High glucose dulbecco s modified eagle s medium dmem

The human enteroendocrine L cell line NCI-H716 (American Type Culture Collection, Manassas, VA, USA) was cultured as described previously [27 (link)]. Briefly, the cells were initially grown in low-glucose RPMI-1640 (Nacalai Tesque, Kyoto, Japan) with 10% fetal bovine serum (FBS) (Biosera, Kansas City, MO, USA) at 37 °C and in the presence of 5% CO₂. Subsequently, in order to generate mature endocrine cells, they were maintained in Matrigel (Corning, Bedford, MA, USA) with high glucose Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque) and 10% FBS for 2 days. Furthermore, 100 U/mL penicillin and 100 mg/mL streptomycin were added to the culture media at a ratio of 1:1000.
LX-2 human stellate cells and HSC-T6 rat stellate cells were purchased from Merck KGaA (Darmstadt, Germany). Both lines were maintained as monolayer cultures in DMEM with 10% FBS and 1% penicillin/streptomycin in an incubator at 37 °C and 5% CO₂. For each assay, cells were pre-incubated for 6 h with 5 ng/mL of human and rat transforming growth factor-β1 (TGF-β1) (Sigma-Aldrich, St. Louis, MO, USA).

Docetaxel was purchased from Sigma-Aldrich Co (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO) which was used as vehicle control. Purified human THBS1 and TNC proteins were purchased from EMD Millipore Corporation (CA, USA). Charcoal stripped fetal bovine serum (CS-FBS) was obtained from Nacalai Tesque (Kyoto, Japan). Human breast cancer cell line MCF-7 (ER+) was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in high glucose Dulbecco's Modified Eagle's Medium (DMEM) (Nacalai Tesque, Japan) supplemented with 10% fetal bovine serum (FBS) and 1X penicillin-streptomycin (Invitrogen, Carlsbad, CA) at 37°C in a humidified atmosphere with 5% CO₂.

The following reagents and drugs were purchased from the commercial sources indicated in parentheses: antibiotic-antimycotic cocktail solution, l-glutamine, high-glucose Dulbecco’s modified Eagle’s medium (DMEM), hygromycin B (Nacalai Tesque, Inc., Kyoto, Japan); fetal bovine serum (FBS) (Equitech-Bio, Inc., Kerrville, TX, USA); and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT reagent) (Sigma-Aldrich Co., St. Louis, MO, USA). SN-38 was generously provided by Yakult Honsha Co., Ltd. (Tokyo, Japan). All other chemicals used were of analytical grade.