I'm attempting to conduct a study involving the epithelial-mesenchymal transition in lung cancer cells. In order to do so, I've been trying to biochemically confirm whether the cells are epithelial or mesenchymal (in addition to morphological confirmation). For some reason, the A549 (ATCC) cells are not expressing E-cadherin, a hallmark protein that is downregulated during EMT, when the literature says they should. I've tried various lysis buffers but to no avail (1X, RIPA, 8M Urea). In addition, I lysed another cell line (MCF7) with the same buffers which did show E-cadherin expression. So I think I can safely rule out problems with lysis. Perhaps it's the media I'm growing my cells in? I've been growing them in DMEM F12. Anybody else work with A549 and could offer some insight?
I have already worked with that cell line and A549 expressed E-cadherin. I used to grow them in RPMI 1640 medium. To exclude for sure your lysis protocol you can plate and fix the cells (with methanol for a maximum of 10 minutes in cold ice for instance) and do an immunofluorescence for E-cadherin.
See:Int J Biochem Cell Biol. 2013 Dec;45(12):2801-7. doi: 10.1016/j.biocel.2013.09.018. Epub 2013 Oct 10
I suspect your A549 is not what you think
Hmm, I cultured some cells in RPMI as well, and the result was the same. Was there anything in particular you wanted me to look at in this article?
TGFbeta is used as a positive control in this report. I am guessing that your A549 are not A549. ATCC may sell primers to confirm identity. But, I recommend purchasing a new A549 stock. Good luck and let us know the results.
I have already worked with that cell line and A549 expressed E-cadherin. I used to grow them in RPMI 1640 medium. To exclude for sure your lysis protocol you can plate and fix the cells (with methanol for a maximum of 10 minutes in cold ice for instance) and do an immunofluorescence for E-cadherin.
Thank you all for your suggestions!
Recent observations suggest that the source of the problem is most likely either the cells themselves or the media. After running an immunoblot on cells treated with TGF-Beta, it was found that they expressed high levels of vimentin, a protein that is upregulated during EMT. In addition, it was found that the controls also expressed vimentin (albeit, at lower levels). This in conjunction with the observation that the untreated cells lack E-cadherin suggests that the A549 cells in the laboratory stock are more-so mesenchymal than expected.
So the next step will probably be to grow the cells in different media, such as the RPMI 1640. If that doesn't work, then it looks like the lab will have to look into purchasing some new stock.
On a side note, won't scanning for E-cadherin via immunofluorescence be messy considering it's a transmembrane/surface protein and may lead to a lot of non-specific binding? Daniela, how was your experience in this regard?
How do your cells look? Do they form colonies or are they growing as single cells? It might be better to obtain some new cells. If they have undergone EMT in the lab you might struggle to find E-Cadherin ever again. I worked with them in the past and growing them in 3D or addition of HGF (or expression of MET) reduces E-Cadherin, but they really should have lots of E-Cadherin to start off with.
They seem to be forming colonies (https://www.dropbox.com/s/a2dh2dqm0fbprzu/a549.jpg). Yeah, from what I've been reading E-cadherin expression should be relatively large, however Westerns are returning nothing. One thing that is being considered is dosing with EGF to stimulate E-cadherin expression.
I agree that you see some colonies and I am pretty sure these are A549 cells. However, they look very similar to when I incubate these cells in HGF. Have a look at the pictures from ATCC : http://www.atcc.org/~/media/Attachments/2/6/1/7/1753.ashx
You can see that the colonies under normal conditions should be a bit tighter unless you made the picture a few hours after plating, but I guess from the size of the few colonies that is not the case. Also I think your cells have rounded up a bit more, which looks as if they are more motile, another feature of having undergone EMT. If you really want to study E-Cadherin in these cells I guess the best thing would be to try to get new cells or maybe try an earlier passage if possible.
The photo was taken roughly 24 hours after passaging the cells (third passage). I'll try blotting again for vimentin before getting new cells. Thanks!
Hello Zubair Amin,
It was completely possible for me to see the staining of E-cadherin in those cells. A lot of papers published already beautiful pictures of E-cadherin staining among different cell lines. I was also inducing EMT (by another process that not TGF-beta) and I could see a considerable reduction of E-cadherin staining. Of course that it's not the best method to quantify it, it only gives you evidence of the cell behaviour and if the cells that have more fibroblastic-like phenotype are the ones that express more vimentin and no E-cadherin.
Hope it helps.
Good luck!!
HI zubair,
I am working with the same model, A549 for induction of EMT with TGF-Beta. And yes A549 express E-cadherin in large amount i have done both western and immunofluorescence and both shows high level e-cadherin as these are alveolar epithelial cells. However, I have observed at high passage no (above 30-40) there is a drastic change in the morphology and the cells looks like mesenchymal. What is the passage no. of your cells?
Hi!
We had a similar problem earlier with A431 cells ..However, we were able to detect E-cadherin after scraping the cells in cold PBS. Are you trypsining ur cells after tgf treatment or scraping them?
Hope this helps.
Good Luck!
Hi Zubair Amin ,
Just wanted to check if addition of EGF helped induce E-cadherin in A549 cells. If yes, what concentration did you use and for how long?
Thank you.
Hi Maithilee Khot ,
I believe we (John Livesey) tried adding EGF along with a few other changes to discourage motility, it didn't make a difference.
Zubair
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