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.
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