Fig 1: The Hedgehog pathway is required for human lung branching morphogenesis. (A) Human fetal lung explant cultures at t = 0, t = 24 h and t = 48 h untreated (in media alone); or treated with 5 µg/mL IgG as negative control or 5 µg/mL 5E1. (B,C) Graphs showing the percentage of distal branching (B) and cyst dilation (C) of 5E1, and IgG treated explants as compared to untreated control (CTL) at t = 24 h and t = 48 h. Results are shown in dots and mean ± SEM (n = 8 for control and 5E1, n = 6 for IgG). (D–F) RT-qPCR for SHH (D), PTCH1 (E), and HHIP (F) performed on control (n = 6), 5E1- (n = 6; red dots) and IgG- treated explants (n = 3). Results are shown as individual data points and mean ± SEM. (G) Representative western blots for GLI2 and GAPDH in human fetal lung explants treated or not with 5E1 or IgG (5 µg/mL) for 48 h. (H) Dot plots (mean ± SEM) of western blot densitometry ratios for GLI2 normalized to GAPDH (n = 3 for each condition). * p < 0.05, ** p < 0.01; *** p < 0.001; ns = no stress.
Fig 2: HH pathway is activated during AEC differentiation. A, Curves representing the relative mRNAs levels normalized to GAPDH obtained during the course of ALI cultures by RT-qPCR (n = 5) for differentiation markers (CK5, non-differentiated cells; FOXJ1, ciliated cells; and MUC5AC/MUC5B, mucous-secreting cells) and HH pathway elements (GLI2/3; SMO and PTCH1). Means ±SEM of 2-??Ct are shown for each ALI time point. B, Representative immunoblots of total proteins extracted from ALI AEC cultures from ALI-7 to ALI-35 for the differentiation markers (Ck5 and Foxj1), HH pathway elements (Gli1/2/3; Smo; Ptch1) and Gapdh (n = 3). C, Histograms showing the relative increased expressions of Foxj1 and Gli2 protein levels as evaluated by western blot analysis after processing on ImageJ (Gapdh normalized ratios) during the course of AEC differentiation (in arbitrary units, AU). Results show mean ±SEM, *p < 0.05, **p < 0.01, ***p < 0.001. D, Representative confocal acquisitions from AEC cultures at ALI-14 and ALI-28 for the core HH pathway components Gli1, Gli2, Gli3, Ptch1 and Smo (all green); cilia (acetylated tubulin, red) and cell nuclei (DAPI, blue). Merged z-projections are shown with a magnification corresponding to the selected area.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig 3: Gli2 expression is decreased in AEC from COPD patients. a. Representative micrograph showing a ROI of a bronchial brushing stained for Gli2 (Gli2, red) and cell nuclei (DAPI, blue) in both non-COPD (upper panel) and COPD patients (lower panel). Magnification corresponding to the selected area is shown. b. Dot plot with median showing the total percentage of Gli2-positive cells in non-COPD (n = 15) and COPD patients (n = 15). *, p < 0.05
Fig 4: Shh depletion decreased Gli2 expression and prevented its nuclear translocation. A, Histograms representing the assessment of fold-change (log2) in the normalized expression to GAPDH of genes during the course of ALI cultures by RT-qPCR (n = 5) for differentiation markers (CK5, non-differentiated cells; FOXJ1, ciliated cells; and MUC5AC/MUC5B, mucous-secreting cells) and HH pathway elements (GLI2/3; SMO and PTCH1). Results show mean±SEM, *p < 0.05 AB5E1 vs CTL. B, Histogram of the ratios of the transcripts levels of GLI2/GLI3 normalized to GAPDH in AB5E1-treated cells in comparison to CTL (median ±SEM are shown). *p < 0.05 AB5E1-treated cells vs CTL. C, Representative immunoblots of total proteins extracted from ALI AEC cultures treated with AB5E1 from ALI-0 to ALI-35 for the differentiation markers (Ck5 and Foxj1), HH pathway elements (Gli1/2/3; Smo; Ptch1) and Gapdh (n = 3). Ratios (R) indicate the normalized quantitative analysis of the expression of each protein compared to CTL. D, Representative Immunoblot of the fractionated protein extractions on CTL and AB5E1 treated-cells at ALI-7. The fractions are in order: S, soluble proteins; Mb, membrane-bound proteins; N, nuclear proteins; Chr, chromatin-bound proteins; Cq, cytoskeleton-bound proteins. E, Representative confocal acquisitions from AEC cultures AB5E1-treated at ALI-14 and ALI-28 for Gli2 (green), cilia (acetylated tubulin, red) and cell nuclei (DAPI, blue). Merged z-projections are shown with a magnification corresponding to the selected area. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig 5: Alteration of Gli2 localization in AEC is associated with COPD. A, Data extracted from the Human Lung Atlas. Left, cellular landscape along the airways in human lung analysed by single-cell RNA sequencing from 26154 cells collected from 17 donors to display specific cluster assignment of epithelial cells; right, identification of GLI2-expressing cells by t-distributed Stochastic Neighbour Embedding (tSNE). A progenitor cluster encompassing basal and club-like cells was added. B, Dot plot (mean) presenting the relative mRNAs levels normalized to GAPDH obtained on isolated AEC from bronchial brushings by RT-qPCR (n = 20 non-COPD and COPD patients) for GLI2. Means ±SEM of 2-??Ct; *p < 0.05 non-COPD vs COPD patients. C, Representative micrographs showing the bronchi epithelia of non-COPD and COPD patients stained for the core HH pathway components Gli1, Gli2, Gli3, Ptch1 and Smo (all green); cilia (acetylated tubulin, red) and cell nuclei (DAPI, blue). Magnification corresponding to the selected area is shown. Cartoons depict the localization of each HH pathway element (in green). D, Dot plots (mean) showing the percentages of Gli2 positive cells in cilia (left panel) and nuclei (right panel) in non-COPD (black) and COPD patients (red). *** p < 0.001 non-COPD vs COPD. E, Linear regression of the percentages of nuclear Gli2 positive cells according to the FEV1/FVC (%) for non-COPD (black) and COPD patients (red). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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