Journal: iScience

Article Title: Analysis of myocardial cellular gene expression during pressure overload reveals matrix based functional intercellular communication

doi: 10.1016/j.isci.2022.103965

Figure Lengend Snippet: Endothelial, fibroblast and cardiomyocyte cell purification from mouse hearts (A) Scheme on the experimental procedures to isolate the indicated cell types from mouse hearts. (B) Heatmap of cell marker gene expression in RNA from isolated cardiomyocytes (CM), endothelial cells (EC) or fibroblasts (FB) after sham or the indicated time point after TAC surgery. (C) Cardiac endothelial cells and fibroblasts were isolated from mouse hearts and stained for the endothelial markers CD31 and CD102, for the leukocyte marker CD45, and the fibroblast marker Mefsk4. Subsequently, flow cytometric analyses were performed and representative results are shown here. The numbers indicated in each quadrant indicates the percentage of cells localized in that particular quadrant. (D) RNA from the different cell types after sham or 1 and 8 weeks after TAC was subjected to RNA sequencing. The differences in overall gene expression patterns were visualized by a principal component analysis.

Article Snippet: CD31 Antibody, anti-mouse , Miltenyi Biotec , Clone 390.

Techniques: Purification, Marker, Gene Expression, Isolation, Staining, RNA Sequencing

Journal: iScience

Article Title: Analysis of myocardial cellular gene expression during pressure overload reveals matrix based functional intercellular communication

doi: 10.1016/j.isci.2022.103965

Figure Lengend Snippet:

Article Snippet: CD31 Antibody, anti-mouse , Miltenyi Biotec , Clone 390.

Techniques: Purification, Plasmid Preparation, Blocking Assay, Magnetic Beads, Recombinant, Clinical Proteomics, Protease Inhibitor, cDNA Synthesis, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: JCI insight

Article Title: α7nAChR activation in AT2 cells promotes alveolar regeneration through WNT7B signaling in acute lung injury.

doi: 10.1172/jci.insight.162547

Figure Lengend Snippet: Figure 4. Activation of α7nAChR on AT2 cells promotes alveolar organoid formation. (A) Schematic of organoid coculture of Sftpc lineage–labeled cells (CD45–CD31–EPCAM+tdTomato+) with lung mesenchymal cells (CD45–CD31–EPCAM–) isolated from α7nAChR-knockout (Chrna7–/–) mice and the interven- tion diagram; LPS (1 μg/mL) was added to simulate lung injury in vitro and methyllycaconitine citrate (MLA; α7nAChR antagonist, 10 μmol/L) 15 minutes before GTS-21 (selective α7nAChR agonist, 10 μmol/L) treatment. Times for renewing organoid medium and adding LPS, GTS-21, and MLA are indicated. The culture time varies in different experiments according to purpose. RS, R-Spondin-1. (B) Representative fluorescence images of AT2 organoids captured on day 10. Scale bars: 400 μm. (C) Statistical quantification of the total colony formation efficiency of alveolar organoids. Each individual dot represents 1 experiment in 1 mouse. (D) Statistical quantification of the size of alveolar organoids. Each individual dot represents 1 organoid. (E) Statistical quantifica- tion of total colony formation efficiency of alveolar organoids of different sizes. (F) Representative fluorescence images showing proliferating cells in AT2 organoids derived from the lungs of lineage-tracing mice. Organoids were treated with 5-ethynyl-2′-deoxyuridine (EdU) at an early time point (day 8) for 3 hours in cultures (tdTomato, red; EdU, green; DAPI, blue). Scale bars: 50 μm. One-way ANOVA with Tukey’s post hoc analysis was used in C–E. Data are representative of at least 3 independent experiments and are presented as mean ± SD (*P < 0.05, ***P < 0.001).

Article Snippet: The antibodies used were as follows: APC/Fire 750 anti-mouse F4/80, BV650 anti-mouse/human CD11b, BV421 anti-mouse Ly6G, PE/Cyanine7 anti-mouse Ly6C, BV421 anti-mouse Ki67, PE anti-mouse PDPN, APC anti-mouse CD31, APC anti-mouse CD45, FITC anti-mouse CD326/EPCAM, BV510 donkey anti-rabbit IgG, AF488 donkey anti-rabbit IgG, rabbit anti-pro-Sftpc (Millipore), rabbit anti-mouse CCSP (Proteintech), CF633-α-Bungarotoxin (Biotium) (all others from Biolegend).

Techniques: Activation Assay, Labeling, Isolation, Knock-Out, In Vitro, Fluorescence, Derivative Assay

Journal: JCI insight

Article Title: α7nAChR activation in AT2 cells promotes alveolar regeneration through WNT7B signaling in acute lung injury.

doi: 10.1172/jci.insight.162547

Figure Lengend Snippet: Figure 6. α7nAChR-driven WNT7B signaling is an essential mediator of α7nAChR-promoted alveolar organoid formation. (A) Schematic of organoid coculture of Sftpc lineage–labeled AT2 cells (CD45–CD31–EPCAM+tdTomato+) isolated from indicated mice, with lung mesenchymal cells (CD45–CD31–EPCAM–) isolated from α7nAChR-knockout (Chrna7–/–) mice, and the intervention diagram. (B) Representative IF images of AT2 organoids. LPS (1 μg/mL) was added to simulate lung injury in vitro, and GTS-21(10 μmol/L) was used to activate α7nAChR in all groups. WNT7B (100 ng/mL) was added as indicated. Scale bars: 400 μm. (C) Statistical quantification of the total colony formation efficiency of alveolar organoids. Each individual dot represents 1 experiment. (D) Statisti- cal quantification of the size of alveolar organoids. Each individual dot represents 1 organoid. One-way ANOVA with Tukey’s post hoc analysis was used in C and D. Data are representative of at least 3 independent experiments and are presented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The antibodies used were as follows: APC/Fire 750 anti-mouse F4/80, BV650 anti-mouse/human CD11b, BV421 anti-mouse Ly6G, PE/Cyanine7 anti-mouse Ly6C, BV421 anti-mouse Ki67, PE anti-mouse PDPN, APC anti-mouse CD31, APC anti-mouse CD45, FITC anti-mouse CD326/EPCAM, BV510 donkey anti-rabbit IgG, AF488 donkey anti-rabbit IgG, rabbit anti-pro-Sftpc (Millipore), rabbit anti-mouse CCSP (Proteintech), CF633-α-Bungarotoxin (Biotium) (all others from Biolegend).

Techniques: Labeling, Isolation, Knock-Out, In Vitro

Journal: JCI insight

Article Title: α7nAChR activation in AT2 cells promotes alveolar regeneration through WNT7B signaling in acute lung injury.

doi: 10.1172/jci.insight.162547

Figure Lengend Snippet: Figure 7. α7nAChR-driven WNT7B signaling is integral for α7nAChR-promoted AT2 cell proliferation and differentiation. (A) Schematic of organoid coculture of Sftpc lineage–labeled cells (CD45–CD31–EPCAM+tdTomato+) isolated from indicated mice, with lung mesenchymal cells (CD45–CD31–EP- CAM–) isolated from α7nAChR-knockout (Chrna7–/–) mice, and the intervention diagram. (B) Effect of selective knockout of α7nAChR on AT2 cells and supplementation of WNT7B (100 ng/mL) on the proliferation of lineage-traced AT2 cells in organoids on day 10, as judged by Ki67 (proliferative marker) (tdTomato, red; Ki67, green; DAPI, blue). Scale bars: 200 μm. (C) Effect of selective knockout of α7nAChR on AT2 cells and supplementation of WNT7B (100 ng/mL) on the differentiation of lineage-traced AT2 cells in organoids on day 12, as judged by PDPN (AT1 marker) (tdTomato, red; PDPN, cyan; DAPI, blue). Scale bars: 200 μm. (D) qPCR analysis of Mki67 from mouse AT2 organoids. (E) qPCR analysis of Pdpn from mouse AT2 organoids. One-way ANOVA with Tukey’s post hoc analysis was used in D and E. Data are presented as mean ± SD (*P < 0.05).

Article Snippet: The antibodies used were as follows: APC/Fire 750 anti-mouse F4/80, BV650 anti-mouse/human CD11b, BV421 anti-mouse Ly6G, PE/Cyanine7 anti-mouse Ly6C, BV421 anti-mouse Ki67, PE anti-mouse PDPN, APC anti-mouse CD31, APC anti-mouse CD45, FITC anti-mouse CD326/EPCAM, BV510 donkey anti-rabbit IgG, AF488 donkey anti-rabbit IgG, rabbit anti-pro-Sftpc (Millipore), rabbit anti-mouse CCSP (Proteintech), CF633-α-Bungarotoxin (Biotium) (all others from Biolegend).

Techniques: Labeling, Isolation, Knock-Out, Marker