Atcc ccl 171

Green Fluorescent Protein-expressing Human Umbilical Vein Endothelial Cells (GFP-HUVEC, cAP-0001GFP) were purchased from Angio-Proteomie, USA, and expanded in Complete Endothelial Cell Growth Medium (EGM-2, Lonza Group AP, Switzerland) in pre-coated culture flasks (Quick Coating Solution, Angio-Proteomie, USA). Human pericytes from placenta (hPC-PL, PromoCell GmbH, Germany) were expanded in complete Pericyte Growth Medium 2 (PromoCell GmbH, Germany). Human normal lung fibroblasts (MRC-5, ATCC® CCL-171™) were cultured in Minimum Essential Medium, alpha modification (α-MEM, Gibco) with 2 ​mM l-Glutamine, supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco) and 1% (v/v) penicillin/streptomycin (P/S). The primary human epithelioid Malignant Pleural Mesothelioma (MPM) cells, obtained after diagnostic thoracoscopies, were kindly provided by the Biologic Bank of Malignant Mesothelioma, S. Antonio e Biagio Hospital (Alessandria, Italy; approval by the local ethical committee: #9/2011) [34 (link)]. MPM cells were expanded in Ham's F-12 ​cell growth medium supplemented with 10% (v/v) FBS and 1% (v/v) P/S. For all cell types, cell culture media were refreshed every 48 ​h, cells were passaged by trypsinization at 90% confluency. Cell culture was maintained at 37 ​°C in a humidified incubator at 5% CO₂.

MRC-5 (lung fibroblast) cells were obtained from ATCC (ATCC CCL-171™). CoronaGrow LLC-MK2 (kidney epithelial) cells, a subclonal line of parental LLC-MK2 cells were obtained from VectorBuilder Inc. (Chicago, IL, Cat. No. CL0004). HEK293T cells expressing the full-length SARS-CoV-2 spike (BEI NR52310) were obtained from BEI Resources (Manassas, VA). HEK-293 T cells expressing the SARS-CoV-2 spike were kindly provided by S. Pöhlmann (Leibniz Institute for Primate Research, Göttingen, Germany). HeLa-ACE2 cells were kindly provided by D.R. Burton (The Scripps Research Institute).

The lung cell lines MRC-5, normal lung fibroblast (ATCC CCL171) and A549, lung adenocarcinoma (ATCC CCL185) were obtained from the American Type Culture Collection (ATCC). MRC-5 and A549 cells were grown in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies, Inc, Rockville, MD) supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, MO) and 1% penicillin and streptomycin (GIBCO). Cells were cultured in 25 cm² cell culture flasks (Corning, USA) and were kept in a CO₂ incubator at 37°C in a humidified atmosphere with 5% CO₂ in air. For experimental purposes, cells cultured to an exponential growth phase (at ~70% confluency) were used. Cells were then serum-starved for 24 h to synchronize the cell cycle. The following day, the cells were pharmacologically treated with either EFV at concentrations of 4, 13, 26, or 50 μM, respectively; or with LPV/r at concentrations of 10, 32, 50, or 80 μM, respectively. Treatment was carried out for 24–72 h. Control cells were exposed to growth medium and vehicle only (methanol 0.1% v/v).

Human lung fibroblast MRC-5 (ATCC® CCL-171™) and 3T3-Swiss albino (ATCC® CCL-92™) cells were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA), and the cells were cultured in Dulbecco’s modified Eagle medium and RPMI 1640 media containing 10% fetal bovine serum. The growth media contained 100 units/mL penicillin and 50 µg/mL streptomycin, respectively. The cells were maintained at 37 °C in a humidified atmosphere in the presence of 5% CO₂.