I had given X drug treatments and got following result. So how i can correlates lc3 and p62 .
Thp-1cell treated with X drug= LC3II increase and p62 increase Thp-1cell treated with X drug and bafilomycin =LC3II increase and p62 slightly decrease.
Is it autophagy inhibit.
Hello Radheshyam,
Autophagy is a multistep phenomenon, hence coming to a conclusion needs several experimental verifications.
Increase in LC3-II means an increase in autophagosome formation. It may also mean an increased accumulation of autophagosomes due to inhibited flux downstream.
p62 binds to ubiquitinated proteins and carries them to the autophagosomes to be degraded. there is always a regular turnover for p62 in the cell. However increased p62 might indicate an accumulation of this protein due to inhibition of flux.
Coming to your observations,
1. Thp-1cell treated with X drug= LC3II increase and p62 increase:
indication of increased autophagosome formation or accumulation of autophagosomes and a dysregulated autophagy flux.
2. Thp-1cell treated with X drug and bafilomycin =LC3II increase and p62 slightly decrease:
Bafilomycin inhibits formation of autolysosomes hence disrupts autophagy flux. Decrease in p62 needs to be analysed further.
Here, I would suggest you perform a time kinetic with the above treatment and check the levels of p62 and LC3-II.
It seems drug X inhibits autophagy, but whether it is the flux or the formation of autophagosomes needs to be verified.
You could check the levels of mTOR, PI3K,Atg5 proteins post treatment with drug X to understand its mechanism of action.
Also you could use 3-MA (inhibitor of autophagosome formation) and check your observations to support your hypothesis.
All the best !
While the new edition is still in the oven, I suggest you study the article below:
Klionsky DJ, Abdelmohsen K, Abe A, et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) [published correction appears in Autophagy. 2016;12(2):443. Autophagy. 2016;12(1):1-222. doi:10.1080/15548627.2015.1100356
The results with X drugs suggest autophagy inhibition. The ratio of LC3II/I is another option to have a look.
I agree with the previous comments, but the literature has shown that the LC3-II / LC3-I ratio has not been considered a good parameter for the interpretation of the autophagy flux. In my previous experience, I obtained results (without the use of drugs that inhibit or activate autophagy) in which there was an increase in LC3-II concomitant with an increase in p62. My choice to better interpret my data was to evaluate other proteins from the different stages of the autophagy flux, such as beclin1, LAMP2 and CHMP2B. As well as immunofluorescence analyzes and transmission electron microscopy.
I reinforce the suggestion of reading made by Prof. Dr. Rafael Linden, authored by Klionsky et. al.
Good luck with your analysis.
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