Hcclm3

LO2, HepG2, Hep3B, MHCC97H, and HCCLM3 cell lines were sourced from Shanghai Zhong Qiao Xin Zhou Biotechnology Co. Ltd. HEK293T cells were stored in the central laboratory of our hospital, Guangzhou, China. The cells were cultured in a DMEM medium containing 10% fetal bovine serum at 5% CO₂ and 37 °C.

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.

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.

Human hepatic carcinoma cell lines SMMC-7721 and HCCLM3 were purchased from Zhong Qiao Xin Zhou Biotechnology (Shanghai, China). SMMC-7721 cells were cultured in RPMI-1640 medium, and HCCLM3 cells were cultured in DMEM. The media were supplemented with 10% fetal bovine serum together with 1% penicillin and streptomycin (P/S). The cells were cultured under standard conditions of 5% CO
₂ 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).