Anxa2 regulates the adhesion of early blood cells to human and murine OBs. (A) KG1a cells (1 × 10⁵) were deposited directly onto SaOS-2 monolayers in the presence or absence of antibodies that blocked the function of Jagged-1, N-cadherin, CD164, or Anxa2 or isotype-matched controls or a synthetic peptide corresponding to the 12 N-terminal amino acids (p1-12 peptide) of Anxa2 or a scrambled control for 15 minutes at 4°C. Where indicated, overexpression vectors or short interfering RNA (siRNA) knock down of Anxa2 was used to alter Anxa2 expression. After removing the nonadherent cells, the number of adherent cells was quantified using a fluorescence plate reader. Data are presented as the mean ± SD percentage of adherent cells. Inserts show the results of the Western blot analysis of overexpression or siRNA knockdown of Anxa2 in MG-63 and SaOS-2 cells. (B) Two hundred murine HSCs were layered on murine wild-type or Anxa2^−/− OBs in the presence or absence of an anti-Anxa2 antibody (1 μg/mL). Adhesion was quantified after washing away the nonadherent cells by direct fluorescent cell enumeration. (C) Lin⁻CD34⁺ bone marrow progenitor cells (1 × 10³) were deposited directly onto primary human OBs in the presence or absence of an antibody to Anxa2 or isotype-matched controls. After removing nonadherent cells, the number of adherent cells was quantified. Approximately one third (32% ± 8% or 320 ± 80 of 1000) of the input cells established functional adhesive interactions after 15 minutes at 4°C that were not disrupted by washing. Progenitor cell assays in methylcellulose were performed on CD34⁺ bone marrow progenitor cells recovered following trypsinization. As the individual frequencies of colony-forming unit–granulocyte-macrophage (CFU-GM), colony-forming unit–granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM), and burst-forming unit–erythroid (BFU-E) did not differ significantly among the groups, data were normalized to the value of the untreated controls and are presented as the mean ± SD total colony-forming units per well as a percentage of the value of the untreated controls (n = 5). *P < .05 versus the no-treatment control, and ^#P < .05 versus the IgG control. Data are presented as the percentage of adherent cells relative to the percentage of cells that adhered under the control conditions (IgG control).