Dulbecco s modified eagle s medium

A375 human melanoma cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco′s modified Eagle′s medium containing 10% foetal bovine serum and 100 U/ml penicillin/streptomycin (all from Sangon Biotech, Shanghai, China) at 37 °C in a 5% CO₂ incubator.
HSDL2-overexpressing lentivirus and shRNA targeting HSDL2 lentivirus was obtained from General Biol (Anhui, China). The sequences were as follows: human HSDL2, shRNA: 5′-CCA GAA GCA GTT AGC AAG AAA-3′; scrambled shRNA, shCtrl: 5′-TTC TCC GAA CGT GTC ACG T-3′. Briefly, cells were seeded in 6-well plates and cultured at 37 °C and 5% CO2 for 24 h. The lentiviral particle suspension was added to the plate (multiplicity of infection = 5, 5 µl lentivirus per well). Following infection at 37 °C for 12 h, the culture medium was replaced. The cells were cultured for 72 h, and infection efficiency was determined by real-time reverse transcription quantitative polymerase chain reaction (RT–qPCR) and western blotting assays.

Dulbecco's modified Eagle's medium (DMEM), Roswell Park Memorial Institute (RPMI 1640) medium, fetal bovine serum (FBS), 4′,6‐diamidino‐2‐phenylindole (DAPI) stain solution, and 0.25% trypsin‐EDTA solution was purchased from Sangon Biotech. Penicillin–streptomycin and Phorbol 12‐myristate 13‐acetate (PMA) were purchased from Solarbio Science & Technology Co., Ltd. Ammonium fluoride and ethylene glycol (Sinopharm chemical reagent). The cell counting kit‐8 (CCK‐8) solution and Matrigel (BD Biosciences). Lactic acid assay Kit (Colorimetric method) and Glucose assay Kit (O‐toluidine method) were purchased from Lengton Biotechnology. Anti‐Glut1, anti‐HK2, anti‐CD86, anti‐CD206, donkey anti‐rabbit secondary antibodies, anti‐AMPK‐α, and AMPK inhibitors were purchased from Abcam. A medical‐grade Ti rod was purchased from Baoti.

Marc-145 cells maintained in Dulbecco's modified Eagle's medium (Sangon Biotech, Shanghai, China) supplemented with 10% fetal calf serum (FCS) (Tianhang Biotech, Huzhou, China) were used to propagate PRRSV and titrate its 50% tissue culture infectious dose (TCID₅₀) employing the Reed–Muench method. PAMs derived from 3 to 6-week-old PRRSV-negative piglets using lung lavage were cultivated in a Roswell Park Memorial Institute (RPMI)-1640 medium (Sangon Biotech) containing 10% FCS plus 100 μg/ml streptomycin and 100 U/ml penicillin (Sangon Biotech). These two cells were kept in a humidified atmosphere with 5% CO₂ at 37°C before use.