Figure 1
Experimental characterization of NF-κB response to TNF and LPS under conditions of normal or inhibited translation. MEFs were stimulated with 10 ng/ml TNF or 1 μg/ml LPS in the absence or presence of 5 μg/ml cycloheximide (CHX). In the CHX + TNF and CHX + LPS costimulation experiments, cells were incubated with 5 μg/ml CHX for 30 min prior to TNF or LPS stimulation. (a) Schematic diagram of the NF-κB pathway. A detailed diagram can be found in Korwek et al.11. (b) Temporal coevolution of nuclear NF-κB and total IκBα. Following stimulation, cells were fixed and stained with antibodies for RelA (a subunit of NF-κB) and for IκBα. Representative excerpts from confocal images show cells at 0 (‘untreated’), 15, 30 and 180 min after TNF stimulation. See Supplementary Data S1 for corresponding uncropped immunostaining images. (c) Histograms (n = 466 for untreated cells and n = 1434 for CHX + TNF costimulated cells) show NF-κB nuclear translocation, defined as nuclear fluorescence normalized to the average whole-cell fluorescence, based on confocal images (see Methods for details). The CHX + TNF histogram is derived from confocal images for the 30-min time point of the CHX + TNF costimulation experiment, which corresponds to the observed peak of NF-κB nuclear translocation. µ denotes the mean values of the distributions. (d) Gene expression profiles of NF-κB inhibitors, IκBα and A20. Time profiles of relative mRNA levels of IκBα and A20 were obtained using digital PCR measurements. (e) Western blot of nuclear RelA (a subunit of NF-κB) in response to TNF and its densitometric quantification with HDAC1 as reference. Average ratio of NF-κBnuc(t = 0)/NF-κBnuc(t = 30 min) calculated based on 3 such independent experiments is 0.03, which allows to estimate the nuclear NF-κB fraction at t = 0, NF-κBnuc(t = 0)/NF-κBtotal, to be below 0.03. See Supplementary Data S2 for full-length Western blots.
