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细胞技术

Quantitative Visualization of Autophagy Induction by mTOR Inhibitors

2025-02-21 细胞技术 加入收藏
Autophagy is a catabolic pathway that degrades bulk cytosol in lysosomal compart

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.

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