Normal goat serum (ngs)

A. satureioides was acquired from Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas (CPQBA) da Universidade Estadual de Campinas (São Paulo, Brazil), via a sample deposited in the herbarium with the number 308. Egg yolk lecithin and medium-chain triglycerides were purchased from Lipoid (Ludwigshafen, Germany), polysorbate 80 was acquired from Vetec (Rio de Janeiro, Brazil), and vitamin E was procured from Alpha Química (Cachoeirinha, Brazil). Ultra-fast liquid chromatography (UFLC) required the following reagents: methanol (J.T. Baker, Philipsburg, NJ, USA), acetonitrile (Tedia, Rio de Janeiro, Brazil), and phosphoric acid (Merck, Rio de Janeiro, Brazil). Carbopol^® Ultrez 20 was kindly donated by Lubrizol do Brasil Aditivos Ltd.a (São Paulo, Brazil). Albumin for enzyme-linked immunosorbent assay (ELISA) was purchased from Calbiochem (San Diego, CA, USA). Normal goat serum was obtained from R&D Systems (Minneapolis, MN, USA). Protease inhibitor cocktail was purchased from VWR Life Science (Radnor Township, PA, USA). Xylazine and ketamine were purchased from Ceva (Paulínea, Brazil). Ketoprofen was obtained from Merial (Paulínea, Brazil). Finally, thiopental was obtained from Cristália (Itapira, Brazil).

RAW 264.7 cells were seeded in an 8-well plate, pretreated with 4-hydroxycinnamaldehyde (12.5 µM and 25 µM) and ECC (200 µg/mL) for 2 h, and then treated with LPS (100 ng/mL) for 22 h. The cells were fixed with 4% paraformaldehyde in PBS for 10 min and blocked with 5% normal goat serum (R&D Systems, Minneapolis, MN, USA) containing 0.3% Triton X-100 in PBS for 1 h. Macrophage cells were probed with primary antibodies overnight at 4 °C. After washing with PBS, the cells were incubated with Alexa Fluor 488-conjugated secondary antibody for 1 h and then counterstained with DAPI (Cell Signaling Technology). Fluorescence images were acquired using an LX51 fluorescence microscope (Olympus, Tokyo, Japan).

Immunohistochemistry was done to visualize WDFY1 protein in colon sections of WT and miR-511-deficient control and DSS induced mice and in human. Non-IBD control (colorectal carcinoma), IBD (Crohn’s) inflamed and non-inflamed colon sections from male adult participants were used. The specimen collection was approved by the biobank review committee under biobank number 178#A201470 and written informed consent was obtained from all study participants. The paraffin embedded sections were deparaffinized in xylene and gradually rehydrated in (v/v) ethanol gradient to PBS followed by antigen retrieval in sodium citrate buffer, pH 6.0 for 20 min at 98 °C and 30 min blocking in 10% normal goat serum (R&D Systems, Abingdon, UK). The slides were incubated overnight at 4 °C with primary antibody (rabbit polyclonal anti-WDFY1, 1:400) in blocking buffer. The slides were blocked in 3% (v/v) hydrogen peroxidase (H₂O₂) in PBS for 20 min followed by a 30 min incubation with secondary antibody (anti-rabbit polyclonal HRP, Brightvision BV, Almere, The Netherlands). Staining was visualized after a 4 min incubation with chromagen substrate diaminobenzidine liquid plus (DAB, Dako Netherlands BV). The sections were finally counter stained with hematoxylin and mounted. Images were processed using the Olympus BX51 microscope.