Figure S2. Characterization of air–liquid interface (ALI)-human nasal epithelial cell (HNEC) and nasal-airway organoid (NAO) swelling assay.

(A) Whole-mount IF confocal images of ALI-HNEC, showing maximal projections of the apical side of the secretory cell markers MUC5AC and club cell protein 10 (CC10), and ciliated cell marker β-tubulin IV (Z-stack at the bottom). The projection of the basal side shows the basal-cell marker p63 (Z-stack at the bottom). All markers are shown in green. (B, C) Open circuit Ussing chamber tracings showing transepithelial voltage measurements (V_te) from healthy control (B) and cystic fibrosis (CF) F508del/F508del (C) ALI-HNEC cultures. CF cultures were pre-treated with VX-809 or vehicle. During measurements, cells were treated sequentially with amiloride, Fsk/IBMX, VX-770, and CFTRi. The bar corresponds to a 10-min frame. (D) IF staining of NAOs for β-tubulin IV, MUC5AC, and p63 (all in green). (E, F) HC NAOs (n = 2 independent donors) were pre-treated with bumetanide or vehicle for 4 h, followed by stimulation with forskolin (Fsk) and assessment of forskolin-induced swelling (FIS). (G, H) HC NAOs (n = 3 independent donors) were pre-treated with CFTR inhibitors or vehicle, followed by assessment of FIS. (I, J) Comparison between HC and CF organoid swelling (both n = 6 independent donors) after stimulation with the calcium-activated chloride channel agonist E_act. (A, B) Data information: DAPI (blue) was used as nuclear staining; phalloidin (red) was used as actin cytoskeleton staining (A, D). (E, F, G, H, I, J) FIS assay results are depicted as the percentage change in surface area relative to t = 0 (normalized area) measured at 15-min time intervals for 2 h (means ± SD) (E, G, I) and as area-under-the-curve (AUC) plots (t = 120 min, means ± SD) (F, H, J). (H, J) Analysis of differences was determined with a paired (H) or unpaired (J) t test. *P < 0.05, ***P < 0.001.