lipofectamine was added to 200 ml of serum free medium (DMEM + F10) per condition for 5 minutes followed by incubation with anti-miR146a LNA or pre-miR146a at a final concentration of 50nM. The volume was adjusted to 1 ml with medium containing 10% FCS and added to the cells. Transfection efficiency was found to be around 80% by flow cytometry in both U373 and primary astrocytes. After 24 hours the IL-1b was added to the wells for 24 hours after which the cells were harvested for RNA or protein isolation. Preparation of Cellular Extracts and Western Blot Analysis Cells were harvested at 24 h after treatments and/or trans- fection. Medium was collected (for the detection of cytokine and HMGB1 release) and glial cells were washed twice with cold PBS. The samples were homogenized in lysis buffer containing 10 mM Tris (pH 8.0), 150 mM NaCl, 10% glycerol, 1% NP-40, Na orthovanadate (10.4 mg/ml), 5 mM EDTA (pH 8.0), 5 mM NaF and protease inhibitor cocktail (Boehringer Mannheim, Germany) by incubating on ice for 15 minutes. The homogenates were centrifuged at 14000 rpm for 10 mins and the supernatant was used for further analysis. Protein content was determined using the bicinchoninic acid method [34]. Western blot analysis was performed, as previously described [35]. For electrophoresis, equal amounts of proteins (15–20 mg/lane) were separated on a 10% sodium dodecylsulfate-polyacrylamide gel electrophoretic (SDS-PAGE) gel. Separated proteins were transferred to nitrocel- lulose paper for 90 min at 100 V, using a wet electroblotting system (BioRad, Hercules, CA, USA). Blots were blocked for 1 hour in 5% non fat dry milk in Tris-buffered saline-Tween (TBST) (20 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.5; for IRAK1 and IRAK2) or in 5% bovine serum albumin (BSA) in TBST (for TRAF6). The blots were incubated overnight with the primary antibody (in 5% milk solution 1:1000 IRAK1 rabbit polyclonal antibody; 1:500 IRAK2 rabbit polyclonal antibody; 1:500 in 5% BSA solution TRAF6 mouse monoclonal, (all from Santa Cruz, CA, USA); COX-2 (1:2500, goat anti-human; Cayman Chemical Company, Ann Arbor, MI, USA); HMGB1 (rabbit polyclonal antibody 1:1000; Pharmingen, San Diego, CA, USA). After several washes in TBST, the membranes were incubated in TBST/5% non fat dry milk, containing the goat anti-rabbit or rabbit anti-mouse coupled to horse radish peroxidase (1:2500; Dako, Denmark) for 1 h. After washes in TBST, immunoreactivity was visualized using ECL PLUS western blotting detection reagent (GE Healthcare Europe, Diegen, Belgium). Expression of b-actin (monoclonal mouse, Sigma, St. Louis, MO, 1:50000) and b- tubulin (1:30000, monoclonal mouse, Sigma, St. Louis, MO, USA) was used as loading control. For the quantification of the blots the band intensities were measured densitometrically Scion Image for Windows (beta 4.02) image-analysis software. A ratio of the integrated band density (IntDen) of the protein of interest to the IntDen of the reference protein was used to normalize band intensities. Detection of Cytokine Release Evaluation of levels of 16 cytokines/chemokines/inflammatory molecules [IL-1b, IL-5, IL-6, IL-8, IL-10, IL-13, IL-17, IL-18, interferon (IFN)-a2, IFN-c, IP-10 (CXCL10), macrophage in- flammatory protein-1b (MIP-1b), G-CSF (granulocyte-colony stimulating factor), Rantes, Tumor necrosis factor (TNF)-a, and VEGF] in the medium of treated cultures was carried out with the human magnetic 10-plex panel (Invitrogen, Camarillo, CA, USA) to which extra cytokine beads were added according to the manufacturer’s protocol. The plate was read on a LuminexH miR-146a and Astrocyte Inflammatory Response 100TM/200TM platform. Data were analyzed and statistics calculated with GraphPad Prism 4 software (San Diego, CA, USA). Levels of MCP-1 were measured using a kit from Ebioscience (Vienna, Austria) according to manufacturer’s instructions. Briefly, flat bottomed ELISA microtiter plates (Costar, Cambridge, MA, USA) were coated overnight with the capture antibody in coating buffer. The plates were washed, preblocked with assay diluent (provided with the kit and incubated with the culture supernatants diluted 1:50 assay diluent or standard antigen dilutions in triplicate. The plates were washed and incubated with biotinylated detection antibody followed by horse radish peroxidase labelled streptavidin. Tetramethyl benzidine (TMB) (Sigma Chemical Co, USA) was used as the colour substrate, and the reaction was stopped with an equal volume of 1 M H2SO4. The absorbance was read in a Titertek Multiskan microplate reader (Germany) at 450 nm with a reference wavelength of 570 nm. Statistical Analysis Statistical analyses were performed with Graphpad PrismH software (Graphpad software Inc., La Jolla, CA, USA) using two- tailed Student’s t-test or, for multiple groups, a non-parametric Kruskal–Wallis test followed by the Dunn’s post hoc test to assess differences between groups. P,0.05 was considered significant. Results miR-146a Expression in GG and FCD Type IIb miR-146a expression was studied using qPCR in control human cerebral cortex samples and in GG and FCD type IIb samples. Expression of miR-146a was significantly increased in GG and FCD specimens compared to control cortex (Fig. 1A). There were no significant differences in miR-146a expression between autopsy and surgical control samples (Figure S1). The cellular distribution of miR-146a in control cortex and FCD type IIb specimens was investigated using in situ hybridization (Fig. 1 B–E). miR-146a was undetectable in glial cells in control grey and white matter specimens (Fig. 1 B–C). In FCD specimens miR-146a expression was detected in glial cells with typical astroglia morphology, particularly in the areas of prominent gliosis (Fig. 1 D). Balloon cells displayed also miR-146a expression (Fig. 1 E). Double labelling confirmed miR-146a expression in GFAP- positive reactive astrocytes (Fig. 1 D, inserts), whereas no detectable expression was observed in HLA-DR positive cells of the microglial/macrophage lineage (not shown). miR-146a expression in reactive astrocytes was also observed in tissue specimens from patients with viral encephalitis and in tumor astrocytes in GG specimens (not shown). Regulation of miR-146a Expression by IL-1b in Human Glial Cells in Culture In the present study we used both U373 glioblastoma cell line and human fetal astrocytes in culture to examine the effect of IL- 1ß and other inflammatory molecules on the expression levels of miR-146a. qPCR demonstrated that exposure to IL-1ß consis- tently increased the miR-146a expression in both cells types (Fig. 2 and 3). The effect of IL-1ß was blocked by the IL-1ß ra, a naturally occurring antagonist of the IL-1 receptor ([36]; Fig. 2 A and Fig. 3 A). Induction of miR-146a (although to a lower extent compared to IL-1ß) was also observed with LPS and blocked by LPS-RS (Fig. 2 A). In contrast, under our culture conditions we did not observe increased miR-146a levels after exposure to IL-6 (10 ng/ ml), TNFa (1 ng/ml;) or HMGB1 (40 nM; Fig. 2 B and Fig. 3 B). IL-1ß induced upregulation of miR-146a at 0.1 ng/ml and its PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e44789