β catenin

Western blotting was carried out as previously described [24 (link)]. The primary antibodies were purchased from Bethyl Laboratories (QKI), Cell Signaling Technology (E-cadherin, N-cadherin, Vimentin, Snail, Slug, p-β-catenin (Ser33/37/Thr41) and p-GSK3β (Ser9)), Santa Cruz Biotechnology (β-catenin, α-tubulin, β-actin, c-myc, cyclin D1, MMP2, DKK3, DVL1, DVL3, GSK3β), Boster (GAPDH) and Sigma (flag).

Rabbit antibodies specific to LC3 (L7543), ZO-1 (SAB5700645), occludin (SAB5700784), claudin 11 (ABT148), and pSQSTM1/P62 (SAB5700845) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit antibodies specific to CX43 (A00599), N-cadherin (BA0673), and β-catenin (BA0426) were purchased from Boster (Wuhan, China). In addition, 3-methyladenine (3-MA) (A8780) was purchased from Solarbio (Beijing, China).

The L5-L6 segments were sliced along the median sagittal plane. After pre-treatment with 5% BAS blocking solution, sections were incubated in diluted antibody (dilution ratio: Coll2 1: 150; MMP-3 1: 120; Aggrecan 1: 150; β-catenin 1: 100) (Boster, Wuhan, China) separately at 4 °C overnight. Then, sections were incubated at 37 °C for 30 min with biotin-coupled secondary antibody diluted 1:150 and SABC. DAB colouration solution was formulated and dropped on sections and sections were observed with a Digital Image Analyzer. All sections were semi-quantitatively analyzed by Image-Pro Plus 6.0 software, and the average optical density was measured on the images at 400× magnification.

All the cells were gathered and total proteins were extracted using RIPA lysis buffer supplemented with protease inhibitor Cocktail (Roche, Switzerland). Protein concentrations were calculated by using BCA protein assay kit (Beyotime, China). Equivalent amounts of protein samples (50 μg) were separated by 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. Nonspecific binding was blocked by incubating the PVDF membranes with 5 % bovine serum albumin (BSA) (Sigma-Aldrich, USA) for 2 h at room temperature. The membrane was then incubated with primary antibodies included p53 (1/1000) (Cell Signaling Technology, USA), p21 (1/2000) (Cell Signaling Technology, USA), Cyclin D1 (1/2000) (Affinity, USA), CDK4 (1/1000) (Affinity, USA), CDK6 (1/2000) (Affinity, USA), E-cadherin (1/1000) (BD Biosciences), β-catenin (1/500) (Boster, China), Vimentin (1/500) (Boster, China), ZEB1 (1/1000) (Cell Signaling Technology, USA), GAPDH (1/500) (Boster, China) and α-tubulin (1/500) (Boster, China) at 4 °C overnight. After several washes, the membranes were incubated with corresponding secondary antibody and detected by enhanced chemiluminescence (ECL) assay kit (Millipore, USA).

3T3-L1 cells were washed three times with PBS before adding RIPA (Beyotime, Shanghai, China) supplemented with protease inhibitors (Pierce, Rockford, IL, USA) at 4 °C. We scraped the lysed cells and then centrifuged (rpm) at 4 °C for 10 min. Supernatant protein concentration was determined by BCA protein assay kit (Cwbio, Beijing, China). A 1/4 volume of 5× loading buffer (Cwbio, Beijing, China) was added to an aliquot of the supernatant, and a 20 µg protein sample was separated using a 12% SDS-polyacrylamide gel. After electrophoresis, the gel was transferred to a polyvinylidene fluoride (PVDF) membrane (CST, Boston, MA, USA) using a current of 250 mA. After 2 h, the membrane was removed and placed in 5% skim milk for 2 h in room temperature. The membrane was then incubated with primary antibody at 4 °C overnight and then incubated with secondary antibody (Boster, Wuhan, China) for two hours in room temperature. Finally, the signals were detected by a gel imaging system (Bio-Rad, CA, USA) and analyzed by Image Lab (Bio-Rad, CA, USA) software. Primary antibodies were used against Cyclin E, Cyclin D1, p27, Bcl-2, Bax, and Caspase-3 (Santa Cruz, Dallas, TX, USA); against Cleaved Caspase-3 (abways, Shanghai, China), against β-actin (CWBio, Beijing, China), and against β-catenin (Boster, Wuhan, China).