Fig 2: The effects of PAR-1 and PAR-2 agonist peptides on cytokine release from HLF-1 cells. Fibroblasts were cultured overnight with serum-free medium, then stimulated with either different concentrations of the PAR-1 or PAR-2 agonist peptides for 24 h (top panels). Fibroblasts were also stimulated with 400 µmol L-1 PAR peptides, or vehicle, for different durations (bottom panels). IL-6 (left panels) and IL-8 (right panels) release was determined by ELISA, and expressed as mean ± SEM per 2.5 × 105 cells from three independent experiments performed in quadruplicate.

Fig 3: Detection of a-smooth muscle actin expression in HLF-1 cells using immunocytochemistry. The fibroblasts were stimulated with 3 U mL-1 thrombin, 12.5 pmol L-1 trypsin, 400 µmol L-1 PAR agonist peptides (PAR-1, PAR-2, PAR-3, and PAR-4), or appropriate control peptides for PAR-1 (PAR1C) and PAR-2 (PAR2C). The cells were subsequently fixed, stained using an anti-a-smooth muscle actin antibody, and visualized using fluorescence microscopy. Bar = 50 µm.

Fig 4: PAR2-Gi activates JNK-SREBP-1c pathways that induce hepatic lipid accumulation in HFD-fed mice. (A) Western blots of liver lysates from WT and PAR2-KO mice fed either normal chow diet (ND) or high fat diet (HFD) for 16 weeks. Densitometric quantification of western blots on left for Phospho (P)-Jnk1/2 normalized to total (T)-Jnk1/2, active 65 kDa proteolytic fragment of SREBP-1c normalized to ß-tubulin, and Phospho (P)-AMPK normalized to total-AMPK. (B) Quantitative PCR (??CT) of mRNA from mouse liver (n = 5) normalized to ß-actin mRNA of Srebp1c and AMPK targets: Pgc1a (peroxisome proliferative activated receptor, gamma, coactivator 1 alpha), Pgc1ß (peroxisome proliferative activated receptor, gamma, coactivator 1 beta) and Ppara (peroxisome proliferator activated receptor alpha). (C–D) Trypsin (10 nM) induces phospho-Jnk1/2 signaling in HuH7 hepatoma cells, which is suppressed by the Gi inhibitor, pertussis toxin (PTX); 15 min time-point used in D. (E) Par2 siRNA (500 nM) knockdown inhibits trypsin-induced phosphorylation of Jnk1/2 (15 min) in HuH7 cells. Trypsin induces nuclear translocation of SREBP-1c, which is inhibited by siPAR2 and by the Jnk1/2 inhibitor SP600125. ß-actin and Lamin A were used as loading controls for whole cell and nuclear lysate western blots, respectively. (F) Mechanism of PAR2-Gi regulation of lipid metabolism in hepatocytes by activating Jnk1/2 and SREBP-1c pathways. One-way ANOVA followed by Tukey's post-hoc test (A) or unpaired 2-tailed T-test (B) was performed, *P < 0.05, **P < 0.01, ***P < 0.001, #P = 0.06.

Fig 5: PAR-2 protein expression in DP treated tendon sections (n = 3) compared to contralateral side (no treatment; n = 3); quantified by the red/green ratio in histograms with 5 FOVs per section (A) and representative samples stained for PAR-2 (B), with areas of higher magnification, taken from the middle of the AT (C). The dark brown staining at 12 weeks does not come from an overexpression of PAR-2 by the tenocytes; it is the partially degraded DegraPol polymer that stains like this (Supporting Information SI Fig. 3). Nevertheless, the mid portion of the AT showed a significant slight increase in PAR-2 expression (+ 4.9%). Scale bars indicate 500 µm; in inserts they indicate 100 µm. Stars, with p-values, indicate pairwise comparison probability: p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***).