Penicillin streptomycin

Chemically pure astragaloside total (AST) and astragaloside IV (ASIV) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) and their purity is greater than 98%. Stock solutions of AST and ASIV were made in DMSO. The final concentration of DMSO added to cells was never greater than 0.1%. Recombinant mouse TNFα was obtained from Calbiochem (San Diego, CA, USA). Cycloheximide (CHX) was purchased from Sigma (St. Louis, MO, USA). TAPI-0 (TNF-α processing inhibitor-0), a synthetic inhibitor of TNFα converting enzyme (TACE), was purchased from Enzo Life Sciences Inc (Farmingdale, NY, USA). RPMI 1640, glutamine, penicillin/streptomycin, and HEPES were purchased from Clontech Laboratories (Palo Alto, CA, USA). Fetal calf serum (FCS) was purchased from Hyclone Laboratories (Logen, UT, USA). Total RNA isolation kits, reverse transcription (RT) kits and SYBR Green PCR Master MIX were all purchased from Takara Bio Inc. (Dalian, China). Cell Surface Protein Isolation Kits were purchased from Pierce (Rockford, IL, USA).

Primary normal human dermal fibroblasts (NHDFs) (Gibco) were cultured in an NHDF medium composed of high glucose DMEM (Life Technologies), 20% FBS (Atlantic bioscience), 2 mM glutamax (Life Technologies), and 1% penicillin/streptomycin (Life Technologies). To confirm the robustness of the transdifferentiation method, NHDFs from two donors were used (lot numbers: 1998537 and 1165554). All NHDFs were subcultured upon 100% confluency with a 1:5 splitting ratio. For primary NHDFs, P3 to P9 were used. The Lenti-X HEK293T cell line (Takara Bio USA) was maintained in a medium composed of high glucose DMEM, 10% FBS, 2 mM glutamax, and 1% penicillin/streptomycin.

psPAX2 (Addgene, no.12260) was a gift from Didier Trono. pCDNA3.3_CoV2_B.1.1.7 (Addgene, no.170451) for Alpha-S and pcDNA3.3-SARS2-B.1.617.2 (Addgene, no.172320) for Delta-S proteins, were gifts from David Nemazee^{36 (link)}. pTwist-SARS-CoV-2 Δ18 B.1.351v1 (Addgene, no.169462) for Beta-S protein was a gift from Alejandro Balazs^{37 (link)}. Lentiviral vector, pWPI-ffLuc-P2A-EGFP for luciferase reporter assay and pTRC2puro-ACE2-P2A-TMPRSS2 for the generation of 293T cell line susceptible to SARS-CoV-2 infection was created from pWPI-IRES-Puro-Ak-ACE2-TMPRSS2, a gift from Sonja Best (Addgene, no.154987) by In-Fusion® technology (Takara Bio). pcDNA3.4 expression plasmids encoding SARS-CoV-2 S proteins with human codon optimization and 19 a.a deletion of C-terminus (C-del19) from Wuhan, D614G, and Omicron were generated by assembly of PCR products, annealed oligonucleotides, or artificial synthetic gene fragments (Integrated DNA Technologies, IDT) using In-Fusion® technology. For Delta plus, Kappa and Lambda variants, S proteins with only RBD, D614, and P681 mutations were created from pcDNA3.4 encoding human codon-optimized Wuhan S protein (C-del19). LentiX-293T cells (Takara Bio) and 293T cells were maintained in culture with Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin-streptomycin (Nacalai tesque), and 25 mM HEPES (Nacalai tesque).

HCSMCs and HCECs were purchased from Clonetics. HCSMCs and HCECs were cultured and used from three to six passages in media supplemented with 5% fetal bovine serum (FBS), penicillin/streptomycin, and smooth muscle cell-growth or endothelial cell-growth supplement (Takara Co., Osaka, Japan) at 37 °C under 5% CO₂. HCSMCs and HCECs without cell-growth supplement were used in the experiments.