MK-1775 induces apoptosis in AML cells. Panel A: Protein extracts from 6 AML cell lines were subjected to Western blotting and probed with anti-Wee1, -Myt1, -p-CDK1 (Y15), -CDK1, -p-CDK2 (Y15), -CDK2, or -β-actin antibody. Panel B: AML cell lines were cultured for 72 h in complete medium with variable concentrations of MK-1775 (MK) and viable cell numbers were determined using MTT assays. IC50 values were calculated as drug concentration necessary to inhibit 50% growth compared to untreated control cells. The data for panels B and C are presented as mean values ± standard errors from at least 3 independent experiments. Panel C-E: AML cells were treated with MK-1775 for 48 h. The percentage of dead cells was determined by Trypan blue exclusion (Panel C). Apoptotic events were determined by annexin V/PI staining and flow cytometry analyses (Panel D). The data for panel D are presented as mean values ± standard errors of triplicates from one representative experiment which was repeated 3 independent times. Whole cell lysates were subjected to Western blotting, and probed with anti-p-CDK1, -CDK1, -p-CDK2, -CDK2, -γH2AX, or -β-actin antibody (Panel E). Representative Western blots are shown.