Figure 1.
Comparison of single-cell NF-κB activation dynamics for three different LPS preparations.
Intensity represents the relative nuclear localization of p65-dsRed fusion protein, calculated as mean nuclear intensity divided by initial mean cytoplasmic intensity. The concentration for all three preparations was 0.5 µg/mL. The black line shows the average time course for all cells; the light blue traces are ten randomly selected individual cells. The number of active cells (N), as well as the maximum peak amplitude (Peak Amp), and time elapsed until the maximum amplitude is reached (Time to Peak) are also shown. The maximum intensity is indicated by a dot and the two dashed lines indicate how Peak Amp and Time to Peak are determined. The duration of the first peak (Peak Width) is also shown. This value is determined by drawing a horizontal line at the intensity that is halfway between the minimum and maximum peak value. The region above the line and shaded in green denotes the time during which the p65-dsRed nuclear intensity is more than half of the maximum p65-dsRed nuclear intensity. Below each plot, corresponding representative microscope images are shown for the first 200 minutes after stimulation, as labeled.
Figure 2.
The potency window for each of the LPS preparations.
The fraction of active cells is plotted as a function of concentration for values spanning nine orders of magnitude, as shown.
Figure 3.
Activation dynamics for each of the LPS preparations and several concentrations and summary statistical comparisons.
(A) As in Figure 1, the average and ten randomly selected traces of active cells are shown, as well as Time to Peak, Peak Amp and N. See Figure 1 legend for more details. Concentrations are indicated at left, and the preparation at top. The very lowest and highest concentrations are not shown here (but appear in Figure 4), because virtually no cells were found to be active in the first case, and the traces are essentially identical to their nearest neighbor in the second. (B) Time-to-peak values for each LPS preparation are shown with standard deviations, across each concentration. LPS preparations are indicated with different colors as labeled in D. (C) Peak amplitude values for each LPS preparation are shown with standard deviations across each concentration. (D) The correspondence between time-to-peak and peak amplitude is shown for each LPS preparation, including all concentrations.
Figure 4.
The similarity between average activation profiles across all concentrations and preparations.
Similarity is calculated as cosine distance between pairs of average activation profiles. Pairs of time courses that are similar have a low vector distance; hence, the dark squares indicate similarity. The activation profiles are grouped by LPS preparation and sorted by concentration.
Figure 5.
Blocking paracrine signaling by TNF across all concentrations and preparations.
The average time course for N number of active cells is plotted for cells stimulated in the absence (blue trace, top value of n) and presence (orange trace, bottom value) of sTNFRII, which competes to bind TNF. Concentrations are indicated at left, and the preparation at top.
Figure 6.
The effect of blocking TNF on the NF-κB activation time courses as a function of concentration and preparation.
The cosine vector distances between the average time courses in the presence and absence of sTNFRII are shown. Almost no cells were activated by UP LPS at low concentrations; hence the dashed light blue line is included for completeness but is not statistically defensible.