Many publications mention that cells were serum starved for 24 hours before adding TGF-beta or EGF to induce EMT. After serum starved, is it possible to treat cells with EMT-inducers in RPMI 5% FCS or 10% FCS?
You have raised an interesting point..!! Serum starvation+TGFb/EGF treatment is to evaluate the specific action of TGFb/EGF in the induction of EMT. However, by withdrawing serum, many cytokines/differentiation factors will be lost and hence TGFb/EGF might induce EMT easily. The ability of TGFb/EGF to induce EMT should be tested in the presence of regular FBS levels to test its robustness (in parallel to serum starved conditions).
You have raised an interesting point..!! Serum starvation+TGFb/EGF treatment is to evaluate the specific action of TGFb/EGF in the induction of EMT. However, by withdrawing serum, many cytokines/differentiation factors will be lost and hence TGFb/EGF might induce EMT easily. The ability of TGFb/EGF to induce EMT should be tested in the presence of regular FBS levels to test its robustness (in parallel to serum starved conditions).
Serum starvation will induce stress to the cells, and it may lead to many alterations including EMT.
Kee, i think you may be misunderstanding why the authors of published papers are starving their cells prior to cytokine treatment. Often, they do this b/c they want to look at downstream signaling events in their cells (Smad phosphorylation, etc) and therefore, to reduce the background level of pSmad, they serum starve the cells b/c the serum has TGF in it. I don't suspect that they serum starved to induce EMT.
I´m agree with Daniela, any stress factor type will induce TGFbeta pathway activation and EMT induction.
I think both, Dr. Freitas and Dr. Marryman, are right.
The question is:
Is it possible to treat cells with EMT-inducers in RPMI 5% FCS or 10% FCS?
Yes it is, of course, but this issue is cell line dependant.... also cell culture substrate stiffness is very important. But if you want to analyse certain concrete pathways or cell responses, serum background affects a lot. More over, albumin, proteases and other factors from serum besides many growth factors could modify the response to certein factors or varables that you try to study.
In some cell lines the cell culture in plastic induces directly this EMT process along passages. Anyway Dr. Tabasum asked a pertinent question... What is the cell line? primary cell culture, epithelial, tumoral, promesenchymal....?
I agree with David, serum has its own TGF beta as well as other factor capable of inducing EMT. So if we are studying specifically TGF beta mediated EMT and the downstream signaling isolated role of TGF beta would be more promising rather than the complex mixture of serum.
In our study (http://stke.sciencemag.org/content/7/345/ra91), we deliberately avoided serum starvation, trying to start with cells in a specific steady state. It will be interesting to compare the results with and without serum starvation.
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