Dysregulated interleukin-8 secretion and NF-κB activity in human cystic fibrosis nasal epithelial cells  Salvatore Carrabino, Daniela Carpani, Alessandra Livraghi, Maurizio Di Cicco, Diana Costantini, Elena Copreni, Carla Colombo, Massimo Conese  Journal of Cystic Fibrosis  Volume 5, Issue 2, Pages 113-119 (May 2006) DOI: 10.1016/j.jcf.2005.12.003 Copyright © 2006 European Cystic Fibrosis Society Terms and Conditions

Fig. 1 Basal and induced NF-κB activity in primary respiratory nasal and A549 cells. (A) Cells were either untreated or treated with IFN-γ (100 U/ml) for 60 min or TNF-α (10 ng/ml) for 30 min prior to the preparation of nuclear extracts. 2.5 μg of each nuclear extract were analyzed for κB-DNA binding activity by EMSA. In lanes 2, 4, 6, 8, 10, 12, and 15, a 50-fold excess of unlabeled NF-κB oligonucleotide was added to the reaction mixture. NF-κB complexes migrated slightly faster in A549 than in nasal cells. (B) Nasal cells (2 weeks-old) were either untreated or incubated with a clinical isolate of P. aeruginosa (PA; 5×104 cfu/ml) for 1 h. The arrows and the asterisks denote the specific band and the free probe, respectively. Journal of Cystic Fibrosis 2006 5, 113-119DOI: (10.1016/j.jcf.2005.12.003) Copyright © 2006 European Cystic Fibrosis Society Terms and Conditions

Fig. 2 Time course of IL-8 secretion by non-CF and CF nasal outgrowths. Nasal polyps (non-CF=4; CF=5) were plated as 1–2 mm2 explants onto 60-mm plate dishes and conditioned medium was collected after 2, 5, 7, 10, and 15 days. ⁎P=0.0001 CF vs. non-CF at each time point. Journal of Cystic Fibrosis 2006 5, 113-119DOI: (10.1016/j.jcf.2005.12.003) Copyright © 2006 European Cystic Fibrosis Society Terms and Conditions

Fig. 3 Evaluation of NF-κB p65 nuclear activity by a colorimetric assay in CF and non-CF nasal epithelial cells under basal conditions and following exposure to P. aeruginosa PAO1 (PA; 1×108 cfu/ml) for 5 h (see Methods) or costimulation with IL-1β (30 ng/ml)/H2O2 (100 μM) for 5 h. ⁎P=0.0001 PA vs. basal and IL-1β/H2O2 vs. basal; §P=0.0004 basal CF vs. basal non-CF; #P=0.0001 PA vs. basal and IL-1β/H2O2 vs. basal. Journal of Cystic Fibrosis 2006 5, 113-119DOI: (10.1016/j.jcf.2005.12.003) Copyright © 2006 European Cystic Fibrosis Society Terms and Conditions

Fig. 4 IL-8 levels in supernatants of CF and non-CF nasal cells under basal condition and following exposure to P. aeruginosa PAO1 (PA; 1×108 cfu/ml) for 5 h (see Methods) or co-stimulation with IL-1β (30 ng/ml)/H2O2 (100 μM). ⁎P=0.0001 PA vs. basal and IL-1β/H2O2 vs. basal; §P=0.0025 basal CF vs. basal non-CF; #P=0.44 PA vs. basal; ⁎⁎P=0.61 IL-1β/H2O2 vs. basal. Journal of Cystic Fibrosis 2006 5, 113-119DOI: (10.1016/j.jcf.2005.12.003) Copyright © 2006 European Cystic Fibrosis Society Terms and Conditions