unconjugated ng2  (R&D Systems)

Journal: Nature

Article Title: Frizzled are colonic epithelial receptors for Clostridium difficile toxin B

doi: 10.1038/nature19799

Figure Lengend Snippet: a, The sensitivities of the indicated HeLa KO cells to TcdB and TcdB 1-1830 were quantified using the cytopathic cell-rounding assay (see ) and normalized to WT HeLa cells as fold-of-resistance. The experiments have been repeated three times. b–c, Immunostaining analysis showed that TcdB binding (10 nM, 10 min) to CSPG4 −/− cells was reduced ( b ). Ectopic expression of NG2 (rat CSPG4) increased binding of TcdB. Transfection of FZD2 also increased TcdB binding to CSPG4 −/− cells ( c ). Scale bar = 20 μm. DIC: differential interference contrast. d, Ectopic expression of CSPG4 or FZD2 restored TcdB entry into CSPG4 −/− cells, resulting in cell-rounding (5 pM, 3 h). GFP marked transfected cells. Scale bar = 50 μm. e, A schematic illustration of FZD (upper panel). Fc-tagged FZD2-CRD binds to GST-tagged TcdB 1501-2366 , but not to GST-tagged CROPs. f–g, FZD2-CRD prevented TcdB (300 pM, 3 h) entry into CSPG4 −/− cells, measured by the cell-rounding assay ( f ) and glucosylation of Rac1 ( g ). Human IgG1-Fc (hIgG1-Fc) is a control. h, Transfection of FZD1, 2, and 7 each increased TcdB binding (10 nM, 10 min) to CSPG4 −/− cells, assayed by immunoblot analysis of cell lysates. Actin is a loading control. i, The sensitivities of FZD1 −/− , FZD2 −/− , FZD7 −/− , and FZD1/2/7 −/− cells to TcdB and TcdB 1-1830 were analyzed as described in panel a. j, Ectopic expression of FZD1, 2, or 7 restored TcdB 1-1830 entry into FZD1/2/7 −/− cells (300 pM, 3 h). Scale bar = 50 μm. k, Characterization of TcdB binding to Fc-tagged CRDs of FZD1, 2, 5, and 7 using the bio-layer interferometry (BLI) assay (see for K D analysis). Representative images are from one of three independent experiments. Error bars indicate mean ± s.d., n = 6, *P < 0.005, t -test.

Article Snippet: The colonic sections were then blocked with 5% goat serum in PBS for 30 min at room temperature and incubated with primary antibodies overnight (anti-TcdB: 1:600; anti-FZDs: 1:250; rabbit anti-NG2: 1:250), followed with biotinylated goat anti-chicken or rabbit IgG secondary antibodies (1:200, Vector Lab) for 1 h at room temperature.

Techniques: Immunostaining, Binding Assay, Expressing, Transfection, Western Blot

Journal: Nature

Article Title: Frizzled are colonic epithelial receptors for Clostridium difficile toxin B

doi: 10.1038/nature19799

Figure Lengend Snippet: a, Schematic drawings of NG2 (rat CSPG4). Two fractions of recombinant extracellular domain (EC) fragments were used: one that does not contain chondroitin sulfate (CS) chains (EC P1), and the other that contains CS (EC P2). TMD-cyto: transmembrane and cytoplasmic domain. b, TcdB, but not TcdB 1-1830 , binds directly to both EC P1 and EC P2 of NG2 in a microtiter plate-based binding assay (error bars indicate mean ± s.d., two independent experiments). c, CSPG4 −/− cells transfected with the indicated constructs were exposed to TcdB (10 nM), TcdB 1-1830 (10 nM), or the receptor-binding domain of botulinum neurotoxin B (BoNT/B, 100 nM) for 10 min. Cell lysates were collected and subjected to immunoblot analysis. IL1RAPL2 and synaptotagmin II (Syt II, a receptor for BoNT/B) served as controls. Transfection of NG2 increased binding of TcdB, but not TcdB 1-1830 , whereas transfection of FZD2 increased binding of both TcdB and TcdB 1-1830 . One of three independent experiments is shown. d, The CROPs domain binds to CSPG4 on cell surfaces in a dose-dependent manner. High concentrations of recombinant CROPs reduced CSPG4-dependent binding of TcdB to cell surfaces, indicating that the CROPs can compete with TcdB for binding to CSPG4 on cell surfaces. One of three independent experiments is shown. e, The CROPs domain reduced cytopathic toxicity of TcdB (5 pM) on WT HeLa cells (error bars indicate mean ± s.d., two independent experiments). f, CSPG4 −/− cells were transfected with FZD2 and then exposed to TcdB or indicated TcdB fragments. FZD2 mediated binding of TcdB, TcdB 1-1830 , and TcdB 1501-2366 , but not the CROPs (TcdB 1831-2366 ). One of three independent experiments is shown.

Article Snippet: The colonic sections were then blocked with 5% goat serum in PBS for 30 min at room temperature and incubated with primary antibodies overnight (anti-TcdB: 1:600; anti-FZDs: 1:250; rabbit anti-NG2: 1:250), followed with biotinylated goat anti-chicken or rabbit IgG secondary antibodies (1:200, Vector Lab) for 1 h at room temperature.

Techniques: Recombinant, Binding Assay, Transfection, Construct, Western Blot

Journal: Nature

Article Title: Frizzled are colonic epithelial receptors for Clostridium difficile toxin B

doi: 10.1038/nature19799

Figure Lengend Snippet: a, NG2-EC was immobilized on microtiter plates, followed by binding of TcdB, washing away unbound TcdB, and addition of FZD-CRD. FZD2-CRD binds robustly to TcdB that is pre-bound by NG2-EC on the microtiter plate. FZD2-CRD did not bind to NG2-EC without TcdB, and FZD5-CRD showed no detectable binding to NG2-TcdB in this assay (error bars indicate mean ± s.d., two independent experiments). b, Experiments are described in on HeLa (5 pM TcdB), HT-29 (50 pM TcdB), and Caco-2 cells (150 pM TcdB). Scale bars = 50 μm (HeLa and Caco-2) or 25 μm (HT-29). Representative images are from one of four independent experiments.

Article Snippet: The colonic sections were then blocked with 5% goat serum in PBS for 30 min at room temperature and incubated with primary antibodies overnight (anti-TcdB: 1:600; anti-FZDs: 1:250; rabbit anti-NG2: 1:250), followed with biotinylated goat anti-chicken or rabbit IgG secondary antibodies (1:200, Vector Lab) for 1 h at room temperature.

Techniques: Binding Assay

Journal: Nature

Article Title: Frizzled are colonic epithelial receptors for Clostridium difficile toxin B

doi: 10.1038/nature19799

Figure Lengend Snippet: a–b, FZD2-CRD protected HT-29 ( a ) and Caco-2 cells ( b ) from TcdB 1-1830 (300 pM, 3 h). Representative images are from one of three independent experiments. Scale bars = 25 μm ( a ) or 50 μm ( b ). c, Expression of CSPG4 in HeLa, HT-29, and Caco-2 cells was examined via immunoblot analysis of cell lysates. One experiment from four is shown. d, Protection from TcdB using FZD2-CRD and NG2-EC on HeLa (5 pM TcdB), HT-29 (50 pM TcdB), and Caco-2 (150 pM TcdB) cells was quantified by the cytopathic cell-rounding assay. Representative images are shown in . Error bars indicate mean ± s.d..

Article Snippet: The colonic sections were then blocked with 5% goat serum in PBS for 30 min at room temperature and incubated with primary antibodies overnight (anti-TcdB: 1:600; anti-FZDs: 1:250; rabbit anti-NG2: 1:250), followed with biotinylated goat anti-chicken or rabbit IgG secondary antibodies (1:200, Vector Lab) for 1 h at room temperature.

Techniques: Expressing, Western Blot

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: Cell Reports

Article Title: PDGFRβ + cells play a dual role as hematopoietic precursors and niche cells during mouse ontogeny

doi: 10.1016/j.celrep.2022.111114

Figure Lengend Snippet: Distinct phenotypic and transcriptomic perivascular cell subsets surround the midgestation DA (A) Immunohistochemistry of E11 WT DA, showing NG2 (i, ii), PDGFRβ (i, ii), and CD31 (ii) expression. Nuclei were counterstained with DAPI. CV, cardinal veins; NC, notochord. (B) t-distributed stochastic neighbor embedding (t-SNE) plots showing 12 E11 WT cell populations and their numbers (42sp). (C) Violin plots showing the expression of genes used to identify the cell clusters DP, PDGFRβ-S, DN, and NG2-S. MP, macrophages; OBC, other blood cells; Ery/EryP, erythroid/progenitors; IAHC, intra-aortic hematopoietic clusters; HEC/EHT, hemogenic endothelial cells/endothelial-to-hematopoietic transition; EC, endothelial cells; SNS, sympathetic nervous system; SkMP, skeletal muscle progenitors. (D and E) Heatmaps showing gene expression of differentially expressed genes encoding surface (D) and intra-/extracellular (E) proteins enriched in each of the NG2 +/− PDGFRβ +/− populations. (F) Isolation of perivascular cells. (G) Fragments per kilobase of transcript per million mapped reads (FPKM) values of selected genes by bulk RNA-seq.

Article Snippet: Briefly, embryos were stained with unconjugated cKit (1:500; BD Bioscience; 553352) or unconjugated NG2 (1:500; Millipore; ab5320) together with biotinylated CD31 (1:500 BD Pharmingen; 553371).

Techniques: Immunohistochemistry, Expressing, Gene Expression, Isolation, RNA Sequencing

Journal: Cell Reports

Article Title: PDGFRβ + cells play a dual role as hematopoietic precursors and niche cells during mouse ontogeny

doi: 10.1016/j.celrep.2022.111114

Figure Lengend Snippet: A subset of ECs, HEC/EHT, and IAHCs derived from PDGFRβ + precursors (A–D) Immunohistochemistry of E11 PDGFRβ-Cre:mTmG AGM stained with NG2 (A and B) and CD31 (C) and at E10 with Runx1 (D). (A and B) Arrowheads: DP cells; stars: GFP + mesenchymal cells. (C) Dashed lines: presumptive separation between DP, PDGFRβ-S, and DN layers; stars: hematopoietic CD31 + GFP + cells. (E–I) Flow cytometry analyses of E10 (n = 7) and E11 (n = 3) PDGFRβ-Cre; tdTomato AGMs, percentage of Tomato +/− cells within (E) PDGFRβ + cells, (F) EC (CD45 − cKit − CD31 + CD41 − ), (G) HEC/EHT-enriched population (CD45 − cKit − CD31 + CD41 + ), (H) IAHC/HSPC (CD31 + cKit + ), and (I) MP-enriched population (CD45 + CD31 − ) live cells; error bars: SD; ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.005, ∗∗∗∗p < 0.0005. (J) CFU-Cs on PDGFRβ +/− cells sorted from E10 (n = 8) and E11 (n = 6) AGMs (see Table S6 ). E11 AGM were ckit + ; error bars: SD; ∗∗∗p < 0.005, ∗∗∗∗p < 0.0005. (K) CFU-Cs on Tomato +/− cells sorted from PDGFRβ-Cre; tdTomato E10 (n = 6) and E11 (n = 4) AGMs (see Table S7 ); error bars: SD; ∗∗∗p < 0.005. (L) PCR on E11 unsorted cells from PDGFRβ-Cre; tdTomato and +; tdTomato WT littermates heads and from sorted AGM cells to detect Cre recombinase expression (324 bp). Unpaired t tests or Mann-Whitney tests were used for the statistics.

Article Snippet: Briefly, embryos were stained with unconjugated cKit (1:500; BD Bioscience; 553352) or unconjugated NG2 (1:500; Millipore; ab5320) together with biotinylated CD31 (1:500 BD Pharmingen; 553371).

Techniques: Derivative Assay, Immunohistochemistry, Staining, Flow Cytometry, Expressing, MANN-WHITNEY

Journal: Cell Reports

Article Title: PDGFRβ + cells play a dual role as hematopoietic precursors and niche cells during mouse ontogeny

doi: 10.1016/j.celrep.2022.111114

Figure Lengend Snippet:

Article Snippet: Briefly, embryos were stained with unconjugated cKit (1:500; BD Bioscience; 553352) or unconjugated NG2 (1:500; Millipore; ab5320) together with biotinylated CD31 (1:500 BD Pharmingen; 553371).

Techniques: Recombinant, Blocking Assay, RNA Sequencing, Software