Mucin 5ac

Anti-integrin α2 (ΙΙΕ10), α3 (X8), α6 (ELE) and β1 (P5D2), along with anti-tetraspanin CD151 (5C11), CD9 (C9BB) and CD82 (M104), were raised in-house as described in prior studies [19 (link)]. The CD151-specific monoclonal antibody used for IHC analyses was obtained from Leica Microsystems, Inc. (Buffalo Grove, IL). Antibodies against fibronectin and Mucin 5Ac were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies recognizing total and phospho-Akt or MAPK or GSK3-β, along with those against Snail, Slug, LRP5, LPR6 and Axin-2, were obtained from Cell Signaling Technology (Danvers, MA). Antibodies against E-cadherin, Twist, Zeb1 and Zeb2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Matrigel invasion chambers and anti-β4 integrin antibody were purchased from BD Biosciences (Franklin Lake, NJ). ICG-001, an inhibitor of canonical Wnt signaling, was obtained from Selleckchem (Houston, TX).

Tracheal RECs were isolated from Lewis rats using methods we described previously (Zang et al., 2012 (link); Zang et al., 2013 (link)). Briefly, RECs were dissociated from the tracheal mucosal lining with 2 mg/mL pronase (Roche Applied Science, Indianapolis, IN) in Ham’s F-12 medium overnight (12 h) at 4°C. Cells were collected by centrifugation at 500 g for 10 min. Cell pellets were re-suspended in DMEM/Ham’s F-12 medium (Sigma) supplemented with 10 μg/mL insulin (Sigma), 0.1 μg/mL hydrocortisone (Sigma), 0.1 μg/mL cholera toxin (Sigma), 5 μg/mL transferrin (BD, Franklin Lakes, NJ), 50 μM phosphoethanolamine (Sigma), 80 μM ethanolamine (Sigma), 25 ng/mL epidermal growth factor (BD), 70 μg/mL bovine pituitary extract (United States Biological, Swampscott, MA), 3 mg/mL bovine serum albumin (Sigma), and 5 nM retinoic acid (Sigma). Epithelial cells at passage 0 were centrifuged with a Shandon Cytospin (GMI, Inc., Ramsey, MN) and stained with antibodies against cytokeratin peptide 17, beta tubulin IV, mucin 5AC, and P63 (all at 1:200; Sigma) to identify their components. We found that the percentage of cells expressing the markers of rat respiratory tract epithelial differentiation decreased during subculture. Because most cells became fibroblast-like cells in passage 2, we used only freshly isolated RECs for cell seeding in the ensuing experiments.