SMI-4a inhibition of Pim protein kinase induces cell-cycle arrest in pre–T-LBL. (A) 6812/2 cells were incubated for 24 hours and Jurkat cells for 48 hours with SMI-4a (10μM) or DMSO in serum-free medium. Propidium iodide staining of these cells was followed by cell-cycle quantification performed using flow cytometric analysis. (B) The effect of 24 hours of treatment with SMI-4a (10μM). After treatment of 6812/2 and Jurkat, the cellular levels of p27^Kip1 were measured by Western blotting using antibodies to this protein and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. (C) 6812/2 and Jurkat cells were incubated with increasing doses of SMI-4a for 24 hours in serum-free medium, and the level of p27^Kip1 was measured by SDS-PAGE followed by Western blotting using β-actin antibodies as a protein loading control. (D) The thymus was harvested from 2 Pim1/2/3 triple knockout (TKO) mice and WT-FVB mouse controls. Extracts of these organs were Western blotted with antibodies to p27^Kip1 and β-actin.