Fig. 4

MUC1 mediates dexamethasone anti-inflammatory effects. a-e Bronchial epithelial Beas2b cells were transiently transfected with siRNA-MUC1 or with control siRNA(−) for 48 h, incubated with dexamethasone (Dex) for 1 h and (a-d) stimulated with cigarette smoke extract (CSE) during 24 h. a, b MKP1 and MUC1 mRNA gene expression was analyzed by real time PCR. c, d IL-8 and GM-CSF release was measured by ELISA. e Glucocorticoid response element (GRE) activation was measured after 4 h of dexamethasone incubation at different concentrations in Beas2b cells transfected with Cignal GRE Reporter Assay. f Confocal co-immunofluorescence analysis of glucocorticoid alpha (GRα) and MUC1-CT co-localization in primary human bronchial epithelial cells (HBECs) treated with dexamethasone during 4 h, and in cells incubated with CSE for 24 h followed by the incubation with dexamethasone for 4 h. DAPI was used to mark cell nuclei. Scale bar: 5 μm. g Protein expression measured by western blot in HBEC stimulated with CSE during 24 h. h HBECs were incubated with MG132 5 μM during 1 h followed by CSE stimulation during 24 h. Western blot of different proteins was analysed. i Immunoprecipitation of MUC1-CT and GRα in HBECs stimulated with different concentrations of CSE. j Immunoprecipitation of MUC1-CT and GRα in HBECs incubated with CSE during 24 h followed by the stimulation with dexamethasone during 4 h. Representative western blot are showed. The results are shown as mean ± SE of n = 4 independent experiments. Gene expression was analyzed by real time PCR using the 2^-ΔCt as described in methods. One-way ANOVA followed by Bonferroni post-hoc tests. a, b *P < 0.05 compared with siRNA(−) transfected cells; #P < 0.05 compared with siRNA-MUC1 + Dex group. c, d) *P < 0.05 compared with siRNA(−) transfected cells; # and ●P < 0.05 compared with siRNA(−) + CSE group; ┴P < 0.05 compared with siRNA(−) + CSE + Dex group. e *P < 0.05 compared with siRNA-MUC1 + Dex group