Galangin

All cell culture supplies were from Invitrogen, Paisley, UK, unless otherwise stated. Caco-2/TC7 cells were kindly donated by Dr M. Rousset, U178 INSERM, Villejuif, France. Millicell-ERS volt ohmmeter was obtained from Millipore corporation, Massachusetts, USA. Transwell inserts and 12 well plates were Costar brand obtained from Fisher Scientific, Loughborough, UK. The MK571 was purchased from Biomol Research Laboratories, Exeter, UK. Mini protease inhibitor cocktail tablets containing EDTA and perfabloc were purchased from Roche, Welwyn Garden City, UK. Galangin, quercetin and kaempferol were purchased from Extrasynthese, 69727 Genay Cedex, France. Alamethacin from Trichoderma viride, uridine 5′-diphosphoglucuronic acid and uridine 5′-diphospho-N-acetyl-glucosamine were purchased from Sigma–Aldrich, Poole, UK. GraFit software was version 4.0 and purchased from Erithacus Software Ltd., East Grinstead, UK. Gemini C18 (150 mm × 2.0 mm i.d., 5 μm) narrow bore HPLC column was purchased from Phenomenex, Macclesfield, UK. Analytical chemistry was performed using ChemStation software on an HP1100 HPLC and single quadrupole MSD SL mass spectrometer from Agilent, Manchester, UK. All solvents and other reagents of analytical grade were obtained from Sigma–Aldrich, Poole, UK.

Galangin (purity ≥ 99%) was purchased from Extrasynthese (Genay, France); dimethylsulfoxide (DMSO), Tris–HCl, ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulphate (SDS), phenylmethylsulfonyl fluoride, bovine serum albumin (BSA), gelatin, leupeptin, Nonidet P-40, deoxycholic acid and sodium orthovanadate were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA); A protein assay kit was obtained from Bio-Rad Labs. (Hercules, CA, USA). Dulbecco’s phosphate buffer solution (PBS), fetal bovine serum (FBS), trypsin-EDTA, and powdered Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco-BRL (Gaithersburg, MD, USA). Matrigel was obtained from BD Transduction Laboratories (San Diego, CA, USA). Antibodies against Akt, ERK1/2, JNK/SAPK, and p38 MAPK, proteins, and phosphorylated proteins were purchased from Cell Signalling Technology (Beverly, MA, USA). An enhanced chemiluminescence (ECL) kit was purchased from Amersham Life Science (Amersham, UK).

In total, 1 × 10⁵ clone 1C116 or 2C21 cells, were cultured with each material (concentration ranges are described below) or vehicle control (dimethyl sulfoxide: DMSO) and 20 ng/mL PMA in 96-well U-bottom tissue culture plates in 200 μL of CCM for 24 h. The materials were 0–100 μg/mL flavonoids (quercetin, isoquercitrin, hyperoside, galangin, kaempferol, morin, myricetin, hesperetin, hesperidin, acacetin, linarin, isorhoifolin, (all purchased from Extrasynthese, Lyon, France)), 0–1000 μM IPP (Sigma-Aldrich), 0–2000 μM risedronate, 0–33.5 μM methylamine (Sigma-Aldrich), 0–33.5 μM ethylamine (Sigma-Aldrich) and 1 μg/mL purified anti-human CD3 mAb (OKT3: Thermo Fisher Scientific). After the incubation, the culture supernatant was corrected, and the concentration of IL-2 was analyzed using an ELISA kit (BD Biosciense).