Plvx tre3g

Half a million HEK293T cells were seeded per well in a 6-well plate (one plate per lentivirus) in 2 ml of High glucose DMEM (Life Technologies, 11995) with 10% FBS (Sigma, F2442). The next evening the medium was replaced with fresh DMEM and cells were transfected with 100 μl of transfection mixture per well. The transfection mixture contained 3 μl X-treme Gene 9 reagent (Roche, 06365787001), 500 ng psPAX2 (psPAX2 was a gift from Didier Trono, Addgene plasmid # 12260), 50 ng pMD2.G (pMD2.G was a gift from Didier Trono, Addgene plasmid # 12259), 500 ng of pLVX-Tet3G (Clontech) or pLVX-TRE3G-Luciferase (Clontech) or pLVX-TRE3G-LbNOX or pLVX-TRE3G-mitoLbNOX or pLVX-TRE3G-TPNOX or pLVX-TRE3G-mitoTPNOX and Opti-MEM medium (Life Technologies, 31985-070) up to 100 μl. To make the transfection mixture, 50 μl solutions of X-treme Gene 9 and DNA mixtures were prepared separately and the DNA solution was added dropwise to the X-treme Gene 9 solution. The mixture was incubated at room temperature for 30 min before adding it to cells. Two days after transfection, medium was collected, centrifuged at 500 x g for 5 min to pellet cells and the supernatant was aliquoted and stored at −80 °C.

A Tet-On 3G inducible expression system for mitochondrial-localized GFP (mitoGFP) and NDI1 was established in NCI-HCC and Nthy-ori 3–1 cells. The NDI1 protein contained an N-terminal FLAG tag after the mitochondrial targeting sequence (39 (link)). Both cell lines were initially infected with the pLVX-Tet3G (Clontech; cat. #631193) regulator vector and then underwent selection with Geneticin (1 mg/mL). Surviving cells were then infected with lentivirus for either mitoGFP or NDI1 expressed in the pLVX-TRE3G (Clontech; cat. #631359) response vector and selected with puromycin (2 μg/mL).

Plasmids used in this study were generated using standard polymerase chain reaction (PCR) techniques and/or type II and IIs restriction enzyme cloning. Restriction enzymes, Phusion DNA polymerase, T4 DNA Ligase, and Antarctic phosphatase were purchased from NEB. psPAX2 and pMD2.G plasmids were gifted by William Miller from Northwestern University and DsRed-Express2 was purchased from (Clontech-Takara). WT-ACE2 and Mut-ACE2 gene fragments were codon optimized and synthesized by Thermo Fisher and cloned into a pGIPZ backbone (Open Biosciences). The Spike protein from pcDNA3.1-SARS2-Spike was a gift from Fang Li (Addgene plasmid # 145032; http://n2t.net/addgene:145032; RRID:Addgene_145032);^{[45 (link)]} this gene was cloned into a modified pcDNA 3.1 backbone (Clontech-Takara) with a beta-globin intron in the 5’ untranslated region for pseudotyping lentivirus. The Tet3G transactivator (pLVX-EF1a-TET3G) and cognate TRE3GV promoter (pLVX-TRE3G) (Takara) were cloned into modified pGIPZ and pLVX (Takara) backbones, respectively. In this context, the Spike protein (Addgene plasmid # 145032) was cloned downstream of the TRE3G promoter. Cloned plasmids used in this study were sequence-verified, and maps are available in Supplementary Data 1. Chemically competent TOP10 Escherichia coli were used for transformation of all plasmids and subsequently grown at 37°C.