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. Author manuscript; available in PMC: 2020 Jun 1.
Published in final edited form as: Stem Cells. 2019 Mar 14;37(6):766–778. doi: 10.1002/stem.2991

Figure 5. In vitro validation of a dual-inducible model to knockout and rescue Tak1 signaling using COSIEN.

Figure 5.

(A) Schematic for Dual-Inducible Model within the Tak1fx-frt/fx-frt mouse: Mutations are introduced into the Lox and Frt sites to allow only one-time inversion and reversion of the targeted gene segment. Initial inversion (loss of gene function) is driven by a Cre/Lox system following addition of Ad.Cre. Rescue reversion (return of gene function) is driven by a Flp/Frt system following addition of Ad.Flp; (B) Normalized quantification of Tak1 gene expression from Ad.LacZ, Ad.Cre, and Ad.Cre+Ad.Flp treated mesenchymal cells (Ad.LacZ: 1.0; Ad.Cre: 0.41; Ad.Cre+Ad.Flp: 0.55). (C) Representative immunoblot of Ad.LacZ, Ad.Cre, and Ad.Cre+Ad.Flp treated mesenchymal cells for pTAK1, pSMAD 1/5, pp38, pSMAD 2/3 and α-tubulin; (D) Normalized quantification of pTAK1 protein expression from Ad.LacZ, Ad.Cre, and Ad.Cre+Ad.Flp treated mesenchymal cells (Ad.LacZ: 1.0; Ad.Cre: 0.66; Ad.Cre+Ad.Flp: 1.2); (E) Normalized quantification of pp38 protein expression from Ad.LacZ, Ad.Cre, and Ad.Cre+Ad.Flp treated mesenchymal cells (Ad.LacZ: 1.0; Ad.Cre: 0.65; Ad.Cre+Ad.Flp: 0.88); (F) Normalized quantification of pSMAD1/5 protein expression from Ad.LacZ, Ad.Cre, and Ad.Cre+Ad.Flp treated mesenchymal cells (Ad.LacZ: 1.0; Ad.Cre: 0.87; Ad.Cre+Ad.Flp: 2.25); (G) Normalized quantification of pSMAD 2/3 protein expression from Ad.LacZ, Ad.Cre, and Ad.Cre+Ad.Flp treated mesenchymal cells (Ad.LacZ: 1.0; Ad.Cre: 0.02; Ad.Cre+Ad.Flp: 4.81); All cells were treated with Ad.Cre (or Ad.LacZ) for 24 hours under serum deprivation conditions followed by 48 hours in serum replete and subsequently treated with Ad.LacZ (Ad.LacZ group), Ad.Cre (Ad.Cre group), or Ad.Flp (Ad.Cre+Ad.Flp) for 24 hours in serum deprived conditions followed by culture for an additional two days in serum replete conditions. Mesenchymal cells described are adipose-derived stem cells (ASCs). * = p<0.05.