Noz cells

NOZ cells were obtained from the Health Science Research Resources Bank (Osaka, Japan), GBC-SD, SGC-996, and EH-GB1 cells were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences, and human embryonic kidney 293 T (HEK293T) cells were purchased from the American Type Culture Collection. NOZ and GBC-SD both were P53 WT genotype (P53^+/+). GBC-SD, SGC-996, EH-GB1, and HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco), and NOZ cells were cultured in William’s E medium (Gibco). All cell lines were supplemented with 10% fetal bovine serum (Gibco), penicillin (100 mg ml⁻¹) and streptomycin (100 mg ml⁻¹) and were incubated in a humidified chamber with 5% CO₂ at 37 °C. All cell cultures were ensured to be mycoplasma-negative cultures by monthly mycoplasma tests and were passaged with 0.25% trypsin containing 2.21 mm ethylenediaminetetraacetic acid (EDTA) in PBS when the cells reached 80~90% confluency. Gemcitabine (GEMZAR) was purchased from Eli Lilly. Cisplatin, cycloheximide, doxorubicin, and puromycin were purchased from MedChem Express.

The GBC cell lines, GBC‐SD, and normal biliary epithelial cell line, HIBEC, were acquired from the Shanghai Institute for Biological Science, Chinese Academy of Science (Shanghai, China). NOZ cells were purchased from the Health Science Research Resources Bank (Osaka, Japan). RPMI‐1640 medium containing 10% dialysed foetal bovine serum (FBS) (HyClone), 100 μg/mL streptomycin and 100 U/mL penicillin was used for all cell culture, and the cells were maintained in a humidified atmosphere of 5% CO₂ at 37°C. MA (C15:1; C22H34O3; molecular weight: 346.50) was purchased from MCE. Gemcitabine was obtained from Selleck Chemicals. Gemcitabine and MA were dissolved in DMSO at the stock concentrations of 10 and 5 mmol/L, respectively. MTT (3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide) and dye of Oil Red O were purchased from Sigma. Working dilutions of Gemcitabine and MA were prepared in the culture medium immediately prior to use, and DMSO was used as the vehicle control. Antibodies targeting SREBP1 and β‐actin were purchased from Santa Cruz Biotechnology; antibodies against FASN (fatty acid synthase), ACC (acetyl‐CoA carboxylase), PCNA (proliferating cell nuclear antigen), Bax, Bcl2, AMPK (AMP‐activated protein kinase), p‐AMPK and Ki‐67 were purchased from Abcam.