Journal: Science Advances

Article Title: Mesenchymal growth hormone receptor deficiency leads to failure of alveolar progenitor cell function and severe pulmonary fibrosis

doi: 10.1126/sciadv.abg6005

Figure Lengend Snippet: ( A ) Representative confocal microscopy images of GHR and COL1A1 coimmunostaining for healthy and IPF human lung tissues. ( B ) Quantification of GHR and COL1A1 coexpression by colocalization calculated from staining in (A). ( C ) GHR and HTII-280 coimmunostaining reveals that a small portion of HTII-280 + ATII cells are GHR positive in normal human lungs and no GHR + HTII-280 + cells in fibrotic human lungs. ( D ) Quantification of GHR and HTII-280 coexpression by flow cytometry or by colocalization calculated from staining in (C). ( E ) Rare CD31 + GHR + endothelial cells were found in normal lungs but not IPF lungs. ( F ) Quantification of GHR and CD31 coexpression by colocalization calculated from staining in (E). Arrows show the coexpressing cells. Scale bars, 100 μm (A to C, lower power images) and 10 μm (A to C, higher power images). Bars represent means ± SEM, n = 7 biological replicates. **** P < 0.0001 by unpaired two-tailed Student’s t test.

Article Snippet: Antibodies used in immunofluorescent staining were Sftpc (Goat and Rabbit; 1:200; B. Stripp laboratory), COL1A1 (NB600-408; 1:200; Novus Biologicals), HTII-280 (1:200; a gift of the L. G. Dobbs laboratory, University of California, San Francisco, San Francisco, CA), CD31 (1:200; mouse, 303101, BioLegend), GHR (Goat, AF1210; 1:200; R&D Systems), red fluorescent protein (Rabbit; 1:200; 600-401-379, Rockland), and correspondent secondary antibodies conjugated to Alexa 546 or 488 (1:500; A-21085, A-11056, and A-11008, Thermo Fisher Scientific).

Techniques: Confocal Microscopy, Staining, Flow Cytometry, Two Tailed Test

Journal: Science Advances

Article Title: Mesenchymal growth hormone receptor deficiency leads to failure of alveolar progenitor cell function and severe pulmonary fibrosis

doi: 10.1126/sciadv.abg6005

Figure Lengend Snippet: ( A ) Single-cell transcriptomic sequencing of wild-type (WT) mouse lung total single cells. Ghr gene expression is mainly in Col1a1 + cell cluster. ( B ) CAMC (mesenchymal cells defined by Col1a1 and Acta2 ) were reclustered and plotted for Ghr and Col1a1 coexpression. Around 88.35% of the total Ghr + cells are Col1a1 + . ( C ) Ghr expression in ATII cells by single-cell RNA-seq analysis. About 6.59% Sftpc + ATII cells are Ghr + . ( D ) ATII expression of Ghr and Sftpc . ( E ) Top up-regulated genes for Ghr -positive mesenchymal cells compared with Ghr -negative mesenchymal cells. ( F ) Fluorescence-activated cell sorting (FACS) analysis of Ghr on cell surface of cultured passage 3 primary lung mesenchymal cells (Epcam + Cd31/45 + cells were excluded) from normal and bleomycin-treated mice (7, 14, and 21 days after bleomycin administration).

Article Snippet: Antibodies used in immunofluorescent staining were Sftpc (Goat and Rabbit; 1:200; B. Stripp laboratory), COL1A1 (NB600-408; 1:200; Novus Biologicals), HTII-280 (1:200; a gift of the L. G. Dobbs laboratory, University of California, San Francisco, San Francisco, CA), CD31 (1:200; mouse, 303101, BioLegend), GHR (Goat, AF1210; 1:200; R&D Systems), red fluorescent protein (Rabbit; 1:200; 600-401-379, Rockland), and correspondent secondary antibodies conjugated to Alexa 546 or 488 (1:500; A-21085, A-11056, and A-11008, Thermo Fisher Scientific).

Techniques: Sequencing, Gene Expression, Expressing, RNA Sequencing, Fluorescence, FACS, Cell Culture

Journal: Science Advances

Article Title: Mesenchymal growth hormone receptor deficiency leads to failure of alveolar progenitor cell function and severe pulmonary fibrosis

doi: 10.1126/sciadv.abg6005

Figure Lengend Snippet: ( A ) Schematic of in vitro coculture colony formation assay for experiments in (B) to (D). ( B ) Scgb1a1 -GFP + (green fluorescent protein–positive) Club cell and Sftpc -GFP + ATII organoids cocultured with mesenchymal cells isolated from Ghr −/− or control mouse lungs. The number and size of Scgb1a1 -GFP + Club cell and Sftpc -GFP + ATII organoids were decreased when cocultured with Ghr −/− mesenchymal cells. ( C ) Expression of Scgb1a1 , Sftpc , Pdpn , and Aqp5 in Scgb1a1 + Club cell organoids cocultured with lung mesenchymal cells isolated from Ghr −/− and control mice. ( D ) Expression of Sftpc , Pdpn , and Cdkn1a in Sftpc + ATII cell organoids cocultured with lung mesenchymal cells isolated from Ghr −/− and control mice. ( E ) Pegvisomant (PEG) treatment for mesenchymal cells cocultured with ATII colonies. ( F ) FACS confirmation of Ghr expression on lung mesenchymal cells from Ghr −/− and control mice. ( G ) Ghr , Ghr TV1 , Ghr TV2 , Tbx4 , Cemip , and Tgfbr3 expression in lung mesenchymal cells from Ghr −/− and control mice. ( H ) Has1 , Has2 , and Has3 expression in lung mesenchymal cells from Ghr −/− and control mice. ( I ) Chemokine gene ( Cxcl1 , Cxcl4 , Cxcl5 , Cxcl12 , Cxcl14 , and Cxcl16 ) expression in Ctrl and Ghr −/− mouse lung mesenchymal cells. ( J ) Il-6 gene expression in lung mesenchymal cells from Ghr −/− and control mice. Scale bars, 1 mm (B). n = 3, * P < 0.05, ** P < 0.005, *** P < 0.001, and **** P < 0.0001 by unpaired two-tailed Student’s t test [B (panels 3 and 4) and J], one-way analysis of variance (ANOVA) (E), and two-way ANOVA [B (panel 2), C, D, G, and H], means ± SEM.

Article Snippet: Antibodies used in immunofluorescent staining were Sftpc (Goat and Rabbit; 1:200; B. Stripp laboratory), COL1A1 (NB600-408; 1:200; Novus Biologicals), HTII-280 (1:200; a gift of the L. G. Dobbs laboratory, University of California, San Francisco, San Francisco, CA), CD31 (1:200; mouse, 303101, BioLegend), GHR (Goat, AF1210; 1:200; R&D Systems), red fluorescent protein (Rabbit; 1:200; 600-401-379, Rockland), and correspondent secondary antibodies conjugated to Alexa 546 or 488 (1:500; A-21085, A-11056, and A-11008, Thermo Fisher Scientific).

Techniques: In Vitro, Colony Assay, Isolation, Control, Expressing, Gene Expression, Two Tailed Test

Journal: Science Advances

Article Title: Mesenchymal growth hormone receptor deficiency leads to failure of alveolar progenitor cell function and severe pulmonary fibrosis

doi: 10.1126/sciadv.abg6005

Figure Lengend Snippet: ( A ) Immunostaining of red fluorescent protein, Scgb1a1, Sftpc, and DAPI in mouse lungs administrated with mCherry sequence containing Ghr vesicles. Yellow solid line, mCherry and Scgb1a1 or Sftpc colocalized cells; white dotted line, Scgb1a1- or Sftpc-negative cells. ( B ) Ghr mRNA expression in ATII cells sorted from mouse lungs treated with Ghr or control vesicles. ( C ) Ghr protein expression in ATII cells sorted from mouse lungs treated with Ghr or control vesicles. ( D ) A schematic diagram depicting the administration of Ghr or control vesicles into the lungs of Ghr ΔBMC mice after bleomycin treatment. Mouse lungs were harvested on day 21. ( E ) Significantly decreased hydroxyproline levels were detected in Ghr vesicles and control vesicle–administered Ghr ΔBMC mouse lungs (Ctrl, n = 18; Ghr ΔBMC + Ctrl vesicle, n = 16; Ghr ΔBMC + Ghr vesicle, n = 18). ( F ) Cxcl1, Cxcl2, Cxcl5, Cxcl10, Cxcl11, Cxcl12, and Cxcl16 protein secretion in BALF of Ghr ΔBMC mice with Ghr or control vesicle treatments. ( G and H ) Representative images of H&E staining (G) and trichrome staining (H) in Ghr or control vesicle–treated Ghr ΔBMC mouse lung 21 days after bleomycin injury. ( I ) A diagram illustrating how subepithelial mesenchymal cells support ATII cells through Ghr vesicle and Ghr-coordinated CXC chemokines in normal and fibrotic conditions in the lung. Scale bars, 10 μm (A), 100 μm (G and H, higher power), and 1 mm (G and H, lower power). * P < 0.05, ** P < 0.005, *** P < 0.001, and **** P < 0.0001 by unpaired two-tailed Student’s t test (B and C) and one-way ANOVA (E), means ± SEM.

Article Snippet: Antibodies used in immunofluorescent staining were Sftpc (Goat and Rabbit; 1:200; B. Stripp laboratory), COL1A1 (NB600-408; 1:200; Novus Biologicals), HTII-280 (1:200; a gift of the L. G. Dobbs laboratory, University of California, San Francisco, San Francisco, CA), CD31 (1:200; mouse, 303101, BioLegend), GHR (Goat, AF1210; 1:200; R&D Systems), red fluorescent protein (Rabbit; 1:200; 600-401-379, Rockland), and correspondent secondary antibodies conjugated to Alexa 546 or 488 (1:500; A-21085, A-11056, and A-11008, Thermo Fisher Scientific).

Techniques: Immunostaining, Sequencing, Expressing, Control, Staining, Two Tailed Test