Fig 2: FIS assay. (A–C) Three representative brightfield and DAPI-stained images at times 0, 1, and 8 h for non-CF (A), F508del/P67L (B), and F508del/F508del (C) CFTR HNE organoids. (D) Average fractional change from baseline over 8 h for each subject, dotted lines highlighting the 1- and 8-h timepoints. (E) 1-h vs. 8-h time points for the average fractional change of non-CF, F508del/P67L, and F508del/F508del organoids (n = 1). (A) Corresponds with Video S5, (B) corresponds with Video S6, and (C) corresponds with Video S7. ns = not significant; * p = 0.0441, ** p = 0.0023, and **** p < 0.0001.

Fig 3: Co-localization of Split-CFTR FH, BH, and R-region Polypeptides in BHK cells. Representative confocal microscopy images of BHK cells co-expressing the split-CFTR front half (FH) or back half (BH) in green and the WT or mutant R-regions in red. Upper rows for each construct show FH and R-region co-localization and bottom rows show BH and R-region co-localization in yellow. FH, BH, and R-region were detected by MM13-4, M3A7, and MAB1660 antibodies, respectively. Negative control images where primary antibodies were omitted or where non-transfected BHK cells were used as indicated

Fig 4: R-region Interaction with the FH and the BH of CFTR. Representative fluorescence microscopy images from in situ PLA experiments showing PLA signals for FH-R (A) and the BH-R (B) complexes (red dots) formed at different phosphorylation conditions. SR-WT cells were either left untreated (Basal) or stimulated with 150 µmol/L CPT-cAMP + 1 mmol/L IBMX + 10 µmol/L FSK (PKA), 20 nmol/L PMA for 2 h (PKC), or with 150 µmol/L CPT-cAMP + 1 mmol/L IBMX + 10 µmol/L FSK + 20 nmol/L PMA for 2 h (PKA + PKC). Cells expressing Split?R (with no R-region) and full-length CFTR were used as negative and positive controls, respectively. Cell nuclei were stained with DAPI (blue). FH, BH, and R-region were detected by H-182, C-19, and MAB1660 antibodies, respectively. C. In situ PLA experiments in cells expressing full-length CFTR showing positive signals due to the presence of the R-region (positive control), and in cells expressing split-?R either untreated or after PKA or PKC stimulation showing negative signals regardless of the phosphorylation condition due to the absence of the R-region (negative control). D. Histogram presenting averages of the corrected total cell fluorescence (CTCF) for the PLA signal for FH-R and BH-R, measured in BHK cells in each condition displayed in A and B (see Section 2 for CTCF calculation). Statistical significance (*P < .01; **P < .001) was calculated in relation to basal values for each half. Values are means ± SEM for a minimum of five independent experiments

Fig 5: Mutant R-region interactions with the FH and the BH of CFTR. Representative fluorescence microscopy images from in situ PLA experiments showing FH-R (upper row) and BH-R (lower row) complexes formed (red dots) for mutant constructs SR-S641A/T682A (A), SR-S686A (B), SR-S686Dm (C), and SR-6CA (D) at basal condition or after PKA or PKA + PKC stimulation as described before. Cell nuclei were stained with DAPI (blue). FH, BH, and R-region were detected by H-182, C-19, and MAB1660 antibodies, respectively. Histograms presenting the average of corrected total cell fluorescence for FH-R (E) and BH-R (F) PLA signals measured in BHK cells from A-D. Statistical significance (*P < .05; **P < .01; ***P < .001) was calculated in relation to values for SR-WT in each phosphorylation condition. Values are means ± SEM for a minimum of 3 + 5 independent experiments