Figure 7.

Effect of the hit compounds on TMEM16A in other in vitro model systems. (A) The effect of the hit compounds (3 µM) on chloride conductance was determined in Ussing chamber measurements with ALI-differentiated CFTR-null nasal cells (n = 3 independent donors; each compound was measured in at least 2 different donors; n = 1–6 measurements per donor). No significant differences were found between one of the compounds and DMSO; (B) assessment whether the hit compounds (3 µM) enhance UTP-induced currents in Ussing chamber measurements. Experiments were conducted by stimulating different concentrations of UTP with hit compounds in ALI-differentiated CFTR-null nasal cells (n = 3 independent donors, each compound was measured in at least 2 different donors; n = 2–3 measurements per donor). No significant results were found between one of the compounds and DMSO, for any concentration of UTP; (C,D) effect of the 12 hit compounds (3 µM) on ionomycin-induced (1 µM) iodide influx was assessed with an YFP-quenching assay in CFBE cells. TMEM16A-dependency was demonstrated with sensitivity for Ani9 (3 and 10 uM, shown in red). DMSO was used as negative control (shown in green) and E_act (3 and 10 µM) as positive control (shown in blue). For quantification, quenching rates were normalized to the control; (E,F) effect of a selection of 6 hit compounds (3 µM) on ATP-induced (5 µM) iodide influx was analyzed in HT-29-YFP cells. TMEM16A-dependency was demonstrated with sensitivity for Ani9 (10 uM, shown in red) and DMSO was used as negative control (shown in green). For quantification, quenching rates were normalized to the control. Analysis of differences were performed with one-way ANOVA and Dunnett’s post hoc test (A,D,F) or a two-way ANOVA with Dunnett’s post hoc test (B). ** p < 0.01, **** p < 0.0001.