The cells were centrifuged and the fixation solution was decanted. herein support a model whereby the activation of Stat3 in the early stage of erythroleukemia induced by FV inhibits terminal differentiation of the infected erythroblasts, at least in part, through the upregulation of Pu.1 expression, thus promoting the polyclonal expansion of infected erythroblasts. The insertional activation of Pu.1 in the late stage of disease could, ultimately, circumvent the requirement for Stat3 in these cells. Results Stat3 is required for the development of Friend erythroleukemia in vivo Contamination of mice with FV drives a rapid polyclonal growth of infected erythroblasts in the spleen of infected animals, resulting in severe splenomegaly. To determine whether Stat3 is required for the progression of Friend erythroleukemia in response to FV contamination. Open in a separate window Physique 1 Deletion of Stat3 renders mice resistant to Friend disease. (a) Wild-type BALB/c and Balb/c-Stat3fl/fl;Mx1Cre mice were injected with phosphate-buffered saline (PBS) or pI/pC followed by infection with Friend computer virus. Two weeks following contamination, spleens were harvested and splenic weights are shown. results in the Epo-independent (Epoind) formation of BFU-E. Previously, we exhibited that bone marrow from Stat3fl/fl mice transduced with Cre recombinase failed to form Epoind colonies in response to FVP contamination (Ni in response to Friend computer virus contamination requires tyrosine phosphorylation, DNA binding and tetramerization of Stat3. (a) Schematic representation of the Stat3 mutants used in this study. (b) Expression of the Stat3 mutants in the 293 packaging cells. (cCe) Bone marrow was extracted from Stat3/ animals and transduced with wild-type or mutant forms of Stat3 followed by contamination with Friend computer virus. The cells were plated in methylcellulose in the presence and absence of Epo, and BFU-E colonies were assessed on day 8 following staining with benzidine. DBD, DNA binding domain name mutant; N, deletion of the first 131 amino acids. **with vector alone (MSCV), WT Stat3 or the indicated mutants was infected with FVP overnight and expression of Pu.1 in infected cells was assessed by circulation cytometry. (d) Bone marrow was harvested from wild-type BALB/c mice and transduced with vacant murine stem cell computer virus (MSCV) or an MSCV-based retroviral vector expressing wild-type or dominant-negative Pu.1 followed by contamination with FVP. The cells were plated in methylcellulose and BFU-E figures were assessed on day 8 following staining with benzidine. (e) Bone marrow from Stat3/ mice was transduced with wild-type or dominant-negative Pu.1 as Piboserod explained above followed by infection with FVP. Cells were plated in methylcellulose and BFU-E figures were assessed on day 8 following benzidine staining. **following contamination with Friend computer virus. (a) Schematic representation Piboserod of the putative Stat3 binding elements in the Pu.1 promoter region. Their positions are shown relative to the start of transcription. (b) The sequences of sites 2 and 3 are aligned from multiple species. The boxed region indicates the putative Stat3 binding sites. Within the boxed region, capital CD3G letters indicate conserved nucleotides and lower case letters indicate nonconserved nucleotides relative to the mouse sequence. (c) Wild-type BALB/c mice were infected with FVA or FVP and Stat3/ mice were infected with FVA. Splenocytes were harvested from these animals Piboserod on day 10 following contamination, and binding of Stat3 to elements in the Pu.1 promoter was assessed by chromatin immunoprecipitation (ChIP) analysis. Pos Cont; input DNA. IL-2, nonspecific control for DNA content. Stat3 is not required for the growth of leukemic cells following FV contamination Following the initial stage of Friend disease, in which infected erythroblasts proliferate and differentiate in the spleen in a polyclonal manner, leukemic clones emerge.
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