Hi All,
I have a bit of headache to find good downstream target gene to track for NFkB and Myc pathway activity.
What I did was treating A549 cell with inhibitors of NFkB (BAY 11-7082) and Myc (10058-F4) for 24 hours and then tracking the downstream gene expression through first generating cDNA (qScript cDNA SuperMix) and real time PCR (Fast SYBR Green Master Mix).
I have tried following list of known NFkB and Myc downstream genes but they show no differences (compare to GAPDH control) or even induction by the inhibitors even though the amplification looks good.
Myc: CEBPB, TERT, CCND2, PTEN, PCNA, TP53, RPL19, E2F1, EIF4A1, EIF4B, EIF4E
NFkB: AGT, CFB, CSF2, CSF3, IFNA1, IFNB1, TNF, LTA, IL6, IL8, ICAM1
I don't know what I did wrong so I get this weird result!
Any suggestion of good downstream targets and possible way for me to fix the experiment would be most appreciated!
Best,
Lyra
Hello
Links here will help you
Good Luck
http://download.springer.com/static/pdf/294/art%253A10.1186%252F1476-4598-12-86.pdf?originUrl=http%3A%2F%2Fmolecular-cancer.biomedcentral.com%2Farticle%2F10.1186%2F1476-4598-12-86&token2=exp=1485885266~acl=%2Fstatic%2Fpdf%2F294%2Fart%25253A10.1186%25252F1476-4598-12-86.pdf*~hmac=5cb001a1cd79b1ef55e2b86605249d5e114f7175e891ee3af2ed95a7d5dc3295
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4300701/
http://www.nature.com/onc/journal/vaop/ncurrent/full/onc2016309a.html
Hello
Links here will help you
Good Luck
http://download.springer.com/static/pdf/294/art%253A10.1186%252F1476-4598-12-86.pdf?originUrl=http%3A%2F%2Fmolecular-cancer.biomedcentral.com%2Farticle%2F10.1186%2F1476-4598-12-86&token2=exp=1485885266~acl=%2Fstatic%2Fpdf%2F294%2Fart%25253A10.1186%25252F1476-4598-12-86.pdf*~hmac=5cb001a1cd79b1ef55e2b86605249d5e114f7175e891ee3af2ed95a7d5dc3295
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4300701/
http://www.nature.com/onc/journal/vaop/ncurrent/full/onc2016309a.html
You need to try the inhibitors in the absence and presence of exogenous factor you know will activate the NFkB and Myc pathways. As Alim mentioned, LPS is a good one. A time course will also allow you to determine when increases in gene expression occur following treatment.
I agree that you may need to activate pathways and then use the inhibitors to look for their downstream targets, at the same time may look at cell proliferation or apoptosis to see the change in kinetics of time or dose response. Both of the mentioned factors may affect complex pathways, as they regulate cell proliferation, differentiation, inflammation and apoptosis and when the various balances are markedly perturbed do play major roles in various cancers and thus are also affected by mutagens and stress factors.
Factors that you may try, TNF, TGFalpha/EGF depending on your cell type.
The links below may be of help. Hope this is of help to you. Good Luck!
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3032584/
http://www.nature.com/cdd/journal/v19/n12/full/cdd201291a.html
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