Occludin

Protein lysates were acquired using RIPA lysis buffer supplemented with both phosphatase and protease inhibitor cocktails (Life Technologies, Thermo Fisher Scientific brand, Waltham, MA, USA). Equal amounts (5 μg) of proteins in RIPA buffer were separated on 10% NuPage Bis-Tris gels (Invitrogen) and transferred to a PVDF membrane (Invitrogen). Antibodies used are as follows: E-Cad (BD), PTP1B (BD, Fisher Scientific brand, Waltham, MA, USA), pSrc529 (Abcam), claudin-1 (Zymed, San Francisco, CA, USA), occludin (Zymed), β-catenin (Biolegend, San Diego, CA, USA), FAK (Cell Signaling, Danvers, MA, USA), pFAK (Cell Signaling) GAPDH (Abcam) and actin (Abcam) were used. In Figures 1,4 and 5, secondary antibodies were horsesadish peroxidase-conjugated anti-mouse or rabbit (BD Scientific; Fisher Scientific brand, Waltham, MA, USA) used at dilution 1:10 000. The protein bands were visualized using enhanced chemiluminescence (ECL) system (Thermo Scientific, Waltham, MA, USA). In Supplementary Figure 4, secondary antibodies were mouse or rabbit IRDye (Li-Cor, Lincoln, NE, USA) used at 1:10 000 and protein bands were detected using the Odyssey Infrared Imaging System (Li-Cor) and then fluorescent images were converted to gray scale.
All western blots are representative of three separate experiments.

To analyze immunofluorescence staining of cell junction proteins, embryos were fixed in 4% paraformaldehyde, followed by permeabilization in PBS containing 0.2% Triton X-100 for 1 h, and blocking in PBS containing 1% Bovine serum albumin and 0.05% Tween 20. The treated embryos were incubated overnight at 4°C with antibodies to either ADAM10 (sc-48400, Santa Cruz Biotechnology; Santa Cruz, CA, USA), CXADR (HPA003342, Sigma, St. Louis, MO, USA), TJP1 (ZO-1; 33–9100, Zymed, San Francisco, CA, USA), OCLN (Occludin; 33–1500, Zymed, San Francisco, CA, USA), CDH1 (E-cadherin; 610182, BD Biosciences, San Jose, CA), or CTNNB1 (β-catenin; sc-7963, Santa Cruz Biotechnology; Santa Cruz, CA, USA) that were diluted into blocking solution (1 in 100). After primary antibody incubation, the embryos were washed three times in PBS containing 0.05% Tween 20 for 10 min, and transferred to PBS containing Alexa-Fluor-488 diluted 1:200. Finally, the samples were stained with Hoechst 33342 (10 mg/mL in PVA-PBS) for 20 min. Before mounting, embryos were washed an additional three times, and mounted onto glass slides with Vectashield (94010, Vector Laboratories, Burlingame, CA). Each slide was examined using a confocal laser-scanning microscope (Zeiss LSM 710 META; Jena, Germany). At least 30 embryos were examined per group.

To examine tight and adherens junctions, polarized epithelial cells were fixed with 4 % paraformaldehyde for 20 min, permeabilized with 0.05 % Triton X-100 and washed three times with PBS. Cells were then incubated with rabbit mAb occludin (Zymed) and rabbit mAb E-cadherin (CSI) for 1 h at room temperature. Cells were washed and incubated for 25 min with secondary antibody DyLight 488 antimouse IgG (Vector), and cell nuclei were stained with DAPI. Cells were analyzed using a Nikon Eclipse E400 fluorescence microscope.

The vaginal tissue was placed in 4% paraformaldehyde fixative for 16 hours and then processed for washing and dehydration. The tissues were embedded in paraffin. Sections of 5 μm thickness were prepared for staining. Immunohistochemistry was performed by using an immunoperoxidase procedure (Vector ABC Kit; Vector Laboratories, Burlingame, CA, USA). The tissue sections were deparaffinized in xylene, dehydrated in a graded series of ethanol, rinsed twice in phosphate-buffered saline (PBS), and then treated with 3% H₂O₂ in 60% methanol for 30 minutes to quench endogenous peroxidase activity. After washing twice (5 minutes) in PBS, the sections were incubated for 12 to 14 hours with antibodies to ZO-1 (1 : 200, Invitrogen), occludin (1 : 200, Zymed), and claudin-1 (1 : 200, Invitrogen) in PBS with 0.3% bovine serum albumin. For a negative control, the sections were incubated in PBS containing 5% normal goat serum only. The sections were then rinsed 3 times in PBS and incubated sequentially for 30 minutes each with the biotinylated secondary antibody and the ABC reagent. The sections were incubated for 5 minutes with a peroxidase substrate solution (diaminobenzidine). Finally, the tissue sections were examined and photographed under a light microscope.

Total protein was extracted from rat ileums, and the concentration of total protein was determined by BCA Protein Assay kit (TransGen, Beijing, China). Western blotting was performed with the following primary antibodies: ACSL4 (Abcam), GPX4 (Abcam), FTH1 (Abcam), ZO-1 (Abcam), occludin (Zymed Laboratories), claudin-1 (Santa Cruz Biotechnology), and claudin-2 (Zymed Laboratories). β-Actin (Sigma) was used as loading control. For protein quantification, the density of Western blot bands was measured by Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).