EP₂, EP₃, and EP₄ are expressed on human mast cells. At low doses prostaglandin E₂ inhibits mannitol mast cell degranulation.

<p>Real time PCR was performed in LAD2 cells using EP receptor probes as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110870#pone-0110870-t001" target="_blank">Table 1</a> (A). The EP receptors expression levels were normalized with the β-actin expression level, EP₁ expression was undetectable. Western blot analysis was carried out with specific antibodies against EP₁, EP₂, EP₃, and EP₄ in whole cell lysates from CD34⁺ derived mast cells and LAD2 cells; blot against β-actin was performed as a loading control (B). PGE₂ titration was carried out before 10% mannitol stimulation in LAD2 cells (C). The experiments are representative of 3 independent assays. Statistical significance (*p≤0.05) is relative to mannitol-stimulated cells.</p

Mast cell degranulation is induced by mannitol in human mast cells.

<p>LAD2 mast cells were stimulated with mannitol (5%, 10%, and 17%) for 30 minutes, and β-hexosaminidase release was measured (A). CD63 staining was performed in LAD2 cells after mannitol stimulation (10%) for 30 minutes. Positive control was conducted with PMA plus ionomycin. Negative control (NC) means isotype staining (B). β-hexosaminidase content was measured in the supernatants from activated LAD2 cells, CD34+ derived human mast cells, and human lung mast cells induced by mannitol (10%) at 37°C for 30 minutes (C). Results are in triplicate and are the mean of three independent experiments expressed as mean ± standard deviation. Statistical significance *p≤0.05, **p≤0.01, ***p≤0.001) is relative to the unstimulated control cells.</p

Primers used to determine prostaglandin E₂ receptor expression on LAD2 cells by quantitative PCR.

<p>*variant number 5 of the ten existing variants of the receptor.</p><p>Primers used to determine prostaglandin E₂ receptor expression on LAD2 cells by quantitative PCR.</p

Calcium mobilization, cytokine production and PGE₂ secretion are induced by mannitol in LAD2 cells.

<p>Determination of calcium flux following mannitol stimulation (10%) was performed in LAD2 cells loaded with Fluo-4 dye, either with calcium containing media (A) or without extracellular calcium (B), as described in material and methods section. After defining basal conditions the stimuli was added (time 0) and measured 10 minutes. Real time polymerase chain reaction was performed in LAD2 cells that were either unstimulated or stimulated with 10% mannitol, using interleukin-8 and tumor necrosis factor alfa as probes. PMA+ Ionomycin (Sigma) were used as positive control stimuli (C). PGE₂ release was measured at different times as indicated in the figure using an ELISA assay. Results are expressed in pg per 1×10⁶ cells (D). Results are triplicates expressed as mean ± standard deviation, and are representative of three independent experiments. Statistical significance (* p≤0.05, **p≤0.01) is relative to unstimulated control cells. RFU: relative fluorescence units.</p

CLM-1 mRNA and protein levels in microglial cells under basal conditions.

<p><b>A)</b> QT-PCR analysis of CLM-1 transcript was performed on primary microglia total RNA. RAW264.7 and NIH3T3 cell lines were used as positive and negative controls respectively. CLM-1 transcript was normalized with 18S RNA levels. <b>B)</b> Surface expression of CLM-1 on primary microglia was monitored by flow cytometry using anti-CLM-1 mAb (white histogram) and an isotypic mAb as a negative control (gray histrogram). <b>C)</b> Immunohistochemistry on microglial primary cultures was performed to analyze the expression of CLM-1 in basal conditions, using an anti-CLM-1 monoclonal antibody. Nuclei were stained with DAPI. No staining was observed in the absence of primary antibody. Scale bar, 20 μM.</p

CLM-1 receptor amplifies LPS-induced TNFα protein levels.

<p>Microglia was stimulated either with anti-CLM-1 Ab or an isotypic control together with LPS (100 ng/mL). Supernatants were recovered 24 hours later and protein levels were measured by ELISA. Data are presented as mean ± SEM of 4 independent experiments. Statistically significant differences between treatments were determined by one-way ANOVA followed by Newman Keuls post-test. *P < 0.05 compared to IgG.</p

Microglia expresses a soluble isoform of CLM-1.

<p><b>A)</b> Genomic organization and splicing pattern of CLM-1. A diagram representing CLM-1 splice variants 1, 2 and 3 is shown. Exons are represented by solid boxes (with their respective lengths in base pairs) and introns by the connecting lines. <b>B)</b> Comparison of the predicted amino acid sequences of CLM-1 variant 1 (NM_001169153) and variant 3 (JX073136). <b>C)</b> mRNA levels of the full length (variant 1) and soluble (variant 3) CLM-1 isoforms in microglia. Total RNA was extracted from primary microglia and RAW264.7 cells and RT-PCR was performed. As controls, cDNAs of both transcripts cloned in the vector pSPARK were used. The image is representative 3 independent experiments. <b>D)</b> Microglia was treated with the TLR agonists LPS (TLR4 agonist, 100 ng/mL), PGN (TLR2/6 heterodimer agonist, 1 μg/mL) and poly I:C (TLR3 agonist, 10 μg/mL) for 24 hours and RT-PCR analysis of the full length and the soluble isoforms of CLM-1 was performed. cDNAs were resolved in 2% agarose gels. The image is representative of 3 independent experiments. Actin mRNA levels were used as a loading control. Signal quantification is graphed bellow. <b>E)</b> Levels of soluble CLM-1 were measured in the supernatants of microglia after treatment with LPS (100 ng/mL) for 24 hours. Data are presented as mean ± SEM of 4 independent experiments. T-test was performed to determine significance and p-value was 0.1953.</p

CLM-1 receptor enhances LPS-induced pro-inflammatory mediators production.

<p>Microglia was stimulated either with anti-CLM-1 Ab or an isotypic control in combination with LPS (100 ng/mL) or IL-4 (10 ng/mL). After 24 hours, total RNA was extracted and QT-PCR analysis was performed. Relative quantification was performed using the condition IgG+LPS as the calibrator condition for NOS-2, COX-2, TNFα, IL-1β, IL-6 and CCL17. For the quantification of RELMα, IgG+mock condition was used as the calibrator condition. Data are presented as mean ± SEM of 3 independent experiments. Statistically significant differences between treatments were determined by one-way ANOVA followed by Newman Keuls post-test, or Kruskal-Wallis test followed by Dunn's Multiple Comparison Test. *P < 0.05, **P<0.01 and ***P<0.001 compared to IgG.</p