Laminarin

PCP was labeled with fluorescein isothiocyanate (FITC) via tyramine reduction method as previously reported [20 (link)]. For uptake assay, DC2.4 cells were seeded in 96-well plates and incubated PCP-FITC at 200, 400 and 800 μg/mL, BMDCs were cultured with PCP-FITC at 50, 100, 200 μg/mL. After 30 min, cells were harvested and stained with anti-mouse CD11c antibody before FACS analysis. For uptake competition assay, BMDCs were pre-incubated with 200 μg/mL Fucoidan (Solarbio, China), 20 μg/mL anti-mouse Dectin-1 antibody (InVivoGen, US), 200 μg/mL Laminarin (InVivoGen, US), 200 μg/mL D-Galactose (Aladdin Bio-Chem Technology, China) or 200 μg/mL Mannan (Solarbio, China). After 30 min, BMDCs were cultured with PCP-FITC (100 μg/mL) for 45 min before FACS analysis. For antigen uptake, BMDCs were first incubated with PCP (200 μg/mL) for 30 min, then DCs were co-cultured with OVA-atto 488 (5 μg/ml), Dextran-FITC (MW 40,000, 100 μg/ml) or Lucifer yellow VS dilithium salt (LY, 100 μg/ml) for 45 min before analysis of antigen uptake by CD11c⁺ BMDCs.

All chemicals used in the study were of cell culture grade and endotoxin-free. Pattern recognition receptor ligands used in this study were purchased from InvivoGen (LPS, Pam₃CSK₄, CpG ODN 2395, zymosan, MDP and flagellin) and curdlan, laminarin and S.c. mannan from Sigma-Aldrich. All samples were prepared in endotoxin-free water. The endocytosis inhibitor Cyt D was purchased from Sigma-Aldrich, the transfection reagent DOTAP from Roche and DNase I from Invitrogen. The chitinase inhibitor Bisdionin C was a kind gift from I. Eggleston (Bath).

Syk inhibitors SykI and BAY 61-3606, JNK inhibitor SP600125, ERK inhibitor U0126, and p38 inhibitor SB203580 were obtained from Calbiochem-Merck. Laminarin and zymosan were purchased from InvivoGen. Peptide inhibitor for NADPH oxidase assembly, gp91ds-tat, was purchased from AnaSpec. Diphenyleneiodonium chloride (DPI) and Mito-TEMPO were obtained from Sigma-Aldrich. LysoTracker Red was obtained from Life Technologies.
Antibodies against LC3, Rubicon, NLRX1, phospho (p)-p40-phox (Thr154), p-JNK (Thr183/Tyr185), p-ERK1/2 (Thr202/Tyr204), p-p38 (Thr180/Tyr182), p-c-Fos (Ser32), p-IKKα/β (Ser176/180), p-IκBα (Ser32), IκBα, and p-NFκBp65 (Ser536) were purchased from Cell Signaling. Antibodies against p-Syk (Tyr525), p-c-Jun (Ser63), and TUFM were obtained from Abcam. Anti-Syk, anti-ATG5, anti-β-actin, HRP-conjugated anti-rabbit IgG, and Rabbit IgG isotype control were purchased from GeneTex Inc. Receptor blocking antibodies against Dectin-1 (clone 2A11), Dectin-2 (clone D2.11E4), and CR3 (clone 5C6) were purchased from Bio-Rad (formerly AbD Serotec) and TLR2 (clone 6C2) was from eBioscience.

BMMs were derived from bone marrow of WT or respective knockout mice as described [58] (link) and stimulated with pathogens (see above) zymosan (Sigma-Aldrich), ultrapure LPS, Curdlan, Pam₃CSK₄ (all Invivogen). Where indicated, cells were pretreated 30 min prior stimulation with the Src kinase inhibitor PP2 (Selleckchem) or its inactive analog PP3 (Calbiochem), the Syk kinase inhibitor R406 (Selleckchem), caspase-1 inhibitor z-YVAD-fmk, caspase-8 inhibitor z-IETD-fmk (both BioVision), anti-Dectin 1 antibody, anti-Mincle antibody, anti-Trem1 antibody (all Invivogen), anti-Dectin 2 antibody (R&D Systems), Laminarin (Invivogen), Cytochalasin D, Bafilomycin A₁, Dynasore or Puromycin (all Sigma-Aldrich).
For RNA-mediated interference, 8-day-old BMMs were transfected with 25 nm siRNA through the use of the transfection reagent DF4 (Dharmacon). 72 h after transfection cells were used for experiments. The following SMARTpool siGENOME siRNAs were used: caspase-8 (M-043044-01), MALT1 (M-051221-01), CARD9 (M-045760-01), Bcl-10 (M-043902-00), caspase-1 (M-043902-00), ASC (M-051439-01) and nontargeting siRNA, as a control (D-001210-01) (all from Dharmacon). Silencing of expression was verified by real-time PCR and immunoblot.

Six glycoprotein receptors present on mBMDC surfaces, namely, Mannose binding receptor, Dectin-1, Dectin-2, DC-SIGN, DEC-205 and DNGR1 were neutralized. Mannan (10 µM) (Sigma-Aldrich, St. Louis, Missouri, USA), obtained from Saccharomyces cerevisiae and Laminarin (10 µM) (InvivoGen, San Diego, CA, USA), prepared from Laminaria digitata were used to block MBRs and Dectin-1 receptors respectively. Neutralizing antibodies (5 µg/ml) were used to block the other four receptor types. After addition of the corresponding inhibitors, each of the groups of cells was incubated within complete RPMI-1640 medium, for one hr at 37 °C, supplied with 5% CO₂. The receptor-neutralizations were done prior to the addition of NLGP or NLGP-FITC to the mBMDCs.