Penicillin and streptomycin

The FaDU cell lines were obtained from the Affiliated Hospital of Changchun University of Chinese Medicine. FaDU cells were cultured in RPMI-1640 (Servicebio, Wuhan, China) supplemented with 10% fetal bovine serum (Servicebio, Wuhan, China), and 1% penicillin and streptomycin (Servicebio, Wuhan, China). The cells were cultured at 37°C in a 5% CO₂ incubator.

In this study, primary hepatocytes of grass carp were isolated and cultured according to a previous study [64 (link)]. Briefly, prior to the isolation of hepatocytes, the blood of the fish was drawn with a syringe. Then, the liver was rapidly isolated and washed several times in ice-cold phosphate-buffered saline (PBS) (Servicebio, Wuhan, China) containing 500 U/mL penicillin and streptomycin. After removal of PBS using sterile pipettes, the samples were cut into small pieces (about 1 mm³). The small pieces of liver were digested with trypsin at 28 °C for 10 min, then the cells were collected, and the process was repeated 3 times. Thereafter, the cell suspension was centrifuged at 400 g for 10 min and washed twice. The harvested cell pellets were resuspended in M199 medium (Gibco, NY, USA) with 10% fetal bovine serum (Gibco, NY, USA) and 100 U/mL penicillin and streptomycin (Gibco, NY, USA) at a density of 1 × 10⁶ cells/mL. Finally, primary hepatocytes were kept at 28 °C in a 5% CO₂ environment.

HepG2 and SMMC-7721 cells were purchased from ATCC. HepG2 cells were cultured in DMEM high glucose medium (Servicebio), and SMMC-7721 cells were cultured in 1640 medium (Servicebio), all containing 10% Foetal Bovine Serum (FBS) and 1% penicillin and streptomycin (Servicebio). Short hairpin (sh)RNAs sh-STK25 were obtained from miaolingbio.lnc, Wuhan, China. HepG2 and SMMC-7721 cells were cultured in 6-well plates (5 × 10⁵/well) and transfected with 2.4 μg siRNA using Attractence Transfection Reagent (Cat. No. 301005, QIAGEN, China).