Occludin

Tissue samples were prepared in the same way as the histopathology observation assay described above. They were fixed in formaldehyde, embedded in paraffin, and sliced. Sectioned samples underwent antigen repair and were then incubated with serum. Afterwards, samples were incubated overnight with specific primary antibodies of ZO-1 (1:100 dilution, ABclonal, Wuhan, China), claudin-1 (1:200 dilution, Bioss, Beijing, China), occludin (1:200 dilution, Bioss, Beijing, China), cleaved caspase-3 (1:500 dilution, CST, Boston, MA, USA), Keap-1 (1:200 dilution, CST, Boston, MA, USA), HO-1 (1:500 dilution, CST, Boston, USA), and NAD (P)H quinone dehydrogenase 1 (NQO1) (1:200 dilution, CST, Boston, MA, USA) in the wet box. After being washed, samples were incubated with luciferin-conjugated goat anti-rabbit IgG (1:2000 dilution, CST, Boston, MA, USA) protected from light for 1 h. Fluorescent signals were visualized using fluorescence microscopy (Leica, Wetzlar, Germany), and quantification was performed by ImageJ 1.51 software (NIH, Bethesda, MD, USA).

Colon tissues were washed with phosphate-buffered saline (PBS) and lysed fully in ice-cold lysis buffer with the Protease Inhibitor Cocktail (MCE, New Jersey, USA) on ice. Lysates were separated by electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, Massachusetts, USA). The membranes were blocked with 5% skim milk powder and incubated with primary antibody against ZO-1 (1:1,000, Bioss), Occludin (1:500, Bioss, Beijing, China), Claudin1 (1:1,000, Bioss), and GAPDH (1:25,000, Proteintech, Chicago, USA) at 4°C overnight and then incubated with a horseradish peroxidase-conjugated goat anti-mouse/rabbit IgG secondary antibody (1:5,000, YEASEN, Shanghai, China) for 1 h at room temperature. The intensity of protein was assessed using a chemiluminescent imaging system (Image Quant LAS 4000, Atlanta, USA).

Cells were cultured and grown to around 80% confluence, then lysed with PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Inc. Gyeonggi-do, South Korea) or PARIS™ Kit. All protein concentrations were detected using a BCA Protein Assay Kit (Beyotime, Shanghai, China), and 30 μg of each sample was loaded. After resolved proteins were blotted onto PVDF transfer membranes and blocked with 10% non-fat milk in TBST for 2 h, the membranes were incubated with primary antibodies against ZO-1 (Bioss, Beijing, China), Occludin (Bioss), Claudin-1 (Bioss), p65 (Santa Cruz, Dallas, TX, USA), phosphorylated p65 (Bioss), GAPDH (Bioss) at 4°C overnight. After washed with PBS subsequently, the membranes were incubated with their corresponding secondary antibodies (Beyotime). In addition, 5% BSA (Beyotime) was used to block the membrane in phosphorylated p65 detection.

Total protein was extracted by lysis buffer for Western blotting with 100 mM of phenylmethanesulfonylfluoride, and 25 μg of total protein sample was subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions. Separated proteins were transferred to nitrocellulose membranes in Tris-glycine buffer containing 20% methanol at 4 °C. The membranes were blocked with 5% skim milk for 2 h, following incubated overnight with diluted primary antibodies against Bcl-xl (1:5000, Abcam, Cambridge, UK), caspase 3 (1:2000, Cell signaling technology, Boston, MA, USA), Bip (1:2000, Cell signaling technology), PERK (1:5000, Abcam), p-PERK (1:1000, Bioss), ATF4 (1:2000, Proteintech, Chicago, IL, USA), ATF6 (1:2000, Proteintech), eIf2α (1:1000, Proteintech, Wuhan, China), p-IRE1(1:1000, Bioss), CHOP (1:1000, Bioss), NF-κB (1:1000, Bioss), IκB (1:2000, Cell signaling technology), ZO-1 (1:1000, Bioss, Beijing, China), Occludin (1:1000, Bioss), Claudin-1 (1:1000, Bioss), and GAPDH (1:1000, Proteintech, China) followed by goat anti-rabbit IgG (H+L; 1:1000, Proteintech, China). The gray values of protein bands were measured by ImageJ software version 6.1 (Bio-Rad Laboratories, Hercules, CA, USA). The cellular protein PERK was used as an internal control for p-PERK, and GAPDH was used as an internal control for other proteins.

Tryptophan, indole, indole-3-propionic acid (IPA), indole-3-aldehyde (IAld), 6-formylindolo[3,2-b]carbazole (Ficz), 2-methyl-2H-pyrazole-3-carboxylic acid (CH223191) were purchased from Sigma Aldrich (St. Louis, MO, USA). The specific primary antibodies of AhR and Cyp1a1 were bought from Affinit Biosciences (OH, USA). Phosphorylation (p-) of p65 and IκB, p65, IκB and β-actin were bought from Cell Signaling Technology (CST, Boston, USA). Occludin and claudin-3 were bought from Bioss (Beijing, China). Enzyme-linked immunosorbent assay (ELISA) kit for TNF-α and IL-1β were bought from Biolegend (CA, USA). Myeloperoxidase (MPO) assay kit was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

STZ was purchased from Sigma Chemicals (St. Louis, MO, USA). Primary antibodies for TLR4, NF-κB, ZO-1, Claudin3, Occludin, and β-actin were obtained from Bioss Biotechnology co. Ltd (Beijing, China). The normal chow diets containing 19.38% (w/w) protein, 45.79% (w/w) carbohydrate, and 4.48% (w/w) fat, and the high-fat diets containing 26.2% (w/w) protein, 26.30% (w/w) carbohydrate, and 34.90% (w/w) fat were purchased from KeAoXieLi Feed Co. Ltd (Beijing, China).

Western blot analysis was carried out as described previously [17 (link)]. Antibodies against total Akt, phospho (Ser473) Akt, total AMPK, phospho (Thr172) AMPK, adiponectin, GAPDH (Cell signaling technology, Beverly, MA), occludin (Bioss antibodies, Woburn, MA), PGC1α (Santa Cruz Biotechnology, Santa Cruz, CA) were used as primary antibodies, with HRP-conjugated anti-rabbit IgG as secondary antibody (Cell signaling technology). Detailed information on the antibodies is available in S3 Table. Immunoblots were visualized by ECL, and densitometric analyses were done using ImageJ software.