Hello,
I am treating my cells with cloroquine to block autophagy at the late stage. Using Western blot, I found that the LC3 II form is accumulating; however, the P62 protein is not changing (it is supposed to accumulate) compared to control cells. Any explanations ?
Thank you.
I am not an expert to answer this but from what I have observed I could say that the level of P62 will be unchanged or increased as Chloroquine blocks the lysosomal activity and P62 is a cargo protein, it might have not been digested in the absence of lysosomal activity. If you could share your WB bands it would have been more easy to analyze. Please correct me if I am wrong.
I suggest you study the article below to gain insight on how to deal with autophagy:
Klionsky DJ. et al - Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy. 2016;12(1):1-222.
Thank you Karthikeyan Vijayakumar . First lane is the control while the third is CQ treated cells. In principle there is basal autophagy that digest the non-specific cargo P62. Upon CQ addition, LC3II should accumulate representing accumulating autophagosomes which is true in my blot. However, should also accumulate due to autophagy blockage, but it didnt!
Hala Shallhoub Autophagy is a complex mechanism, various other complex formation also needed in order for the entire autophagy flux to complete. From your blot what I could say is that in your control LC3II has been expressed. But with only that we could not come to a conclusion that basal level autohpagy is happening. LC3II is needed to be recruited to the autophagosomes in order to recruit P62 and degrade it, which might not happened in this case. In my experience in cell lines autophagy occurs only during the presence of any stressors like H2O2 or anything. As autophagy is a stress response mechanism. Looking for the expression of Atg9 or Atg5 or Atg12 will enlighten us in this situation. What I can observe from your data is that LC3II conversion has been seen but this LC3II might not have been recruited to the Autophagosome or the Autophagosome might not formed. Therefore the P62 level is unchanged. I'll attach my Western blot image in which the control didn't even express LC3II.
Karthikeyan Vijayakumar . Thank you. I see what do you mean, but isn't LC3II a marker of autophagosome formation? I mean LC3I (cytosolic) conversion into LC3 II indicates autophagosome formation which I have seen using IF also ( autophagosomes accumulated in CQ treated cell compared to control) . Is it possible that basal autophagy is taking place; however, it doesnt involve the non-specific digestion of P62? so that upon its blocking, P62 level will not change?
Hi Hala Shallhoub I am sorry, I assumed that the 1st lane is control, 2nd lane is some drug treated and the 3rd lane is drug+CQ treated. If the 3rd lane is just CQ treated then we should go with your interpretation. And also we could not say that Autophagosome is formed after seeing the presence of LC3II in our Western blot. LC3II is also present in cytosol, from where it is recruited to the phagophore and elongates to autophagosome. Reading the paper shared by Prof. Rafeal Linden will be more helpful in the interpretation.
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