Endotoxin free water

A limulus amoebocyte lysate (LAL) assay was conducted to assess the interaction of surface bound AMPs with lipopolysaccharides (LPS) of P. aeruginosa using a chromogenic assay (Cape Cod, E. Flamouth, MA, United States) (Gustafsson et al., 2010 (link)). LPS (8 × 10^–4 nmol/ml) from P. aeruginosa 10 (Sigma Aldrich, St. Louis, MO, United States) was dissolved in endotoxin free water (Sigma Aldrich) and incubated with surface bound AMPs at 37°C for 4 h. Following addition of LAL reagent, any decrease in OD_405nm was measured and compared with control surface (without peptides), and results expressed as a percentage reduction compared to the control surface.

Medium-chain polyP (polyP75) with average 75 Pi units long and long-chain polyP (polyP1150) with average 1150 Pi units long were purchased from Kerafast (Boston, MA, USA). The pNPP was obtained from Sigma-Aldrich (St. Louis, MO, USA).
Wheat phytase (Sigma-Aldrich, USA) was reconstituted in endotoxin-free water (Sigma-Aldrich, USA). The enzyme was then dialyzed against 50 mM Tris-HCl (pH 8.0) at 4°C overnight. Its inorganic phosphate background was removed by using Pi-bond resin (Innova Biosciences, Cambridge, UK) according to the manufacturer’s instructions.

Phenol-extracted LPS from E. coli O157:B8 (ECLPS) was purchased from Sigma. Ultrapure LPS from Rhodobacter sphaeroides (RSLPS) was purchased from Invivogen. Lipid IVa was purchased from Pepta Nova. RSLPS and ECLPS were reconstituted in sterile endotoxin-free water (Sigma) to 1 mg/ml. Lipid IVa was reconstituted in sterile DMSO to 1 mg/ml. All LPS derivatives were sonicated prior to freezing at −20°C in aliquots and then re-sonicated for one minute following thawing before use.

Nucleotides (ATP and UDP) and wheat phytase used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA). wheat phytase was reconstituted in endotoxin-free water (Sigma-Aldrich, USA) and residual inorganic phosphate was removed through Pi-bond resin (Innova Biosciences, Cambridge, UK). A malachite green-based Pi Color Lock gold phosphate detection kit was procured from Innova Biosciences (UK). L-phenylalanine and L-homoarginine as inhibitors of dephosphorylation, were procured from Sigma-Aldrich (USA). For cell viability assay, an EZ-CYTOX kit was purchased from DogenBio (Seoul, Korea). Cymax human interleukin-6 (IL-6) and IL-8 enzyme-linked immunosorbent assay (ELISA) kits used for IL-6 and IL-8 secretion assay were obtained from Ab FRONTIER (Seoul, Korea). A caspase-3/CPP32 colorimetric assay kit used to measure the activity of caspase-3, marker of programmed cell death, was sourced from BioVision (Milpitas, CA, USA). Human colorectal adenocarcinoma cell line, HT-29, was obtained from ATCC (Manassas, VA, USA). Cells were cultured in McCoy’s 5A medium purchased from Gibco Life technologies (Carlsbad, CA, USA). The medium was supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution, both of which were purchased from Gibco Life technologies (USA). These cells were cultured at 37°C in a humidified air incubator with 5% CO₂.

All peptides, including three linear peptides (SP27, SP27’ and L2), a multiple antigenic peptide (named MAP27), and the attaching backbone of MAP (MAPctrl) were synthesized by Hybio Pharmaceutical (Shenzhen, China). Sequence accuracy of these peptides was confirmed by mass spectrometry and high performance liquid chromatography. Peptide purity was above 95% and the residual endotoxin was measured below 0.025 EU/ml. All of the peptides, provided as lyophilized powders, were dissolved in endotoxin-free water (Sigma) at 20mg/ml and stored at -70°C.

The sources of the TAK1 inhibitor NG25 (Dzamko et al, 2012 (link); Tan et al, 2015 (link)), the IKKβ inhibitor BI605906 (Clark et al, 2011 (link)), the TBK1/IKKɛ inhibitor MRT67307 (Clark et al, 2011 (link)) and the LRRK2 inhibitor GSK2578215A (Reith et al, 2012 (link)) have been described. The IKKβ inhibitors PS1145 (Castro et al, 2003 (link)) and TPCA‐1 (Podolin et al, 2005 (link)) were from Sigma and Calbiochem, respectively. The NLRP3 inhibitor MCC950 (Coll et al, 2015 (link)) was obtained from Selleckchem. The TLR ligands Pam₃CSK₄ and R848 and the inflammasome agonists ATP, nigericin and poly(dA:dT) were from InvivoGen. LPS (lipopolysaccharide; Escherichia coli 055:B5) was from Alexis Biochemicals (ALX‐581‐001). A stock ATP solution (200 mM) was prepared in endotoxin‐free water (Sigma), and the pH of the solution was adjusted to 7.4 using NaOH. Actinomycin D and cycloheximide were from Sigma, disuccinimidyl suberate (DSS) from Thermo Fisher Scientific and murine TNF‐α (315‐01a) from PeproTech.