(A) Inhibition of LSK cell adhesion onto immobilized Del-1 by anti-CD61 (anti-β3) but not by anti-CD11a antibody or anti-CD49d antibody using a static adhesion assay (n = 3–4 independent experiments). Appropriate isotype control antibodies were used; cell adhesion in the presence of isotype controls was set as 1. (B and C) Expression of CD61 (β3 integrin) on hematopoietic progenitor cells was studied by flow cytometry. (B) Representative histograms and (C) median fluorescent intensity (MFI) are shown (n = 5). (D) Ccnd1 expression in LSK cells cultured for 3 hours on Fc-control protein, Del-1−Fc, or Del-1−Fc mutated in the RGD motif (Del-1[RGE]–Fc) (n = 5 independent experiments). Ccnd1 expression in LSK cells is shown relative to control (Fc-control protein [Fc-ctrl]). (E) Effect of ILK inhibition [ILK inh] in Del-1–dependent upregulation of Ccnd1 expression (n = 4 independent experiments). DMSO was used as control. Ccnd1 expression in LSK cells is shown relative to Del-1–Fc in the presence of DMSO. (F) Experimental design for the differentiation assay. LT-HSCs from mice were cultured with Del-1−Fc or Fc-control or Del-1[RGE]–Fc (500 ng/ml each) in cell suspension cultures, and analysis was performed after 7 days. (G) Representative images of the colonies, (H) representative flow cytometry plots, and (I) percentage of GMPs (n = 4 cultures). Scale bars: 200 μm. (J) LT-HSCs were cultured with Del-1−Fc in the presence of a β3 inhibitor or control peptide (25 μg/ml each) for 7 days, as described in F, and the percentage of GMPs was assessed by flow cytometry (n = 5 cultures). (K) Experimental design for the differentiation assay. LT-HSCs were cultured with Del-1−Fc or Fc-control (500 ng/ml each) for 48 hours, and (L) the percentage of MPPs was analyzed by flow cytometry (n = 5 cultures). (M) Accumulation of CFSE⁺ LSK cells in the BM of non-irradiated recipient Edil3^–/– (n = 9) or Edil3^+/+ mice (n = 11) 20 hours after adoptive transfer of CFSE-labeled Lin^– cells from WT mice. Cell accumulation in the BM is expressed as the percentage of CFSE⁺ LSK in total LSK cells. (N) Accumulation of CFSE⁺ LSK cells in the BM of non-irradiated recipient Edil3^+/+ mice at 20 hours after adoptive transfer of CFSE-labeled Lin^– cells from WT mice pretreated with a β3 inhibitor (n = 6) or control peptide (n = 8 mice), as described in Methods. (O) Accumulation of CFSE⁺ LSK cells in the BM of non-irradiated recipient Edil3^–/– mice at 20 hours after adoptive transfer of CFSE-labeled Lin^– cells from WT mice pretreated with a β3 inhibitor or control peptide (n = 7 mice per group). Cell accumulation in the BM is shown as relative to control peptide (ctrl) in N and O. Data are presented as mean ± SEM. Student’s paired t test was used in A, J, and L. One-way ANOVA followed by Holm-Šidák’s multiple comparison test was used in D, E, and I. Mann-Whitney U test was used in M–O. *P < 0.05, **P < 0.01.