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BioSpherix hyperoxia animal chamber

( A ) Wildtype C57BL/6 mice were treated in 75% <t>hyperoxia</t> versus normoxia from P0-P10 and recovered in room air until harvest at P40 for analysis by either histology or lung physiology. ( B ) H&E sections of representative lungs from ( A ) harvested at P40 (left) with quantification of mean linear intercept (right). ( C ) Mice treated as in ( A ) and harvested for lung physiology measurements of compliance, elastance and lung capacity. ( D ) Tgfbr2 F/F and Tgfbr2 F/F ;Nkx2.1-cre littermates were treated in hyperoxia versus normoxia from P0-P10 and recovered in room air until harvest at P40 for analysis by histology. ( E ) H&E sections of representative lungs from ( D ) harvested at P40 (left) with quantification of mean linear intercept (right). ( F ) Normoxia cohort treated as in ( D ) and harvested for lung physiology measurements of compliance, elastance and lung capacity. Data in ( B ), ( C ), and ( F ) compared by two-tailed unpaired Student’s t-test. Data in ( E ) compared by ANOVA with Fisher’s post hoc test. Error bars depict mean ± SEM. **p<0.01, ***p<0.001, ****p<0.0001. Scale bars = 100 μm.

Hyperoxia Animal Chamber, supplied by BioSpherix, used in various techniques. Bioz Stars score: 96/100, based on 417 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Impaired myofibroblast proliferation is a central feature of pathologic post-natal alveolar simplification"

Article Title: Impaired myofibroblast proliferation is a central feature of pathologic post-natal alveolar simplification

Journal: eLife

doi: 10.7554/eLife.94425

( A ) Wildtype C57BL/6 mice were treated in 75% hyperoxia versus normoxia from P0-P10 and recovered in room air until harvest at P40 for analysis by either histology or lung physiology. ( B ) H&E sections of representative lungs from ( A ) harvested at P40 (left) with quantification of mean linear intercept (right). ( C ) Mice treated as in ( A ) and harvested for lung physiology measurements of compliance, elastance and lung capacity. ( D ) Tgfbr2 F/F and Tgfbr2 F/F ;Nkx2.1-cre littermates were treated in hyperoxia versus normoxia from P0-P10 and recovered in room air until harvest at P40 for analysis by histology. ( E ) H&E sections of representative lungs from ( D ) harvested at P40 (left) with quantification of mean linear intercept (right). ( F ) Normoxia cohort treated as in ( D ) and harvested for lung physiology measurements of compliance, elastance and lung capacity. Data in ( B ), ( C ), and ( F ) compared by two-tailed unpaired Student’s t-test. Data in ( E ) compared by ANOVA with Fisher’s post hoc test. Error bars depict mean ± SEM. **p<0.01, ***p<0.001, ****p<0.0001. Scale bars = 100 μm.

Figure Legend Snippet: ( A ) Wildtype C57BL/6 mice were treated in 75% hyperoxia versus normoxia from P0-P10 and recovered in room air until harvest at P40 for analysis by either histology or lung physiology. ( B ) H&E sections of representative lungs from ( A ) harvested at P40 (left) with quantification of mean linear intercept (right). ( C ) Mice treated as in ( A ) and harvested for lung physiology measurements of compliance, elastance and lung capacity. ( D ) Tgfbr2 F/F and Tgfbr2 F/F ;Nkx2.1-cre littermates were treated in hyperoxia versus normoxia from P0-P10 and recovered in room air until harvest at P40 for analysis by histology. ( E ) H&E sections of representative lungs from ( D ) harvested at P40 (left) with quantification of mean linear intercept (right). ( F ) Normoxia cohort treated as in ( D ) and harvested for lung physiology measurements of compliance, elastance and lung capacity. Data in ( B ), ( C ), and ( F ) compared by two-tailed unpaired Student’s t-test. Data in ( E ) compared by ANOVA with Fisher’s post hoc test. Error bars depict mean ± SEM. **p<0.01, ***p<0.001, ****p<0.0001. Scale bars = 100 μm.

Techniques Used: Two Tailed Test

( A ) Wildtype C57BL/6 mice were treated in 75% hyperoxia versus normoxia from P0-P10 and harvested on P10 for analysis by flow cytometry. Graph on right shows total cells per left lung as quantified by flow cytometry. ( B ) Representative flow cytometry plots of the lung mesenchyme (live, CD45-, CD31-, and Epcam-) with gates depicting PDGFRα+ cells (left). Major cell populations of the lung were defined by the indicated cell surface markers and shown as either a percentage of all cells (top) or as absolute number (bottom). ( C ) Tgfb2 F/F and Tgfbr2 F/F ;Nkx2.1-cre littermates were maintained in normoxic conditions from P0-P10 and harvested on P10 for analysis by flow cytometry. Graph on right shows total cells per left lung as quantified by flow cytometry. ( D ) Flow cytometry plots for ( C ) using the same gating strategy as described above. Data in ( A–D ) analyzed by two-tailed unpaired Student’s t-test. Error bars depict mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure Legend Snippet: ( A ) Wildtype C57BL/6 mice were treated in 75% hyperoxia versus normoxia from P0-P10 and harvested on P10 for analysis by flow cytometry. Graph on right shows total cells per left lung as quantified by flow cytometry. ( B ) Representative flow cytometry plots of the lung mesenchyme (live, CD45-, CD31-, and Epcam-) with gates depicting PDGFRα+ cells (left). Major cell populations of the lung were defined by the indicated cell surface markers and shown as either a percentage of all cells (top) or as absolute number (bottom). ( C ) Tgfb2 F/F and Tgfbr2 F/F ;Nkx2.1-cre littermates were maintained in normoxic conditions from P0-P10 and harvested on P10 for analysis by flow cytometry. Graph on right shows total cells per left lung as quantified by flow cytometry. ( D ) Flow cytometry plots for ( C ) using the same gating strategy as described above. Data in ( A–D ) analyzed by two-tailed unpaired Student’s t-test. Error bars depict mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Techniques Used: Flow Cytometry, Two Tailed Test

( A ) Schematic of scRNA-seq project showing CTRL ( Tgfbr2 F/F ) and cKO ( Tgfbr2 F/F ;Nkx2.1-cre) littermates treated in 75% hyperoxia versus normoxia from P0-P10 and harvested on P7 or P14 for FACS-purification and analysis by scRNA-seq. n=2 mice harvested for each genotype, treatment condition and timepoint. ( B ) Venn-diagram depicting strategy to compare these two models to identify shared features of alveolar simplification. ( C ) UMAP projection of mesenchymal cells from scRNA-seq as outlined in ( A ). ( D ) Bar graphs depicting the frequency of each cell type by treatment condition, genotype, and timepoint. ( E ) The frequency of alveolar and ductal myofibroblasts within the mesenchyme as depicted in ( D ) with each data point representing either a biologic or technical replicate. Because the data depicts both biologic and technical replicates, no statistics were performed on this data set. ( F ) Differentially expressed genes in myofibroblasts were identified by comparing either CTRL RA vs O2 cells or RA CTRL vs cKO cells. These lists were subsequently analyzed by Qiagen IPA to identify predicted upstream regulators for each comparison. The Venn-diagram on left depicts the number of overlapping predicted upstream regulators with z-score >1.5 (upregulated), while the table on right lists these 32 shared upstream regulators. Blue = TGFβ signaling, red = inhibitors of cell cycle, green = Wnt signaling.

Figure Legend Snippet: ( A ) Schematic of scRNA-seq project showing CTRL ( Tgfbr2 F/F ) and cKO ( Tgfbr2 F/F ;Nkx2.1-cre) littermates treated in 75% hyperoxia versus normoxia from P0-P10 and harvested on P7 or P14 for FACS-purification and analysis by scRNA-seq. n=2 mice harvested for each genotype, treatment condition and timepoint. ( B ) Venn-diagram depicting strategy to compare these two models to identify shared features of alveolar simplification. ( C ) UMAP projection of mesenchymal cells from scRNA-seq as outlined in ( A ). ( D ) Bar graphs depicting the frequency of each cell type by treatment condition, genotype, and timepoint. ( E ) The frequency of alveolar and ductal myofibroblasts within the mesenchyme as depicted in ( D ) with each data point representing either a biologic or technical replicate. Because the data depicts both biologic and technical replicates, no statistics were performed on this data set. ( F ) Differentially expressed genes in myofibroblasts were identified by comparing either CTRL RA vs O2 cells or RA CTRL vs cKO cells. These lists were subsequently analyzed by Qiagen IPA to identify predicted upstream regulators for each comparison. The Venn-diagram on left depicts the number of overlapping predicted upstream regulators with z-score >1.5 (upregulated), while the table on right lists these 32 shared upstream regulators. Blue = TGFβ signaling, red = inhibitors of cell cycle, green = Wnt signaling.

Techniques Used: Purification, Comparison

( A ) Hurskainen et al. treated C57BL/6 wildtype mice with 85% hyperoxia versus normoxia from P0-P14 and analyzed the lungs by scRNA-seq at P3, P7, and P14 . The Seurat object used for publication was provided by the authors. UMAP projection shows the mesenchymal populations as defined by Hurskainen et al. ( B ) By using metadata within the Seurat object, we graphed the frequency of each mesenchymal population by treatment condition and time point. ( C ) The frequency of myofibroblasts within the mesenchyme as depicted in ( B ). ( D ) Xia et al. treated C57BL/6 wildtype mice with 85% hyperoxia versus normoxia from P0-P14 and analyzed the lungs by scRNA-seq at P14 . The Seurat object used for publication was provided by the authors. UMAP projection shows the mesenchymal populations as defined by Xia et al. ( E ) By using metadata within the Seurat object, we graphed the frequency of each mesenchymal population by treatment condition and time point. ( F ) The frequency of alveolar and ductal myofibroblasts within the mesenchyme as depicted in ( E ). Graphs in ( C ) and ( F ) depict one value for each condition because we were unable to extract replicate values from the data provided.

Figure Legend Snippet: ( A ) Hurskainen et al. treated C57BL/6 wildtype mice with 85% hyperoxia versus normoxia from P0-P14 and analyzed the lungs by scRNA-seq at P3, P7, and P14 . The Seurat object used for publication was provided by the authors. UMAP projection shows the mesenchymal populations as defined by Hurskainen et al. ( B ) By using metadata within the Seurat object, we graphed the frequency of each mesenchymal population by treatment condition and time point. ( C ) The frequency of myofibroblasts within the mesenchyme as depicted in ( B ). ( D ) Xia et al. treated C57BL/6 wildtype mice with 85% hyperoxia versus normoxia from P0-P14 and analyzed the lungs by scRNA-seq at P14 . The Seurat object used for publication was provided by the authors. UMAP projection shows the mesenchymal populations as defined by Xia et al. ( E ) By using metadata within the Seurat object, we graphed the frequency of each mesenchymal population by treatment condition and time point. ( F ) The frequency of alveolar and ductal myofibroblasts within the mesenchyme as depicted in ( E ). Graphs in ( C ) and ( F ) depict one value for each condition because we were unable to extract replicate values from the data provided.

Techniques Used:

scRNA-seq data was analyzed using the NicheNet software package. ( A ) Alveolar epithelial clusters were pooled to define sender population and myofibroblast clusters were pooled to define receiver population. By analyzing differential expression of ligands, receptors, and downstream gene expression changes in myofibroblasts, NicheNet predicted increased (left) or decreased (right) ligand-receptor signaling in each comparison of interest. Gray boxes show ligands expressed on the epithelium and the purple-shaded boxes indicate the corresponding receptors expressed on myofibroblasts. Upper panels compare hyperoxia versus normoxia in CTRL cells. Lower panels compare CTRL versus cKO cells in normoxia. ( B ) List of ligand-receptor pairs predicted to be increased in both injury models. Tgfb1-Tgfbr2 pairings highlighted in blue. ( C ) List of ligand-receptor pairs predicted to be decreased in both injury models. Pdgfa-Pdgfra and Pdgfb-Pdgfrb pairings highlighted in red.

Figure Legend Snippet: scRNA-seq data was analyzed using the NicheNet software package. ( A ) Alveolar epithelial clusters were pooled to define sender population and myofibroblast clusters were pooled to define receiver population. By analyzing differential expression of ligands, receptors, and downstream gene expression changes in myofibroblasts, NicheNet predicted increased (left) or decreased (right) ligand-receptor signaling in each comparison of interest. Gray boxes show ligands expressed on the epithelium and the purple-shaded boxes indicate the corresponding receptors expressed on myofibroblasts. Upper panels compare hyperoxia versus normoxia in CTRL cells. Lower panels compare CTRL versus cKO cells in normoxia. ( B ) List of ligand-receptor pairs predicted to be increased in both injury models. Tgfb1-Tgfbr2 pairings highlighted in blue. ( C ) List of ligand-receptor pairs predicted to be decreased in both injury models. Pdgfa-Pdgfra and Pdgfb-Pdgfrb pairings highlighted in red.

Techniques Used: Software, Expressing, Comparison

( A ) Wildtype C57BL/6 mice were injected every other day from P2-P10 with PBS or 1D11 (pan- TGFβ-blocking antibody), treated in 75% hyperoxia treatment versus normoxia from P0-P10, and recovered in room air until harvest at P40 for analysis by either histology or lung physiology. ( B ) H&E sections of representative lungs from ( A ) harvested at P40 (left). Images shown are from PBS and 30 mg/kg 1D11 treatment groups. Mean linear intercepts calculated for all treatment groups (right). ( C ) PBS- and 30 mg/kg 1D11-treated mice treated in normoxic conditions as in ( A ) and harvested for lung physiology measurements of compliance, elastance and lung capacity at P40. Data in ( B ) compared by ANOVA with Fisher’s post hoc test; for readability and limitations of graphing, only the statistical significance values within normoxia or hyperoxia cohorts are plotted. Data in ( C ) compared by two-tailed unpaired Student’s t-test. Error bars depict mean ± SEM. *p<0.05, **p<0.01, ****p<0.0001. Scale bars = 100 μm.

Figure Legend Snippet: ( A ) Wildtype C57BL/6 mice were injected every other day from P2-P10 with PBS or 1D11 (pan- TGFβ-blocking antibody), treated in 75% hyperoxia treatment versus normoxia from P0-P10, and recovered in room air until harvest at P40 for analysis by either histology or lung physiology. ( B ) H&E sections of representative lungs from ( A ) harvested at P40 (left). Images shown are from PBS and 30 mg/kg 1D11 treatment groups. Mean linear intercepts calculated for all treatment groups (right). ( C ) PBS- and 30 mg/kg 1D11-treated mice treated in normoxic conditions as in ( A ) and harvested for lung physiology measurements of compliance, elastance and lung capacity at P40. Data in ( B ) compared by ANOVA with Fisher’s post hoc test; for readability and limitations of graphing, only the statistical significance values within normoxia or hyperoxia cohorts are plotted. Data in ( C ) compared by two-tailed unpaired Student’s t-test. Error bars depict mean ± SEM. *p<0.05, **p<0.01, ****p<0.0001. Scale bars = 100 μm.

Techniques Used: Injection, Blocking Assay, Two Tailed Test

( A ) Either Gli1-CreERT2 or Pdgfra-CreERT2 mice and their cre-negative littermates were injected with tamoxifen on P2 and P4, treated in 75% hyperoxia versus normoxia from P0-P10, and recovered in room air until harvest at P40 for analysis by histology. ( B ) Mean linear intercepts of Gli1 CreER/+ and Gli1 +/+ mice treated as outlined in ( A ) and harvested at P40. ( C ) Mean linear intercepts of Pdgfra CreER/+ and Pdgfra +/+ mice treated as outlined in ( A ) and harvested at P40. Data compared by ANOVA with Fisher’s post hoc test. Error bars depict mean ± SEM. ***p<0.001, ****p<0.0001.

Figure Legend Snippet: ( A ) Either Gli1-CreERT2 or Pdgfra-CreERT2 mice and their cre-negative littermates were injected with tamoxifen on P2 and P4, treated in 75% hyperoxia versus normoxia from P0-P10, and recovered in room air until harvest at P40 for analysis by histology. ( B ) Mean linear intercepts of Gli1 CreER/+ and Gli1 +/+ mice treated as outlined in ( A ) and harvested at P40. ( C ) Mean linear intercepts of Pdgfra CreER/+ and Pdgfra +/+ mice treated as outlined in ( A ) and harvested at P40. Data compared by ANOVA with Fisher’s post hoc test. Error bars depict mean ± SEM. ***p<0.001, ****p<0.0001.

Techniques Used: Injection

( A ) Tgfbr2 F/F and Tgfbr2 F/F ;Gli1-CreERT2 littermates were injected with tamoxifen on P2 and P4, treated in 75% hyperoxia versus normoxia from P0-P10, and recovered in room air until harvest at P40 for analysis by histology. ( B ) H&E sections of representative lungs from ( A ) harvested at P40 (left). Mean linear intercepts calculated for all treatment groups (right). ( C ) Itgb6 F/F and Itgb6 F/F ;Nkx2.1-cre littermates were treated in 75% hyperoxia versus normoxia from P0-P10, and recovered in room air until harvest at P40 for analysis by histology ( D ) H&E sections of representative lungs from ( C ) harvested at P40 (left). Mean linear intercepts calculated for all treatment groups (right). Data compared by ANOVA with Fisher’s post hoc test. Error bars depict mean ± SEM. *p<0.05, ***p<0.001, ****p<0.0001. Scale bars = 100 μm.

Figure Legend Snippet: ( A ) Tgfbr2 F/F and Tgfbr2 F/F ;Gli1-CreERT2 littermates were injected with tamoxifen on P2 and P4, treated in 75% hyperoxia versus normoxia from P0-P10, and recovered in room air until harvest at P40 for analysis by histology. ( B ) H&E sections of representative lungs from ( A ) harvested at P40 (left). Mean linear intercepts calculated for all treatment groups (right). ( C ) Itgb6 F/F and Itgb6 F/F ;Nkx2.1-cre littermates were treated in 75% hyperoxia versus normoxia from P0-P10, and recovered in room air until harvest at P40 for analysis by histology ( D ) H&E sections of representative lungs from ( C ) harvested at P40 (left). Mean linear intercepts calculated for all treatment groups (right). Data compared by ANOVA with Fisher’s post hoc test. Error bars depict mean ± SEM. *p<0.05, ***p<0.001, ****p<0.0001. Scale bars = 100 μm.

Techniques Used: Injection

( A ) Itgav F/F and Itgav F/F ;Gli1-CreERT2 littermates were injected with tamoxifen on P2 and P4, treated in 75% hyperoxia versus normoxia from P0-P10, and recovered in room air until harvest at P40 for analysis by either histology or lung physiology. ( B ) H&E sections of representative lungs from ( A ) harvested at P40 (left). Mean linear intercepts calculated for all treatment groups (right). ( C ) Normoxia cohort treated as in ( A ) and harvested for lung physiology measurements of compliance, elastance and lung capacity. Data in ( B ) compared by ANOVA with Fisher’s post hoc test. Data in ( C ) compared by two-tailed unpaired Student’s t-test. Error bars depict mean ± SEM. *p<0.05, **p<0.01, ****p<0.0001. Scale bars = 100 μm.

Figure Legend Snippet: ( A ) Itgav F/F and Itgav F/F ;Gli1-CreERT2 littermates were injected with tamoxifen on P2 and P4, treated in 75% hyperoxia versus normoxia from P0-P10, and recovered in room air until harvest at P40 for analysis by either histology or lung physiology. ( B ) H&E sections of representative lungs from ( A ) harvested at P40 (left). Mean linear intercepts calculated for all treatment groups (right). ( C ) Normoxia cohort treated as in ( A ) and harvested for lung physiology measurements of compliance, elastance and lung capacity. Data in ( B ) compared by ANOVA with Fisher’s post hoc test. Data in ( C ) compared by two-tailed unpaired Student’s t-test. Error bars depict mean ± SEM. *p<0.05, **p<0.01, ****p<0.0001. Scale bars = 100 μm.

Techniques Used: Injection, Two Tailed Test

( A ) Wildtype C57BL/6 mice were treated in 75% hyperoxia versus normoxia from P0-P10 and recovered in room air until indicated timepoints for analysis. Mice were injected with EdU 24 hr prior to each analysis timepoint. ( B ) Flow cytometry plots of lung mesenchyme (CD45-, CD31-, Epcam-, MCAM-) show gating of PDGFRα+ cells (upper panels) and subsequent identification of EdU+/PDGFRα+ cells (lower panels). Panels on far-right show an EdU-untreated littermate used to define EdU + gate. ( C ) Time course graphs showing indicated populations of the lung as percent of lung (top), total cells in left lung (middle), and percent EdU + cells (bottom). Data graphed as mean ± with exception of EdU + panel in which each animal is plotted individually. Data compared by two-tailed unpaired Student’s t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure Legend Snippet: ( A ) Wildtype C57BL/6 mice were treated in 75% hyperoxia versus normoxia from P0-P10 and recovered in room air until indicated timepoints for analysis. Mice were injected with EdU 24 hr prior to each analysis timepoint. ( B ) Flow cytometry plots of lung mesenchyme (CD45-, CD31-, Epcam-, MCAM-) show gating of PDGFRα+ cells (upper panels) and subsequent identification of EdU+/PDGFRα+ cells (lower panels). Panels on far-right show an EdU-untreated littermate used to define EdU + gate. ( C ) Time course graphs showing indicated populations of the lung as percent of lung (top), total cells in left lung (middle), and percent EdU + cells (bottom). Data graphed as mean ± with exception of EdU + panel in which each animal is plotted individually. Data compared by two-tailed unpaired Student’s t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Techniques Used: Injection, Flow Cytometry, Two Tailed Test

( A ) Mean fluorescence intensity (geometric mean, MFI) of PDGFRα antibody staining on MCAM-negative mesenchymal cells (CD45-, CD31-, Epcam-) and PDGFRa + cells (CD45-, CD31-, Epcam-, MCAM-, PDGFRα+) as quantified by flow cytometry. Each column represents an experiment shown earlier in this study: normoxia vs hyperoxia at P10 , Tgfbr2 F/F vs Tgfbr2 F/F ;Nkx2.1-cre at P10 , PBS vs 1D11 at P10 , Ect2 F/F vs Ect2 F/F ;Pdgfra-CreERT2 at P14 . To compare values across multiple experiments, MFI’s were normalized to the control condition within each experiment. Data compared by ANOVA with Fisher’s post hoc test. Error bars depict mean ± SEM. ***p<0.001, ****p<0.0001.

Figure Legend Snippet: ( A ) Mean fluorescence intensity (geometric mean, MFI) of PDGFRα antibody staining on MCAM-negative mesenchymal cells (CD45-, CD31-, Epcam-) and PDGFRa + cells (CD45-, CD31-, Epcam-, MCAM-, PDGFRα+) as quantified by flow cytometry. Each column represents an experiment shown earlier in this study: normoxia vs hyperoxia at P10 , Tgfbr2 F/F vs Tgfbr2 F/F ;Nkx2.1-cre at P10 , PBS vs 1D11 at P10 , Ect2 F/F vs Ect2 F/F ;Pdgfra-CreERT2 at P14 . To compare values across multiple experiments, MFI’s were normalized to the control condition within each experiment. Data compared by ANOVA with Fisher’s post hoc test. Error bars depict mean ± SEM. ***p<0.001, ****p<0.0001.

Techniques Used: Fluorescence, Staining, Flow Cytometry, Control

Image Search Results

Journal: eLife

Article Title: Impaired myofibroblast proliferation is a central feature of pathologic post-natal alveolar simplification

doi: 10.7554/eLife.94425

Figure Lengend Snippet: ( A ) Wildtype C57BL/6 mice were treated in 75% hyperoxia versus normoxia from P0-P10 and recovered in room air until harvest at P40 for analysis by either histology or lung physiology. ( B ) H&E sections of representative lungs from ( A ) harvested at P40 (left) with quantification of mean linear intercept (right). ( C ) Mice treated as in ( A ) and harvested for lung physiology measurements of compliance, elastance and lung capacity. ( D ) Tgfbr2 F/F and Tgfbr2 F/F ;Nkx2.1-cre littermates were treated in hyperoxia versus normoxia from P0-P10 and recovered in room air until harvest at P40 for analysis by histology. ( E ) H&E sections of representative lungs from ( D ) harvested at P40 (left) with quantification of mean linear intercept (right). ( F ) Normoxia cohort treated as in ( D ) and harvested for lung physiology measurements of compliance, elastance and lung capacity. Data in ( B ), ( C ), and ( F ) compared by two-tailed unpaired Student’s t-test. Data in ( E ) compared by ANOVA with Fisher’s post hoc test. Error bars depict mean ± SEM. **p<0.01, ***p<0.001, ****p<0.0001. Scale bars = 100 μm.

Article Snippet: The hyperoxia animal chamber (BioSpherix) was attached to a medical oxygen source controlled by a ProOx single gas controller (BioSpherix) set to maintain 75% oxygen under normobaric conditions.

Techniques: Two Tailed Test

Journal: eLife

Article Title: Impaired myofibroblast proliferation is a central feature of pathologic post-natal alveolar simplification

doi: 10.7554/eLife.94425

Figure Lengend Snippet: ( A ) Wildtype C57BL/6 mice were treated in 75% hyperoxia versus normoxia from P0-P10 and harvested on P10 for analysis by flow cytometry. Graph on right shows total cells per left lung as quantified by flow cytometry. ( B ) Representative flow cytometry plots of the lung mesenchyme (live, CD45-, CD31-, and Epcam-) with gates depicting PDGFRα+ cells (left). Major cell populations of the lung were defined by the indicated cell surface markers and shown as either a percentage of all cells (top) or as absolute number (bottom). ( C ) Tgfb2 F/F and Tgfbr2 F/F ;Nkx2.1-cre littermates were maintained in normoxic conditions from P0-P10 and harvested on P10 for analysis by flow cytometry. Graph on right shows total cells per left lung as quantified by flow cytometry. ( D ) Flow cytometry plots for ( C ) using the same gating strategy as described above. Data in ( A–D ) analyzed by two-tailed unpaired Student’s t-test. Error bars depict mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Techniques: Flow Cytometry, Two Tailed Test

Journal: eLife

Article Title: Impaired myofibroblast proliferation is a central feature of pathologic post-natal alveolar simplification

doi: 10.7554/eLife.94425

Figure Lengend Snippet: ( A ) Schematic of scRNA-seq project showing CTRL ( Tgfbr2 F/F ) and cKO ( Tgfbr2 F/F ;Nkx2.1-cre) littermates treated in 75% hyperoxia versus normoxia from P0-P10 and harvested on P7 or P14 for FACS-purification and analysis by scRNA-seq. n=2 mice harvested for each genotype, treatment condition and timepoint. ( B ) Venn-diagram depicting strategy to compare these two models to identify shared features of alveolar simplification. ( C ) UMAP projection of mesenchymal cells from scRNA-seq as outlined in ( A ). ( D ) Bar graphs depicting the frequency of each cell type by treatment condition, genotype, and timepoint. ( E ) The frequency of alveolar and ductal myofibroblasts within the mesenchyme as depicted in ( D ) with each data point representing either a biologic or technical replicate. Because the data depicts both biologic and technical replicates, no statistics were performed on this data set. ( F ) Differentially expressed genes in myofibroblasts were identified by comparing either CTRL RA vs O2 cells or RA CTRL vs cKO cells. These lists were subsequently analyzed by Qiagen IPA to identify predicted upstream regulators for each comparison. The Venn-diagram on left depicts the number of overlapping predicted upstream regulators with z-score >1.5 (upregulated), while the table on right lists these 32 shared upstream regulators. Blue = TGFβ signaling, red = inhibitors of cell cycle, green = Wnt signaling.

Techniques: Purification, Comparison

Journal: eLife

Article Title: Impaired myofibroblast proliferation is a central feature of pathologic post-natal alveolar simplification

doi: 10.7554/eLife.94425

Figure Lengend Snippet: ( A ) Hurskainen et al. treated C57BL/6 wildtype mice with 85% hyperoxia versus normoxia from P0-P14 and analyzed the lungs by scRNA-seq at P3, P7, and P14 . The Seurat object used for publication was provided by the authors. UMAP projection shows the mesenchymal populations as defined by Hurskainen et al. ( B ) By using metadata within the Seurat object, we graphed the frequency of each mesenchymal population by treatment condition and time point. ( C ) The frequency of myofibroblasts within the mesenchyme as depicted in ( B ). ( D ) Xia et al. treated C57BL/6 wildtype mice with 85% hyperoxia versus normoxia from P0-P14 and analyzed the lungs by scRNA-seq at P14 . The Seurat object used for publication was provided by the authors. UMAP projection shows the mesenchymal populations as defined by Xia et al. ( E ) By using metadata within the Seurat object, we graphed the frequency of each mesenchymal population by treatment condition and time point. ( F ) The frequency of alveolar and ductal myofibroblasts within the mesenchyme as depicted in ( E ). Graphs in ( C ) and ( F ) depict one value for each condition because we were unable to extract replicate values from the data provided.

Techniques:

Journal: eLife

Article Title: Impaired myofibroblast proliferation is a central feature of pathologic post-natal alveolar simplification

doi: 10.7554/eLife.94425

Figure Lengend Snippet: scRNA-seq data was analyzed using the NicheNet software package. ( A ) Alveolar epithelial clusters were pooled to define sender population and myofibroblast clusters were pooled to define receiver population. By analyzing differential expression of ligands, receptors, and downstream gene expression changes in myofibroblasts, NicheNet predicted increased (left) or decreased (right) ligand-receptor signaling in each comparison of interest. Gray boxes show ligands expressed on the epithelium and the purple-shaded boxes indicate the corresponding receptors expressed on myofibroblasts. Upper panels compare hyperoxia versus normoxia in CTRL cells. Lower panels compare CTRL versus cKO cells in normoxia. ( B ) List of ligand-receptor pairs predicted to be increased in both injury models. Tgfb1-Tgfbr2 pairings highlighted in blue. ( C ) List of ligand-receptor pairs predicted to be decreased in both injury models. Pdgfa-Pdgfra and Pdgfb-Pdgfrb pairings highlighted in red.

Techniques: Software, Expressing, Comparison

Journal: eLife

Article Title: Impaired myofibroblast proliferation is a central feature of pathologic post-natal alveolar simplification

doi: 10.7554/eLife.94425

Figure Lengend Snippet: ( A ) Wildtype C57BL/6 mice were injected every other day from P2-P10 with PBS or 1D11 (pan- TGFβ-blocking antibody), treated in 75% hyperoxia treatment versus normoxia from P0-P10, and recovered in room air until harvest at P40 for analysis by either histology or lung physiology. ( B ) H&E sections of representative lungs from ( A ) harvested at P40 (left). Images shown are from PBS and 30 mg/kg 1D11 treatment groups. Mean linear intercepts calculated for all treatment groups (right). ( C ) PBS- and 30 mg/kg 1D11-treated mice treated in normoxic conditions as in ( A ) and harvested for lung physiology measurements of compliance, elastance and lung capacity at P40. Data in ( B ) compared by ANOVA with Fisher’s post hoc test; for readability and limitations of graphing, only the statistical significance values within normoxia or hyperoxia cohorts are plotted. Data in ( C ) compared by two-tailed unpaired Student’s t-test. Error bars depict mean ± SEM. *p<0.05, **p<0.01, ****p<0.0001. Scale bars = 100 μm.

Techniques: Injection, Blocking Assay, Two Tailed Test

Journal: eLife

Article Title: Impaired myofibroblast proliferation is a central feature of pathologic post-natal alveolar simplification

doi: 10.7554/eLife.94425

Figure Lengend Snippet: ( A ) Either Gli1-CreERT2 or Pdgfra-CreERT2 mice and their cre-negative littermates were injected with tamoxifen on P2 and P4, treated in 75% hyperoxia versus normoxia from P0-P10, and recovered in room air until harvest at P40 for analysis by histology. ( B ) Mean linear intercepts of Gli1 CreER/+ and Gli1 +/+ mice treated as outlined in ( A ) and harvested at P40. ( C ) Mean linear intercepts of Pdgfra CreER/+ and Pdgfra +/+ mice treated as outlined in ( A ) and harvested at P40. Data compared by ANOVA with Fisher’s post hoc test. Error bars depict mean ± SEM. ***p<0.001, ****p<0.0001.

Techniques: Injection

Journal: eLife

Article Title: Impaired myofibroblast proliferation is a central feature of pathologic post-natal alveolar simplification

doi: 10.7554/eLife.94425

Figure Lengend Snippet: ( A ) Tgfbr2 F/F and Tgfbr2 F/F ;Gli1-CreERT2 littermates were injected with tamoxifen on P2 and P4, treated in 75% hyperoxia versus normoxia from P0-P10, and recovered in room air until harvest at P40 for analysis by histology. ( B ) H&E sections of representative lungs from ( A ) harvested at P40 (left). Mean linear intercepts calculated for all treatment groups (right). ( C ) Itgb6 F/F and Itgb6 F/F ;Nkx2.1-cre littermates were treated in 75% hyperoxia versus normoxia from P0-P10, and recovered in room air until harvest at P40 for analysis by histology ( D ) H&E sections of representative lungs from ( C ) harvested at P40 (left). Mean linear intercepts calculated for all treatment groups (right). Data compared by ANOVA with Fisher’s post hoc test. Error bars depict mean ± SEM. *p<0.05, ***p<0.001, ****p<0.0001. Scale bars = 100 μm.

Techniques: Injection

Journal: eLife

Article Title: Impaired myofibroblast proliferation is a central feature of pathologic post-natal alveolar simplification

doi: 10.7554/eLife.94425

Figure Lengend Snippet: ( A ) Itgav F/F and Itgav F/F ;Gli1-CreERT2 littermates were injected with tamoxifen on P2 and P4, treated in 75% hyperoxia versus normoxia from P0-P10, and recovered in room air until harvest at P40 for analysis by either histology or lung physiology. ( B ) H&E sections of representative lungs from ( A ) harvested at P40 (left). Mean linear intercepts calculated for all treatment groups (right). ( C ) Normoxia cohort treated as in ( A ) and harvested for lung physiology measurements of compliance, elastance and lung capacity. Data in ( B ) compared by ANOVA with Fisher’s post hoc test. Data in ( C ) compared by two-tailed unpaired Student’s t-test. Error bars depict mean ± SEM. *p<0.05, **p<0.01, ****p<0.0001. Scale bars = 100 μm.

Techniques: Injection, Two Tailed Test

Journal: eLife

Article Title: Impaired myofibroblast proliferation is a central feature of pathologic post-natal alveolar simplification

doi: 10.7554/eLife.94425

Figure Lengend Snippet: ( A ) Wildtype C57BL/6 mice were treated in 75% hyperoxia versus normoxia from P0-P10 and recovered in room air until indicated timepoints for analysis. Mice were injected with EdU 24 hr prior to each analysis timepoint. ( B ) Flow cytometry plots of lung mesenchyme (CD45-, CD31-, Epcam-, MCAM-) show gating of PDGFRα+ cells (upper panels) and subsequent identification of EdU+/PDGFRα+ cells (lower panels). Panels on far-right show an EdU-untreated littermate used to define EdU + gate. ( C ) Time course graphs showing indicated populations of the lung as percent of lung (top), total cells in left lung (middle), and percent EdU + cells (bottom). Data graphed as mean ± with exception of EdU + panel in which each animal is plotted individually. Data compared by two-tailed unpaired Student’s t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Techniques: Injection, Flow Cytometry, Two Tailed Test

Journal: eLife

Article Title: Impaired myofibroblast proliferation is a central feature of pathologic post-natal alveolar simplification

doi: 10.7554/eLife.94425

Figure Lengend Snippet: ( A ) Mean fluorescence intensity (geometric mean, MFI) of PDGFRα antibody staining on MCAM-negative mesenchymal cells (CD45-, CD31-, Epcam-) and PDGFRa + cells (CD45-, CD31-, Epcam-, MCAM-, PDGFRα+) as quantified by flow cytometry. Each column represents an experiment shown earlier in this study: normoxia vs hyperoxia at P10 , Tgfbr2 F/F vs Tgfbr2 F/F ;Nkx2.1-cre at P10 , PBS vs 1D11 at P10 , Ect2 F/F vs Ect2 F/F ;Pdgfra-CreERT2 at P14 . To compare values across multiple experiments, MFI’s were normalized to the control condition within each experiment. Data compared by ANOVA with Fisher’s post hoc test. Error bars depict mean ± SEM. ***p<0.001, ****p<0.0001.

Techniques: Fluorescence, Staining, Flow Cytometry, Control

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