I need to induce autophagy in hela cells for my experiment. Theoretically it is simple. 4 hours of starvation in EBSS medium should do the trick according to literature. However, after the starvation treatment I do not see increase in LC3 I to II conversion in my blot. I did not transfect the cells with GFP LC3 yet but it should show a difference even with basal LC3 level.
What can I be doing wrong that the autophagy does not induce in the cells?
Any suggestions?
Thanks!
For autophagy study, you could try mRFP-GFP-LC3 and GFP-LC3 labeling system, which can detect the intensity of autophagy flux in real-time, in which GFP and/or RFP tags are fused at the N-termini of the autophagosome marker LC3. These biosensors provide an enhanced dissection of the maturation of the autophagosome to the autolysosome, which capitalizes on the pH difference between the acidic autolysosome and the neutral autophagosome. The acid-sensitive GFP will be degraded in autolysosome whereas the acid-insensitive RFP will not. Therefore, the change from autophagosome to autolysosome can be visualized by imaging the specific loss of the GFP fluorescence, leaving only red fluorescence.
You could find more on this website: www.genemedi.net/i/autophagy
Genemedi provides the production service of AAV, adenovirus and lentivirus encoding mRFP-GFP-LC3 or GFP-LC3, suitable for monitoring the intensity of autophagy flux in real-time in vivo or in vitro.
Attached file is an image of mRFP-GFP-LC3 labeled A549 cells with nutrition deprivation.
Hello Mohammad,
I am not sure about LC3, may be for changes at protein level, you might require longer treatment durations. Autophagy at 4 hrs can be seen nicely upon staining with acridine orange and mdc staining. Go for that..It will work fine.
Hope it helps !
LC3 Western is tricky as the bands come close to the bottom of gels. Consider changing gel running duration and gel percentage to prevent run out of processed LC3 bands.
I would suggest a time course (0, 30 min, 1 h) for starvation after rinsing of cells with large excess of PBS. In most cell types starvation for 20-30 min is sufficient to initiate autophagy and detect LC3 conversion using antibodies from Cell Signaling.
In the autophagy paper "Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)", HeLa cells does not show the LC3-II increase after starvation without lysosome inhibitor. This result suggests that autophagic flux was triggered after starvation in HeLa cells.
If starvation didn't work, you may wish to consider exposing them to a cationic lysgomogenic drug like chloroquine. It works in cultured primary rat hepatocytes.
I also think you should do a time course with starvation, 1, 4, 7 hrs. or such. It is always good to see the kinetics of the response. May be, also check if you have washed the cells several times with PBS before putting them on starvation. Also, you could try time course and dose response with Rapamycin in comparison.
May be links below will be of help.
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0061020
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633384/
I suggest you have a look at this paper:
http://www.tandfonline.com/doi/pdf/10.4161/auto.4600
It should be helpful.
Kind regards,
Aleksandra
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