Dulbecco s modified eagle s medium

We have established human esophageal cancer cell lines YES-2, YES-3, YES-5, and YES-6 (22 (link)) and YPK-1 and YPK-4 (23 (link)) in our department. We purchased SNV398, MKN28 and MKN74 from American Type Culture Collection (ATCC, Sumitomo Farma International, Tokyo, Japan). These human cancer cell lines were maintained in Dulbecco’s modified Eagle’s medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U/ml penicillin G, and 100 μg/ml streptomycin. Colon 26, a murine colon adenocarcinoma cell line derived from BALB/c mice (24 (link)), and normal human fibroblasts derived from the epidermis of surgical specimens were maintained in RPMI-1640 medium (Life Technologies) supplemented with 10% FCS. Subconfluent cultures in 33-mm 6-well plates were transfected with 2 μg of each plasmid vector by using Lipofectamine 2000 reagent (Life Technologies) according to the manufacturer’s instructions. G418 (100 mg/ml) (Life Technologies) was added to the cells 48 h later. G418-resistant clones were isolated and expanded in culture medium containing 100 mg/ml of G418.

pET expression system 3 (code 69410‐3) was purchased from Novagen, Inc. (Madison, MI, USA); and maleimide‐activated keyhole limpet hemocyanin (code 77606), from Pierce Biotechnology, Inc. (Rockford, IL, USA). An ECL kit (code RPN2106) and anti‐rabbit IgG‐conjugated peroxidase (code NA934‐1ML) were obtained from GE Healthcare Life Science (Bucks, UK). Polyclonal antibody against human MCU (code HPA016480) was purchased from Sigma‐Aldrich Japan (Osaka, Japan). Polyclonal antibody against human EMRE (code sc‐86337) was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). HeLa cells were procured from the JCRB (Japanese Collection of Research Bioresources) Cell Bank. Dulbecco’s modified Eagle’s medium (code 05919) was purchased from Nissui (Tokyo, Japan).

RD cells and Vero cells were cultured in Dulbecco's modified Eagle's medium (Nissui) supplemented with 5% fetal bovine serum (FBS) and penicillin-streptomycin (Life Technologies) (5% FBS-DMEM). RD-hSCARB2 cells (generously gifted by Dr. K. Fujii) were cultured in 5% FBS-DMEM supplemented with 4 μg/ml puromycin (Calbiochem). Cells had been tested for contamination and mycoplasma infection.

HepG2 cells were maintained and cultured in Dulbecco’s modified Eagle’s medium (Nissui Pharmaceutical, Tokyo, Japan) containing 10% (v/v) fetal bovine serum (Sigma-Aldrich), 4 mM l-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin under a humidified atmosphere of 95% (v/v) air and 5% (v/v) CO₂ at 37°C. The cells were grown to 80% confluence and incubated in serum-free medium overnight before treatment. The cells were simultaneously treated with 1.2 mM fatty acids mixture (palmitic acid:oleic acid = 1:2 ratio) and various concentrations of bisacurone and curcumin for 24 h. Dimethyl sulfoxide (DMSO) [final concentration is 0.1% (v/v)] was used for a vehicle control. A stock solution of each fatty acid was dissolved in 99% (v/v) methanol and diluted in culture medium containing 1% (w/v) BSA to the final concentration.

MCF-7 human breast cancer cells provided by the American Type Culture Collection (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (Nissui Pharmaceutical) supplemented with 10% of foetal bovine serum (Sigma-Aldrich) and 1% of a penicillin/streptomycin solution (Wako Pure Chemical Industries).