(A–C) Double-fluorescent immunostaining of a longitudinal section through the femur, followed by nuclear DAPI staining (blue), revealed the expression of α₄β₁ (green; A) and ^MuPAR (red; B) near the endosteal bone. The merged image (C) shows coexpression of α₄β₁ and ^MuPAR on the same cells near the endosteal bone (yellow). Dashed line demarcates the border between cortical bone (B) and BM; dotted line distinguishes the bone (left) from the bone marrow cavity (right). Scale bars: 10 μm. (D) Homing and early engraftment of Ly5.1⁺ Lin^–cKit⁺ HSPCs to the BM of lethally irradiated splenectomized Ly5.2⁺ recipient mice. Compared with control IgG, pretreatment with 2 μg anti-α₄β₁ per 10⁶ cells inhibited the homing and early engraftment of HSPCs to the BM 5 days after transplantation. Anti-^MuPAR did not further reduce homing and early engraftment when α₄β₁ was inhibited, which suggests that ^MuPAR and α₄β₁ act in the same pathway. Data with anti-^MuPAR from Figure 3B are repeated for comparison. *P < 0.05 versus control IgG (n = 5–6). (E–G) Adhesion of 5 × 10⁴ WT Lin^–cKit⁺ HSPCs to OP9 mouse BM stromal cells (E), sVCAM-1 (F), or fibronectin (G). Compared with control IgG, pretreatment with anti-α₄β₁ inhibited HSPC adhesion. Anti-^MuPAR did not further reduce adhesion when α₄β₁ was inhibited, which suggests that ^MuPAR and α₄β₁ act in the same pathway. Data with anti-^MuPAR from Figure 3, C–E, are repeated for comparison. *P < 0.05 versus control IgG (n = 8).