VX-680 induces G2/M arrest and disturbs mitotic spindle assembly. HL-60 cells were released from double thymidine-induced G1/S block in the presence of 5 nM VX-680 or DMSO. (A) DNA content of cells collected at the indicated time points was assessed by flow cytometric analysis of cells labeled with PI. Percentage of the cell population in G2/M phase was shown. (B) Cyclin B1 expression at these time points was assessed by Western blot analysis. GAPDH was used as a loading control. A representative of 3 independent experiments was shown. (C) Samples were taken during mitosis and stained with anti-α-tubulin antibody. The percentage of distinct spindle structures in mitosis was averaged from 3 independent experiments. At each time point, more than 100 spindle structures were counted (P < .001). (D) The morphology of mitotic spindles and chromosomes was shown by immunofluorescence staining with anti–α-tubulin antibody and anti–Aur-A antibody. Microtubules are stained as green, Aur-A protein as red, and chromosomes as blue. Bars represent 10 μm. (E) Aur-A expression was suppressed by RNAi in HL-60 cells. (F) The morphology of mitotic spindles and chromosomes in siRNA-transfected HL-60 cells was similar to that in VX-680–treated cells. Microtubules are stained as green and chromatosomes as blue. Bars represent 10 μm.
