Vx 661

Primary human nasal epithelial cells (pHNEs) were obtained from nasal brushing as before [37 (link)]. After expansion, cells were seeded on porous membranes and cultured under air–liquid interface (ALI) conditions for 21 days following protocols by Jeffrey Beekman’s lab (Utrecht, The Netherlands, submitted to Cell Rep Med). Differentiated monolayers of pHNEs were incubated with 3 µM VX809, 5 µM VX661 (Selleckchem, TX, USA), or DMSO alone for 48 h prior to measurements in an Ussing Chamber. The pHNE monolayers were mounted on micro-Ussing chambers and analyzed under open-circuit conditions, as described previously [16 (link)]. CFTR was stimulated by the presence of forskolin (2 µM) + IBMX (100 µM), and specificity was evaluated after using CFTR inhibitor 172 (25 µM). Experiments were performed in the presence of amiloride (20 µM) to inhibit the Epithelial Sodium (Na+) Channel (ENaC) and thus avoid interference of ENaC-mediated currents. In pHNEs, the maximum CFTR activation corresponds to the sum of IBMX/Fsk and IBMX/Fsk+VX-770 responses [16 (link)]. CFTR rescue by CFTR modulators in pHNEs was assessed by comparison of the maximum activation of CFTR between control pHNE (DMSO) and treated pHNE (VX809 or VX661). The individuals were considered responders when this difference was statistically different (p-value < 0.05). Each experiment was performed at least three times.

The assay was performed as previously described (Botelho et al, 2015 (link)) CFBE cells expressing the inducible mCherry‐Flag‐CFTR reporter (WT‐ or F508del‐CFTR variants) were grown to confluence and split to 50% confluency. Twenty‐four hours later, the cells were trypsinized to antibiotic‐free medium and seeded in siRNA coated 384‐well plates (1,000 cells/well) using a Multidrop™ Combi peristaltic dispenser (Thermo Scientific 5840300). CFTR expression was induced for 48 h (24 h after seeding) by supplementing the medium with 1 μg/ml of doxycycline (Sigma 9891). At this point, VX‐809 (3 µM) or VX‐661 (5 µM) (Selleckchem (S7059)) were added to selected wells containing Neg1 siRNA as a positive control for F508del‐CFTR traffic rescue. Extracellular Flag tags were immunostained in non‐permeabilized cells 72 h after seeding. The primary anti‐Flag antibody (Sigma F‐1804, 1:500) was incubated 1 h at 4°C, the cells were fixed with PFA 3% for 20 min at 4°C, an anti‐mouse IgG antibody conjugated with Alexa 647 (Invitrogen A‐31571. 1:500) was incubated 1 h at room temperature and Hoechst 33342 (200 ng/ml, Sigma B2261) was incubated 1 h at room temperature. Four independent biological replicates were performed.

The 3-[6-[[[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl]amino]-3-methyl-2-pyridinyl]-benzoic acid (Lumacaftor, VX809), 1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-[1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(2-hydroxy-1,1-dimethylethyl)-1H-indol-5-yl]-cyclopropanecarboxamide (Tezacaftor, VX661), N-[2-(5-chloro-2-methoxyphenylamino)-2 0-yl]benzamide (corr4a), 4-(cyclohexyloxy)-2-{1-[4-(4-metho-xybenzenesulfonyl)piperazin-1-yl]ethyl}quinazoline (VX325) correctors were purchased from Selleck Chemicals (Munich, Germany). If not explicitly indicated in the text, all other chemicals and culture media components were provided by Sigma-Aldrich (Milan, Italy).

For the FIS assay, organoids were seeded into 96-well plates. After 24 h, the organoids were stained with Calcein AM (Biotium) and stimulated with forskolin (Sigma-Aldrich, USA) at concentrations of 0.128 and 5 µM. The processing lasted for 60 min, and the “fixed” fields were photographed using the Observer. D1 fluorescence microscope (Zeiss, Germany) at 10-min intervals. CFTR correctors VX-809, VX-661 and VX-445 were all added at a concentration of 3.5 µM (Selleckchem, Houston, TX, USA) at the stage of organoids seeding, and CFTR potentiator VX-770 (3.5 µM; Selleckchem, USA)—simultaneously with forskolin. Quantitative analysis of the FIS was carried out using the Image J program (v1.52n state version). The Sigma Plot 12.5 program was used for plotting.

Apical CFTR-mediated chloride conductance was measured as previously described^{18 (link)}. Briefly, hPSC-derived cholangiocytes were grown in 96-well plates and treated for 24 h with either or combination of CFTR modulators, 3 μM VX809, 3 μM VX661 (Selleck), 3 μM R and S-VX445 (MedChemExpress), 0.5 μM AC1 (X281602), 3 μM AC2-1 (X281632), 3 μM AC2-2 (X300549) (Abbvie), or DMSO. Cells were labeled using blue membrane potential sensitive FLIPR dye dissolved in sodium and chloride-free buffer (150 mM NMDG, 150 mM gluconolactone, 10 mM HEPES, pH 7.4, 300 mOsm) at a concentration of 0.5 mg/ml. Cells were incubated for 30 min at 37 °C. The plate was read in a fluorescence plate reader (FLIPR® Tetra System or SpectraMax i3; Molecular Devices) at 37 °C. After reading baseline fluorescence, CFTR was stimulated with a combination of the cAMP agonist Forskolin (10 μM) and potentiators, 1 μM VX770 (Selleck) or 1.5 μM AP2 (X300529) (Abbvie). CFTR-mediated depolarization was detected as an increase in fluorescence, and repolarization was detected as a decrease with addition of 10 μM CFTR-specific inhibitor CFTR_Inh-172 to all wells.

3 μM VX-445 (S8851; SelleckChem), 5 μM VX-661 (S7059; SelleckChem), or 3 μM VX-809 (S1565; SelleckChem) were added to CFBE cells 48 h after transfection with NCtrl or siYAP1 (1) and left for 24 h. Compounds were dissolved in DMSO which was used as a negative control in the experiment.