Journal: Science advances

Article Title: Hedgehog-interacting protein orchestrates alveologenesis and protects against bronchopulmonary dysplasia and emphysema.

doi: 10.1126/sciadv.adu2958

Figure Lengend Snippet: Fig. 2. Hhip deletion induces the persistence of myofibroblasts. (A) IF analysis of SMA in the alveoli of Hhip-deleted (HHIP CKO) and control mice at P14. (B) Number of myofibroblasts per unit alveolar area of Hhip-deleted and control mice. (C and D) UMAP showing cell clusters in the lung fibroblasts of Hhip-deleted and control mice at P14. (E) Violin plots showing the expression of Pdgfra, Acta2, Hhip, and Cdh4 in alveolar myofibroblasts (ALMF), ductal myofibroblasts (DMF), peribronchial fibroblasts (Perib), adventitial fibroblasts (Adv), and alveolar fibroblasts (Alv). (F) Expression of Acta2, Myh11, Tagln, and Igf1 in ALMFs and DMFs of Hhip-deleted and control mice. (G) qPCR analysis of Igf1 expression in the lung stromal cells isolated from Hhip-deleted and control mice. (H) Analysis of Igf1 (RNA in situ) and SMA expression in the alveoli. (I) Number of SMA+ Igf1+ cells per unit alveolar area of Hhip-deleted and control mice at P14. (J) Percentage of SMA+ Igf1+ cells in total Igf1+ cells. (K) Top 10 activated pathways in Hhip-deleted, relative to control myofibroblasts, analyzed with IPA. (L) qPCR analysis of Gli1, Igf1, and Acta2 expression in the lung stromal cells treated with PBS, SHH, and SHH plus HHIP. (M) qPCR analysis of Acta2 expression in SHH-stimulated lung stromal cells treated with vehicle or IGF1R inhibitor. (N) IF analysis of SMA expression in the alveoli of Hhip-deleted mice administered with vehicle or IGF1R inhibitor. (O) Number of myofibroblasts per unit alveolar area of Hhip-deleted mice administered with vehicle or IGF1R inhibitor. All in vitro experiments have been repeated at least one time with consistent results for validation. Each data point represents one mouse [(B), (G), (I), (J), and (O)] of an individual experiment. Data are expressed as mean ± SD. *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001.

Article Snippet: The following primary antibodies were used for mouse tissue: goat anti- HHIP (catalog no. AF1568; R&D Systems; used 1: 250), rat anti- CDH4 (catalog no. MRCD5; DSHB; used 1: 20), rabbit anti- CDH4 (catalog no. BS40099; Bioworld; used 1: 100), rabbit anti- SMA (catalog no. ab5694; Abcam; used 1:400), mouse anti- SMA (catalog no. AB7817; Abcam; used 1:500), goat anti- tdTomato (catalog no. AB8181- 200; OriGene; used 1:500), rabbit anti- RFP (catalog no. 600- 401- 379; Rockland; used 1:500), rabbit anti- SPC (catalog no. AB3786; Millipore Sigma; used 1:2000), rat anti- Ki67 (catalog no. 14- 5698- 82; Thermo Fisher Scientific; used 1:100), rabbit anti- γH2A (catalog no. 9718T; Cell Signaling Technology; used at 1:250), rat anti- p21 (catalog no. ab107099; Abcam; used 1:100), rat anti– DC- LAMP (catalog no. DDX0191P- 100; Novus; used 1:250), goat anti- PDGFRα (catalog no. AF1062; R&D Systems; used 1:200), and rabbit anti- PDGFRα (catalog no. 3174; Cell Signaling Technology; used 1:200).

Techniques: Control, Expressing, Isolation, In Situ, In Vitro, Biomarker Discovery

Journal: Science advances

Article Title: Hedgehog-interacting protein orchestrates alveologenesis and protects against bronchopulmonary dysplasia and emphysema.

doi: 10.1126/sciadv.adu2958

Figure Lengend Snippet: Fig. 3. Deletion of Hhip induces cell senescence. (A) Violin plots showing the expression of Cdkn1a in ALMFs and DMFs of Hhip-deleted and control mice. (B) Senescence β-galactosidase staining of Hhip-deleted and control lungs. (C) IF analysis of SMA, p21, and CDH4 in Hhip-deleted and control lungs. Arrow: p21+ DMFs; arrowhead: p21+ ALMFs. (D and E) Number of p21+ ALMFs (D) and DMFs (E) per unit alveolar area of Hhip-deleted and control mice at P14. (F) IF analysis of SPC and p21 in Hhip-deleted and control lungs. Arrow: p21+ SPC+ cells. (G) Percentage of p21+ cells in AT2s. (H) Activation of senescence pathways in the AT2s of Hhip-deleted mice, relative to control AT2s, and analyzed with IPA. (I) Top 5 upstream regulators in the AT2s of Hhip-deleted mice, relative to control AT2s, analyzed with IPA. (J) AT2 organoids cocultured with lung stromal cells (R26RSmoM2/+) pre-infected with adenovirus-empty and adenovirus-Cre, treated with anti-IGF1 antibody and IgG. (K and L) Quantification of colony- forming efficiency (CFE) and organoid size. (M) IF analysis of SPC and p21 in AT2 organoids. Arrow: p21+ SPC+ cells. (N) Percentage of p21+ cells in AT2s in the organoid assay. All in vitro experiments have been repeated at least one time with consistent results for validation. Each data point represents one mouse [(D), (E), and (G)] of an individual experiment. Data are expressed as Mean ± SD. *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001.

Article Snippet: The following primary antibodies were used for mouse tissue: goat anti- HHIP (catalog no. AF1568; R&D Systems; used 1: 250), rat anti- CDH4 (catalog no. MRCD5; DSHB; used 1: 20), rabbit anti- CDH4 (catalog no. BS40099; Bioworld; used 1: 100), rabbit anti- SMA (catalog no. ab5694; Abcam; used 1:400), mouse anti- SMA (catalog no. AB7817; Abcam; used 1:500), goat anti- tdTomato (catalog no. AB8181- 200; OriGene; used 1:500), rabbit anti- RFP (catalog no. 600- 401- 379; Rockland; used 1:500), rabbit anti- SPC (catalog no. AB3786; Millipore Sigma; used 1:2000), rat anti- Ki67 (catalog no. 14- 5698- 82; Thermo Fisher Scientific; used 1:100), rabbit anti- γH2A (catalog no. 9718T; Cell Signaling Technology; used at 1:250), rat anti- p21 (catalog no. ab107099; Abcam; used 1:100), rat anti– DC- LAMP (catalog no. DDX0191P- 100; Novus; used 1:250), goat anti- PDGFRα (catalog no. AF1062; R&D Systems; used 1:200), and rabbit anti- PDGFRα (catalog no. 3174; Cell Signaling Technology; used 1:200).

Techniques: Expressing, Control, Staining, Activation Assay, Infection, In Vitro, Biomarker Discovery