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RKIP loss aggravates RAS-driven leukemogenesis
with our findings in HL-60 and in healthy human individ- uals, RKIP mRNA levels were high in HSPC and lympho- cytes but significantly reduced in more differentiated cells of the myeloid lineage (P<0.001) (Figure 1C). Of note, by focusing on specific HSPC compartments within GDS3997 and three additional datasets,31-34 we observed that RKIP expression remains high until the granulocyte- macrophage-progenitor (GMP) stage and decreases during the terminal stages of myelomonocytic differentiation (Online Supplementary Figure S1).
Knockdown of RAF kinase inhibitor protein amplifies the myelomonocytic differentiation of hematopoietic stem and progenitor cells
Having shown that RKIP levels are high in HSPC and reduced in differentiated myeloid cells, we next aimed to investigate whether RKIP expression levels are of func- tional relevance for the myeloid lineage commitment of HSPC. Therefore, we performed lentiviral knockdown of RKIP in CD34+ human HSPC isolated from umbilical cord blood specimens (Figure 2A). Subsequently, cells were
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Figure 3. Rkip deletion causes increased myeloid lineage commitment in a murine model by increasing the sensitivity to granulocyte-macrophage colony-stimu- lating factor (GM-CSF). (A) To assess the effects of GM-CSF in vivo, four mice with a deletion of Rkip (Rkip-/-), as well as four control mice (Rkip+/+) were injected intraperitoneally with 500ng GM-CSF twice a day for four days and were analyzed on day 5. (B) Cytological analysis of peripheral blood smears from these mice revealed a significant increase in the number of myelomonocytic cells (MMC) in Rkip-/- animals. A representative peripheral blood smear picture is shown. Lymphoid cells are shown as LYM. Graphs show the average ±Standard Deviation (SD). Statistical significance was evaluated by Student’s t-test. (C) Representative flow cyto- metric plots of the mice presented in (B) showing an increased percentage of CD11b+ and CD11b+ Ly6G+ cells in the bone marrow (BM), peritoneal cavity (PC), and peripheral blood (PB) of Rkip-/- mice. Graphs show the average ±SD. Statistical significance was evaluated by Student’s t-test. RKIP: RAF kinase inhibitor protein.
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