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( A ) Schematic of flow cytometric analysis and quantification of immune cells in abdominal aortic tissue from the AAA model after 1 week of AngII infusion. ( B ) Representative flow cytometry plots of digested abdominal aortic tissue 1 week after AngII infusion ( n = 5 mice per group). ( C ) Quantification of immune cell populations in digested abdominal aortic tissue (absolute number per 100 mg). Neut, neutrophils; Ly6C hi Mono, Ly6C hi monocytes; Mac, macrophages; CCR2 + Mac, CCR2 + macrophages. Saline, n = 6; AngII, n = 5; B cells and T cells, n = 5 per group. * P < 0.05 and ** P < 0.01, by Mann-Whitney U test. ( D ) Representative images of CD68, α–smooth muscle actin (α-SMA), CD31/platelet endothelial cell adhesion molecule 1 (PECAM-1), and DAPI immunofluorescence staining of abdominal aortic tissue from aortas of control and 20% Tet2 -KO BM recipient mice after 1 week of AngII infusion. Images are representative of 8 mice per genotype. Scale bars: 100 μm. ( E ) Schematic of RNA-seq analysis of sorted macrophages from abdominal aortas of 100% Tet2 -KO BM recipient mice after 1 week of AngII infusion. ( F and G ) Upregulated and downregulated genes are presented as a heatmap ( F ) and volcano plot ( G ). ( H ) Gene Ontology enrichment analysis of upregulated genes in Tet2 -deficient macrophages. The table lists the predominant genes ranked by q value among significantly enriched pathways. ( I and J ) In vitro differentiation of BM-derived macrophages. ( I and J ) Representative images and quantification of the TRAP + cell-to-macrophage ratio after RANKL stimulation from 8 independent biological replicates per genotype. *** P < 0.001, by Mann-Whitney U test. ( K <t>)</t> <t>qRT-PCR</t> analysis of BM-derived macrophages 6 hours after stimulation with 10 ng/mL LPS ( n = 6 independent biological replicates per genotype). **** P < 0.0001, by Mann-Whitney U test.
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( A ) Schematic of flow cytometric analysis and quantification of immune cells in abdominal aortic tissue from the AAA model after 1 week of AngII infusion. ( B ) Representative flow cytometry plots of digested abdominal aortic tissue 1 week after AngII infusion ( n = 5 mice per group). ( C ) Quantification of immune cell populations in digested abdominal aortic tissue (absolute number per 100 mg). Neut, neutrophils; Ly6C hi Mono, Ly6C hi monocytes; Mac, macrophages; CCR2 + Mac, CCR2 + macrophages. Saline, n = 6; AngII, n = 5; B cells and T cells, n = 5 per group. * P < 0.05 and ** P < 0.01, by Mann-Whitney U test. ( D ) Representative images of CD68, α–smooth muscle actin (α-SMA), CD31/platelet endothelial cell adhesion molecule 1 (PECAM-1), and DAPI immunofluorescence staining of abdominal aortic tissue from aortas of control and 20% Tet2 -KO BM recipient mice after 1 week of AngII infusion. Images are representative of 8 mice per genotype. Scale bars: 100 μm. ( E ) Schematic of RNA-seq analysis of sorted macrophages from abdominal aortas of 100% Tet2 -KO BM recipient mice after 1 week of AngII infusion. ( F and G ) Upregulated and downregulated genes are presented as a heatmap ( F ) and volcano plot ( G ). ( H ) Gene Ontology enrichment analysis of upregulated genes in Tet2 -deficient macrophages. The table lists the predominant genes ranked by q value among significantly enriched pathways. ( I and J ) In vitro differentiation of BM-derived macrophages. ( I and J ) Representative images and quantification of the TRAP + cell-to-macrophage ratio after RANKL stimulation from 8 independent biological replicates per genotype. *** P < 0.001, by Mann-Whitney U test. ( K <t>)</t> <t>qRT-PCR</t> analysis of BM-derived macrophages 6 hours after stimulation with 10 ng/mL LPS ( n = 6 independent biological replicates per genotype). **** P < 0.0001, by Mann-Whitney U test.
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( A ) Schematic of flow cytometric analysis and quantification of immune cells in abdominal aortic tissue from the AAA model after 1 week of AngII infusion. ( B ) Representative flow cytometry plots of digested abdominal aortic tissue 1 week after AngII infusion ( n = 5 mice per group). ( C ) Quantification of immune cell populations in digested abdominal aortic tissue (absolute number per 100 mg). Neut, neutrophils; Ly6C hi Mono, Ly6C hi monocytes; Mac, macrophages; CCR2 + Mac, CCR2 + macrophages. Saline, n = 6; AngII, n = 5; B cells and T cells, n = 5 per group. * P < 0.05 and ** P < 0.01, by Mann-Whitney U test. ( D ) Representative images of CD68, α–smooth muscle actin (α-SMA), CD31/platelet endothelial cell adhesion molecule 1 (PECAM-1), and DAPI immunofluorescence staining of abdominal aortic tissue from aortas of control and 20% Tet2 -KO BM recipient mice after 1 week of AngII infusion. Images are representative of 8 mice per genotype. Scale bars: 100 μm. ( E ) Schematic of RNA-seq analysis of sorted macrophages from abdominal aortas of 100% Tet2 -KO BM recipient mice after 1 week of AngII infusion. ( F and G ) Upregulated and downregulated genes are presented as a heatmap ( F ) and volcano plot ( G ). ( H ) Gene Ontology enrichment analysis of upregulated genes in Tet2 -deficient macrophages. The table lists the predominant genes ranked by q value among significantly enriched pathways. ( I and J ) In vitro differentiation of BM-derived macrophages. ( I and J ) Representative images and quantification of the TRAP + cell-to-macrophage ratio after RANKL stimulation from 8 independent biological replicates per genotype. *** P < 0.001, by Mann-Whitney U test. ( K <t>)</t> <t>qRT-PCR</t> analysis of BM-derived macrophages 6 hours after stimulation with 10 ng/mL LPS ( n = 6 independent biological replicates per genotype). **** P < 0.0001, by Mann-Whitney U test.
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( A ) Schematic of flow cytometric analysis and quantification of immune cells in abdominal aortic tissue from the AAA model after 1 week of AngII infusion. ( B ) Representative flow cytometry plots of digested abdominal aortic tissue 1 week after AngII infusion ( n = 5 mice per group). ( C ) Quantification of immune cell populations in digested abdominal aortic tissue (absolute number per 100 mg). Neut, neutrophils; Ly6C hi Mono, Ly6C hi monocytes; Mac, macrophages; CCR2 + Mac, CCR2 + macrophages. Saline, n = 6; AngII, n = 5; B cells and T cells, n = 5 per group. * P < 0.05 and ** P < 0.01, by Mann-Whitney U test. ( D ) Representative images of CD68, α–smooth muscle actin (α-SMA), CD31/platelet endothelial cell adhesion molecule 1 (PECAM-1), and DAPI immunofluorescence staining of abdominal aortic tissue from aortas of control and 20% Tet2 -KO BM recipient mice after 1 week of AngII infusion. Images are representative of 8 mice per genotype. Scale bars: 100 μm. ( E ) Schematic of RNA-seq analysis of sorted macrophages from abdominal aortas of 100% Tet2 -KO BM recipient mice after 1 week of AngII infusion. ( F and G ) Upregulated and downregulated genes are presented as a heatmap ( F ) and volcano plot ( G ). ( H ) Gene Ontology enrichment analysis of upregulated genes in Tet2 -deficient macrophages. The table lists the predominant genes ranked by q value among significantly enriched pathways. ( I and J ) In vitro differentiation of BM-derived macrophages. ( I and J ) Representative images and quantification of the TRAP + cell-to-macrophage ratio after RANKL stimulation from 8 independent biological replicates per genotype. *** P < 0.001, by Mann-Whitney U test. ( K <t>)</t> <t>qRT-PCR</t> analysis of BM-derived macrophages 6 hours after stimulation with 10 ng/mL LPS ( n = 6 independent biological replicates per genotype). **** P < 0.0001, by Mann-Whitney U test.
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( A ) Schematic of flow cytometric analysis and quantification of immune cells in abdominal aortic tissue from the AAA model after 1 week of AngII infusion. ( B ) Representative flow cytometry plots of digested abdominal aortic tissue 1 week after AngII infusion ( n = 5 mice per group). ( C ) Quantification of immune cell populations in digested abdominal aortic tissue (absolute number per 100 mg). Neut, neutrophils; Ly6C hi Mono, Ly6C hi monocytes; Mac, macrophages; CCR2 + Mac, CCR2 + macrophages. Saline, n = 6; AngII, n = 5; B cells and T cells, n = 5 per group. * P < 0.05 and ** P < 0.01, by Mann-Whitney U test. ( D ) Representative images of CD68, α–smooth muscle actin (α-SMA), CD31/platelet endothelial cell adhesion molecule 1 (PECAM-1), and DAPI immunofluorescence staining of abdominal aortic tissue from aortas of control and 20% Tet2 -KO BM recipient mice after 1 week of AngII infusion. Images are representative of 8 mice per genotype. Scale bars: 100 μm. ( E ) Schematic of RNA-seq analysis of sorted macrophages from abdominal aortas of 100% Tet2 -KO BM recipient mice after 1 week of AngII infusion. ( F and G ) Upregulated and downregulated genes are presented as a heatmap ( F ) and volcano plot ( G ). ( H ) Gene Ontology enrichment analysis of upregulated genes in Tet2 -deficient macrophages. The table lists the predominant genes ranked by q value among significantly enriched pathways. ( I and J ) In vitro differentiation of BM-derived macrophages. ( I and J ) Representative images and quantification of the TRAP + cell-to-macrophage ratio after RANKL stimulation from 8 independent biological replicates per genotype. *** P < 0.001, by Mann-Whitney U test. ( K ) qRT-PCR analysis of BM-derived macrophages 6 hours after stimulation with 10 ng/mL LPS ( n = 6 independent biological replicates per genotype). **** P < 0.0001, by Mann-Whitney U test.

Journal: The Journal of Clinical Investigation

Article Title: Tet2-driven clonal hematopoiesis drives aortic aneurysm via macrophage-to-osteoclast–like differentiation

doi: 10.1172/JCI198708

Figure Lengend Snippet: ( A ) Schematic of flow cytometric analysis and quantification of immune cells in abdominal aortic tissue from the AAA model after 1 week of AngII infusion. ( B ) Representative flow cytometry plots of digested abdominal aortic tissue 1 week after AngII infusion ( n = 5 mice per group). ( C ) Quantification of immune cell populations in digested abdominal aortic tissue (absolute number per 100 mg). Neut, neutrophils; Ly6C hi Mono, Ly6C hi monocytes; Mac, macrophages; CCR2 + Mac, CCR2 + macrophages. Saline, n = 6; AngII, n = 5; B cells and T cells, n = 5 per group. * P < 0.05 and ** P < 0.01, by Mann-Whitney U test. ( D ) Representative images of CD68, α–smooth muscle actin (α-SMA), CD31/platelet endothelial cell adhesion molecule 1 (PECAM-1), and DAPI immunofluorescence staining of abdominal aortic tissue from aortas of control and 20% Tet2 -KO BM recipient mice after 1 week of AngII infusion. Images are representative of 8 mice per genotype. Scale bars: 100 μm. ( E ) Schematic of RNA-seq analysis of sorted macrophages from abdominal aortas of 100% Tet2 -KO BM recipient mice after 1 week of AngII infusion. ( F and G ) Upregulated and downregulated genes are presented as a heatmap ( F ) and volcano plot ( G ). ( H ) Gene Ontology enrichment analysis of upregulated genes in Tet2 -deficient macrophages. The table lists the predominant genes ranked by q value among significantly enriched pathways. ( I and J ) In vitro differentiation of BM-derived macrophages. ( I and J ) Representative images and quantification of the TRAP + cell-to-macrophage ratio after RANKL stimulation from 8 independent biological replicates per genotype. *** P < 0.001, by Mann-Whitney U test. ( K ) qRT-PCR analysis of BM-derived macrophages 6 hours after stimulation with 10 ng/mL LPS ( n = 6 independent biological replicates per genotype). **** P < 0.0001, by Mann-Whitney U test.

Article Snippet: Lentiviral titers were determined using a Lenti-X quantitative reverse transcription PCR (qRT-PCR) Titration Kit (catalog 631235, Clontech).

Techniques: Flow Cytometry, Saline, MANN-WHITNEY, Immunofluorescence, Staining, Control, RNA Sequencing, In Vitro, Derivative Assay, Quantitative RT-PCR