Dynamic, large-scale profiling of transcription factor activity from live cells in 3D culture.
Extracellular activation of signal transduction pathways and their downstream target transcription factors (TFs) are critical regulators of cellular processes and tissue development. The intracellular signaling network is complex, and techniques that quantify the activities of numerous pathways and...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2010-11-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC2984444?pdf=render |
| _version_ | 1818542249739288576 |
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| author | Michael S Weiss Beatriz Peñalver Bernabé Abigail D Bellis Linda J Broadbelt Jacqueline S Jeruss Lonnie D Shea |
| author_facet | Michael S Weiss Beatriz Peñalver Bernabé Abigail D Bellis Linda J Broadbelt Jacqueline S Jeruss Lonnie D Shea |
| author_sort | Michael S Weiss |
| collection | DOAJ |
| description | Extracellular activation of signal transduction pathways and their downstream target transcription factors (TFs) are critical regulators of cellular processes and tissue development. The intracellular signaling network is complex, and techniques that quantify the activities of numerous pathways and connect their activities to the resulting phenotype would identify the signals and mechanisms regulating tissue development. The ability to investigate tissue development should capture the dynamic pathway activity and requires an environment that supports cellular organization into structures that mimic in vivo phenotypes. Taken together, our objective was to develop cellular arrays for dynamic, large-scale quantification of TF activity as cells organized into spherical structures within 3D culture.TF-specific and normalization reporter constructs were delivered in parallel to a cellular array containing a well-established breast cancer cell line cultured in Matrigel. Bioluminescence imaging provided a rapid, non-invasive, and sensitive method to quantify luciferase levels, and was applied repeatedly on each sample to monitor dynamic activity. Arrays measuring 28 TFs identified up to 19 active, with 13 factors changing significantly over time. Stimulation of cells with β-estradiol or activin A resulted in differential TF activity profiles evolving from initial stimulation of the ligand. Many TFs changed as expected based on previous reports, yet arrays were able to replicate these results in a single experiment. Additionally, arrays identified TFs that had not previously been linked with activin A.This system provides a method for large-scale, non-invasive, and dynamic quantification of signaling pathway activity as cells organize into structures. The arrays may find utility for investigating mechanisms regulating normal and abnormal tissue growth, biomaterial design, or as a platform for screening therapeutics. |
| first_indexed | 2024-12-11T22:19:37Z |
| format | Article |
| id | doaj.art-5c9e36bd41ed4331b62c96938779860c |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-11T22:19:37Z |
| publishDate | 2010-11-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-5c9e36bd41ed4331b62c96938779860c2022-12-22T00:48:29ZengPublic Library of Science (PLoS)PLoS ONE1932-62032010-11-01511e1402610.1371/journal.pone.0014026Dynamic, large-scale profiling of transcription factor activity from live cells in 3D culture.Michael S WeissBeatriz Peñalver BernabéAbigail D BellisLinda J BroadbeltJacqueline S JerussLonnie D SheaExtracellular activation of signal transduction pathways and their downstream target transcription factors (TFs) are critical regulators of cellular processes and tissue development. The intracellular signaling network is complex, and techniques that quantify the activities of numerous pathways and connect their activities to the resulting phenotype would identify the signals and mechanisms regulating tissue development. The ability to investigate tissue development should capture the dynamic pathway activity and requires an environment that supports cellular organization into structures that mimic in vivo phenotypes. Taken together, our objective was to develop cellular arrays for dynamic, large-scale quantification of TF activity as cells organized into spherical structures within 3D culture.TF-specific and normalization reporter constructs were delivered in parallel to a cellular array containing a well-established breast cancer cell line cultured in Matrigel. Bioluminescence imaging provided a rapid, non-invasive, and sensitive method to quantify luciferase levels, and was applied repeatedly on each sample to monitor dynamic activity. Arrays measuring 28 TFs identified up to 19 active, with 13 factors changing significantly over time. Stimulation of cells with β-estradiol or activin A resulted in differential TF activity profiles evolving from initial stimulation of the ligand. Many TFs changed as expected based on previous reports, yet arrays were able to replicate these results in a single experiment. Additionally, arrays identified TFs that had not previously been linked with activin A.This system provides a method for large-scale, non-invasive, and dynamic quantification of signaling pathway activity as cells organize into structures. The arrays may find utility for investigating mechanisms regulating normal and abnormal tissue growth, biomaterial design, or as a platform for screening therapeutics.http://europepmc.org/articles/PMC2984444?pdf=render |
| spellingShingle | Michael S Weiss Beatriz Peñalver Bernabé Abigail D Bellis Linda J Broadbelt Jacqueline S Jeruss Lonnie D Shea Dynamic, large-scale profiling of transcription factor activity from live cells in 3D culture. PLoS ONE |
| title | Dynamic, large-scale profiling of transcription factor activity from live cells in 3D culture. |
| title_full | Dynamic, large-scale profiling of transcription factor activity from live cells in 3D culture. |
| title_fullStr | Dynamic, large-scale profiling of transcription factor activity from live cells in 3D culture. |
| title_full_unstemmed | Dynamic, large-scale profiling of transcription factor activity from live cells in 3D culture. |
| title_short | Dynamic, large-scale profiling of transcription factor activity from live cells in 3D culture. |
| title_sort | dynamic large scale profiling of transcription factor activity from live cells in 3d culture |
| url | http://europepmc.org/articles/PMC2984444?pdf=render |
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