Hello,
I am studying autophagic activity by using LC3B marker. I already know that my patient cell lines have higher levels of LC3BI and II forms compared to the control. However, these data by themselves are not really explicatory, so i would like to try and measure autophagic flux. In particular, firstly, I would like to determine if there is a difference in terms of flux in control vs patient cell lines, and then see if a treatment with a drug has an effect on the flux.
From the literature, I gathered that the lipidated form LC3B-II is the one I should be focusing on, as it is the form that better reflects the degradation of substrates through lysosomes. I have read that the best technique to measure this flux is by treating cells with a lysosomal inhibitor and then comparing the change in the LC3BI-II accumulation.
What is not really clear to me is what is the best way to "compare" these LC3B-II levels, as I have seen different things reported. Is it better to calculate and compare the difference in LC3B-II [AF = (sample + Baf) – (sample – Baf)] or the ratio [AF=(sample + BafA1)/ (sample – BafA1)]?
Any suggestion is very welcome, and thank you in advance!
In my opinion, up-regulated LC3B-II suggested increased autophagic onset or disruption of autophagic flux. To determine whether autophagy increases on the onset, you could investigate whether the inhibitor of autophagic onset, for example, the mTORC1, is inhibited. Or, you could examine whether some of the ATGs are overexpressed. Besides, if you observe at the same time that the LC3B-II level is further increased upon disruption of autophagic flux by BafA1 or CQ, I would like to consider the autophagic flux is enhanced on the onset. It might be difficult to assess the autophagic flux only by the change in the LC3B-II. Try to investigate different aspects, and make the right judgment.
Thanks Xianjing Han !
I was considering adding p62 also, to have another autophagic substrate besides LC3B, that could help nudge in the right direction of data intepretation. But yeah, yours is a great suggestion! I will for sure try to investigate also mTORC, to see all the different "angles".
Do you have any suggestion regarding what's the best method to analyze these changes in LC3B when Bafilomycin is used?
Try the WB for detecting the LC3B-II or the IF for detecting the LC3B-II puncta.
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