Fig. 3.
VX-809 increased CFTR maturation and chloride secretion in cultured F508del-HBE. (A) Glycosylation pattern of CFTR (Upper) and F508del-CFTR pretreated for 48 h with VX-809 at the indicated concentrations (Lower). (B) Quantification of the data in A (n = 3) expressed as a percentage of the mature/total CFTR in the absence of VX-809 (as percent of control). (C) Representative recording of the forskolin (10 μM)-stimulated IT in F508del-HBE pretreated for 48 h with VX-809 at the indicated concentrations. Before adding forskolin, amiloride was added to block the epithelial Na+ channel. A basolateral-to-apical chloride gradient was used for Ussing chamber experiments. (D) Quantification of the forskolin-stimulated IT in F508del-HBE isolated from seven patients with CF homozygous for the F508del-CFTR mutation (left y axis). Right y axis shows the IT normalized to the 10 μM forskolin-stimulated IT in non-CF HBE. (E) The onset of VX-809 action was determined by measuring the CFTR-mediated IT in F508del-HBE pretreated with 3 μM VX-809 for the indicated times (n = 6; data from single donor lung). (F) Cell surface turnover of F508del-CFTR was determined by first incubating F508del-HBE for 48 h with 3 μM VX-809 and then measuring the forskolin-stimulated IT at the indicated times after VX-809 washout (data from single donor lung; n = 6). (G) Concentration response curve for VX-809 in the absence (•) and presence of 1 μM VX-770 (○) in F508del-HBE from a single donor. (H) Mean (±SEM) forskolin-stimulated IT values in F508del-HBE pretreated for 48 h with VX-809 (3 μM), 4-phenylbutyrate (4-PB; 1,500 μM), Corr-4a (10 μM), and VRT-325 (6.7 μM) for 8 d with 1 μM SAHA or 2 to 4 h with 100 μM miglustat. The concentration and treatment duration for each compound were based on the maximally effective experimental conditions published for the previously described CFTR corrector (18, 30, 43, 44). Asterisks indicate significant (P < 0.05; paired t test) increase in IT vs. untreated levels.