19 research outputs found
Loss of IRAK-1 causes decreased expression of <i>Lcn2</i> mRNA and protein <i>in vivo</i>.
<p>WT and IRAK-1<sup>ā/ā</sup> 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<sup>ā/ā</sup>). *<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 Ī²-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>TnfĪ±</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 +/ā 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>TnfĪ±</i> and the persistent induction of <i>Lcn2</i>. (B) Computational simulation of the transient induction of <i>TnfĪ±</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/ebpĪ“</i> does not. C/EBPĪ“ 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/ebpĪ“</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/EBPĪ“ to the promoter of LCN2 upon LPS stimulation depends upon IRAK-1. Nuclear lysates were subject to ChIP analysis using a C/EBPĪ“ specific antibody. The arrow points to the specific amplification signal for <i>C/ebpĪ“</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 +/ā 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>TnfĪ±</i> and <i>C/ebpĪ“</i>, and induces prolonged recruitment of C/EBPĪ“ to the promoter of <i>C/ebpĪ“</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/ebpĪ“</i> (A) or <i>TnfĪ±</i> (B).</p
Ordinary Differential Equations used in the model.
<p>Subscripts of parameters denote the direction of regulations. For example, <i>k</i><sub>61</sub> represents the threshold for c-Jun (x<sub>1</sub>) to activate C/EBPĪ“ (x<sub>6</sub>).</p
LPS induces transient activation of AP-1 and persistent induction of C/EBPĪ“ in kidney fibroblasts.
<p>WT fibroblasts were treated with LPS (100 ng/ml) for the indicated time periods. (A) LPS fails to significantly induce IĪŗBĪ± degradation. Whole cell lysates were harvested and analyzed by Western blot using an IĪŗBĪ± 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/EBPĪ“ protein. Whole cell lysates were harvested and analyzed by Western blot. (F) LPS significantly induces the expression of <i>C/ebpĪ“</i> dependent upon IRAK-1. WT and IRAK-1 deficient cells were treated with LPS for indicated time period. Message levels of <i>C/ebpĪ“</i> were determined by RT-PCR. nā=ā3; *<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>Ļ<sub>ji</sub></i> among data sets under each priming mechanism. The āslowā and āfastā time scales reflect the values of Ī³<i><sub>j</sub></i> in comparison to Ī³<sub>3</sub>ā=ā1. (BāD) 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