Loss of IRAK-1 causes decreased expression of <i>Lcn2</i> mRNA and protein <i>in vivo</i>.

<p>WT and IRAK-1^−/− C57/BL/6 female mice of 12 weeks old (6 each) were intraperitoneally injected with either 30 mg/kg of LPS or PBS for 6 hours. (A) Kidney tissues were extracted and subject to RNA extraction. <i>Lcn2</i> transcripts were measured by qRT-PCR assays and standardized against their respective controls (mice receiving PBS injections). Data is depicted as three separate mice (both WT and IRAK-1^−/−). *<i>P</i><0.05 (B) Protein lysates were extracted from the kidney tissues and subjected to Western blot. Blots were analyzed using LCN2 specific antibodies. The same blot was probed with β-actin as a loading control.</p

LPS stimulation induces a persistent induction of LCN2 in kidney fibroblasts.

<p>(A) LPS (100 ng/ml) induces a transient induction of <i>Tnfα</i> mRNA. (B) LPS (100 ng/ml) induces a persistent induction of <i>Lcn2</i> mRNA Transcript levels were measured by qRT-PCR as described above. The results are expressed as means +/− standard deviation performed in triplicate. (C) LCN2 protein levels persist after 24 hours of LPS stimulation. The levels of LCN2 were visualized by western blot.</p

Computational simulation captures the dynamic features of the experimental data.

<p>(A) The network motif responsible for the transient induction of <i>Tnfα</i> and the persistent induction of <i>Lcn2</i>. (B) Computational simulation of the transient induction of <i>Tnfα</i> and persistent induction of <i>Lcn2</i>. Circles denote actual experimental data. Lines represent computational simulations.</p

<i>Lcn2</i> transcription requires new protein synthesis, but <i>C/ebpδ</i> does not. C/EBPδ is recruited to the promoter of <i>LCN2</i> upon LPS stimulation in an IRAK-1 dependent manner.

<p>(A) Cyclohexamide, a protein synthesis inhibitor, blocks <i>Lcn2</i> transcription upon LPS stimulation. (B) The induction of <i>C/ebpδ</i> does not require new protein synthesis. (B) Wild-type kidney fibroblasts were untreated (DMSO) or pretreated with cyclohexamide 5 ug/mL for 1 hour. Immediately following pretreatment, the cells were stimulated with or without LPS for 4 hours. Transcripts were quantitated using qRT-PCR and standardized using <i>Gapdh</i> as the internal loading control. Each experiment was performed in triplicate. *P<0.05 (C) The recruitment of C/EBPδ to the promoter of LCN2 upon LPS stimulation depends upon IRAK-1. Nuclear lysates were subject to ChIP analysis using a C/EBPδ specific antibody. The arrow points to the specific amplification signal for <i>C/ebpδ</i> promoter. The * sign indicates a non-specific amplification.</p

<i>Socs1</i> and <i>Atf3</i>, negative regulators of TLR4 signaling, are not induced and IRAK-1 remains intact upon LPS stimulation in kidney fibroblasts.

<p>(A) The expressions of <i>Socs1</i> and <i>Atf3</i> were not induced after stimulation with LPS. Wild-type kidney fibroblasts were either untreated or treated with 100 ng/mL LPS for 4, 6, or 10 hours. <i>Socs1</i>, <i>Atf3</i>, and <i>Il-6</i> transcripts were measured by real time RT-PCR assays and standardized against <i>Gapdh</i> levels. Each experiment was performed in triplicate. Data is depicted as means +/− standard deviation. (B) LPS does not cause the degradation of IRAK-1. Wild-type kidney fibroblasts were either untreated or treated with 100 ng/mL LPS for either 2 or 4 hours. Whole cell lysates were harvested and analyzed by Western blot with IRAK-1 specific antibodies.</p

LPS induces transient recruitment of c-Jun to the promoters of <i>Tnfα</i> and <i>C/ebpδ</i>, and induces prolonged recruitment of C/EBPδ to the promoter of <i>C/ebpδ</i>.

<p>WT kidney fibroblasts were treated with or without LPS for a time course. Nuclear lysates were then subjected to ChIP analysis to examine relative binding to the promoter of <i>C/ebpδ</i> (A) or <i>Tnfα</i> (B).</p

Ordinary Differential Equations used in the model.

<p>Subscripts of parameters denote the direction of regulations. For example, <i>k</i>₆₁ represents the threshold for c-Jun (x₁) to activate C/EBPδ (x₆).</p

LPS induces transient activation of AP-1 and persistent induction of C/EBPδ in kidney fibroblasts.

<p>WT fibroblasts were treated with LPS (100 ng/ml) for the indicated time periods. (A) LPS fails to significantly induce IκBα degradation. Whole cell lysates were harvested and analyzed by Western blot using an IκBα specific antibody. (B) LPS fails to significantly induce p65 nuclear translocation in kidney fibroblasts. Nuclear cell lysates were harvested and analyzed by Western blot using an anti-p65 antibody. (C) LPS induces the active translocation of AP-1/c-Jun. Nuclear lysates were harvested and analyzed by Western blot. (D&E) LPS induces a persistent increase of C/EBPδ protein. Whole cell lysates were harvested and analyzed by Western blot. (F) LPS significantly induces the expression of <i>C/ebpδ</i> dependent upon IRAK-1. WT and IRAK-1 deficient cells were treated with LPS for indicated time period. Message levels of <i>C/ebpδ</i> were determined by RT-PCR. n = 3; *<i>P</i><0.05.</p

Details of the three priming mechanisms.

<p>(A) Backbone motifs (topological features shared by most of the good parameter sets) of each priming mechanism (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002526#pcbi.1002526.s003" target="_blank">Figure S3</a> and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002526#pcbi.1002526.s012" target="_blank">Text S1</a> for details). The width of a line is proportional to the mean value of the corresponding <i>ω_ji</i> among data sets under each priming mechanism. The “slow” and “fast” time scales reflect the values of γ<i>_j</i> in comparison to γ₃ = 1. (B–D) Typical time courses and corresponding phase space trajectories with or without LD pretreatment. Bistable results for AI and SD are shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002526#pcbi.1002526.s005" target="_blank">Figure S5</a>.</p

Description of modeling parameters.

<p>Description of modeling parameters.</p