Fig. 2.

A35 leads to significant G2/M phase arrest in different cancer cells independent of p53. a DNA content in K562 cells treated with 1 μM A35 for 0, 3, 8 and 24 h as determined by propidium iodide staining and flow cytometry. b Quantitative analysis of (a). c K562 cells were incubated with colchicine or A35 (0.5 or 1 μM) for 24 h. Cells were subjected to immunofluorescence with histone H3 (pSer10), and the mitotic index was calculated. d Cancer cell lines of diverse origins expressing wild type p53, mutated p53 or deleted p53 were treated with 1 μM A35 for 24 h. DNA content was analyzed by propidium iodide staining and flow cytometry. e HCT116 cells were treated by A35 for the indicated time and G2/M arrest-associated proteins were detected by immunoblot. f HCT116 cells were treated for 24 h with A35 and CyclinB1/CDK1 complex was detected by co-immunoprecipitation assay. Cyclin B1 construct was transfected into HCT116 cells, after 24 h A35 was added to incubate for an additional 24 h, and CyclinB1/CDK1 complex was detected by co-immunoprecipitation (g), and cell cycle was detected by flow cytometry (h).*P < 0.05; ** P < 0.01