Dmem high glucose medium

Human colorectal adenocarcinoma (HT-29) and human metastatic melanoma (A2058) cells were cultured at 37 °C and 5% CO2. A2058 cells were grown in DMEM high-glucose medium (Welgene Inc., Gyeongsan, Korea), and HT-29 cells were grown in RPMI1640 medium (Welgene Inc., Gyeongsan, Korea). All media were supplemented with 10% FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin, and all cells were purchased from the Korean Cell line Bank (Seoul, Korea).

Human hepatoblastoma (HepG2) cells were purchased from the Korean Cell Line Bank (Seoul, Korea) and grown in 5% CO₂ at 37°C in high-glucose DMEM medium (Welgene, Daegu, Korea) containing 10% (v/v) FBS (Cellgro, Manassas, VA, USA) and 5% AA (Invitrogen, Carlsbad, CA, USA). For experiments, cells were plated at a density of 1.6×10⁶ cells per 60 mm dish. The next day, the medium was changed to a serum-starvation medium containing 0.5% (v/v) FBS. After overnight incubation, the cells were treated with genistein at a final concentration of 40 µM for the indicated periods of time. For JNK inhibition experiments, cells were pre-treated with the JNK inhibitor SP600126 (Calbiochem, Darmstadt, Germany) at a concentration of 10 µM for 30 min prior to the addition of genistein.

A human immortalized keratinocyte cell line (HaCaT cells) was provided by Korea Institute of Oriental Medicine (Daegu, Korea) and maintained in high glucose DMEM medium (Welgene Inc., Gyeongsangbuk, Korea) supplemented with 10% FBS and 1% antibiotics (10,000 μg/ml of streptomycin and 10,000 units/ml of penicillin) (Invitrogen Inc., Carlsbad, CA, USA) and incubated at 37°C with 5% CO₂. After serum-starvation for 24 h, HaCaT cells were pretreated with SMGGT (10 and 100 μg/ml), puerarin, paeoniflorin (1, 10 μM), or DEX (10 μM) for 1 h and then incubated with TNF-α and IFN-γ (TI; 10 ng/ml each) for the indicated times. Recombinant human TNF-α and IFN-γ were obtained from Koma Biotech Inc. (Seoul, Korea).
A human mast cell line (HMC-1 cells) was purchased from Merck Millipore (Darmstadt, Germany) and grown in Iscove's Modified Dulbecco's Medium (IMDM) (Merck Millipore) supplemented with 10% FBS, 1% antibiotics, and 1.2 mM 1-thioglycerol (Sigma-Aldrich, St. Louis, MO, USA), at 37°C with 5% CO₂. The cells were stimulated with 10 μM SP for 48 h, followed with 1 nM CRH for 24 h, as described previously [21 (link)]; SMGGT (10 and 100 μg/ml), puerarin, paeoniflorin (1, 10 μM), or DEX (10 μM) was treated 1 h before addition of CRH. SP and CRH were obtained from Sigma-Aldrich (St. Louis, MO, USA).