Protein lysate

The intracellular protein was extracted with protein lysate (Beijing Solarbio Science & Technology Co., Ltd.), and the protein concentration was measured employing Bicinchoninic Acid (BCA) assay kit (BOSTER). A total of 50 µg protein was segregated on 8‑10% SDS‑PAGE and diverted to polyvinylidene difluoride membranes (PVDF), then sealed with 5% skim milk at room temperature for 2 hours. Primary antibodies: SNAI1 (dilution,1:500; cat. no. ab82846; Abcam), E-cadherin (dilution, 1:1000; cat. no. ab15148; Abcam), N-cadherin (dilution, 1:1000; cat. no. ab18203; Abcam), MMP-9 (dilution, 1:500; cat. no. ab38898; Abcam) and β-actin (cat. no. ab8227; Abcam 1:5000). The primary antibody was incubated overnight in a refrigerator at 4°C. Next, the horseradish peroxidase labeled secondary antibody (dilution 1:5000; cat. No. ab6721; Abcam) was added and incubated at room temperature for another hour. Visualization of protein bands were obtained based on an Odyssey infrared laser imaging system. Lastly, using β-actin as internal reference, the protein relative expressions of SNAI1, E-cadherin, N-cadherin and MMP-9 were calculated using Image J [22 (link)].

The cancer tissues and adjacent tissues were added with liquid nitrogen and ground until the tissues were homogeneous. Then, the tissues were added with protein lysate (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), centrifuged at 12000 rev/min at 4°C for 20 min, and the supernatant was obtained and stored for further use. The cells were collected, lysed, and centrifuged, and the total protein was collected. The total protein concentration was measured by BCA Kit (Thermo Fisher Scientific, Carlsbad, California, U.S.A.). After taking the total protein of the cell and separating by SDS/PAGE (12% gel), the protein was transferred on to PVDF membrane and sealed by skimmed milk at room temperature for 1 h. The following primary antibodies were added – EZH2 (1:1000, ab186006), SMYD3 (1:2000, ab187149), and GAPDH (1:1000, ab8245) – and cultured at 4°C overnight. All antibodies were purchased from Abcam Inc. (Cambridge, MA, U.S.A.). The membrane was washed three times to remove the primary antibodies, and added with second antibody and cultured at room temperature for 1 h. The membrane was washed three times again and ECL reagent was applied to reveal Western blotting bands. Using GAPDH as a reference, the relative expression of protein was analyzed by Western blotting image (ImageJ2x software).