Mhcc97h

Hepatocellular carcinoma cell lines, HepG2, SK‐hep‐1, Huh‐7, and the normal liver epithelium cell line LO2, were purchased from the Cell Bank of the Chinese Academy of Sciences. Meanwhile, another HCC cell line, MHCC‐97H, was purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Company. All cell lines were cultured in high‐glucose Dulbecco's modified eagle medium (Gibco) supplemented with 10% fetal bovine serum (Biological Industries), 100 U/mL penicillin, and 100 mg/mL streptomycin. All cells were maintained at 37.0°C in an atmosphere containing 5% CO₂.

The HCC cell lines Hep G2, Hep 3B, Huh7, and MHCC-97H, as well as normal hepatic epithelial cell line (LO2), were obtained from Zhong Qiao Xin Zhou Biotechnology (Shanghai, China). All cells were cultured in cell culture dishes (Guangzhou Jet Bio-Filtration, Guangzhou, China) and maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% (v/v) fetal bovine serum and 5 mg/mL penicillin/streptomycin at 37°C with 5% CO₂. EFNA4-targeting siRNA and scramble control siRNA were purchased from RiboBio (Guangzhou, China). EFNA4-targeting sequences were as follows: siRNA #1, 5′-GGGCCTCAACGATTACCTA-3′; siRNA #2, 5′-GGAGAGACTTACTACTACA-3′. PIK3R2-targeting sequences were as follows: siRNA #1, 5′-GCACCTATGTGGAGTTCCT-3′; siRNA #2, 5′-GGCCAGACTCAAGAGAAAT-3′. β-catenin-targeting sequences were as follows: siRNA #1, 5′-GCCACAAGATTACAAGAAA-3′; siRNA #2, 5′-GACTACCAGTTGTGGTTAA-3′. The overexpression plasmids pcDNA3.1-EFNA4 as well as the empty vector (pcDNA3.1) were obtained from Sino Biological (Beijing, China). Cell transfection was performed using Lipofectamine 3000 (Thermo Scientific, Waltham, MA, USA) according to the instructions provided by the manufacturer. The expression level of EFNA4 was detected by quantitative real-time PCR.

Human normal hepatocyte NHC (Jining Shiye, Shanghai, China) and HCC cell lines MHCC97H (Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd., Shanghai, China), Huh7, and Hep3B (National Collection of Authenticated Cell Cultures, Shanghai, China) were included in this study. The Hep3B cells were cultured in minimal essential medium (Gibco, Carlsbad, CA, USA) plus 10% FBS (Gibco), 50 U/mL penicillin (Gibco), and 100 μg/mL streptomycin (Gibco). NHC, MHCC97H, and Huh7 cells were cultured with Dulbecco's Modified Eagle's Medium (DMEM). The culture condition was at 37°C in a 5% CO₂ cell incubator (Thermo Fisher Scientific Inc., Waltham, MA, USA). The media were renewed daily. After 3-4 d, the cells were subcultured, and the cells in logarithmic growth phase and in good growth condition were selected for the following assays.

HCC cell lines HepG2 (catalog number, #SCSP-510), Hep3B (catalog number, #SCSP-5045), and BEL-7404 (catalog number, #TCHu64) were provided by the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). MHCC97H (catalog number, #ZQ0020), and HCCLM3 (catalog number, #ZQ0023) were provided by Zhong Qiao Xin Zhou Biotechnology (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) was used to culture the cells, supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin/streptomycin solution. Plasmids coding for CSNK1D (pTSB-CMV-CSNK1D-copGFP-F2A-PuroR) were designed and purchased from TransheepBio-Tech CO, LTD (Shanghai, China). To overexpress CSNK1D, HepG2 cells were transfected with OE-CSNK1D plasmids or control vector plasmids by Lipofectamine 3000(Invitrogen). Forty-eight hours later, 5 µg/ml of puromycin (Invitrogen) was added to the medium and maintained for another 14 days until the stably-transfected cell lines were established. The sequence of short hairpin RNA(ShRNA) with highest intervening efficacy targeting CSNK1D was CUAUCUCGGUACGGACAUUTTAAUGUCCGUACCGAGAUAGTT. The sequence of small interfering RNA (siRNA) with highest intervening efficacy targeting Dishevelled Segment Polarity Protein 3 (DVL3) was GCUCCCUUUCACCAUUUAUAUAAAUGGUGAAAGGGAGC.