Dynamic Visualization of TGF-β/SMAD3 Transcriptional Responses in Single Living Cells.
Dieuwke Louise MarvinLi YouLaura BornesMaarten van DintherNiek PetersHao DangSarah K HakunoMarten HornsveldOnno W KranenburgJacco van RheenenJos H T RohlingMiao-Ping ChienPeter Ten DijkeLaila RitsmaPublished in: Cancers (2022)
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.
Keyphrases
- transforming growth factor
- epithelial mesenchymal transition
- living cells
- single cell
- crispr cas
- gene expression
- transcription factor
- high resolution
- binding protein
- poor prognosis
- fluorescent probe
- induced apoptosis
- quantum dots
- oxidative stress
- mass spectrometry
- small molecule
- risk assessment
- protein protein
- cell death
- dna binding
- heat stress
- fluorescence imaging
- label free