Fig 2: Western blot analysis of relaxin (a), LGR7 (b), and LGR8 (c) protein expression in four CCLr tissue samples and in five CCLc tissues. The bar graphics show the mean ± SEM of relative relaxin, LGR7, and LGR8 protein expression in the CCLr and CCLc samples. Plots show densitometric analysis of relaxin, LGR7, and LGR8 protein bands expressed as relaxin, LGR7, or LGR8/GAPDH densitometry ratio for each tissue sample. Note that GAPDH was identical solo for relaxin and LGR7; for LGR8, the same GAPDH was applied but on a different membrane. M = molecular weight marker.

Fig 3: Double immunofluorescence (IF) staining: relaxin = cytoplasmic red IF; LGR7 = (membranous green IF) relaxin/LGR7 co-localization = membranous yellow IF, and relaxin/LGR8 = cytoplasmic yellow IF; for textual explanation see Results above; CCLc = control dog ligaments in (a,c), and normal synovial membranes in (e,g); CCLr ruptured ligaments in (b,d), and altered synovial membranes of dogs with CCLr in (f,h). Full original blots can be found in Figure S1.

Fig 4: Immunohistochemical labeling of relaxin, LGR7, and LGR8 receptors in canine cruciate ligaments; controls (CCLc) and ruptured ligaments (CCLr): (a) CCLc, case 5, relaxin IHC, IRS = 1, weak, diffuse cytoplasmatic immunoreactivity in 9.5% of fibrocytes. (b) CCLr, case 17, relaxin IHC, IRS = 9, strong immunoreactivity, characterized by few, small cytoplasmic granules in 38.4% of fibroblast-like cells. (c) CCLc, dog 6, LGR7 IHC, IRS = 4, moderate immunoreactivity localized on cell membranes in 12.7% of fibrocytes. (d) CCLr, case 17, LGR7 IHC, IRS = 6, moderate immunoreactivity, characterized cytoplasmic granules in 38.5% of fibroblast-like cells. (e) CCLc, dog 6, LGR8 IHC, IRS = 2, moderate diffuse cytoplasmatic immunoreactivity in 10.7% of fibrocytes. (f) CCLr, case 17, LGR8 IHC, IRS = 9, strong immunoreactivity, diffuse and cytoplasmatic in 48.4% of fibroblast-like cells. Scale bars 50 µm.

Fig 5: Bar graphs showing expression of relaxin (REL), LGR7, and LGR8 in synovial membranes and cruciate ligaments in the control group (n = 7), SMc, and CCLc columns, respectively, and in dogs with CCLD (n = 18), SMr and CCLr columns, respectively; values are expressed as mean immunoreactivity score (IRS) with standard error bars; significance levels of REL CCLc/CCLr p = 0.001 (**), LGR7 CCLc/CCLr p < 0.001 (****), LGR8 CCLc/CCLr p = 0.001 (**), REL SMc/SMr p = 0.02 (**), LGR7 SMc/SMr p = 0.001 (**), LGR8 SMc/SMr p = 0.003 (**).