How do you analyze autophagy?
Autophagy is a dynamic process essential for skin homeostasis. Autophagy can be measured at a single time point by observation of autophagy proteins or autophagy structures using techniques such as western blotting, immunofluorescence, immunohistochemistry, or electron microscopy.
What is marker for autophagy?
LC3 is currently the most widely used autophagosome marker because the amount of LC3-II reflects the number of autophagosomes and autophagy-related structures.
How is autophagy flux measured?
Autophagosome flux can be quantified experimentally by completely blocking one of the steps in the pathway and measuring the initial rate of accumulation in the substrate for the inhibited step, such as, for example, the rate of autophagosome accumulation upon complete inhibition of the lysosomal fusion step.
What is an autophagy assay?
Autophagy Assay Kit ab139484 measures autophagic vacuoles and monitors autophagic flux in live cells using a dye that selectively labels autophagic vacuoles. It can be analyzed using flow cytometry, fluorescent microscopy, or using a microplate reader.
What is autophagy?
A process by which a cell breaks down and destroys old, damaged, or abnormal proteins and other substances in its cytoplasm (the fluid inside a cell). The breakdown products are then recycled for important cell functions, especially during periods of stress or starvation.
What is autophagy flux?
The term “autophagic flux” is used to represent the dynamic process of autophagy. In detail, autophagic flux refers to the whole process of autophagy, including autophagosome formation, maturation, fusion with lysosomes, subsequent breakdown and the release of macromolecules back into the cytosol (Figure 1).
What is an example of autophagy?
Autophagy has roles in various cellular functions. One particular example is in yeasts, where the nutrient starvation induces a high level of autophagy. This allows unneeded proteins to be degraded and the amino acids recycled for the synthesis of proteins that are essential for survival.
Can we assess autophagy markers LC3B and p62 By immunohistochemistry?
Here we show the feasibility of specifically and reliably assessing the autophagy markers LC3B and p62 (SQSTM1) in formalin fixed and paraffin embedded human tissue by immunohistochemistry. Preceding functional experiments consisted of depleting LC3B and p62 in H1299 lung cancer cells with subsequent induction of autophagy.
How is autophagic flux monitored in autophagy?
The ubiquitin-associated protein p62, which binds to LC3, is also used to monitor autophagic flux. Cells undergoing autophagy should demonstrate the co-localization of p62, LC3, and lysosomal markers.
What is the most upstream autophagy factor?
The most upstream autophagy factors are the ULK1 complex, consisting of ULK1, ATG13, FIP200, and ATG101, and ATG9A on vesicles [18].
Does autophagy have a role in cancer development?
Autophagy assures cellular homeostasis, and gains increasing importance in cancer, where it impacts on carcinogenesis, propagation of the malignant phenotype and development of resistance. To date, its tissue-based analysis by immunohistochemistry remains poorly standardized.