Dear All,
I am study FasL protein expression using western blot in oxidative stress-induced cell death in SH-SY5Y. My results mostly shows that FasL expression is non-significant changed or reduced in H2O2-treated group compare to Ctrl group. The H2O2-treated group is death around 50% after 24h treatment by MTT. So according to theory FasL should be increased, shouldn't it.
I alos notice that many papers usually measure FasL mRNA rather than its protein. Not sure western blot may not be a good method for detecting FasL.
Thank you
Probably your oxidative stress conditions are too harsh so that you get necrotic cells instead of apoptotic cells. What is the hydrogen peroxide concentration you apply?
Regards, Christian
Dear Christian, I use 200uM of H2O2, and rum experiment on time dependent effect of H2O2 in SH-SY5Y for 30m, 1h, 2h, 4h,6h, and 24h. Collect the cell, determine its protein with western blot. However, the signal doesn't significantly increased comparison to the control/untreated group.
I suspect that the SH-SY5Y cells react very differently to apoptosis stimulation. I found an interesting publication (please see attachment) that shows that SH-SY5Y cells show no TNFalpha-induced increase of Fas levels nor sensitivity to the apoptosis inducers etoposide/cisplatin whereas SK-N-AS and SK-N-SH cells react in a more conventional manner. Maybe you could use a different cell line to see whether it displays an up-regulation of Fas levels under the conditions you use?
Cheers, Christian
Article TNFα sensitizes neuroblastoma cells to FasL-, cisplatin- and...
Dear Asa, many neuroblastoma cells are known to have silenced expression of caspase-8 (nevertheless caspase-10 expression might still be present, however little publications are available on this subject). Therefore, if you want to assess the role of FasL in oxidative stress-induced cell death, the neuroblastoma cell line SH-SY5Y is probably not the best model since these cells are frequently described to lack capsase-8 expression.
In your experiments, the cell death you are observing is likely induced by another pathway than the Fas / death receptor activated pathways. For example, activation of the intrinsic/mitochondrial apoptotic pathway, necroptosis, etc
To asses whether you are dealing with apoptosis or necroptosis you might want to use specific inhibitors of these pathways such as qVD (apoptosis/ pan caspase), IETD (apoptosis/caspase-8), or Nec-1 (necroptosis).
My recommendation for if you want to study the role of FasL in oxidative stress-induced cell death is to use another cell line, due to the complications you will have explaining how FasL induces cell death in a cell line that lacks caspase-8 expression (SH-SY5Y). As mentioned by Christian, if you want to use neuroblastoma cells, SK-N-AS and SK-N-SH cells are probably the way to go.
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