Sicrnde 1

Normal human intrahepatic biliary epithelial cell (HIBEpic) and four human IHCC cells (HuCCT1, RBE, HCCC9810 and HUH28) were all purchased from the American Type Culture Collection. These cell lines were routinely cultured in DMEM (HIBEpic, HuCCT1, HUH28 and RBE) or RPMI-1640 (HCCC9810), supplemented with 10% fetal bovine serum (FBS) (Sigma, St. Louis, MO, USA), 100 U/ml penicillin sodium, and 100 mg/ml streptomycin sulfate. All cells were cultured in a humidified atmosphere containing 5% CO₂ at 37°C.
HuCCT1 cells were transfected with siRNAs targeting CRNDE (siCRNDE-1 and siCRNDE-2) or a scrambled negative control (siNC) (GenePharma, Shanghai, China). HCCC9810 cells were transfected with CRNDE overexpressing vector (pcDNA3.1-CRNDE) or its control vector (pcDNA3.1-Vector) (GenePharma, China). The transfection was conducted with Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's protocol.

Colorectal neoplasia differentially expressed (CRNDE) small interfering RNA (siRNA), si-CRNDE-1 (5′-GGTGTTAAGTGTGATGCTTCC-3′) or siCRNDE-2 (5′-GGATGCTGTCAGCTAAGTTCA-3′), and negative control siRNA (5′-UUCUCCGAACGUGUCACGUTT-3′) were purchased from Shanghai GenePharma Co., Ltd (cat. no. A10001). CRNDE expression in SHG-44 cells was silenced by siRNA transfection using Lipofectamine^® RNAiMax reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The transfection compound was prepared at room temperature for 20 min. The concentration of siRNA used was 50 nM. After culturing in the incubator (37°C) for 48 h, the cells and cell cultures were harvested for subsequent experiments, and the transfection efficiency was evaluated by reverse transcription-quantitative PCR (RT-qPCR).