E cadherin

After the embedded tissue was cut into 4 μm sections and deparaffinized, the sections were treated with 3% H₂O₂ for 10 min to block endogenous peroxidase activity. After antigen retrieval in a microwave oven, the sections were incubated with normal goat serum at 37 °C for 10 min. Afterwards, the sections were incubated with the following primary antibodies: SMA (Affinity, Changzhou, China), HIF-1α (Affinity, Changzhou, China), E-cadherin (Affinity, Changzhou, China) and Vimentin (Affinity, Changzhou, China). After 2 h, the sections were washed and incubated with a secondary antibody (Zhongshan Biotechnology Co., Ltd., Beijing, China) at 37 °C for 30 min. After staining with 3,3′-diaminobenzidine, the tissues sections were incubated with hematoxylin staining solution for 20 s, washed with tap water for 5 min, and observed for staining intensity under a microscope. Each experiment was performed in triplicate.

The cells and tissue specimens were lysed with radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing phenylmethanesulfonyl fluoride (Beyotime) and a protease inhibitor (CoWin Biosciences). The protein concentration was determined using a bicinchoninic acid protein assay kit (Beijing Solarbio Science and Technology Co., Ltd. Beijing, China). Each sample with equal amounts of protein were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. After running 1–2 h under 120V, the protein was transferred onto a polyvinylidene difluoride membrane. Next, the membranes were blocked with 5% not-fat milk for 1 h at room temperature and incubated with primary antibodies c-Myc, HIF-1α, CXCR4, SDF-1, vimentin, E-cadherin, and β-actin overnight at 4°C (Affinity Biosciences). Subsequently, the membranes were washed 3 times with tris-buffered saline and Tween-20 and incubated with a horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibody (Cell Signaling Technology, Beverly, MA, USA). Finally, the membranes were treated with a chemiluminescence reagent and exposed to X-ray. The band intensities were quantified using image J software (National Institutes of Health, Bethesda, MD, USA). Relative protein expression was quantified using control protein β-actin.

Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, MEM medium, Trypsin-EDTA, and phosphate-buffered saline (PBS) buffer were purchased from M&C Gene Technology (Beijing) Ltd. TRIzol was obtained from Invitrogen (Carlsbad, CA). A reverse transcription kit was purchased from Thermo Fisher Scientific Inc. (Waltham, MA). Sohlh2 and Klotho antibodies were purchased from Abcam Inc. (Cambridge, MA). Ki67, DNMT3a, E-cadherin, ZO-1, N-cadherin, GAPDH, and Actin antibodies were bought from Affinity Biosciences Ltd.