Hlmvec

Human lung microvascular endothelial cells (HLMVECs) (Cell Applications Inc., CA, USA) were cultured in EBM-2 (containing endothelial growth factor, CC3162, Lonza, MD, USA) supplemented with 5% FBS (Gibco, CA, USA) at 37°C, 5% CO₂, and 95% humidity and passaged every 3–5 days. P4–P7 cells were utilized in the next stage of the experiment. Lipopolysaccharide (LPS) (Sigma-Aldrich, MO, USA; 10 μg/mL) treatment for 1 day was applied on HLMVECs for the in vitro model. Then, 50 μM HYP or 3 mM 3-MA was used to investigate the regulatory role of HYP on autophagy. For Atg13 silencing (si-Atg13 : 5′-AAGUCCCUUCUUGCUAUAACUAGTTCUAGUUAUAGCAAGAAGGGACUUTT-3′), siRNA against Atg13 was used to transfect into HLMVECs using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). After transfection for 48 hr, HLMVECs cells were harvested for use in subsequent experiments.

Primary cultures of human lung microvascular endothelial cells (HLMVECs) were purchased from Cell Applications (San Diego, CA). Cells were cultured in VascuLife medium (Frederick, MD) supplemented VascuLife VEGF-Mv LifeFactors Kit (Frederick, Maryland) and maintained at 37°C in a humidifier with 5% CO₂.

THP-1 cells (TIB-202, American Type Culture Collection [ATCC], Manassas, VA, USA) were cultured in 1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Gibco) at 37 °C, 5% CO₂, and 95% humidity (Thermo Fisher, Austin, TX, USA). The medium was replaced with fresh medium every 3–4 days. The cells were collected for passage with a pipette without trypsinization due to their suspension properties. THP-1 cells were subsequently treated with 100 ng/mL phorbol 12-myristate 13-acetate to induce cell adhesion to the surface and macrophage differentiation for further experiments.
The human bronchial epithelium cell line (BEAS-2B cells, # CRL-9609; ATCC) was cultured in Dulbecco’s Modified Eagle Medium/F12 (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin–streptomycin (Gibco) at 37 °C, 5% CO₂, and 95% humidity.
Human lung microvascular endothelial cells (HLMVECs, #540–05a; Cell Applications Inc., San Diego, CA, USA) were cultured in EBM-2 (containing endothelial growth factor, CC3162, Lonza, Walkersville, MD, USA) supplemented with 5% FBS (Gibco) at 37 °C, 5% CO₂, and 95% humidity and passaged every 3–5 days. P4–P7 cells were used in the next phase of the experiment.

HLMVECs (Cell Applications Inc., San Diego, CA, USA, or Promo Cell, Heidelberg, Germany) were cultured in HMVEC Growth Medium (Table 1). All experiments were performed with cells passaged four to six times. Primary HLMVECs were seeded at a density of 40,000 cells/cm² on plates coated with collagen as specified by the manufacturer. All experiments were performed 12–16 h after seeding. ANDV (strain Chile-9717869), HTNV (strain 76–118), and PHV were a kind gift of Connie Schmaljohn at the United States Medical Research Institute of Infectious Diseases. Viruses were amplified in Vero E6 cells (ATCC CRL-1586) in Earls’ Modified Essential Medium (EMEM, Corning, Corning, NY, USA) with 10% FBS (Gibco, Waltham, MA, USA), 5 mM L-glutamine (Corning), and 1% penicillin–streptomycin (Gibco). Virus titers were determined by plaque assay [48 ]. Cells and viruses were checked for Mycoplasma spp. using LookOut Mycoplasma PCR Detection Kit (Sigma-Aldrich, St. Louis, MO, USA). All other reagents used were purchased from Thermo Fisher unless specified. All work with viruses was conducted at the UTHSC Regional Biocontainment Laboratory (RBL) in BSL-3.