(A-14) NF-κB Dynamics in Response to Dual Stimulation

Cells are influenced by their environment through the activation of signaling pathways.  The inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1β (IL-1) are two separate signaling pathways where both activate transcription factor nuclear factor κB (NF-κB) via distinct receptor complexes to regulate cell fate decisions.  Upon binding to their respective receptors, both cytokines recruit and activate multiple copies of the IκB kinase (IKK) complex to receptor-associated protein assemblies  Subsequent phosphorylation and degradation of NF-κB inhibitor proteins (IκB) leads to accumulation of NF-κB in the nucleus and transcription of NF-κB inducible genes.  

Previously, CRISPR-Cas9 technology was used to generate double knock-in U2OS cells that express fluorescent protein fusions from the endogenous loci. The NF-κB essential modulator (NEMO) subunit of the IKK was tagged with eGFP, and RelA, a subunit of NF-κB, was tagged with mCherry.  Upon stimulation with TNF or IL-1, EGFP-NEMO is observed to form fluorescent puncta, which represents the recruitment and activation of IKK at receptor-associated protein assemblies.  Similarly, mCherry-RelA is observed to translocate from the cytoplasm to the nucleus when NF-κB is activated.  It was previously shown that IL-1 stimulation produces a stronger response than TNF stimulation, but cells may be exposed to multiple cytokines in their natural environment.  This work uses concurrent treatment with IL-1 and TNF to study the effects of dual-stimulation on NF-κB signaling.

CRISPR-modified U2OS cells were cultured in McCoy’s 5A media, supplemented with 10% Corning regular fetal bovine serum, penicillin (100 U/ml), streptomycin (100 U/ml), and 0.2mM L-glutamine (Invitrogen).  Cells were seeded in no. 1.5 glass-bottom 96-well imaging plates 48 hours before cytokine treatment at a density of 4000 cells per well.  On the day of the experiment, media was changed to phenol red-free FluoroBrite DMEM.  Live cells were imaged in an environmentally controlled chamber (37°C and 5% CO₂) on a DeltaVision Elite microscope.  Cells were stimulated with 100 ng/ml IL-1, 100 ng/ml TNF, or both.  Cells were imaged for 3 hours at a frequency of 4 minutes after treatment.  

EGFP-NEMO spots were detected and quantified using dNEMO, a custom computational tool for detection and measurement of fluorescent puncta.  Nuclear mCherry-RelA was quantified as the relative fold change of the nuclear intensity using ImageJ.  For each cell, the number of IKK puncta over the observation period can be calculated as the area under the curve (AUCNEMO) of single-cell IKK spot trajectories.  AUCNEMO can be used to describe the overall activity of IKK in response to cytokine stimulation.  Further, the area under the curve of single-cell RelA trajectories (AUCRelA) represents the overall activation of NF-κB.