Induction of apoptosis by SGI-1776 in AML primary cells. (A) Flow cytometry analysis of annexin-FITC/propidium iodide staining of AML primary cells (patient 1) that were either untreated, treated with 0.1% DMSO vehicle alone, 1, 3, 10μM SGI-1776 or 10μM SGI-1776 with 25μM ZVAD for 24 hours. (B) Graphical representation of annexin/PI positive cells in AML cells cultured with increasing concentrations of SGI-1776 in media supplemented with autologous plasma (patients 1 and 2 left of dotted line) or with FBS (patients 3, 4, 5, and 6 right of dotted line). (C) Immunoblot analysis of PARP protein using GAPDH as a loading control in AML primary cells (patient 1). (D) Summary of relevant AML pathway signaling pathways (solid arrows) and Pim kinase phosphorylation targets (dashed arrows). In all 3 AML cell lines and primary AML cells, inhibition of Pim kinase function by SGI-1776 results in the decrease of downstream target protein Mcl-1 and induction of apoptosis.