Fap α

The human CAC tissue chips were purchased from Alenabio (Xi’an, China), containing normal colon tissue, chronic colitis, colonic tubular adenoma, and well-differentiated CAC. The tissue chip was dewaxed using conventional methods and then incubated with hydrogen peroxide for 15 min. The first antibodies, including FAPα (1:300, Abcam), OXTR (1:300, Abcam), OXT (1:200, Abcam), and CCL-2 (1:200, Bioss) were added to the incubation overnight at 4°C. The corresponding secondary antibodies (50 μl, 1:1000) were added to the reaction that was incubated for 30 min at room temperature (21–23°C). After coloration, the photographs of the images were taken with an imaging system (Life Science), and the positive immunohistochemical staining was represented with brown stains visible under a light microscope.

Chrysin was obtained from Sigma-Aldrich (Sigma-Aldrich, Cat No. C80105, St. Louis, MO, USA).BrMC was synthesized with previously described protocols [20 (link)].EGF (Carlsbad, CA, USA, 0.1 μg/mL), bFGF (0.1 μg/mL) and B27 (without Vitamin A) from Invitrogen. Accutase from PromoCell (Cat No. C-41310, Germany), insulin was purchased from Sigma-Aldrich. Additional reagents were: horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibodies (1:2000) (Cat. No. A0216 and A0239, respectively; Beyotime Institute of Biotechnology, Shang Hai, China); Rabbit anti-human CD133 polyclonal antibody (1:2000), CD44 and α-SMA(1:2000), No. ab66141, No. ab41478, No.ab75273,Abcam,Inc.,Burlingame,CA,USA; β-actin (1:2000), FAP-α(1:2000) and IL-6 neutralizing antibody, Cat. No. sc-7210, Cat.No.sc-54,538,Cat.No.sc-57,315, Santa Cruz Biotechnology, Inc. Beverly, MA, USA; HGF neutralizing antibody, Cat.No.ab24865, Abcam Company, Cambridge, MA, USA; human recombinant IL-6 and human recombinant HGF, Cat. No. 200–06, Cat.No.100–39, PeproTech Inc. Rocky Hill, NJ, USA; and enhanced chemiluminescence detection reagent (ECL), Amersham Pharmacia Biotech, USA; ELISA kits, Neobioscience, Shenzhen, Guangdong, China.

Both cryosections and paraffin sections were used in the study. Following hydration, the paraffin sections were treated consequently with citrate buffer (pH = 6.0 for 30 min at about 100°C), 3% hydrogen peroxide blocker, and 10% blocking solution (BioVitrum, Russia). Immunohistochemical assay was performed with antibodies to differentiation-specific and cell state-specific markers including connexin-43 (the marker of cardiac myocytes), CD34 (the marker of endothelial cells), α-SMA (α-smooth muscle actin, the marker of smooth muscle cells, and myofibroblasts), Fapα (the marker of reactive fibroblasts), CD68 (the marker of macrophages), and Ki67 (the marker of cell proliferation), all manufactured by Abcam, Cambridge, UK. Incubation with primary antibodies was carried out at 4°C overnight, in concentrations recommended by the manufacturer. The excess of primary antibodies was removed by three consequent washes with PBS; the sections were further incubated in 1 : 200 dilutions of FITC-conjugated secondary antibodies at room temperature for 1 hour. The sections were additionally stained with DAPI (Sigma Aldrich, USA) to label the nuclei. The number of positively stained cells was counted in 50 fields of view per experimental group.