| Autor: |
Marvin DL; Oncode Institute and Department of Cell and Chemical Biology, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands., You L; Oncode Institute and Department of Molecular Genetics, Erasmus University Medical Center, Erasmus MC Cancer Institute, 3015 GD Rotterdam, The Netherlands., Bornes L; Oncode Institute and Department of Molecular Pathology, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands., van Dinther M; Oncode Institute and Department of Cell and Chemical Biology, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands., Peters N; UMC Utrecht Cancer Center, University Medical Center Utrecht, Utrecht University, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands., Dang H; Department of Gastroenterology and Hepatology, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands., Hakuno SK; Department of Gastroenterology and Hepatology, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands., Hornsveld M; Oncode Institute and Department of Cell and Chemical Biology, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands.; Department of Gastroenterology and Hepatology, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands., Kranenburg O; UMC Utrecht Cancer Center, University Medical Center Utrecht, Utrecht University, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands., van Rheenen J; Oncode Institute and Department of Molecular Pathology, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands., Rohling JHT; Department of Cell and Chemical Biology, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands., Chien MP; Oncode Institute and Department of Molecular Genetics, Erasmus University Medical Center, Erasmus MC Cancer Institute, 3015 GD Rotterdam, The Netherlands., Ten Dijke P; Oncode Institute and Department of Cell and Chemical Biology, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands., Ritsma L; Oncode Institute and Department of Cell and Chemical Biology, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands. |
| Abstrakt: |
Transforming growth factor-β (TGF-β) signaling is tightly controlled in duration and intensity during embryonic development and in the adult to maintain tissue homeostasis. To visualize the TGF-β/SMAD3 signaling kinetics, we developed a dynamic TGF-β/SMAD3 transcriptional fluorescent reporter using multimerized SMAD3/4 binding elements driving the expression of a quickly folded and highly unstable GFP protein. We demonstrate the specificity and sensitivity of this reporter and its wide application to monitor dynamic TGF-β/SMAD3 transcriptional responses in both 2D and 3D systems in vitro, as well as in vivo, using live-cell and intravital imaging. Using this reporter in B16F10 cells, we observed single cell heterogeneity in response to TGF-β challenge, which can be categorized into early, late, and non-responders. Because of its broad application potential, this reporter allows for new discoveries into how TGF-β/SMAD3-dependent transcriptional dynamics are affected during multistep and reversible biological processes. |
