Hi all
I use a compound to induce cell death and found it could be enhanced by zVAD-FMK. So we could exclude the apoptosis and proptosis and considered it might be necroptosis. However, necrostatin-1 had no effect on it.
According to current cell death types we knew, can I confirm it is autophagy if it can be inhibited by PI3K inhibitor? Any other experiment I should do to confirm it?
Thank you so much.
some more information:
TUNEL: negative
caspase 3/7 activity: negative
zVAD: up to 50uM, pretreatment for 1h before compound
Necrostatin-1: up to 50uM, pretreatment for 1h before compound
You also have to exclude the possibility of PARP-mediated necrotic cell death due to the exhaustion of NAD and ATP. PARP-mediated necrosis can be analyzed with DPQ, which is a specific inhibitor to PARP. Furthermore, WB analysis of PAR (poly-ADP ribose) should be performed. When DNA damage is the critical cause, the smear band of high molecular weight can be detected.
Even when you can observe attenuated degree of cell death with 3-MA treatment, you have to keep in mind that 3-MA inhibits class III PI3K, which influences other biological responses than autophagy. Given that bafilomycinA1 itself is very cytotoxic, the treatment with chloroquine might support the possibility of autophagic cell death.
Article Zong WX, Ditsworth D, Bauer DE, Wang ZQ and Thompson CBAlkyl...
I think that you should have a more than in depth reading of the attached articles.
"Then", I am sure that you will have a pretty more good idea ...
Best regards
Robert
PS for Go: thank you Go for sharing with us this interesting article!
Thanks so much Dr. Kiss and Yoshida
Actually I am new for cell death field. Thanks again for your kind suggestions and papers. It's really helpful.
Dear Shujun,
I actually wish you good luck in this more than complex scientific area!
Best regards
Robert
Hi Shujun,
Yes I think you can test if the mode of death is autophagic if you inhibit autophagy pharmacologically or genetically (ATG5 KO or KD). Try looking at autophagy markers too like LC3-II or if you want to be a bit sophisticated probe for p62. I had encountered a similar scenario before when working with viruses (please see https://www.researchgate.net/publication/260112146_MTORp70S6K_signaling_distinguishes_routine_maintenance-level_autophagy_from_autophagic_cell_death_during_influenza_A_infection or http://www.sciencedirect.com/science/article/pii/S0042682214000117). It was a difficult project but with team work we were able to demonstrate some mechanism behind the pathology we were studying. Wishing you best and let me know if you have more questions!
-Emmanuel
Article MTOR/p70S6K signaling distinguishes routine, maintenance-lev...
Hi Shujun,
Note also that in some cases zVAD-fmk can upregulates caspase-9 cleavage and activity and stimulate an apoptotic like cell death.
http://www.ncbi.nlm.nih.gov/pubmed/22613767
http://www.ncbi.nlm.nih.gov/pubmed/10544235
Best regards
Hi Emmanuel and Bernard
Thanks you for the suggestions and papers. I'll read them carefully. Cell death is so complicated and always impact on each other.
Good luck to all of you!
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