned chromosome(s) in the bipolar spindle were quantified in control and KT-NB treated cells (n > 50, three independent experiments). (E) All phases of mitotic cells were analyzed. The percentage of mitotic cells with multipolar spindles or misaligned chromosome(s) was quantified in control and KT-NB treated cells (n > 100, three independent experiments). Bi-mis: cells with bipolar spindle and misaligned chromosomes. Multi: cells with multipolar spindles.