Figure 3.

Combination of OSI-906 and ZM447439 causes severe defect in chromosome alignment. HeLa S3 cells were treated with 6 µM RO-3306 for 20 h, released by washing out this drug, and then cultured without RO-3306. After 60-min culture, the cells were fixed and stained for α-tubulin and DNA. Based on morphologies of spindle and chromosomes, mitotic cells were classified into four groups: prophase/prometaphase (P/PM, red), metaphase (M, yellow), anaphase/telophase (A/T, green), and cytokinesis (Cyto, blue). Cells were treated with 3 µM OSI-906, 1 µM ZM447439, or their combination (OSI + ZM) for 1 h at the end of the RO-3306 treatment and were continuously treated during the 60-minute release. Dimethyl sulfoxide was used as solvent control (DMSO). Representative images are shown. Arrows indicate cells after the onset of anaphase. The average value of each group and the mitotic index, a ratio of mitotic cells among total cell numbers, were calculated from three independent experiments (n > 222 in each experiment) and expressed as mean ± SD. p-values were calculated using Tukey’s multiple comparison test. *** p < 0.001. NS, not significant.