Fig. 2: Induction of Slpi enhances osteoblast differentiation and proliferation.

a Representative images of MC3T3-E1 and primary mouse calvarial osteoblasts transfected with either pMX-Slpi-IRES-puro or pMX-IRES-puro by alkaline phosphatase (ALP) staining (upper) and Alizarin Red S staining (lower) (n = 3 biological replicates per group). β-glycerophosphate was used at a concentration of 10 mM. b, Immunoblot of SLPI in conditioned medium from control MC3T3-E1 and Slpi-overexpressing cells (Representative blot, n = 2 biologically independent experiments). c Typical images of ALP staining of MC3T3-E1 cells. Control (mock) or Slpi overexpressing (Slpi) MC3T3-E1 cells were treated with mock or Slpi conditioned medium under osteogenic conditions for 3 days. d ALP-stained images of MC3T3-E1 cells treated with vehicle or human recombinant SLPI (10 µg/mL) under osteogenic conditions for 3 days. e qPCR analysis of MC3T3-E1 cells transfected with pMX-Slpi-IRES-puro or pMX-IRES-puro (n = 3 biological replicates per group). Cells were cultured in osteogenic differentiation medium for 3 days, and the expression levels of osteoblast transcript regulators (Runx2, Sp7) and differentiation marker genes (Bglap, Col1a1) were quantified. f Proliferation of MC3T3-E1 cells transfected with pMX-Slpi-IRES-puro or pMX-IRES-puro. Cells were cultured in α- MEM for 72 or 120 h after adherence (n = 3 biological replicates per group). **P = 0.0071. Data are means ± SEM. NS, not significant. Statistical significance was determined by ANOVA with Dunnett’s test (a), and two-tailed Student’s t-test (e, f). a P₁ approximate value < E⁻¹⁵, P₂ approximate value < E⁻¹⁵, P₃ approximate value < E⁻¹⁵, and P₄ approximate value < E⁻¹⁵.