Figure 8. Mutant F508del-CFTR function in double mutant murine organoids can be enhanced by directly enhancing PKG-mediated phosphorylation.

(a) Split-open organoids from double mutant (Cftr^{F508del/F508del}; Slc6a14^(-/y)) mice were used to perform ACC assay, after low temperature (27°C) rescue of the mutant F508del CFTR protein. Line graph represents change in fluorescence from baseline (ΔF/F₀) relative to DMSO vehicle addition. After capturing baseline read, cells were acutely treated with 8Br-cGMP (10 µM) or vehicle, followed by addition of CFTR cAMP agonist FSK (10 µM). All wells received CFTRinh-172 (10 µM) at the end. (b) Bar graph represents maximum change in ACC fluorescence from baseline (ΔF/F₀) after acute addition of FSK, following low temperature (27°C) rescue of F508del-CFTR protein in double mutant spilt-open organoids (mean ± SEM). Stippled line represents ACC response to FSK in temperature rescued (27°C) CF (Cftr^{F508del/F508del}) split-open murine organoids, in absence of 8Br-cGMP. Paired t-test was performed (**p=0.0029, n = 5 mice for each genotype). (c) Organoids from double mutant (Cftr^{F508del/F508del}; Slc6a14^(-/y)) mice were stained with Calcein AM and after capturing baseline images, organoids were acutely treated with 8Bromo-cGMP (8Br-cGMP 10 µM) for 15 mins, followed by FSK (1 µM) for 30 mins. Representative images of forskolin-induced swelling (FIS) are shown for organoids pre-treated with 8Br-cGMP or vehicle (control). Experiments were performed at physiological temperature of 37°C. (d) Bar graph represents FIS response in double mutant (Cftr^{F508del/F508del}; Slc6a14^(-/y)) murine organoids, as measured by change in area from baseline after 30 mins of FSK addition (ΔA/A₀). Stippled line represents FIS response in CF (Cftr^{F508del/F508del}) organoids in absence of 8Br-cGMP. Graph represents mean ± SEM. Experiments were performed at physiological temperature of 37°C. Unpaired t-test was performed (***p=0.0005, n = 4 mice).