Ivacaftor

48 h after transfection, cells were exposed to the CFTR potentiator ivacaftor (Sigma) and/or the anthracycline doxorubicin as indicated. The final concentration of DMSO in all treatment conditions and the control group was 0.3% v/v. Following exposure, cells were immediately placed in an incubator outfitted with an IncuCyte S3 (ESSEN Bioscience) microscope and phase-contrast images of 16 fields per well were automatically acquired every 4 h over an 80 h time course and confluence was determined automatically using the integrated software (S3 2019B Rev3). All conditions were performed in biological triplicates. Cell numbers were normalized to the first scan of the well right after the exposure. Statistical significance was determined using heteroscedastic two-tailed t tests or F tests as indicated. P < 0.05 was considered significant.

Cultures were treated with 3 µM elexacaftor (E) and 18 µM tezacaftor (T) or with 0.07% dimethyl sulfoxide (DMSO) (vehicle control) for 24 before Ussing chamber analysis. ivacaftor was added acutely during transepithelial short-circuit current (I_sc) measurements to ET pre-treated cultures, to provide conditions that maximally enhance CFTR function. CFTR modulators were obtained from Selleck Chemicals (Planegg, Germany), DMSO was obtained from Sigma-Aldrich (St Louis, MO, USA). I_sc was measured by EasyMount Ussing chambers (Physiologic Instruments, San Diego, CA, USA) as previously described, in presence of a chloride gradient (basolateral 145 mM vs. apical 5 mM) [22 (link),42 (link)]. After 10–20 min equilibration, amiloride (100 µM) was added to inhibit sodium absorption via the epithelial sodium channel (ENaC). To assess CFTR-mediated chloride secretion, forskolin (Fsk, 10 µM) and 3-isobutyl-1-methylxanthin (IBMX, 100 µM) were applied together, then ivacaftor (2.5 µM) was added, followed by CFTR inhibitor-172 (CFTRinh-172, 20 µM, TargetMol chemicals, Boston, MA, USA). All Ussing chamber chemicals and reagents, apart from CFTRinh-172 and ivacaftor, were obtained from Sigma-Aldrich (St Louis, MO, USA) at the highest level of available purity. Bioelectric responses were quantified by LabChart8 (AF Instruments, Oxfordshire, UK).