CFTR protein expression in Calu-3 cells in the presence of cycloheximide with or without dexamethasone.

<p>Calu-3 cells were treated (<b>A</b>) with (+) or (<b>B</b>) without (−) 10 nM Dx for 24 h, and then incubated in the presence of 100 µg/ml cycloheximide (CHX) for the indicated times (0.5, 1, 2, 4, and 8 h). In the graphs, CFTR protein expression is represented by the densitometric values of each blot using GAPDH as a housekeeper protein. For CHX+Dx, the protein expression was normalized by the group treated with dexamethasone for 24 h and for CHX the normalization was related to the C group. *Significantly different from the respective 24-h group; <i>p</i><0.05, <i>n</i> = 4.</p

Total protein expression and surface protein biotinylation following treatment with dexamethasone (Dx, 10 nM) 24 h.

<p>(<b>A</b>) Graphs represent the means ± SE for densitometric values of CFTR total (<b>A</b>) and surface (<b>B</b>) protein expression and respective blots showing CFTR and GAPDH total and surface protein expression for the control, C (total), and after treatment with dexamethasone (10 nM), Dx (total), for 24 h. *<i>p</i><0.05, <i>n</i> = 3 (pooled with 2 samples per N).</p

Effect of dexamethasone on CFTR and αENaC mRNA expression.

<p>Graphs represent the mean ± SE for mRNA relative expression of CFTR and αENaC acquired by real-time PCR. Dose–response curves for dexamethasone on (<b>A</b>) CFTR and (<b>B</b>) αENaC mRNA expressions. *Significant difference compared with the control (C) group #significant difference compared with the group treated with 1 nM dexamethasone; <i>p</i><0.05, <i>n</i> = 5. Time course of (<b>C</b>) CFTR and (<b>D</b>) αENaC mRNA expression in response to treatment with 10 nM dexamethasone for 3, 24, and 48 h. *Significant difference compared with the control group; §significant difference compared with the group treated for 3 h; <i>p</i><0.05, <i>n</i> = 4.</p

Glucocorticoid and mineralocorticoid receptor inhibition by mifepristone and spironolactone, with or without dexamethasone (Dx) following 24 h of treatment.

<p>(<b>A</b>) Graphs representing the densitometric values of protein expression from the blots normalized by GAPDH and the respective blots showing CFTR and GAPDH protein expression in the experimental groups (<b>B</b>). *<i>p</i><0.05, <i>n</i> = 4.</p

Inhibition of protein synthesis by cycloheximide and treatment with dexamethasone for 24 h in Calu-3 cells.

<p>(<b>A</b>) Graphs representing means ± SE for densitometric values of respective blots showing CFTR and GAPDH protein expression in C, control group; Dx, dexamethasone 10 nM; CHX, cycloheximide (50 µg/ml) treated group; and CHX+Dx, cycloheximide (50 µg/ml) plus dexamethasone (10 nM) treated group. *Significantly different from the C group; #significantly different from the Dx group; <i>p</i><0.05, <i>n</i> = 4.</p

CFTR protein expression in Calu-3 using [35S]methionine pulse-labeling analysis.

<p>Pulse labeling with 35S for 0, 5, and 15 min after a 24-h cell treatment with or without 10 nM dexamethasone. (<b>A</b>) Blot representing the CFTR band (<b>B</b>) protein labeled with 35S on a radiographic film. The graphs represent the densitometric values of the bands shown in the blot for CFTR. <i>n</i> = 3, *<i>p</i><0.05.</p

Immunoprecipitation assay for CFTR protein followed by Western blot for HSP90 or HSP70 in Calu-3 cells.

<p>Cells were treated with (Dx) or without (C group) dexamethasone or with mifepristone (Dx+Mif) for 24 h and total cell lysate. (<b>A</b>) Immunoprecipitation with CFTR Ab and subsequent Western blot for HSP90 or HSP70. The graphs represent the densitometric values of the bands shown in the blots for (<b>B</b>) CFTR, (<b>C</b>), HSP90, and (<b>D</b>) HSP70. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047405#s3" target="_blank">Results</a> are presented as the average and SE. <i>n</i> = 4, <i>p</i><0.05.</p

In CFBE41 cells, PGE₂ stimulated HCO₃^- secretion is completely CFTR dependent.

<p><b>A.</b> Representative <i>I</i>_<i>sc</i> trace with vertical deflections indicating the change in <i>I</i>_<i>sc</i> after a 1 mV pulse was applied (every 1 minute). CFBE41 WT cells were exposed to serosal to mucosal HCO₃^- gradient with equivalent bilateral Cl^-. PGE₂ (1 μM, serosal) was added to CFBE41 WT cells after a baseline period of ≥ 10 minutes, with CFTR_inh-172 (20 μM, mucosal) added afterwards. <b>B.</b> Representative <i>I</i>_<i>sc</i> trace from a similar experiment with CFBE41 CF cells. To verify cell viability, ATP (500 μM, mucosal) was added. <b>C.</b> Representative <i>I</i>_<i>sc</i> trace from a similar experiment as Fig 5A with CFBE41 WT cells, except experiments were performed in HCO₃^--free conditions. <b>D.</b> Change in PGE₂-stimulated <i>I</i>_<i>sc</i> (mean ± SEM, n = 3) in CFBE41 WT and CF cells in HCO₃^- containing and HCO₃^--free conditions. Asterisks denote significance by Student’s t-test (**, P < 0.01). Mean percent inhibition compared to CFBE41 WT noted.</p

PGE₂-stimulated mucociliary transport in ferret trachea.

<p><b>A.</b> PGE₂ stimulates a dose-dependent increase in MCC in ferret trachea. Each tissue was exposed to 2–3 doses of PGE₂ for 30 minutes each (n = 3 each dose). Data are shown as the mean PGE₂-stimulated increase in MCC over baseline ± SEM. The half-maximal effective concentration (EC₅₀) is noted in lower right corner. <b>B.</b> Timecourse of PGE₂-stimulated MCC with and without CFTR inhibition (n ≥ 6 each). For CFTR inhibition, tissues were bathed in apical and serosal solution for 30 minutes with CFTR_inh-172 (20 μM) prior to the 15-minute period and kept in the serosal bath for the length of the experiment. PGE₂ (1 μM) was added to the serosal bath. Circles represent means with bars indicating SEM. Asterisks represent P < 0.05 by ANOVA.</p

In Calu-3 cells, PGE₂ stimulated Cl^- secretion is mediated by CFTR and Ca²⁺-activated Cl^- channels.

<p><b>A.</b> Representative <i>I</i>_<i>sc</i> trace with vertical deflections indicating the change in <i>I</i>_<i>sc</i> after a 1 mV pulse was applied (every 1 minute). Calu-3 cells were exposed to serosal to mucosal Cl^- gradient with equivalent bilateral HCO₃^-. PGE₂ (1 μM, serosal) was added to Calu-3 cells after a baseline period of ≥ 10 minutes, with CFTR_inh-172 (20 μM, mucosal) added after 30 minutes. <b>B.</b> Representative <i>I</i>_<i>sc</i> trace of Calu-3 cells incubated in CFTR_inh-172 (20 μM, mucosal) for at least 30 minutes prior to PGE₂ (1 μM, serosal) stimulation. After 30 minutes, niflumic acid (100 μM, mucosal) was added. <b>C.</b> Change in PGE₂-stimulated <i>I</i>_<i>sc</i> (mean ± SEM, n ≥ 4) in Calu-3 cells, with comparisons between no inhibition, CFTR inhibition, or CFTR and Ca²⁺-activated Cl^- inhibition. Asterisks denote significance by Student’s t-test (*, P < 0.05). Mean percent inhibition compared to PGE₂ stimulation alone noted.</p