I performed an immunoblotting with LC3 antibody (please see the image) upon siRNA-mediated knockdown of two different proteins, A and/or B, with or without Bafilomycin A1 treatment. I would like to know how to interpret this result, especially regarding the autophagic function upon protein A knockdown. Protein A knockdown seems to show a decrease of LC3II as compared with the control siRNA; and Bafilomycin A1 treatment elevates the LC3II level in the knockdown cells, although still lower than the control siRNA cells with Bafilomycin A1 treatment.
Thanks a lot in advance!
LC3-II is lipidated LC3 whos upregulation is a positive marker of autophagy
Protein A knockdown alone is not sufficient to cause autophagy
but in the presence of bafilomycin and A protein autophagy is induced
The following observations can be made for the controls:
- Protein A knockdown leads to decreased LC3-II levels (compared to control siRNA), indicating that the presence of protein B alone decreases autophagosome numbers
- Protein B knockdown leads to increased LC3-II levels (compared to control siRNA), indicating that the presence of protein A alone increases autophagosome numbers
- Combined knockdown of proteins A + B, however, slightly decreases LC3-II levels, indicating that the effect of protein B blocks the effect of protein A.
Autophagic flux analysis (by additional bafilomycin treatment) reveals an increase in LC3-II levels for all samples compared to their equivalent controls, suggesting that both protein A (protein B knockdown) as well as protein B (protein A knockdown) rather lead to induction of autophagy, with protein A (protein B knockdown) being a stronger inducer.
If this is true, why are LC3-II levels after protein A knockdown lower compared to the control siRNA? And: Why are LC3-II levels substantially higher for the siRNA control after additional treatment with bafilomycin?
In my opinion, you should confirm the results with other inhibitors like pepstatin A/E-64d, chloroquine, NH4Cl, or vinblastine in addition to bafilomycin. Furthermore, you could check LC3-II levels in the presence of autophagy inhibitors, e.g. wortmannin, which would be of special interest for the control siRNA. But most important, you should verify that the control siRNA itself does not lead to autophagy induction. Therefore, include a non-treated control and make sure, nutrient levels are sufficient throughout the entire duration of the experiment.
Further help for interpretation of LC3 immunoblotting may be found in the publication of Mizushima & Yoshimori (doi.org/10.4161/auto.4600).
Please keep us updated. - CL
Maybe I need to make the question more clearly. My main interest is to interpret the result from protein A knockdown.
From the WB result, what I can see is that protein A knockdown showed a decrease of LC3II; and with BafA1 treatment, the LC3II levels of both ctrl and protein A knockdown cells could be enhanced.
For decreased LC3II upon protein A knockdown: since LC3I level did not decrease, the reason to LC3II decrease may not be a high turn over rate (i.e. high lysosomal degradation). Possibilities could be inhibition of upstream autophagy or inhibition of LC3I to LC3II transition.
My main question is: BafA1 treatment did not inhibit LC3II increase upon protein A knockdown, how should I interpret this? Very specifically, what about the lysosomal degradation function in this case? Is it definitely normal with the lysosomal function or it could be any other possibilities?
Thanks!
BafA1 treatment is not supposed to inhibit LC3-II increase, but to further increase LC3-II levels. BafA1 blocks the final degradation step by inhibiting vATPase acidification of lysosomes. Therefore, LC3-II accumulates due to the blocked degradation in the lysosomes as well as the blocked recycling back to LC3-I. As expected, LC3-II levels are raised after treatment with BafA1 in all of your samples, indicating autophagy induction. There's nothing to worry about.
What I indeed worry about is your control siRNA sample, as it seems to have surprisingly high levels of LC3-II, which are markedly increased by BafA1 as well.
Is there anything known about high basal autophagy in the cell line you used? Can you exclude nutritional deprivation of the cells during cultivation?
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