Tnf α

Cells were plated in 24 well plates with 1 × 10⁵ cells per well and treated for 48 h with 10 ng/ml TNF-a (Cell Signaling Technology, Danvers, MA) 1 d post-transfection.

DK-MG cells were seeded in 6-well plates at 2×10⁵ cells per well for western blot analysis or at 4×10⁴ cells per well for real-time observation, incubated overnight to allow for adhesion and serum starved for subsequent 24h. Serum free medium containing appropriate inhibitors was applied onto cells and incubated for indicated amount of time, prior to stimulation with 20ng/mL EGF (Sigma-Aldrich) or 5ng/mL TNFa (Cell Signaling Technology) for 20 or 30min, as indicated, prior to lysis for western blotting or left for real-time observation. Following concentrations of inhibitors were used: DMSO (Sigma, Cat. No.D2438, solvent control), 10 μM Erlotinib (Molecula; Cat. No. 89983631), 2.5 μM ACHP (Tocris Bioscience, Cat. No. 4547), 1 μM CID2858522 (Tocris Bioscience, Cat. No. 4246).

Western blot assays were performed as described before [29 (link)]. Briefly, liver tissues were homogenized in RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA). Proteins were collected by centrifuging at 12,000 g at 4°C. The sample of total protein was separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). These membranes were rinsed briefly in tris-buffered saline containing 0.05% Tween 20 (TBST), blocked in blocking buffer (5% milk and 0.5% BSA) for 1 h, and then incubated with different primary antibodies overnight at 4°C, followed by three washes with TBST and incubation with secondary horseradish peroxidase-conjugated antibody for 1 h at room temperature. Antigen-antibody complexes were then visualized using ECL kit (Amersham, Piscataway, NJ). The primary antibodies used here include those against 3-nitrotyrosine (3-NT, 1 : 2000, Millipore, Billerica, MA), 4-hydroxynonenal (4-HNE, 1 : 2000, Alpha Diagnostic International, San Antonio, TX), ICAM-1 (1 : 500), CTGF and β-actin (1 : 1000, Santa Cruz Biotechnology, Santa Cruz, CA), PAI-1 (1 : 2000, BD Biosciences, Sparks, MD), TNF-a (1 : 500), and TGF-β1 (1 : 1000; Cell Signaling, Danvers, MA).