Fig 2: PAR2 activation protects CASKI cells against cisplatin-induced apoptosis through an EGFR-dependent mechanismCASKI cells were starved in serum-free medium for 4 h, followed by treatment with cetuximab (100 µg/mL) when indicated. One hour later, cells were stimulated with FVIIa (20 nM, 50 nM or 100 nM) or PAR2-AP (50 µM) for 60 minutes. Next, cells were treated with cisplatin (CDDP, 20 µM) for 48 h. (A) FVIIa-mediated chemoresistance was evaluated by propidium iodide staining, followed by flow cytometry. Cells with fragmented DNA (sub-G1 peak) were considered apoptotic cells. Values represent mean + SD of three independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA). (B) PAR2-AP-mediated chemoresistance was evaluated by propidium iodide staining, followed by flow cytometry. Values represent mean + SD of six independent experiments; ***P < 0.001 (one-way ANOVA). (C) PAR2-AP-mediated chemoresistance was also evaluated by western blot for cleaved caspase-3 and cleaved PARP. GAPDH was used as a loading control. Representative image from three experiments. (D) Cetuximab reversed the resistance to cisplatin promoted by the PAR2 activation. Apoptosis was evaluated by propidium iodide staining, followed by flow cytometry. Values represent mean + SD of three independent experiments; ***P < 0.001 (one-way ANOVA).

Fig 3: Effects of WAS on the expression of PAR2 receptor in the colon, ileal VE and FAE. (A–F) The PAR2 receptor was located on the epithelial layer of the colon, ileum FAE and adjacent VE, which upregulated in WAS rats, and may be downregulated by cromolyn. SED, subepithelial dome; LF, lymphoid follicles. Scale bars: 50 µm for (A–C) and 100 µm for (D–F). (G,H) Higher expression of PRA2 was observed in WAS rats in colonic epithelium as well as the ileum FAE and VE. Cromolyn treatment significantly reduced the levels of PAR2 in FAE. N = 10 for each group. Data are displayed as the mean ± SD. *P < 0.05, compared with SS group; #P < 0.05, compared with WAS group. The gels were run under the same experimental conditions. Cropped blots are presented and full-length blots are shown in the supplementary information.

Fig 4: Decreased PAR2 represses inflammation responses in HBx overexpressed LO2 cells. (A) The mRNA expression of TNF-a, IL-6, and CXCL-2 in LO2 cells under different treatments was detected by qRT-PCR. (B) The protein level of TNF-a, IL-6, and CXCL-2 in LO2 cells under different treatments was measured by ELISA. *P < 0.05 vs the HBx(-) + FSL(-) group. #P < 0.05 vs the HBx(+) + FSL(-) group. The experiments were performed in triplicate.

Fig 5: Inhibition of PAR2 reverses the influences of HBx overexpression on the proliferation and apoptosis of LO2 cells. (A) The proliferation of LO2 cells under different treatments was measured by EdU assay. (B) The apoptosis of LO2 cells under different treatments was analysed using a flow cytometer. *P < 0.05 vs the HBx(-) + FSL(-) group. #P < 0.05 vs the HBx(+) + FSL(-) group. The experiments were performed in triplicate.