HOXA9 induces Pim1-associated phosphorylation of the proapoptotic BAD protein, and Pim1 partially rescues the apoptotic defect in Hoxa9^−/− primitive cells. (A) Protein isolated from U937 cells transduced with the 3 vectors described in Figure 1 was subjected to Western-blot analysis. As expected, Pim1 protein was induced by wild-type HOXA9 but not by the GFP or HOXA9NS vectors. BAD protein levels were similar in cells transduced with each of the 3 vectors (second row). However wild-type HOXA9 induced increased phosphorylation of BAD protein at serine 112 as shown by a pS112-specific antibody (top row). Actin levels were measured to ensure equal loading. (B) Wild-type and Hoxa9^−/− marrow cells from 5-FU–treated animals were transduced with vectors expressing either GFP alone or GFP together with Pim1. FACS analysis of cell cycle revealed that Hoxa9^−/− cells transduced with the MIG control vector showed significantly increased percentages of apoptotic sub-G₀ cells compared with wild-type control cells (8.6% vs 1.2%). This apoptotic defect was reduced approximately 2-fold by the introduction of the Pim1 expression vector. Hoxa9^−/− cells also showed a significantly reduced fraction of cells in the S and G₂/M phases of the cell cycle compared with wild-type cells (10.3% vs 30.1%) and this fraction was increased (16.1% vs 10.3%) by the expression of Pim1.