The coexpression of a truncated Smad4 blocks immortalization of primitive hematopoietic cells. (A) Depiction of the different portions of MH1 used to construct the retroviral plasmids. (B) Findings from the colony-forming unit assay obtained after 4 rounds of plating, with Wt or Smad4^−/− HSPCs transduced with the NUP98-HOXA9 oncogene and different portions of MH1. (Data show mean ± SEM, n = 3, *P < .01 measured by Student unpaired t test). (C) Representative immunoblots showing binding of different portions of MH1 with the Hoxa9 protein in Wt HSPCs transduced with NUP98-HOXA9. Input with the anti-FLAG antibody (top panel). After immunoprecipitation with the anti-FLAG antibody, MH1/Hoxa9 complexes were analyzed by immunoblot with the anti-Hoxa9 antibody (bottom panel, 1 representative experiment of 2 independent experiments). (D) Analysis of cells from the second round of plating by Western blot showing an important activation of apoptosis in primitive hematopoietic cells transduced with MH1-c, assessed by an up-regulation of the pro- and cleaved-Caspase 3 (CASP3) and a down regulation of BCL2. (E) TUNEL assay of Wt and Smad4^−/− HSPCs transduced with the NUP98-HOXA9 oncogene and different portions of MH1. (F) Confocal microscopy in sequential scanning mode, using a 63× oil-immersion objective showing that the expression of MH1-c changes the subcellular distribution of the Smad4 protein, while the cytoplasmic stabilization is still observed under the expression of MH1-e. Only Smad4 translocates to the nucleus, while Hoxa9 stays in the cytoplasm (experiments on Lin⁻ cells). (G) Increased presence of Smad4 was confirmed on the chromatin of cells transduced with MH1-c, compared with cells transduced with the MH1-e control. Chromatin was immunoprecipitated (ChIP) using the anti-Smad4 antibody. IgG antibody was used as control. The relative amount of precipitated p21 and p15 promoter DNA was determined by PCR amplification. (H) Increase in transcription of specific targets of the Smad4 pathway (p15, p21, and p27) in cells transduced with MH1-c. Relative fold change in mRNA expression between the empty (green) and MH1-c (orange) is shown (data show mean ± SEM, n = 4, *P < .01 measured by Student unpaired t test). (I) Kaplan-Meier plot of primary recipient mice showing that mice transplanted with primitive hematopoietic cells coexpressing HOXA9 and MH1-e developed AML, while all mice transplanted with MH1-c– and HOXA9-transduced primitive hematopoietic cells stayed healthy over 1 year (n = 7 for Wt; n = 6 for Smad4^−/−; P value measured by Mantel Haenszel logrank test). (J) Primary recipient mice transplanted with HSPCs transduced with NUP98-HOXA9 and MH1-c (left panel) stayed healthy and no detectable GFP⁺ RFP⁺ cells in BM and no splenomegaly were found of killed mice. In contrast, mice transplanted with primitive hematopoietic cells cotransduced with NUP98-HOXA9 and the empty control vector MH1-e (right panel) developed myeloproliferative disease with splenomegaly (analysis representative of 6 mice).