Plasmid pcDNA3-TEV-FlipGFP-T2A-mCherry was ordered from Addgene (catalog No.124429). pcDNA3 FlipGFP-Mpro plasmid and pcDNA3 FlipGFP-PLpro plasmid were constructed by introducing SARS-CoV-2 Mpro cleavage site AVLQSGFR and SARS-CoV-2 PLpro cleavage site LRGGAPTK, respectively, via overlapping PCRs. pLVX SARS-CoV-2 Mpro and pcDNA3.1 SARS-CoV-2 PLpro plasmids was ordered from Genescript (Piscataway NJ) with codon optimization.
The Flip-GFP Mpro and PLpro assays were performed as previous reported [15 (link), 23 (link), 24 (link), 37 (link)]. Briefly, the assay started with seeding 293T-ACE2 in 96-well, black, clear bottomed plate (Greiner, catalog No. 655090) and incubating overnight to allow cells to reach 70–80% confluency. 50 ng of pLVX SARS-CoV-2 Mpro (or pcDNA3.1 SARS-CoV-2 PLpro) and 50 ng of pcDNA3 FlipGFP- Mpro (or pcDNA3 FlipGFP- PLpro) reporter plasmid was mixed with transfection reagent TransIT-293 (Mirus, catalog No. MIR 2700). The mixture was then transfected to each well according to manufacturer’s instructions. After 2.5–3 hours of incubation in 37 °C, 1 μL of testing compound was added into each well directly and mixed by gentle plate shaking. 48 h post transfection, fluorescence was quantified using SpectraMax iD3 plate reader (Molecular Devices) and images were taken using BZ-X800E fluorescence microscope (Keyence) in GFP and mCherry channels at 4X objective lens.
The Flip-GFP Mpro and PLpro assays were performed as previous reported [15 (link), 23 (link), 24 (link), 37 (link)]. Briefly, the assay started with seeding 293T-ACE2 in 96-well, black, clear bottomed plate (Greiner, catalog No. 655090) and incubating overnight to allow cells to reach 70–80% confluency. 50 ng of pLVX SARS-CoV-2 Mpro (or pcDNA3.1 SARS-CoV-2 PLpro) and 50 ng of pcDNA3 FlipGFP- Mpro (or pcDNA3 FlipGFP- PLpro) reporter plasmid was mixed with transfection reagent TransIT-293 (Mirus, catalog No. MIR 2700). The mixture was then transfected to each well according to manufacturer’s instructions. After 2.5–3 hours of incubation in 37 °C, 1 μL of testing compound was added into each well directly and mixed by gentle plate shaking. 48 h post transfection, fluorescence was quantified using SpectraMax iD3 plate reader (Molecular Devices) and images were taken using BZ-X800E fluorescence microscope (Keyence) in GFP and mCherry channels at 4X objective lens.
