β glucan peptide

HEK-Blue hDectin-1a cells (InvivoGen; catalog hkb-hdect1a) were maintained in growth medium containing DMEM (4.5 g/L glucose), 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 μg/mL Normocin (InvivoGen; catalog ant-nr-05), 2 mM l-glutamine, 1 μg/mL puromycin (InvivoGen; catalog ant-pr-1), and 1× HEK-Blue CLR Selection (InvivoGen; catalog hb-csm). This cell line expresses the Dectin-1a isoform and genes involved in the Dectin-1/NF-κB/SEAP signaling pathway. On the assay day, 180 μL/well of cells at a concentration of 2.8 × 10⁵ cells/mL in HEK-Blue Detection media (InvivoGen; catalog hb-det2) were plated in a 96-well tissue culture plate. Plasma samples (20 μL) were added to each well with and without piceatannol (250 nM). The plates were then incubated at 37°C and 5% CO₂ for 24 hours. Levels of SEAP were monitored and measured spectrophotometrically at 620 nm. As controls, 20 μL of water, piceatannol (250 nM), 2 μL of DMSO (piceatannol solvent), 20 μL of 10 μg/mL β-glucan peptides (InvivoGen; catalog tlrl-bgp), or 20 μL of β-glucan peptides + piceatannol (250 nM) were added in separated wells.

Mice were intratracheally instilled with bleomycin (3 U kg⁻¹; Sigma). After 3 d, the mice were intraperitoneally injected with β‐glucan peptide (25 mg kg⁻¹; InvivoGen) or PBS. The survival rates of the mice were monitored until 37 dpbi. Mice were sacrificed at 37 dpbi and the lungs were subjected to the hydroxyproline assay.

Antibodies used in this study include: BCL10 Rabbit polyclonal Ab (Cell Signaling Technology, C78F1), CARD9 mouse monoclonal Ab (Santa Cruz Biotechnology, A-8: sc-374569), and MALT1 mouse monoclonal Ab (Santa Cruz Biotechnology, B-12: sc-46677). β-Actin mouse monoclonal Ab (Santa Cruz Biotechnology, C4: sc-47778). mEos3.1 Rabbit polyclonal Ab (Halfmann laboratory, #4530). Alexa-488 Goat anti-Rabbit IgG (Thermo Fisher Scientific, A32731). Anti-mouse IgG Ab HRP (Cell Signaling Technology, 7076), mouse anti-rabbit IgG-HRP (Santa Cruz Biotechnology, sc-2357). Other reagents include: FuGENE HD (promega, E2311), β-glucan peptide (Invivogen, tlrl-bn, ant-zn-05), Hygromycin B (Invivogen, ant-hg-1), Penicillin–Streptomycin (Thermo Fisher, 1514014 gp), LPS (Sigma-Aldrich, L2880), PMA (BioVision, 1544–5), Halt protease inhibitor (Thermo Fisher, 78439), Puromycin (Invivogen, ant-pr-1), and Zeocin (Invivogen, ant-zn-05).