Extended Data Fig. 2. FOXI1-Cre^ERT2 and CFTR conditional KO (CFTR^L/L) ferret models demonstrate CFTR is required for ionocyte apical uptake of anions.

CRISPR homology-directed repair (HDR) was used to generate FOXI1-Cre^ERT2 and CFTR^L/L ferrets. a, Schematic of the strategy for generating transgenic ferrets with an IRES-CreERT2 insertion in the FOXI1 3’-UTR. b, Schematic of the floxed exon-16 in CFTR^L/L ferrets and strategy for deletion of CFTR in FOXI1-Cre^ERT2::CFTR^L/L (CFTR-cKO) ferrets. c, Primary FOXI1-Cre^ERT2 airway basal cells transduced with a lentivirus encoding LoxP-dsRED-stop-LoxP-YFP-H148Q/I152L cassette (herein called FOXI1-Cre^ERT2::YFP^H148Q/I152L) and differentiated at ALI, treated with hydroxy-tamoxifen (OH-Tam) and then used for functional studies of halide transport. d, Scattered YFP-positive ionocytes were observed in the pseudostratified airway epithelium of only OH-Tam treated differentiated FOXI1-Cre^ERT2::YFP^H148Q/I152L ALI airway cultures. Representative images from 3 independent ferret donors. e, Representative images of ASL height from differentiated WT, CFTR-KO, and FOXI1-KO ALI cultures challenged with 18 μl of Alexa-dye containing buffer (time zero) and following equilibration 24 hrs later. Representative images from n = 11 (WT), n = 10 (FOXI1-KO) and n = 9 (CFTR-KO) independent cultures. f, Dehydration experiment on WT ALI cultures monitoring the ASL height following apical perfusion of non-humidified 5% CO₂ for the indicated times. 20 min of dehydration was chosen for basolateral halide sensor assays (Extended Data Fig. 3) since the ASL height approached that observed CFTR-KO and FOXI1-KO cultures. Representative images from 3 independent experiments. g–k, Representative images and traces of apical I^– uptake in YFP halide sensor expression ionocytes of ALI cultures. g, No YFP quenching is observed in WT ionocytes after the addition of apical Cl^– buffer (18 μl) with Forskolin/IBMX (F&I) to stimulate CFTR (negative control). Mean ± s.e.m.; n = 28 ionocytes. h, YFP quenching is observed in WT ionocytes following the addition of apical I^– buffer with F&I. Mean ± s.e.m.; n = 38 ionocytes. i, YFP quenching, as shown in (h), is not observed following the addition of apical Na-Gluconate (Na-Gluc) buffer with F&I (negative control). Mean ± s.e.m.; n = 11 ionocytes. j, YFP quenching, as shown in (h), is reduced following the addition of apical I^– buffer, F&I, and GlyH101 CFTR inhibitor (negative control). Mean ± s.e.m.; n = 38 ionocytes. k, YFP quenching is not observed in CFTR-cKO ionocytes (FOXI1-Cre^ERT2::CFTR^L/L) following application of apical I^– buffer with F&I. Mean ± s.e.m.; n = 33 ionocytes.

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