Fig. 3: SLPI is associated with adhesion between osteoblasts and osteoclasts.

a TRAP-stained cells showing the effect of recombinant human SLPI on osteoclastogenesis. Scale bar, 100 µm. Right, numbers of TRAP⁺ cells (n = 3 biological replicates per group). b Effects of SLPI on the generation of bone resorption pits. Mature osteoclasts were transferred to a Corning Osteoassay Surface in the presence or absence of recombinant human SLPI. After 48 h, cells were removed, and resorption pits were visualized. Scale bar, 200 µm. Right, area of resorption pits (%) (n = 3 biological replicates per group). c Fluorescence images of mock (upper panel) or Slpi-overexpressing (lower panel) MC3T3-E1-EGFP cells in contact with TRAP-tdTomato⁺ primary osteoclasts (Representative image, n = 3 biologically independent experiments). Confocal fluorescence microscopy images were acquired at 0, 120, 240, 360, and 480 min. White triangles, direct cell–cell contact. Scale bar, 10 µm. d, e Analysis of osteoblast–osteoclast contact duration based on the data in c (Mock: n = 67 cells examined over six independent experiments, Slpi: n = 31 cells examined over 5 independent experiments). f Relative expression levels of Slpi in enhanced green fluorescent protein (EGFP)-labeled MC3T3-E1 cells and tdTomato-labeled osteoclasts. After coculture, MC3T3-E1 cells and osteoclasts were separated (n = 1 experiment per group). g Left, diagrammatic representation of in vitro coculture of osteoclasts with MC3T3-E1-EGFP cells. Right panel shows the effects of coculture on Slpi and Fgf2 expression in MC3T3-E1-EGFP cells (n = 3 biological replicates per group). 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 (b, e, g).