3t3 l1 cells

Epidermoid carcinoma KB cells were provided by DS Pharma Biomedical Co., Ltd. (Suita, Japan). Mouse fibroblast-like 3T3-L1 cells were obtained from the JCRB Cell Bank (Tokyo, Japan). KB cells were cultured in Eagle’s minimal essential medium supplemented with 10% fetal bovine serum. The 3T3-L1 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% bovine calf serum. All media were supplemented with 100 IU/mL penicillin and 100 μg/mL streptomycin. The cells were maintained in a humidified atmosphere containing 5% CO₂ at 37 °C.

Transformed African green monkey kidney fibroblast COS-7 cells (RCB0539, Riken BioResource Research Center, Tsukuba, Japan) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (FUJIFILM Wako, Osaka, Japan) supplemented with 10% (v/v) fetal bovine serum (FBS) (Biofluids, Fleming Island, FL) and nonessential amino acids (FUJIFILM Wako). Human breast cancer MCF-7 cells (Health Science Research Resources Bank, Osaka, Japan) were maintained in DMEM supplemented with 15% (v/v) FBS. A knockout cell line for human GDE7 gene (GDPD3) was previously established^{18 (link)}. 3T3-L1 cells (Japanese Collection of Research Bioresources Cell Bank, Ibaraki, Japan) were maintained in DMEM (1 g L⁻¹ glucose) supplemented with 10% (v/v) calf serum (Cytiva, Marlborough, MA). These cell lines were cultured at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air.

Murine preadipocyte 3T3-L1 cells were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan). Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 4.5 g/L glucose, 10% bovine serum (16170–078; Life Technologies, Carlsbad, CA, USA), and antibiotics (100 U/mL penicillin G and 100 μg/mL streptomycin sulfate). 3T3-L1 preadipocytes were differentiated to adipocytes using a routine procedure [27 (link)]. Briefly, at 2 days post-confluency (day 0), the medium was exchanged to differentiation medium containing 4.5 g/L glucose, 10% fetal bovine serum, and the above antibiotics, and the cells were stimulated with 10 μg/mL insulin, 1 μM dexamethasone (DEX), and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX). The medium was replaced by fresh differentiation medium containing 10 μg/mL insulin every 2 days, and the cells were harvested on day 8. Control cells were not exposed to differentiation stimuli and harvested on day 8. Cells were pretreated with mogrol for 30 min before stimulation with the inducers (on day 0) and treated with mogrol at every exchange of medium (on days 2, 4, and 6). Cells were maintained at 37°C in a 5% CO₂/95% air atmosphere with 98% humidity.