Hello to all autophagy experts, or those who know a bit more deeply about autophagy.
Currently, we are using LC3 to estimate the autophagic activity of HEK293T cells in different aggregative conditions.
As I have read in the literature, people have many approaches to estimating autophagy, some use the ratio of LC3-II/ LC3-I as an indicator of the basal autophagic activity, others use the autophagic flux indicated by the subtraction of LC3-II levels (with no autophagic inhibitor) from LC3-II levels (with autophagic inhibitor) and so.
This issue has been tricky a bit since decreased levels of LC3-II might be due to impaired autophagosome formation by inhibition of LC3-II synthesis, or to increased degradation rates.
Any comments? tips to consider?
Hello Amal,
an increase in LC3-II levels is a sign of autophagy induction, but as you correctly mentioned, simply analyzing LC3-II levels is not enough for a complete analysis. For instance, LC3-II is recycled back to LC3-I after autophagolysosom formation and also degraded during autophagy by lysosomal proteases, which lowers LC3-II levels. Therefore, autophagic flux should be analyzed in the absence and presence of specific inhibitors, e.g. PSA/E-64d for inhibition of lysosomal proteases. If you are new to the field, have a look at Daniel Klionsky's community-approved guidelines for autophagy analysis (10.1080/15548627.2020.1797280), which gives a good overview and also answers a lot of questions.
Good luck - CL
I agree with Carsten Lange and I recommend you to read this article:
https://pubmed.ncbi.nlm.nih.gov/17611390/
Increase in LC3-II level does not always indicate that there is an autophagic induction. Sometimes, LC3-II accumulates in the cell due to autophagosome-lysosome fusion inhibition. Besides using an autophagy inhibitor, I also recommend you to do Western blotting experiments with p62 (SQSTM1). Since it accumulates when the autophagic flux is inhibited, it will help you to interprete your LC3 turnover results.
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