Fig 1: PTCH1 protein is up-regulated in the airway epithelium of patients with COPD compared to subjects without COPD. Paraffin-embedded human kidney tissues stained with (A) secondary only and (B) PTCH1 antibody were shown. Paraffin-embedded human lung tissues from subjects (C) without COPD, (D) COPD GOLD STAGE 2, and (E) COPD GOLD STAGE 3 were stained with PTCH1 antibody. Airway epithelium-specific PTCH1 protein expression was normalized to the length of basement membrane (µm) in (F) non-COPD, COPD GOLD STAGE 2 and GOLD STAGE 3/4, and (G) with COPD stratified by smoking status (current vs ex-smokers). (H) PTCH1 mRNA expression was normalized to GAPDH and expressed as ??ct in human bronchial brushings from subjects with or without COPD. (I) Airway epithelial thickness and (J) total airway epithelial cell counts were quantified in subjects without COPD, COPD GOLD STAGE 2 and GOLD STAGE 3/4. Values were expressed as mean ± SEM. Kruskal–Wallis test with Dunnett’s multiple comparisons test was used in panels F,G. A two-tailed unpaired parametric t test was used in panel H. One-way analysis of variance was used in panels I,J. Correlations between total epithelial-specific PTCH1 protein expression (data log-transformed) with (K) epithelial thickness and (L) total epithelial cell count were shown. Linear regression analyses were used in panels K and L. Red dot = non-COPD, blue dot = COPD GOLD STAGE 2, orange dot = COLD GOLD STAGE 3/4. Representative immunofluorescence images of lung tissues from a patient with COPD GOLD STAGE 2 stained with (M) PTCH1 and MUC5AC (goblet cell) antibodies, (N) PTCH1 and FOXJ1 (ciliated cell) and (O) secondary only were shown.
Fig 2: EGF-induced MUC5AC expression is mediated through PTCH1-SMO axis in vitro. (A) PTCH1, SMO, SHH, GLI1, GLI2 and GLI3 mRNA expressions were assessed in NCI-H292 cells treated with EGF only. (B) Percent change in PTCH1, SMO, SHH, GLI1, GLI2 and GLI3 mRNA expressions were assessed in NCI-H292 cells treated with scrambled or PTCH1 siRNA for 48 h. (C) MUC5AC and D) MUC5B mRNA expressions were assessed in cells pre-treated with scrambled or PTCH1 siRNA followed by EGF stimulation. Values were expressed as mean ± SEM (N = 3 independent experiments). A two-tailed unpaired parametric t-test was used for each gene in panels A,B. One-way analysis of variance with Bonferroni’s multiple comparisons test was used in panels C,D. (E) PTCH1, SMO, SHH, GLI1, GLI2 and GLI3 mRNA expressions were assessed in primary bronchial epithelial cells differentiated in ALI (N = 1 replicate). Immunofluorescence images showing goblet cell expression (MUC5AC) with nuclei counterstain (DAPI) in (F) DMSO vehicle, (G) DMSO + 100 ng/ml EGF, H) and 100 ng/ml EGF + 300 nM vismodegib (SMO inhibitor) (N = 1 replicate). Cross-sections through both axes of the membrane are shown by the red (x-axis) and white (y-axis) lines.
Fig 3: PTCH1 expression regulates cell proliferation and adhesion. Representative images of cross-hatched wounds in (A) scrambled siRNA- or (B) PTCH1 siRNA-treated 1HAE0 cells imaged at day 0, 1 and 2 post-injury were shown. Red line indicated the wound edge boundary. Yellow box standardized the area of field. Black dots indicated pen marks of the same representative wound images taken over time. (C) Wound area remaining in scrambled siRNA- or PTCH1 siRNA-treated cells was expressed as a percentage of the total area measured at day 0 in each individual wound. (D) Wound area remaining in untreated controls, lipofectamine (lipo)- and scrambled siRNA-treated cells was expressed as a percentage of the total area measured at day 0 in each individual wound. E) PTCH1 mRNA expression in scrambled siRNA- and PTCH1 siRNA-treated cells was normalized to 100% scrambled siRNA-treated cells for each time point. Values were expressed as mean ± SEM (N = 3–4 independent experiments). Two-way analysis of variance with Bonferroni’s multiple comparisons test was used in panel C,D. (F) Representative protein blot showing PTCH1 antibody expression and specificity in PTCH1-overexpressing lysate and untreated human airway epithelial cell lysate. Cell morphology was imaged 24 h post mitomycin C treatment in (G) scrambled siRNA- or (H) PTCH1 siRNA-treated cells. Yellow boxes indicated 2 magnified regions in each condition. Red line indicated the wound edge boundary 24 h post injury showing normal wound closing ability in scrambled siRNA-treated cells.
Fig 4: Ptch1+/- mice were partially protected from HDM-induced mucous expression compared to HDM-exposed Wt mice. (A) Periodic acid Schiff (PAS)-, B) MUC5AC- and (C) Ki67-stained sections of paraffin-embedded mouse lung tissues from Wt and Ptch1+/- mice after PBS or HDM exposures were shown. (D) Airway-specific PAS staining was normalized to length of basement membrane (BM). (E) Ki67+ epithelial cells and (G) airway epithelial thickness were normalized to length of BM. Scale bar = 50 µm. Values were expressed as mean ± SEM. One-way analysis of variance with Bonferroni’s multiple comparisons test was used in panels D–F. (G–H) Representative images and quantification of epithelial-specific Ptch1 protein expression in all visible distal airways in a representative Wt PBS- and Ptch1+/- PBS-treated mouse.
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