Fig 1: Functional Effect of ASO Combination with Corrector Compounds in Cell Lines with Defective CFTR(A–C) Representative traces of functional response in Ussing chambers after ASO treatment in CFF-16HBEge-F508del (A), CFF-16HBEge-N1303K (B), and CFF-16HBEge-W1282X (C) cells. Amiloride (100 µM) was added to block the epithelial sodium channel, followed by forskolin (10 µM) to stimulate CFTR, VX-770 (10 µM) to potentiate CFTR, and the inhibitor 172 (CFTRinh-172, 50 µM) to inhibit CFTR. F, forskolin; I, CFTR inhibitor 172. (D) Mean electrophysiological data for all CFF-16HBEge cell lines tested. Data is normalized to untreated control (UTC). Two-way ANOVA analysis was conducted with Dunnett’s multiple-comparison test between each mean and the mean of the untreated control. Data are mean + SEM. ***p < 0.0005, **p < 0.005.
Fig 2: Effect of ASOs to Upregulate CFTR in 16HBE14o- Cells(A) CFTR protein expression after indicated ASO treatments, normalized to untreated control (UTC). *p < 0.05, **p < 0.005, ***p < 0.0005 by two-way ANOVA with Dunnett’s multiple-comparison test. (B) CFTR surface-localized protein expression. *p < 0.05, ***p < 0.0005 by Holm-Sidak method. (C) CFTR function, measured by Ussing chamber and quantified relative to untreated control. Mean electrophysiological data for ASO-treated 16HBE14o- cells. Data is normalized to untreated control (UTC). Ctl, Control ASO; CFTR ASO, CFTR gapmer ASO. Amiloride (100 µM) was added to block the epithelial sodium channel, followed by forskolin (10 µM) to stimulate CFTR and the inhibitor 172 (CFTRinh-172, 50 µM) to inhibit CFTR. Data are mean + SEM. *p < 0.05, **p < 0.005 by Holm-Sidak method, a = 0.05. (D) Representative traces of functional response in Ussing chambers after the indicated ASO treatments in 16HBE14o- cells.
Fig 3: Let-7b-5p targets and regulates CFTR in a hyperglycemic microenvironment. (a) Intersections of CFTR-related miRNAs, T2DM-related miRNAs, and TDSCs-related miRNAs were shown by the VEEN plot. (b) A volcanic map of miRNAs related to TDSCs. (c) Relative expression levels of let-7b-5p in the diabetic and normal groups. (d) Expressions of let-7b-5p were significantly upregulated after high glucose (15 mM and 25 mM) treatment of TDSCs, compared to the control (5.5 mM), p < 0.05. (e). Let-7b-5p targeting regulated CFTR transcription, as predicted by RNAhybrid (minimum free energy = -28.9 kcal/mol). (f). TDSCs were transfected with the 3'UTR CFTR luciferase reporter gene along with a let-7b-5p mimic or let-7b-5p inhibitor, respectively. (g) Western blot assays demonstrated that the let-7b-5p mimic significantly downregulated CFTR protein levels in TDSCs.
Fig 4: Identification of Regulatory Elements in the CFTR 5' UTR(A) Structure prediction of the human CFTR 5' UTR. Structures of potential interest are boxed in green; the uAUG site is boxed in blue. Red arrows indicate regions of overlap with the ASO screen. (B) Schematic of reporter construct. CMV, CMV promoter; HSV TK, HSV TK promoter. (C) Representative reporter assay data in three cell lines for various mutations introduced into the CFTR 5' UTR. Data are mean + SEM. *p < 0.05, **p < 0.005 by Holm-Sidak method, a = 0.05.
Fig 5: Effect of ASOs on CFTR Function in CF Patient-Derived Primary CellsMean electrophysiological data for ASO-treated primary hBE cells from A455E/2183delAA > G (A), F508del/A455E (B), R117H 5T/F508del (C), and F508del/F508del (D) donors. Data are represented as percent of WT donor function. n = 5 per donor. Data are mean + SEM. *p < 0.05, **p < 0.005, ***p < 0.0005 by Holm-Sidak method, a = 0.05.
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