Synergistic effects of CX-4945 with doxorubicin on cell proliferation arrest and apoptosis in B-ALL cells. (A-C) Effects (left) and synergistic analysis (right) of doxorubicin (gray line) and the combination of doxorubicin and CX-4945 (red line) on proliferation of Nalm6 (A) and 697 (B), and drug-resistant N/6 B-ALL (C) cells. Cells were treated with the indicated doses of drugs for 2 days. Cellular proliferation was measured by WST-1 assay. Synergistic analysis was performed with Calcusyn; where combination index value of: 1.15 to 0.8 is considered additive effect; 0.85 to 0.7 is moderately synergistic; and <0.7 is very synergistic effect, respectively. ***P < .001. (D) Effect of CX-4945 (4 μM), doxorubicin (DOX, 3.2 nM), and the combination of doxorubicin (3.2 nM) plus CX-4945 (4 μM) on apoptosis in Nalm6 (left) and patient 1 (right) B-ALL cells. Cells were treated for 2 days and stained with 7-AAD and annexin V for flow cytometry to assess apoptosis. The percentage of cells in the lower right quadrant and upper right quadrant of each flowchart represents the percentage of early apoptotic or late apoptotic cells, respectively, in samples treated with the indicated drugs. Mean ± SD of triplicates representative of 1 of 3 independent experiments (A-C). Flow cytometry plots in panel D depict a representative experiment and percentages are the mean ± SD of 3 independent experiments.