Claudin 1

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.

The harvested colon was fixed in formalin (4%), and paraffin was used for embedding samples. Serial sections were used for immunohistochemical analysis every 25 sections. For antigen retreat, Tissue sections were boiled in sodium citrate (10 mmol/L, pH 6.0) for 10 min at 100°C and sections were blocked with BSA (Sigma, St. Louis Missouri, United States). The sections were then incubated with primary antibody solutions for ZO-1 (Affinity, Jiangsu, China), Claudin-1 (Bioss, Beijing, China), and Occludin (Affinity, Jiangsu, China). Anti-rabbit antibodies conjugated with fluorescence dyes (Alexa 594 and Alexa 488) were used as the secondary antibody (Boster, Wuhan, China).

Protein expressions in cecal tissues were detected by Western blotting (operation details in Supplemental Methods), as previously described [44 (link)]. Primary antibodies against TFF3, occludin, claudin-1, ZO-1, TGF-β1, Smad2, Phospho-Smad2 (Ser465 + Ser467), Smad3, Phospho-Smad3 (Ser423 + Ser425), Smad4, and Smad7 were obtained from BIOSS (Beijing, China). Blots were quantified with Fusion 16.0.9.0 software (Vilber Lourmat, Paris, France).

Cells were rinsed twice in ice-cold PBS. The RIPA buffer (Solarbio), protease inhibitor mixture (Solarbio), and protein phosphatase inhibitor (Solarbio) were subsequently added to the extract protein. Supernatants were used to quantify the protein concentration via the Bradford protein kit (Tiandz, Beijing, China). Proteins were separated by sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE) at 80 V for 40 min and 120 V for 50 min, and subsequently electrotransferred to polyvinylidene fluoride (PVDF) membranes at 200 mA for 80 min. Membranes were incubated with primary antibody P21 (Proteintech, Wuhan, China), P53 (Bioss, Beijing, China), PCNA (Bioss), p-Cyclin B1 (Bioss), Claudin1 (Bioss), Occludin (Bioss), ZO-1 (Bioss), Cdc25c (CST, Boston, MA, USA), and GAPDH (Servicebio, Wuhan, China) overnight at 4 °C. After incubation with corresponding secondary antibodies, the protein band densities were quantified using Bio-Rad ChemiDoc™ XRS and Image Lab™ Software.

Immunohistochemistry (IHC) was conducted according to the previously former method (15 (link)). Colon tissue embedded in paraffin was sliced (3 μm) and then deparaffinized and rehydrated with dimethyl-benzene and alcohol. Next, samples were incubated with 3% H₂O₂ for 10 min to block endogenous peroxidase activity and then blocked with goat serum (Proteintech, Chicago, USA) for 1 h. After being blocked, the samples were incubated with the following primary antibodies overnight at 4°C: ZO-1 (1:250, Bioss, Beijing, China), Occludin (1:400, Bioss), and Claudin1 (1:200, Bioss). The tissues were washed three times with PBS for 5 min each time. After that, the primary antibody in the negative group was displaced by PBS. The secondary antibody was subsequently added with the horseradish peroxidase-polymer anti-mouse/rabbit IHC kit (Maixin Biotech, Fuzhou, China) for 1 h at room temperature. 3,3′-Diaminobenzidine-tetrahydrochloride (DAB) was used to stain the tissues and counterstained with hematoxylin. All the samples were observed with a microscope (Olympus, Tokyo, Japan). The immunohistochemistry score was calculated by multiplying the intensity (0 = negative, 1 = canary yellow, 2 = claybank, 3 = brown) and the positive cell percentage scores (1 = less than 25%, 2 = 25–50%, 3 = 51–75%, 4 = more than 75%).