Fig 2: Effect of SFPQ overexpression on CFTR expression. Overexpression of SFPQ in F508del-CFTR CF lung epithelial cell line (CFBE41o-) was performed using a vector encoding SFPQ. (A) CFBE41o- cells containing the F508del mutation show low levels of CFTR labeling (green) that is faintly perinuclear and punctate. Overexpression of SFPQ in CFBE41o- causes a large increase in the amount of CFTR labeling (green). Green = CFTR, red = phalloidin, blue = DAPI. (B) Expression levels of CFTR protein was analyzed by immunoblot and CFTR mRNA were measured by RT-qPCR analysis using GAPDH mRNA as an endogenous control. (C) Total and surface expression of CFTR was analyzed using a FAP assay in F508del-CFTR CFBE410- cells transfected with the adenoviral vector encoding SFPQ with GFP-tag or control vector encoding GFP alone. The graph indicates data normalized from 4 independent experiments. (D) F508del-CFTR function was analyzed in CFBE cells by Ussing chamber assays. CFBE cells were cultured at the air–liquid interface (ALI), and after differentiation, they were transfected for 48 h with the adenoviral vector encoding SFPQ with GFP-tag or control vector encoding GFP alone. Forty-eight hours post infection, cells were analyzed for short circuit current. The data are representative of two or more independent experiments.

Fig 3: Cellular thermal stability assay (CETSA) to test the binding of correctors to F508del-CFTR. (A) Representative immunoblots of the supernatants and pellets after cell lysates are incubated for 10 minutes at different temperatures (33, 38, 43, 47, 52, 57 and 61 °C) in the presence of putative pharmacological chaperones. All correctors were tested at 10 µM except VX-809 (1 µM). Blots were probed with a monoclonal anti-CFTR antibody. (B) Graphical representation of the results shown in part A.

Fig 4: Protein trafficking assays, immunoblotting, FLIPR membrane potential (FMP) assays and electrophysiology demonstrate the rescue of F508del-CFTR. (A) Graph of concentration dependence of correction in BHK cells by MCG1516A after 24 h incubation. (B) Immunoblot of F508del-CFTR in BHK cells after 24 h treatment with MCG1516A (10 µM) VX-809 (1 µM) and wild-type CFTR (WT). (C) Relative band intensities for bands B and C in each lane in panel B. (D) FMP assay (FMP) that monitors membrane depolarization induced by forskolin + genistein when cells are pretreated for 24 h with VX-809 (1 µM), RDR1 (10 µM) or MCG1516A (10 µM). (E) F508del-CFTR functional expression was assayed as ? Isc when well differentiated HBE cells are stimulated with forksolin + genistein after 24 h pretreatment with pharmacological chaperones. The basolateral membrane was permeabilized using nystatin and an apical-to-basolateral chloride gradient was imposed. Panels A,C,E, and F show means ± SEM, n = 4, *p < 0.05.

Fig 5: CETSA with deletion mutations identify the domain of F508del-CFTR where pharmacological chaperones act. (A) Schematic of full length CFTR (1-1480aa) and 5 deletion mutants used to localize corrector sites of action: A1 (1-1218aa), B1 (389-1480aa), C1 (679-1480aa), D1 (835-1480aa), and E1 (1-694aa). All constructs were HA tagged and expressed in BHK cells using the pNUT vector. (B) Immunoblots of supernatants and pellets from after lysates are incubated at different temperatures (33, 38, 43, 47, 52, 57 and 61) in the presence of the RDR1, MCG1516A or latonduine (each at 10 µM) or VX-809 (1 µM). Blots were probed with anti-HA antibody. (For graphical representation see Suppl. Fig. 7).