Effect of PD0332991 on primary AML cells. CD34⁺ and primary AML cells from patients 28 (Flt3 wt) and 82 (Flt3 ITD) were plated in methylcellulose and treated with PD0332991 (500 nM) or DMSO (vehicle control) for 4 days. The cells were fixed and stained with propidium iodide, and cell-cycle analysis was performed by flow cytometry. (A) Representative flow cytometric cell-cycle analysis of 1 experiment with CD34⁺ cells as well as cells from patients 28 and 82. (B) Bar graph analysis of the percentage of PD0332991-treated cells in S and G₂ compared with control cells using data from 3 independent experiments (mean ± SE). (C) Western blot. CD34⁺ and primary AML cells from patients 28 and 82 were treated as in (A). Cell lysates were prepared and resolved by SDS/PAGE, and an immunoblot was performed with the indicated antibodies. (D) Cells were treated as in (panel A). Cell lysates were prepared and normalized by protein concentration. CDK2 was immunoprecipitated to perform the in vitro kinase assay. Incorporation of ³²P in the CDK2 substrate histone H1 was assessed by SDS-PAGE followed by autoradiography. As a control for the specificity of the kinase assay, a lysate from CD34⁺ cells was incubated with an isotype-matched antibody to form an immunocomplex. To confirm equal protein amounts in each normalized extract, aliquots were separated by SDS-PAGE and analyzed by Western blot with an anti-Grb-2 antibody. (E) Quantitative representation of ³²P-histone H1(cpm) of the experiment depicted in (panel D).