MDP-induced mRNA expression of NALP3 and caspase 1.

<p>RT-qPCR of mRNA of inflammasome member NALP3 (A) and caspase 1 (B). Arbitrary units. Isolated monocytes were stimulated for 24 hours. Stimulation with MDP increased the level of caspase 1 expression in control cells (p<0.03), whereas such an increase was not found in CD monocytes.</p

Kinetics of MDP-induced mRNA expression of TNF-α and IL-1β.

<p>RT-qPCR of mRNA of TNF-α (A) and IL-1β (B). Kinetic studies of mRNA-response (C and D). Isolated monocytes were stimulated for 5 minutes (0.08 hours), 4 hours and 24 hours. Stimulation with MDP increased the level of mRNA transcription in monocytes from control subjects (p<0.01 and p<0.02 for TNF-α and IL-1β, respectively), whereas an upregulation was not seen in monocytes isolated from CARD15 non-mutated CD patients. Arbitrary units. Due to high variability in IL1β expression in both control and CD patients, these expression data were normalised to expression values at t = 0 hours. *p<0.05; **p<0.01.</p

Expression of inflammasome related proteins.

<p>Immunoblotting of NALP3, ASC, and CARD8. Monocytes expressed all these proteins involved in caspase 1 maturation.</p

MDP-induced expression IL-1β and activation of p38 and IKKα/β.

<p>Immunoblotting of IL-1β (A), p38 (B), and IKKα/β (C). Quatitative IKKα/β phosphorylation, bars represent ranges (D). Monocytes were stimulated for 24 hours with MDP. IL-1β expression was increased in CD patients, but no response was seen to MDP. Monocytes stimulated with MDP had increased p38 phosphorylation, regardless of disease status. Contrary to the p38 response, reduced IKKα/β phosphorylation was seen in CD regardless of CARD15 status. Control monocytes did respond to MDP stimulation by increasing IKKα/β phosphorylation. CD: Crohn's disease. Crohn*: SNP8 homozygote.</p

Caspase 1 processing and IL-1β release.

<p>Immunoblotting of caspase 1 (A). CD patients had increased basal expression of pro-caspase 1 and active cleaved caspase 1. No response was seen to MDP stimulation. ELISA measurement of released active IL-1β (B). Media was taken from monocytes stimulated for 24 hours with MDP. No differences in IL-1β secretion were detected.</p

Overrepresentation analysis for predicted transcription factor binding sites using Primo on the 1^st principal component.

<p>Overrepresentation analysis for predicted transcription factor binding sites using Primo on the 1^st principal component.</p

The three most important PC1 and PC2 probes in both the positive and negative directions.

<p>The probes have been selected by sorting the loadings from the PCA. The confidence intervals for the mean of each group (control, serumInhib and serumOnly) are plotted for each probe set ID.</p

Description of the PRIMO algorithm.

<p>This figure shows the calculation of a position weight matrix (PWM) score for a specific DNA sequence. The sequence window under calculation is shown at the bottom in capital letters. To the left is the PWM, which can be obtained from the Transfac or Jaspar databases. A value for each position was calculated based on the PWM value for the specific base. Underflow and the trivial zero result (if zero occurs in the PWM) were avoided as indicated.</p

Selfcontained test for overrepresentation (p<0.001) of GO terms in genes with higher expression in serum stimulated cells compared to controls (FDR<0.05).

<p>Selfcontained test for overrepresentation (p<0.001) of GO terms in genes with higher expression in serum stimulated cells compared to controls (FDR<0.05).</p

Principal component analysis score plot using pcaInfoPlot().

<p>This plot shows the output from the function pcaInfoPlot(). This function makes a principal component analysis score plot and applies functional annotation to the axis. The plot shows the experiments of the three experimental groups (control, serum only and serum with inhibitor) separated into three clusters. The 1^st principal component (PC1), which contained 21% of the variance, shows the differences in gene expression caused by the inhibitor and the serum. The serum-only group (serumOnly) is in the most negative direction. The control group is in the middle. The serum with inhibitor (serumInhib) group is in the positive direction. The 2^nd principal component (PC2), which contained 16% of the variance, shows the effect of the added serum. The control group is in the positive direction, and the serum-supplemented groups (serumOnly and serumInhib) are in the negative direction. Each axis is functionally annotated with the five most significant GO terms (biological processes) and the five most significant overrepresented predicted transcription factor binding sites.</p