I found differences concerning cell migration in my treated melanoma cells by chance. Unfortunately, I only have some weeks left in the lab and want to check for RNA and proteins, that are possibly influenced over Christmas and New Years.
I tried to read into it but there is just too many information to narrow it down to 10-15 tragets. Has anyone a suggestion what I could look for.
I work with human melanoma cells (SK-Mel-28). I saw changes in a wound healing assay and in the linked assay. There are also changes in colony formation. I repeated each experiment at least 7 times and each run was done in dupicates or even more. Proliferation is not affected.
I'm grateful for any suggestions.
Article An agarose spot chemotaxis assay for chemokine receptor antagonists
can check cytoskeleton proteins genes as actin tubulin being rearranged, likely signal transduction molecules as well and general morphology - nucleus can sometime go annular when moving
Hi Bettina,
I suggest you to look at the protein and mRNA expression levels of E-cadherin, in the first instance. It has been extensively demonstrated that it is clearly and deeply involved in melanoma cell migration.
The paper below could help you to figure out how to get quickly some results.
Just tell me if you need further help.
http://www.ncbi.nlm.nih.gov/pubmed/24035834
In my opinion you should focus on the genes/proteins associated with EMT (Epithelial to mesenchymal activation). To start with, check the protein levels for EMT markers like as Damiano said E cadherin (it goes down in the cells acquired the migratory behaviour i.e acquiring mesenchymal traits),Vimentin(goes up, highly studied protein in migratory and invasive behaviour), N-Cadherin (reported to goes up in EMT), cell junction proteins (this may go down as well, as cell loosing its epithelial phenotype and acquiring the migratory behaviour...so these may include checking protein expression from Desmoplakin 1/2, plakoglobins etc).....Thats it!...sufficient to start with for the welcoming year!
Hi,
You can also check for changes in the expression of Paxillin and phospho-Paxillin Y118, Src and Focal adhesion kinase and its phosphorylated forms. These proteins play important roles in cell adhesion and migration .
All the best to you.
Thank you all for your answers, its more or less what I found so far but its good to have some conformation. I'm going to order the primers today and hope I'll be done in two weeks.
I wish you all more relaxing holidays :D
other than those mentioned above i would like to suggest you to check beta catenin, vimentin, zo-1.integrins depending the cell lines. you can check various papers to conclude which integring you should check. some time it may be an epigenetic alterations which lead to migration of cells. so you may better check ROS and alterations in e-cadherin . if its altered then go for methylation experiment
what is the mechanism you focus on? and the TGF-beta singnaling pathway is a key pathway, and in the other the important factors such as MMPs, and so on.
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