INCB053914 synergizes with ruxolitinib to induce apoptosis and inhibit phosphorylation of BAD. (A) UKE1, SET2, and BaF3-JAK2^VF cells were incubated with DMSO (0.1%), INCB053914, ruxolitinib, or the combination of the 2 drugs for 72 hours, and annexin V⁺ cells were detected by flow cytometry. The percent of annexin V⁺ cells induced over DMSO-treated cells is plotted for each treatment. Error bars indicate standard deviation (SD) of biological triplicates, and P values were determined using one-way analysis of variance with multiple comparisons. Drug concentrations used were 0.5 μM INCB053914 and 0.25 μM ruxolitinib for UKE1 cells, 1 μM INCB053914 and 0.04 μM ruxolitinib for SET2 cells, and 0.05 μM INCB053914 and 0.1 μM ruxolitinib for BaF3-JAK2^VF cells. Cell lysates of SET2 cells (B) and BaF3-JAK2^VF cells (C) incubated with INCB053914 and ruxolitinib, alone and in combination as indicated for 4 hours (B) or 8 hours (C) were immunoblotted to assess effects on the levels of the indicated proteins. PARP, poly adenosine diphosphate (ADP)–ribose polymerase.