X tremegene 360 transfection reagent

Details of the cells used in this study are shown in the table below.
HEK 293T and COS-7 cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin. iHTMCs were cultured in DMEM/F-12 medium supplemented with 15% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. All cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Cells were transfected with plasmids containing wild-type (WT) or mutant MYOC cDNA by transfection reagents (Lipofectamine 2000, Invitrogen, CA, United States, Cat# 11668-019 or X-tremeGene™ 360 Transfection Reagent, Roche, Mannheim, Germany, Cat# 8724105001) following the manufacturer’s instructions. To ensure the stability and consistency of the transfection efficiency of various plasmids, cells were simultaneously transfected with a cDNA construct encoding GFP and the transfection rate was measured via calculating the ratio of GFP-positive cells under a fluorescence microscope.

Calu-3 cells (100 μL of 1.5 × 10⁵ cells/mL) were mixed with 100 ng of the pCMV-SARS-CoV-2 subreplicon RNA expression vector and 0.3 μL of X-tremeGene 360 transfection reagent (8724121001, Roche, Basel, Switzerland) and plated on a 96-well white cell culture plate (655083, Greiner Bio-One, Kremsmunster, Austria) and a 96-well clear cell culture plate (92096, TPP, Trasadingen, Switzerland). After 1 day of transfection, the cells were treated with 1–10,000 IU/mL IFN-α and 0.001–10 µM CDM-3008 for 3 days.