Pcdna3.1 c k dyk ace2

The expression plasmids used in this study were constructed using standard protocols, as described in our previous studies [42 (link)]. The PCR primer sequences used for cloning are available upon request. Renilla luciferase expression plasmid pLAS3w-Rluc was cloned by using PCR to amplify the RLuc sequence from pRL-TK (Promega, Madison, WI, USA) into pLAS3w-pPuro vector (RNAi core, Taipei City, Taiwan). SARS-CoV-2 spike-protein-expressing plasmid was constructed using the chemically synthesized codon-optimized sequence for expression in human cells [43 (link)], while the SARS-CoV S gene was derived from pcDNA3.1-S [42 (link)]. Human ACE2-expressing plasmid was constructed by cloning the ACE2 sequence from pcDNA3.1+/C-(K)DYK-ACE2 (GenScript) into pcDNA3.1-V5-HisA.

Huh7.5 cells were plated in a 24-well plate, 18 to 24 h prior to transfection. Fresh culture medium was added to each well 1 h before transfection. Thereafter, the cells were transfected with 0.5 μg pcDNA3.1+/C-(K)DYK-ACE2 (GenScript) diluted in 25 μL serum-free DMEM, or mock transfected, and PolyJet reagent (SignaGen Laboratories), according to the manufacturer’s instructions. Medium was replaced after 6 h and 48 h posttransfection, both transfected and non-transfected cells were infected with HCV at an MOI of 0.1. Cells were harvested 72 h postinfection for RNA isolation.
For knockdown experiments, Huh7.5 cells were seeded in a 24-well plate and incubated until they reached 80% confluence, which was generally 24 h after seeding. Cells were then transfected with ON-TARGETplus Human ACE2 siRNA (Dharmacon) or Non-targeting Pool (Dharmacon) using RNAiMAX (Invitrogen), incubated for 24 h and then infected with HCV at an MOI of 0.1. Cells were harvested 72 h postinfection for RNA isolation.

Huh7.5 cells were plated in a 24-well plate, 18 to 24 h prior to transfection. Fresh culture medium was added to each well 1 h before transfection. Thereafter, the cells were transfected with 0.5 μg pcDNA3.1+/C-(K)DYK-ACE2 (GenScript) diluted in 25 μL serum-free DMEM, or mock transfected, and PolyJet reagent (SignaGen Laboratories), according to the manufacturer’s instructions. Medium was replaced after 6 h and 48 h posttransfection, both transfected and non-transfected cells were infected with HCV at an MOI of 0.1. Cells were harvested 72 h postinfection for RNA isolation.
For knockdown experiments, Huh7.5 cells were seeded in a 24-well plate and incubated until they reached 80% confluence, which was generally 24 h after seeding. Cells were then transfected with ON-TARGETplus Human ACE2 siRNA (Dharmacon) or Non-targeting Pool (Dharmacon) using RNAiMAX (Invitrogen), incubated for 24 h and then infected with HCV at an MOI of 0.1. Cells were harvested 72 h postinfection for RNA isolation.