Journal: International Journal of Molecular Sciences

Article Title: Mucus Release and Airway Constriction by TMEM16A May Worsen Pathology in Inflammatory Lung Disease

doi: 10.3390/ijms22157852

Figure Lengend Snippet: Expression of TMEM16A in human healthy, asthmatic, and CF lungs. Lung slices of normal airways indicate little expression of TMEM16A in both surface airway epithelium and airway submucosal glands. Pronounced expression of TMEM16A in apical membranes of surface epithelial cells and airway submucosal glands of patients with asthma and CF (brown precipitation, DAP staining). Submucosal glands were often found to be hypertrophic. Representative stainings of four patients each. Bars indicate 20 µm.

Article Snippet: The primary antibody, anti-DOG1/TMEM16A (SP31, Novus Biologicals, USA) was incubated overnight at 4 °C.

Techniques: Expressing, Staining

Journal: International Journal of Molecular Sciences

Article Title: Mucus Release and Airway Constriction by TMEM16A May Worsen Pathology in Inflammatory Lung Disease

doi: 10.3390/ijms22157852

Figure Lengend Snippet: Activation of TMEM16A by Eact induces mucus release and airway contraction. ( A ) Mucus staining by alcian blue in OVA-treated asthmatic mice. Acute application of the activator of TMEM16A, Eact (4.8 µg/mL intratracheal), induced acute release of mucus from goblet cells and increased intraluminal mucus. Bars indicate 50 µm. ( B ) Summary of intracellular and intraluminal mucus. ( C ) Summary of cross sectional area indicating airway contraction by Eact. Mean ± SEM (number of animals/number of measurements). # significant difference when compared to control ( p < 0.05, unpaired t -test).

Article Snippet: The primary antibody, anti-DOG1/TMEM16A (SP31, Novus Biologicals, USA) was incubated overnight at 4 °C.

Techniques: Activation Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: Mucus Release and Airway Constriction by TMEM16A May Worsen Pathology in Inflammatory Lung Disease

doi: 10.3390/ijms22157852

Figure Lengend Snippet: Expression of MUC5AC and activation of TMEM16A in Calu-3 airway epithelial cells is inhibited by niclosamide. ( A ) Expression of MUC5AC induced by IL-13 (20 ng/mL; 72 h) in Calu-3 airway epithelial cells was inhibited by simultaneous incubation with niclosamide (1 µM). Bar = 100 µm. ( B ) Quantification of MUC5AC expression indicating inhibition by niclosamide (Niclo). ( C ) Current overlays from whole-cell patch clamp experiments before and after induction of MUC5AC expression by IL-13. Activation of whole-cell currents by purinergic stimulation (ATP, 100 µM) was enhanced by IL-13, which was completely inhibited by acute application of niclosamide (1 µM). ( D ) Corresponding current/voltage relationships. The inhibitor of Ca 2+ -activated KCNN4 K + channels, TRAM-34 (100 nM), was present in all patch clamp experiments to avoid potential activation of Ca 2+ -activated K + channels. Mean ± SEM (number of cells). * significant activation by ATP ( p < 0.05, paired t -test). # significant difference when compared to the absence of IL-13 ( p < 0.05, unpaired t -test).

Article Snippet: The primary antibody, anti-DOG1/TMEM16A (SP31, Novus Biologicals, USA) was incubated overnight at 4 °C.

Techniques: Expressing, Activation Assay, Incubation, Inhibition, Patch Clamp

Journal: International Journal of Molecular Sciences

Article Title: Mucus Release and Airway Constriction by TMEM16A May Worsen Pathology in Inflammatory Lung Disease

doi: 10.3390/ijms22157852

Figure Lengend Snippet: Niclosamide inhibits expression of MUC5AC and SPDEF in Calu-3 cells. ( A ) Western blot indicating upregulation of TMEM16A in Calu-3 cells by IL-13 (20 ng/mL, 72 h) and inhibition of expression by niclosamide (1 µM). Blots were performed in triplicates. ( B ) RT-PCR analysis of the expression of MUC5AC, TMEM16A, and SAM pointed domain-containing ETS transcription factor (SPDEF) in Calu-3 airway epithelial cells. SPDEF is an integrator of goblet cell differentiation and pulmonary Th2 inflammation. ( C ) Low cycle numbers (20×) were chosen for quantification of expression by relating specific signals to expression of the housekeeper protein GAPDH. IL-13 (20 ng/mL) leads to upregulation of expression of MUC5AC, TMEM16A, and SPDEF. Niclosamide (Niclo, 1 µM, 72 h) strongly inhibits expression of MUC5AC, TMEM16A, and SPDEF. Mean ± SEM (number of assays). #,§ significant increase by IL-13 and inhibition by niclosamide, respectively ( p < 0.05, ANOVA).

Article Snippet: The primary antibody, anti-DOG1/TMEM16A (SP31, Novus Biologicals, USA) was incubated overnight at 4 °C.

Techniques: Expressing, Western Blot, Inhibition, Reverse Transcription Polymerase Chain Reaction, Cell Differentiation

Journal: International Journal of Molecular Sciences

Article Title: Mucus Release and Airway Constriction by TMEM16A May Worsen Pathology in Inflammatory Lung Disease

doi: 10.3390/ijms22157852

Figure Lengend Snippet: Activation of TMEM16A whole-cell currents is potentiated by brevenal. ( A ) Dose-dependent activation of endogenous TMEM16A whole-cell currents in CFBE airway epithelial cells, by the purinergic agonist ATP. Clamp voltages ± 100 mV in steps of 20 mV. ( B ) Corresponding current/voltage relationships. ( C , D ) Activation of whole-cell currents obtained from cells pre-incubated with brevenal (500 nM, 15 min) and corresponding current/voltage relationships. ( E ) Activation of whole-cell currents in brevenal-incubated cells, in which expression of TMEM16A has been knocked down by treatment with siRNA-TMEM16A (c.f. ). ( F ) Corresponding current/voltage relationships. Mean ± SEM (number of cells). * significant activation by ATP ( p < 0.05, ANOVA). The inhibitor of Ca 2+ -activated KCNN4 K + channels, TRAM-34 (100 nM), was present in all patch clamp experiments to avoid potential activation of Ca 2+ -activated K + channels. # significant difference when compared to the absence of brevenal ( p < 0.05, ANOVA). No currents were activated in siRNA-TMEM16A-treated cells.

Article Snippet: The primary antibody, anti-DOG1/TMEM16A (SP31, Novus Biologicals, USA) was incubated overnight at 4 °C.

Techniques: Activation Assay, Incubation, Expressing, Patch Clamp

Journal: International Journal of Molecular Sciences

Article Title: Mucus Release and Airway Constriction by TMEM16A May Worsen Pathology in Inflammatory Lung Disease

doi: 10.3390/ijms22157852

Figure Lengend Snippet: RT-PCR Primer.

Article Snippet: The primary antibody, anti-DOG1/TMEM16A (SP31, Novus Biologicals, USA) was incubated overnight at 4 °C.

Techniques: