Dulbecco s modified eagle s medium

HEK293T cells (ATCC) were cultured in Dulbecco's modified Eagle's medium (Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS) and 1 × penicillin/streptomycin (P/S) (Nacalai Tesque). MT4 cells (ATCC) were cultured in RPMI supplemented with 10% FBS and 1 × P/S.

H4IIE cell (DS Pharma Biomedical Co., Ltd.) was used between passage numbers 12 and 45. Cells were cultured in 6-well tissue culture plates (Becton, Dickinson and Company, Japan) and grown to near confluence in Dulbecco's modified Eagle's medium (Nacalai Tesque, Kyoto) containing 10 % fetal bovine serum at 37 °C under a 5 % CO₂ atmosphere. Cells were pretreated in the presence or absence of 250 μM or 100 μM L-Cit (supplied by Protein Chemical Co., Ltd., Japan) in serum-free medium for 1 h and were then incubated for 10 min in the presence or absence of 0.1 nM insulin. In order to measure phosphoenolpyruvate carboxykinase (PEPCK) gene expression, cells were treated for 6 h with 500nM Dexamethasone and 0.1 mM cAMP (Dex/cAMP) to induce PEPCK gene expression together with 250 μM L-Cit and/or 10 nM insulin during the same time frame.

Human coronary artery smooth muscle cells (Lonza, Basel, Switzerland) were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (Nacalai, Kyoto, Japan) (glucose; 5.5 mM) supplemented with 15 % fetal bovine serum (Life Technologies, Carlsbad, CA, USA), 100 IU/ml penicillin and 100 g/ml streptomycin (Nacalai) (normal glucose-containing media). High glucose-containing media (final 25 mM) was made by addition of 19.5 mM glucose to normal glucose-containing media. In like manner, 19.5 mM mannitol (Sigma-Aldrich, St. Louis, MO, USA) was added to normal glucose-containing media as an osmolality control (mannitol-containing media). Cells between passages 8 and 14 were used for the experiments. Unless otherwise noted, cells were cultured for the indicated time periods without changing media. In some experiments, HCASMCs were cultured with rosuvastatin (10 µM), mevalonate (Sigma-Aldrich) (100 µM), FPP (Sigma-Aldrich) (10 µM), GGPP (Sigma-Aldrich) (10 µM), fasudil (Wako) (10 µM) and Y-27632 (Wako) (10 µM). Human umbilical vein endothelial cells (HUVECs) (Lonza) were cultured at 37 °C in the EGM-2 BulletKit (Lonza).

The H-1 mESC line was originally isolated from C3H/He mice [23 (link)]. mESCs were maintained in Dulbecco’s modified Eagle’s medium (4.5 g/l glucose) with L-glutamine, without sodium pyruvate, (Nacalai Tesque, Japan) supplemented with 15% foetal bovine serum (Gibco, USA), 1000 U/ml Stem Sure Leukemia Inhibitory Factor (mouse recombinant solution; Wako), 0.1 mM Stem Sure 2-mercaptoethanol solution (Wako), and penicillin–streptomycin (Gibco). Cells were grown on mitomycin C (Kyowa Kirin, Japan)-treated mouse embryonic fibroblast feeder cells (C57BL/6J) at 37°C in a humidified incubator with 5% CO₂. For chemical stress treatments, mESCs were cultured in ESGRO Complete Plus serum-free clonal grade medium (Merck Millipore, Germany) on gelatine (Sigma, USA)-coated dishes without feeder cells.

Dulbecco's Modified Eagle's Medium (DMEM), trypsin-EDTA, antibiotics (penicillin and streptomycin), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-di-phenyltetrazolium bromide (MTT) powder, and trypan blue dye solution were purchased from Nacalai Tesque (Kyoto, Japan). Dimethyl sulfoxide (DMSO) was purchased from Fisher Sc. (UK) and the phosphate buffer saline tablet was purchased from Oxoid (England). Fetal Bovine Serum (FBS) was purchased from iDNA (South America Origin). Propidium iodide was purchased from Sigma (St. Louis, USA). Other kits used were the Glutathione Assay Kit (Cayman, USA) and Annexin V/FITC Kit (BD Bioscience, USA). Primary rabbit antibodies anti-Nrf2 (C-20: sc-720), anti-Keap1 (H-190: sc-33569), caspase-3 (H-277: sc-7148), caspase-7 p20 (H-65: sc-33773), and anti-beta-actin (sc-47778) were purchased from Santa Cruz Biotechnology (CA, USA). Horseradish peroxidized conjugated anti-rabbit (ab6721) was purchased from ABCAM (Cambridge, MA, USA). TQ and TQ-NLC were provided by Assoc. Prof. Dr. Latifah Saiful Yazan, Laboratory of Molecular Biomedicine, Institute of Bioscience (IBS), Universiti Putra Malaysia.

Two human endometrioid carcinoma cell lines (HEC1 and HEC116) were provided by the Japanese Collection of Research Bioresources (Osaka, Japan) and were cultured in Dulbecco’s modified Eagle’s medium (Nacalai tesque, Kyoto, Japan) containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA). Both cell lines were authenticated by genetic profiling using polymorphic short tandem repeat analysis.

MC3T3-E1 cells, an immortalized cell line, were purchased from RIKEN BioResource Research Center (Tsukuba, Ibaraki, Japan) and cultured in Dulbecco’s Modified Eagle’s Medium (Nacalai Tesque, Kyoto, Japan) with or without the nine EAAs and supplemented with 10% fetal bovine serum (Hyclone, Thermo Fisher Scientific, Waltham, MA, USA).