Fig 2: Characterization of nasal-airway organoids (NAOs) cultured with NR/IL-1ß.(A) Brightfield images of cystic fibrosis (CF) F508del/F508del NAOs cultured in control conditions or with NR+IL-1ß for 5 d. (B) Quantification of the mean organoid area (µm2, means ± SD) of control and NR+IL-1ß cultured CF F508del/F508del NAOs (n = 3 independent donors). (C, D) mRNA expression analysis of CF F508del/F508del NAOs (n = 3–6 independent) that were left unstimulated (control) or cultured with NR+IL-1ß for 5 d. (C, D) mRNA expression was determined of (C) CFTR, ANO1, SLC26A9 (all chloride channels), (D) SPDEF (secretory cells), and FOXJ1 (ciliated cells). Results represent target mRNA expression normalized for the geometric mean expression of the housekeeping genes ATP5B and RPL13A (means ± SD). (E) Immunofluorescence staining of CF organoids (control and cultured with NR+IL-1ß for 5 d) was conducted of ß-tubulin IV (ciliated cell) and MUC5AC (goblet cell) (green). DAPI (blue) was used for nuclear staining. Phalloidin (red) was used for actin cytoskeleton staining. (B, C, D) Analysis of differences was determined with a paired t test (B, C, D). *P < 0.05, **P < 0.01.

Fig 3: P300 downregulates protein expression levels of MUC5AC in A549 cells. Following co-transfection with the empty vector, untransfected control, P300 wt plasmid, P300 mut plasmid, P300 siRNA and control siRNA for 24 h, protein expression levels of MUC5AC were detected by immunofluorescence. MUC5AC, mucin 5AC; wt wild-type; mut, mutant; siRNA, small interfering RNA.

Fig 4: Formononetin inhibits mucus formation in IL-13-stimulated JME/CF15 cells. Relative mRNA and protein expression levels of MUC5AC in IL-13-stimulated JME/CF15 cells treated with 0, 0.1, 1 or 10 µM formononetin were detected using (A) reverse transcription-quantitative PCR and (B) western blotting, respectively. **P<0.01, ***P<0.001 vs. control; #P<0.05, ###P<0.001 vs. IL-13; @@P<0.01 vs. IL-13 + 0.1 µM; ???P<0.001 vs. IL-13 + 1 µM. (C) Fluorescence intensity of MUC5AC in JME/CF15 cells treated with control, IL-13 or IL-13 + formononetin (10 µM) was detected using immunofluorescence. MUC5AC is shown as the green fluorescence Scale bar, 50 µm. MUC5AC, mucin 5AC oligomeric mucus/gel-forming.

Fig 5: Characterization of basal cells derived from normal-looking regions of TO patients.a HE staining showing distinct cell commitments of patient-matched (PM) normal-looking region-derived TBBCs (PM n = 6 independent biological samples). Differentiation efficiency was assessed by IF staining on xenograft sections with Ace-Tub (ciliated) and Muc5AC (goblet) cell-type-specific markers. Scale bar, 100 µm. b PM-TBBCs prior to and post 20-day ALI differentiation were subjected to RNA-Seq. Differential gene expression profiles were visualized by heatmap with hierarchical clustering. c GO and KEGG enrichment analyses were performed to reveal molecular characteristics of three sub-groups, corresponding to normal-like (NL), goblet-metaplasia (GM), and cilium dysplasia (CD) phenotypes.