14-1449-37

antibody from Invitrogen Antibodies

Targeting: CDH5 7B4, CD144

Provider product page for 14-1449-37

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Antibody Data
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References [35]
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Western blot [1]
Immunocytochemistry [1]
Other assay [37]

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Product number: 14-1449-37 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD144 (VE-cadherin) Monoclonal Antibody (16B1), eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The 16B1 antibody reacts with human CD144, also known as VE-cadherin and cadherin-5. The cadherin family of receptors, which are calcium-dependent adhesion molecules, is known to be involved in homophilic cell interactions. VE-cadherin, which is 140 kDa, is localized at the intercellular boundaries of endothelial cells in blood and lymphatic vessels in several tissues. It is thought to play a role in vascular permeability and remodeling. Applications Reported: This 16B1 antibody has been reported for use in flow cytometric analysis, immunoblotting (WB), microscopy, and immunocytochemistry without fixation. Applications Tested: This 16B1 antibody has been tested by immunoblotting Human Umbilical Vein Endothelial Cell (HUVEC) lysates. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest. Purity: Greater than 90%, as determined by SDS-PAGE. Aggregation: Less than 10%, as determined by HPLC. Filtration: 0.2 µm post-manufacturing filtered.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: 16B1
Vial size: 2 mg
Concentration: 0.5 mg/mL
Storage: 4° C

CDH5 protein structure - 14-1449-37 shown in red.

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Experimental details: Fig 3 False positive CTCs identified by ISET filtration and morphology criteria in healthy volunteers. Presumed false positive ''CTC'' (blue arrow head). Cell staining positive for CD45/CD144, consistent with white blood cell or endothelial cell (yellow arrow). The dark circles are 8mum filter pores (black arrow).

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Experimental details: Figure 5 FACS and genomic analysis of VE-cadherin-positive CDX tumour cells. ( a ) Schematic representing generation of CDX3 and subsequent processing of samples for FACS of human (Mouse MHC1 -VE ) subpopulations based on VE-cadherin +VE and VE-cadherin -VE expression. ( b ) Flow Cytometry dot plots showing gating strategies used for cell sorting. Left panels, forward light scatter versus anti-mouse anti-MHC1 staining. Right panels, side light scatter versus anti-human, anti-VE-cadherin staining. Gating strategies set according to relevant controls (see the 'Methods' section). ( c ) CNA profiles from mice bearing CDX3 tumours (CDX3(a) and CDX3(b)) FACS-sorted mouse MHC -VE , bulk tumour/VE-cadherin +VE /VE-cadherin -VE and patient 3 germline control (white blood cells). The GC-normalized and mappability corrected read counts (log 2 scale) were segmented using Hidden Markov Model (HMM), red=copy-number gains, blue=copy-number losses. ( d ) VAF of TP53 (p.Y220C) mutation in sorted cell subpopulations. ( e ) Read alignment plots from representative samples demonstrate the presence of variant TP53 allele (p.Y220C) in bulk tumour, VE-cadherin +VE and VE-cadherin -VE fractions, but absent in germline patient control.

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Experimental details: Figure 6 Single-cell CNA analysis of VE-cadherin-negative and -positive CTCs from an ES SCLC patient. ( a ) CTCs identified using the HD-SCA assay: cells were stained with DAPI (white) and antibodies to CKs (green), CD45 (red) and VE-cadherin (blue) and pseudo-coloured. Top panel, DAPI +VE /CD45 -VE /CK +VE /VE-cadherin +VE CTC; middle panel, DAPI +VE /CD45 -VE /CK +VE /VE-cadherin -VE CTC; bottom panel, DAPI +VE /CD45 +VE /CK -VE /VE-cadherin -VE white blood cell (WBC). Arrows link to the respective CNA analysis of the indicated cell, showing loss of 3p, gain of 5p including TERT and hemizygous loss of RB1 on 13 in CTC. Major chromosome losses (blue arrow) and gains (red arrow) highlighted above. The selected WBC in contrast has a characteristic flat CNA profile indicative of healthy somatic cells. Representative images and profiles are shown. Scaled x 10 images from the scanner are shown for CD45, while remaining images were acquired at x 40. Scale bar, 10 mum. ( b ) CNA analysis of matched patient ctDNA: the GC-normalized and mappability corrected read counts (log 2 scale) were segmented using Hidden Markov Model (HMM), red=copy-number gains, blue=copy-number losses. Major chromosome losses (blue arrow) and gains (red arrow) highlighted above and match pattern identified by single CTC CNA.

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Experimental details: Fig. 3 ATR2 siRNA transfection and immunophenotyping for EC markers. I Concentration selection for siRNA transfection. Three different concentrations (10, 35, and 50 nM) of ATR2 siRNA were used according to the manufacturer's protocol. Western blot analysis showed inhibition of ATR2 by 10, 35, and 50 nM of ATR2 siRNA. However, 50 nM of ATR2 siRNA showed the highest inhibition among all three different concentrations ( A ). ATR2 silencing by siRNA transfection with EGM compared with AMSCs with EGM and EGM + scrambled siRNA (negative control) ( B ). GAPDH was used as a housekeeping gene. II Flow cytometric analysis of PECAM1 (CD31) in four different groups; control group with EGM ( A ), AMSCs with EGM and MMP-2 siRNA ( B ), AMSCs with EGM and MMP-14 siRNA ( C ), and HUVECs as the positive control ( D ). Cell transfection with 5 muM of ATR2 siRNA for EGM ( E ), AMSCs with EGM and MMP-2 siRNA ( F ), and AMSCs with EGM and MMP-14 siRNA ( G ). Flow cytometry data were analyzed to show the significant differences between the groups ( H ). III Flow cytometric analysis of VE-cadherin (CD144) in four different groups: control group AMSCs with EGM ( A ), AMSCs with EGM and MMP-2 siRNA ( B ), AMSCs with EGM and MMP-14 siRNA ( C ), and HUVECs as the positive control ( D ). Cell transfection with 5 muM of ATR2 siRNA for EGM ( E ), AMSCs with EGM and MMP-2 siRNA ( F ), and AMSCs with EGM and MMP-14 siRNA ( G ). Flow cytometry data were analyzed to show the significant differences betwe

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Experimental details: Fig. 5 Inhibition of ERK phosphorylation and immunophenotyping for EC markers. I Concentration -dependent effect of ERK inhibitor (U0126). Three different concentrations (0.5, 1.0, and 5.0 muM) of U0126 were used. Western blot analysis showed significant inhibition of p-ERK by 1.0 and 5.0 muM of U0126. However, 5.0 muM of U0126 showed the highest inhibition among all three different concentrations. Phospho-ERK was normalized to its total protein expression. II Flow cytometric analysis of PECAM1 (CD31) with ERK inhibitor (U0126). Three different groups treated with 5.0 muM of U0126: AMSCs with EGM ( A ), AMSCs with EGM and MMP-2 siRNA ( B ), and AMSCs with EGM and MMP-14 siRNA ( C ). Flow cytometry data were analyzed to show the significant differences between the groups ( D ). III Flow cytometric analysis of VE-cadherin (CD144) with ERK inhibitor (U0126). Three different groups were treated with 5.0 muM of U0126: AMSCs with EGM ( A ), AMSCs with EGM and MMP-2 siRNA ( B ), and AMSCs with EGM and MMP-14 siRNA ( C ). Flow cytometry data were analyzed to show the significant differences with or without U0126 ( D ). * p < 0.05, ** p < 0.01, *** p < 0.001. EBM endothelial cell basal medium, EGM endothelial cell growth medium, MMP matrix metalloproteinase, ERK extracellular signal-regulated kinase, HUVEC human umbilical vein endothelial cell

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Experimental details: Fig 3 Identifying human pulmonary artery endothelial cells (HPAECs) by flow cytometry. (A) Commercially purchased HPAECs were analyzed by flow cytometry using anti-CD31 and anti-CD144 Abs #12 and #13. Compared with HPASMCs and HLFs, high expression of CD31 and CD144 was observed only in HPAECs. These results served as a positive control for further experiments aiming to confirm that cells isolated from rat lungs ex vivo were, in fact, endothelial. (B) Commercially purchased HPAECs were used to test the generalizability of these results. Alternative anti-CD31 and anti-CD144 antibodies did not reliably identify endothelial cell surface markers, supporting our earlier findings indicating variability in reactivity (i.e., quality) of tested antibodies across experimental methods, including flow cytometry. Representative plots and histograms shown. Means +- standard error, % CD31 or CD144 positive, N = 4-5/condition. Ab, antibody; Iso, Isotype control.

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Experimental details: Fig 4 Identifying rat pulmonary endothelial cells by flow cytometry. Presumed rat PMVECs isolated by mechanical and enzymatic dissociation of peripheral lung and culture in endothelial-selective medium, commercial rat pulmonary artery endothelial cells (RPAECs), or rat pulmonary artery smooth muscle cells (RPASMCs) were labeled with antibodies against endothelial surface markers. (A) Anti-CD31 (Ab #12 and #16) and CD144 (Ab #13) signal was not observed in presumed rat PMVECs by flow cytometry (N = 3/condition). (B) Labeling of RPAECs was also not observed for anti-CD31 antibodies #12 (N = 4/condition) and (C) #16, respectively (N = 3/condition). (D) False-positive signal was detected in RPASMCs labeled with anti-CD144 Ab #19 (N = 4/condition). Representative plots and histograms shown. Means +- standard error, % CD31 or CD144 positive. Ab, antibody; Iso, Isotype control.

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Experimental details: Representative images of DM2 effect on endothelial and pericyte differentiation of hMSC (A-E). Flow cytometry analysis confirmed DM2 decreased the endothelial differentiation of hMSCs indicated by a reduction in VE-cadherin levels (n = 3) (A and B). DM2 is also able to decrease Dil-Ac-LDL uptake capacity in hMSCs (C). The level of NG2, a pericyte marker, was also decreased during hMSC differentiation into pericyte-like cells under diabetic condition (n = 6) (D).

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Experimental details: Figure 2 Generation and characterization of endothelial-specific YY1 deficient mice for the tumor angiogenesis. ( A ) Schematic diagram for the endothelial cell specific deletion of YY1 in mice (Ve-Cad-CreER T2 ; YY1 flox/flox , YY1 iDeltaEC ) and the strategy of the tumor angiogenesis study. The tumor was induced by melanoma B16-F10 cells (5 x 10 cells per mouse) with subcutaneously transplanted into 8-week-old WT or YY1 iEC mice. ( B ) PCR analysis for the genotyping of Ve-Cad-CreER T2 ; YY1 flox/flox mice (YY1 iDeltaEC ) and YY1 flox/flox (WT) mice. ( C ) Immunofluorescence image of endothelial cell marker VE-Cadherin in lung endothelial cells isolated from YY1 iDeltaEC mice. Nuclei were labeled by DAPI (blue) (Left panel) and Western blot analysis of endothelial YY1 expression in mouse lung endothelial cells isolated from WT and YY1 iDeltaEC mice (n = 3) (Right panel). ( D ) Dual immunostaining analysis of YY1 (red) and endothelial cell marker CD31 (green) in melanoma tumor tissues isolated from WT and YY1 iDeltaEC mice, (n = 7). Scale bars: 20 mum.

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Experimental details: Figure 3. ETV2 overexpression enhanced endothelial differentiation of DPSCs. (A) Effects of ETV2 overexpression on the protein expression levels of VE-Cadherin, as detected by immunofluorescence staining and quantitative analysis of fluorescence intensity. Scale bar: 50 mum. (B) Evaluation of endothelial-specific marker expression by flow cytometry. (C) Results of tube formation assay in vitro and quantification of tubule length and branching point numbers. Scale bar: 50 mum, * P < 0.05, ** P < 0.01. DPSCs: dental pulp stem cells.

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Experimental details: Figure 4 ZIKV and IEVs induce structural rearrangements of VE-cadherin at the early time points. ( A ) Alterations in the architecture of VE-cadherin (white arrows) are observed at 30 min in ZIKV-infected and IEV-treated hcMEC/D3 cells. These changes are restored at later time points, except for ZIKV-infected cells, where reorganization of VE- cadherin is still detectable at 1 h post infection. TNF-alpha (100 ng/mL) is used as a positive control (40x objective). ( B ) Expression levels of VE-cadherin are not significantly changed in ZIKV- and EV-treated cells after 30 min, 1 h, and 24 h. Clathrin is used as an internal loading control in the Western blot analysis.

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Experimental details: FIGURE 8 Descriptive images of immunohistochemically stained pulmonary arteries for VE-cadherin (magnification 20x): a) normal control (NC), b) idiopathic pulmonary fibrosis (IPF), in insets junctional and cytoplasmic expression of VE-cadherin in NC and IPF, respectively (100x). Staining images for: N-cadherin c) NC and d) IPF (20x); S100A4 e) NC and f) IPF; vimentin g) NC and h) IPF; alpha-SMA i) NC and j) IPF; collagen-I k) NC and l) IPF; and collagen-IV m) NC and n) IPF (all images taken in 20x magnification for medium-size arteries). The black arrows indicate mesenchymal protein expression in the intima, and the red arrows indicate alpha-SMA+ myofibroblast (in inset intima) and ECM protein: collagen I and collagen IV deposition (in inset intima).

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Experimental details: Figure 4 LCM and genomic analysis of VE-cadherin-positive VM vessels in CDX. ( a ) Schematic representation of generation of CDX3 and subsequent samples used for VM analysis, genomic analysis and targeted sequencing of LCM regions. White blood cells (WBC) used to generate germline control DNA. ( b ) IHC staining for PAS/CD31, VE-cadherin and (non DNA damaging) Cresyl Violet with VM-low/-high LCM area from CDX3 tumour. Scale bar, 100 mum. ( c ) Sequencing data from the 8 different samples used for genomic analysis demonstrating minimal mouse cell contamination and predominantly human genomes in VM structures in vivo . All reads were aligned to both human and mouse genome separately.

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Experimental details: Figure 7 Functional significance of VE-cadherin expression for SCLC VM in vitro and in vivo . ( a ) Western blot analysis of VE-cadherin expression levels in VM proficient C8161 melanoma cells and H446, H446 non-silencing empty vector controls, H446 VE-cadherin shRNA knockdown and H1048 SCLC cells. ( b ) VM-like network formation on matrigel for VE-cadherin +VE cells (C8161, H446 and H446 E.V) and VE-cadherin -VE cells (H1048 and H446 VE-cadherin KD) lacking network formation. Representative images are shown for n >3 experiments. Scale bar, 200 mum. ( c ) Left panel, representative images of anti-human anti-VE-cadherin staining in H446 parental and H446 VE-cadherin KD xenografts. Scale bar, 50 mum. ( c ) Right panel percentage of cells positive for VE-cadherin in H446 and H446 VE-cadherin KD xenografts (*** P =0.0001, two-tailed t -test), n =10 animals per group. ( d ) Western blot analysis VE-cadherin expression in H446 and H446 VE-cadherin KD xenografts ( n =6 tumours for each group). ( e ) Left panel, representative images of anti-mouse anti-CD31/PAS staining in H446 Parental and H446 VE-cadherin KD tumours. Scale bars, 50 mum. ( e ) Right panel VM ratio in H446 parental ( n =10) and H446 VE-cadherin KD xenografts ( n =8; *** P =0.0005, two-tailed Mann-Whitney's test). ( f ) Tumour growth rates in H446 (black) and H446 VE-cadherin KD (grey) tumours ( n =10 animals per group). ( g ) Days to 200 mm 3 volume tumours in H446 and H446 VE-cadherin xenografts KD ( n =20 anim

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Experimental details: Fig 1 Circulating Tumour Cell (CTC) detected by Immunocytochemical (ICC)-Staining of ISET Filters. Large cells from blood samples are enriched by ISET filtration and immunocytochemically stained for the White Blood Cell marker CD45 and the Endothelial Cell marker CD144 (yellow arrow). Circulating Tumour Cells (CTCs) are identified as >=16mum diameter cells with a hyperchromatic nucleus and negative for CD45/CD144 brown chromogen staining (blue arrowhead). The dark circles are 8mum filter pores (black arrow).

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Experimental details: Fig 3 False positive CTCs identified by ISET filtration and morphology criteria in healthy volunteers. Presumed false positive ''CTC'' (blue arrow head). Cell staining positive for CD45/CD144, consistent with white blood cell or endothelial cell (yellow arrow). The dark circles are 8mum filter pores (black arrow).

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Experimental details: Figure 3 HEB -/- hESCs Display Defects in Mesoendodermal Induction and Early Hematopoietic Differentiation (A) Experimental overview of embryoid body (EB) formation and differentiation. BMP4, bone morphogenetic protein 4; bFGF, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; IL, interleukin; EPO, erythropoietin; SCF, stem cell factor; IGF1, insulin-like growth factor 1; FLT3L, FMS-like tyrosine kinase 3 ligand; TPO, thrombopoietin. (B) Reverse-transcriptase PCR analysis of HEB transcript (HEBCan, canonical; HEBAlt, alternative) expression at various stages of EB differentiation, and in sorted day-8 (d8) CD34 + cells (last column). GAPDH was measured as a loading control. (C) qRT-PCR analysis for the expression of pluripotency and differentiation markers in undifferentiated hESCs (day 0 [d0]) versus d4 EB-derived cells. (D) Flow-cytometric analysis of CD34 and KDR, CD144, and CD31 expression on d8 EB-derived cells. (E and F) Percentages (E) and numbers (F) of CD34 + cells in d8 EBs. (G) qRT-PCR analysis of the expression of mesodermal and hematopoietic genes in CD34 + cells. For qRT-PCR graphs, mRNA levels are shown relative to GAPDH. Error bars represent mean +- SD (n = 3 independent experiments). ** p < 0.01; *** p < 0.005 by Student''s t test. Images in (B) and plots in (D) are representative of three independent experiments. See also Figure S4 .

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Experimental details: Figure 6 Ectopic Expression of HEBCan in HEB -/- hESCs Restores Lineage-Specific Gene Expression and Hematopoietic Specification (A) Western blot analysis for HEB expression in WT, KO (HEB -/- ), KO + GFP (HEB -/- hESCs transduced with GFP control vector) and KO + HEBCan (HEB -/- hESCs transduced with HEBCan-encoding vector) hESCs. (B) Bright-field (top) and fluorescent (bottom) images of day-8 (d8) EBs derived from HEB -/- hESCs transduced with control or HEBCan-expressing lentiviral particles. Scale bar, 100 mum. (C and D) qRT-PCR analysis for the expression of pluripotency-associated genes (C) and mesoendodermal genes (D) in WT, KO + GFP, and KO + HEBCan hESC-derived cells at d0 and d4 of EB culture. mRNA levels are shown relative to GAPDH. (E) Flow-cytometric analysis of CD34 and KDR, CD144, and CD31 on WT, KO + GFP, and KO + HEBCan d8 EB-derived cells. (F and G) Percentages (F) and numbers (G) of CD34 + cells in WT, KO + GFP, and KO + HEBCan d8 EBs. (H) Flow-cytometric analysis for CD34 and CD45 on WT, KO + GFP, and KO + HEBCan d18 EB-derived cells. (I and J) Percentages (I) and numbers (J) of CD34/CD45 subsets in WT, KO + GFP, and KO + HEBCan d18 EB-derived cells. (K) Numbers of erythroid (BFU-E) and myeloid (CFU-GM) arising from unfractionated WT, KO + GFP, and KO + HEBCan d18 EBs. Error bars represent mean +- SD (n = 3 independent experiments). * p < 0.05, ** p < 0.01, *** p < 0.005 by Student's t test. Images in (A) and (B) and plots in (E) and (H) are represent

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Experimental details: siRNA transfection and immunophenotyping of differentiated adipose-derived mesenchymal stem cells (AMSCs); (I) : MMP-2 (A) and MMP-14 (B) silencing by siRNA transfection with EGM compared to AMSCs with EGM and EGM plus scrambled siRNA (negative control). GAPDH was used as a housekeeping gene (*, p < .05; **, p < .01; ***, p < .001). (II) : Flow cytometric analysis of PECAM1 (CD31) in five different groups; control group was the undifferentiated cells with EBM (A), AMSCs with differentiation medium EGM (B), AMSCs with differentiation medium EGM and MMP-2 siRNA (C), AMSCs with differentiation medium EGM and MMP-14 siRNA (D), and HUVECs as the positive control (E). Flow cytometry data were analyzed to show the significant differences between the groups (F). (III) : Flow cytometric analysis of VE-Cadherin (CD144) in five different groups; control group was the undifferentiated cells with EBM (A), AMSCs with differentiation medium EGM (B), AMSCs with differentiation medium EGM and MMP-2 siRNA (C), AMSCs with differentiation medium EGM and MMP-14 siRNA (D), and HUVECs as the positive control (E). Flow cytometry data were analyzed to show the significant differences between the groups (F). (*, p < .05; **, p < .01; ***, p < .001). Abbreviations: CD, cluster of differentiation; EBM, endothelial basal medium; EGM, endothelial growth medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HUVECs, human umbilical vein endothelial cells; MMP, matrix metalloproteinases.

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Experimental details: Immunostaining of VEGFR2 and immunophenotyping of differentiated adipose-derived mesenchymal stem cells (AMSCs) after VEGFR2 kinase inhibition; (I) : Immunofluorescence staining for VEGFR2. AMSCs in EGM showed significant increases in the expression of VEGFR2 (B & E) compared to endothelial basal medium group (A & E). AMSCs cultured with EGM and MMP-2 siRNA showed significantly higher fluorescence intensity of VEGFR2 in comparison to the EGM cultured cells (C & E). AMSCs cultured with EGM and MMP-14 siRNA showed the greatest positive staining of VEGFR2 compared to that of EGM and EGM plus MMP2 siRNA (D & E). Fluorescence intensity was measured to show the significant differences between the groups using ImageJ software (E). (II) : Flow cytometric analysis of PECAM1 (CD31) in three different groups; control group was the differentiated cells with EGM and 5 muM of VEGFR2 inhibitor (A), AMSCs with differentiation medium EGM, MMP-2 siRNA and 5 muM of VEGFR2 inhibitor (B) and AMSCs with EGM, MMP-14 siRNA and 5 muM of VEGFR2 inhibitor (C). Flow cytometry data were analyzed to show the significant differences between the groups in comparison to the same groups without VEGFR2 inhibitor (D). (III) : Flow cytometric analysis of VE-Cadherin (CD144) in three different groups; the differentiated cells with EGM and 5 muM of VEGFR2 inhibitor (A), AMSCs with differentiation medium EGM, MMP-2 siRNA and 5 muM of VEGFR2 inhibitor (B) and AMSCs with EGM, MMP-14 siRNA and 5 muM of VEGFR2 inhibito

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Experimental details: Figure 1 Expression of KDM4A and KDM4C following mESC Differentiation into Endothelial Cells (A) Flow sorting diagram of mESCs differentiated to endothelial cells stained with anti-FLK1 and anti-VE-cadherin antibodies. Cells were separated into FLK1/VE-cadherin-double positive endothelial cells or double-negative control cells. (B) Time course of gene expression assessed by qRT-PCR for the pluripotency genes Oct4 and Nanog as well as for endothelial genes Flk1 and VE-cadherin during the 6-day differentiation period (D1 to D6; ES are mESCs); error bars represent mean +- SD (n = 3 independent experiments, p < 0.05 by ANOVA). (C) qRT-PCR screen for 28 known histone demethylases was carried out in mESCs differentiated into FLK1/VE-cadherin-double positive endothelial cells (D6), double-negative control cells (D6), and adult primary mouse endothelial cells. Expression of KDM4a and KDM4c was markedly increased in mESC-derived and mouse endothelial cells as assessed by two-way ANOVA (all values are normalized to 18S in undifferentiated mESCs). Values are mean +- SD (n = 3 independent experiments, * p < 0.001 by two-way ANOVA). (D) Western blotting carried out using anti-KDM4A and anti-KDM4C antibodies in sorted FLK1/VE-cadherin-double-positive cells derived from mESCs at day 6 of differentiation. Representative examples from three independent experiments are shown. (E) Time course of Kdm4a and Kdm4c expression determined by qRT-PCR during the day 1 to day 6 period of endothelial cel

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Experimental details: Figure 2 KDM4A and KDM4C Mediate mESC Differentiation to Endothelial Cells (A) Diagram showing experimental protocol for siRNA treatment. mESC were transfected on days 1 and 3 to achieve maximum knockdown of protein. The cells were differentiated in medium containing BMP-4, bFGF, and VEGF for 6 days. (B) The effect of Kdm4a and Kdm4c knockdown on expression of endothelial-specific markers. Efficiency of Kdm4a and Kdm4c knockdown was 60%-80%. Oct4 expression was not suppressed by either Kdm4a or Kdm4c knockdown (n = 4 per group). Both Kdm4a and Kdm4c knockdown resulted in 90% and 80%, respectively, reduction in Flk1 and VE-cadherin expression on day 6 (n = 3 independent experiments). (C) Flow cytometry assessment of cell surface expression of Flk1 and VE-cadherin on day 6 of differentiation following treatment with scrambled, Kdm4a , Kdm4c , or Kdm4a and Kdm4c siRNAs. Quantification in the bar graph shows that the percentage of FLK1 and VE-cadherin double positive cells was reduced from 17% to less than 5%; FACS profile is representative (n = 3 independent experiments). Quantification values are mean +- SD; p < 0.05. Combining Kdm4a with Kdm4c siRNAs did not further suppress differentiation. (D) Treatment of mESCs with BMP-4 alone (2 ng/ml) is sufficient to induced Kdm4a expression at 24-hr post-stimulation. Treatment with bFGF alone (10 ng/ml) induced Kdm4c expression at 48-hr post-stimulation. Error bars represent mean +- SD (n = 3 independent experiments). VEGF stimulation

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Experimental details: Figure 1 Differentiation of FOP hiPSCs into ECs and Pericytes (A) Total SMAD1/5 and phospho-SMAD1/5 (pSMAD1/5) level in control hiPSCs (C3-3 and UE017C1) and FOP hiPSCs (F2-6 and F3-8). Note that the antibodies used here also may recognize SMAD8 and pSMAD8 bands. (B) FACS analysis of EC marker (CD31) or pericyte marker (PDGFRbeta) expression at differentiation days 10-12 is shown. (C) Quantification of the FACS analysis data for relative percentage of CD31 and VE-cadherin double-positive ECs and PDGFRbeta-positive pericytes. All values were adjusted to the control colony C3-3, which is defined as 1. (D) Relative gene expression at different time points during the differentiation. ACTIN was used to normalize gene expression. Data are presented as mean and SD from three independent experiments in (A), (C), and (D).

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Experimental details: Fig. 3 Human iPS cells differentiation toward ECs. Morphology of hiPSCs and their EC differentiated counterparts are shown by bright field microscopy. Scale bar: 50 mum ( a ). Flow cytometry showed the pure population of hiPS-derived ECs after MACS selection using CD144 magnetic beads ( b ). Immunofluorescence confocal image showing that the differentiated ECs expressed the EC-specific markers CD31, CD144, and ZO-1 localizing to cell-cell junction. QKI-7 displayed perinuclear cytoplasm localization. Scale bar: 25 mum ( c ). The expression of EC marker proteins CD31, CD144, KDR, and eNOS was shown by western blot ( d ). hiPS-ECs formed tube structure indicating their angiogenic capacity. Scale bar: 200 mum ( e ). Data are from n = 3 representative images. Source data are provided as a Source data file.

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Experimental details: Figure 2. Epithelial/mesenchymal nature of patient CTCs and CDX. CTCs were enriched via ISET filtration from a parallel blood sample to that which generated the CDX. The ISET filter was stained for pan-CK (green), vimentin (blue), CD45 and CD144 (pink), and DAPI (white). (A) Representative images and (B) quantitation of cells classed as epithelial, mixed epithelial/mesenchymal (E/M), and mesenchymal. Also shown is a circulating tumour microemboli (CTM) containing cells of all three phenotypes. *Leucocyte or endothelial cell; scale bar, 10 um. Quantitation carried out by two independent scorers on two ISET spots, each counting >150 CTCs per spot. Error bars, SEM. (C) Single cells from the ISET filter shown in A classed as a leucocyte/endothelial cell, epithelial CTC, mixed E/M CTC, or a mesenchymal CTC were captured by laser capture microdissection. The PACRG locus, shown by WES to be mutated in the CDX, was Sanger sequenced in DNA extracted from the captured CTCs and green fluorescent cells (leucocytes or endothelial cells), from the patients PBMCs (germline sample), from their primary tumour biopsy and from the passage 2 CDX shown in Figure 1 A-C. Highlighted base represents the mutated base. (D) RNAseq was carried out on passage 2 NSCLC CDX tumours and passage 3 CDX from two SCLC CDX models. Expression of EPCAM, KRT8, VIM, S100A4, CD44, and CD24 is displayed as RPKM values. Data represent mean of three independent tumours +- SEM. * P < 0.05; *** P < 0.001 according to two-t