Hi,
I have been confused of the way many authors interpret their LC3 turn over assay. To my understanding, the read out of this assay is the difference between the intensity of the sample in the presence of bafilomycin and in the absence of it. Then you'll compare this subtract among treatments. (Of course the intensity is already normalized to loading control)
For example: I have 4 samples: Control; Control + Baf; Treated; Treated + Baf.
I'll get the X = (Treated + Baf) - Treated. X indicates the amount of LC3B which are recruited for the degradation.
Y = (Control + Baf) - Control
If X >Y: Autophagic flux is induced in treated samples.
However, in many literature I've found, they showed the charts to indicate that samples with Baf have increasing level of LC3II and concluded that autophagic flux is induced. I thought an increase level in Baf treated samples only indicate that there IS autophagic flux IN SOME EXTENT but you can't tell whether your treatment increases autophagic flux compared to control group. So how do you interpret this assay?
It is also mentioned in this review but so far I'm still confused about the way I should incorporate it into publication.
http://www.ncbi.nlm.nih.gov/pubmed/20144757
Hi,
The problem with LC3 assays is that you can't make a difference between an accumulation of autophagosomes and an actual incrase in autophagic flux.
Here the bafilomycin results in an increased pH in the lysosome and so an absence of degradation of the autophagolysosome content. So you have an inhibition of autophagy at the end of the process. It results in an accumulation of auphagosomes and thus of LC3-II.
But, if you stop autophagy at earlier stages, with Atgs KO for example, you will avoid the formation of the autophagosome and then have less LC3-II. However, concerning the autophagy, the results ill be the same: an inhibition.
Thus, I would probably add some more analysis to your experiment, like checking the levels of P62 (sequestrosome-1,I use an Ab that works nicely with WB and IHC if you want the reference), which accumulates upon autophagy inhibition, and maybe some electronic microscopy to chek if there is an accumulation of autophagosomes in the cytosol.
Hope, I have been useful, good luck!
Perfect will be to show western blot data for lc3 and p62 witn control, stimuli+ and - lysosomal proteolysis blockade ( with additional quantified data). P62 levels reducing with stimuli and this bloched in combination with lysosomal blockade will be good confirmation for induction of autophagy.
As u rightly said stimuli/ control alone are necessary to show, but will vary greatly depending on time of treatment( especially lc3 -2). Please refer to the guidelines for acceptable methods and details (Klionsky et al 2012). There are very few things for which we have such descriptive guidelines available!
As previously commented, bafilomycin treatment affects the fusion of autophagonsomes with lysosomes. Importantly, bafilomycin also influences on the endosome function, so that this agent has a high cellular toxicity. That is why I strongly recommend you to check LAMP1/2 in WB experiments after the treatment with bafilomycin.
This publication was written by the authorities in the autophagy field and is recognized as the standard in the field:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404883/
It will guide you through the proper procedure and interpretation of autophagy-related assays, including the LC3 turnover assay. One of the reasons for its publication is because of the many different ways that different labs interpret their results. The publication gives clear direction for proper interpretation.
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