I have perform Hypoxic treatment on AML-12 cells for 12hrs and there is significant up regulation of RIP3 in western blot and no expression of cleaved casp3. I did flow cytometry with PI annexin and in Q2 theres more necrotic cells in hypoxic samples. I used gsk inhibitor to inhibit rip3 and it showed decrease in flow cytometry. So is it apoptosis or necroptosis. Also i havnot been able to see TNFA changes in treated cells. If its necroptosis, which other pathway or cell death initiator it can be?
Hello Raussi
I suggest you take a look at the most recent account of cell death modes and mechanisms, just out. This might help you navigate through the multiple and often mixed cell death pathways that tend to coexist more often than not.
Cell Death Differ. 2018 Mar;25(3):486-541. doi: 10.1038/s41418-017-0012-4. Epub 2018 Jan 23.
Molecular mechanisms of cell death: recommendations of the NomenclatureCommittee on Cell Death 2018.
Galluzzi L et al.
Hi Raussie,
Did you try to use zVAD alone or in combination with the GSK inhibitor? This might help in figuring it out. Or not -it's possible that you have both going on. Good luck!
Hello Raussi
I suggest you take a look at the most recent account of cell death modes and mechanisms, just out. This might help you navigate through the multiple and often mixed cell death pathways that tend to coexist more often than not.
Cell Death Differ. 2018 Mar;25(3):486-541. doi: 10.1038/s41418-017-0012-4. Epub 2018 Jan 23.
Molecular mechanisms of cell death: recommendations of the NomenclatureCommittee on Cell Death 2018.
Galluzzi L et al.
Hi! I would say that your case looks more like necroptosis than apoptosis. RIPK1 and RIPK3 activation without caspases activation is features of necroptosis. But it is important to show that there is no activation of caspase-8 (if you talk about necroptosis). And caspases activation is almost permanent feature of apoptosis.
Some additional tests to exclude (or to confirm) apoptosis might be made for example revealing of cytochrom C or apaf-1 in cytoplasm or activation of different caspases (-7, -8, -9)...or if it possible examine the morphology of cells after treating...necroptosis looks like necrosis and it can be easilly differentiated from apoptosis.
There is a number of receptors which can induce necroptosis, for example CD95L, TRAIL, Tweak...
Here is a couple of links that can be useful in your researching:
https://www.nature.com/articles/nrm3737
https://www.nature.com/articles/s41418-017-0012-4
Thank you Anastasiya. I have looked into cleaved caspase 3 and is it not expressed in my samples. I haven't looked to caspase 8. I am in process to look into RIP1. Thank you very much for your recommendation. I will certainly look into those.
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