C/EBPα-C^m, but not C/EBPα-N^m, collaborated with Flt3-ITD in inducing aggressive AML. (A) Kaplan-Meier analysis for the survival of mice that received transplants of BM cells transduced with both Flt3-ITD-IG and pMYs-IR (FLT/pMYs-IR, n = 5), both Flt3-ITD-IG and C/EBPα-C^m-IR (FLT/C^m, n = 8), both Flt3-ITD-IG and C/EBPα-N^m-IR (FLT/N^m, n = 8), or mock (pMYs-IG/pMYs-IR, n = 8). (B) Cytospin preparations of BM cells derived from mice/FLT/pMYs-IR or mice/FLT/C^m were stained with Giemsa. Images were obtained with a BX51 microscope and a DP12 camera (Olympus); objective lens, UplanFl (Olympus); original magnification ×40 (top), ×100 (bottom). (C) Flow cytometric analysis of BM cells derived from mice/pMYs-IG/pMYs-IR, mice/FLT/pMYs-IR, or mice/FLT/C^m. The dot plots show expression of dsRED versus expression of GFP (first panel). In the indicated gating, the dot plots show expression of Gr-1, CD11b, c-kit, CD34, B220, or CD19 labeled with phycoerythrin-Cy5–conjugated streptavidin versus expression of GFP. (D) Expression of C/EBPα-C^m or Flt3-ITD in spleen cells of mice/pMYs-IG/pMYs-IR (lane 1), mice/FLT/pMYs-IG (lanes 2-3), or mice/FLT/C^m (lanes 4-5) was detected by using anti-C/EBPα (14AA) Ab (middle) or anti-Flt3 Ab (top), respectively, in Western blot analysis. Equal loading was evaluated by probing the immunoblots with anti-tubulin Ab. Data are representative of 3 independent experiments. (E) Immortalized leukemic cells derived from mice/FLT/C^m had increased phosphorylation of STAT5, AKT, STAT3, and ERK compared with those from mice/C^m. Whole-cell extracts of the former cells (immortalized in the absence of IL-3) or the latter (immortalized in the presence of IL-3) were immunoblotted with phospho-specific Abs as described in the Methods. Equal loading was evaluated by reprobing the immunoblots with anti-STAT5, anti-AKT, anti-STAT3, or anti-ERK Abs. Data are representative of 3 independent experiments.