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. 2021 Sep 10;9:690397. doi: 10.3389/fcell.2021.690397

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Copyright © 2021 Bertolio, La Colla, Carrea, Romo, Canziani, Echarte, Campisano, Barletta, Monzon, Rodríguez, Chisari and Dewey.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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TβRII-SE/Fc overexpressed in human cells is secreted to the extracellular milieu as a disulfide-linked homodimer and inhibits Smad2/3 activation. (A) Either coTβRII-SE/Fc or TβRII-EC was ligated to the human IgG1 Fc coding sequence and cloned into a self-inactivating (SIN) bicistronic lentiviral vectors making Lv.TβRII-SE/Fc and Lv.TβRII-Fc, respectively. The proviral lentiviral vectors contain long terminal repeat sequences (LTRs) on both ends, devoid of LTR promoter/enhancer sequences (ΔU3) after reverse transcription. The cytomegalovirus (CMV) promoter was used as internal promoter to drive the expression of either TβRII-SE/Fc or TβRII-Fc. Furthermore, the vectors include the safety-improved woodchuck hepatitis virus post-transcriptional regulatory element (wPRE), a splice donor (SD), a splice acceptor (SA) and the packaging signal (Ψ), together with the Rev responsive element (RRE), and the central polypurine tract (PPT). (B) SDS-PAGE and Coomassie blue staining of Protein A/G purified supernatants of TβRII-SE/Fc-overexpressing 293T cells under reducing (R) and non-reducing (NR) conditions. (C) Dimeric 3D model of TβRII-SE/Fc using the structures 2PJY (TβRII) and 1L6X (Fc), as templates (middle panel). TβRII-SE is shown magnified and rotated 90° (left panel) with atomic representation wrapped in its solvent-accessible surface area. Amino acids with solvent-accessible surface (highlighted in gray) are part of the protein-to-protein interface. Also, TβRII-SE/Fc 3D model is shown rotated 90° (right panel). (D) Representative immunoblotting (top panel) and semi-quantitative densitometry of three independent experiments (bottom panel) of P-Smad2/3 and total Smad2/3 in HCT116 cells overexpressing TβRII-SE/Fc (+) and control (–), in the presence (+) and absence (–) of TGF-β1 stimulation. *p < 0.05.