Penicillin

Large yellow croakers were treated in seawater containing 1000 IU/mL penicillin (Sangon Biotech, China) and 1.2 mg/mL streptomycin (Sangon Biotech, China) for 24 h. The livers were dissected under sterile conditions and collected in cold sterile PBS supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. After washing with Dulbecco’s modified Eagle’s medium (DMEM/F12) (Biological Industries, Beit Haemek, Israel), liver tissue was minced into 1 mm³ pieces and digested with 0.25% trypsin-EDTA (Gibco, USA) at room temperature for 10 min. The reaction was terminated by adding DMEM/F12 medium containing 15% fetal bovine serum (FBS), and the cell suspension was purified through a cell strainer with 70 μm mesh. The isolated cells were collected in a 15 ml sterile centrifuge tube and centrifuged at 500 g for 10 min at 4°C. Cell pellets were resuspended in complete medium (DMEM/F12 medium containing 15% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin). Primary hepatocytes were seeded into six-well culture dishes at a density of 2×10⁶ cells/well and incubated at 28°C in 5% CO₂.

HEK293T, U2OS, SW480, SW620,HCT116, ZR-75-30, and HeLa cells were purchased from the American Type Culture Collection (ATCC). HeLa, U2OS, HEK293T, Sirt3^−/− MEFs, SW620, SW480 and HCT116 cells were cultured in DMEM/ high glucose medium (HyClone) supplemented with 10% fetal bovine serum (BI), 100 units ml⁻¹ penicillin and 100 µg ml⁻¹ streptomycin (Sangon Biotech). ZR-75-30 cells were cultured in RPMI-1640 (GIBCO) with supplemented with 10% fetal bovine serum (BI), MEM NEAA (GIBCO), 100 units ml⁻¹ penicillin and 100 µg ml⁻¹ streptomycin (Sangon Biotech). Cells were maintained in an incubator at 37 °C, in a humidified atmosphere containing 5% CO₂. Cell transfection was performed using PEI (Polysciences) based on a 3:1 ratio of PEI (μg): total DNA (μg). Cell transfection for siRNA was carried out by Lipofectamine RNAiMAX according to the manufacturer’s protocol. Synthetic siRNA oligo nucleotides were obtained commercially from Shanghai Genepharma Co, Ltd. siATG5: 5ʹ-GGTTTGGACGAATTCCAACTTGTTT-3ʹ; siATG7: 5ʹ-GCTTTGGGATTTGACACATTT-3ʹ.

MARC-145, HEK-293T, HeLa, and CRL-2843-CD163 cell lines were stored in our laboratory (71 (link)). MARC-145 and HEK-293T cells were maintained in DMEM (Solarbio, Cat. No. 12100) supplemented with 10% heat-inactivated FBS (Gibco, Cat. No. 10270–106), and 100 units mL⁻¹ penicillin and 100 mg mL⁻¹ streptomycin sulfate (Sangon, Cat. No. B540732) at 37°C in 5% CO₂. HeLa cells were routinely maintained in modified Eagle medium (iCell Bioscience Inc., Cat. No. 138–0012) supplemented with 10% heat-inactivated FBS and 100 units mL⁻¹ penicillin and 100 mg mL⁻¹ streptomycin sulfate at 37°C in 5% CO₂. CRL-2843-CD163 cells were maintained in Roswell Park Memorial Institute 1640 medium (Solarbio, Cat. No. 31800) supplemented with 10% FBS and 100 units mL⁻¹ penicillin and 100 mg mL⁻¹ streptomycin sulfate at 37°C in 5% CO₂.
HP-PRRSV strain HN07-1 (GenBank: KX766378.1) and NADC30-like PRRSV strain HNhx (GenBank: KX766379) were previously isolated by our laboratory (72 (link), 73 (link)). Low-pathogenic PRRSV strain BJ-4 (GenBank: AF331831) was kindly provided by Professor Hanchun Yang of China Agricultural University. The HP-PRRSV strain HN07-1 was utilized in the current study unless otherwise stated.