Plc prf 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.

PLC/PRF/5, Huh-7 and THLE-3 cells were purchased from Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (Shanghai, China). All cell lines were cultured in MEM medium (Gibco, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA), 100 μg/mL streptomycin and 100 IU/mL penicillin (Gibco, USA). They were cultured at 37°C, 5% CO2.