Hi,

I recommend to look into this guide:

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).

doi: 10.1080/15548627.2015.1100356

did you check autophagic flux?

Dear Veronika,

Definitely higher expression of LC3-II signifies autophagy. As autophagy leads to degradation of SQSTM1 (p62), its expression always inversely related to LC3-II thats what you are observing. But at the same time other autophagic marker protein should also increase. Now coming to lysosomal membrane protein LAMP1 and LAMP2 can also be used as a marker for macro autophagy and chaperon mediated autophagy (CMA) respetively. So whenever there is a induction of CMA, LAMP2 will be higher similarly LAMP1 in case of macroautophagy. Thus i would suggest you can also go with immuno-fluoroscence staining using tf- LC3 which will give you a clear idea about autophagy status.

Best;

Prakash

In our hands, LAMP1 and LAMP2 can be quite stable when autophagy is induced and don't give great readouts of macroautophagy. As suggested above, tf-LC3 is a useful marker when used correctly.

Have you performed the classic with and without chloroquine assay with western blot for LC3 to get a better handle on flux of LC3II through the lysosomal system?

To further refine the conversation around LAMP2 and CMA, the specific splice variant that is supposed to mediate CMA is LAMP2A. You would have to dig through quite a bit of literature to determine whether you thought higher expression of LAMP2A reasonably equates to increased chaperone mediated autophagy, or whether you would have to set up a more function-based assay.

Further, don't neglect mTORC1 and AMPK targets (western blotting for phospho-epitopes) to give further information on the likely activity of macroautophagy.

Tim

Dear all,

Thanks a lof for your answers/suggestions-I really appreciate it.

Katarzyna, I did not measure the flux, I should mention it before - it is not possible since I am working with the human tissue. This also answer to your question Tim regarding chloroquine experiments. I did measure, however, phospho mTORC1 and phospho AMPK, but could see only significant change in case of mTORC1, although the change was much smaller than in the case of LC3B-II. I will perform IF experiment for LC3B soon -but how this will really help me? Isn't it the way that LC3B "dots" will reflect only if there is an increase in the number of autophagosomes but will not tell me if it is due to the autophagy induction or blocked flux??? With LC3B IF experiments I can only detect the level of LC3B-total, not separetly LC3B-II, right? But I might be wrong -I am really a novice in this field and autophagy is only small part of my project.

Does anybody know if there is any other circumstances when the level of LC3B-II increases besides autophagy? I should also mention, that unfortunately I don't have the oportunity to measure mRNA level for LC3B because of not enough material. But could the high level of LC3B-II be explained by transcriptional upregulation if the level of LC3B-I is not increased? I don't think so...but again, I might be mistaken...

Veronika