Treatment with DZNep induces apoptosis in a dose- and time-dependent manner and markedly reduces clonogenic survival of AML cells. (A) OCI-AML3 cells were treated with the indicated concentrations of DZNep for 24 hours, then fixed and stained with propidium iodide, and cell-cycle status was determined by flow cytometry. *G₀/G₁ values significantly different from untreated cells; †S-phase values significantly different from untreated cells; ‡G₂/M values significantly different from untreated cells. (B) HL-60 and OCI-AML3 cells were treated with the indicated concentrations of DZNep for 72 hours. Then, the cells were stained with annexin V, and the percentages of apoptotic cells were determined by flow cytometry. Columns represent the mean of 3 independent experiments; bars represent SEM. (C) OCI-AML3 and HL-60 cells were left untreated, or treated with 500 nmol/L DZNep, for 48 and 72 hours. After this, total cell lysates were prepared and immunoblot analysis was performed for PARP. The expression levels of β-actin in the lysates served as the loading control. A vertical line has been inserted to indicate a repositioned gel lane. (D) OCI-AML3 and HL-60 cells were treated with the indicated concentrations of DZNep for 48 hours. After treatment, colony growth in semisolid media was assessed after 7 days. Bar graphs represent the mean percentage values ± SEM of untreated colony growth.