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The human gallbladder cancer cell lines GBC-SD, NOZ, SGC-996, OCUG, EHGB-1 and EHGB-2 were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (CAS, Shanghai, China). The GBC-SD cells were grown in high-glucose DMEM (Gibco, Grand Island, NY, USA) supplemented with 10 % fetal bovine serum (Gibco) and 1 % penicillin-streptomycin(Hyclone, Logan, UT, USA). The NOZ cells were grown in William’s medium (Gibco) supplemented with 10 % fetal bovine serum (Gibco) and 1 % penicillin-streptomycin (Hyclone). The SGC cells were grown in 1640 medium (Gibco) supplemented with 10 % fetal bovine serum (Gibco) and 1 % penicillin-streptomycin (Hyclone), The OCUG, EHGB-1 and EHGB-2 cells were grown in high-glucose DMEM (Gibco) supplemented with 15 % fetal bovine serum (Gibco) and 1 % penicillin-streptomycin (Hyclone). All cells lines were maintained at 37 °C in a humidified atmosphere with 5 % CO₂. The A66 used in the in vitro and in vivo experiments was purchased from Selleck Chemicals (Houston, USA). Before conducting the CO-IP experiments, the GBC-SD and NOZ cells were starved for 12 h and 1 ng/mL EGF (Sangon Biotech, Shanghai, China) was added to the cell culture medium. The cells were maintained for 12 h to eliminate other confounding growth factor signals.

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.