mouse anti human antibody  (R&D Systems)

Journal: The Journal of Pathology

Article Title: Macrophages producing chondroitin sulfate proteoglycan‐4 induce neuro‐cardiac junction impairment in Duchenne muscular dystrophy

doi: 10.1002/path.6362

Figure Lengend Snippet: Cardiac fibrosis and innervation. (A) Masson's trichrome staining of young (3‐month‐old) and old (10‐month‐old) WT and mdx mouse hearts. Scale bars, 100 μm. The graph shows the area as a percentage of the fibrosis index (fibrotic area/whole area). N = 3 biological replicates. (B) qPCR for fibrosis‐related transcripts in 3‐ and 10‐month‐old mdx and WT hearts ( Col1a1 , Col3a1 , Fn1 , Tgfb1 , Twist 1 , Twist 2 ). N = 3 biological replicates. (C) Confocal images for tyrosine hydroxylase (TH), Synapsin 1 (SYN1), MPs (F4/80), proteoglycan CSPG4, and fibronectin 1 (FN1) on myocardium sections of 3‐ and 10‐month‐old mice. The markers TH, SYN1, and CSPG4 are labeled in yellow, cardiac troponin (cTNNT) and F4/80 are in magenta, while FN1 is in white. Scale bars, 10 μm. Nuclei are counterstained with DAPI in blue. (D) Charts indicate positive area expressed as percentage of TH, SYN1, and F4/80 on whole area. N = 3 sections per N = 3 biological replicates. (E and F) Western blot analysis of TH, SYN1, and CSPG4 in mdx and WT hearts of young and old mice. Graphs show optical density (OD) of protein bands normalized to vinculin (VCL). N = 3 biological replicates. Error bars show SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 were calculated using Student's t ‐test.

Article Snippet: Frozen and paraffin sections were blocked for 30 min at RT with 5% bovine serum albumin (BSA, Catalogue No. A1470, Sigma‐Aldrich, Saint Louis, MO, USA), incubated overnight at 4 °C with primary antibodies: anti‐cardiac troponin T (cTNNT, 1:100, Abcam, ab33589, Cambridge, UK), anti‐Connexin 43 (Cx43, 1:100, Cell Signaling Technology (CST), #3512, Danvers, MA, USA), anti‐tyrosine hydroxylase (TH, 1:100, Sigma‐Aldrich, ab152), anti‐Synapsin 1 (SYN1, 1:100, CST, #6710, Danvers, MA, USA), anti‐MPs F4/80 (F4/80, 1:100, Biorad, MCA497G, Segrate, Milan, Italy), anti‐CSPG4 (1:200, Novus Biologicals, NBP266979 or 1:50, Abingdon, Oxfordshire, UK, Santa Cruz, SC‐80003, Dallas, TX, USA), and anti‐fibronectin 1 (FN1; 1:50, Santa Cruz, SC‐271098) and incubated with fluorescent‐conjugated secondary antibodies (1:1000, Invitrogen, Waltham, MA, USA) for 1 h. Nuclei were counterstained with DAPI (Catalogue No. D1306, Invitrogen).

Techniques: Staining, Labeling, Western Blot

Journal: The Journal of Pathology

Article Title: Macrophages producing chondroitin sulfate proteoglycan‐4 induce neuro‐cardiac junction impairment in Duchenne muscular dystrophy

doi: 10.1002/path.6362

Figure Lengend Snippet: MPs as main producers of CSPG4. (A) Graphic illustration of CSPG4‐CAR‐Ts cells against MPs expressing CSPG4. Created with Biorender.com . Diagrams quantifying the percentage of live cells of bone marrow‐derived MPs of both WT and mdx M0 (nonactivated), M1 (pro‐inflammatory) and M2 (anti‐inflammatory), or CFs after exposure to CSPG4.CARTs compared to cells co‐cultured with control CART (cCART). N = 3 biological replicates. (B) ELISA assay for CSPG4 in supernatants (SUP) isolated from WT and mdx MPs. The chart illustrates the optical density (OD) of CSPG4 in the two groups. Each dot represents the eluted fractions, N = 16. (C) FACSymphony of cardiac tissues. The first chart represents a scatter plot of t‐SNE relative to concatenated total cells from 3‐ or 10‐month‐old WT and mdx hearts. The second chart illustrates the distribution of CD45 neg cells (light pink cluster) and myeloid cells (dark pink cluster) in all groups. (D) Graph of FACS analysis relative to number of total MPs (F4/80) positive for CSPG4 in 3‐ or 10‐month‐old WT and mdx hearts. (E) Expression of CSPG4 in subpopulations of MPs: CD80 pos (M1), CD206 pos (M2), Ly6C low (resident), and Ly6C high (monocytes) in 3‐ or 10‐month‐old WT and mdx hearts. Error bars show SEM. * p < 0.05, ** p < 0.01 calculated using Student's t ‐test and one‐way ANOVA.

Article Snippet: Frozen and paraffin sections were blocked for 30 min at RT with 5% bovine serum albumin (BSA, Catalogue No. A1470, Sigma‐Aldrich, Saint Louis, MO, USA), incubated overnight at 4 °C with primary antibodies: anti‐cardiac troponin T (cTNNT, 1:100, Abcam, ab33589, Cambridge, UK), anti‐Connexin 43 (Cx43, 1:100, Cell Signaling Technology (CST), #3512, Danvers, MA, USA), anti‐tyrosine hydroxylase (TH, 1:100, Sigma‐Aldrich, ab152), anti‐Synapsin 1 (SYN1, 1:100, CST, #6710, Danvers, MA, USA), anti‐MPs F4/80 (F4/80, 1:100, Biorad, MCA497G, Segrate, Milan, Italy), anti‐CSPG4 (1:200, Novus Biologicals, NBP266979 or 1:50, Abingdon, Oxfordshire, UK, Santa Cruz, SC‐80003, Dallas, TX, USA), and anti‐fibronectin 1 (FN1; 1:50, Santa Cruz, SC‐271098) and incubated with fluorescent‐conjugated secondary antibodies (1:1000, Invitrogen, Waltham, MA, USA) for 1 h. Nuclei were counterstained with DAPI (Catalogue No. D1306, Invitrogen).

Techniques: Expressing, Derivative Assay, Cell Culture, Control, Enzyme-linked Immunosorbent Assay, Isolation

Journal: The Journal of Pathology

Article Title: Macrophages producing chondroitin sulfate proteoglycan‐4 induce neuro‐cardiac junction impairment in Duchenne muscular dystrophy

doi: 10.1002/path.6362

Figure Lengend Snippet: MPs release CSPG4 in media and inhibit axon growth of dorsal root ganglia. (A) Rendering of 3D NCJ generation. The picture was created with Biorender.com and illustrates the composition of the 3D NCJ model: WT dorsal root ganglia (DRG), WT cardiomyocytes (CMs), and WT CFs were encapsulated in PEG‐FB hydrogel conditioned with the supernatants derived from MP cultures of WT, WT + CSPG4 blocking antibody (BAb), mdx , and mdx + CSPG4 (BAb). (B) Brightfield images of CFs, CMs, and DRG after 1 day of culture (upper panel) and 3D constructs of NCJ models after 7 days (lower panel). Scale bars, 100 μm. (C) Confocal images of 3D NCJ models: WT, WT + CSPG4 BAb, mdx , mdx + CSPG4 BAb. DRG are labeled with tubulin beta 3 (TUBB3, in yellow), CMs with cardiac troponin (cTNNT, in magenta), and CFs with fibroblast marker (ER‐TR7, in white). (D) The graph indicates the area (%) occupied by neuron endings labeled by TUBB3 inside the 3D NCJ models. N = 3 biological replicates, N = 2 sections/constructs. Scale bars, 10 μm. ** p < 0.01, *** p < 0.001 were determined using one‐way ANOVA.

Article Snippet: Frozen and paraffin sections were blocked for 30 min at RT with 5% bovine serum albumin (BSA, Catalogue No. A1470, Sigma‐Aldrich, Saint Louis, MO, USA), incubated overnight at 4 °C with primary antibodies: anti‐cardiac troponin T (cTNNT, 1:100, Abcam, ab33589, Cambridge, UK), anti‐Connexin 43 (Cx43, 1:100, Cell Signaling Technology (CST), #3512, Danvers, MA, USA), anti‐tyrosine hydroxylase (TH, 1:100, Sigma‐Aldrich, ab152), anti‐Synapsin 1 (SYN1, 1:100, CST, #6710, Danvers, MA, USA), anti‐MPs F4/80 (F4/80, 1:100, Biorad, MCA497G, Segrate, Milan, Italy), anti‐CSPG4 (1:200, Novus Biologicals, NBP266979 or 1:50, Abingdon, Oxfordshire, UK, Santa Cruz, SC‐80003, Dallas, TX, USA), and anti‐fibronectin 1 (FN1; 1:50, Santa Cruz, SC‐271098) and incubated with fluorescent‐conjugated secondary antibodies (1:1000, Invitrogen, Waltham, MA, USA) for 1 h. Nuclei were counterstained with DAPI (Catalogue No. D1306, Invitrogen).

Techniques: Derivative Assay, Blocking Assay, Construct, Labeling, Marker

Journal: The Journal of Pathology

Article Title: Macrophages producing chondroitin sulfate proteoglycan‐4 induce neuro‐cardiac junction impairment in Duchenne muscular dystrophy

doi: 10.1002/path.6362

Figure Lengend Snippet: Cardiac innervation in old mdx and WT mice. (A) Immunofluorescence staining for tyrosine hydroxylase (TH), Synapsin 1 (SYN1), total MPs (F4/80) and proteoglycan (CSPG4), fibronectin 1 (FN1) on cardiac sections from 10‐month‐old mice. The markers TH and SYN1 are labeled in yellow, while cardiac troponin (cTNNT) and F4/80 are in magenta, FN1 in white, and CSPG4 in yellow or white. DAPI nuclear counterstaining is in blue. Scale bars, 10 μm. (B) Charts indicate area expressed as percentage of TH, SYN1, and F4/80 (positive area/whole area). N = 3 sections per N = 3 biological replicates. (C and D) Western blot analysis for TH, SYN1, and CSPG4 quantified as optical density (OD) of protein bands normalized to vinculin (VCL). N = 3 biological replicates. (E) qPCR relative to markers for neurotransmitter production ( Th ), survival ( Ngf , Ntrk1 , and Ngfr ) and axon guidance ( Tubb3 ) in DRG. N = 3 biological replicates. Error bars show SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 were calculated using one‐way ANOVA.

Article Snippet: Frozen and paraffin sections were blocked for 30 min at RT with 5% bovine serum albumin (BSA, Catalogue No. A1470, Sigma‐Aldrich, Saint Louis, MO, USA), incubated overnight at 4 °C with primary antibodies: anti‐cardiac troponin T (cTNNT, 1:100, Abcam, ab33589, Cambridge, UK), anti‐Connexin 43 (Cx43, 1:100, Cell Signaling Technology (CST), #3512, Danvers, MA, USA), anti‐tyrosine hydroxylase (TH, 1:100, Sigma‐Aldrich, ab152), anti‐Synapsin 1 (SYN1, 1:100, CST, #6710, Danvers, MA, USA), anti‐MPs F4/80 (F4/80, 1:100, Biorad, MCA497G, Segrate, Milan, Italy), anti‐CSPG4 (1:200, Novus Biologicals, NBP266979 or 1:50, Abingdon, Oxfordshire, UK, Santa Cruz, SC‐80003, Dallas, TX, USA), and anti‐fibronectin 1 (FN1; 1:50, Santa Cruz, SC‐271098) and incubated with fluorescent‐conjugated secondary antibodies (1:1000, Invitrogen, Waltham, MA, USA) for 1 h. Nuclei were counterstained with DAPI (Catalogue No. D1306, Invitrogen).

Techniques: Immunofluorescence, Staining, Labeling, Western Blot

Journal: The Journal of Pathology

Article Title: Macrophages producing chondroitin sulfate proteoglycan‐4 induce neuro‐cardiac junction impairment in Duchenne muscular dystrophy

doi: 10.1002/path.6362

Figure Lengend Snippet: Histological analysis and gene profile of cardiac tissue after treatment with givinostat in old mdx and WT mice. (A) Confocal images of immunostaining for gap junctions (CX43) and cardiomyocytes positive for cardiac troponin (cTNNT), N = 3 sections per N = 3 biological replicates. Scale bars, 50 μm. (B) Charts indicate area as percentage of CX43 (positive area/whole area). (C and D) Representative Masson's trichrome images of WT, mdx saline, and mdx Giv hearts and fibrosis index quantification (fibrotic area/whole area) after 60 days of treatment. Scale bars, 100 μm. N = 3 biological replicates. (E) qPCR of fibrosis‐related genes ( Tgfb1 , Col1a1 , Col3a1 , Twist1 , Twist2 , Fn1 ), cell surface proteoglycan ( Cspg4 ); the enzyme catalyzes the transfer of sulfate groups on CSPG4 chains ( Chst11 ) and inflammatory markers ( Adgre1 and Mmp9 ). N = 3 biological replicates. Error bars show SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 were calculated using one‐way ANOVA and Student's t ‐test.

Article Snippet: Frozen and paraffin sections were blocked for 30 min at RT with 5% bovine serum albumin (BSA, Catalogue No. A1470, Sigma‐Aldrich, Saint Louis, MO, USA), incubated overnight at 4 °C with primary antibodies: anti‐cardiac troponin T (cTNNT, 1:100, Abcam, ab33589, Cambridge, UK), anti‐Connexin 43 (Cx43, 1:100, Cell Signaling Technology (CST), #3512, Danvers, MA, USA), anti‐tyrosine hydroxylase (TH, 1:100, Sigma‐Aldrich, ab152), anti‐Synapsin 1 (SYN1, 1:100, CST, #6710, Danvers, MA, USA), anti‐MPs F4/80 (F4/80, 1:100, Biorad, MCA497G, Segrate, Milan, Italy), anti‐CSPG4 (1:200, Novus Biologicals, NBP266979 or 1:50, Abingdon, Oxfordshire, UK, Santa Cruz, SC‐80003, Dallas, TX, USA), and anti‐fibronectin 1 (FN1; 1:50, Santa Cruz, SC‐271098) and incubated with fluorescent‐conjugated secondary antibodies (1:1000, Invitrogen, Waltham, MA, USA) for 1 h. Nuclei were counterstained with DAPI (Catalogue No. D1306, Invitrogen).

Techniques: Immunostaining, Saline

Journal: Nature Communications

Article Title: Runx1+ vascular smooth muscle cells are essential for hematopoietic stem and progenitor cell development in vivo

doi: 10.1038/s41467-024-44913-z

Figure Lengend Snippet: a Three-dimensional (3D) wholemount immunostaining with αSMA, CD31 and NG2 of E10.5 (31–38 somite pairs (sp)) WT dorsal aorta; b NG2 and Runx1 expression on single plane wholemount WT E10.5 sections. NG2 + Runx1 + vSMCs (arrows), hemogenic endothelial cells (arrowheads) and intra-aortic hematopoietic clusters (IAHCs, stars) (Table ); c Representative example of flow cytometric analysis of NG2 + Runx1(GFP) + (green box) in E10.5 Runx1-IRES-GFP AGM and E10.5 WT control. d Percentages of NG2 + Runx1(GFP) + cells in E9 (21-25sp) body ( n = 6), E10/E10.5/E11 AGMs ( n = 8/7/7), N = 5, Kruskal-Wallis and Dunn’s post-hoc test. e Representative examples of wholemount 3D-images showing αSMA, CD31 and NG2 in E10.5 cKO dorsal aortae; f αSMA, Runx1 and CD31 immunofluorescence of E11 WT and cKO transversal frozen sections; n = WT/cKO: 2/2, N = 2. g cKit and CD31 wholemount 3D-images in E10.5 WT and cKO AGM; h Number of intra-aortic hematopoietic clusters (IAHCs) in E10.5 AGM; n = WT/KO: 5/4, N = 4. Number of colony forming unit-culture (CFU-C) in i E10.5 (31-38sp) AGM; n = WT/HET/KO: 14/10/5 embryos; N = 7 and j E11 (43–52sp) AGM; n = WT/HET/KO: 22/8/19 embryos; N = 11; one-way ANOVA and Tukey’s post-hoc test (Table ). k Percentages of donor cell chimerism 4-months post-transplantation of 6 E11 WT (NG2 +/+ ;Runx1 fl/+ or NG2 +/+ ;Runx1 fl/fl ) , 7 HET ( NG2-Cre;Runx1 fl/+ ) and 6 cKO AGMs ( NG2-Cre;Runx1 fl/fl ) into sub-lethally adult irradiated recipients (1xAGM cells transplanted/recipient; N = 4). Each dot represents one recipient. Mice are reconstituted when ≥5% donor cells are found in the host peripheral blood (dashed line) ; one-tailed Z score test for two population proportions (Tables and ). For wholemount staining in a , b , e , g : WT/cKO ( N = 6/4): αSMA ( n = 9/7), CD31 ( n = 10/7), cKit ( n = 3/2), NG2 ( n = 3/1) and WT Runx1 ( n = 4) in 3 distinct combinations (Table ). D = dorsal, V = ventral. N = number of independent experiments; n = number of biological samples (embryos). All data are presented as mean values ± SEM. Source data for d , h , i , j and k are provided as a file.

Article Snippet: Embryos were stained for several days with unconjugated cKit (1:500, BD Bioscience, 553352), biotinylated CD31 (1:500, BD Pharmingen, 553371), unconjugated NG2 (1:500, R&D Systems, MAB6689), Runx1,2,3 (1:250, Abcam, ab92336) and αSMA FITC (1:500, Sigma, F3777).

Techniques: Immunostaining, Expressing, Control, Immunofluorescence, Transplantation Assay, Irradiation, One-tailed Test, Staining

Journal: Nature Communications

Article Title: Runx1+ vascular smooth muscle cells are essential for hematopoietic stem and progenitor cell development in vivo

doi: 10.1038/s41467-024-44913-z

Figure Lengend Snippet: a t-SNE plot highlighting eight populations of interest identified in the E11 WT AGM. Each dot represents one cell and colours represent cell clusters as indicated. The number of cells in each population is shown in brackets. MP (macrophages); Ery/EryP (erythroid/progenitors); IAHC (intra-aortic hematopoietic clusters); HEC/EHT (hemogenic endothelial cells including those that enter endothelial-to-hematopoietic transition); EC (endothelial cells); SNS (sympathetic nervous system); SkMP (skeletal muscle progenitors), PC/vSMC (pericytes/vascular smooth muscle cells, NG2 + Acta2 + ). Other cells (OC) are coloured in grey. b t-SNE plot highlighting the eight populations identified after excluding all other (grey) cells. c Zoom into PC/vSMC cluster (black rectangle) further show the presence or the absence of selected genes that characterise this population and confirms the presence of Runx1 in a subset of cells. d Violin plots showing distribution of expression for selected genes that contributed to the identification of cell clusters. Immunohistochemistry on frozen E11 WT sections stained with e CD146/CD31/DAPI and f CD146/αSMA/DAPI, n = 2 samples tested, N = 2 independent experiments. Arrows: vascular cells, asterisks: perivascular cells. DA: dorsal aorta, CV: cardinal veins, NC: notochord. Source data for e (first column, 20X) is provided as a file.

Article Snippet: Embryos were stained for several days with unconjugated cKit (1:500, BD Bioscience, 553352), biotinylated CD31 (1:500, BD Pharmingen, 553371), unconjugated NG2 (1:500, R&D Systems, MAB6689), Runx1,2,3 (1:250, Abcam, ab92336) and αSMA FITC (1:500, Sigma, F3777).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Nature Communications

Article Title: Runx1+ vascular smooth muscle cells are essential for hematopoietic stem and progenitor cell development in vivo

doi: 10.1038/s41467-024-44913-z

Figure Lengend Snippet: a t-SNE plots showing the distribution of Runx1 and Acta2 expression in NG2 + Runx1 + cells in the WT E11 AGM after excluding all other (grey) cells found in the Fig. . b Zoom into NG2 + Runx1 + cluster (black rectangle) shows the presence or the absence of Acta2 . c Heatmap showing the expression of Cspg4 and Runx1 and 15 selected genes out of 25 top significantly upregulated genes in WT NG2 + Runx1 + Acta2 + cells (upper half) and NG2 + Runx1 + Acta2 - cells (bottom half) at single cell level; *Runx1 potential target genes. Pdgfra and Ptn genes were next added to inform their expression in both populations. Barplot of fold enrichment for selected GO biological processes significantly overrepresented in genes significantly upregulated in both d WT NG2 + Runx1 + Acta2 + and e NG2 + Runx1 + Acta2 - cells. f t-SNE of WT E11 AGM cells, overlaid with principal pseudotime curve inferred by Slingshot, predicting a lineage from NG2 + Runx1 + Acta2 - cells to NG2 + Runx1 + Acta2 + cells. g WT NG2 + Runx1 + cells arranged in pseudotime (x-axis) based on the inferred curve. Y-axis represents log normalised gene expression.

Article Snippet: Embryos were stained for several days with unconjugated cKit (1:500, BD Bioscience, 553352), biotinylated CD31 (1:500, BD Pharmingen, 553371), unconjugated NG2 (1:500, R&D Systems, MAB6689), Runx1,2,3 (1:250, Abcam, ab92336) and αSMA FITC (1:500, Sigma, F3777).

Techniques: Expressing, Gene Expression

Journal: Nature Communications

Article Title: Runx1+ vascular smooth muscle cells are essential for hematopoietic stem and progenitor cell development in vivo

doi: 10.1038/s41467-024-44913-z

Figure Lengend Snippet: a t-SNE plot showing eight populations of interest found in the E11 cKO AGM. Each dot represents one cell and colours represent cell clusters as indicated. MP (macrophages); Ery/EryP (erythroid/progenitors); IAHC (intra-aortic hematopoietic clusters); HEC/EHT (hemogenic endothelial cells including those that enter endothelial-to-hematopoietic transition), EC (endothelial cells); SNS (sympathetic nervous system); SkMP (skeletal muscle progenitors); PC/vSMC (pericytes/vascular smooth muscle cells, NG2 + Acta2 + ) . Other cells (OC) are coloured in grey. The number of cells in each cluster is shown in brackets. b t-SNE plot highlighting the eight populations identified after excluding all other (grey) cells. c Percentage of single live cells found in each E11 AGM sample (cell number/total cells) defined by scRNA-seq in WT (full bars) and cKO (empty bars) AGMs. Colours and numbers correspond to each population defined in a ; chi-squared two-tailed test was used for comparison. d Barplot of fold enrichment for selected GO biological processes significantly overrepresented in genes significantly downregulated in cKO PC/vSMCs compared to their WT counterparts. Heatmap of ligand-receptor interactions inferred by NicheNet from e WT and f cKO E11 AGM cells. Colour represents the interaction potential score between the 10 top-ranked ligands expressed in ECs and their inferred targets expressed in PC/vSMCs. Ligands and receptors are ordered by hierarchical clustering. g Scatter plots of AUC vs –log10(FDR) showing downregulated genes associated with selected GO terms in cKO PC/vSMCs. Red dots represent significantly downregulated genes (FDR<0.05); dashed line shows FDR = 0.05. Gene labels with red borders represent potential Runx1 target genes.

Article Snippet: Embryos were stained for several days with unconjugated cKit (1:500, BD Bioscience, 553352), biotinylated CD31 (1:500, BD Pharmingen, 553371), unconjugated NG2 (1:500, R&D Systems, MAB6689), Runx1,2,3 (1:250, Abcam, ab92336) and αSMA FITC (1:500, Sigma, F3777).

Techniques: Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Runx1+ vascular smooth muscle cells are essential for hematopoietic stem and progenitor cell development in vivo

doi: 10.1038/s41467-024-44913-z

Figure Lengend Snippet: a , b Representative plots and percentages of Lin - Sca1 + cKit + (LSK) and c , d LSK CD150 + CD48 - (SLAM) bone marrow (BM) cells by flow cytometry of WT/ NG2+/+;Runx1 fl/+ ,NG2+/+;Runx1 fl/fl ( n = 9), HET NG2-Cre;Runx1 fl/+ ( n = 4) and cKO NG2-Cre;Runx1 fl/fl ( n = 4) adult mice is shown. e Colony-forming unit-culture (CFU-C) numbers per 10 adult BM cells; n = WT/HET/cKO: 13/7/8 mice. N = 7 independent experiments. Data are mean ± SEM (Table ). f Hematopoietic stem cell repopulating potential and donor chimerism of WT and mutant BM cells in vivo. 5 × 10 5 BM donor WT, HET and cKO cells were injected into 29, 11 and 20 Ly5.1 HET recipients, respectively, with 18, 3 and 4 found to be reconstituted respectively (Table S3, p = 0.024 (WT/HET) and p = 0.002 (WT/cKO) by Z score test for 2 population proportions). Mice are reconstituted when ≥5% donor cells are found in the host peripheral blood; p = 0.002 (WT/cKO) by Kruskal-Wallis and Dunn’s post-hoc test (Table ). g Histograms showing the contribution of CD45.2 + CD45.1 - donor cells to myeloid cells (CD11b + Gr1 +/- ), B cells (CD19 + ) and T cells (CD4/8 + ) in all reconstituted host mice from ( f ). ( n = WT/HET/cKO = 18/3/4), p = 0.019 (WT/HET) for B cells by one-way ANOVA and Tukey’s post-hoc test. h BM cells from selected reconstituted primary recipients (found in f ) were transplanted into multiple irradiated secondary recipients. Mice are reconstituted when ≥5% donor cells are found in the host peripheral blood (Table – ). i Representative flow cytometric analysis plot of NG2 in Runx1-IRES-GFP adult BM ( n = 6). All data are presented as Mean values+/-SEM. N = number of independent experiments; n = number of biological samples. Source data for b , d , e , f , g and h are provided as a file.

Article Snippet: Embryos were stained for several days with unconjugated cKit (1:500, BD Bioscience, 553352), biotinylated CD31 (1:500, BD Pharmingen, 553371), unconjugated NG2 (1:500, R&D Systems, MAB6689), Runx1,2,3 (1:250, Abcam, ab92336) and αSMA FITC (1:500, Sigma, F3777).

Techniques: Flow Cytometry, Mutagenesis, In Vivo, Injection, Irradiation

Journal: Nature Communications

Article Title: Runx1+ vascular smooth muscle cells are essential for hematopoietic stem and progenitor cell development in vivo

doi: 10.1038/s41467-024-44913-z

Figure Lengend Snippet: a Three-dimensional (3D) wholemount immunostaining with αSMA, CD31 and NG2 of E10.5 (31–38 somite pairs (sp)) WT dorsal aorta; b NG2 and Runx1 expression on single plane wholemount WT E10.5 sections. NG2 + Runx1 + vSMCs (arrows), hemogenic endothelial cells (arrowheads) and intra-aortic hematopoietic clusters (IAHCs, stars) (Table ); c Representative example of flow cytometric analysis of NG2 + Runx1(GFP) + (green box) in E10.5 Runx1-IRES-GFP AGM and E10.5 WT control. d Percentages of NG2 + Runx1(GFP) + cells in E9 (21-25sp) body ( n = 6), E10/E10.5/E11 AGMs ( n = 8/7/7), N = 5, Kruskal-Wallis and Dunn’s post-hoc test. e Representative examples of wholemount 3D-images showing αSMA, CD31 and NG2 in E10.5 cKO dorsal aortae; f αSMA, Runx1 and CD31 immunofluorescence of E11 WT and cKO transversal frozen sections; n = WT/cKO: 2/2, N = 2. g cKit and CD31 wholemount 3D-images in E10.5 WT and cKO AGM; h Number of intra-aortic hematopoietic clusters (IAHCs) in E10.5 AGM; n = WT/KO: 5/4, N = 4. Number of colony forming unit-culture (CFU-C) in i E10.5 (31-38sp) AGM; n = WT/HET/KO: 14/10/5 embryos; N = 7 and j E11 (43–52sp) AGM; n = WT/HET/KO: 22/8/19 embryos; N = 11; one-way ANOVA and Tukey’s post-hoc test (Table ). k Percentages of donor cell chimerism 4-months post-transplantation of 6 E11 WT (NG2 +/+ ;Runx1 fl/+ or NG2 +/+ ;Runx1 fl/fl ) , 7 HET ( NG2-Cre;Runx1 fl/+ ) and 6 cKO AGMs ( NG2-Cre;Runx1 fl/fl ) into sub-lethally adult irradiated recipients (1xAGM cells transplanted/recipient; N = 4). Each dot represents one recipient. Mice are reconstituted when ≥5% donor cells are found in the host peripheral blood (dashed line) ; one-tailed Z score test for two population proportions (Tables and ). For wholemount staining in a , b , e , g : WT/cKO ( N = 6/4): αSMA ( n = 9/7), CD31 ( n = 10/7), cKit ( n = 3/2), NG2 ( n = 3/1) and WT Runx1 ( n = 4) in 3 distinct combinations (Table ). D = dorsal, V = ventral. N = number of independent experiments; n = number of biological samples (embryos). All data are presented as mean values ± SEM. Source data for d , h , i , j and k are provided as a file.

Article Snippet: The following unconjugated primary antibodies were incubated overnight at 4 °C: NG2 (1:100, Rabbit polyclonal, Millipore, ab5320), NG2 (1:50, Rat anti-mouse, R&D systems, MAB6689), Rabbit anti-RFP to detect TdTomato (1:100, Rockland, 600-401-379), CD45 (1:100, Goat anti-mouse, R&D systems, AF114), F4/80 (1:50, Rat anti-mouse, Abcam, ab6640), and Runx1,2,3 (1:100, Rabbit anti-mouse, Abcam, ab92336).

Techniques: Immunostaining, Expressing, Control, Immunofluorescence, Transplantation Assay, Irradiation, One-tailed Test, Staining

Journal: Nature Communications

Article Title: Runx1+ vascular smooth muscle cells are essential for hematopoietic stem and progenitor cell development in vivo

doi: 10.1038/s41467-024-44913-z

Figure Lengend Snippet: a t-SNE plot highlighting eight populations of interest identified in the E11 WT AGM. Each dot represents one cell and colours represent cell clusters as indicated. The number of cells in each population is shown in brackets. MP (macrophages); Ery/EryP (erythroid/progenitors); IAHC (intra-aortic hematopoietic clusters); HEC/EHT (hemogenic endothelial cells including those that enter endothelial-to-hematopoietic transition); EC (endothelial cells); SNS (sympathetic nervous system); SkMP (skeletal muscle progenitors), PC/vSMC (pericytes/vascular smooth muscle cells, NG2 + Acta2 + ). Other cells (OC) are coloured in grey. b t-SNE plot highlighting the eight populations identified after excluding all other (grey) cells. c Zoom into PC/vSMC cluster (black rectangle) further show the presence or the absence of selected genes that characterise this population and confirms the presence of Runx1 in a subset of cells. d Violin plots showing distribution of expression for selected genes that contributed to the identification of cell clusters. Immunohistochemistry on frozen E11 WT sections stained with e CD146/CD31/DAPI and f CD146/αSMA/DAPI, n = 2 samples tested, N = 2 independent experiments. Arrows: vascular cells, asterisks: perivascular cells. DA: dorsal aorta, CV: cardinal veins, NC: notochord. Source data for e (first column, 20X) is provided as a file.

Article Snippet: The following unconjugated primary antibodies were incubated overnight at 4 °C: NG2 (1:100, Rabbit polyclonal, Millipore, ab5320), NG2 (1:50, Rat anti-mouse, R&D systems, MAB6689), Rabbit anti-RFP to detect TdTomato (1:100, Rockland, 600-401-379), CD45 (1:100, Goat anti-mouse, R&D systems, AF114), F4/80 (1:50, Rat anti-mouse, Abcam, ab6640), and Runx1,2,3 (1:100, Rabbit anti-mouse, Abcam, ab92336).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Nature Communications

Article Title: Runx1+ vascular smooth muscle cells are essential for hematopoietic stem and progenitor cell development in vivo

doi: 10.1038/s41467-024-44913-z

Figure Lengend Snippet: a t-SNE plots showing the distribution of Runx1 and Acta2 expression in NG2 + Runx1 + cells in the WT E11 AGM after excluding all other (grey) cells found in the Fig. . b Zoom into NG2 + Runx1 + cluster (black rectangle) shows the presence or the absence of Acta2 . c Heatmap showing the expression of Cspg4 and Runx1 and 15 selected genes out of 25 top significantly upregulated genes in WT NG2 + Runx1 + Acta2 + cells (upper half) and NG2 + Runx1 + Acta2 - cells (bottom half) at single cell level; *Runx1 potential target genes. Pdgfra and Ptn genes were next added to inform their expression in both populations. Barplot of fold enrichment for selected GO biological processes significantly overrepresented in genes significantly upregulated in both d WT NG2 + Runx1 + Acta2 + and e NG2 + Runx1 + Acta2 - cells. f t-SNE of WT E11 AGM cells, overlaid with principal pseudotime curve inferred by Slingshot, predicting a lineage from NG2 + Runx1 + Acta2 - cells to NG2 + Runx1 + Acta2 + cells. g WT NG2 + Runx1 + cells arranged in pseudotime (x-axis) based on the inferred curve. Y-axis represents log normalised gene expression.

Article Snippet: The following unconjugated primary antibodies were incubated overnight at 4 °C: NG2 (1:100, Rabbit polyclonal, Millipore, ab5320), NG2 (1:50, Rat anti-mouse, R&D systems, MAB6689), Rabbit anti-RFP to detect TdTomato (1:100, Rockland, 600-401-379), CD45 (1:100, Goat anti-mouse, R&D systems, AF114), F4/80 (1:50, Rat anti-mouse, Abcam, ab6640), and Runx1,2,3 (1:100, Rabbit anti-mouse, Abcam, ab92336).

Techniques: Expressing, Gene Expression

Journal: Nature Communications

Article Title: Runx1+ vascular smooth muscle cells are essential for hematopoietic stem and progenitor cell development in vivo

doi: 10.1038/s41467-024-44913-z

Figure Lengend Snippet: a t-SNE plot showing eight populations of interest found in the E11 cKO AGM. Each dot represents one cell and colours represent cell clusters as indicated. MP (macrophages); Ery/EryP (erythroid/progenitors); IAHC (intra-aortic hematopoietic clusters); HEC/EHT (hemogenic endothelial cells including those that enter endothelial-to-hematopoietic transition), EC (endothelial cells); SNS (sympathetic nervous system); SkMP (skeletal muscle progenitors); PC/vSMC (pericytes/vascular smooth muscle cells, NG2 + Acta2 + ) . Other cells (OC) are coloured in grey. The number of cells in each cluster is shown in brackets. b t-SNE plot highlighting the eight populations identified after excluding all other (grey) cells. c Percentage of single live cells found in each E11 AGM sample (cell number/total cells) defined by scRNA-seq in WT (full bars) and cKO (empty bars) AGMs. Colours and numbers correspond to each population defined in a ; chi-squared two-tailed test was used for comparison. d Barplot of fold enrichment for selected GO biological processes significantly overrepresented in genes significantly downregulated in cKO PC/vSMCs compared to their WT counterparts. Heatmap of ligand-receptor interactions inferred by NicheNet from e WT and f cKO E11 AGM cells. Colour represents the interaction potential score between the 10 top-ranked ligands expressed in ECs and their inferred targets expressed in PC/vSMCs. Ligands and receptors are ordered by hierarchical clustering. g Scatter plots of AUC vs –log10(FDR) showing downregulated genes associated with selected GO terms in cKO PC/vSMCs. Red dots represent significantly downregulated genes (FDR<0.05); dashed line shows FDR = 0.05. Gene labels with red borders represent potential Runx1 target genes.

Article Snippet: The following unconjugated primary antibodies were incubated overnight at 4 °C: NG2 (1:100, Rabbit polyclonal, Millipore, ab5320), NG2 (1:50, Rat anti-mouse, R&D systems, MAB6689), Rabbit anti-RFP to detect TdTomato (1:100, Rockland, 600-401-379), CD45 (1:100, Goat anti-mouse, R&D systems, AF114), F4/80 (1:50, Rat anti-mouse, Abcam, ab6640), and Runx1,2,3 (1:100, Rabbit anti-mouse, Abcam, ab92336).

Techniques: Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Runx1+ vascular smooth muscle cells are essential for hematopoietic stem and progenitor cell development in vivo

doi: 10.1038/s41467-024-44913-z

Figure Lengend Snippet: a , b Representative plots and percentages of Lin - Sca1 + cKit + (LSK) and c , d LSK CD150 + CD48 - (SLAM) bone marrow (BM) cells by flow cytometry of WT/ NG2+/+;Runx1 fl/+ ,NG2+/+;Runx1 fl/fl ( n = 9), HET NG2-Cre;Runx1 fl/+ ( n = 4) and cKO NG2-Cre;Runx1 fl/fl ( n = 4) adult mice is shown. e Colony-forming unit-culture (CFU-C) numbers per 10 adult BM cells; n = WT/HET/cKO: 13/7/8 mice. N = 7 independent experiments. Data are mean ± SEM (Table ). f Hematopoietic stem cell repopulating potential and donor chimerism of WT and mutant BM cells in vivo. 5 × 10 5 BM donor WT, HET and cKO cells were injected into 29, 11 and 20 Ly5.1 HET recipients, respectively, with 18, 3 and 4 found to be reconstituted respectively (Table S3, p = 0.024 (WT/HET) and p = 0.002 (WT/cKO) by Z score test for 2 population proportions). Mice are reconstituted when ≥5% donor cells are found in the host peripheral blood; p = 0.002 (WT/cKO) by Kruskal-Wallis and Dunn’s post-hoc test (Table ). g Histograms showing the contribution of CD45.2 + CD45.1 - donor cells to myeloid cells (CD11b + Gr1 +/- ), B cells (CD19 + ) and T cells (CD4/8 + ) in all reconstituted host mice from ( f ). ( n = WT/HET/cKO = 18/3/4), p = 0.019 (WT/HET) for B cells by one-way ANOVA and Tukey’s post-hoc test. h BM cells from selected reconstituted primary recipients (found in f ) were transplanted into multiple irradiated secondary recipients. Mice are reconstituted when ≥5% donor cells are found in the host peripheral blood (Table – ). i Representative flow cytometric analysis plot of NG2 in Runx1-IRES-GFP adult BM ( n = 6). All data are presented as Mean values+/-SEM. N = number of independent experiments; n = number of biological samples. Source data for b , d , e , f , g and h are provided as a file.

Article Snippet: The following unconjugated primary antibodies were incubated overnight at 4 °C: NG2 (1:100, Rabbit polyclonal, Millipore, ab5320), NG2 (1:50, Rat anti-mouse, R&D systems, MAB6689), Rabbit anti-RFP to detect TdTomato (1:100, Rockland, 600-401-379), CD45 (1:100, Goat anti-mouse, R&D systems, AF114), F4/80 (1:50, Rat anti-mouse, Abcam, ab6640), and Runx1,2,3 (1:100, Rabbit anti-mouse, Abcam, ab92336).

Techniques: Flow Cytometry, Mutagenesis, In Vivo, Injection, Irradiation

Journal: Biosensors & bioelectronics

Article Title: Tracking the EMT-like phenotype switching during targeted therapy in melanoma by analyzing extracellular vesicle phenotypes.

doi: 10.1016/j.bios.2023.115819

Figure Lengend Snippet: Fig. 1. Working scheme of tracking EMT-like phenotype switching during MAPKi therapy. (A) Melanoma cells undergo EMT-like phenotype switching in response to MAPKi therapy and secret EVs into blood circulation. (B) Serum EVs are captured by anti-MCSP and anti-MCAM antibodies immobilized on an electrode and subsequently labeled with SERS nanotags against EMT-associated biomarkers N-cadherin, E-cadherin, THBS1 and ABCB5 under the applied ac-EHD field. (C) EV phenotypes are characterized by SERS mapping. Under the laser excitation, SERS nanotags with MBA, TFMBA, DTNB, and MPY molecules generate characteristic peaks at 1075, 1375, 1335, and 1005 cm−1, respectively. The false-color SERS spectral images are established based on the characteristic Raman signals of four SERS nanotags, indicating the expression of four biomarkers (N-cadherin-MBA, red; E-cadherin-TFMBA, blue; THBS1-DTNB, green; ABCB5-MPY, yellow). The expression levels of four target biomarkers are determined by calculating the average signal spectra of false-color SERS spectral images. (D) EMT-associated EV phenotypic evolution in response to MAPKi treatment.

Article Snippet: The primary antibodies were mouse anti-human MCSP (R&D Systems, MAB2585) and MCAM (R&D Systems, MAB932) antibodies.

Techniques: Labeling, Expressing

Journal: Biosensors & bioelectronics

Article Title: Tracking the EMT-like phenotype switching during targeted therapy in melanoma by analyzing extracellular vesicle phenotypes.

doi: 10.1016/j.bios.2023.115819

Figure Lengend Snippet: Fig. 3. Specificity of the SERS-based microfluidic biosensor. Representative SERS false-color spectral images and average SERS intensities of EVs derived from SK-MEL-28 (high expression of MCSP and MCAM) and MCF7 (low expression of MCSP and MCAM) cell lines, and control experiments including (++) PBS, (-+) without capture antibodies and (+-) with nontarget IgG antibody on SERS nanotags. EV biomarkers are represented by red (N-cadherin), blue (E-cadherin), green (THBS1) and yellow (ABCB5). Average SERS intensities were generated at 1075 cm−1 (N-cadherin), 1375 cm−1 (E-cadherin), 1335 cm−1 (THBS1), and 1005 cm−1

Article Snippet: The primary antibodies were mouse anti-human MCSP (R&D Systems, MAB2585) and MCAM (R&D Systems, MAB932) antibodies.

Techniques: Derivative Assay, Expressing, Control, Generated

Journal: Biosensors & bioelectronics

Article Title: Tracking the EMT-like phenotype switching during targeted therapy in melanoma by analyzing extracellular vesicle phenotypes.

doi: 10.1016/j.bios.2023.115819

Figure Lengend Snippet: Fig. 2. Characterization of EVs derived from melanoma (SK-MEL-28) and breast cancer (MCF7) cell lines. (A) The expression of canonical tetraspanin CD63 and (B) size distributions of SK-MEL-28 and MCF7 EVs using nanoflow cytometry. (C) The expression of melanoma-specific biomarkers MCSP and MCAM on SK-MEL- 28 and MCF7 EVs, measured by western blot.

Article Snippet: The primary antibodies were mouse anti-human MCSP (R&D Systems, MAB2585) and MCAM (R&D Systems, MAB932) antibodies.

Techniques: Derivative Assay, Expressing, Cytometry, Western Blot