Figure 6. SLC6A14 expression enhances residual cAMP-dependent F508del channel function in murine intestinal tissues via arginine-mediated NO signaling.

(a) Hypothetical model depicting the role of SLC6A14 in transporting arginine across the apical surface thereby increasing intracellular nitric oxide (NO) levels. (b) Split-open colonic organoids from CF (Cftr^{F508del/F508del}) mice were studied using the ACC assay at physiological temperature (37°C). Line graph represents change in fluorescence relative to baseline (ΔF/F₀) as a measure of F508del-CFTR, after pre-incubation with vehicle or inducible Nitric-oxide Synthase (iNOS) inhibitor 1400W (100 µM) for 30 mins. (c) Bar graph represents maximum change in ACC fluorescence from baseline (ΔF/F₀) after acute addition of FSK, at physiological temperature (37°C) in F508del-CFTR split-open murine organoids (mean ± SEM), after 30 mins pre-incubation with vehicle or iNOS inhibitor 1400W (100 µM). (**p=0.006, ns = not significant, n = 4 biological replicates for each genotype). (d) Epithelial basal NO levels measured using DAF-FM fluorophore, by splitting open colonic organoids derived from Wt and Slc6a14^(-/y) mice. Bar graph represents mean ± SEM. Unpaired t-test was performed (*p=0.022, n = 5 biological replicates). (e) Line graph represents change in NO levels upon acute addition of L-arginine (1 mM), in split-open colonic organoids derived from C57Bl/6 Wt and Slc6a14^(-/y) mice. Two-way ANOVA with Sidak’s multiple comparison test was performed (p<0.0001, n ≥ 4 for each genotype, for t = 0, 5, 10 mins *p<0.05, for t = 15, 20, 25 mins **p=0.006) (f) Bar graph represents maximum change in intracellular NO levels upon acute addition of L-arginine (1 mM), in split-open colonic organoids from Wt and Slc6a14^(-/y) mice (mean ± SD). Unpaired t-test was performed (*p=0.009, n ≥ 4 biological replicates for each genotype). (g) Line graph represents basal [NO] levels and change in [NO] after addition of SLC6A14 agonist L-arginine (1 mM) or control (buffer alone), in split-open murine FVB Cftr^{F508del/F508del} colonic organoids transduced with human SLC6A14-GFP or control GFP. (h) Bar graph represents basal [NO] levels in split-open murine organoids transduced with SLC6A14-GFP or just GFP. Mean ± SEM is plotted. Unpaired t-test was performed (*p<0.0001, n = 3 biological replicates). (i) Bar graph represents change in [NO] levels (Δ[NO]) after addition of SLC6A14 agonist L-arginine (1 mM) or control (buffer alone), in split open FVB Cftr^{F508del/F508del} colonic organoids transduced with human SLC6A14-GFP or control GFP. Mean ± SEM is plotted. One-way ANOVA with Tukey’s multiple comparison test was performed (*p=0.02, **p=0.006, n ≥ 3 biological replicates for each condition).

Figure 6—figure supplement 1. iNOS expression in primary murine colonic tissue.

Fresh murine colonic tissue was homogenized and RNA was extracted. iNOS gene and housekeeping gene Tbp mRNA expression was measured using PCR (35 cycles), in presence or absence of reverse transcriptase (RT) enzyme. Expected product size for iNOS cDNA amplification is 115 bp and for Tbp cDNA is 131 bp.

Figure 6—figure supplement 2. NO-mediated signaling potentiates mutant F508del CFTR function in intestinal epithelial cells.

(a) Split-open ileal organoids from F508del-CFTR (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 reads, cells were acutely treated with GSNO (10 µM) or vehicle, followed by addition of CFTR cAMP agonist FSK (10 µM) or DMSO. All wells received CFTRinh-172 (10 µM) at the end. (b) Each point on the scatter plot represents maximum change in ACC fluorescence from baseline (ΔF/F₀) after acute addition of FSK. Paired t-test was performed (*p=0.005, n = 3 biological replicates defined as different passages of murine ileal organoids, and n ≥ 2 technical replicates for each biological replicate).

Figure 6—figure supplement 3. Standard curve for Nitric-Oxide (NO) measurement.

(a) Split-open organoids from Wt mice were used to measure NO levels with DAF-FM fluorescence. Increasing levels of NO in the epithelium were achieved by addition of known NO donor (Proli NONOate). Linear regression was used to fit the data. Goodness of the fit was R² = 0.84 (n = 3 biological replicates). (b) Split-open organoids from Wt C57Bl/6 mice, and transduced with GFP, were used to measure NO levels with DAF-FM fluorescence, as described in the methods. A standard curve was generated by increasing intracellular NO levels using a NO donor (Proli NONOate). Linear regression was used to fit the data. Goodness of the fit was R² = 0.95 (n = 3 biological replicates).