We found that konockdown of a gene didn't cause apoptosis in cancer cells, but its specific inhibitor treatment induced massive apoptosis. How to explain this phenomonon? Could this result be simply due to a not very effective knockdown of this gene? Or its inhibitor not so specific? Does anyone meet with this situation,too?
Hi,
If you could be more specific about which target? Which inhibitor? Is it p53 prot?
Thanks
I had never had a situation like that, but I will think that your inhibitor is not very specific and could have used a concentration that it start hitting other proteins. Also you could had used a too high of a concentration, try lowering the dose.
The other option could be the sh or si RNA wasn't effective. Did you check by WB if your gene if your protein is completely gone, partially gone or still there compared to your control?
Good luck!
Hi Zhen, I have some questions regarding your issues:
1) How much you knock down your target protein? 50%? or less? Is there any difference of the cell phenotype compared to your control?
2) What's the expression level of your protein when using the inhibitor? If the protein is totally gone when using the inhibitor, which probably means the leftover protein in your knockdown cells still works.
3) What's the mechanism of the inhibitor? Does it block protein synthesis or just inhibit its activity? For example, if your target protein is an enzyme, only a little bit of it is enough for the whole cell's use. in this case, generating knockout cells might be one solution; furthermore, other family member(s) could compensate for the loss of the target protein.
4) Did the cells exhibit dose- or time-dependent response when using the inhibitor? It might be the nonspecific effect of the inhibitor.
Hi Han, Thanks a lot for your constructive suggestions. Our target is an enzyme that participate into biosynthesis of nucleotide. And efficiency of the target protein loss is about 70%. So it might be the situation as you said that only a little bit of it was enough for the cell's use. I should have heard your suggesions earlier. But our project is coming to an end. In our study, we used doxorubicin or actinomycin D or temozolomide to treat the silenced cells and the control groups, and the result showed that silenced cells was more sensitive to these drugs-induced apoptosis. Do you think it is meaningful to do research like that? Now, a reviewer asked a question like I asked as above. How can I explain it in our disscussion and rebuttal letter? Could you give me some suggestions? Thank you very much!
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