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

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

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

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

Ultrasonographic Diagnosis of Thoracic Outlet Syndrome Secondary to Brachial Plexus Piercing Variation

Structural variations of the thoracic outlet create a unique risk for neurogenic thoracic outlet syndrome (nTOS) that is difficult to diagnose clinically. Common anatomical variations in brachial plexus (BP) branching were recently discovered in which portions of the proximal plexus pierce the anterior scalene. This results in possible impingement of BP nerves within the muscle belly and, therefore, predisposition for nTOS. We hypothesized that some cases of disputed nTOS result from these BP branching variants. We tested the association between BP piercing and nTOS symptoms, and evaluated the capability of ultrasonographic identification of patients with clinically relevant variations. Eighty-two cadaveric necks were first dissected to assess BP variation frequency. In 62.1%, C5, superior trunk, or superior + middle trunks pierced the anterior scalene. Subsequently, 22 student subjects underwent screening with detailed questionnaires, provocative tests, and BP ultrasonography. Twenty-one percent demonstrated atypical BP branching anatomy on ultrasound; of these, 50% reported symptoms consistent with nTOS, significantly higher than subjects with classic BP anatomy (14%). This group, categorized as a typical TOS, would be missed by provocative testing alone. The addition of ultrasonography to nTOS diagnosis, especially for patients with BP branching variation, would allow clinicians to visualize and identify atypical patient anatomy

Time course and reversibility of epithelial stratification by the hydrostatic pressure.

<p>(A) A vertical section of MDCK I cell sheets. Hydrostatic pressure from basal to apical side was applied to the MDCK I cell sheets at two days after seeding on filters, and the vertical section of cell sheets was observed at Day 2 and 1–12 days after application of the hydrostatic pressure (Days 3–14). MDCK I cells showed gradual development of epithelial stratification with time. (B) The reversibility of epithelial stratification by the hydrostatic pressure. Hydrostatic pressure from basal to apical side was applied to the MDCK I cell sheets for four days, and the hydrostatic pressure gradient was then eliminated by the increase of the culture medium in the apical side. (C) Stratification index under the conditions in (A) and (B). The upper side is apical side and the lower side is basal side. Scale bars = 20 μm.</p

Effects of signal inhibitors and activators on cell proliferation and TER.

<p>(A) Effects of signal inhibitors and activators on the cell number in MDCK I cells. MDCK I cells were seeded at a density of 5.0 × 10⁴ cells/well in a 12-well plate, cultured for 48 h in the presence of the inhibitor or activator, and the cell number was counted with counting chamber after the trypsinization of the cells. (B) Effects of signal inhibitors and activators on the doubling time of MDCK I cells. The doubling time was calculated from the results in (A) on the assumption that the rate of cell proliferation was constant during the 48 h of the culture. (C) Effects of signal inhibitors and activators on TER. MDCK I cells were cultured on filters in the presence of the signal inhibitor or activator, and the TER was measured at each time point.</p