Hep3b

A human normal hepatic epithelial cell line (LO2) and human lung cancer cell lines (HepG2, Hep3B, 97H, and LM3) were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., and cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Grand Island, NY, USA).
Total RNA was routinely extracted after adding TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Shanghai, China) to the cells, and the RNA concentration was determined. cDNA was synthesized and analyzed by qRT-PCR using SYBR® Green (Roche, Basel, Switzerland) [24 (link)]. The PCR conditions were 95°C for 10 minutes, 95°C for 15 seconds, and 60°C for 30 seconds for 40 cycles. GAPDH was used as the internal control, and the relative changes between two groups were analyzed by the 2^-ΔΔCt method. The following primers were used for qRT-PCR: CCDC45 forward, 5′-AAAAGCCTTAGCCTCACCAAG-3′; CCDC45 reverse, 5′-CTCCCCTAGCTTCCTAGCATT-3′; GAPDH forward, 5′-ATCTTCCAGGAGCGAGATCC-3′; and GAPDH reverse, 3′-ACCACTGACACGTTGGCAGT-5′.

The HCC cell lines PLC/PRF/5, HCCLM3, Huh-7, Hep3B, and HepG2, and the control liver epithelial cell line LO2 were purchased from Zhong Qiao Xin Zhou Biotechnology (Shanghai, China). The cells were inoculated into culture dishes purchased from Guangzhou Jet Biofiltration (Guangzhou, China) and added to Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum to maintain growth. The growth environment temperature was maintained at 37 °C with 5% CO₂. The overexpression plasmid pcDNA3.1-AHSA1, empty vector pcDNA3.1, shRNAs targeting AHSA1, and sh-control were purchased from OBiO Technology (Shanghai, China). The sequences of shRNAs targeting AHSA1 and sh-control were as follows: sh-AHSA1-1, GCATGATCTTACCTACAAT; sh-AHSA1-2, CCATCACCTTGACCTTCAT; sh-control, CCTAAGGTTAAGTCGCCCTCG. The siRNAs targeting CALD1 and si-NC were obtained from RIBOBIO (Guangzhou, China). The sequences of siRNAs targeting CALD1 were as follows: si-CALD1-1, AGAGCTTCATGGATCGAAA; si-CALD1-2, GTACGCAACATCAAGA GTA; si-CALD1-3, GAAGGAGTTCGACCCAACA. Lipofectamine 3000 (Thermo Scientific, Waltham, MA, USA) was used for conventional cell transfection for 72 h. AHSA1 expression was detected by western blotting.

The human HCC cell lines HepG2, Hep3B, Huh7, and HCCLM3 were purchased from Zhong Qiao Xin Zhou Biotechnology (ShangHai, China). The HepG2, Huh7, and HCCLM3 cells were cultured in Dulbecco's modified Eagle medium (DMEM, Gibco, USA), and the Hep3B cells were cultured in minimum essential medium (MEM, Gibco, USA). All the media were supplemented with 10% fetal bovine serum (FBS, A0500-3011, Cegrogen Biotech, Germany) and 1% penicillin-streptomycin (Meilunbio, China), and the cells were cultured at 37°C in a 5% CO₂ air atmosphere.

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.

Hepatitis B virus‐infected cell line Hep3B 21 and noninfected cell line Huh7 22 were obtained from Shanghai Zhong Qiao Xin Zhou Biotechnology Co.,Ltd. (Shanghai, China) and used in the present experiments within 6 months after receipt. A cell line CSQT‐2 (HBV‐infected) with high metastatic potential was kindly provided by Prof. Shuqun Cheng (Eastern Hepatobiliary Surgery Hospital, Second Military Medical University) and used as an in vitro model for PVTT. Cells were cultured in DMEM (Gibco, Grand Island, NY) with 10% fetal bovine serum (HyClone, Logan, UT) in a humidified 5% CO₂/95% air environment at 37°C.

The human normal liver cell line, THLE-2, was obtained from Shanghai Academy of Life Science (Shanghai, China). Human hepatoblastoma cell line HepG2 (catalog No. ZQ0022), human HCC cell lines, (HCCLM3 (catalog No. ZQ0023), Hep3B (catalog No. ZQ0024), and Huh7 (catalog No. ZQ0025) were purchased from Zhong Qiao Xin Zhou Biotechnology (Shanghai, China). All cell lines were inoculated into culture dishes and added to Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) to maintain growth. The cell culture medium was also supplemented with 1% penicillin/streptomycin. The growth environment temperature was maintained at 37 °C with 5% CO₂. The culture dishes were purchased from Guangzhou Jet Biofiltration (Guangzhou, China). FBS, and penicillin/streptomycin were purchased from BI (BI, Ridgefield, CT, USA).