I have been trying to show the difference in TGF-treated "activated" fibroblast from untreated mouse embryonic fibroblast (MEF) and according to literature, the difference in alpha smooth muscle actin protein profile in western blot is one thing that differentiate treated MEF from untreated MEF. I could not get that expression profile so I was curious if there are any other markers I can used to see a difference between TGF treated and untreated MEF?
First check SMAD activation - phosphorylation of SMAD2 and expression level of SMAD6 (TGFb target gene, inhibitory SMAD). Maybe just treatment did not work.
Will checking of Smad7 (inhibitory Smad) real-time PCR works? I have that primer with me.
Hi Daves,
as Jan said, it might be very useful to verify if your treatment worked or not, but doing a PCR isn't the best method at all. In my opinion you have to check the phosphorylation levels of SMAD 2/3 or their nuclear translocation. Regarding as your question, the main markers for activated CAF are α-SMA, fibroblast specific protein (FSP), platelet-derived growth factor (PDGF) receptors-β and fibroblast activation protein (FAP).
The attached papers may be useful.
Article Cancer associated fibroblasts: The dark side of the coin
Article Cancer-associated fibroblasts and macrophages: Friendly cons...
Oh great! I have already cloned my own Smad 2, smad3 and smad4 gfp construct. Hopefully that can tell me something. Regarding the markers, I am interested in S1004A (FSP-1) antibody. Just wondering if you have done Western blot on this before and does the FSP-1 expression reduces after Tgf beta 1 treatment?
No I haven't. I'm really sorry but I have never done it. I'm use to work with TGF beta but mainly focusing on its EMT-inducing activity.
Oh. I understand. My lab is focusing on the same thing too. I am the only one dealing with CAF. Thanks for taking time to answer Alessio! Thanks!
Hi Daves,
Besides checking that TGFb was truly active and verifying that your MEFs are responsive to it by using another cell type as a control, you should also consider that very ofter cultured MEFs differentiate into myofibroblasts. This is due to the stiffness of the plastic and, in practical terms, it means that SMA could be upregulated even in your control MEFs.
If this was what you have observed, you may want to repeat these experiments using fresh MEFs cultured on a soft substrate.
Good luck!
Metello
I have recently used another immortalised mouse embryonic fibroblast cell line from another lab and I have managed to obtain an increase in Smooth Muscle Actin expression using Western blot . I carried out the same experiment for my own lab's mouse embryonic fibroblast but for this I was unable to get an increase in the smooth muscle actin protein expression using the same membrane for western blot.
I am beginning to suspect something has happened to my cell line which goes along with what Metallica suspected. How do I check this? May I know what is the protocol used to grow MEF on soft substrate? What do you mean by fresh? Do you mean primary cell?
Thanks!
Yes. I have read up that CAFs expresses reduced amount FSP-1 as it becomes less fibroblastic and more myofibroblast.
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