Fig. 3

Inhibition of phosphorylated ENSA via MKI-2 delays mitotic entry and disrupts spindle pole integrity. A-C HeLa cells were incubated in DMSO or 20 nM MKI-2 for 24 h and stained using the indicated antibodies. Spindles and chromosomes were visualized using α-tubulin and Hoechst staining, respectively. Scale bar, 5 μm. D The percentage of metaphase cells with misaligned chromosomes among the total metaphase cells. E Quantification of spindle pole abnormalities during mitosis. F Quantification of spindle abnormalities during mitosis. G The percentage of centrosome defects. Data are presented as the mean ± SEM of values from at least three independent experiments (n = 50 miotic cells for each quantification and group). ***p < 0.0001; **p < 0.001. H and I HeLa cells synchronized at the G1/S stage by double thymidine treatment and cultured in DMSO or 20 nM MKI-2 after replacement of the medium. Cells were harvested at the indicated times and analyzed by immunoblotting with the indicated antibodies. Asynchronized cells (Asy) were not arrested by thymidine (H). Quantification of the intensities of the indicated protein bands (I). The data represent typical results and are presented as the mean ± SD of values from three independent experiments; ***p < 0.0001