I want to lead HEK293 cell into apoptosis pathway via TNFR1. TNF alpha cannot affect into apoptosis pathway alone.
Tran, I'm not sure this helps, but many previous studies using TNFalpha to induce apoptotis used actinomycin D, cyclohexaminde or INFgamma to pre-treat the cells. As TNFalpha induces both pro-death and pro-survival signaling, the pre-treatment with these agents acts to block the synthesis of pro-survival factors (NFkappa B, IAP1,2, ect) among other things, thus allowing for the pro-apoptotic factors to complete their task. I always preferred INFgamma as it, along with TNFalpha, are normally found in the circulation during inflammatory pathologies. As camptothecin is a TOP1 blocker, I'm not sure you will get the same effect.
Thank you for your help. There are some studies co-treat TNF alpha and camptothecin to induce apoptosis, but in another cell lines, not HEK293.
I will try TNF gamma as your suggestion.
I used PARP as a marker of apoptosis, and checking its increasing after treatment.
And I also treat only TNF alpha and checking ikB alpha for survival pathway.
Could you give me some experience about it?
Best.
Gin
Previously I treated MEFs with 20 ng/ml interferon-gamma for 18-24 hrs and then treated with 40 ng/ml TNFalpha. 30 min after TNFalpha, IkBalpha was mostly degraded. By 24 hrs the cells were 50% dead as determined by TUNEL. One might assume PARP to be cleaved at this point as well, but I did not measure this.
Tran Hue Vy An William Blalock
I tried treating human fibroblasts with IFN-y and TNF-a. What are the concentrations that worked for you Tran? I tried with 40 ng/ml IFN-y and 20-80 TNF-a, but they only died a little bit.
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