hhip lacz  (Jackson Laboratory)

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: ( A ) Quantification of SMA + and HHIP + cells in the alveoli during alveologenesis. ( B ) Immunofluorescence (IF) analysis of SMA-lineage cells (staining tdT) and SMA in the alveoli of Acta2 DreER/+ :R26R RSR-tdT/+ mice at P7 and P14. ( C ) Histology quantification of the percentage of SMA + myofibroblasts in total SMA-lineage cells. ( D ) IF analysis of HHIP, PDGFRα, and SMA expression in the alveoli. ( E ) Histology quantification of the percentage of myofibroblasts in HHIP + cells. ( F ) X-gal staining of Gli1 lacZ/+ reporter demonstrates Gli1 expression in the alveoli of P7 and P14 mice. ( G ) qPCR analysis of Hhip and Gli1 expression in the lung stromal cells of P7 and P14 mice. ( H ) IF analysis of GLI1-lineage cells (staining tdT) and SMA in the alveoli of Gli1 CreER/+ :R26R tdT/+ mice. ( I ) Histology quantification of the percentage of myofibroblasts in total GLI1-lineage cells. Each data point represents one mouse [(A), (C), (E), (G), and (I)] of an individual experiment. Data are expressed as mean ± SD. * P < 0.05, ** P < 0.005, and **** P < 0.0001.

Article Snippet: Generation and genotyping of the Gli1 CreER , Gli1 LacZ , Hhip LacZ , R26R SmoM2 , and R26R tdTomato lines were performed as previously described by The Jackson Laboratory.

Techniques: Immunofluorescence, Staining, Expressing

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: ( 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: Generation and genotyping of the Gli1 CreER , Gli1 LacZ , Hhip LacZ , R26R SmoM2 , and R26R tdTomato lines were performed as previously described by The Jackson Laboratory.

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: ( 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 ( R26R SmoM2/+ ) 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: Generation and genotyping of the Gli1 CreER , Gli1 LacZ , Hhip LacZ , R26R SmoM2 , and R26R tdTomato lines were performed as previously described by The Jackson Laboratory.

Techniques: Expressing, Control, Staining, Activation Assay, Infection, 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: ( A ) H&E images of the lungs of Hhip -deleted and control mice. ( B to D ) Quantification of MLI, mean alveolus size, and alveolar density of Hhip -deleted and control lungs. ( E ) H&E images of the lungs of Hhip -deleted mice treated with vehicle or IGF1R inhibitor. ( F to H ) Quantification of MLI, mean alveolus size, and alveolar density of Hhip -deleted mice treated with vehicle or IGF1R inhibitor. ( I ) IF analysis of SPC and p21 in the lungs of Hhip -deleted mice treated with vehicle or IGF1R inhibitor. Arrow: p21 + SPC + cells. ( J and K ) Quantification of the number of AT2s and percentage of p21 + AT2s in Hhip -deleted mice treated with vehicle or IGF1R inhibitor. Each data point represents one mouse [(B) to (D), (F) to (H), and (K)] of an individual experiment. Data are expressed as mean ± SD. * P < 0.05, ** P < 0.005, and **** P < 0.0001.

Article Snippet: Generation and genotyping of the Gli1 CreER , Gli1 LacZ , Hhip LacZ , R26R SmoM2 , and R26R tdTomato lines were performed as previously described by The Jackson Laboratory.

Techniques: Control

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: ( A ) snRNA-seq analysis of GLI1 , PATCH1 , IGF1 , and ACTA2 expression in ALMFs and DMFs of human BPD. ( B ) H&E images of the lungs of hyperoxia-treated and control mice. ( C ) Quantification of MLI of hyperoxia-treated and control lungs. ( D ) qPCR analysis of Hhip , Gli1 , and Igf1 expression in the lung stromal cells isolated from hyperoxia-treated and control mice. ( E ) IF analysis of SMA in the lungs of hyperoxia-treated and control mice. ( F ) Histology quantification of the number of myofibroblasts of hyperoxia-treated and control mice. ( G ) qPCR analysis of Acta2 expression in the lung stromal cells isolated from hyperoxia-treated and control mice. ( H ) qPCR analysis of Cdkn1a expression in the lung epithelial cells isolated from hyperoxia-treated and control mice. ( I ) IF analysis of p21 and SPC in the lungs of hyperoxia-treated and control mice. Arrow: p21 + SPC + cells. ( J and K ) Quantification of the percentage of p21 + AT2s and number of AT2s in hyperoxia-treated and control mice. Each data point represents one mouse [(C), (D), (F) to (H), (J), and (K)] of an individual experiment. Data are expressed as mean ± SD. * P < 0.05, ** P < 0.005, and **** P < 0.0001.

Article Snippet: Generation and genotyping of the Gli1 CreER , Gli1 LacZ , Hhip LacZ , R26R SmoM2 , and R26R tdTomato lines were performed as previously described by The Jackson Laboratory.

Techniques: Expressing, Control, Isolation

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: ( A ) Design strategy for HHIP-Fc recombinant protein and its analysis by Western blotting. ( B ) qPCR analysis of Gli1 expression in the lung stromal cells isolated from neonatal mice 3 and 5 days after one dose of HHIP or HHIP-Fc treatment. ( C ) H&E images of hyperoxia-exposed lungs, treated with HHIP-Fc or Fc fragment control. ( D ) MLI quantification of hyperoxia-exposed lungs, treated with HHIP-Fc or Fc. ( E and F ) IF analysis and quantification of myofibroblasts in hyperoxia-exposed lungs, treated with HHIP-Fc or Fc. ( G to I ) IF analysis and quantification of AT2 number and p21 + AT2 percentage in hyperoxia-exposed lungs, treated with HHIP-Fc or Fc. Arrow: p21 + SPC + cells. ( J ) qPCR analysis of Igf1 expression in the lung stromal cells isolated from hyperoxia-exposed mice, treated with HHIP-Fc or Fc. Each data point represents one mouse [(B), (D), (F), and (H) to (J)] 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: Generation and genotyping of the Gli1 CreER , Gli1 LacZ , Hhip LacZ , R26R SmoM2 , and R26R tdTomato lines were performed as previously described by The Jackson Laboratory.

Techniques: Recombinant, Western Blot, Expressing, Isolation, Control

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: ( A and B ) H&E images and MLI quantification of the lungs from adult Hhip LacZ/+ and WT mice. ( C ) IF analysis of PDGFRα and SMA in the lungs of adult Hhip LacZ/+ and WT mice. Arrow: SMA + PDGFRα + cells. ( D and E ) Quantification of the number and percentage of myofibroblasts in adult Hhip LacZ/+ and WT mice. ( F and G ) IF analysis of SPC and quantification of AT2s in the lungs of adult Hhip LacZ/+ and WT mice. ( H and I ) H&E images and MLI quantification of the lungs from adult Hhip LacZ/+ and WT mice treated with HHIP-Fc or Fc. ( J and K ) IF analysis and quantification of myofibroblasts in the lungs of adult Hhip LacZ/+ and WT mice treated with HHIP-Fc or Fc. Arrow: SMA + cells. ( L and M ) IF analysis and quantification of AT2s in the lungs of adult Hhip LacZ/+ and WT mice treated with HHIP-Fc or Fc. Each data point represents one mouse [(B), (D), (E), (G), (I), (K), and (M)] 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: Generation and genotyping of the Gli1 CreER , Gli1 LacZ , Hhip LacZ , R26R SmoM2 , and R26R tdTomato lines were performed as previously described by The Jackson Laboratory.

Techniques: