Induction of osteogenesis by bone-targeted Notch activation
- Max Planck Institute for Molecular Biomedicine, Department of Tissue Morphogenesis, and University of Münster, Faculty of Medicine, Germany
- Flow Cytometry Unit, Max Planck Institute for Molecular Biomedicine, Germany
- Embryonic Self-Organization Research Group, Max Planck Institute for Molecular Biomedicine, Germany
Figures
Figure 1 with 5 supplements
Recombinant Dll4(E12) increases bone formation in vivo.
(A) Schematic diagram showing the domain organization of murine Dll4 full-length protein and recombinant Dll4-Asp8x and Dll4(E12) fusion proteins. The latter contain 6x His epitope tags (green …
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Figure 1—source data 1
Source data for Figure 1E and F.
- https://cdn.elifesciences.org/articles/60183/elife-60183-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Generation of Dll4-Asp8x recombinant proteins.
(A) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified Dll4-Asp8x recombinant proteins. From left to right, protein molecular mass marker (Marker), extract of …
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Figure 1—figure supplement 1—source data 1
Source data for Figure 1—figure supplement 1B,C.
- https://cdn.elifesciences.org/articles/60183/elife-60183-fig1-figsupp1-data1-v2.xlsx
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Figure 1—figure supplement 1—source data 2
Images of uncropped blots and gels with the relevant bands labeled for Figure 1—figure supplement 1, Figure 1—figure supplement 4, and Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/60183/elife-60183-fig1-figsupp1-data2-v2.pdf
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Figure 1—figure supplement 1—source data 3
ZIP file containing the full raw unedited blots and gels for Figure 1—figure supplement 1, Figure 1—figure supplement 4, and Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/60183/elife-60183-fig1-figsupp1-data3-v2.zip
Figure 1—figure supplement 2
Dll4-Asp8x detection in bone.
(A, B) Confocal images showing Dll4 (cyan) and Emcn (red) staining in femoral metaphysis (A) and endosteum and cortical bone (B) of pLIVE-Dll4-Asp8x and control-injected 11-week-old mice. Nuclei, …
Figure 1—figure supplement 3
Bone formation is not increased by Dll4-Asp8x.
(A) Tile scan images of Dll4 (cyan) staining in sections of pLIVE-Dll4-Asp8x and control femur. Nuclei, DAPI (blue). Images on the right show higher magnifications of insets in metaphysis (1), bone …
Figure 1—figure supplement 4
Generation of Dll4(E12).
(A) Diagram showing the amino acid sequence of mouse and rat Dll4 extracellular domain (ECD). Amino acid replacements shown in red were introduced into the murine sequence for the generation of Dll4(…
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Figure 1—figure supplement 4—source data 1
Source data for Figure 1—figure supplement 1C,D.
- https://cdn.elifesciences.org/articles/60183/elife-60183-fig1-figsupp4-data1-v2.xlsx
Figure 1—figure supplement 5
Dll4(E12) immunoreactivity in vivo.
(A–C) Representative confocal images showing Dll4(E12) immunoreactivity after pLIVE-Dll4(E12) or control injection in liver (A), spleen, (B) and lung (C). Dll4(E12) signal (green) is prominent in …
Figure 2 with 2 supplements
Notch-dependent processes are not blocked by Dll4(E12)in vivo.
(A) Alcian blue and nuclear fast red double staining of small intestine. Secretory goblet cells are labeled by Alcian blue. (B) Hematoxylin and eosin staining of liver sections of 11-week-old pLIVE-D…
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Figure 2—source data 1
Source data for Figure 2D and F.
- https://cdn.elifesciences.org/articles/60183/elife-60183-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Inflammatory cytokines are not induced by Dll4(E12).
Quantification of inflammatory cytokines in the plasma of pLIVE-Dll4(E12)- and control-injected mice. Data represent mean ± s.e.m. (n = 3 mice) (p-values determined by multiple unpaired t-test with …
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Figure 2—figure supplement 1—source data 1
Source data for Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/60183/elife-60183-fig2-figsupp1-data1-v2.xlsx
Figure 2—figure supplement 2
Dll4(E12) does not induce vascular leakage.
(A–C) Representative confocal images and corresponding quantitation showing comparable extravasation of Texas Red-coupled fluorescent Dextran (purple) in liver (A), spleen, (B) and lung (C) of pLIVE-…
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Figure 2—figure supplement 2—source data 1
Source data for Figure 2—figure supplement 2A-C.
- https://cdn.elifesciences.org/articles/60183/elife-60183-fig2-figsupp2-data1-v2.xlsx
Figure 3 with 2 supplements
Recombinant Dll4(E12) increases osteogenesis.
(A) Tile scan confocal images of Osterix (OSX) staining (green) in femurs from pLIVE-Dll4(E12) and control-injected mice. Nuclei, DAPI (blue). Graph shows quantitation of OSX+ cells. Data represent …
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Figure 3—source data 1
Source data for Figure 3A–E.
- https://cdn.elifesciences.org/articles/60183/elife-60183-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Activation of the Hey1-EGFP reporter by Dll4(E12).
Expression of the Tg(Hey1-EGFP)ID40Gsat reporter (green fluorescent protein [GFP], green) in 11-week-old pLIVE-Dll4(E12) and control femur. Note that Dll4(E12) increases the number of Osterix+ (OSX+)…
Figure 3—figure supplement 2
Effect of Dll4(E12) on metaphyseal mesenchymal stromal cells (MSCs) and chondrocytes.
(A, B) Tile scan confocal images showing that PDGFRβ (A) and NG2 (B) immunostaining in the adult metaphysis is increased by recombinant Dll4(E12). Nuclei, DAPI (blue). Graph shows quantitation of …
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Figure 3—figure supplement 2—source data 1
Source data for Figure 3—figure supplement 2A-D.
- https://cdn.elifesciences.org/articles/60183/elife-60183-fig3-figsupp2-data1-v2.xlsx
Figure 4
Effect of Dll4(E12) on bone vasculature.
(A) Tile scan images of Emcn (red), VEGFR3 (green), and Osterix (OSX)- (cyan) stained sections of pLIVE-Dll4(E12) and control femur at 11 weeks of age. Dashed line indicates the border of VEGFR3high …
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Figure 4—source data 1
Source data for Figure 4C and D.
- https://cdn.elifesciences.org/articles/60183/elife-60183-fig4-data1-v2.xlsx
Figure 5 with 2 supplements
Bone formation is not changed by recombinant Jag1-Asp8x and Jag1(JV1).
(A) Schematic diagram showing the domain organization of murine Jag1 full-length protein and recombinant Jag1-Asp8x and Jag1(JV1) fusion proteins. Fusion proteins contain 6x His epitope tag (green …
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Figure 5—source data 1
Source data for Figure 5C.
- https://cdn.elifesciences.org/articles/60183/elife-60183-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Generation of Jag1-Asp8x and Jag1(JV1) recombinant proteins.
(A) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified Jag1-Asp8x recombinant protein. From left to right, protein molecular mass marker (Marker), extract of …
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Figure 5—figure supplement 1—source data 1
Source data for Figure 5—figure supplement 1D,E.
- https://cdn.elifesciences.org/articles/60183/elife-60183-fig5-figsupp1-data1-v2.xlsx
Figure 5—figure supplement 2
Detection of Jag1-Asp8x and Jag1(JV1) in the bone.
(A) Tile scan confocal images of Jag1 (cyan) immunostaining in sections from control, pLIVE-Jag1-Asp8x and pLIVE-Jag1(JV1) femur. Nuclei, DAPI (blue). (B) Representative higher magnification images …
Figure 6 with 1 supplement
Bone formation is not increased by Dll4(E12) in ovariectomized female mice.
(A, B) Representative 3D micro-computed tomography (μ-CT) images of trabecular bone in the distal tibial metaphysis (A) and cortical bone in the mid-tibial diaphysis (B) of sham or ovariectomized …
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Figure 6—source data 1
Source data for Figure 6C–G.
- https://cdn.elifesciences.org/articles/60183/elife-60183-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Reduced Dll4(E12) immunoreactivity and trabecular bone in female mice compared to male mice.
(A, B) Representative overview confocal images of Collagen I alpha one chain (Col1a1) staining (green) in the femurs of wild-type male and female mice treated with vehicle (A) or pLIVE-Dll4(E12) (B).…
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Figure 6—figure supplement 1—source data 1
Source data for Figure 6—figure supplement 1A-D.
- https://cdn.elifesciences.org/articles/60183/elife-60183-fig6-figsupp1-data1-v2.xlsx
Figure 7 with 2 supplements
Synergistic action of recombinant Dll4(E12) and parathyroid hormone (PTH).
(A) Representative micro-computed tomography (μ-CT) images of trabecular bone in the distal femoral metaphysis of female wild-type mice treated with vehicle, pLIVE-Dll4(E12), PTH, or the combination …
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Figure 7—source data 1
Source data for Figure 7B–G,I-K.
- https://cdn.elifesciences.org/articles/60183/elife-60183-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
Effects of combined parathyroid hormone (PTH) and Dll4(E12) administration.
(A) Time course of pLIVE-Dll4(E12) and PTH administration to adult wild-type female mice. (B–E) Representative overview confocal images and corresponding quantification of immunostainings for Dll4 (B…
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Figure 7—figure supplement 1—source data 1
Source data for Figure 7—figure supplement 1B-E.
- https://cdn.elifesciences.org/articles/60183/elife-60183-fig7-figsupp1-data1-v2.xlsx
Figure 7—figure supplement 2
Synergistic effects of Dll4(E12) and parathyroid hormone (PTH) on osteogenesis.
Representative overview confocal images and corresponding quantifications for Osterix (OSX) (A, white) and Opn (B, green) immunostaining in wild-type female femur after treatment with vehicle, pLIVE-…
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Figure 7—figure supplement 2—source data 1
Source data for Figure 7—figure supplement 2A,B.
- https://cdn.elifesciences.org/articles/60183/elife-60183-fig7-figsupp2-data1-v2.xlsx
Figure 8 with 2 supplements
Single cell RNA sequencing (scRNA-seq) analysis of control and Dll4(E12)-treated bone.
(A) Overview of the sample processing and scRNA-seq procedure. (B) UMAP projection of all cells in pLIVE-Dll4(E12) and control, colored by Louvain clusters. Endothelial cells (ECs), mesenchymal …
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Figure 8—source data 1
Source data for Figure 8F–H.
- https://cdn.elifesciences.org/articles/60183/elife-60183-fig8-data1-v2.xlsx
Figure 8—figure supplement 1
Single cell RNA sequencing (scRNA) quality control statistics.
(A–D) Quality control plots. In each panel, the top plot shows distribution of count depths, middle plot shows knee plot, and the bottom plot shows cumulative count depth plotted as a function of …
Figure 8—figure supplement 2
Extended analysis of differentially expressed genes.
(A) Differential expression volcano plots showing -log10(adjusted p-value) against average log-fold change of pLIVE-Dll4(E12) vs. control for Notch pathway related genes (parent Gene Ontology [GO] …
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Figure 8—figure supplement 2—source data 1
Source data for Figure 8—figure supplement 2A-E.
- https://cdn.elifesciences.org/articles/60183/elife-60183-fig8-figsupp2-data1-v2.xlsx
Figure 9
Single cell RNA sequencing (scRNA-seq) analysis of mesenchymal stromal cell (MSC) subclusters.
(A) UMAP projection of MSCs in both control and pLIVE-Dll4(E12) samples colored by Louvain clusters. Metaphyseal MSCs (mpMSCs), diaphyseal MSCs (dpMSCs), and fibroblasts are indicated. (B) Barplot …
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Figure 9—source data 1
Source data for Figure 9D–G.
- https://cdn.elifesciences.org/articles/60183/elife-60183-fig9-data1-v2.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus, C57BL/6JRj) | WT | Janvier Labs | ||
| Genetic reagent (Mus musculus) | Tg(Hey1-EGFP)ID40Gsat | GENSAT | MGI:4847129 | |
| Recombinant DNA reagent | pcDNA3.1- Dll4(ECD)- His(6x)-Asp(8x)(plasmid) | This paper | Dll4-Asp(8x) | |
| Recombinant DNA reagent | pcDNA3.1- Dll4(ECD)- Variant- His(6x)-Asp(8x) (plasmid) | This paper | Dll4(E12) | |
| Recombinant DNA reagent | pLIVE-Dll4(ECD)-His(6x)-Asp(8x) (plasmid) | This paper | pLIVE-Dll4-Asp(8x) | |
| Recombinant DNA reagent | pLIVE-Dll4(ECD) Variant- His(6x)-Asp(8x) (plasmid) | This paper | pLIVE-Dll4(E12) | |
| Recombinant DNA reagent | pcDNA3.1- Jag1 (ECD)- His(6x)-Asp(8x)(plasmid) | This paper | Jag1- Asp(8x) | |
| Recombinant DNA reagent | pcDNA3.1- Jag1 (ECD)- JV1- His(6x)-Asp(8x)(plasmid) | This paper | Jag1(JV1) | |
| Recombinant DNA reagent | pLIVE- Jag1 (ECD)- His(6x)-Asp(8x) (plasmid) | This paper | pLIVE-Jag1- Asp(8x) | |
| Recombinant DNA reagent | pLIVE- Jag1 (ECD)- JV1- His(6x)-Asp(8x) (plasmid) | This paper | pLIVE-Jag1(JV1) | |
| Cell line (Homo sapiens) | Human Umbilical Vein Endothelial Cells (HUVEC) | ThermoFisher | Cat# C0035C | Cell identity and absence of mycoplasma contamination or human pathogens were certified by the supplier |
| Cell line (Homo sapiens) | Human Embryonal kidney –293(HEK293) | DSMZ | Cat# ACC 305 | Cell identity and absence of mycoplasma contamination were certified by the supplier |
| Antibody | Anti-Endomucin (Rat, monoclonal) | Santa Cruz | Cat# SC-65495 | IF (1:100) |
| Antibody | Anti-PECAM-1(Rat, monoclonal) | Pharmigen | Cat# 553,370 | IF (1:100) |
| Antibody | Anti-CD31(Goat, polyclonal) | R&D Systems | Cat# FAB3628 | IF (1:100) |
| Antibody | Anti-Pdgfrβ (Goat, polyclonal) | R&D Systems | Cat# AF1042 | IF (1:100) |
| Antibody | Anti-NG2 (Rabbit, polyclonal) | Millipore | Cat# AB5320 | IF (1:100) |
| Antibody | Anti-BCAM (Goat, polyclonal) | R&D Systems | Cat# AF8299 | IF (1:50) |
| Antibody | Anti-ATP6V1B1+ ATP6V1B2 (Rabbit, polyclonal) | Abcam | Cat# ab200839 | IF (1:100) |
| Antibody | Anti-Aggrecan (Rabbit, polyclonal) | Millipore | Cat# AB1031 | IF (1:100) |
| Antibody | Anti-Sox9 (Goat, polyclonal) | R&D Systems | Cat# AF3075 | IF (1:100) |
| Antibody | Anti-Perilipin (Rabbit, polyclonal) | Cell Signaling | Cat# 9349 | IF (1:100) |
| Antibody | Anti-Osterix (Rabbit, polyclonal) | Abcam | Cat# ab22552 | IF (1:1000) |
| Antibody | Anti-Collagen Type I (Rabbit, polyclonal) | Millipore | Cat# AB765P | IF (1:200) |
| Antibody | Anti-Osteopontin (Goat, polyclonal) | R&D Systems | Cat# AF808 | IF (1:100) |
| Antibody | Anti-Osteocalcin (Rabbit, polyclonal) | LifeSpan BioSciences | Cat# LS-C17044 | IF (1:100) |
| Antibody | Anti-Runx2 (Rabbit, monoclonal) | R&D Systems | Cat# MAB2006 | IF (1:50) |
| Antibody | Anti-Dll4 (Goat, polyclonal) | R&D Systems | Cat# AF1389 | IF (1:100)WB (1:200) |
| Antibody | Anti-Jag1 (Goat, polyclonal) | Sigma | Cat# J4127 | IF (1:100)WB (1:500) |
| Antibody | Anti-GAPDH (Rabbit, monoclonal) | Ambion | Cat# AM4300 | WB (1:1000) |
| Antibody | Anti-CD45-FITC (Rat, monoclonal) | eBioscience | Cat# 11–0451 | |
| Antibody | Anti-CD8-Biotin (Rat, monoclonal) | eBioscience | Cat# 13–0081 | |
| Antibody | Anti-CD4-APC (Rat, monoclonal) | eBioscience | Cat# 17-0042-82 | |
| Antibody | Streptavidin PE/Cy7 | Thermo Scientific | Cat# SA1012 | |
| Antibody | Lineage Cell Depletion Kit | Miltenyi Biotec | Cat# 130-090-858 | |
| Antibody | CD45 Microbeads | Miltenyi Biotec | Cat# 130-052-301 | |
| Antibody | CD117 Microbeads | Miltenyi Biotec | Cat# 130-091-224 | |
| Antibody | Ter-119 Microbeads | Miltenyi Biotec | Cat# 130-049-901 | |
| Antibody | Anti-rabbit Alexa Fluor-488 (Donkey, polyclonal) | Invitrogen | Cat# A21206 | IF (1:500) |
| Antibody | Anti-rabbit Alexa Fluor-594 (Donkey, polyclonal) | Invitrogen | Cat# A21207 | IF (1:500) |
| Antibody | Anti-rabbit Alexa Fluor-647 (Donkey, polyclonal) | Invitrogen | Cat# A31573 | IF (1:500) |
| Antibody | Anti-rat Alexa Fluor-488 (Donkey, polyclonal) | Invitrogen | Cat# A21208 | IF (1:500) |
| Antibody | Anti-rat Alexa Fluor-594 (Donkey, polyclonal) | Invitrogen | Cat# A21209 | IF (1:500) |
| Antibody | Anti-rat Alexa Fluor-647 (Donkey, polyclonal) | Jackson ImmunoResearch | Cat# 712-605-153 | IF (1:500) |
| Antibody | Anti-rat Alexa Fluor-647 (Donkey, polyclonal) | Invitrogen | Cat# A21247 | IF (1:500) |
| Antibody | Anti-goat Alexa Fluor-488 (Donkey, polyclonal) | Invitrogen | Cat# A11055 | IF (1:500) |
| Antibody | Anti-goat Alexa Fluor-647 (Donkey, polyclonal) | Invitrogen | Cat# A21447 | IF (1:500) |
| Antibody | Anti-goat Alexa Fluor-647 (Donkey, polyclonal) | Thermo Scientific | Cat# A32849 | IF (1:500) |
| Antibody | Anti-rabbit IgG HRP-linked (Goat) | Cell Signaling | Cat# 7074 | WB (1:15000) |
| Antibody | Anti-goat IgG HRP-linked (Donkey) | Antibody online | Cat# ABIN1536502 | WB (1:15000) |
| Antibody | Anti-mouse IgG 656G HRP-linked (Sheep) | GE Healthcare | Cat# NA931 | WB (1:40000) |
| Antibody | Anti-goat IgG (H + L) Peroxidase AffiniPure Bovine | Jackson ImmunoResearch | Cat# 805-035-180 | WB (1:15000) |
| Sequence-based reagent | Human GAPDH Endogenous Control (VIC/MGB probe, primer limited) | Applied Biosystems | Cat# 4326317E | TaqMan probe Hs99999905_m1 |
| Sequence-based reagent | Human ACTB Endogenous Control (VIC/MGB probe, primer limited) | Applied Biosystems | Cat# 4326315E | TaqMan probe Hs99999903_m1 |
| Sequence-based reagent | Human HEY1 TaqMan Gene Expression Assay (FAM) | Applied Biosystems | Cat# 4331182 | TaqMan probe Hs00232618_m1 |
| Sequence-based reagent | Human DLL4 TaqMan Gene Expression Assay (FAM) | Applied Biosystems | Cat# 4331182 | TaqMan probe Hs00184092_m1 |
| Sequence-based reagent | Human HEY2 TaqMan Gene Expression Assay (FAM) | Applied Biosystems | Cat# 4331182 | TaqMan probe Hs00232622_m1 |
| Sequence-based reagent | Human HES1 TaqMan Gene Expression Assay (FAM) | Applied Biosystems | Cat# 4331182 | TaqMan probe Hs00172878_m1 |
| Sequence-based reagent | Human EFNB2 TaqMan Gene Expression Assay (FAM) | Applied Biosystems | Cat# 4331182 | TaqMan probe Hs00187950_m1 |
| Sequence-based reagent | Human NRARP TaqMan Gene Expression Assay (FAM) | Applied Biosystems | Cat# 4331182 | TaqMan probe Hs01104102_s1 |
| Peptide, recombinant protein | Recombinant Human PTH (1-34) | BACHEM | Cat# H-4835-GMP, 4033364 | |
| Commercial assay or kit | BCA Protein Assay Kit | Pierce | Cat# 23225 | |
| Commercial assay or kit | RNeasy Plus Micro Kit | QIAGEN | Cat# 74034 | |
| Commercial assay or kit | iScript cDNA Synthesis Kit | BIO-RAD | Cat# 170–8891 | |
| Commercial assay or kit | SsoAdvanced Universal Probes Supermix | BIO-RAD | Cat# 172–5284 | |
| Commercial assay or kit | LEGENDplex Mouse Inflammation Panel (13-plex) with V-bottom plates | BioLegend | Cat# 740446 | |
| Commercial assay or kit | Anticoagulant EDTA-treated Microvettes | Sarstedt | Cat# 20.1341 | |
| Commercial assay or kit | BD Rhapsody Whole Transcriptome Analysis (WTA) Amplification kit | BD Biosciences | Cat# 633,801 | |
| Commercial assay or kit | Agencourt AMPure XP magnetic beads | Beckman Coulter Life Sciences | Cat# A638880 | |
| Commercial assay or kit | Disposable polystyrene columns | Thermo Scientific | Cat# 29922 | |
| Commercial assay or kit | Amicon Ultra-0.5 Centrifugal Filter | Millipore | Cat# UFC500396 | |
| Commercial assay or kit | Pierce Slide-A-Lyzer 10K MWCO Dialysis Cassettes | Thermo Scientific | Cat# 66380 | |
| Chemical compound, drug | Ni-NTA agarose resin | Qiagen | Cat# 302010 | |
| Chemical compound, drug | Sucrose | Sigma | Cat# S0389 | |
| Chemical compound, drug | cOmplete ULTRA Tablets Protease Inhibitor Cocktail | Roche | Cat# 05892970001 | |
| Chemical compound, drug | Phosphatase inhibitor cocktail set V | EMD Millipore | Cat# 524629 | |
| Chemical compound, drug | SimplyBlue SafeStain | Invitrogen | Cat# LC6060 | |
| Chemical compound, drug | Gelatine | Sigma | Cat# G1890 | |
| Chemical compound, drug | Polyvinylpyrrolidone | Sigma | Cat# P5288 | |
| Chemical compound, drug | Trypsin-EDTA solution | Sigma | Cat# T3924 | |
| Chemical compound, drug | Paraformaldehyde | Sigma | Cat# P6148 | |
| Chemical compound, drug | 70 kDa Dextran, Texas Red Lysine fixable | Molecular Probes | Cat# D1864 | |
| Chemical compound, drug | Calcein | Sigma | Cat# C0875 | |
| Chemical compound, drug | Fluoromount-G | Southern Biotech | Cat# 0100–01 | |
| Chemical compound, drug | ECL Prime Western Blotting Detection Reagent | GE-Healthcare | Cat# RPN2236 | |
| Chemical compound, drug | 4-Hydroxy tamoxifen | Sigma | Cat# H7904 | |
| Chemical compound, drug | Hematoxilin | Sigma | Cat# MHS16 | |
| Chemical compound, drug | Dimethyl sulfoxide | Sigma | Cat# D8418 | |
| Chemical compound, drug | MEM200 endothelial cells medium | ThermoFisher | Cat# M200500 | |
| Chemical compound, drug | LSGS | ThermoFisher | Cat# S00310 | |
| Chemical compound, drug | EBM-2 endothelial cells medium | Lonza | Cat# CC-3156 | |
| Chemical compound, drug | EGM-2 Single Quots | Lonza | Cat# CC-4176 | |
| Chemical compound, drug | Opti-MEM | Gibco | Cat# 31985–047 | |
| Chemical compound, drug | Poly-L-lysine | Sigma | Cat# P6282 | |
| Chemical compound, drug | HEPES | Sigma | Cat# H3537 | |
| Chemical compound, drug | Ketamine | Zoetis | Cat# 344771 | |
| Chemical compound, drug | Rompum | Bayer HealthCare | Cat# D-51368 | |
| Chemical compound, drug | Collagenase I | Gibco | Cat# 17100–017 | |
| Chemical compound, drug | Collagenase IV | Gibco | Cat# 17104–019 | |
| Chemical compound, drug | Dispase | Gibco | Cat# 17105–041 | |
| Software, algorithm | ImageJ (v2.0.0 Fiji) | Schindelin et al., 2012 | ||
| Software, algorithm | Volocity (v6.3) | Perkin Elmer | ||
| Software, algorithm | Illustrator (vCC2018) | Adobe | ||
| Software, algorithm | GraphPad Prism7 | GraphPad Software | ||
| Software, algorithm | FlowJo (v10.3) | BD Life Sciences | ||
| Other | DAPI stain | Sigma | Cat# D9542 | (1 mg/mL) |
