Figure 1.
Effect of dexamethasone on CFTR and αENaC mRNA expression.
Graphs represent the mean ± SE for mRNA relative expression of CFTR and αENaC acquired by real-time PCR. Dose–response curves for dexamethasone on (A) CFTR and (B) αENaC mRNA expressions. *Significant difference compared with the control (C) group #significant difference compared with the group treated with 1 nM dexamethasone; p<0.05, n = 5. Time course of (C) CFTR and (D) 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; p<0.05, n = 4.
Figure 2.
Glucocorticoid and mineralocorticoid receptor inhibition by mifepristone and spironolactone, with or without dexamethasone (Dx) following 24 h of treatment.
(A) 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). *p<0.05, n = 4.
Figure 3.
Total protein expression and surface protein biotinylation following treatment with dexamethasone (Dx, 10 nM) 24 h.
(A) Graphs represent the means ± SE for densitometric values of CFTR total (A) and surface (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. *p<0.05, n = 3 (pooled with 2 samples per N).
Figure 4.
Inhibition of protein synthesis by cycloheximide and treatment with dexamethasone for 24 h in Calu-3 cells.
(A) 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; p<0.05, n = 4.
Figure 5.
CFTR protein expression in Calu-3 cells in the presence of cycloheximide with or without dexamethasone.
Calu-3 cells were treated (A) with (+) or (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; p<0.05, n = 4.
Figure 6.
CFTR protein expression in Calu-3 using [35S]methionine pulse-labeling analysis.
Pulse labeling with 35S for 0, 5, and 15 min after a 24-h cell treatment with or without 10 nM dexamethasone. (A) Blot representing the CFTR band (B) protein labeled with 35S on a radiographic film. The graphs represent the densitometric values of the bands shown in the blot for CFTR. n = 3, *p<0.05.
Figure 7.
Immunoprecipitation assay for CFTR protein followed by Western blot for HSP90 or HSP70 in Calu-3 cells.
Cells were treated with (Dx) or without (C group) dexamethasone or with mifepristone (Dx+Mif) for 24 h and total cell lysate. (A) 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) CFTR, (C), HSP90, and (D) HSP70. Results are presented as the average and SE. n = 4, p<0.05.