Mmp 2

Cells were lysed in RIPA lysis buffer (Beyotime Biotechnology, China) containing protease inhibitor cocktail. BCA protein quantification kit (Vazame, China) was used to detect the protein concentration. Equal amounts of protein were separated by SDS-PAGE and then transferred to PVDF membrane. After blocking with 5% BSA for 1 h, the membranes were incubated overnight at 4 °C with the indicated primary antibodies, followed by incubation with HRP-labeled secondary antibody for 1 h at room temperature. Primary antibodies were purchased from Cell Signaling Technology (E-cadherin, 1:1000, #3195; N-Cadherin, 1:1000, #13,116; Snail, 1:1000, #3879; MMP-2, 1:1000, #40,994; MMP-9, 1:1000, #13,667; Phospho-Akt, 1:1000, #4060; Akt, 1:1000, #4691; PI3K, 1:1000, #3011), Bioss (Phospho-PI3KCA, 1:1000, bs-5570R), Proteintech (TEAD4, 1:2000, 12,418–1-AP; Vimentin, 1:1000, 10,366–1-AP), and ImmunoWay (GAPDH, 1:10,000, YM3209). Secondary antibodies were purchased from Elabscience (Goat Anti-Rabbit IgG (H + L), 1:10,000, E-AB-1003; Goat Anti-Mouse IgG(H + L), 1:10,000, E-AB-1001).

Total proteins were extracted from liver tissue and cells using radio-immunoprecipitation buffer (RIPA). 80 μg of sample proteins were separated by in 10 or 12% SDS-PAGE gel, and transferred onto PVDF membranes (Millipore Corporation, Billerica, MA, USA) by electrobloting. The membranes were blocked for 60 min in a buffer containing 0.1% Tween-20 and 5% milk. The membranes were incubated overnight at 4 °C with primary antibodies against α-SMA, Col-1, MMP-2 (Bioss, Beijing, China), Smad7 (Novus Biologicals, Littleton, USA), Wnt1 (Abcam, Cambridge, MA, USA), Wnt5a (Novus Biologicals, Littleton, USA), β-catenin, GSK-3β (ProteinTech Group, Chicago, USA), NFAT5 (Santa Cruz, CA, USA). Immune complexes were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies (ProteinTech Group, Chicago, USA), and then were visualized by ECL method. β-actin (Boster, Wuhan, China) was served as a loading control. Intensity of each protein band of interest was quantified by densitometry using Quantity One 4.6.3 software (Bio Rad).

A total of 3×10⁵ cells were seeded on glass coverslips overnight and then fixed for 15 min with 4% paraformaldehyde. Blocking buffer (5% normal serum and 0.5% Triton™ X−100) was used for blocking for 1 h. Next, coverslips were incubated with specific primary antibodies overnight at 4°C. The primary antibodies were as follows: KLF4 (#BS90773, BioWorld, Nanjing, China), BMI1 (#10832-1-AP, Proteintech, Rosemont, IL, USA), MMP2 (#bs-0412R, Bioss, London, UK), MMP9 (#bs-4593R, Bioss, London, UK), Ki67 (#ab15580, Cambridge, UK). The cells were then incubated in fluorochrome−conjugated secondary antibody and stained with DAPI (Cell Signalling Technology, Danvers, MA, USA), and slides were examined under a slide scanner.