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Published on: July 21, 2017
AUTOCOUNTER, an ImageJ JavaScript to analyze LC3B-GFP expression dynamics in autophagy-induced astrocytoma cells
L Fassina1, G Magenes, A Inzaghi
1Department of Industrial Engineering and Information, University of Pavia, Italy.
European Journal of Histochemistry : EJH
|January 31, 2013
Summary
AUTOCOUNTER, an ImageJ JavaScript, quantifies LC3B-GFP expression in astrocytoma cells to monitor autophagy. This tool tracks autophagosomal-like vesicles, aiding in vitro autophagy modulation studies.
Area of Science:
- Cell Biology
- Biochemistry
- Bioinformatics
Background:
- Autophagy is a crucial cellular process involving the degradation of damaged components.
- LC3B-GFP is a widely used marker for monitoring autophagosomes.
- Quantifying dynamic changes in LC3B-GFP puncta is essential for studying autophagy.
Purpose of the Study:
- To develop and validate an ImageJ JavaScript tool, AUTOCOUNTER, for quantifying LC3B-GFP expression.
- To monitor dynamic changes in autophagosomal-like vesicles in living human astrocytoma cells.
- To assess the utility of AUTOCOUNTER in autophagy modulation studies.
Main Methods:
- Development of AUTOCOUNTER, an ImageJ JavaScript for ImageJ software.
- Transduction of T98G and U373-MG astrocytoma cells with BacMam LC3B-GFP.
- Induction of autophagy using Rapamycin.
- Assays including immunoblotting (ph-p70S6K/p70S6K, LC3B) and electron microscopy.
- Utilizing a mutated LC3B transgene as a negative control.
Main Results:
- AUTOCOUNTER successfully quantified LC3B-GFP expression in astrocytoma cells.
- The tool enabled scoring of dynamic changes in the number and area of LC3B-GFP puncta.
- Results were validated against immunoblotting and electron microscopy data.
- A negative control confirmed that puncta were related to lipidation, not aggregation.
Conclusions:
- AUTOCOUNTER is a suitable algorithmic tool for quantifying LC3B-GFP expression.
- The JavaScript enables monitoring of dynamic changes in autophagosomal-like vesicles.
- AUTOCOUNTER facilitates in vitro autophagy modulation studies.
