Related Experiment Video
Updated: Sep 18, 2025
08:07
Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence
Published on: April 28, 2023
1.6K
Quantitative visualization of autophagy induction by mTOR inhibitors
Beat Nyfeler1, Philip Bergman, Christopher J Wilson
1Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, MA, USA.
Methods in Molecular Biology (Clifton, N.J.)
|November 30, 2011
Summary
This study quantifies autophagy induction by mammalian target of rapamycin (mTOR) inhibitors using a novel reporter system. We developed an imaging protocol and algorithm to measure changes in the autophagic pathway, aiding drug discovery.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Autophagy is a vital catabolic process for cellular recycling.
- The mammalian target of rapamycin (mTOR) pathway regulates autophagy initiation.
- mTOR inhibition activates autophagy, making it a therapeutic target.
Purpose of the Study:
- To develop a quantitative method for measuring autophagy induction by mTOR inhibitors.
- To describe an imaging protocol and image recognition algorithm for autophagy analysis.
- To facilitate the study of autophagy modulation in mammalian cells.
Main Methods:
- Utilized an mCherry-GFP-LC3 reporter system for autophagy visualization.
- Employed retroviral delivery for stable expression in mammalian cells.
- Applied fluorescence microscopy and a custom image recognition algorithm for quantification.
Main Results:
- Successfully visualized and quantified autophagy induction.
- Demonstrated the utility of the mCherry-GFP-LC3 reporter system.
- Established a robust protocol for analyzing autophagic flux.
Conclusions:
- The developed imaging protocol and algorithm effectively quantify autophagy induction by mTOR inhibitors.
- The mCherry-GFP-LC3 reporter system provides a reliable tool for studying autophagy.
- This methodology can advance research in autophagy-related diseases and drug development.
Related Concept Videos
03:03mTOR Signaling and Cancer Progression
3.9K
The mammalian target of rapamycin or mTOR protein was discovered in 1994 due to its direct interaction with rapamycin. The protein gets its name from a yeast homolog called TOR. The mTOR protein complex in mammalian cells plays a major role in balancing anabolic processes such as the synthesis of proteins, lipids, and nucleotides and catabolic processes, such as autophagy in response to environmental cues, such as availability of nutrients and growth factors.
The mTOR pathway or the...
The mTOR pathway or the...
3.9K
01:22PI3K/mTOR/AKT Signaling Pathway
4.0K
The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates growth, proliferation, and cell survival in response to hormones, growth factors, or nutrient availability. This kinase exists in two structurally and functionally distinct forms: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). The first form (mTORC1) is composed of a rapamycin-sensitive Raptor and proline-rich Akt substrate, PRAS40. In contrast, mTORC2 consists of a...
4.0K
01:27Autophagy
4.6K
Autophagy is a self-digesting process by which a cell protects itself from threats both within and outside the cell, ranging from abnormal proteins to invading bacteria. In this process, obsolete components of the cell and invading microbes are degraded by hydrolytic enzymes active in an acidic environment of the lysosomal lumen.
An autophagic pathway consists of a series of signaling events activated in response to diverse stress and physiological conditions such as food deprivation,...
An autophagic pathway consists of a series of signaling events activated in response to diverse stress and physiological conditions such as food deprivation,...
4.6K