Bhk 21 wi 2 cells

Vesicular Stomatitis Virus (VSV) pseudotyped with spike (S) proteins of MERS and SARS-CoV-2 were generated according to a published protocol^{45 (link)}. Briefly, BHK-21/WI-2 cells (Kerafast, MA) transfected to express the S proteins were inoculated with VSV-G pseudotyped ΔG-luciferase VSV (Kerafast, MA). After a 2-hour incubation at 37 °C, the inoculum was removed and DMEM supplemented with 5 % FBS, 50 U/mL penicillin, and 50 μg/mL streptomycin were added back to cells. Pseudotyped particles were collected 24 h post-inoculation, then centrifuged at 1,320×g to remove cell debris and stored at −80 °C until use. To determine the effect of the selected compounds on viral entry, Vero E6 cells were treated with each compound at a concentration of 2.5 μM for 1 h prior to inoculation with respective pseudotyped VSV. After 2 hours inoculation in the presence of the compounds, the inoculum was removed, and fresh medium was added to cells for further culture. The activity of firefly luciferase as a readout of infected cells was measured using the bright-Glo™ luciferase assay (Promega) for quantitative determination at 16 hours post-transduction.

HEK293T human fetal kidney and HeLa human cervix epithelial cells were obtained from ATCC (cat. nos. CRL-3216 and CCL-2). Both cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with fetal bovine serum (FBS) and antibiotic 1% penicillin/streptomycin. BHK-21-/WI-2 cells (Kerafast, EH1011, Boston, MA) were grown in DMEM supplemented with 5% FBS and 1% penicillin/streptomycin. All cells were incubated in a 37°C incubator under 5% CO₂ atmosphere.
For most experiments, 96-well plates were used. For mRNA stability, a 24-well plate was used. Cells resuspended (in DMEM plus 10% FBS) were seeded (3–4 × 10⁴ cells/well for a 96-well plate or 1–2 × 10⁵ cells/well for a 24-well plate) the night before the transfections. For transfections, Transporter 5 transfection reagent (TP5) (Polysciences, Warrington, PA, cat. no. 26008) was used at 1 to 4 ratios of DNA/reagent. Typically, 50–100 ng plasmid DNA per well for 96-well plates was mixed with 0.2–0.4 μL TP5 in 0.9% NaCl solution and incubated at room temperature for 20 min. The transfection reagent and DNA solution were mixed again and added to each well dropwise. The transfections were incubated at 37°C in 5% CO₂ overnight (16–18 h), and the medium was replaced with DMEM plus 10% FBS.