NF-κB and STAT3 are essential transcription factors in immunity and act at the interface of the transition from chronic inflammation to cancer. Different functional crosstalks between NF-κB and STAT3 have been recently described arguing for a direct interaction of both proteins. During a systematic analysis of NF-κB/STAT3 crosstalk we observed that appearance of the subcellular distribution of NF-κB and STAT3 in immunofluorescence heavily depends on the fixation procedure. Therefore, we established an optimized fixation protocol for the reliable simultaneous analysis of the subcellular distributions of both transcription factors. Using this protocol we found that cytokine-induced nuclear accumulation of NF-κB or STAT3 did not alter the subcellular distribution of the other transcription factor. Both knockout and overexpression of STAT3 does not have any major effect on canonical TNFα-NF-κB signalling in MEF or HeLa cells. Similarly, knockout of p65 did not alter nuclear accumulation of STAT3 in response to IL-6. However, p65 expression correlates with elevated total cellular levels of STAT3 and STAT1 and supports activation of these transcription factors. Our findings in MEF cells argue against a direct physical interaction of free cellular NF-κB and STAT3 but point to more intricate functional interactions.

Interferons exhibit their key role of immune modulators through activation of the Jak-Stat signalling pathway. We know substantial amount of molecular details regarding functioning of the pathway. However, to what extend the action of the pathway is dose dependent at the single cell level remains largely unclear. Specifically it is not know if single cells respond in a digital fashion or their output is continuously dependent on the stimulant’s concentration. We have combined an information-theoretic framework with high-throughput confocal imaging of mouse embryonic fibroblasts to provide a thorough, single-cell analysis of the Jak-Stat signalling in response to interferon beta and interferon gamma. We showed that in a baseline state single cells have information hardly sufficient to distinguish between presence or absence of interferons. However they can be put in an alert state by an action of interferons, which allows them to respond more in an analogous fashion. Our results show that the accuracy with which signalling pathways transmit information is not fixed but can be modulated on the contextual basis.