I am checking the apoptosis and Necrosis -inducing effect of a drug on a MCF-7 cancer cell line.
I have done the Annexin staining showed that the drug induced both apoptosis and necrosis with treatment.
Please suggest which main proteins I should check for confirmation of necroptosis.
Hi Nikhil.
There is substantial interplay between the apoptosis and necroptosis pathways. At multiple stages of their respective signalling cascades, the two pathways can regulate each other, therefore there is no specific protein.
However, RIPK1, RIPK3 and MLKL including its phosphorylation status are good targets.
RIPK1 and RIPK3 auto- and transphosphorylate each other, leading to the formation of the necrosome.The necrosome then activates the pro-necroptotic protein MLKL via phosphorylation.
You could also use Necrostatin-1, which is an allosteric inhibitor of RIPK1, and see, if there is any difference in outcome.
We used it in one of our projects for hepatic I/R.
Article RIP1-Dependent Programmed Necrosis is Negatively Regulated b...
Dear Nikhil,
You can go for Bax, cytochrome c and caspases for apoptosis detection. However cyclophilin A could be used as a marker for necrosis.
I would also like to suggest you to go for nuclear fragmentation and DNA fragmentation assay to further distinguish between the apoptosis and necrosis.
Dear Nikhil,
cleaved caspase-3 (c-cas-3) can be used for apoptosis and cyclophilin A for necrosis
Good luck
cyclophilin A for necrosis
cleaved caspase-3
cytochrome-c in cytosol as well as in mitochondrial fractions
Bax/Bcl2 ratio
All these parameters will help you to confirm mitochondria mediated apoptosis.
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