For example, I treat cells with "X" - performed immunoblotting for LC3-II - found elevated autophagy, then did the same treatment in GFP-LC3 transfected cells followed by confocal imaging - found no difference in number of puncta quantification -compared to Mock treated control. Is it possible? (In my understanding these two methods are complementary to each other, in this context). If yes, please explain or provide any reference.
Thanks in advance.
Dear Sasikala Muthusamy,
You did not mention what type of LC3 (LC3-I/LC3-II) which you measured by immunoblotting and what kind of data (fluorescent signal, intracellular location of LC3, etc.). I'm not sure that you could apply the same parameter for both immunoblotting and microscopy. LC3-II could possibly be up-regulated during the autophagy; however, we should not rely on LC3 only to measure the autophagy activity because its properties change quite much during the activity. For example, I also used immunoblotting for LC3 in hepatocarcinoma (HCC) tumor tissue, but needed to check other parameters (autophagy-related genes, in vitro assay on HCC cell line.
You could also check for LC3's intracellular location following your treatment. Hope that you would find the attached article useful :)
Best regards
@Huy Quang Do, Hi. Thank you for your response. I edited my question based on your queries. In-vitro assay, you mean is qPCR for autophagy gene expression relative quantification right? Thanks I will consider this point.
Hi @Sasikala. We have seen in plants (GFP-ATG8 transgenic plants) that GFP immunoblot (gives an estimation of autophagic flux) is a more sensitive method than autophagosomes analysis by confocal microscopy for the analysis of autophagic activity.
As @Huy suggested you, always better to check by different methods.
Good luck!
Hi Sasikala Muthusamy
I suggest you need to measure autophagy flux with lysosomal inhibitor.
You may misinterpret only with the data at steady-state level whatever it is LC3-II-based immunoblotting or immunofluorescence analysis with GFP-LC3B, since both increase of autophagosome formation and decrease of lysosomal-mediated clearance result in an accumulation of LC3-II. You can easily find many references describing how to measure autophagy flux with lysosomal inhibitor.
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