TLR6 transcript, protein and function during hypoxia.

<p><i>A</i> and <i>B</i>, Quantification of TLR6 transcripit levels in freshly purified blood DCs, MM6 cells, confluent HMEC-1 monolayers and confluent Caco-2 monolayers. Cells were exposed to normoxia and hypoxia for indicated time points. Total RNA was isolated and TLR6 mRNA levels were determined by real-time RT-PCR. Data were calculated relative to ß-actin and expressed as fold change relative to normoxia±SEM, where transcript levels in normoxic cells were normalized to 1. Results are derived from three different experiments. *, significant differences from normoxic cells (p<0.01). <i>C</i>, Confluent HMEC-1 cells were grown to confluence and exposed to indicated periods of hypoxia. Result depicts a representative TLR6 Western blot from three separate experiments. The same blot was probed for ß-actin expression as a control for protein loading. <i>D</i>, Same amount of HMEC-1 cells were grown to confluence in 24-well plates. Cells were then stimulated with TLR2/6 agonist (FSL-1) at the indicated concentrations and exposed to hypoxia or normoxia for 24 h. After 24 h, generation of IL6 was measured by ELISA in the cell supernatant. Data are mean±SEM from 3 separate replicates. *, significant differences from untreated cells (p<0.05); #, significant differences from normoxia and untreated cells (p<0.05).</p

Influence of hypoxia inducible factor (HIF)-1α on TLR6 expression during hypoxia.

<p><i>A</i>, Map of TLR6 promoter region showing positions of the putative HIF binding sites and the binding site for NFκB relative to the transcription start site (TSS). <i>B</i>, Stable transfected HMEC-1 monolayers containing either HIF-1α siRNA or control-siRNA were exposed to normoxia or hypoxia for indicated time points. Total RNA was isolated, and 1 µg of RNA was transcribed into first strand cDNA. Relative expressional levels of TLR6 transcripts were compared to normoxic controls by real-time RT-PCR. Data were calculated relative to internal control gene (ß-actin), and are expressed as fold change over normoxia±SEM, *, significant differences from normoxia and control cells. Results are derived from three different experiments in each condition. <i>C</i>, Total RNA of normoxic monolayers of either wildtype (WT) or oxygen-stable HIF-1α expressing (HIF^+/+) HMEC-1 cells was isolated and realt-time RT-PCR was performed as described above. *, significant differences from wildtype cells. <i>D</i>, Western blot analysis of TLR6 protein of normoxic HMEC-1 wildtype (WT) and oxygen-stable HIF-1α expressing (HIF+/+) cells. The same blot was probed for ß-actin expression as a control for protein loading. <i>E</i>, HMEC-1 monolayers were treated with 1mM of dimethyloxalylglycine (DMOG) for 24 hours. Afterwards transcript levels of TLR6 where quantified by real-time RT-PCR as described above. *, significant differences from untreated cells. <i>F</i>, ChIP assay was utilized to examine HIF-1α binding to the TLR6 promoter in normoxic and hypoxic HMEC-1 cells. Reaction controls included immunoprecipitations using a nonspecific igG monoclonal antibody (IgG) and PCR performed using HMEC-1 DNA (input). An example of three experiments is shown.</p

Influence of hypoxia on TLR mRNA expression in murine dendritic cells.

<p>Isolated murine bone-marrow-derived dendritic cells (BMDCs) were exposed to normoxia or hypoxia for 24 hours. Total RNA was isolated, and quantitative mRNA levels of TLR1-9 and TLR11-13 were assessed by real-time RT-PCR. Data were calculated relative to ß-actin and expressed as fold change relative to normoxia±SEM and transcript levels in normoxic BMDCs were normalized to 1. Results are derived from three different experiments (*p<0.05, significant differences from normoxia).</p

Role of hypoxia inducible factor (HIF)-1 in TLR2 and TLR6 expression during hypoxia in vivo.

<p>Real-time RT-PCR analysis of murine epithelial TLR2 and TLR6 mRNA in conditional HIF-1α mutant (HIF-/-) and littermate control (WT) animals subjected to normoxia or hypoxia. Data were calculated relative to ß-actin and are expressed as fold change over normoxia±SEM, where transcript levels of control animals were normalized to 1. *, significant differences from normoxic control animals (p<0.05). Results are derived from 8 animals in each condition.</p

TLR2 transcript, protein and function during hypoxia.

<p>TLR2 transcript, protein and function during hypoxia.</p

Human and murine Primer pairs as used for real-time RT-PCR.

<p>Human and murine Primer pairs as used for real-time RT-PCR.</p