Dynamic transcription factor activity profiling in 2D and 3D cell cultures

Area of Science:

• Cell biology
• Biotechnology
• Molecular imaging

Background:

• Live-cell assays in 3D cultures are crucial for understanding disease and tissue formation.
• Cells in 3D cultures remodel their microenvironment and form complex structures.
• Dynamic monitoring of cellular functions is essential for biological research.

Purpose of the Study:

• To develop a non-invasive method for imaging transcription factor (TF) activity in 2D and 3D cultures.
• To evaluate different normalization strategies for dynamic TF activity measurements.
• To characterize TF activity dynamics in response to stimuli and in different culture formats.

Main Methods:

• Development of a transcription factor (TF) activity array using lentiviral delivered luminescent reporter constructs.
• Utilized bioluminescence imaging (BLI) for non-invasive, repeated imaging of TF activity.
• Investigated in-well (Gaussia, Renilla luciferase) and external well (firefly luciferase) normalization techniques.

Main Results:

• External well normalization using firefly luciferase provided consistent and low-variability measurements of dynamic TF activity.
• In-well normalization methods (Gaussia, Renilla) were insufficient for long-term TF activity tracking.
• The developed array successfully captured known TF responses to stimuli and revealed novel dynamic TF activity profiles in 2D and 3D cultures.

Conclusions:

• The developed TF activity array enables dynamic, non-invasive monitoring of TF activity in both 2D and 3D cell cultures.
• External normalization is a reliable method for quantifying dynamic TF activity over time.
• This technology offers a powerful tool for gaining deeper insights into complex cellular processes and disease mechanisms.