Hepg2

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.

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′.

SK-HEP1(catalog number, #ZQ0030), HepG2 (#ZQ0022), Hep3B (#ZQ0024), Huh7 (#ZQ0025), SMMC-7721 (#ZQ0029), HCCLM3 (#ZQ0023), and LO2 (#ZQ0031) were purchased from Zhong Qiao Xin Zhou Biotechnology (Shanghai, China). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Hyclone, CA, USA) or RIPM1640 medium supplemented with fetal bovine serum (FBS; Gibco, CA, USA) and penicillin/streptomycin at 37 °C under the condition of 5% CO2. Human FEN1 cDNA was amplified by PCR and cloned into the pTSB02-GFP-PURO vector constructed by Transheep (Shanghai, China). siRNA Smartpool targeting FEN1 was designed and constructed by Dharmocon (NY, USA). Plasmids or siRNAs were transfected with Lipofectamine 3000 reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. Cells transfected with pTSB02-GFP-PURO plasmids were selected with puromycin (5 μg/mL, Sigma-Aldrich, CA, USA) for 30 days to obtain stably transfected cells.

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.