Figure 5
The mechanism of glafenine-mediated CFTR correction works via the arachidonic acid pathway. (A) A cartoon of the arachidonic acid pathway with specific enzyme inhibitors marked in red. (B) Cell-based HTS assay measuring surface F508del-CFTR in BHK cells after 24 h of treatment with glafenine, 10 μM and Trikafta in the presence and absence of PGH2 at 1 μM (n = 4). (C) Cell-based HTS assay measuring surface F508del-CFTR in BHK cells after 24 h of treatment with glafenine (10 μM), MF63 (10 μM), picotamide (1 μM), tranyl cypromine (10 μM), sorbinil (10 μM), and suramin (5 μM) (n = 4). (D) FMP assay (FMP) that monitors membrane depolarization induced by forskolin + VX-770 when cells are pretreated for 24 h with MF63 (10 μM) and Trikafta in the presence and absence of CFTR172inh (10 μM). Additionally, MF63 (10 μM) and Trikafta were added together for 24 h (n = 4). (E) Representative Isc responses of primary HBE cells expressing F508del-CFTR functional expression in well-differentiated primary human bronchial epithelial (HBE) cells determined from the increase in short-circuit current stimulated by acute addition of forskolin + genistein (ΔIsc). The basolateral membrane was permeabilized using nystatin, and an apical-to-basolateral chloride gradient was imposed by sequential addition of 10 µM forskolin, 100 nM vx-770, and 10 µM CFTR inh-172 after 24 h of preincubation with 0.1% dimethyl sulfoxide (vehicle) and MF63 (10 µM). (F) Graph for each compound for the data attained from the Ussing chamber (E) (n = 4). Data in (B), (C), (D) and (F) are presented as the means ± SEM, n = 4, *p < 0.05, **p < 0.01 and ***p < 0.001.
