Fig 2: Effects of EEDAR on signaling molecules that regulate TJ structure and function in spleen-deficiency diarrhea rats (n = 3). The phosphorylation levels of p38 and PAR-2 in total colon homogenates were measured by Western blot analysis (A,B). Representative images of immunohistochemistry sections (200× magnification) from PAR-2 and P-P38 (C) colons indicated different pathological changes in spleen deficiency diarrhea rats treated with EEDAR-L (1.85 g·kg−1), EEDAR-H (7.40 g·kg−1), and SLBZS. Total protein levels of P-MLC and MLCK were measured by Western blot analysis (E,F). Bands were quantified and values normalized to non-phosphorylated protein or β-actin levels. Values represent means ± SEM. # p < 0.05 vs. Control; * p < 0.05 vs. Model. (Scale bar 1:100).

Fig 3: IL-22 inhibits PAR-2 expression in HaCaT keratinocytes(A) Addition of IL-22 (20 ng/mL) to keratinocytes for 24 h inhibited PAR-2 expression. (B) Addition of IL-22 (20 ng/mL) to keratinocytes induced phosphorylation of Stat3, Akt. Western blot of melanocytes lysates after 0, 5, 10, 20, 40, 60 min of IL-22 treatment (20 ng/mL). (C) Cultured cells were pre-treated with 10 μM of LY294002 for 60 min, followed by exposure to IL-22 (20 ng/mL) for 60 min. The effect of IL-22 on phosphorylation of Akt was partially suppressed by LY294002. Cultured cells were pre-treated with 10 μM of LY294002 for 60 min, followed by exposure to IL-22 (20 ng/mL) for 12 or 24 h, the expression of PAR-2 were examined. (D) Cultured cells were pre-treated with 10 μM of Stattic for 60 min, followed by exposure to IL-22 (20 ng/mL) for 12 h and 24 h. The inhibition of IL-22 on PAR-2 expression can be interfered by Stattic. Results were normalized against β-actin expression.

Fig 4: Effect of PAR-2 shRNA on the cell cycle of different EC109 cell lines by flow cytometry. (A) EC109 cell line treated with PAR-2 shRNA for 24 h. (B) EC109 cell line treated with PAR-2 shRNA for 48. (C) EC109 cell line treated with PAR-2 shRNA for 72 h. (D) Control group (without transfection) for 24 h. (E) Control group (without transfection) for 48 h. (F) Control group (without transfection) for 72 h. PAR-2, protease-activated receptor 2; shRNA, small hairpin RNA.

Fig 5: Matrigel and Transwell assays to detect the effect of PAR-2 shRNA on EC109 cell invasion and migration. Cells were stained using hematoxylin and eosin. (A) Cell invasion analysis was measured by Matrigel method on the EC109 cell and PAR-2 shRNA groups (magnification, ×200). (B) Cell migration analysis was measured by Transwell method on EC109 cells group and PAR-2 shRNA group (magnification, ×200). PAR-2, protease-activated receptor 2; shRNA, small hairpin RNA.