Does anyone have cell culture experience w/ human mammary epithelial cells &/or has tried to induce EMT in any cell line including epithelial cells?
Also any suggestions on doing scratch wound assays with human mammary epithelial cells would be helpful.
Thank you.
I have the following experimental experiences;
4T1 murine breast cancer cells were treated with recombinant TGF-beta for 12-96 hours and checked by quantitative RT-PCR and WB (E-cadherin, CD44 variant isoform, N-cadherin, vimentin, CD44 standard isoform, ZEB1)
MDA-MB-231 human triple negative breast cancer cell line was exposed to H2O2 and checked by by quantitative RT-PCR, FACS and WB (E-cadherin, CD44 variant isoform, N-cadherin, vimentin, CD44 standard isoform, ZEB1, Twist)
Adding EGF to the media can induce epithelial cells to undergo EMT. In this paper EGF induces EMT in MCF7 cells, which are probably the most commonly used breast epithelial cell line.
Article EGF induces epithelial-mesenchymal transition through phosph...
MCF7s are a breast cancer cell line, so if you want to use a non-cancer breast epithelial cell line, then MCF10A are an alternative option. MCF10A cells can be a pain to work with because they grow very slowly, take a relatively long time to trypsinise, and require many supplements for growth.
https://brugge.med.harvard.edu/system/files/protocols/MediaRecipesForMCF10ACells.pdf
Your life will be a lot easier if you can use MCF7s.
For scratch assays, here is a nice nature protocols paper which should be a good place to start.
https://www.nature.com/articles/nprot.2007.30
Best of luck!
Go J Yoshida , what was best concentration of TGF beta that you found induced EMT in the 4T1 cell line?
Eoghan Patrick McGrath , I completely believe you on the MCF7s as I have worked with them in the past and they are very hardy cells. Thank you for the link on the migration assay. I will take a look!
Do either of you have experience trying to induce EMT in a transfected epithelial cell line, particularly a primary epithelial cell line Go J Yoshida and Eoghan Patrick McGrath
About the concentration of recombinant mouse TGF-beta for the induction of EMT in 4T1 cells: mouse TGF-β1 5-10ng/ml
The period of the treatment with recombinant mouse TGF-beta for the induction of EMT in 4T1 cells: If you perform qRT-PCR, 24-48hours is recommended, but when you check the down-regulation of E-cadherin by Western blot analysis, you should treat 72-96 hours.
there is a range of materials imposing EMT on cancer cell lines; besides, I am fond of Go J Yoshida researches and I have read some of your articles they are great but I have a question about EMT subject. if I am not mistaken by TGF-β and all materials which can ignite reprogramming genes we could achieve EMT phenomenon, and it can be proved by phenotypically or genetically dissection (which is limited to some remarkable features). the question is; do you have any article or experience that shows in vivo EMT transformation by these materials? because I feel in order to have EMT cells, it has to be elicited from the blood flow (during migration), this form of EMT cell is genuine.
BEst,
pejman
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