I am investigating a process called epithelial-mesenchymal transition (EMT) in which epithelial cells lose their typical characteristic and acquire mesenchymal markers. Cells undergo EMT disorganize, lose cell-cell adhesion junctions, assume a spindle-shape morphology. Changes are evident also in cytoskeletal reorganization (vimentin, alpha-SMA, catenin..). Therein, I am wondering if classical actin or tubulin could be right housekeeping for proteins expression evaluated by western blotting. Red ponceau could be an alternative? Anyone working with cell morphology alteration?
Hi,
We use Actin and it works fine with cells before and after EMT induction in breast cancer cells.
Best
You may also try to evaluate the ration of CD44/CD24 cells in the lesion.
Thank you Konrad! I have the same doubts about actin and CD44. Do you think that GAPDH is not affected by metabolism change in EMT?
dear,
you can try RPLO (mitochondrial protein), less sensitive to actin remodeling and study.
best
Yes I know Konrad. :)
Thank you Yves, I don't know it, but I will try.
I wouldn't trust actin as a loading control if you're looking at EMT. Actin stress fibers are dramatically remodeled during mesenchymal transition.
http://www.molbiolcell.org/content/22/24/4750.long
In my experiments, i've seen consistent actin increase upon treatment with TGFB1. Also see figure 3 in the above link. Having said that, i don't think there is an upregulation of actin expression per se. It might be that the thick stress fibers formed during EMT are not being completely disassembled upon cell lysis and some of it pellets down along with the cell debris during centrifugation. I have been using GAPDH as a loading control and it seems quite consistent even after lengthy TGFB1 treatments (up to 10 days).
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