I generated GFP-LC3 HEK-A cell lines to study autophagy. I can detect GFP-LC3 signal on western blot. I want to measure autophagy flux based on level of free GFP in the cells after autophagy induction. I used both HBSS starvation and Rapamycin treatment for 2 hours but I can't see any free GFP signal. All I have is endogenous LC3 I and II as well as GFP-LC3 signal.
Any thoughts on this?
Thanks!
Is your cell line based on HEK293 or a different one? Because these two papers (though from the same author) seem to suggest that carboxyl termini cleavage of LC3 does not occur in HEK293 cells.
Hi Mohammad
You should take a look at the attached publication. As we know, in yeast cells the accumulation of cleaved free GFP in the vacuole from GFP-Atg8 is a reliable assay used to measure autophagy. The authors of this study propose that in mammalian cells this assay is complicated by the fact that lysosomes have a lower pH and degrade free GFP more efficiently. Therefore in mammalian cells, accumulation of free GFP from GFP-LC3 could either reflect increased delivery or reduced lysosomal activity. In contrast, an absence of free GFP could be caused by either increased autophagy and efficient lysosome activity, or reduced delivery of GFP-LC3 to the lysosome. It is suggested to combine this assay with other measures of autophagy.
In your case, you could try reproducing the assay conditions that are used to detect free GFP in this paper. Note that concentration of autophagy inhibitors used is very important.
Article Dissecting the dynamic turnover of GFP-LC3 in the autolysosome
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