I tested 3-MA on human podocytes for the first time but couldn't see any downregulation in LC-II levels. Actually, there was a minor increase in band intensities of 3-MA-treated cells. I tried 2h and 4h incubation with concentrations of 1mM, 2mM and 4mM. Is the incubation time too short? What else could be wrong?
3-MA is very tricky if you use the correct concentration and also exact time point you will see the changes in LC-II/LC-I ratio. You have to try a broad range of concentration starting from 1 mM to 15 mM. I hope I could help you.
I think increase time course will be helpful. Some time LC3-II levels changed a lot dependending upon the condition of cells in culture. As Mr yubin mentioned you should check p62 levels.
I think 3MA should be used in the 5 mM to 10mM range. If you increase the incubation time, you may loose the inhibitory effect of 3MA. In my experience, an overnight treatment with 3MA alone leads to a mild increase in LC3II. This effect may be due to the fact that 3MA is loosing activity, and at lower concentrations it can inhibit PI3K type I (and not PI3K type III anymore), thus leading to a mild induction of autophagy.
Did you use lysosomal inhibitor? Without it you usually can not assess changes in LC3 II. Also, in some cells 3-MA is toxic. This can increase autophagy in type III PI3k independent manner. Check your cell viability. You can also use Spautin instead of 3-MA. For most cell lines its a much better drug.
Dar Sonja; 3MA is a very strange molecule. We have used at 5 mM, diluting it directly with the medium for 15-24 hours. I should use another inhibitors, such as chloroquine or bafilomycin. Good luck!!!
Agreed with many of the above answers. 3-MA can have multiple effects so, depending on the goal, a much more specific inhibitor would be preferable. With 3-MA, one can never be certain which of its many properties is responsible for the effects observed. I'd recommend consulting a recent report by Klionsky et al on methods for assessing autophagy.
First you can check if your cells use autophagy with a late stage inhibitor (bafilomycin),
when your cells are positive than you can use 3-MA, but i use 6h - 24h incubation time.
I think that if you use 3MA you block autophagy in the initial step, so maybe you can't see the formation of autophagosome. In this case the level of LC3 II is the same.
This paper might be helpful.
Juenemann et. al., 2012, Alternative Macroautophagic Pathways.
The best and the only acceptable way to block autophagy is to knockdown ATG7. 3MA has numerous other effects apart from inhibiting PI3K CI and CIII activities.
Hi,
thanks for your answers. Autophagy is a new topic for me but I've been reading a lot of autophagy-related papers. I'm actually alsohaving problembs with inducing autophagy. Actually the only thing that's working nicely is Bafilomycin. It clearly increases LC-II levels as it should.
Actually, I can't see any clear trend of increase or decrease. The best decrease is with 4mM for 4h (0,85-fold). In Fang's paper they have shown a nice decrease by using 1-5 mM 3-MA for 24h (figure: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3623813/figure/pone-0060546-g002/). However, they are using a mouse podocyte line instead of human. I can of course try 24 h's incubation or higher concentration.
The podocytes exhibit a high level of of basal autophagy so LC-II can be seen as a strong band without any induction. Thus, I would expect that 3-MA would decrease LC-II levels.
Paresh Kulkarni and Marta Lipinski, do you mean that I check the effect of 3-MA with and without Bafilomycin?
I haven't heard about Spautin before - I'll check it!
I'll let you know whether that helps when I have my cells ready, thanks!
Hi everyone,
I’m in similar situation, I attempted to block autophagy induction during starvation. Cells (rat primary astrocytes) were incubated in medium containing 3MA at concentration of 1 mM for 30 min before and during exposition to stress conditions (1, 4, 8, 24 h). Immunobloting results didn’t show downregulation in LC-II but p62 levels were similar to those in control cells (cultured in normal conditions, no autophagy induction), whereas cells exposed to stress without 3MA exhibited strong p62 degradation. Do you think I can assume that autophagy is (at least partially) blocked in my cells? There is no possibility to use another autophagy inhibitor. What else can I check?
3-MA has (as pointed out above) too many inhibitory properties to be useful. I doubt that one could get a paper published in a reputable journal using 3-MA as an autophagy antagonist. Knockdowns can be useful, e.g., ATG5 or ATG7, but these can have unexpected effects. ATG5, for example, can be cleaved to form a pro-apoptotic fragment so its loss might impair apoptosis. There are several recent papers published in the journal 'Autophagy' that deal with methods for assessing autophagy. My preference for initial studies is to use either ammonium chloride or bafilomycin to block the digestion phase. But check the references to see how the data is best interpreted.
Hello ,
Check this article: http://www.jbc.org/content/285/14/10850.abstract
I think it'll help you if it's not too late.
http://www.jbc.org/content/285/14/10850.abstract
3MA is not sufficiently specific for the results to be readily interpreted. There are better procedures that are amply described in the literature.
3-MA inhibits only starvation induced autophagy however, in complete medium it actually induces the LC3-|| conversion as reported in JBC http://www.jbc.org/content/285/14/10850.long however in my case this is right so if you want to use it as an inhibitor provide starvation condition or better to use Wortmannin..
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