N cadherin

Antibodies against ADAM9, phospho-AKT (Ser473), β-actin, phospho-FAK (Tyr397), ubiquitin and phospho-GSK3b (Ser9) were purchased from Cell Signaling (Boston, MA). Antibodies against laminin-5 (γ² chain), Sox2, Oct-3/4, Nanog and E-cadherin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against fibronectin, vimentin and N-cadherin were purchased from GeneTex (San Antonio, TX). Antibodies against Col XVII, Col XVII (NC16A-3) and Snail were purchased from Abcam (Cambridge, MA). Antibodies against ADAM10 were from Millipore (Amersham Pharmacia Biotech, Piscataway, NJ). Antibodies against phospho-Snail (Ser246) were from OriGene Technologies, Inc. (Rockville, MD).

Western blotting staining and immunohistochemical (fluorescence) staining were performed as described previously 17, 19. The primary antibodies used in this study were Snail (1:1,000 dilution; MABE167; Merck, Darmstadt, Germany), E‐cadherin (1:1,000 dilution; #3195S; Cell Signaling Technology), PCAF (1:1,000 dilution; #3378S; Cell Signaling Technology), vimentin (1:2,000 dilution; GTX100619; GeneTex), β‐actin (1:10,000 dilution; #4967L; Cell Signaling Technology), HA (1:1,000 dilution; #3724S; Cell Signaling Technology), GFP (1:500 dilution; SC‐9996; Santa Cruz Biotechnology), ISX (1:200 dilution; sc‐398934; Santa Cruz Biotechnology), TWIST1 (1:200 dilution; ab49254; Abcam), BRD4 (1:1,000 dilution; #13440S; Cell Signaling Technology), acetylated lysine (1:500 dilution; #9441S Cell Signaling Technology), fibronectin (1:500 dilution; GTX112794; GeneTex), mCherry (1:500 dilution; GTX128508; GeneTex), Slug (1:1,000 dilution; GTX128796; GeneTex), VEGF (1:200 dilution; sc‐7269; Santa Cruz Biotechnology), and N‐cadherin (1:1,000 dilution; GTX127345; GeneTex). FITC‐conjugated anti‐rabbit IgG, rhodamine‐conjugated anti‐mouse IgG, and alkaline phosphatase‐conjugated anti‐rabbit IgG antibody (1:500 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were also used. All experiments were repeated at least three times.

Cultured cells were lysed in a buffer containing 150 mM KCl, 10 mM Tris pH 7.4 and 1% Triton X-100 together with phosphatase inhibitor and protease inhibitors cocktail (Complete Mini; Roche, Mannheim, Germany). The protein concentrations of the cell homogenates were measured using the Bradford method [23 (link)]. Thirty microgram of proteins were separated using 10% SDS-PAGE and then transferred to a nitrocellulose membrane (Hybond-C; Amersham Biosciences, NJ, USA). The membrane was blocked with 5% bovine serum albumin, which was followed by probing with various specific antibodies such as Src antibody, C-term (GTX61220, GeneTex, Texas, USA), p190-B RhoGAP antibody (GTX61259, GeneTex, Texas, USA), ERα (GTX100634, GeneTex, San Antonio, Texas), anti-α-tubulin 1A (GTX109832, GeneTex, San Antonio, Texas), and anti-β-actin (GTX109639, GeneTex, San Antonio, Texas), N-cadherin (C-terminus clone EPR1792Y. #04-1126 Merck Millipore, Billerica, Massachusetts), E-Cadherin E (#3195) and Vimentin (GTX100619, GeneTex, Texas, USA) and Snail (#AP2054a, ABGENT, San Diego, CA) antibodies. These were purchased commercially (Cell signaling, Danvers, Massachusetts).

The HCC cell lines J7 and SK-Hep1 were cultured in DMEM containing 10% fetal bovine serum at 37°C in an atmosphere containing 5% CO₂. Polyclonal antibodies against E-cadherin, N-cadherin, vimentin, twist, snail and β-actin were purchased from Genetex (Irvine, CA, USA) or Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibodies were purchased from Santa Cruz Biotechnology. All siRNAs were purchased from Applied Biosystems (Foster City, CA, USA). pCDNA3.1-AOC4P, a neomycin-selective CMV-based expression plasmid that contains the AOC4P lncRNA sequence, was constructed by GenScript (Piscataway, NJ, USA).

Magnolol was purchased from Shanghai BS Bio-Tech Co., Ltd. (Shanghai, China). MM1 was prepared as described by Lin et al. (43 (link)). The purity of magnolol and MM1 was <99%, as determined by high-precision liquid chromatography (HPLC) analysis. Magnolol and MM1 were each dissolved in dimethyl sulfoxide (DMSO) to obtain a stock concentration of 100 mM, which was then stored at −20°C before use. DMSO 0.1% v/v was used as the vehicle control. Sorafenib was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against human class I HDACs (HDAC1, HDAC2, HDAC3, and HDAC8), acetyl-histone H3, acetyl-p53, p53, p21, Ki-67, E-cadherin, N-cadherin, vimentin, Snail, Slug, and β-actin were purchased from GeneTex (Irvine, CA, USA) and Cell Signaling Technology (Beverly, MA, USA). The antibody to cyclin D1 was purchased from ABclonal Technology (Woburn, MA, USA), and the antibodies against CDK4 were purchased from Proteintech (Rosemont, IL, USA). Secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).