An in vitro CFTR membrane potential assay was performed on Calu-3 cells and primary human bronchial epithelial cells, from two independent donors, cultured at 37 °C as previously described14. All human samples were collected from consented individuals under ethics approval granted by Hamilton Integrated Research Ethics Board (Project Number 5099-T).

Calu-3 cells were grown for 21 days on 96-well plates and primary HBECs were grown for 14 days under air–liquid interface conditions on collagen-coated (Sigma-Aldrich) 6.5 mm Transwell Inserts (Corning). All cells were washed with HBSS prior to experimental conditions. After washing with HBSS, cells were loaded with BLUE Membrane Potential Dye (Molecular Devices, #R8042) dissolved in 37 °C chloride-free buffer (NMDG Gluconate Buffer – 150 mM NMDG-gluconate, 3 mM KCl, 10 mM HEPES, pH 7.35, 300 mOsm). Immediately after dye loading, CFTR activity measurements were taken. CFTR activity measurements are fluorescent readouts for detecting ion channel activity. CFTR inhibition was performed with CFTRinh-172 (10 μM) for all experiments to attribute changes in fluorescence as CFTR channel activity. This assay has been validated using proof of concept studies where results obtained from this assay were able to recapitulate findings obtained from direct measurements of ion channel activity using Ussing chambers14. At the end of this assay, raw data was exported for statistical analysis. Tracings demonstrating the variation in CFTR Activity as a function of time, for key experiments, have been included in the supplementary information (Supplementary Fig. 5).

For Calu-3 cells, measurements were taken during baseline (40 min), cAMP elevation (30 min), and CFTR inhibition (20 min). cAMP elevation was performed using forskolin or isoproterenol (0.0005–50 μM). For assays with ABCC4 or PDE-4 inhibitors, a pre-incubation with the inhibitors (30 min) in the plate reader was performed prior to cAMP elevation. CFTR modulator VX-770 was used at 1 μM. CFTR activity was determined by dividing a single membrane potential peak measurement after the ABCC4 or PDE-4 inhibition and cAMP elevation additions to the stabilized baseline over DMSO control.

For primary HBECs, measurements were taken during baseline (8 min), drug additions (40 min), and CFTR inhibitions (18 min). cAMP elevation was performed using forskolin (10 μM). The same concentrations used in Calu-3 cells for ABCC4 and PDE4 inhibitors, and VX-770 were used for primary HBECs, however the pre-incubation was skipped and instead, all drug combinations were added once at the beginning of the drug addition step. CFTR activity was determined by averaging six independent measurements within a single Transwell insert. Measurements were normalized to the averaged baseline over DMSO control.

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