mTORC1 inhibition fails to drive activated SSC accumulation and early progenitor depletion in the absence of intercellular bridges (A and B) WM-IIF of PLZF (green, spermatogonia), RARgamma (red, late progenitors), SOX3 (white, early/late progenitors) or KIT (white, differentiating spermatogonia), and Ki67 (blue, proliferation) in seminiferous tubules from P36 Tex14 -/- mice. h Cell cycle analysis with Ki-67/DAPI staining of MPPs (left panel) and LT-HSCs (right panel) of young Vav-Cre ASXL1-MT KI mice ( n = 5). g Apoptosis analysis of HSPCs of young Vav-Cre ASXL1-MT KI mice ( n = 7). f Levels of donor chimerism in peripheral blood were analyzed at the indicated weeks after transplantation ( n = 3 (Control), 4 (ASXL1-MT)). 3000 MPPs or 200 LT-HSCs isolated from young control or young Vav-Cre ASXL1-MT KI mice were transplanted into lethally irradiated recipient mice with 4 x 10 5 whole bone marrow cells. e The experimental design for competitive transplantation assays. d Frequency of LSK cells, multipotent progenitors (MPPs), short-term HSCs (ST-HSCs) and long-term HSCs (LT-HSCs) in bone marrow cells of young Vav-Cre ASXL1-MT KI mice ( n = 5). c Absolute numbers of bone marrow cells per leg in young Vav-Cre ASXL1-MT KI mice ( n = 5). b Frequency of myeloid cells (CD11b + ), B cells (B220 + ), and T cells (CD3 + ) in peripheral white blood cells of young Vav-Cre ASXL1-MT KI mice ( n = 13 (Control), 11 (ASXL1-MT)). a Enumeration of white blood cells (WBC), hemoglobin (Hb), and platelets (Plt) in peripheral blood of young Vav-Cre ASXL1-MT KI mice ( n = 13 (Control), 11 (ASXL1-MT)). 1 ASXL1-MT causes dysfunction of HSPCs associated with increased apoptosis and altered cell cycle status. MLL, lysine methyltransferase 2A APm-1, cells with MLL/AF10(OM-LZ) and oncogenic PTPN11 G503A sh, sįig. Allele burden of the 12G-H11-1 clone in BM (left column) or spleen (right column) was determined by PCR-DNA sequencing. The mice were sacrificed at 43 and 57 days post-transplantation (n=2 and n=7, respectively). 12G-V1 and 12G-H11-1 cells were mixed in a 1:1 ratio and i.p. (F) Competitive engraftment and clonal expansion ability between 12G-V1 and 12G-H11-1 cells in vivo. Data shown are representative of three mice with similar results. (E) Flow cytometry analyses was performed to determine Ki-67 and Mac-1 expression in the BM cells obtained from 12G-V1 and 12G-H11-1 leukemia mice at moribund stage. Survival analysis was conducted according to the Kaplan-Meier method. Assays were performed in triplicate and data shown are representative of three independent experiments. (A and C) Reverse transcription-quantitative PCR analyses were performed to determine Hoxa11 expression in (A) Hoxa11 -knockdown APm-1 (APm-1-shH11-1, APm-1-shH11-2) and control (APm-1-shV) cell lines, or in (C) Hoxa11 -overexpression 12G (12G-H11-1, 12G-H11-2) and control (12G-V1 and 12G-V3) cell lines. Expression of Hoxa11 affects survival of MLL/AF10 leukemia mice.
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