Figure 8

Late gene expression requires the NF-κB oscillatory mode. (a) Experimental Strategy. Schematic diagram of the tonic and pulse stimulation paradigm. Parallel plates of cells were stimulated with TNF continuously ("tonic" treatment), without removing the agonist. Pulse stimulated cells were exposed to TNF to activate the NF-κB pathway (activation is maximal within 15 min of stimulation), whereupon the agonist is removed from the medium. At identical times after application of the stimulus, cells are harvested for gel shift (Figure 8b) or Q-RT-PCR (Figures 8c, d). (b) NF-κB-binding in tonic- vs pulse-stimulated cells. Nuclear extracts from tonic- or pulse stimulated HeLa cells were prepared and NF-κB-binding measured. Shown is an autoradiogram of the bound NF-κB complexes by EMSA. The specific NF-κB/Rel A and NF-κB1 complexes previously identified by supershift analyses are indicated at left (see Ref [21] for further details). (c) IκB proteolysis and resynthesis in tonic- vs pulse-stimulated cells. Cytoplasmic extracts from tonic- or pulse stimulated HeLa cells were prepared and abundance of IκB determined by Western blot. IκB is rapidly proteolyzed, with both treatments, however, the steady state levels are reduced 3 and 6 h in tonic treated cells compared to those pulse-treated. (d) Early gene expression profiles. HeLa cells were treated as in Figure 8a, total RNA extracted and mRNA abundance (normalized by 18S) determined by Q-RT- PCR. For each of the indicated mRNA transcripts, values are expressed as fold change relative to unstimulated cells and plotted on a logarithmic scale. (e) Late gene expression profiles. Samples obtained as in Figure 8d. The mRNA transcript measured is indicated for each plot.