RT-PCR primers and annealing temperature.

<p>RT-PCR primers and annealing temperature.</p

Fringe Controls Naïve CD4⁺T Cells Differentiation through Modulating Notch Signaling in Asthmatic Rat Models

<div><p>The ability of Notch signaling to regulate T helper cell development and differentiation has been widely accepted. Fringe, <em>O-fucose-β1,3-N</em>-acetylglucosaminyltransferases modulate Notch receptor expression and promote the Notch signaling pathway through receptor-ligand binding. In this study, we assayed the expression levels of three Fringe homologs in naive CD4⁺T cells in asthmatic rats. We found that Radical Fringe (Rfng) was highly expressed, whereas both Lunatic Fringe (Lfng) and Manic Fringe (Mfng) were expressed at low levels. Down-regulation of Rfng using siRNA, and overexpression of Lfng or Mfng enhanced Th1 subset lineages and diminished Th2 subset lineages. Notch signaling was more activated in asthmatic naïve CD4⁺T cells than in control cells, and Lfng, but not Mfng or Rfng, partly inhibited Notch signaling in asthmatic naïve CD4⁺T lymphocytes. Lfng overexpression resulted in significantly decreased Th2 cytokine production in asthma, which was the same effect as the GSI (γ-secretase inhibitor) treatment alone, but had an increased effect on Th1 cytokines than GSI treatment. Collectively, these data identify the essential role of Fringe modulating naïve CD4⁺T cells differentiation through Notch signaling. Lfng regulated Th2 cells differentiation via a Notch-dependent manner and Th1 cells differentiation via a Notch-independent manner. Fringe could be a therapeutic strategy for the management and prevention of allergic asthma.</p> </div

Addition of BMP-2 (40 ng/ml) in GM increased PTEN gene expression of PASMCs.

<p>BMP-2 significantly increased PTEN expression at 8 and 24 hours after treatment. (&: vs any other time point, p<0.05; *: vs 0, 1, and 4 hours, p<0.05).</p

HE staining of lung tissue in OVA-sensitized asthmatic group and control group.

<p><b>A</b>, control group; <b>B</b>, asthmatic group (Original magnification×400). Asthmatic group showed bronchial inflammation that exsisted in the forms of epithelial damage, wall thickening of the tunica mucosa bronchiorum, inflammatory cell infiltration into the lower layer of the mucous membrane and the surrounding bronchus, and accrementition and spasm of airway smooth muscle.</p

Addition of BMP-2 (40 ng/ml) in GM did not significantly change AKT-1 and AKT-2 gene expressions of PASMCs.

<p>No significant reduction or increment of AKT-1 gene expression was found when PASMCs were cultured in GM supplemented with BMP-2 (<b>A</b>). Similarly, no significant change of AKT-2 gene expression was found when PASMCs were cultured in GM supplemented with BMP-2 (<b>B</b>).</p

Typical pictures of EGFP expression from pEGFP lipoplexes transfected PASMC when the ratio between Lipofectamine-2000 and plasmid DNA was at (A) 1∶1, (B) 2∶1, (C) 3∶1 and (D) 4∶1, when 2 µg plasmid DNA was used per 1×10⁵ cells.

<p>(Magnification = 40×).</p

Western blot analysis of PASMCs cultured in GM supplemented with BMP-2 under hypoxia.

<p>(<b>A</b>) A dose dependent effect of BMP-2 on PTEN protein expression when BMP-2 was supplemented up to 80 ng/ml in GM. (<b>B</b>) Quantification of PTEN protein expression when BMP-2 was increased from 0–80 ng/ml after normalized to GAPDH (consider as 100%). (<b>C</b>) Typical pictures of PTEN and pAKT protein expressions as a function of time when BMP-2 was supplemented at 40 ng/ml in GM. Quantification of PTEN (<b>D</b>) and pAKT (<b>E</b>) protein expressions after normalized to GAPDH (consider as 100%). Reduced PTEN protein expression was found when GW9662 was added alone (<b>F</b>) or combined with BMP-2 (<b>G</b>). Reduced PTEN protein expression was also found when bpV (HOpic) was added alone (<b>H</b>) or combined with BMP-2 (<b>I</b>). (*: vs 0 ng/ml BMP-2, p<0.05; &: vs 0 hour, p<0.05).</p

Toxicity of BMP-2 towards PASMCs when PASMCs were cultured in GM supplemented with 0−80 ng/ml BMP-2 under normoxia.

<p>It appears that only at 80 ng/ml concentration of BMP-2 resulted in significantly increased LDH leakage as compared with GM with 0 ng/ml BMP-2. The percentage of LDH leakage was normalized to fresh GM (consider as 0%). (*: vs 0 ng/ml, p<0.05).</p

OVA-sensitized asthmatic rat models were established successfully.

<p>Airway function was detected and BAL fluid and serum were collected and evaluated by ELISA. <b>A</b>, R_L and Cdyn values were obtained in response to increasing concentrations of inhaled MCh, as described in material and methods. Data represented mean±SEM (n = 20 in each group). *<i>p</i><0.05. <b>B</b>, Cellular composition of BAL fluid. <b>C</b>, Cytokine levels in BAL fluid and serum. <b>D</b>, Eotaxin levels in BAL fluid and serum. IgE levels in serum. Total, total cells; Epi, epithelium; Mac, macrophages; Lym, lymphocytes; Neu, neutrophils; Eos, eosinophils. Date represent the mean±SEM (n = 20 in each group). *<i>p</i><0.05, **<i>p</i><0.01 significant differences comparing asthmatic group and control group.</p

Growth profile of PASMCs cultured in GM supplemented with BMP-2, GW9662 (PPARγ antagonist) or bpV(HOpic) (PTEN inhibitor).

<p>PPARγ antagonist and PTEN inhibitor were added into respective cell culture medium 1 hour before adding BMP-2 (40 ng/ml). (The number of PASMCs after cultured in GM only was considered as 100%). (*: vs GM only) (Lipo = Lipofetamine −2000).</p