Pi bond resin

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.

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.