Silencing STAT3 abrogates IL-9 mediated CCL11 expression.

<p><b>A</b>. Efficiency of Lentiviral transduction in human ASM cells. Human ASM cells were transduced by infecting with lentivirus containing scramble sequence or STAT3-shRNA sequence and examined by flow cytometry for tGFP expression. <b>B</b>. Total STAT3 in mock, scramble and STAT3-ShRNA transduced ASM cells as analyzed by Western blot <b>C</b>. ASM cells stably expressing scramble or STAT3-shRNA was transfected with CCL11 promoter luciferase reporter plasmid and stimulated with IL-9 (10 ng/ml) or IL-4 (10 ng/ml) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009178#s4" target="_blank">Methods</a>. The mean ±SEM of three independent experiments are shown. * P<0.05 compared to scramble lentiviral transduced ASM cells stimulated with IL-9.</p

IL-9 mediated CCL11 expression is not affected in STAT6 silenced ASM cells.

<p><b>A</b>. Human ASM cells were transduced by infecting with lentivirus containing scramble sequence, STAT6-shRNA sequence or mock and examined by flow cytometry for turbo GFP (tGFP) expression. <b>B</b>. Effect of STAT6-shRNA on STAT6 expression by ASM cells. Expression of total STAT6 in mock, scramble or STAT6-shRNA transduced ASM cells was analyzed by western blot. <b>C</b>. Stably silenced STAT6 ASM cells was co-transfected with CCL11 promoter then stimulated with IL-9 or IL-4 (both at 10 ng/ml) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009178#s4" target="_blank">Materials and Methods</a>. The mean ±SE of three independent experiments are shown.</p

STAT3 inhibitory peptide decreases IL-9 mediated CCL11 transcriptional activity.

<p>Human primary ASM cells were growth-arrested, transfected with CCL11 promoter for 24 h. Cells were then incubated with inhibitory or control peptide for 1 h before stimulation for 12 hour with IL-4 or IL-9 after which transcriptional activation was measured by luciferase activity. Fold induction represents luciferase activity in cytokine treated cells compared to transfected untreated cells, and is the mean of five independent experiments. * P<0.05.</p

IL-9 induces STAT3 phosphorylation and <i>in vivo</i> binding to CCL11 promoter in ASM cells.

<p>A–B. Cells were stimulated with IL-9 (A) or IL-4 (only 20 min is shown in B) and analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009178#pone-0009178-g001" target="_blank">Figure 1</a>. The data represent one of similar results from 5 independent experiments. C. IL-9 induced STAT3 binding to CCL11 promoter <i>in vivo</i>. Confluent and serum starved human ASM cells were treated with IL-4 (10 ng/ml) or IL-9 (10 ng/ml). The <i>in vivo</i> STAT3 binding to the CCL11 promoter was analyzed by ChIP assay as described under Material and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009178#s4" target="_blank">Methods</a>. The input represents PCR products from chromatin pellets prior to immunoprecipitation. The results are representative of three independent experiments with similar results. D. IL-9 driven STAT3 reporter gene activity. STAT3-specific reporter plasmid (p<i>Luc</i>TKS3) which harbors seven copies of a sequence corresponding to the STAT3-specific binding site was transfected into ASM cells or with pLucSRE serum response element (SRE) of the c-fos promoter (data not shown) following stimulation by IL-9 or IL-4 as described above. Data in D represent the mean ± SEM from a total of 5 independent experiments.</p

IL-9 does not induce STAT6 nuclei translocation in human ASM cells.

<p>Growth arrested semi-confluent ASM cells were stimulated with IL-4 (A) or IL-9 (B) both at 10 ng/ml in 8 wells slide. Slides were stained with specific anti- phospho tyrosine STAT-6 mAb or isotype matched control, followed by goat anti-mouse IgG F(ab')₂ AlexaFluor® 488. Nuclear counter-staining was performed using TOTO-1 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009178#s4" target="_blank">Materials and Methods</a>. Original magnification 400X.</p

Effect of DN STAT3β, STAT3 Ser 727, or DN STAT6 over-expression on IL-9 induced CCL-11 promoter activity.

<p>Human primary ASM cells were co-transfected with WT-CCL-11 promoter and DN STAT3, STAT3β, STAT3 mutant at Ser 727 or DN STAT6. 24 h after transfection, cells were stimulated for 12 h with IL-4 or IL-9 (all at 10 ng/ml). Fold induction represents luciferase activity in cytokine treated cells compared to transfected untreated cells, and is the mean of five independent experiments. * P<0.05.</p

IL-9 does not induce STAT6 phosphorylation in human ASM cells.

<p>Growth arrested ASM cells were stimulated with IL-9 (A) or IL-4 (20 min,B) both at 10 ng/ml. Lysates were immunoblotted with phospho-specific Abs and detected by enhanced chemiluminescence as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009178#s4" target="_blank">Methods</a>. Total STAT6 and β actin Abs was used for loading control. The results represent one of similar results from four independent experiments.</p

IgE-sensitization induces the chemokines promoter activity in human B/TSM cells.

<p>Cultured human B/TSM cells were transiently transfected with luciferase reporter constructs driven by wild-type promoters for human eotaxin-1/CCL11, IL-8/CXCL8, IP-10/CXCL10, and RANTES/CCL5; stimulated with mIgG1-MOPC21, IgE, or IL-1β; and respective chemokine promoter activity was measured as mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006153#s2" target="_blank"><i>Materials and Methods</i></a>. Data were normalized according to the <i>Renilla</i> luciferase activity, and presented as fold-increase over unstimulated control. Data represents the mean±SD of three separate experiments. Mann-Whitney <i>U</i> test was performed to analyze the differences between the samples. *<i>P</i><0.05, **<i>P</i><0.01 compared to unstimulated control.</p

FcεRI-α protein expression (24, 72 h) is upregulated by proinflammatory and Th-2 cytokines.

<p>Two-day serum-deprived primary B/TSM cells were cultured in presence or absence of IL-1β, TNF-α or IL-4. FcεRI-α protein from (A) 24 h and (B) 72 h culture cell lysates was Immunoprecipitated (IP) followed by Western blotting, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006153#s2" target="_blank"><i>Materials and Methods</i></a> section. FcεRI-α protein was immunoprecipitated with either anti-FcεRIα mAb 15/1 or with isotype antibody mouse IgG1 (MOPC21) for negative control. Non-specific bands from the same gels were used as loading control. (C) FcεRI-γ protein was analyzed by Western blotting and (D) presented as the ratio of 9 KDa FcεRI-γ-specific protein bands intensity over GAPDH (as internal control). Human basophilic KU812 cells were used as a positive control. Figures represent three separate experiments. <i>ns</i>, non-significant (p>0.05).</p

TNF-α pre-sensitization augments multiple CC and CXC chemokines release following IgE stimulation.

<p>Primary human B/TSM cells were cultured in presence of 10 ng/ml each of TNF-α, IL-1β, or IL-4 for 48 h. Cells were then washed twice, and stimulated with either IgE (1 µg/ml), mIgG1-MOPC21 (1 µg/ml) or left unstimulated (medium alone) for another 24 h. Culture supernatants were used for (A) eotaxin-1/CCL11, (B) IL-8/CXCL8, (C) IP-10/CXCL10, and (D) RANTES/CCL5 measurement by ELISA. Data represents mean±SD of three independent experiments performed under the same conditions. Mann-Whitney <i>U</i> test was performed to analyze the differences between the samples. *<i>P</i><0.05, **<i>P</i><0.01.</p