Figure 1.
ANGPTL2 is highly expressed in endothelial cells and important for HSC stemness maintenance. (A) The relative mRNA levels of Angptl2 were measured in total bone marrow (BM), macrophages, erythroid cells, B cells, T cells, megakaryocytes (MKs), multipotent progenitors (MPPs), megakaryocyte-erythroid progenitors (MEPs), common myeloid progenitors (CMPs), granulocyte-monocyte progenitors (GMPs), lymphoid progenitors (CLPs), osteoblasts (OBs), mesenchymal stem cells (MSCs), and endothelial cells (ECs) by qRT-PCR (n = 3). (B) Schematic diagram for the strategy for the construction of the Angptl2 conditional knockout mouse lines. (C) Representative electrophoretic images for the genotyping of WT allele, Angptl2fl/fl allele (A2fl/fl) and Angptl2fl/fl deleted allele (A2-deleted) transgenic mice. (D) The frequencies of immunophenotypic Lin−Sca-1+c-Kit+CD34−CD135− LT-HSCs in the BM of Angptl2fl/fl and Cdh5-Cre; Angptl2fl/fl mice as measured by flow cytometric analysis (n = 7). (E) Schematic diagram for the strategy for a serial BM transplantation. (F-G) Representative flow cytometric analyses (F) and quantification data (G) for the competitive reconstitution assay with donor cells isolated from Angptl2fl/fl and Cdh5-Cre; Angptl2fl/fl recipient mice receiving CD45.1 BM cells 6 months after transplantation. The repopulation of CD45.1 donor cells was measured 4 to 16 weeks after transplantation (n = 5). (H) The multilineage contribution of donor cells to T cells (CD3), B cells (B220), and myeloid cells (Mac-1 and Gr-1) in peripheral blood was quantitated by flow cytometric analysis at 16 weeks posttransplantation (n = 5). (I) The frequencies of LT-HSCs in the BM of Angptl2fl/fl and Tie2-Cre; Angptl2fl/fl mice as measured by flow cytometric analysis (n = 7). (J-L) The repopulation ability of donor cells isolated from primary Angptl2fl/fl and Tie2-Cre; Angptl2fl/fl recipient mice was determined by flow cytometric analyses at the indicated time points (J) using a serial transplantation strategy (K). The multilineage contribution of donor cells in peripheral blood was quantified at 16 weeks after transplantation (L; n = 5). (M) Angptl2fl/fl and Cdh5-Cre; Angptl2fl/fl mice were treated with 5-fluorouracil and overall survival was analyzed (log-rank test, n = 8). *P < .05; **P < .01; ***P < .001.

ANGPTL2 is highly expressed in endothelial cells and important for HSC stemness maintenance. (A) The relative mRNA levels of Angptl2 were measured in total bone marrow (BM), macrophages, erythroid cells, B cells, T cells, megakaryocytes (MKs), multipotent progenitors (MPPs), megakaryocyte-erythroid progenitors (MEPs), common myeloid progenitors (CMPs), granulocyte-monocyte progenitors (GMPs), lymphoid progenitors (CLPs), osteoblasts (OBs), mesenchymal stem cells (MSCs), and endothelial cells (ECs) by qRT-PCR (n = 3). (B) Schematic diagram for the strategy for the construction of the Angptl2 conditional knockout mouse lines. (C) Representative electrophoretic images for the genotyping of WT allele, Angptl2fl/fl allele (A2fl/fl) and Angptl2fl/fl deleted allele (A2-deleted) transgenic mice. (D) The frequencies of immunophenotypic Lin−Sca-1+c-Kit+CD34−CD135− LT-HSCs in the BM of Angptl2fl/fl and Cdh5-Cre; Angptl2fl/fl mice as measured by flow cytometric analysis (n = 7). (E) Schematic diagram for the strategy for a serial BM transplantation. (F-G) Representative flow cytometric analyses (F) and quantification data (G) for the competitive reconstitution assay with donor cells isolated from Angptl2fl/fl and Cdh5-Cre; Angptl2fl/fl recipient mice receiving CD45.1 BM cells 6 months after transplantation. The repopulation of CD45.1 donor cells was measured 4 to 16 weeks after transplantation (n = 5). (H) The multilineage contribution of donor cells to T cells (CD3), B cells (B220), and myeloid cells (Mac-1 and Gr-1) in peripheral blood was quantitated by flow cytometric analysis at 16 weeks posttransplantation (n = 5). (I) The frequencies of LT-HSCs in the BM of Angptl2fl/fl and Tie2-Cre; Angptl2fl/fl mice as measured by flow cytometric analysis (n = 7). (J-L) The repopulation ability of donor cells isolated from primary Angptl2fl/fl and Tie2-Cre; Angptl2fl/fl recipient mice was determined by flow cytometric analyses at the indicated time points (J) using a serial transplantation strategy (K). The multilineage contribution of donor cells in peripheral blood was quantified at 16 weeks after transplantation (L; n = 5). (M) Angptl2fl/fl and Cdh5-Cre; Angptl2fl/fl mice were treated with 5-fluorouracil and overall survival was analyzed (log-rank test, n = 8). *P < .05; **P < .01; ***P < .001.

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