I am targeting NF-kB pathway in cancer cells, and my drug may block this pathway. According to my hypothesis, I will use western blot and qRT-PCR to confirm this effect. Should I use a positive control to study its effect in this pathway? if yes, which positive control I should select?
Dear Bashir,
If you are studying NF-kB then the toll like receptor (TLR) pathway should be expressed in your system. If that is the case, the best NF-kB activator I know is Lipopoysaccharide (LPS), that is recognized through TLR4. This is the strongest activator of NF-kB I am aware of. Upon activation, you could easily detect a many different molecules that are expressed downstream NF-kB, such as TNF-alpha, IL1-beta, CD69 and many others.
It would be very interesting to see if your treatment blocks the activation promoted by LPS! If you could combine both your treatment and LPS stimulation, you would unequivocally show that your drug blocks NF-kB activation, upon a strong stimulus.
Finally, I would never use a different cell line as a positive control, because NF-kB expression depends on the cells you are using!
Good luck.
Cheers!
Paulo
Hi Bashir,
Epithelial (HeLa), gastric (NCI-N87) or colorectal (MDST8) cancer cells could be good positive controls for your experiment to prove the hypothesis.
For further reading you can go through the attached review article.
Best-
Hi Bashir,
I agree with Amritlal. The cell line where you are using the drug, the no-drug negative control can also be your positive control for the cell population. If you are talking about WB and qRT pcr, I guess betaactin should be a good choice because others like tubulin or may be GAPDH does show some alternating effects also. So, bactin should be your molecular positive control.
I recommend you to do a combined NFkB and phospho-NFkB WB.
There is a basal level of NFkB activity in many cells, which can be induced by TNFa (this could be used as positive control).
qPCR can be used to monitor regulation of the NFkB target genes rather than NFkB itself. There are many target genes you may choose depending on your cells/tissue of interest.
Dear Bashir,
If you are studying NF-kB then the toll like receptor (TLR) pathway should be expressed in your system. If that is the case, the best NF-kB activator I know is Lipopoysaccharide (LPS), that is recognized through TLR4. This is the strongest activator of NF-kB I am aware of. Upon activation, you could easily detect a many different molecules that are expressed downstream NF-kB, such as TNF-alpha, IL1-beta, CD69 and many others.
It would be very interesting to see if your treatment blocks the activation promoted by LPS! If you could combine both your treatment and LPS stimulation, you would unequivocally show that your drug blocks NF-kB activation, upon a strong stimulus.
Finally, I would never use a different cell line as a positive control, because NF-kB expression depends on the cells you are using!
Good luck.
Cheers!
Paulo
I agree with Paulo. LPS is the best; try 100-300 ng/ml, you'll see NF-kB in the nucleus as early as 10 and 20 min. If you will analyze by western blot you'll be able to study prompt responses which can be useful when standardizing protocols.
Good luck.
Thanks for the useful and helpful suggestions and comments,
Paulo and Esmeralda, can I use PMA (Phorbol 12-myristate 13-acetate) instead of LPS? because it is avaliable in our lab.
other question: Should I compare the effect of my drug with a known drug that block NF-kB pathway as a positive control? if yes, what the suitable drug I should select?
Any suggestions please.
Dear Bashir,
I am glad I can help you.
First question: PMA stimulates other mechanisms apart from NF-kB. I would avoid using PMA for that purpose. Most immunology labs have LPS. You may ask a collaborator or a nearby lab for an aliquot.
Second question: Yes, using a NF-kB inhibitors could be a good control. There are many different pharmacological NF-kB inhibitors with varying degrees of activities. However this inhibition is partial and not total. I would rather prefer a biological inhibition based on siRNA, miRNA or others.
Good luck!
Cheers,
Paulo
Dear Paulo,
Thanks v much for your valuable answers, and it would help me a lot.
I am using colon cancer cell line HCT-116 and COLO-205, and I am not sure LPS could be a good NF-kB activator for these 2 cell lines or not?
Can u help me please?
Regards,
Bashir
Dear Bashir,
Yes, LPS is a good NF-kB activator in both cell lines you mentioned. But don't take my word for it. Please verify it on PubMed, by searching for your cell lines and LPS. You will find other papers that did similar work to yours. Also you can get nice ideas for experiments by looking at those papers.
Good luck!
Paulo
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