Expression of Pim kinase and the effect of SGI-1776 on apoptosis induction in CLL primary cells by SGI-1776. (A) Protein expression of Pim-1, -2, and -3 in untreated CLL primary cells. CLL lymphocytes were lysed and analyzed by immunoblot for Pim kinases with GAPDH as a loading control. (B) Elevated Pim-2 protein levels in CLL primary cells compared with normal lymphocytes. CLL lymphocytes and normal lymphocytes obtained from healthy donors were analyzed by immunoblot for Pim-2 protein as described in panel A. The IgV_H mutation status for the CLL patients were as follows: unmutated (nos. 1, 4, 5, and 6), mutated (nos. 3, 7, 8), and undetermined (no. 2). (C) Flow cytometry analysis of annexin-FITC/PI staining of CLL primary cells (patient no. 1) that were either untreated; treated with 0.1% DMSO vehicle alone; or with 0.3, 1, 3, or 10 μmol/L SGI-1776 for 24 hours. (D) Graph of cell death in CLL samples (n = 7) from treatment with either 0, 0.3, 1, 3, or 10 μmol/L SGI-1776 for 24 hours. (E) Graph of cell death in untreated samples or samples treated with 10 μmol/L SGI-1776 for 24 hours in CLL (n = 19) and lymphocytes from healthy donors (n = 3). (F) Graph of the increase in apoptosis in CLL cells treated with 10 μmol/L SGI-1776 for 24 hours cultured in either media supplemented with autologous human plasma or FBS. Annexin V binding assay was used for apoptosis assay as described in “Apoptosis assay.”