Dmem medium

Cells were differentiated and treated as described above. On the 4^th day of differentiation, mitochondrial oxidative phosphorylation from mouse differentiated myotubes was analyzed using extracellular flux analysis (XF24; Seahorse Biosciences) in DMEM medium (pH 7.4; Sigma-Aldrich). Unless otherwise stated, the Mitochondrial Stress Test assay was performed with 1 mM pyruvate and 25 mM glucose (Sigma-Aldrich). Baseline oxygen consumption rates (OCR) and extracellular acidification rate (ECAR) were measured every 7th minute. Following baseline measurements, oligomycin (1 μM), Carbonyl cyanide-4 (trifluoromethoxy)phenylhydrazone (FCCP) (1 μM), and antimycin A (2 μM) were sequentially injected to measure OCR and ECAR. The glycolytic flux was measured using the Glycolytic Stress Test assay performed in DMEM medium (pH 7.4; Sigma-Aldrich). Baseline ECAR was measured every 7th minute. Following baseline measurements, glucose (10 mM), oligomycin (1 μM), and 2-deoxyglucose (100 mM) were sequentially injected followed by ECAR measurements.

Male ANP32A^−/− or ANP32B^−/− mice and the wild type male litter mates thereof were briefly placed in isoflurane narcosis, final retrobulbary bleeding was performed and mice were euthanized by cervical dislocation. Lungs from two mice of each genotype were removed, washed once with PBS, cut into small parts and mixed together. The tissue was digested with collagenase D (in DMEM medium; Sigma-Aldrich/Merck) for 1 h at 37°C, followed by digestion with DNase I (in DMEM medium; Sigma-Aldrich/Merck) for 10 min at room temperature (RT). Then, the tissue was squeezed through a 45 μM mesh filter using DMEM medium, and the cell suspension was centrifuged at 300 × g for 10 min at 4°C. The pelleted cells were resuspended in LF growth medium and seeded into 24-well plates. Cells were incubated for 72 h at 37°C. After 72 h, cells were trypsinized for a few minutes to separate the fibroblasts from the epithelial cells, which are more resistant to trypsin. Cells were then passaged twice a week by differential trypsination until only fibroblast cells remained. These fibroblast cells were further maintained in culture until spontaneous immortalization occurred after ~25 passages, as evident by fast and contact-independent growth of the cells. Knockout of the respective ANP32 protein was verified by Western blotting every five to six passages.

Compounds 1a, 1b, and 2–14 as well as the positive control edaravone (Aladdin, Shanghai, China) were dissolved in DMSO (Sigma-Aldrich, Shanghai, China) as a stock, and the tested compounds was further diluted by DMEM medium (Gibco, Beijing, China) into three gradient concentrations (2.5, 5, and 10 μM). PC12 cells were digested and seeded into 96-well plates at a density of 5 ×10³ cells per well and cultured in DMEM medium with 5% CO₂ for 24 h. Then the cell culture medium was replaced by DMEM medium containing different concentrations of compounds for pretreatment for 2 h and then treated with 700 μM SNP (Sigma-Aldrich, Shanghai, China) for another 24 h. About 10 µL of MTT (Beyotime Institute of Biotechnology, Shanghai, China) (5 mg/mL) was added into each well and incubated at 37 °C for 3.5 h. Afterwards, the supernatant was removed and the crystals were dissolved in 100 µL DMSO. The optical absorbance at 570 nm was read with an EPOCH 2 microplate reader (BioTek Devices, San Mateo, CA, USA). The experiments were repeated three times.

BeWo cells were purchased from the Wuhan Cell Banker (Guangdong Peiyu) and cultured in Ham’s F-12K medium (Pythonbio) supplemented with 15% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco). HTR-8/SVneo cells were a gift from Prof. Xue-Song Yang (Jinan University) and cultured in DMEM medium (Sigma) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco). Human umbilical vein endothelial cells (HUVECs) were a gift from Prof. Xiao-Jun Yang (Shantou University Medical College) and cultured in DMEM medium (Sigma) supplemented with 15% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco). Transfection of HERV-W siRNA (GenePharma) was performed using Lipofectamine 2000, the sequences were 5′-GGC​GGU​AUC​ACA​ACC​UCU​ATT-3′ and 5′-UAG​AGG​UUG​UGA​UAC​CGC​CAA-3’. Forty-8 h after transfection, transfection efficiency was detected by using real-time quantitative PCR. All siRNA experiments were conducted in both BeWo and HTR-8/SVneo trophoblast cells, and data showed in this work was from BeWo cells.

The HEK293T cell line (ATCC) was cultured in DMEM medium (Sigma) containing 10% FBS (Invitrogen), 2 mM L-glutamine, 100 mg/ml penicillin and 100 mg/ml streptomycin. HEK Blue hTLR2 cell line (Invivogen), the HEK293 cell line expressing human TLR2, CD14 and NF-κB-SEAP (secreted embryonic alkaline phosphatase) reporter gene was cultured in DMEM medium (Sigma) containing 10% FBS (Invitrogen), 2 mM L-glutamine, 100 μg/ml Normocin (Invivogen) and 1X HEK-Blue Selection (Invitrogen).

IHH-4 cell lines were cultured under standard conditions at 37°C, 5% CO2 atmosphere, and saturated humidity in RPMI-1640 and DMEM medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum. Cells were maintained in logarithmic phase of growth. TPC-1 cell lines were cultured under standard conditions at 37°C, 5% CO2 atmosphere, and saturated humidity in DMEM medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum. Cells were maintained in logarithmic phase of growth. The IHH4 cell lines was obtained from the Health Science Research Resources Bank (Osaka, Japan). The TPC1 cell line was a gift from Professor Meiping Shen (Department of General Surgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu).

All lung tissues were homogenized in the DMEM medium and filtered with 0.2μm filter (Millipore, Billerica, MA). Total RNA was extracted from rodent lung tissues using the QIAamp Viral RNA Mini Kit. The hantaviruses were detected by the Hantavirus Type I and II Universal Nucleic Acid Kit using fluorescence PCR method. The PCR reaction was run under the following conditions: 1 cycle, 10 min at 45°C and 15 min at 95°C; 40 cycles, each cycle consisting of 15 s at 95°C and 1 min at 60°C. Samples with a CT value ≤ 38 were considered positive for hantavirus. The positive samples of hantavirus were sent to Beijing Macro & Micro-test Bio-Tech Co., Ltd. for sequencing by Next generation sequencing(NGS).