Vitamin D receptor agonists inhibit pro-inflammatory cytokine production from the respiratory epithelium in cystic fibrosis P. McNally, C. Coughlan,

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Vitamin D receptor agonists inhibit pro-inflammatory cytokine production from the respiratory epithelium in cystic fibrosis P. McNally, C. Coughlan, G. Bergsson, M. Doyle, C. Taggart, L. Adorini, M.R. Uskokovic, B. El-Nazir, P. Murphy, P. Greally, C.M. Greene, N.G. McElvaney Journal of Cystic Fibrosis Volume 10, Issue 6, Pages 428-434 (December 2011) DOI: 10.1016/j.jcf.2011.06.013 Copyright © 2011 European Cystic Fibrosis Society Terms and Conditions

Fig. 1 A) Western blot for vitamin D receptor (VDR) in cytoplasm (C) and nucleus (N) of CFTE cells at baseline (CTR) and after 10min, 30min, 1h, 2h and 4h of incubation with 10−9M 1,25(OH)2D3. The experiment was repeated 3 times. A representative blot is shown. B) Densitometric analysis of the blot in A. Journal of Cystic Fibrosis 2011 10, 428-434DOI: (10.1016/j.jcf.2011.06.013) Copyright © 2011 European Cystic Fibrosis Society Terms and Conditions

Fig. 2 IL-8 concentrations in the supernatants of CFTE (A) and CFBE (B) cells after treatment with either 1% media (control), LPS (1%) or Pseudomonas PAO1 culture media (1%). The cells were either untreated or pre-treated (for 1h) with 1,25(OH)2D3. Assays were preformed in duplicate, 3 times. All results are shown. Error bars reflect standard error of the mean. Differences in individual means were assessed by Student's t test. Journal of Cystic Fibrosis 2011 10, 428-434DOI: (10.1016/j.jcf.2011.06.013) Copyright © 2011 European Cystic Fibrosis Society Terms and Conditions

Fig. 3 Mean A) IL-8 and B) IL-6 levels in the supernatants of CFTE cells after treatment with 0, 0.1, 0.5 or 1.0μg/ml LPS for 4h, 8h or 24h. The cells were either pre-treated (for 1h) with 10−9M 1,25(OH)2D3 or untreated. Assays were performed in duplicate 3 times. All results are shown. Error bars reflect standard error of the mean. Differences in individual means were assessed by Student's t test. Journal of Cystic Fibrosis 2011 10, 428-434DOI: (10.1016/j.jcf.2011.06.013) Copyright © 2011 European Cystic Fibrosis Society Terms and Conditions

Fig. 4 Relative IL-8 (A) and IL-6 (B) concentrations in the supernatants of CFTE cells after treatment with 1.0μg/ml LPS for 4h (relative to controls). The cells were either untreated or pre-treated (for 1h) with 1,25(OH)2D3, or the VDR agonists A1–A5 at 10−9M. Assays were performed in duplicate 3 times. All results are shown. Error bars reflect standard error of the mean. Differences in individual means were assessed by Student's t test. Differences in means between the groups were assessed by ANOVA. Journal of Cystic Fibrosis 2011 10, 428-434DOI: (10.1016/j.jcf.2011.06.013) Copyright © 2011 European Cystic Fibrosis Society Terms and Conditions

Fig. 5 Relative absorbance values reflecting concentrations of total cellular IκBα from CASE® assay (relative to controls at Time 0). Wells containing CFTE cells were treated with LPS 1μg/ml or control, in the presence or absence of 10−9M 1,25(OH)2D3. Differences in mean values were measured between 1) control and 1,25(OH)2D3, 2) control and LPS and 3) LPS and LPS with 1,25(OH)2D3. Assays were performed in duplicate twice. All results are shown. Error bars reflect standard error of the mean. Differences in individual means were assessed by Student's t test. Journal of Cystic Fibrosis 2011 10, 428-434DOI: (10.1016/j.jcf.2011.06.013) Copyright © 2011 European Cystic Fibrosis Society Terms and Conditions

Fig. 6 Relative absorbance values reflecting concentrations of cellular phospho-IκBα from CASE® assay (relative to controls at Time 0). Wells containing CFTE cells were treated with LPS 1μg/ml, in the presence or absence of 10−9M 1,25(OH)2D3. Differences in mean values were measured between 1) control and 1,25(OH)2D3, 2) control and LPS and 3) LPS and LPS with 1,25(OH)2D3. Assays were performed in duplicate twice. All results are shown. Error bars reflect standard error of the mean. Differences in individual means were assessed by Student's t test. Journal of Cystic Fibrosis 2011 10, 428-434DOI: (10.1016/j.jcf.2011.06.013) Copyright © 2011 European Cystic Fibrosis Society Terms and Conditions

Fig. 7 (A) Western blot for L-37 in CFTE cells (1×106) under control conditions and after incubation for 24h with 1,25(OH)2D3. (B) Expression levels of hCAP18/LL-37 mRNA normalised to GAPDH in CFTE cells in the presence and absence of 1,25(OH)2D3. Journal of Cystic Fibrosis 2011 10, 428-434DOI: (10.1016/j.jcf.2011.06.013) Copyright © 2011 European Cystic Fibrosis Society Terms and Conditions