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. 2022 Mar 10;139(10):1529–1540. doi: 10.1182/blood.2021011644

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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, Angptl2^fl/fl allele (A2^fl/fl) and Angptl2^fl/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 Angptl2^fl/fl and Cdh5-Cre; Angptl2^fl/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 Angptl2^fl/fl and Cdh5-Cre; Angptl2^fl/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 Angptl2^fl/fl and Tie2-Cre; Angptl2^fl/fl mice as measured by flow cytometric analysis (n = 7). (J-L) The repopulation ability of donor cells isolated from primary Angptl2^fl/fl and Tie2-Cre; Angptl2^fl/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) Angptl2^fl/fl and Cdh5-Cre; Angptl2^fl/fl mice were treated with 5-fluorouracil and overall survival was analyzed (log-rank test, n = 8). *P < .05; **P < .01; ***P < .001.