Effect of VX-770 (Ivacaftor) and OAG on Ca2+ influx and CFTR activity in G551D and F508del-CFTR expressing cells Laura Vachel, Caroline Norez, Frédéric.

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Effect of VX-770 (Ivacaftor) and OAG on Ca2+ influx and CFTR activity in G551D and F508del-CFTR expressing cells Laura Vachel, Caroline Norez, Frédéric Becq, Clarisse Vandebrouck Journal of Cystic Fibrosis Volume 12, Issue 6, Pages 584-591 (December 2013) DOI: 10.1016/j.jcf.2013.05.008 Copyright © 2013 European Cystic Fibrosis Society. Terms and Conditions

Fig. 1 Expression of CFTR and TRPC6 proteins in CHO-K1, CHO-WT, CHO-F508del and CH-G551D cells. (A) TRPC6 and Hsp70 proteins detected by Western blot performed for the four cell lines. Hsp70 protein was used as a control protein. (B) Ratio of Hsp70 band intensity on TRPC6 band intensity (N=2). (C) CFTR protein detected by Western blot performed for the four cell lines. ns, no significant difference. Journal of Cystic Fibrosis 2013 12, 584-591DOI: (10.1016/j.jcf.2013.05.008) Copyright © 2013 European Cystic Fibrosis Society. Terms and Conditions

Fig. 2 Functional characterization of Ca2+ influx and CFTR activity in CHO-WT cells, CHO-G551D cells and CHO-F508del cells, corrected or not with 100μm miglustat. (A) Representative traces of iodide efflux curves after 3μm forskolin+30μm genistein stimulation in CHO-WT and CHO-G551D cells. (B) Representative traces of iodide efflux curves after forskolin 3μm+genistein 30μm stimulation in CHO-F508del cells corrected or not with 100μm miglustat. (C) Histograms show the mean relative rate of CFTR activity in each cell line (n=4). (D) Representative traces of Ca2+ mobilization induced by 100μm of OAG for 10min in CHO-WT, CHO-G551D, and CHO-F508del cells. (E) Representative traces of Ca2+ mobilization during 10min of stimulation by 100μm of OAG in CHO-F508del cells after being treated for 2h with 100μm miglustat. (F) Histogram shows normalized area under the curve (AUC) corresponding to Ca2+ mobilization induced by 100μm OAG (n, number of cells recorded/N, number of cell passages). Results are presented as mean±SEM. ***p<0.001. ns, no significant difference. Journal of Cystic Fibrosis 2013 12, 584-591DOI: (10.1016/j.jcf.2013.05.008) Copyright © 2013 European Cystic Fibrosis Society. Terms and Conditions

Fig. 3 Functional coupling of Ca2+ influx and CFTR in non-CF cells (CHO-WT cells). (A) Representative traces of iodide efflux curves after CFTR stimulation with forskolin 3μm and OAG 100μm or SKF96365 10μm. (B) Histograms show the mean relative rate of CFTR activity for the experimental condition indicated below each bar (n=4). (C) Representative traces of Ca2+ mobilization induced by 100μm OAG after modulation of CFTR activity with 3μm forskolin or 1μm GPinh5a. (D) Histogram shows normalized AUC corresponding to Ca2+ mobilization induced by 100μm of OAG (n, number of cells recorded/N, number of cell passages). Results are presented as mean±SEM. ***p<0.001. ns, no significant difference. Journal of Cystic Fibrosis 2013 12, 584-591DOI: (10.1016/j.jcf.2013.05.008) Copyright © 2013 European Cystic Fibrosis Society. Terms and Conditions

Fig. 4 Functional coupling of Ca2+ influx and CFTR in CF cells (CHO-G551D cells). (A) Representative traces of Ca2+ mobilization induced by 100μm of OAG after modulation of CFTR activity with 10μm VX-770. (B) Histograms show normalized AUC corresponding to Ca2+ mobilization induced by 100μm OAG (n, number of cells recorded/N, number of cell passages). (C) Representative traces of iodide efflux curves after CFTR stimulation with 10μm VX-770 or 100μm OAG. (D) Histograms show the mean relative rate of CFTR activity for the experimental condition indicated below each bar (n=4). Results are presented as mean±SEM. ***p<0.001. **p<0.01. ns, no significant difference. Journal of Cystic Fibrosis 2013 12, 584-591DOI: (10.1016/j.jcf.2013.05.008) Copyright © 2013 European Cystic Fibrosis Society. Terms and Conditions

Fig. 5 Relative comparison of CFTR and OAG-Ca2+ activities. Ratio=(kpeak−kbasal)/AUC. Journal of Cystic Fibrosis 2013 12, 584-591DOI: (10.1016/j.jcf.2013.05.008) Copyright © 2013 European Cystic Fibrosis Society. Terms and Conditions