Cc 3170

BEAS-2B cells (ATCC CRL-9609) were cultured from cryopreserved stocks in collagen-coated T-75 culture flasks according to ATCC guidelines. Briefly, cells were seeded at 3,000 cells/cm² and cultured in 23 mL of bronchial epithelial cell growth medium (BEGM) (Lonza, CC-3170), omitting the addition of the gentamicin–amphotericin aliquot to the medium, as recommended by ATCC. Cells were grown at 37°C in a humidified incubator with a 5% CO₂ atmosphere, and complete media exchanges were performed every 48 h. After approximately 4 days, the cultures reached ∼70% confluency, and cells were subcultured into 6-well or 96-well plates coated with type 1 collagen (Advanced BioMatrix, Cat#5005) at 1,500 cells/cm², and allowed to attach to the growth surface for 24 h before exposure to PM.

A549 human lung adenocarcinoma epithelial cells (RCB0098, provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan) were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin at 37°C in a humidified 5% CO₂ atmosphere. NHBE normal human bronchial epithelial cells (CC-2540, Lonza, Basel, Switzerland) were cultured in bronchial epithelial cell growth medium (BEGM, CC-3170, Lonza) at 37°C in a humidified 5% CO₂ atmosphere.

Primary normal human bronchial epithelial (NHBE) cells (CC-2540, Lonza, Walkersville, MD) (3500cells/cm²) were maintained as previously described (19 (link)). Cells were placed in bronchial epithelial cell growth medium (BEGM, CC-3170, Lonza) without supplements for 24 h and then stimulated with 10μg/ml Dermatophagoides pteronyssinus 1 (Der p1, Arthropods of Medical Importance Resource Bank, Institute of Tropical Medicine, Yonsei University). In separated tests, NHBE were transfected with small interfering RNA (siRNA) duplexes designed against Nectin4 or nonspecific siRNA control (1027418, Qiagen, CA, USA). NHBE cells cultured in 6-well plates were transfected with 100 nM siRNA or negative control using Lipofectamine 2000 (11668019, Invitrogen, CA, USA). After 24hr, cells were treated with 10 μg/ml Der p1 and harvested for western blotting. Trans-epithelial electrical resistance (TEER) was measured using an Epithelial Volt/Ohm Meter (EVOM) (EVOM2, World Precision Instruments, FL). TEER values are expressed by raw oh m values minus the oh m value of a blank insert, multiplied by the area of the insert (1.12 cm²).

293T, HeLa, HepG-2, Huh-7, Calu-3, and BEAS-2B cells were cultured in DMEM (Gibco, C11995500BT) with 10% fetal bovine serum (FBS) (ExCell Bio, FSP500) and 1% penicillin/streptomycin (P/S) (Gibco, 15140-122) in a 37 °C incubator containing 5% CO2. THLE-2 cells were exposed to odium oleate (OA) dissolved in 0.5% fatty acid-free bovine serum albumin (BSA) at a final concentration of 0.3 mmol/L for 24 h to establish an in vitro non-alcoholic fatty liver disease model. THLE-2 and primary hepatocytes were cultured in BEGM (Lonza, CC-3170) in 5 ng/mL EGF, 70 ng/mL Phosphoethanolamine, and 10% FBS in a 37 °C incubator containing 5% CO2. The SARS-CoV-2 strain (GenBank accession number: 622319) was isolated from a laboratory-confirmed COVID-19 patient. All experiments involving live SARS-CoV-2 were performed in a Biosafety Level 3 (BLS-3) laboratory of the Navy Medical University.

The human lung epithelial cell line Calu-3 (UC Berkeley Cell Culture Facility) was maintained in RPMI media supplemented with GlutaMAX (Thermo Fisher Scientific), 10% fetal bovine serum (FBS, HyClone) and 100 μg ml⁻¹ penicillin and streptomycin (Gibco; R10 medium). Cells were grown in T225 flasks (Thermo Fisher Scientific) and regularly passaged at 80–90% confluence with 10× concentrated TrypLE Express (Thermo Fisher Scientific). After lentiviral transductions with Cas9 and dCas9–VP64 constructs, Calu-3 cells were cultured in R10 media additionally supplemented with 16 ng ml⁻¹ of hepatocyte growth factor (HGF, Stem Cell Technologies) to preserve viability and support robust growth^{67 (link)}. NHBE were purchased from Lonza (CC-2541) and cultivated in bronchial epithelial cell growth medium (CC-3170) as per the manufacturer’s instructions.

Human normal bronchial epithelial cell line BEAS-2B and LC cell lines Calu3, A549 and HCC827 were bought from American Type Culture Collection (ATCC; ATCC CRL-9609, ATCC HTB-55, ATCC CCL-185, ATCC CRL-2868, Rockefeller, MD, USA). A separate LC cell line, PC-9, was obtained from Shanghai Yaji Biological Technology Co., Ltd. (YS319C, China, http://www.yajimall.com/). BEAS-2B was incubated in bronchial epithelial growth medium (BEGM; CC-3170, Lonza/Clonetics Corporation, USA) in which we removed the Gentamycin-Amphotericin B mix (GA-1000). Calu3 cells were cultured in Eaglés Minimum Essential Medium (EMEM; ATCC 30-2003, ATCC, USA) while A549 cells were incubated in Kaighn's Modification of Ham's F-12 (F-12K) Medium (ATCC 30-2004). PC-9 and HCC827 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (ATCC 30-2001, ATCC). All media except BEGM were supplemented with 10% fetal bovine serum (FBS; ATCC 30-2020, USA) and 1% Penicillin-Streptomycin Solution (ATCC 30-2300, ATCC). All cells were incubated in a humidified atmosphere at 37°C with 5% CO₂.

For BEAS-2B infection experiments, bacteria were inoculated into fresh BEGM (Lonza, CC-3170) medium according to desired multiplicity of infection (MOI). Alternatively, cells were treated with the putative TLR2 ligand lipoteichoic acid from Staphylococcus aureus (LTA, 1 µg/ml, Invivogen, tlrl-pslta), the pore-forming cytolysin pneumolysin (Ply), Ruxolitinib (10 µM, SelleckChem, S1378), FK-866 (1 µM, R&D Systems, 4808) or SBI-797812 (1 µM, Merck, SML2791) whereas control cells were treated with the respective dissolvent.