The document discusses the detection of autophagy, focusing on the role of LC3 proteins in the formation and maturation of autophagosomes. It outlines various methods for assessing autophagic flux, including immunoblotting, fluorescence microscopy, and flow cytometry, as well as the significance of p62 accumulation in autophagy suppression. Additionally, it evaluates the suitability of fluorescent probes for assessing mitochondrial mass during oxidative stress.

1. Detection of Autophagy
Ekeh Chukwuemeka Obinna

2. Autophagy
● Nutrient starvation
● Response to hypoxia
● ER stress
● Cell death
● Cell damage
● Oxidative stress

3. Detection of Autophagy Dependent on LC3
● Principle: LC3 is a member of the ATG family. ATG proteins mediate
autophagosome formation which involves nucleation and elongation of the
isolation membrane. During autophagy cytosolic LC3(LC3-1) is conjugated to
phosphatidylethaloamine to form lipidated LC3(LC3-11). LC3(LC3-11) binds
to expanding isolation membrane and remains bound to complete
autophagosome.
LC3-11 can be used to analyse autophagic flux which include
autophagosome formation, maturation, fusion with lysosomes and breakdown
of autophagic substrates inside the lysosomes.
P62 possess ubiquitin binding domain and LC3 interacting region sequence.
It binds an enable their clearance in lysosome so are part of lysosomal
degradation. Accumulation of p62 signifies suppression of autophagy

4. Methods
● Monitoring LC3-11 turnover by immunoblotting to assess autophagic flux: The
number of LC3-11 = number of autophagosomes. The conversion of LC3-1
TO LC3-11 and lysosomal degradation of LC3-11 = progression of
autophagy.
● Western blotting of LC3 can detect the autophagic flux at every stage of
autophagy. LC3-11 has larger molecular weight (PE conjugated) than LC3-1
thus migrates faster on SDS-PAGE due to extreme hydrophobicity. The
densitometric units of both with or without lysosomal inhibitors are compared.
● Western blotting of p62 provides information for amount and rate of
autophagic substrates sequestered and degraded.

6. Monitoring the number of autophagosomes
● Visualizing and localization of LC3-11 punta using fluorescence microscopy:
The change in the subcellular distribution of LC3 can be observed through
either indirect immunofluorescnce or by examining the signal of fluorescent
protein tagged to LC3-11.Endogenous LC3-11 and anti LC3-11 antibodies in
immunocytochemistry.
● Monitoring autophagic flux using tandem fluorescent tagged LC3 to assess
autophagic flux: Tandem monomeric RFP-GFP-tagged LC3 maintain red
fluorescence in acidic compartments while GFP fluorescent are quenched in
acidic ph in lysosomes. This helps in colocalization of GFP and mRFP to
localize LC3 fused to lysosome.

7. Detection of p62 and LC3 marker in paraffin
embedded and formalin fixed tissues
P62 accumulation in tissues with deficient autophagy can be detected by electron
microscopy which is the gold standard. Other methods include Indirect
Histochemical staining of p62 antigens. Using antibody LC3 marked with
fluorescence dye activity can be observed after autophagy induction. Dot like
staining was observed which are positive for p62 antigen.

8. Observation of mitochondria mass, damage and
ROS by flow cytometry
MitoTracker green (MTG) and nonyl acridine orange (NAO) are fluorescent probes which have been
commonly used to assess mitochondrial mass. This is based on the assumption that both MTG and NAO
accumulate in mitochondria regardless of the mitochondrial transmembrane potential MTG probe is
oxidized by superoxide to red fluorescence.. Here, we utilized flow cytometry to evaluate the performance
of these probes for assessment of mitochondrial mass relative to forward (FSC) and side scatter (SSC).
In isolated mitochondria, two subpopulations were identified by FSC and SSC measurements which were
matched to subpopulations stained by MTG and NAO. The performance of these dyes was examined
under oxidative and nitrosative stress induced by rotenone and NOC-18 while N-acetylcysteine (NAC) was
employed as an antioxidant. Production of reactive oxygen species (ROS) and antioxidants were
monitored in parallel. However, MTG showed significant correlation with cytosolic and mitochondrial ROS
production and nitrosative stress. Our data suggest that NAO may be more suitable than MTG for
assessment of mitochondrial mass by flow cytometry during oxidative stress.

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