Fig. 4: The PTH-induced direct association between osteoblasts and osteoclasts was disrupted in the absence of SLPI.
From: SLPI is a critical mediator that controls PTH-induced bone formation
a Maximum-intensity projection (MIP) images of skull tissues obtained by intravital microscopy (Representative image, n = 6 biologically independent experiments). Left panel, tiling image of Col2.3-ECFP/TRAP-tdTomato/SlpiWT/WT mice; right panel, image of Col2.3-ECFP/TRAP-tdTomato/SlpiKO/KO mice at 3 weeks after intermittent PBS (upper panel) or PTH (lower panel) treatment. Cyan, mature osteoblasts; red, mature osteoclasts. Scale bar, 200 µm. b Areas of ECFP+ mature osteoblasts per visual field in the tiling image. Cyan+ (mature osteoblast) areas were binarized using Otsu’s thresholding method. c Areas of tdTomato+ mature osteoclasts per visual field in the tiling image. tdTomato+ (mature osteoclast) areas were binarized using Otsu’s thresholding method. d Cell mixture index (CMI) values per visual field in WT mice. b–d WT PBS: n = 103 images from six mice, WT PTH: 80 images from five mice, Slpi-KO PBS: 64 images from five mice, Slpi-KO PTH: 93 images from six mice. e, MIP images of 3D colocalization. Left panel, Col2.3-ECFP/TRAP-tdTomato/SlpiWT/WT mice; right panel, Col2.3-ECFP/TRAP-tdTomato/SlpiKO/KO mice, at 3 weeks after intermittent PBS (upper panel) or PTH (lower panel) treatment (Representative image, n = 6 biologically independent experiments). Cyan, mature osteoblasts; red, mature osteoclasts. Contact areas (yellow) were defined as the areas of osteoblast and osteoclast colocalization. Scale bar, 200 µm.Numbers of contacts between osteoblasts and osteoclasts; data were normalized to the surface area of ECFP+ osteoblasts (f), and tdTomato+ osteoclasts (g), respectively (WT PBS: n = 11 mice, WT PTH: 11 mice, Slpi-KO PBS: 16 mice, Slpi-KO PTH: 13 mice). Data are means ± SEM. NS, not significant. Statistical significance was determined by ANOVA with Tukey’s test (b, c, f, g), and by Welch’s ANOVA followed by Bonferroni’s multiple comparisons test (d). b P1 exact value = 3.4E−13, P2 exact value = 9.6E−9. c P3 exact value = 4.3E−13, P4 exact value = 4.9E−5. d P5 approximate value < E−15. f P6 exact value = 2.1E−12, P7 exact value = 4.2E−6. g P8 exact value = 2.5E−7.
