Cell culture plastics

MDCK (Madin-Darby Canine Kidney) cell line was purchased from the Bioresource Collection and Research Center, Hsinchu, Taiwan. The cell line was subculture according to the procedures of providers. Dulbecco’s Modified Eagle Medium (DMEM; Thermo Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum, and 100 µL/mL penicillin was prepared for cell culture at 37 °C and 5% CO₂ humidified incubator. Cell culture plastics were obtained from Corning Inc. (Corning, NY, USA). Cells were maintained for high glucose challenge assays. Briefly, the MDCK (1 × 10⁶/mL) cells were seeded in high glucose DMEM supplemented with 2 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mL/L penicillin and 10% fetal bovine serum (Sigma-Aldrich, USA). MDCK cells cultured in the high glucose condition was termed HG-MDCK cells.

Luteolin (98% HPLC purity) was provided by Prof. Jian-Guo Xing from the Xinjiang Institute of Materia Medica of China (Urumqi, China). Luteolin was isolated from total flavonoids extracted from the aerial part of Dracoephalum moldavica L. (Patent No. CN 200710203385.1; specimen ID 20100708). Cell culture plastics were purchased from Corning (Corning Co., Corning, NY, USA). All other reagents were purchased from Sigma Chemical Company (St. Louis, MO, USA) unless stated otherwise.

Quercetin (QT), sodium azide, phosphotungstic acid (PTA, H₃PW₁₂O₄₀), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), sodium chloride (NaCl), and peptone were purchased from Sigma-Aldrich Chemicals, St. Louis, MO, USA. 2,7-Dichlorodihydrofluorescein diacetate (H2DCFDA) was obtained from Invitrogen, Waltham, MA, USA. Capmul MCM NF (CAP MCM NF) and cremophor RH40 (CR RH 40) were ex gratis from Gattefosse (Saint-Priest, Cedex, France) and BASF (Ludwigshafen, Germany), respectively. Sodium hydroxide (NaOH), doxorubicin hydrochloride (DOX), and organic solvents were procured by Merck, India. Agar was purchased from HIMEDIA, Mumbai, India. Glassware was acquired from Borosil, India. A549 (lung carcinoma epithelial) cells, MIA PaCa-2 (human pancreatic cancer) cells, and HeLa (cervical cancer) cells were purchased from the National Centre for Cell Science, India. These cells were maintained in 10% fetal bovine serum (FBS) comprising Roswell Park Memorial Institute (RPMI) media (Gibco, Waltham, MA, USA) and Dulbecco’s Modified Eagle Medium (DMEM) in a humidified CO₂ incubator (Thermo Scientific, Waltham, MA, USA) at 37 °C. The cell culture plastics were procured from Corning, Corning, NY, USA. All other chemicals and reagents were of analytical grade and were used as such.

Unless indicated otherwise, cell culture reagents were from Life Technologies, cell culture plastics were from Corning, and all other materials were from Sigma. All reagents were of the lowest available endotoxin grade. PI3K inhibitors (Selleck Chemicals) and final concentrations used were as follows: pan-PI3K, wortmannin (50 nM); PI3Kα, BYL-719 (0.25 μM); PI3Kβ, TGX-221 (40 nM); and PI3Kδ, IC87114 (1 μM).

Rat jaws were only collected for this study. Wistar male rats (28 ± 2 days old) were humane killed in-advance in the Central Biomedical Services (University of Leeds, Leeds, United Kingdom) as per the institutional animal use and care committee at the University of Leeds and according to guidelines laid down by the Animals (Scientific Procedures) Act 1986. Dental pulp tissues were aseptically retrieved from the rat mandibular incisors and stored in 0.1 M phosphate-buffered saline (PBS, 17-516F, Lonza, Slough, United Kingdom; pH 7.4) at − 80 °C for up to 4 weeks. All reagents and solutions were purchased from Sigma-Aldrich (Poole, United Kingdom) and cell culture plastics were obtained from Corning, Inc. (Amsterdam, The Netherlands) unless stated otherwise.