Calu 3

Human bronchial EC lines BEAS-2B (CRL-9609) [10 (link)] and Calu-3 (HTB-55) [11 (link)] were purchased from ATCC (Manassas, VA, USA) and maintained in BEGM with BulletKit (#CC-3170; Lonza, Walkersville, MD, USA) and EMEM, respectively, in a humidified atmosphere containing 5% CO₂ at 37°C. S1P induced interleukin-8 release via S1PR2 and nuclear factor κB in BEAS-2B cells [12 (link)]. In addition, the production of interleukin-8 was also observed in Calu-3 [13 (link)]. Therefore, these bronchial ECs were treated with or without 1 μM S1P for 2 h.

All studies were performed based on the approved IRB protocols by the University of California (UCSD), San Diego. The human carcinoma cell lines Calu-3 and Caco-2 were purchased from ATCC and were cultured in MEM medium supplemented with 1×NEAA, 100 IU/mL penicillin–streptomycin, and 20% FBS. HEK293T (293T), A549 and Vero cells were maintained in our lab and were cultured in DMEM medium supplemented with 10% FBS. MT4 cell was cultured in RPMI medium 1640 with 10% FBS. 293T cells transduced with lentivirus encoding human ACE2 and TMPRSS2 was maintained in DMEM medium with 10% FBS and 2 ug/ml puromycin. Brefeldin A was purchased from BioLegend; kifunensine, swainsonine, decanoyl-RVKR-CMK, and NGI-1 were purchased from Cayman Chemical; Endo H and PNGase F were purchased from New England Biolabs; The DNA fragments encoding spike of SARS-CoV-2 strains USA-WA1/2020 (WT), B. 1. 351 (Beta) were inserted into pLVX-puro and C-terminal was tagged with Flag, no organelle localization signal was included in this vector . The spike plasmids of B.1.617.2 (Delta), and B.1.1.529 (Omicron) were obtained from InvivoGen. The plasmids of SARS-CoV (CUHK-W1) spike, MERS-CoV (HCoV-EMC/2012) spike, IAV H5N1 (Thailand/KAN-1/2004) HA and NA were purchased from Sino Biological. All the plasmids of viral glycoproteins above were codon-optimized.

All cells were maintained in humidified 37°C incubators with 5% CO₂ and 20% O₂. Vero E6 (ATCC), HEK293T cells (ATCC), HeLa Tet-Off cells (Clontech) and HeLa Flp-In TREx GFP-Dcp1a cells (a generous gift from Dr. Anne-Claude Gingras) were cultured in DMEM (Thermo Fisher) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine (Thermo Fisher) and 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher). Calu3 (ATCC) and MRC-5 cells (ATCC, a generous gift from Dr. David Proud) were cultured in EMEM (ATCC) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine and 10% FBS (Thermo Fisher). HUVECs (Lonza) were cultured in endothelial cell growth medium (EGM-2) (Lonza). HUVECs were seeded onto tissue culture plates or glass coverslips coated with 0.1% (w/v) porcine gelatin (Sigma) in 1x PBS. For sodium arsenite treatment, HUVECs were treated with 0.25 mM sodium arsenite (Sigma) or a vehicle control for 30 min.

AU565, HCC1419, NCI-H2170, HCC202, HCC1954, NCI-N87, ZR75-1, SKOV3, ZR75-30, MDAMB175VII, CALU3, MDAMB453, MDAMB361, JIMT1, SKBR3 and HCC2218 cells were obtained from ATCC. OE19 and OE33 were obtained from ECCC; COLO-678 was obtained from DSMZ, and KYSE-410 from Sigma-Aldrich. BT-474-M3 cells (hereafter simply referred to as BT-474) were obtained from Hermes biosciences. All cell lines were maintained in RPMI supplemented with 10% FBS, penicillin, and streptomycin. GSK-1120212 and MK-2206 were purchased from Selleckchem. Recombinant human HRG-β1 (EGF domain) was from R&D Systems.