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Methods Mol Biol. 2012;821:239-50. doi: 10.1007/978-1-61779-430-8_14.

Quantitative visualization of autophagy induction by mTOR inhibitors.

Author information

1
Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, MA, USA.

Abstract

Autophagy is a catabolic pathway that degrades bulk cytosol in lysosomal compartments enabling amino acids and fatty acids to be recycled. One of the key regulators of autophagy is the mammalian target of rapamycin (mTOR), a conserved serine/threonine kinase which suppresses the initiation of the autophagic process when nutrients, growth factors, and energy are available. Inhibition of mTOR, e.g., by small molecules such as rapamycin, results in activation of autophagy. To quantify autophagy induction by mTOR inhibitors, we use an mCherry-GFP-LC3 reporter which is amenable to retroviral delivery into mammalian cells, stable expression, and analysis by fluorescence microscopy. Here, we describe our imaging protocol and image recognition algorithm to visualize and measure changes in the autophagic pathway.

PMID:
22125069
DOI:
10.1007/978-1-61779-430-8_14
[Indexed for MEDLINE]

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