Analysis of genes dysregulated upon over-expression, or knock-down, of miR-146a in stimulated EOC 13.31 cells using either miRNA mimics or anti-miRs.

<p><b>A.</b> Venn diagram to show the intersection between genes down-regulated in LPS stimulated EOC 13.31 cells by miR-146a over-expression, and those up-regulated upon miR-146a knock-down. Also shown are those targets bioinformatically predicted using the TargetScan 5.1 program and IPA software. <b>B.</b> Schematic showing alterations in expression in key inflammatory response-related genes in LPS stimulated EOC 13.31 cells following miR-146a over-expression. Colored green are those down-regulated genes, colored red are up-regulated genes, while those highlighted in blue are bioinformatically predicted targets of miR-146a using the TargetScan 5.1 program and IPA software.</p

EOC 13.31 cells were stimulated with 100 ng/ml semi-pure LPS and temporal changes in cytokine gene expression relative to mock-treated control were measured by microarray analysis.

<p>MiR-146a expression relative to mock-treated control was measured by TaqMan® qRT-PCR and expression is shown as grey bars.</p

Hierarchical cluster plot generated from miRNA expression profiling of a repertoire of a variety of CNS cell lineages (neuronal, microglia, astrocytes).

<p>A microglia specific cluster of miRNAs is indicated. Red indicates high levels of miRNA expression, green low levels and gray indicates expression that was undetected by the microarray used.</p

MiR-146a induction in microglia.

<p><b>A.</b> LPS at concentrations ranging from 0.1–100 ng/ml was used to stimulate BV-2 cells. After 8 hours, RNA was collected and TaqMan® qRT-PCR was used to determine miR-146a expression relative to PBS treated (unstimulated) control cells. The experiment was performed in triplicate and the average fold induction is shown. <b>B.</b> EOC 13.31 cells were stimulated with 100 ng/ml semi-pure LPS, 100 ng ultra-pure LPS or PBS alone (unstimulated). RNA was collected after 8 hours and miR-146a expression was measured by TaqMan® qRT-PCR. Fold induction relative to untreated cells is shown. The experiment was performed in triplicate and the average fold change is shown. <b>C.</b> EOC 13.31 cells were incubated with increasing concentrations of an anti-TLR2 antibody for 30 minutes prior to stimulation with 100 ng/ml LPS. MiR-146a expression relative to mock-treated control cells was measured by TaqMan® qRT-PCR. Inhibition of miR-146a expression following anti-TLR2 antibody treatment was significant at all concentrations; 10 and 50 ng/ml * p<0.01, 100 ng/ml ** p<0.005. Treatment of BV-2 cells with 100 ng/ml anti-TLR-2 antibody prior to LPS stimulation failed to inhibit miR-146a induction. The experiment was performed in triplicate and the average fold change is shown. <b>D.</b> EOC 13.31 and BV-2 cells were stimulated with with 10⁸ heat-killed <i>Listeria monocytogenes</i> (HKLM) cells/ml. RNA was collected at various time-points over 72 hours and miR-146a expression measured by TaqMan® qRT-PCR. Fold induction relative to unstimulated cells (US) is shown. The experiment was performed in triplicate and the average fold change is shown.</p

Analysis of genes dysregulated by over-expression, or knock-down of miR-146a in resting EOC 13.31 cells using either miRNA mimics or anti-miRs.

<p><b>A.</b> Venn diagram to show the intersection between genes down-regulated by miR-146a over-expression, up-regulated on miR-146a knock-down and those targets bioinformatically predicted using the TargetScan 5.1 program and IPA software. <b>B.</b> Networks showing the interactions between several predicted miR-146a target genes. Shaded grey are those genes also bioinformatically predicted using the TargetScan 5.1 program and IPA software.</p

Altering the expression of miR-146a in microglia.

<p><b>A.</b> MiR-146a over-expression as measured by TaqMan® qRT-PCR in microglia cells after 8, 24, and 48 hrs of treatment with 30 nM (previously optimized, data not shown) of transfected pre-miR-146a in comparison to 30 nM of transfected scrambled negative control miRNA. An average fold change and standard deviation were measured from triplicate experiments. <b>B.</b> MiR-146a knock-down as measured by TaqMan® qRT-PCR in microglia cells after 8, 24, and 48 hrs of treatment with 50 nM (previously optimized, data not shown) of transfected anti-miR-146a in comparison to 50 nM of transfected scrambled negative control miRNA. An average fold change and standard deviation were measured from triplicate experiments.</p

Analysis of genes dysregulated by over-expression, or knock-down, in resting EOC 13.31 cells using either miR-146a mimics or anti-miRs.

<p>Genes listed in bold are those that are bioinformatically predicted to be targets of miR-146a using the TargetScan 5.1 program and IPA software.</p

A summary showing key inflammatory response-related genes whose expression is modulated upon the over-expression of miR-146a.

<p>A summary showing key inflammatory response-related genes whose expression is modulated upon the over-expression of miR-146a.</p

EOC 13.31 cells were transfected with 30 nM of miR-146a mimic or anti-miR and 16 hrs later stimulated with 10 ng/ml LPS.

<p>At 24 hours post LPS stimulation, culture supernatant was collected and the levels of IL-6 and GM-CSF (coded for by the gene CSF2) were determined by ELISA; significance: ** p<0.001, * p<0.01.</p

Expression levels of microglial cell surface receptors in hippocampal tissue of RML infected, symptomatic mice as determined by microarray analysis.

<p>Expression levels of microglial cell surface receptors in hippocampal tissue of RML infected, symptomatic mice as determined by microarray analysis.</p