Transient Transfection of HEK293-EcI Cells

HEK293-EcI cells were transfected 2 days after seeding at a density of 3 × 10⁵ cells/mL in 6-well plates in DMEM with 4.5 g/L glucose, L-glutamine, and sodium pyruvate with 10% HI FBS, and 1% penicillin-streptomycin. The culture medium was refreshed on the day of transfection. Transfection was performed using Lipofectamine 3000 following the manufacturer’s instructions. For all experiments, 1.5 μg/well of pERV3 receptor plasmid (Agilent Technologies, 217468) was transfected along with 750 ng/well of pEGSH-LUC luciferase reporter plasmid (Agilent Technologies, 217468), and 250 ng/well of pRL-CMV Renilla luciferase reporter plasmid (Promega, E2261) as a reference. After 2 days of incubation in the conditions described above, the cells were washed twice with PBS and then transferred to a clear 96-well plate in DMEM with 4.5 g/L glucose, sodium pyruvate; without L-glutamine, phenol red at 100 μL/well. Final concentrations ranging from 300 to 300 μM of 20E or CF were added immediately after, with a final volume of 125 μL/well. The treated cells were then incubated for 1 day in the same conditions. The Dual-Luciferase Reporter Assay was performed as described previously (Okamoto et al., 2018 (link)).