I am treating adipocytes with IL-6 and TNFalpha to mimic obese stress conditions (in a VERY simplified manner). How can I prove that these two cytokines are effective on the day that I am treating my cells? (I guess I am looking for independent positive controls?) Can I just show activation of downstream signaling molecules, such as JAK/STAT and MAPK or NFkB? And should I show this in another cell type or via my adipocytes?
Thanks!
I think kinetics will be the best approach to understand that effect of cytokines on your adipocyte cells; And to estimate the result, checking of downstream protein marker would be a great idea (as you mentioned). The approach will get you an idea about the timings and the concentration required to generate the stress condition. Further, you can use the same to mimic the desired condition and treat accordingly!
Hope, the idea can be of any use.
I think that JAK1/2 and Stat3 should be good IL-6 downstream targets to look at. If you detect activation of JAk and stat that should prove the effect of IL-6, at least partially. It would be kind of positive controls. You can also use inhibitors of JAK as negative controls in the same set of experiments. For TNFalpha - Nf-kB should be a good target to check. You can also check PKC activation or ceramide production. It depends on your research question which targets are more appropriate. are you looking at the link from adipose tissue to cancer? Or at the link between adipose tissue and cancer microenvironment/inflammation?
STAT3 phosphorylation using a phospospecific antibody for IL-6. You have to check if you need to add soluble IL-6R to the media for your cells. Better to stay in starving conditions to limit basal activation. For TNFalpha, look at NF-kB (nice if possible, to analyze NFkB nuclear translocation in confocal, or phospho-IKB o NF-kB ser 536 in WB), or phospho ERK1/2
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