H1299 and H1975 lung epithelial cells were grown at 37 °C and 5% CO2 in RPMI-1640 (Gibco) with 10% FCS and 2 mM l -glutamine (Gibco).
Lyophilized, recombinant LecB (Dr. Anne Imberty) was dissolved in PBS (with Ca/Mg) and used at concentrations of 0.09 μM, 0.9 μM and 4.3 μM for indicated time periods. Starvation in serum-free medium was performed, when indicated, for 2 h before treatment and maintained during stimulation. In order to block fucose-binding sites of LecB,l -fucose (Sigma-Aldrich) was directly dissolved in medium, sterile filtered and used at a final concentration of 43 mM for the same time periods as LecB. Other proteins for stimulation purposes were used at the following concentrations: Wnt3a (400 ng/ml, R&D Systems), TNF-α (10 ng/ml, Life Technologies) and staurosporine (Stauro, 1 μM, Sigma-Aldrich). The used inhibitors were pre-incubated for 30 min at 37 °C and maintained during stimulation at following concentrations: lactacystin (Lact, 10 μM, Biomol), bafilomycin (Baf, 0.2 μM, Invivogen), LY294002 (LY, 100 μM, Sigma-Aldrich), triciribine (TCN, 10 μM, Sigma-Aldrich), Akt1/2 inhibitor (Akti, 10 μM, Sigma-Aldrich), TWS119 (TWS, 20 μM, Selleckchem), lithium chloride (LiCl, 20 mM, Roth) and Cycloheximide (Cyclo, 100 μg/ml, Sigma-Aldrich).
Lyophilized, recombinant LecB (Dr. Anne Imberty) was dissolved in PBS (with Ca/Mg) and used at concentrations of 0.09 μM, 0.9 μM and 4.3 μM for indicated time periods. Starvation in serum-free medium was performed, when indicated, for 2 h before treatment and maintained during stimulation. In order to block fucose-binding sites of LecB,