Molecular mechanism of action of SGI-1776 in primary AML cells. (A) Immunoblot analysis of Pim kinase targets in AML primary cells treated with SGI-1776. AML blasts were treated with 0.1% vehicle DMSO alone, 0.3, 1, 3, or 10μM SGI-1776 for 24 hours, then harvested and lysed. Immunoblot analysis of the total and phospho-protein levels of (A) histone H3 (Ser10), c-Myc(Ser62), and Bad(Ser112) from lysates (patient 1). (B) Immunoblot analysis of the total and phospho-protein levels of 4E-BP1 (Thr37/46) from AML primary cells (patients 4 and 5). (C) Quantitation of phospho-4E–BP1 protein levels normalized to total 4E-BP1 levels in AML primary cells (patients 1, 2, 3, 4, and 5). (D) Immunoblot analysis of Mcl-1 protein using GAPDH as a loading control in AML primary cells (patients 4, 5). (E) Quantification of Mcl-1 protein levels normalized to GAPDH levels in 5 AML primary samples (patients 1, 2, 3, 4, and 5) treated with SGI-1776. (F) Inhibition of RNA synthesis in AML primary cells treated with SGI-1776. AML primary cells (patients 1, 2, 3, 4, and 6) were incubated with 0.1% DMSO alone, 1, 3, or 10μM SGI-1776 for 24 hours, then 1 hour before harvesting the cells, [³H]uridine was added to the cell culture as described in “RNA/protein synthesis assay.” (G) Immunoblot of anti- and proapoptotic proteins Bcl-x_L, Bcl-2, Bak, and Bax in AML primary cells (patients 4 and 5).