Analysis of Plk1 expression and inhibition in AML cell lines. (A) Exponentially growing AML cell lines (left panel) and primary samples from patients with AML (patients 1-22) or from healthy donor (CD34⁺ and peripheral blood leukocytes [PBL]; right panel) were processed for Western blot analysis of Plk1. Samples 1, 10, and 12 were used as standards to allow comparison of Plk1 levels between the 4 membranes. The membranes were stripped and reprobed for actin detection as a loading control. Data in the left panel are representative of 3 independent experiments. (B) KG1 and U937 cells were cultured with increasing concentrations of BI2536 (1 nM, 10 nM, and 100 nM) for 3 days. C indicates control curve. Cell viability was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide. Results are mean ± SD of 3 experiments performed in triplicate. (C) Top left: KG1 cells were grown in clonogenic assays in the presence of increasing concentrations of BI2536 (control, ■; 1 nM, ; 10 nM, ▧). Colonies were scored at day 7 after seeding. Results are presented as percentage of CFU-L for each BI2536 concentration relative to untreated cells and are mean ± SD of 2 independent experiments performed in duplicate. (Bottom left) To check the efficiency of Plk1 inhibition, KG1 cells were treated for 4 hours with increasing concentrations (1 nM and 10 nM) of BI2536. The corresponding fractions were then analyzed by Western blot with an antibody against the Wee1 kinase, whose stability is reduced via phosphorylation by Plk1. Western blot against actin was used as a loading control. Data are representative of 3 independent experiments. (Top right) KG1 cells were cultured for 3 days after electroporation with Plk1 or control siRNA. Every day, the number of viable cells was assessed by trypan blue staining. Results shown are representative of 3 independent experiments. (Bottom right) Western blot analysis of Plk1 in KG1 cells transfected with control (C) or Plk1 siRNA. (D) KG1 and U937 cells were treated with increasing concentrations of BI2536 (1 nM, 10 nM, and 100 nM) for 48 hours, and analyzed for apoptosis induction by annexin V staining. Results are mean ± SD of 3 independent experiments performed in duplicate.