huvecs  (Lonza)


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    Structured Review

    Lonza huvecs
    Role of adrenomedullin in flow-induced eNOS regulation. ( A ) BAECs were treated with adrenomedullin (ADM, 10 nM, 5 minutes), calcitonin gene–related peptide (CGRP; 10 nM, 10 minutes), or adrenomedullin-2 (ADM2, 1 nM, 3 minutes), and phosphorylation of eNOS S635 was determined by immunoblotting. Bar diagram shows the densitometric evaluation ( n = 3). ( B – D and F – H ) BAECs ( B , C , and F – H ) or <t>HAECs</t> ( D ) were transfected with scrambled (control) siRNA or siRNA directed against Gα s , CALCRL, eNOS, or ADM as indicated. In D , eNOS WT or the eNOS phospho-site mutants S1177A and S633A were expressed by lentiviral transduction. Cells were treated with adrenomedullin (ADM, 10 nM, 5 minutes [ B ] or 30 minutes [ D ]) or adrenomedullin-2 (ADM2, 1 nM, 3 minutes, C ) or were exposed to 15 dyn/cm 2 for 30 minutes or for the indicated time periods ( F – H ). Phosphorylation of eNOS at serine 635 and serine 1179 was determined by immunoblotting ( B , C , and F ). Intracellular cAMP concentration ( n = 7, control; n = 6, CALCRL; n = 8, ADM) ( G ) or nitrate and nitrite concentration in the cell culture medium ( n = 6 [ D ]; n = 13, control; n = 4, CALCRL; n = 5, ADM [ H ]) was determined. Bar diagrams in B , C , and F show densitometric evaluation of immunoblots ( n = 3). ( E ) Expression of ADM, CGRP (CALCA), ADM2, and RAMP1–3 RNA in BAECs and <t>HUVECs</t> ( n = 4). Data represent the mean ± SEM; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.00, 2-way ANOVA with Bonferroni’s post hoc test ( A – D , G , and H ) or 1-way ANOVA with Tukey’s post hoc test ( F ).
    Huvecs, supplied by Lonza, used in various techniques. Bioz Stars score: 97/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Shear stress–induced endothelial adrenomedullin signaling regulates vascular tone and blood pressure"

    Article Title: Shear stress–induced endothelial adrenomedullin signaling regulates vascular tone and blood pressure

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI123825

    Role of adrenomedullin in flow-induced eNOS regulation. ( A ) BAECs were treated with adrenomedullin (ADM, 10 nM, 5 minutes), calcitonin gene–related peptide (CGRP; 10 nM, 10 minutes), or adrenomedullin-2 (ADM2, 1 nM, 3 minutes), and phosphorylation of eNOS S635 was determined by immunoblotting. Bar diagram shows the densitometric evaluation ( n = 3). ( B – D and F – H ) BAECs ( B , C , and F – H ) or HAECs ( D ) were transfected with scrambled (control) siRNA or siRNA directed against Gα s , CALCRL, eNOS, or ADM as indicated. In D , eNOS WT or the eNOS phospho-site mutants S1177A and S633A were expressed by lentiviral transduction. Cells were treated with adrenomedullin (ADM, 10 nM, 5 minutes [ B ] or 30 minutes [ D ]) or adrenomedullin-2 (ADM2, 1 nM, 3 minutes, C ) or were exposed to 15 dyn/cm 2 for 30 minutes or for the indicated time periods ( F – H ). Phosphorylation of eNOS at serine 635 and serine 1179 was determined by immunoblotting ( B , C , and F ). Intracellular cAMP concentration ( n = 7, control; n = 6, CALCRL; n = 8, ADM) ( G ) or nitrate and nitrite concentration in the cell culture medium ( n = 6 [ D ]; n = 13, control; n = 4, CALCRL; n = 5, ADM [ H ]) was determined. Bar diagrams in B , C , and F show densitometric evaluation of immunoblots ( n = 3). ( E ) Expression of ADM, CGRP (CALCA), ADM2, and RAMP1–3 RNA in BAECs and HUVECs ( n = 4). Data represent the mean ± SEM; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.00, 2-way ANOVA with Bonferroni’s post hoc test ( A – D , G , and H ) or 1-way ANOVA with Tukey’s post hoc test ( F ).
    Figure Legend Snippet: Role of adrenomedullin in flow-induced eNOS regulation. ( A ) BAECs were treated with adrenomedullin (ADM, 10 nM, 5 minutes), calcitonin gene–related peptide (CGRP; 10 nM, 10 minutes), or adrenomedullin-2 (ADM2, 1 nM, 3 minutes), and phosphorylation of eNOS S635 was determined by immunoblotting. Bar diagram shows the densitometric evaluation ( n = 3). ( B – D and F – H ) BAECs ( B , C , and F – H ) or HAECs ( D ) were transfected with scrambled (control) siRNA or siRNA directed against Gα s , CALCRL, eNOS, or ADM as indicated. In D , eNOS WT or the eNOS phospho-site mutants S1177A and S633A were expressed by lentiviral transduction. Cells were treated with adrenomedullin (ADM, 10 nM, 5 minutes [ B ] or 30 minutes [ D ]) or adrenomedullin-2 (ADM2, 1 nM, 3 minutes, C ) or were exposed to 15 dyn/cm 2 for 30 minutes or for the indicated time periods ( F – H ). Phosphorylation of eNOS at serine 635 and serine 1179 was determined by immunoblotting ( B , C , and F ). Intracellular cAMP concentration ( n = 7, control; n = 6, CALCRL; n = 8, ADM) ( G ) or nitrate and nitrite concentration in the cell culture medium ( n = 6 [ D ]; n = 13, control; n = 4, CALCRL; n = 5, ADM [ H ]) was determined. Bar diagrams in B , C , and F show densitometric evaluation of immunoblots ( n = 3). ( E ) Expression of ADM, CGRP (CALCA), ADM2, and RAMP1–3 RNA in BAECs and HUVECs ( n = 4). Data represent the mean ± SEM; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.00, 2-way ANOVA with Bonferroni’s post hoc test ( A – D , G , and H ) or 1-way ANOVA with Tukey’s post hoc test ( F ).

    Techniques Used: Flow Cytometry, Transfection, Transduction, Concentration Assay, Cell Culture, Western Blot, Expressing

    2) Product Images from "Optimal Hypoxia Regulates Human iPSC-Derived Liver Bud Differentiation through Intercellular TGFB Signaling"

    Article Title: Optimal Hypoxia Regulates Human iPSC-Derived Liver Bud Differentiation through Intercellular TGFB Signaling

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2018.06.015

    Hif1a Is Positively Correlated with Tgfbs and Biliary Markers and Negatively Correlated with Hepatocyte Markers in Mouse Liver Development (A) Microarray gene expression analysis of mouse fetal liver from E9.5 to 17.5. (B) Immunofluorescence staining for HIF1A (red), HIF2A (red), DLK1 (green), and nuclei (blue, DAPI) in E10.5 mouse liver. Scale bar, 100 μm (upper) or 20 μm (lower). (C) Correlation analysis of hypoxia- ( Hif1a ), hepatocyte- ( Alb and Rbp4 ), and cholangiocyte- (others) associated markers in mouse livers from E9.5 to 8-week-old mice. (D) hiPSC-LBs cultured for 10 days (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar, 250 μm). (E) ELISA on protein secretion in hiPSC-LBs cultured for 10 days (mean ± SD; n = 15 independent experiments; ∗∗ p
    Figure Legend Snippet: Hif1a Is Positively Correlated with Tgfbs and Biliary Markers and Negatively Correlated with Hepatocyte Markers in Mouse Liver Development (A) Microarray gene expression analysis of mouse fetal liver from E9.5 to 17.5. (B) Immunofluorescence staining for HIF1A (red), HIF2A (red), DLK1 (green), and nuclei (blue, DAPI) in E10.5 mouse liver. Scale bar, 100 μm (upper) or 20 μm (lower). (C) Correlation analysis of hypoxia- ( Hif1a ), hepatocyte- ( Alb and Rbp4 ), and cholangiocyte- (others) associated markers in mouse livers from E9.5 to 8-week-old mice. (D) hiPSC-LBs cultured for 10 days (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar, 250 μm). (E) ELISA on protein secretion in hiPSC-LBs cultured for 10 days (mean ± SD; n = 15 independent experiments; ∗∗ p

    Techniques Used: Microarray, Expressing, Immunofluorescence, Staining, Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    TGFB Signal Inhibition Promotes Hepatocyte Differentiation in Liver Buds (A) Confocal imaging of hiPSC-LBs cultured with various concentrations of A83-01 for 15 days in Excess-hypoxia group (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar from left to right, 250, 100, and 100 μm). (B) Image analysis of HUVEC abundance in hiPSC-LBs cultured with various A83-01 concentrations for 15 days in Excess-hypoxia group. Fluorescence intensity of KO1 protein expression in HUVECs was evaluated as HUVEC abundance in hiPSC-LBs (left: mean ± SD; n = 9–17 independent experiments; ∗∗ p
    Figure Legend Snippet: TGFB Signal Inhibition Promotes Hepatocyte Differentiation in Liver Buds (A) Confocal imaging of hiPSC-LBs cultured with various concentrations of A83-01 for 15 days in Excess-hypoxia group (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar from left to right, 250, 100, and 100 μm). (B) Image analysis of HUVEC abundance in hiPSC-LBs cultured with various A83-01 concentrations for 15 days in Excess-hypoxia group. Fluorescence intensity of KO1 protein expression in HUVECs was evaluated as HUVEC abundance in hiPSC-LBs (left: mean ± SD; n = 9–17 independent experiments; ∗∗ p

    Techniques Used: Inhibition, Imaging, Cell Culture, Fluorescence, Expressing

    TGFB Signals from the Mesenchyme and Endothelium Are Candidate Regulators of O 2 -Dependent Hepatocyte Differentiation in Liver Buds (A) Phase-contrast and confocal images of hiPSC-LBs cultured for 1 (phase) or 5 (confocal) days (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar, 250 μm). (B) Boxplots of TGFB family gene expression in hiPSC-LBs cultured for 5 and 15 days. The error bars represent the maximum and minimum values; n = 9 (day 5) and 10 (day 15) independent experiments; ∗ p
    Figure Legend Snippet: TGFB Signals from the Mesenchyme and Endothelium Are Candidate Regulators of O 2 -Dependent Hepatocyte Differentiation in Liver Buds (A) Phase-contrast and confocal images of hiPSC-LBs cultured for 1 (phase) or 5 (confocal) days (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar, 250 μm). (B) Boxplots of TGFB family gene expression in hiPSC-LBs cultured for 5 and 15 days. The error bars represent the maximum and minimum values; n = 9 (day 5) and 10 (day 15) independent experiments; ∗ p

    Techniques Used: Cell Culture, Expressing

    3) Product Images from "EphA1 activation promotes the homing of endothelial progenitor cells to hepatocellular carcinoma for tumor neovascularization through the SDF-1/CXCR4 signaling pathway"

    Article Title: EphA1 activation promotes the homing of endothelial progenitor cells to hepatocellular carcinoma for tumor neovascularization through the SDF-1/CXCR4 signaling pathway

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-016-0339-6

    EphA1 regulates EPC proangiogenic potency in a paracrine fashion in vitro. a A1: WB assay showing the EphA1 protein expression in HCC cells. A2: EPC incorporation into HUVECs, EPC’s uptake of DiI-ac-LDL ( red ) together with HUVEC tube formation ( blue ). A3: EPC incorporation assay analysis data (** P
    Figure Legend Snippet: EphA1 regulates EPC proangiogenic potency in a paracrine fashion in vitro. a A1: WB assay showing the EphA1 protein expression in HCC cells. A2: EPC incorporation into HUVECs, EPC’s uptake of DiI-ac-LDL ( red ) together with HUVEC tube formation ( blue ). A3: EPC incorporation assay analysis data (** P

    Techniques Used: In Vitro, Western Blot, Expressing

    4) Product Images from "LncRNA VEAL2 regulates PRKCB2 to modulate endothelial permeability in diabetic retinopathy"

    Article Title: LncRNA VEAL2 regulates PRKCB2 to modulate endothelial permeability in diabetic retinopathy

    Journal: The EMBO Journal

    doi: 10.15252/embj.2020107134

    Overexpression and knockdown of VEAL2 regulate migration and proliferation in HUVECs Representative images showing wound closure rate in overexpressed VEAL2 and control plasmid‐transfected HUVEC monolayer at 0, 9, and 24 h post‐scratch. Images taken at 10× magnification with scale bar representing 50 μm. Dot plot representing wound closure rate at 0, 9, and 24 h post‐scratch in control cells and veal2 ‐overexpressed HUVEC monolayer. Data from 3 different technical replicates of 3 biological replicates are presented. Data are plotted as individual values; the middle bar represents the mean, and the error bar represents ± standard deviation. Representative images showing wound closure rate in control siRNA‐ and VEAL2 siRNA‐transfected HUVEC monolayer at 0, 9, and 24 h post‐scratch. Images taken at 10× magnification with scale bar representing 50 μm. Dot plot representing wound closure rate at initial time, 9, and 24 h post‐scratch in control siRNA‐ and VEAL2 siRNA‐transfected HUVEC monolayer. Data from 3 different technical replicates of 3 biological replicates are presented. Data are plotted as individual values; the middle bar represents the mean, and the error bar represents ± standard deviation. smFISH of VEAL2 in HUVECs transfected with control siRNA and VEAL2 siRNA shows specificity of cytoplasmic signal of VEAL2 . VEAL2 in CAL Fluor Red (610 nM). Merged image for VEAL2 and DAPI. Magnification‐100× and scale bar‐5 μm. Data information: All the experiments N ≥ 3. ** P ‐value
    Figure Legend Snippet: Overexpression and knockdown of VEAL2 regulate migration and proliferation in HUVECs Representative images showing wound closure rate in overexpressed VEAL2 and control plasmid‐transfected HUVEC monolayer at 0, 9, and 24 h post‐scratch. Images taken at 10× magnification with scale bar representing 50 μm. Dot plot representing wound closure rate at 0, 9, and 24 h post‐scratch in control cells and veal2 ‐overexpressed HUVEC monolayer. Data from 3 different technical replicates of 3 biological replicates are presented. Data are plotted as individual values; the middle bar represents the mean, and the error bar represents ± standard deviation. Representative images showing wound closure rate in control siRNA‐ and VEAL2 siRNA‐transfected HUVEC monolayer at 0, 9, and 24 h post‐scratch. Images taken at 10× magnification with scale bar representing 50 μm. Dot plot representing wound closure rate at initial time, 9, and 24 h post‐scratch in control siRNA‐ and VEAL2 siRNA‐transfected HUVEC monolayer. Data from 3 different technical replicates of 3 biological replicates are presented. Data are plotted as individual values; the middle bar represents the mean, and the error bar represents ± standard deviation. smFISH of VEAL2 in HUVECs transfected with control siRNA and VEAL2 siRNA shows specificity of cytoplasmic signal of VEAL2 . VEAL2 in CAL Fluor Red (610 nM). Merged image for VEAL2 and DAPI. Magnification‐100× and scale bar‐5 μm. Data information: All the experiments N ≥ 3. ** P ‐value

    Techniques Used: Over Expression, Migration, Plasmid Preparation, Transfection, Standard Deviation

    VEAL2 is involved in diabetic retinopathy and can recover associated microvascular complications Bar graph representing relative expression of VEAL2 (in fold change) in choroid tissue isolated from control and diabetic retinopathy (DR) patients. Data obtained from 8 biological replicates (patients) and represented as individual values with mean fold change ± standard deviation. Bar graph representing relative fold change of VEAL2 expression in blood samples of patients with different diabetic stages with aggravating vascular dysfunctions from diabetic mellitus (DM) to non‐proliferative diabetic retinopathy (NPDR) to proliferative diabetic retinopathy (PDR) compared to control patients. Data were collected from 50 different patients in each condition and represented as individual values with mean fold change ± standard deviation. ROC curve shows sensitivity and specificity of VEAL2 as a diagnostic biomarker for proliferative diabetic retinopathy with endothelial dysfunction. Complementation of VEAL2 and veal2 reverted increased permeability levels in the HUVEC monolayer model for hyperglycemia. Bar graph representing the effect of overexpression of VEAL2 and veal2 on permeability levels in hyperglycemia disease model, measured as efflux of dextran‐conjugated FITC. Data obtained from 3 different biological replicates and plotted as mean percentage fold change values ± standard deviation. Modeling hyperglycemia in HUVEC resulted in dysregulation of junctional assembly of CDH5 and CTNNB1 proteins and increased membrane localization of PRKCB protein. Complementation of VEAL2 and veal2 in hyperglycemic conditions reverted junctional disassembly of CDH5 and CTNNB1 and also kept PRKCB in cytoplasm to mitigate pathological conditions associated with hyperglycemia. (E–H, Q) CDH5 protein, (I–L, R) CTNNB1 protein, and (M–P, S) PRKCB protein. (E–P) Magnification‐60× and scale bar‐15 μm. Arrowheads indicate representation of signals of proteins in HUVECs. (Q–S) Data from cells of different fields of 3 technical replicates of 1 biological replicate are presented as representation. Data are shown as individual values; the middle bar represents the mean, and the error bar represents ± standard deviation. Data information: All the experiments N ≥ 3. ** P ‐value
    Figure Legend Snippet: VEAL2 is involved in diabetic retinopathy and can recover associated microvascular complications Bar graph representing relative expression of VEAL2 (in fold change) in choroid tissue isolated from control and diabetic retinopathy (DR) patients. Data obtained from 8 biological replicates (patients) and represented as individual values with mean fold change ± standard deviation. Bar graph representing relative fold change of VEAL2 expression in blood samples of patients with different diabetic stages with aggravating vascular dysfunctions from diabetic mellitus (DM) to non‐proliferative diabetic retinopathy (NPDR) to proliferative diabetic retinopathy (PDR) compared to control patients. Data were collected from 50 different patients in each condition and represented as individual values with mean fold change ± standard deviation. ROC curve shows sensitivity and specificity of VEAL2 as a diagnostic biomarker for proliferative diabetic retinopathy with endothelial dysfunction. Complementation of VEAL2 and veal2 reverted increased permeability levels in the HUVEC monolayer model for hyperglycemia. Bar graph representing the effect of overexpression of VEAL2 and veal2 on permeability levels in hyperglycemia disease model, measured as efflux of dextran‐conjugated FITC. Data obtained from 3 different biological replicates and plotted as mean percentage fold change values ± standard deviation. Modeling hyperglycemia in HUVEC resulted in dysregulation of junctional assembly of CDH5 and CTNNB1 proteins and increased membrane localization of PRKCB protein. Complementation of VEAL2 and veal2 in hyperglycemic conditions reverted junctional disassembly of CDH5 and CTNNB1 and also kept PRKCB in cytoplasm to mitigate pathological conditions associated with hyperglycemia. (E–H, Q) CDH5 protein, (I–L, R) CTNNB1 protein, and (M–P, S) PRKCB protein. (E–P) Magnification‐60× and scale bar‐15 μm. Arrowheads indicate representation of signals of proteins in HUVECs. (Q–S) Data from cells of different fields of 3 technical replicates of 1 biological replicate are presented as representation. Data are shown as individual values; the middle bar represents the mean, and the error bar represents ± standard deviation. Data information: All the experiments N ≥ 3. ** P ‐value

    Techniques Used: Expressing, Isolation, Standard Deviation, Diagnostic Assay, Biomarker Assay, Permeability, Over Expression

    5) Product Images from "Three-Dimensional Vascularized Lung Cancer-on-a-Chip with Lung Extracellular Matrix Hydrogels for In Vitro Screening"

    Article Title: Three-Dimensional Vascularized Lung Cancer-on-a-Chip with Lung Extracellular Matrix Hydrogels for In Vitro Screening

    Journal: Cancers

    doi: 10.3390/cancers13163930

    Optimization of tumor spheroid formation. ( A ) Fluorescent image of tri-cellular spheroids at 1, 3 and 5 days after seeding. Scale bar: 100 μM. ( B ) Quantification of expression area of A549 cells, HUVECs and HLFs. * p
    Figure Legend Snippet: Optimization of tumor spheroid formation. ( A ) Fluorescent image of tri-cellular spheroids at 1, 3 and 5 days after seeding. Scale bar: 100 μM. ( B ) Quantification of expression area of A549 cells, HUVECs and HLFs. * p

    Techniques Used: Expressing

    Angiogenic sprouting in VLCC. ( A ) Representative confocal microscope images from the top and cross-section of VLCC on day 1, 3, and 5. ( B ) z-stack images on day 5 (red color denotes the HUVECs in the channel, green color denotes the A549 cells in the spheroids, blue color denotes the HLFs in the spheroids, magenta color denotes the HUVECs in the spheroids). Scale bar: 200 μM. ( C ) Quantification of the average length of blood vessels. *** p
    Figure Legend Snippet: Angiogenic sprouting in VLCC. ( A ) Representative confocal microscope images from the top and cross-section of VLCC on day 1, 3, and 5. ( B ) z-stack images on day 5 (red color denotes the HUVECs in the channel, green color denotes the A549 cells in the spheroids, blue color denotes the HLFs in the spheroids, magenta color denotes the HUVECs in the spheroids). Scale bar: 200 μM. ( C ) Quantification of the average length of blood vessels. *** p

    Techniques Used: Microscopy

    6) Product Images from "Surface Tethering of Inflammation-Modulatory Nanostimulators to Stem Cells for Ischemic Muscle Repair"

    Article Title: Surface Tethering of Inflammation-Modulatory Nanostimulators to Stem Cells for Ischemic Muscle Repair

    Journal: ACS nano

    doi: 10.1021/acsnano.9b04926

    In vitro angiogenesis assay with a 3D microvascular device. (A) Schematic illustration of the device fabricated with polydimethylsiloxane using soft lithography. (B) The central portion features five channels. ADSCs in fibrin gels were seeded in the outer channels ① and ⑤; cell culture medium was filled in channels ② and ④; and HUVECs in the fibrin gel were seeded in the center channel ③. (C) Confocal laser scanning microscope images of immunostained HUVECs with CD31 (in green) in channel ③ after 5 days of incubation with the cell culture media only, with untreated ADSCs only, or ADSCs tethered with TNF α -releasing HA-liposomes. Cell nuclei were stained with Hoechst dye (in blue). Scale bar represents 200 μ m. The lower panels display an overview of the selected region. (D) Quantification of the tubule length and (E) the number of interconnected junctions. Data points represent the mean, and error bars represent standard deviations. N = 3, * represents the statistical significant between the conditions indicated. * p
    Figure Legend Snippet: In vitro angiogenesis assay with a 3D microvascular device. (A) Schematic illustration of the device fabricated with polydimethylsiloxane using soft lithography. (B) The central portion features five channels. ADSCs in fibrin gels were seeded in the outer channels ① and ⑤; cell culture medium was filled in channels ② and ④; and HUVECs in the fibrin gel were seeded in the center channel ③. (C) Confocal laser scanning microscope images of immunostained HUVECs with CD31 (in green) in channel ③ after 5 days of incubation with the cell culture media only, with untreated ADSCs only, or ADSCs tethered with TNF α -releasing HA-liposomes. Cell nuclei were stained with Hoechst dye (in blue). Scale bar represents 200 μ m. The lower panels display an overview of the selected region. (D) Quantification of the tubule length and (E) the number of interconnected junctions. Data points represent the mean, and error bars represent standard deviations. N = 3, * represents the statistical significant between the conditions indicated. * p

    Techniques Used: In Vitro, Angiogenesis Assay, Cell Culture, Laser-Scanning Microscopy, Incubation, Staining

    7) Product Images from "Functional characterization of iPSC-derived arterial- and venous-like endothelial cells"

    Article Title: Functional characterization of iPSC-derived arterial- and venous-like endothelial cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-40417-9

    Functional characterization of iPSCs-derived AELCs and VELCs. ( a . 1 ) Representative images of AELCs or VELCs cultured under flow shear stress values for 24 h. Cells were stained for VE-cadherin. ( a . 2 ) Elongation ratio (calculated by the ratio of cell length to width) of AELCs, VELCs, HUAECs and HUVECs cultured under different flow shear stress values for 24 h. Quantifications were done in 3 microfluidic systems per experimental condition (approximately 150 cells were counted per each microfluidic system). Statistical analyses were performed using a Kruskal-Wallis test, followed by a Dunn’s multiple comparisons test. ( b ) Nitric Oxide (NO) production on AELCs, VELCs, HUVECs and HCAECs was measured using the probe DAF-FM. DAF-FM is non-fluorescent until it reacts with NO forming benzotriazole, a fluorescent compound. Fluorescence intensity was analyzed by flow cytometry. In ( a – c ) results are mean ± SEM, n = 3. *p
    Figure Legend Snippet: Functional characterization of iPSCs-derived AELCs and VELCs. ( a . 1 ) Representative images of AELCs or VELCs cultured under flow shear stress values for 24 h. Cells were stained for VE-cadherin. ( a . 2 ) Elongation ratio (calculated by the ratio of cell length to width) of AELCs, VELCs, HUAECs and HUVECs cultured under different flow shear stress values for 24 h. Quantifications were done in 3 microfluidic systems per experimental condition (approximately 150 cells were counted per each microfluidic system). Statistical analyses were performed using a Kruskal-Wallis test, followed by a Dunn’s multiple comparisons test. ( b ) Nitric Oxide (NO) production on AELCs, VELCs, HUVECs and HCAECs was measured using the probe DAF-FM. DAF-FM is non-fluorescent until it reacts with NO forming benzotriazole, a fluorescent compound. Fluorescence intensity was analyzed by flow cytometry. In ( a – c ) results are mean ± SEM, n = 3. *p

    Techniques Used: Functional Assay, Derivative Assay, Cell Culture, Flow Cytometry, Staining, Fluorescence, Cytometry

    Derivation and characterization of VELCs. ( a ) Schematic representation of the protocol. The EPCs were cultured in serum free medium supplemented with VEGF (10 ng/ml) for 10–15 days (4 passages). ( b ) qRT-PCR of general EC markers. Data were normalized by GAPDH and expressed relatively to HUVECs (n = 4). ( c ) FC analyses of EC markers (n = 3). ( d ) Expression and localization of general (CD31, KDR, VE-cadherin, vWF) and specification EC markers (COUP-TFII and Ephrin B2) by immunofluorescence. Functional activity of ECs measured by their capacity to form cord-like structures in Matrigel and incorporate ac-LDL. ( e ) qRT-PCR for venous markers. Data was normalized by GAPDH and expressed relatively to HUVECs (n = 3). ( f ) qRT-PCR of arterial markers. Data were normalized by GAPDH and expressed relatively to HUAECs (n = 3). ( g ) Quantification of COUP-TFII. Images were acquired in 6 different fields that contained an average of 300 cells. ( h ) Flow cytometry analyses of the arterial marker EphB2 in ECs (n = 3). In ( e – h ) statistical analyses were performed by Kruskal-Wallis test followed by a Dunn’s multiple comparisons test. *p
    Figure Legend Snippet: Derivation and characterization of VELCs. ( a ) Schematic representation of the protocol. The EPCs were cultured in serum free medium supplemented with VEGF (10 ng/ml) for 10–15 days (4 passages). ( b ) qRT-PCR of general EC markers. Data were normalized by GAPDH and expressed relatively to HUVECs (n = 4). ( c ) FC analyses of EC markers (n = 3). ( d ) Expression and localization of general (CD31, KDR, VE-cadherin, vWF) and specification EC markers (COUP-TFII and Ephrin B2) by immunofluorescence. Functional activity of ECs measured by their capacity to form cord-like structures in Matrigel and incorporate ac-LDL. ( e ) qRT-PCR for venous markers. Data was normalized by GAPDH and expressed relatively to HUVECs (n = 3). ( f ) qRT-PCR of arterial markers. Data were normalized by GAPDH and expressed relatively to HUAECs (n = 3). ( g ) Quantification of COUP-TFII. Images were acquired in 6 different fields that contained an average of 300 cells. ( h ) Flow cytometry analyses of the arterial marker EphB2 in ECs (n = 3). In ( e – h ) statistical analyses were performed by Kruskal-Wallis test followed by a Dunn’s multiple comparisons test. *p

    Techniques Used: Cell Culture, Quantitative RT-PCR, Expressing, Immunofluorescence, Functional Assay, Activity Assay, Flow Cytometry, Cytometry, Marker

    Derivation and characterization of AELCs. ( a ) Schematic representation of the protocol. EPCs were cultured in serum free medium supplemented with VEGF (50 ng/ml) for 10–15 days (4 passages). ( b ) qRT-PCR of general EC markers. Results were normalized by GAPDH and expressed relatively to HUAECs (n = 4). ( c ) FC analyses of EC markers (n = 3). ( d ) Expression and localization of general (CD31, KDR, VE-cadherin, vWF) as well as EC specification markers (COUP-TFII and Ephrin B2) by immunofluorescence. Functional activity of ECs as measured by their capacity to form cord-like structures in Matrigel and incorporate ac-LDL. ( e ) qRT-PCR of arterial markers (n = 4). Data was normalized by GAPDH and expressed relatively to HUAECs. ( f ) qRT-PCR of venous markers (n = 6). Data was normalized by GAPDH and expressed relatively to HUVECs. ( g ) Flow cytometry analyses of the arterial marker EphB2 (n = 3). ( h ) Quantification of the nuclei positive for COUP-TFII. Images were acquired in 6 different fields that contained an average of 300 cells. In ( e , f , g and h ) statistical analyses was performed by Kruskal-Wallis test followed by a Dunn’s multiple comparisons test. *p
    Figure Legend Snippet: Derivation and characterization of AELCs. ( a ) Schematic representation of the protocol. EPCs were cultured in serum free medium supplemented with VEGF (50 ng/ml) for 10–15 days (4 passages). ( b ) qRT-PCR of general EC markers. Results were normalized by GAPDH and expressed relatively to HUAECs (n = 4). ( c ) FC analyses of EC markers (n = 3). ( d ) Expression and localization of general (CD31, KDR, VE-cadherin, vWF) as well as EC specification markers (COUP-TFII and Ephrin B2) by immunofluorescence. Functional activity of ECs as measured by their capacity to form cord-like structures in Matrigel and incorporate ac-LDL. ( e ) qRT-PCR of arterial markers (n = 4). Data was normalized by GAPDH and expressed relatively to HUAECs. ( f ) qRT-PCR of venous markers (n = 6). Data was normalized by GAPDH and expressed relatively to HUVECs. ( g ) Flow cytometry analyses of the arterial marker EphB2 (n = 3). ( h ) Quantification of the nuclei positive for COUP-TFII. Images were acquired in 6 different fields that contained an average of 300 cells. In ( e , f , g and h ) statistical analyses was performed by Kruskal-Wallis test followed by a Dunn’s multiple comparisons test. *p

    Techniques Used: Cell Culture, Quantitative RT-PCR, Expressing, Immunofluorescence, Functional Assay, Activity Assay, Flow Cytometry, Cytometry, Marker

    8) Product Images from "TRIM14 promotes endothelial activation via activating NF-κB signaling pathway"

    Article Title: TRIM14 promotes endothelial activation via activating NF-κB signaling pathway

    Journal: Journal of Molecular Cell Biology

    doi: 10.1093/jmcb/mjz040

    Knockdown of TRIM14 attenuated the expression of adhesion molecules and cytokines and monocyte adherence to HUVEC. ( A ) HUVECs were transfected with TRIM14 siRNAs or control siRNAs. Twenty-four hours later, the transfected cells were treated with 10 ng/ml TNF-α for 0, 4, 8, and 24 h. Expression of VCAM-1, ICAM-1, E-selectin, and VE-cadherin were determined by western blot analysis. ( B ) Relative fold changes of proteins after 8 h treatment of TNF-α were determined by densitometry and normalized to β-actin. Data are presented as mean ± SD ( n = 3); * P
    Figure Legend Snippet: Knockdown of TRIM14 attenuated the expression of adhesion molecules and cytokines and monocyte adherence to HUVEC. ( A ) HUVECs were transfected with TRIM14 siRNAs or control siRNAs. Twenty-four hours later, the transfected cells were treated with 10 ng/ml TNF-α for 0, 4, 8, and 24 h. Expression of VCAM-1, ICAM-1, E-selectin, and VE-cadherin were determined by western blot analysis. ( B ) Relative fold changes of proteins after 8 h treatment of TNF-α were determined by densitometry and normalized to β-actin. Data are presented as mean ± SD ( n = 3); * P

    Techniques Used: Expressing, Transfection, Western Blot

    Overexpression of TRIM14 increased the expression of adhesion molecules and cytokines and monocyte adherence to HUVEC. ( A ) HUVECs were transiently transfected with Flag-TRIM14 or empty vector for 24 h and then treated with 10 ng/ml TNF-α for 0, 4, 8, and 24 h. Cell lysates were extracted and used to detect the protein levels of VCAM-1, ICAM-1, E-selectin, and VE-cadherin. ( B ) Relative fold changes of proteins after 8 h treatment of TNF-α were determined by densitometry and normalized to β-actin. Data are presented as mean ± SD ( n = 3); * P
    Figure Legend Snippet: Overexpression of TRIM14 increased the expression of adhesion molecules and cytokines and monocyte adherence to HUVEC. ( A ) HUVECs were transiently transfected with Flag-TRIM14 or empty vector for 24 h and then treated with 10 ng/ml TNF-α for 0, 4, 8, and 24 h. Cell lysates were extracted and used to detect the protein levels of VCAM-1, ICAM-1, E-selectin, and VE-cadherin. ( B ) Relative fold changes of proteins after 8 h treatment of TNF-α were determined by densitometry and normalized to β-actin. Data are presented as mean ± SD ( n = 3); * P

    Techniques Used: Over Expression, Expressing, Transfection, Plasmid Preparation

    TRIM14 facilitated IκBα phosphorylation and degradation in activated ECs. ( A ) HUVECs were transiently transfected with 50 ng or 100 ng Flag-TRIM14 or empty vector for 24 h and then treated with TNF-α for 15 min. Cell lysates were collected, and western blots were performed to detect phosphorylation of IκBα and total protein levels of IκBα. ( B ) Relative fold changes of phosphorylated protein was determined by densitometry and normalized to β-actin. Data are presented as mean ± SD ( n = 3); ** P
    Figure Legend Snippet: TRIM14 facilitated IκBα phosphorylation and degradation in activated ECs. ( A ) HUVECs were transiently transfected with 50 ng or 100 ng Flag-TRIM14 or empty vector for 24 h and then treated with TNF-α for 15 min. Cell lysates were collected, and western blots were performed to detect phosphorylation of IκBα and total protein levels of IκBα. ( B ) Relative fold changes of phosphorylated protein was determined by densitometry and normalized to β-actin. Data are presented as mean ± SD ( n = 3); ** P

    Techniques Used: Transfection, Plasmid Preparation, Western Blot

    TRIM14 facilitated the activation of NF-κB signaling pathway. ( A ) HUVECs were transiently transfected with Flag-TRIM14 or Flag vector (control) for 24 h and then stimulated with 10 ng/ml TNF-α for 0, 15, 30, and 60 min. Cell lysates were harvested for detection of the phosphorylated protein levels by western blot. ( B ) Relative fold changes of phosphorylated protein levels after 15 min treatment of TNF-α were determined by densitometry and normalized to its total protein levels. Phosphorylation of p38, p65, c-JUN, ERK1/2, IκBα, IKKα/β, and JNK were determined and quantified. Data are presented as mean ± SD ( n = 3); * P
    Figure Legend Snippet: TRIM14 facilitated the activation of NF-κB signaling pathway. ( A ) HUVECs were transiently transfected with Flag-TRIM14 or Flag vector (control) for 24 h and then stimulated with 10 ng/ml TNF-α for 0, 15, 30, and 60 min. Cell lysates were harvested for detection of the phosphorylated protein levels by western blot. ( B ) Relative fold changes of phosphorylated protein levels after 15 min treatment of TNF-α were determined by densitometry and normalized to its total protein levels. Phosphorylation of p38, p65, c-JUN, ERK1/2, IκBα, IKKα/β, and JNK were determined and quantified. Data are presented as mean ± SD ( n = 3); * P

    Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Western Blot

    The K63-linked ubiquitination of TRIM14 is required for its function. ( A ) HUVECs were stimulated with 10 ng/ml TNF-α for 0, 5, and 15 min. Cell lysates then were immunoprecipitated using TRIM14 antibody, followed by western blot analysis using the indicated antibodies. ( B and C ) HUVECs were transiently transfected with TRIM14, TRIM14 mutant or empty vector for 24 h and then treated with TNF-α for 15 min. Whole-cell lysates were immunoprecipitated with Flag antibody and the precipitates were immunoblotted with TAK1, NEMO, TRAF2, TRAF5, and Ub antibodies ( B ). Cell lysates were collected, and western blots were performed to detect phosphorylation of IκBα and total protein levels of IκBα ( C ). Relative fold changes of phosphorylated protein was determined by densitometry and normalized to β-actin. Data are presented as mean ± SD ( n = 3); ** P
    Figure Legend Snippet: The K63-linked ubiquitination of TRIM14 is required for its function. ( A ) HUVECs were stimulated with 10 ng/ml TNF-α for 0, 5, and 15 min. Cell lysates then were immunoprecipitated using TRIM14 antibody, followed by western blot analysis using the indicated antibodies. ( B and C ) HUVECs were transiently transfected with TRIM14, TRIM14 mutant or empty vector for 24 h and then treated with TNF-α for 15 min. Whole-cell lysates were immunoprecipitated with Flag antibody and the precipitates were immunoblotted with TAK1, NEMO, TRAF2, TRAF5, and Ub antibodies ( B ). Cell lysates were collected, and western blots were performed to detect phosphorylation of IκBα and total protein levels of IκBα ( C ). Relative fold changes of phosphorylated protein was determined by densitometry and normalized to β-actin. Data are presented as mean ± SD ( n = 3); ** P

    Techniques Used: Immunoprecipitation, Western Blot, Transfection, Mutagenesis, Plasmid Preparation

    Expression of TRIM14 in human ECs. ( A ) Whole-cell lysates were isolated from human primary vascular ECs including HAEC, HCAEC, HDMEC, HLMEC, HUVEC, and other human cell lines as indicated and used for western blot assays to detect TRIM14. β-actin served as a loading control. Relative fold changes were determined by densitometry and normalized to β-actin. ( B – G ) HUVECs were treated with 10 ng/ml TNF-α ( B ), 10 ng/ml IL-1β ( D ), or 1 μg/ml LPS ( F ) for 0, 2, 4, 6, 8, 16, and 24 h, respectively. HUVECs were also treated with TNF-α ( C ), IL-1β ( E ), or LPS ( G ) in different doses as indicated for 24 h. Whole-cell lysates were harvested for detection of TRIM14 protein levels, β-actin served as a loading control. Western blot bands were quantified using Gel-Pro Analyzer software and presented as fold changes under the images. * P
    Figure Legend Snippet: Expression of TRIM14 in human ECs. ( A ) Whole-cell lysates were isolated from human primary vascular ECs including HAEC, HCAEC, HDMEC, HLMEC, HUVEC, and other human cell lines as indicated and used for western blot assays to detect TRIM14. β-actin served as a loading control. Relative fold changes were determined by densitometry and normalized to β-actin. ( B – G ) HUVECs were treated with 10 ng/ml TNF-α ( B ), 10 ng/ml IL-1β ( D ), or 1 μg/ml LPS ( F ) for 0, 2, 4, 6, 8, 16, and 24 h, respectively. HUVECs were also treated with TNF-α ( C ), IL-1β ( E ), or LPS ( G ) in different doses as indicated for 24 h. Whole-cell lysates were harvested for detection of TRIM14 protein levels, β-actin served as a loading control. Western blot bands were quantified using Gel-Pro Analyzer software and presented as fold changes under the images. * P

    Techniques Used: Expressing, Isolation, Western Blot, Software

    TRIM14 is a target gene of transcription factor p65. ( A ) Prediction of p65 binding sites in human TRIM14 promoter region using JASPAR database. p65 consensus sequence is shown on the left, and the predicted binding sites are on the right. Nucleotide in red, consensus index value > 60. Capitals, core sequence, highest conserved, consecutive positions of the matrix. ( B ) HUVECs were co-transfected with TRIM14 promoter-reporter vector and different amounts of p65 expression plasmid (0, 30, and 60 ng), and the transfected cells were treated with or without 10 ng/ml TNF-α for 15 min. Relative luciferase activity values are presented as the means ± SEM of three independent experiments. * P
    Figure Legend Snippet: TRIM14 is a target gene of transcription factor p65. ( A ) Prediction of p65 binding sites in human TRIM14 promoter region using JASPAR database. p65 consensus sequence is shown on the left, and the predicted binding sites are on the right. Nucleotide in red, consensus index value > 60. Capitals, core sequence, highest conserved, consecutive positions of the matrix. ( B ) HUVECs were co-transfected with TRIM14 promoter-reporter vector and different amounts of p65 expression plasmid (0, 30, and 60 ng), and the transfected cells were treated with or without 10 ng/ml TNF-α for 15 min. Relative luciferase activity values are presented as the means ± SEM of three independent experiments. * P

    Techniques Used: Binding Assay, Sequencing, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay

    9) Product Images from "Kif26b controls endothelial cell polarity through the Dishevelled/Daam1-dependent planar cell polarity–signaling pathway"

    Article Title: Kif26b controls endothelial cell polarity through the Dishevelled/Daam1-dependent planar cell polarity–signaling pathway

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E14-08-1332

    Kif26b is expressed in ECs and interacts with the Dvl3/Daam1 complex. (A) Quantitative real-time PCR of Kif26b transcripts in HUVECs, HMVECs, HeLa, immortalized HMVECs, and HEK293T (HEK) cells. (B) Endogenous Kif26b appears to colocalize with MTs in ECs. HUVECs immunostained with the antibodies against Kif26b (green) and α-tubulin (red). Bottom, high-magnification images of MTs. Scale bar, 15 μm (top), 5 μm (bottom). (C) Confocal analysis of HUVECs transfected with plasmids coding for Dvl3 with a Myc tag and immunostained with anti-Kif26b (green), anti-Daam1 (red), or anti-Myc (blue) antibodies. Bottom, high-magnification images. Scale bar, 10 μm (top), 3 μm (bottom). (D) Extracts from HeLa cells transfected with each indicated vector were immunoprecipitated with antibody against V5 (for V5-tagged N-MD mutant), Kif26b, or nonspecific immunoglobulin. Immunoprecipitates (IPs) and lysates were then immunoblotted with anti-Myc (for Myc-tagged Kif26b), anti-Kif26b, anti-V5, anti-Daam1, and anti-Dvl3 antibodies. (E) HeLa cells cotransfected with Kif26b-Myc and Dvl3 were treated with either si Control or si Daam1. Lysates were then immunoprecipitated with anti-Kif26b. IPs and lysates were immunoblotted with anti-Myc, anti-Daam1, and anti Dvl3 antibodies.
    Figure Legend Snippet: Kif26b is expressed in ECs and interacts with the Dvl3/Daam1 complex. (A) Quantitative real-time PCR of Kif26b transcripts in HUVECs, HMVECs, HeLa, immortalized HMVECs, and HEK293T (HEK) cells. (B) Endogenous Kif26b appears to colocalize with MTs in ECs. HUVECs immunostained with the antibodies against Kif26b (green) and α-tubulin (red). Bottom, high-magnification images of MTs. Scale bar, 15 μm (top), 5 μm (bottom). (C) Confocal analysis of HUVECs transfected with plasmids coding for Dvl3 with a Myc tag and immunostained with anti-Kif26b (green), anti-Daam1 (red), or anti-Myc (blue) antibodies. Bottom, high-magnification images. Scale bar, 10 μm (top), 3 μm (bottom). (D) Extracts from HeLa cells transfected with each indicated vector were immunoprecipitated with antibody against V5 (for V5-tagged N-MD mutant), Kif26b, or nonspecific immunoglobulin. Immunoprecipitates (IPs) and lysates were then immunoblotted with anti-Myc (for Myc-tagged Kif26b), anti-Kif26b, anti-V5, anti-Daam1, and anti-Dvl3 antibodies. (E) HeLa cells cotransfected with Kif26b-Myc and Dvl3 were treated with either si Control or si Daam1. Lysates were then immunoprecipitated with anti-Kif26b. IPs and lysates were immunoblotted with anti-Myc, anti-Daam1, and anti Dvl3 antibodies.

    Techniques Used: Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Immunoprecipitation, Mutagenesis

    10) Product Images from "NRF2 is a key regulator of endothelial microRNA expression under proatherogenic stimuli"

    Article Title: NRF2 is a key regulator of endothelial microRNA expression under proatherogenic stimuli

    Journal: Cardiovascular Research

    doi: 10.1093/cvr/cvaa219

    NRF2-regulated miRNAs involved in athero sclerosis-related functions. ( A ) IPA’s comparison analysis for HUVECs and HAECs showing the changes in atherosclerosis-related functions. ( B ) IPA’s VEGF pathway from HUVEC data. Stars mark the molecules directly affected by miRNAs. Red marks up-regulation and cyan down-regulation. ( C ) Overview of the observed changes in atherosclerosis context.
    Figure Legend Snippet: NRF2-regulated miRNAs involved in athero sclerosis-related functions. ( A ) IPA’s comparison analysis for HUVECs and HAECs showing the changes in atherosclerosis-related functions. ( B ) IPA’s VEGF pathway from HUVEC data. Stars mark the molecules directly affected by miRNAs. Red marks up-regulation and cyan down-regulation. ( C ) Overview of the observed changes in atherosclerosis context.

    Techniques Used: Indirect Immunoperoxidase Assay

    Global characterization of NRF2-regulated transcriptional mechanisms in human vascular endothelial cells. ( A ) Motif enrichment in up-regulated and down-regulated transcripts from the GRO-seq data in HUVECs, HAECs, HASMCs, and CD14+ macrophages under oxPAPC stimuli. ( B ) Ingenuity pathway upstream regulator analysis of differentially expressed genes (FDR
    Figure Legend Snippet: Global characterization of NRF2-regulated transcriptional mechanisms in human vascular endothelial cells. ( A ) Motif enrichment in up-regulated and down-regulated transcripts from the GRO-seq data in HUVECs, HAECs, HASMCs, and CD14+ macrophages under oxPAPC stimuli. ( B ) Ingenuity pathway upstream regulator analysis of differentially expressed genes (FDR

    Techniques Used:

    Identification of NRF2-regulated miRNome in vascular cells. ( A ) Global expression of HUVEC and HAEC miRNAs, those found in both, and putative and confirmed NRF2-regulated miRNAs. ( B ) Percentages of NRF2-regulated miRNAs of the total miRNA expression in HUVECs and HAECs. For all: n = 2, mean ± SD. Genomic loci of mir-22 ( C ) and mir-106b/25/93 cluster ( D ). Histone and chromatin segmentation data are ENCODE data 32 from UCSC Genome Browser. 24 Chromatin segmentation track shows promoters in red, enhancers in orange, and active chromatin regions in green. AREs are determined using previously published tool. 11 HiC interactions were visualized with WashU Epigenome Browser. 25
    Figure Legend Snippet: Identification of NRF2-regulated miRNome in vascular cells. ( A ) Global expression of HUVEC and HAEC miRNAs, those found in both, and putative and confirmed NRF2-regulated miRNAs. ( B ) Percentages of NRF2-regulated miRNAs of the total miRNA expression in HUVECs and HAECs. For all: n = 2, mean ± SD. Genomic loci of mir-22 ( C ) and mir-106b/25/93 cluster ( D ). Histone and chromatin segmentation data are ENCODE data 32 from UCSC Genome Browser. 24 Chromatin segmentation track shows promoters in red, enhancers in orange, and active chromatin regions in green. AREs are determined using previously published tool. 11 HiC interactions were visualized with WashU Epigenome Browser. 25

    Techniques Used: Expressing

    11) Product Images from "KSHV MicroRNAs Repress Tropomyosin 1 and Increase Anchorage-Independent Growth and Endothelial Tube Formation"

    Article Title: KSHV MicroRNAs Repress Tropomyosin 1 and Increase Anchorage-Independent Growth and Endothelial Tube Formation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0135560

    miR-K2 and miR-K5 down-regulate the expression of HMW-TPM1. HUVECs were transfected with individual KSHV miRNA. After 48 hours, expression of TPM1 and actin was analyzed by quantitative Western blot. (A) Representative images for TPM1 and actin expression are shown. (B) The average change in TPM1 protein expression levels was normalized to actin and to TPM1 levels in cells transfected with miR-Neg (based on four independent experiments). (C) HUVECs were transfected with a LNA control (LNA-Neg), inhibitor of miR-K2 (LNAαK2), or inhibitor of miR-K5 (LNAαK5). At 24 hpt, the cells were infected with KSHV for 48 more hours. After 72 hpt (2 dpi), total cell lysates were harvested and the expression of TPM1 was analyzed by quantitative immunoblot. Results are presented as the average change in TPM1 protein expression levels normalized to actin and relative to the levels of LNA-Neg in de novo infected cells. Average and SD values were calculated from four independent experiments. (D) Relative KSHV miRNA expression level determined by Taqman qPCR in de novo infected HUVEC. (E) Relative protein level changes from cells infected with wild-type KSHV or mutant version lacking miR-K2 (Kd2) or mirR-K5 (Kd5). (F) Immunoblot for data in (E). (G) Relative miRNA level changes from cells infected with mutant viruses used in (E) and (F).
    Figure Legend Snippet: miR-K2 and miR-K5 down-regulate the expression of HMW-TPM1. HUVECs were transfected with individual KSHV miRNA. After 48 hours, expression of TPM1 and actin was analyzed by quantitative Western blot. (A) Representative images for TPM1 and actin expression are shown. (B) The average change in TPM1 protein expression levels was normalized to actin and to TPM1 levels in cells transfected with miR-Neg (based on four independent experiments). (C) HUVECs were transfected with a LNA control (LNA-Neg), inhibitor of miR-K2 (LNAαK2), or inhibitor of miR-K5 (LNAαK5). At 24 hpt, the cells were infected with KSHV for 48 more hours. After 72 hpt (2 dpi), total cell lysates were harvested and the expression of TPM1 was analyzed by quantitative immunoblot. Results are presented as the average change in TPM1 protein expression levels normalized to actin and relative to the levels of LNA-Neg in de novo infected cells. Average and SD values were calculated from four independent experiments. (D) Relative KSHV miRNA expression level determined by Taqman qPCR in de novo infected HUVEC. (E) Relative protein level changes from cells infected with wild-type KSHV or mutant version lacking miR-K2 (Kd2) or mirR-K5 (Kd5). (F) Immunoblot for data in (E). (G) Relative miRNA level changes from cells infected with mutant viruses used in (E) and (F).

    Techniques Used: Expressing, Transfection, Western Blot, Infection, Real-time Polymerase Chain Reaction, Mutagenesis

    miR-K2 indirectly regulates expression levels of HMW-TPM1s. (A) Luciferase reporter carrying the exon-1 and exon-3 of TPM1 gene (pLuc-Exon1-Exon3) was transfected in HEK-293 cells in the presence of miR-K2 mimics, miR-K5 mimics or a mimic control (miR-Neg). Luciferase activity was measured at 24 and 48 hpt. Results are presented as the change in normalized RFU relative to negative-control miRNA (miR-Neg). Average and SD were calculated from three independent experiments. (B) The putative binding site for miR-K2 in the 3'UTR of hnRNP-H1 was introduced downstream of a luciferase reporter (pLuc-hnRNP-H1 K2BS wt). The same oligonucleotide with three base pairs substitutions ( ata ) in the seed-matching region of miR-K2 was also introduced in a luciferase reporter (pLuc-hnRNP-H1 K2BS mut). (C) Co-transfection in HEK-293 of miR-K2 mimics with the luciferase reporter carrying the wild type or mutated sequence of the putative miR-K2 binding site of hnRNP-H1 3’UTR. Luciferase activity was measured 24 and 48 hpt. Results are presented as the change in normalized RFU relative to negative-control miRNA (miR-Neg). Average and SD values were calculated from four independent experiments. (D) HUVECs were transfected with a control (miR-Neg) or miR-K2 mimics and protein levels were measured by quantitative immunoblotting in six experiments. Statistically significant repression (p
    Figure Legend Snippet: miR-K2 indirectly regulates expression levels of HMW-TPM1s. (A) Luciferase reporter carrying the exon-1 and exon-3 of TPM1 gene (pLuc-Exon1-Exon3) was transfected in HEK-293 cells in the presence of miR-K2 mimics, miR-K5 mimics or a mimic control (miR-Neg). Luciferase activity was measured at 24 and 48 hpt. Results are presented as the change in normalized RFU relative to negative-control miRNA (miR-Neg). Average and SD were calculated from three independent experiments. (B) The putative binding site for miR-K2 in the 3'UTR of hnRNP-H1 was introduced downstream of a luciferase reporter (pLuc-hnRNP-H1 K2BS wt). The same oligonucleotide with three base pairs substitutions ( ata ) in the seed-matching region of miR-K2 was also introduced in a luciferase reporter (pLuc-hnRNP-H1 K2BS mut). (C) Co-transfection in HEK-293 of miR-K2 mimics with the luciferase reporter carrying the wild type or mutated sequence of the putative miR-K2 binding site of hnRNP-H1 3’UTR. Luciferase activity was measured 24 and 48 hpt. Results are presented as the change in normalized RFU relative to negative-control miRNA (miR-Neg). Average and SD values were calculated from four independent experiments. (D) HUVECs were transfected with a control (miR-Neg) or miR-K2 mimics and protein levels were measured by quantitative immunoblotting in six experiments. Statistically significant repression (p

    Techniques Used: Expressing, Luciferase, Transfection, Activity Assay, Negative Control, Binding Assay, Cotransfection, Sequencing

    Down-regulation of HMW-TPM1 by miR-K2 and miR-K5 enhances tube formation in HUVECs. Tube formation assays were performed with HUVEC transfected by the indicated RNA. At 36 hpt, HUVECs were seeded on basement membrane matrix extract for 12 hours and stained using calcein AM. The entire well surface was imaged. Whole well images were analyzed using the angiogenesis tube formation module in MetaMorph 7.7 software (A) MetaMorph quantification of total tube length (total microns of vessel length excluding nodes), segments (total number of vessel segments connecting branch points and/or ends), branch points (total number of junctions connecting segments, nodes are not considered branches) and total node area (total square microns of connected junctions) formed by HUVECs transfected with the indicated RNA. Average and SD were calculated from four independent experiments. Statistically significant data (p
    Figure Legend Snippet: Down-regulation of HMW-TPM1 by miR-K2 and miR-K5 enhances tube formation in HUVECs. Tube formation assays were performed with HUVEC transfected by the indicated RNA. At 36 hpt, HUVECs were seeded on basement membrane matrix extract for 12 hours and stained using calcein AM. The entire well surface was imaged. Whole well images were analyzed using the angiogenesis tube formation module in MetaMorph 7.7 software (A) MetaMorph quantification of total tube length (total microns of vessel length excluding nodes), segments (total number of vessel segments connecting branch points and/or ends), branch points (total number of junctions connecting segments, nodes are not considered branches) and total node area (total square microns of connected junctions) formed by HUVECs transfected with the indicated RNA. Average and SD were calculated from four independent experiments. Statistically significant data (p

    Techniques Used: Transfection, Staining, Software

    12) Product Images from "3D Bioprinted perfusable and vascularized breast tumor model for dynamic screening of chemotherapeutics and CAR-T cells"

    Article Title: 3D Bioprinted perfusable and vascularized breast tumor model for dynamic screening of chemotherapeutics and CAR-T cells

    Journal: bioRxiv

    doi: 10.1101/2022.03.15.484485

    Evaluation of spheroid and hydrogel properties for aspiration-assisted bioprinting. Three different tumor spheroids were used to compare the mechanical and structural properties. Tumor spheroids used were spheroids formed by only MDA-MB-231 cells (MDA-MB-231-only), combination of HUVECs and MDA-MB-231s (H231) and combination of HUVECs, MDA-MB-231 and HDFs (H231F). (A1) Tumor spheroids were aspirated with a glass micropipette for 10 min to analyze their structural integrity under aspiration. (A2) Graphical representation of elastic modulus measured for the tumor spheroids ( n =3 for all, p ***
    Figure Legend Snippet: Evaluation of spheroid and hydrogel properties for aspiration-assisted bioprinting. Three different tumor spheroids were used to compare the mechanical and structural properties. Tumor spheroids used were spheroids formed by only MDA-MB-231 cells (MDA-MB-231-only), combination of HUVECs and MDA-MB-231s (H231) and combination of HUVECs, MDA-MB-231 and HDFs (H231F). (A1) Tumor spheroids were aspirated with a glass micropipette for 10 min to analyze their structural integrity under aspiration. (A2) Graphical representation of elastic modulus measured for the tumor spheroids ( n =3 for all, p ***

    Techniques Used: Multiple Displacement Amplification

    13) Product Images from "Desert Hedgehog-driven endothelium integrity is enhanced by Gas1 but negatively regulated by Cdon"

    Article Title: Desert Hedgehog-driven endothelium integrity is enhanced by Gas1 but negatively regulated by Cdon

    Journal: bioRxiv

    doi: 10.1101/2020.04.20.050542

    ECs express Cdon, Gas1 and Hhip. ( A ) Cdon, Boc, Gas1 and Hhip mRNA expression was evaluated via RT-PCR HUVECs, HMVECs-D and HBMECs. ( B ) Heart cross section from wild type mice were co-immunostained with anti-Cdon (in red) or anti-Gas1 (in red) antibodies together with anti-CD31 (in green) antibodies to identify Cdon or Gas1 expression in ECs respectively. ( C ) HeLa were co-transfected with Gas1 and myc-tagged Dhh encoding vectors. Gas1 interaction with Dhh was evaluated by co-immunoprecipitation assay. ( D ) HeLa were co-transfected with Cdon and myc-tagged Dhh encoding vectors. Cdon interaction with Dhh was evaluated by co-immunoprecipitation assay. ( E ) HeLa were co-transfected with Cdon, Ptch1 and Dhh encoding vectors. Cdon interaction with Dhh was evaluated by co-immunoprecipitation assay.
    Figure Legend Snippet: ECs express Cdon, Gas1 and Hhip. ( A ) Cdon, Boc, Gas1 and Hhip mRNA expression was evaluated via RT-PCR HUVECs, HMVECs-D and HBMECs. ( B ) Heart cross section from wild type mice were co-immunostained with anti-Cdon (in red) or anti-Gas1 (in red) antibodies together with anti-CD31 (in green) antibodies to identify Cdon or Gas1 expression in ECs respectively. ( C ) HeLa were co-transfected with Gas1 and myc-tagged Dhh encoding vectors. Gas1 interaction with Dhh was evaluated by co-immunoprecipitation assay. ( D ) HeLa were co-transfected with Cdon and myc-tagged Dhh encoding vectors. Cdon interaction with Dhh was evaluated by co-immunoprecipitation assay. ( E ) HeLa were co-transfected with Cdon, Ptch1 and Dhh encoding vectors. Cdon interaction with Dhh was evaluated by co-immunoprecipitation assay.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Transfection, Co-Immunoprecipitation Assay

    Cdon deficiency in ECs prevents Il1β-induced BBB disruption. HUVECs ( A ) and HBMECs ( B ) were treated or not with 10 ng/mL Il1β for 6 hours. Cdon mRNA expression was quantified by via RT-qPCR. Experiments were repeated 3 times, each experiment included triplicates. ( C-F ) Both Cdh5-Cre ERT2 ; Cdon Flox/Flox (Cdon ECKO ) and Cdon Flox/Flox (control) mice were administered in the cerebral cortex with adenoviruses encoding Il1β (n=7 and 5 mice respectively). Mice were sacrificed 7 days later. ( C ) Brain sagittal sections were immunostained with anti-Cdh5 (in green), anti-Fibrinogen (in red) or anti-IgG (in red) antibodies. Representative confocal images are shown. ( D ) Cdh5 expression was quantified as the Cdh5+ surface area. ( E ) Fibrinogen extravasation was quantified as the fibrinogen+ surface area. ( F ) IgG extravasation was quantified as the fibrinogen+ surface area. *: p≤0.05; **: p≤0.01; ***: p≤0.001. Mann Withney test.
    Figure Legend Snippet: Cdon deficiency in ECs prevents Il1β-induced BBB disruption. HUVECs ( A ) and HBMECs ( B ) were treated or not with 10 ng/mL Il1β for 6 hours. Cdon mRNA expression was quantified by via RT-qPCR. Experiments were repeated 3 times, each experiment included triplicates. ( C-F ) Both Cdh5-Cre ERT2 ; Cdon Flox/Flox (Cdon ECKO ) and Cdon Flox/Flox (control) mice were administered in the cerebral cortex with adenoviruses encoding Il1β (n=7 and 5 mice respectively). Mice were sacrificed 7 days later. ( C ) Brain sagittal sections were immunostained with anti-Cdh5 (in green), anti-Fibrinogen (in red) or anti-IgG (in red) antibodies. Representative confocal images are shown. ( D ) Cdh5 expression was quantified as the Cdh5+ surface area. ( E ) Fibrinogen extravasation was quantified as the fibrinogen+ surface area. ( F ) IgG extravasation was quantified as the fibrinogen+ surface area. *: p≤0.05; **: p≤0.01; ***: p≤0.001. Mann Withney test.

    Techniques Used: Expressing, Quantitative RT-PCR, Mouse Assay

    14) Product Images from "Cell Type-Specific Interferon-γ-mediated Antagonism of KSHV Lytic Replication"

    Article Title: Cell Type-Specific Interferon-γ-mediated Antagonism of KSHV Lytic Replication

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-38870-7

    IFN-γ and TNF-α impair the production of infectious viruses in LECs. Human primary cells (LECs, HUVECs, TIME) were infected with rKSHV.219 at MOI = 3 in the absence/presence of IFN-γ ( A ) or TNF-α ( B ) (0 to 5000 units/ml) for the indicated time period. Infectious viruses in the culture supernatant were determined. The means ± SEM of three independent experiments is plotted. * P
    Figure Legend Snippet: IFN-γ and TNF-α impair the production of infectious viruses in LECs. Human primary cells (LECs, HUVECs, TIME) were infected with rKSHV.219 at MOI = 3 in the absence/presence of IFN-γ ( A ) or TNF-α ( B ) (0 to 5000 units/ml) for the indicated time period. Infectious viruses in the culture supernatant were determined. The means ± SEM of three independent experiments is plotted. * P

    Techniques Used: Infection

    15) Product Images from "Preparation of iPS cell-derived CD31+ endothelial cells using three-dimensional suspension culture"

    Article Title: Preparation of iPS cell-derived CD31+ endothelial cells using three-dimensional suspension culture

    Journal: Regenerative Therapy

    doi: 10.1016/j.reth.2018.06.004

    Pre-vascular network structure formed in co-culture. (A) Either hiPS cell-derived CD31 + cells or HUVECs were co-cultured with NHDFs. Images of the CD31 + cell network structure were collected by ImageXpress Ultra confocal high-content screening system (Molecular Devices) and analysed by MetaXpress software (Molecular Devices). Scale bars = 400 μm. (B) The software scored total tube length and branch point of the CD31 + cell network structure. (C) Comparison of cell proliferation between hiPS cell-derived CD31 + cells and HUVECs. Growth rate was calculated by comparing the total cell number on day 0 with that on day 3. Values are shown as mean ± standard deviation for three separate experiments. Values are shown as mean ± standard deviation for five separate experiments. NS, not significant.
    Figure Legend Snippet: Pre-vascular network structure formed in co-culture. (A) Either hiPS cell-derived CD31 + cells or HUVECs were co-cultured with NHDFs. Images of the CD31 + cell network structure were collected by ImageXpress Ultra confocal high-content screening system (Molecular Devices) and analysed by MetaXpress software (Molecular Devices). Scale bars = 400 μm. (B) The software scored total tube length and branch point of the CD31 + cell network structure. (C) Comparison of cell proliferation between hiPS cell-derived CD31 + cells and HUVECs. Growth rate was calculated by comparing the total cell number on day 0 with that on day 3. Values are shown as mean ± standard deviation for three separate experiments. Values are shown as mean ± standard deviation for five separate experiments. NS, not significant.

    Techniques Used: Co-Culture Assay, Derivative Assay, Cell Culture, High Content Screening, Software, Standard Deviation

    16) Product Images from "A small molecule targeting ALK1 prevents Notch cooperativity and inhibits functional angiogenesis"

    Article Title: A small molecule targeting ALK1 prevents Notch cooperativity and inhibits functional angiogenesis

    Journal: Angiogenesis

    doi: 10.1007/s10456-014-9457-y

    K02288 inhibits BMP9-ALK1 signalling in HUVECs. a HUVECs were seeded onto BSA- or Dll4-coated plates and treated with the indicated inhibitors and 10 ng/mL BMP9 for 1 h before collecting and analysing by Western blot. b HUVECs on uncoated plates were treated as indicated with 1 ng/mL BMP9 in the presence or absence of 100 ng/mL ALK1-Fc and similarly analysed by Western blot. c HUVECs on uncoated plates were starved in low serum medium (EGM2 without serum) for 6 h after which the medium was replaced with complete EGM2 in the presence or absence of the indicated inhibitors. Samples were analysed by Western blot. d – f Expression levels of BMP-responsive ( d ), Notch-responsive ( e ) and tip cell-specific ( f ) genes were determined in HUVECs seeded onto BSA- or Dll4-coated plates, 4 h after treatment with 1 μM K02288 and 1 ng/mL BMP9 ( d ) or 10 ng/mL BMP9 ( e , f ). g HUVECs were transiently transfected with a RBPJ-responsive luciferase construct for 24 h, re-plated onto BSA- or Dll4-coated plates in the presence or absence of K02288 and luciferase activity determined after 24 h
    Figure Legend Snippet: K02288 inhibits BMP9-ALK1 signalling in HUVECs. a HUVECs were seeded onto BSA- or Dll4-coated plates and treated with the indicated inhibitors and 10 ng/mL BMP9 for 1 h before collecting and analysing by Western blot. b HUVECs on uncoated plates were treated as indicated with 1 ng/mL BMP9 in the presence or absence of 100 ng/mL ALK1-Fc and similarly analysed by Western blot. c HUVECs on uncoated plates were starved in low serum medium (EGM2 without serum) for 6 h after which the medium was replaced with complete EGM2 in the presence or absence of the indicated inhibitors. Samples were analysed by Western blot. d – f Expression levels of BMP-responsive ( d ), Notch-responsive ( e ) and tip cell-specific ( f ) genes were determined in HUVECs seeded onto BSA- or Dll4-coated plates, 4 h after treatment with 1 μM K02288 and 1 ng/mL BMP9 ( d ) or 10 ng/mL BMP9 ( e , f ). g HUVECs were transiently transfected with a RBPJ-responsive luciferase construct for 24 h, re-plated onto BSA- or Dll4-coated plates in the presence or absence of K02288 and luciferase activity determined after 24 h

    Techniques Used: Western Blot, Expressing, Transfection, Luciferase, Construct, Activity Assay

    17) Product Images from "Augmentation of radiation response by motesanib, a multikinase inhibitor that targets vascular endothelial growth factor receptors"

    Article Title: Augmentation of radiation response by motesanib, a multikinase inhibitor that targets vascular endothelial growth factor receptors

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    doi: 10.1158/1078-0432.CCR-09-3385

    Motesanib in vitro activity on VEGFR2 signaling and interaction with radiation. (A) Impact of motesanib on VEGF-stimulated HUVEC proliferation. Cells were grown in EBM-2 basal medium with 2% FBS, without exogenous growth factors. VEGF stimulation was
    Figure Legend Snippet: Motesanib in vitro activity on VEGFR2 signaling and interaction with radiation. (A) Impact of motesanib on VEGF-stimulated HUVEC proliferation. Cells were grown in EBM-2 basal medium with 2% FBS, without exogenous growth factors. VEGF stimulation was

    Techniques Used: In Vitro, Activity Assay

    18) Product Images from "Biodegradable organic acid-crosslinked alkali-treated gelatins with anti-thrombogenic and endothelialization properties"

    Article Title: Biodegradable organic acid-crosslinked alkali-treated gelatins with anti-thrombogenic and endothelialization properties

    Journal: Science and Technology of Advanced Materials

    doi: 10.1088/1468-6996/13/6/064215

    Surface density of HUVECs on the AlGelatin matrices at different crosslinker concentrations, after 1 day (a) or 7 day (b) culture in EBM-2. Error bars represent the standard deviations ( n = 5).
    Figure Legend Snippet: Surface density of HUVECs on the AlGelatin matrices at different crosslinker concentrations, after 1 day (a) or 7 day (b) culture in EBM-2. Error bars represent the standard deviations ( n = 5).

    Techniques Used:

    19) Product Images from "Methacrylated gelatin/hyaluronan-based hydrogels for soft tissue engineering"

    Article Title: Methacrylated gelatin/hyaluronan-based hydrogels for soft tissue engineering

    Journal: Journal of Tissue Engineering

    doi: 10.1177/2041731417744157

    In vitro tube formation—HUVEC were seeded on hydrogels, and tube formation was analyzed after 20 h by staining with Calcein AM: (a) cells on quattroGel show changed morphology and branches (arrows), (b) analysis of mesh area showed increased tube formation on control ( p
    Figure Legend Snippet: In vitro tube formation—HUVEC were seeded on hydrogels, and tube formation was analyzed after 20 h by staining with Calcein AM: (a) cells on quattroGel show changed morphology and branches (arrows), (b) analysis of mesh area showed increased tube formation on control ( p

    Techniques Used: In Vitro, Staining

    20) Product Images from "CDK4/6 inhibitors sensitize gammaherpesvirus-infected tumor cells to T-cell killing by enhancing expression of immune surface molecules"

    Article Title: CDK4/6 inhibitors sensitize gammaherpesvirus-infected tumor cells to T-cell killing by enhancing expression of immune surface molecules

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-022-03400-z

    CDK4/6 inhibitors inhibit growth of KSHV + cells and EBV + cells. JSC-1 ( a ), BCBL-1 ( b ), Akata ( c ), and Raji ( d ) cells were treated in triplicate with tenfold increasing concentrations of Abe, Pal, or Rib, or with RPMI medium control for 72 h. HUVEC ( e ) and HUVEC.BAC16 ( f ) were treated in triplicate with fivefold increasing concentrations of abemaciclib, or with RPMI medium control for 4 or 7 days. The number of viable cells was assessed at different timepoints using CellTiter-Glo® Luminescent Cell Viability Assay. Shown are mean fold change in relative light units (RLU) from 3 experiments. Error bars indicate the standard deviations. Statistics were done using unpaired two-tailed t -test. Asterisks indicate p values: *p
    Figure Legend Snippet: CDK4/6 inhibitors inhibit growth of KSHV + cells and EBV + cells. JSC-1 ( a ), BCBL-1 ( b ), Akata ( c ), and Raji ( d ) cells were treated in triplicate with tenfold increasing concentrations of Abe, Pal, or Rib, or with RPMI medium control for 72 h. HUVEC ( e ) and HUVEC.BAC16 ( f ) were treated in triplicate with fivefold increasing concentrations of abemaciclib, or with RPMI medium control for 4 or 7 days. The number of viable cells was assessed at different timepoints using CellTiter-Glo® Luminescent Cell Viability Assay. Shown are mean fold change in relative light units (RLU) from 3 experiments. Error bars indicate the standard deviations. Statistics were done using unpaired two-tailed t -test. Asterisks indicate p values: *p

    Techniques Used: Cell Viability Assay, Two Tailed Test

    CDK4/6 inhibitors increase cell surface expression of ICAM-1, B7-2, and PD-L1 in KSHV- and EBV- uninfected and infected cells. BJAB, JSC-1, BCBL-1, BC-1, BC-2, Akata, Raji, and Daudi cells were treated with 1 μM Abe, 1 μM Pal, 5 μM Rib, or RPMI medium control for 72 h. HUVEC and HUVEC.BAC16 cells were treated with 0.5 μM Abe, 0.5 μM Pal, 2.5 μM Rib, or EGM-2 medium control for 4 days. Surface expression of ICAM-1, B7-2 and PD-L for the lymphoma cells ( a – c ) and endothelial cells ( d – f ) were analyzed by flow cytometry. Results shown are mean fold changes relative to untreated cells from 3 experiments. Statistics were done using unpaired two-tailed t -test. Asterisks indicate p values: *p
    Figure Legend Snippet: CDK4/6 inhibitors increase cell surface expression of ICAM-1, B7-2, and PD-L1 in KSHV- and EBV- uninfected and infected cells. BJAB, JSC-1, BCBL-1, BC-1, BC-2, Akata, Raji, and Daudi cells were treated with 1 μM Abe, 1 μM Pal, 5 μM Rib, or RPMI medium control for 72 h. HUVEC and HUVEC.BAC16 cells were treated with 0.5 μM Abe, 0.5 μM Pal, 2.5 μM Rib, or EGM-2 medium control for 4 days. Surface expression of ICAM-1, B7-2 and PD-L for the lymphoma cells ( a – c ) and endothelial cells ( d – f ) were analyzed by flow cytometry. Results shown are mean fold changes relative to untreated cells from 3 experiments. Statistics were done using unpaired two-tailed t -test. Asterisks indicate p values: *p

    Techniques Used: Expressing, Infection, Flow Cytometry, Two Tailed Test

    21) Product Images from "Sox9 regulates cell state and activity of embryonic mouse mammary progenitor cells"

    Article Title: Sox9 regulates cell state and activity of embryonic mouse mammary progenitor cells

    Journal: Communications Biology

    doi: 10.1038/s42003-018-0215-3

    eMPCs harbour varying potential for differentiation into mesodermal lineage. a In vitro differentiation of eMPC clones and positive control (MSC) with adipogenic stimuli. Neutral lipid staining merged with bright-field image shows accumulation of lipid droplets (red) within cells after 6 days of induction. Scale bar, 100 μm. b Vasculogenesis assay results of eMPCs and positive control (HUVEC). Tubes formed in medium with growth factors (EGM) and without growth factors were stained with Calcein AM (white) after 24 h incubation. The total area of networks (blue, yellow and green lines) was analysed and presented in box plots with whiskers denoting minimum and maximum values ( n = 4 mean ± s.e.m.). Statistical significance was computed using one-way analysis of variance (ANOVA) and Dunnett’s multiple comparisons test as **** P ≤ 0.0001, *** P ≤ 0.001, NS = no significance. Two-tailed P values are ePool EGM 0.0001, e1 EGM 0.0001, e2 EGM 0.1607, eG1 EGM 0.9994, eG2 EGM 0.0005, MSC EGM 0.9999 and HUVEC EGM 0.0001, when compared to control HUVEC EBM. Scale bar, 200 μm. eMPC, embryonic mammary progenitor cell; MSC, mesenchymal stem cell; HUVEC, human umbilical vein endothelial cells; EGM, endothelial cell growth media; EBM, endothelial basal growth media
    Figure Legend Snippet: eMPCs harbour varying potential for differentiation into mesodermal lineage. a In vitro differentiation of eMPC clones and positive control (MSC) with adipogenic stimuli. Neutral lipid staining merged with bright-field image shows accumulation of lipid droplets (red) within cells after 6 days of induction. Scale bar, 100 μm. b Vasculogenesis assay results of eMPCs and positive control (HUVEC). Tubes formed in medium with growth factors (EGM) and without growth factors were stained with Calcein AM (white) after 24 h incubation. The total area of networks (blue, yellow and green lines) was analysed and presented in box plots with whiskers denoting minimum and maximum values ( n = 4 mean ± s.e.m.). Statistical significance was computed using one-way analysis of variance (ANOVA) and Dunnett’s multiple comparisons test as **** P ≤ 0.0001, *** P ≤ 0.001, NS = no significance. Two-tailed P values are ePool EGM 0.0001, e1 EGM 0.0001, e2 EGM 0.1607, eG1 EGM 0.9994, eG2 EGM 0.0005, MSC EGM 0.9999 and HUVEC EGM 0.0001, when compared to control HUVEC EBM. Scale bar, 200 μm. eMPC, embryonic mammary progenitor cell; MSC, mesenchymal stem cell; HUVEC, human umbilical vein endothelial cells; EGM, endothelial cell growth media; EBM, endothelial basal growth media

    Techniques Used: In Vitro, Clone Assay, Positive Control, Staining, Incubation, Two Tailed Test

    22) Product Images from "The nucleus of endothelial cell as a sensor of blood flow direction"

    Article Title: The nucleus of endothelial cell as a sensor of blood flow direction

    Journal: Biology Open

    doi: 10.1242/bio.20134622

    The actin cytoskeleton blocks polarization against the flow in non-confluent endothelial cells. (a) The device designed for controlled addition of pharmacological reagents without changing shear applied to ECs. Plain medium is fed from inlet 1 (normally open); medium with reagent is fed from inlet 2 (normally blocked); outlet 2 (normally open) is used to fill the device, such that a reagent can be rapidly applied when needed, but without its premature leaking into the test channels. Inlets 1 and 2 are equally pressurized, so flow of media with and without a reagent are at the same shear stress. All channels are 73 µm deep. (b,c) Rapid change of the relative positioning of the nucleus and the MTOC in HUVEC exposed to shear stress τ = 7.2 dyn/cm 2 after transient depolymerization of F-actin due to the treatment with 1 µM latrunculin A for 15 min (b) or after inhibition of myosin II due to the treatment with 30 µM blebbistatin for 30 min (c). Inversed fluorescent signal of GFP–α-tubulin is depicted in grey . Red dashed lines outline nuclei. Yellow dots indicate position of MTOCs. Flow is directed from left to right ( blue arrow ). Scale bars: 20 µm. (d,e) Mean degree of cell polarization, β , as a function of time for cells along the upstream ( blue ) and downstream ( red ) sides of the wound. Wounded HUVECs monolayer was exposed to shear stress τ = 7.2 dyn/cm 2 for 310 min (d) or 70 min (e) followed by transient application of 1 µM of latrunculin A for 15 min (d) or blebbistatin for 30 min (e). Error bars are SEM.
    Figure Legend Snippet: The actin cytoskeleton blocks polarization against the flow in non-confluent endothelial cells. (a) The device designed for controlled addition of pharmacological reagents without changing shear applied to ECs. Plain medium is fed from inlet 1 (normally open); medium with reagent is fed from inlet 2 (normally blocked); outlet 2 (normally open) is used to fill the device, such that a reagent can be rapidly applied when needed, but without its premature leaking into the test channels. Inlets 1 and 2 are equally pressurized, so flow of media with and without a reagent are at the same shear stress. All channels are 73 µm deep. (b,c) Rapid change of the relative positioning of the nucleus and the MTOC in HUVEC exposed to shear stress τ = 7.2 dyn/cm 2 after transient depolymerization of F-actin due to the treatment with 1 µM latrunculin A for 15 min (b) or after inhibition of myosin II due to the treatment with 30 µM blebbistatin for 30 min (c). Inversed fluorescent signal of GFP–α-tubulin is depicted in grey . Red dashed lines outline nuclei. Yellow dots indicate position of MTOCs. Flow is directed from left to right ( blue arrow ). Scale bars: 20 µm. (d,e) Mean degree of cell polarization, β , as a function of time for cells along the upstream ( blue ) and downstream ( red ) sides of the wound. Wounded HUVECs monolayer was exposed to shear stress τ = 7.2 dyn/cm 2 for 310 min (d) or 70 min (e) followed by transient application of 1 µM of latrunculin A for 15 min (d) or blebbistatin for 30 min (e). Error bars are SEM.

    Techniques Used: Flow Cytometry, Inhibition

    Hydrodynamic drag mechanically displaces the nucleus downstream, inducing planar polarization. HUVECs were exposed to a short-term hydrodynamic drag from a passing air bubble. (a) The device was designed to introduce an air bubble into microchannels seeded with cells. A constant pressure is maintained at inlet 1 resulting in a shear stress τ = 0.14 dyn/cm 2 in the test channel. Inlet 2 is fed by pressure regulated compressed air, which is used to form a bubble. This bubble invades into test channel and is pinched off by depressurizing the air. All channels are 75 µm deep. (b) Mechanical displacements of nuclei in HUVECs under an advancing air bubble ( boundary seen as a black line ). Direction of the air bubble passage is from top to bottom. Dashed outline shows the positions of nuclei before ( red ) and after ( brown ) passage of the bubble. Inversed fluorescent signal of GFP–α-tubulin is depicted in grey . Yellow dots indicate positions of MTOCs. Scale bar: 30 µm. (c) Mean cell polarizations, mean β , with respect to the direction of passing of the air bubble as a function of time ( green oval ). Control ( orange triangles ): cells exposed to continuous perfusion ( τ = 0.14 dyn/cm 2 ) without bubble. Error bars indicate SEM. (d) Establishment of flow-induced planar cell polarity in endothelial monolayer. Rearward mechanical displacement of nuclei under a direct action of hydrodynamic drag results in consistent polarization of confluent ECs against the flow.
    Figure Legend Snippet: Hydrodynamic drag mechanically displaces the nucleus downstream, inducing planar polarization. HUVECs were exposed to a short-term hydrodynamic drag from a passing air bubble. (a) The device was designed to introduce an air bubble into microchannels seeded with cells. A constant pressure is maintained at inlet 1 resulting in a shear stress τ = 0.14 dyn/cm 2 in the test channel. Inlet 2 is fed by pressure regulated compressed air, which is used to form a bubble. This bubble invades into test channel and is pinched off by depressurizing the air. All channels are 75 µm deep. (b) Mechanical displacements of nuclei in HUVECs under an advancing air bubble ( boundary seen as a black line ). Direction of the air bubble passage is from top to bottom. Dashed outline shows the positions of nuclei before ( red ) and after ( brown ) passage of the bubble. Inversed fluorescent signal of GFP–α-tubulin is depicted in grey . Yellow dots indicate positions of MTOCs. Scale bar: 30 µm. (c) Mean cell polarizations, mean β , with respect to the direction of passing of the air bubble as a function of time ( green oval ). Control ( orange triangles ): cells exposed to continuous perfusion ( τ = 0.14 dyn/cm 2 ) without bubble. Error bars indicate SEM. (d) Establishment of flow-induced planar cell polarity in endothelial monolayer. Rearward mechanical displacement of nuclei under a direct action of hydrodynamic drag results in consistent polarization of confluent ECs against the flow.

    Techniques Used: Introduce, Flow Cytometry

    23) Product Images from "Human adult mesangiogenic progenitor cells reveal an early angiogenic potential, which is lost after mesengenic differentiation"

    Article Title: Human adult mesangiogenic progenitor cells reveal an early angiogenic potential, which is lost after mesengenic differentiation

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-017-0562-x

    Sprouting from 3D spheroids. Substantial differences among cell populations were found in culturing spheroids on Matrigel®. A front of gel invasion was rapidly (24 h) detected in MPC-derived spheroids while spheroids from P2-MSCs and HUVECs showed more compact structures with sharper edges ( a ). After 1 week, MPC spheroids showed loose network structures and sprouting over 250 μm from the edge. Sprouting from P2-MSC spheroids was definitely reduced with only few cells detected within a shorter (35 μm) distance from the edge. Conversely, HUVECs showed no invading capacity ( b ). HUVEC human umbilical vein endothelial cell, MPC mesangiogenic progenitor cell, MSC mesenchymal stromal cell, N.D. not detected
    Figure Legend Snippet: Sprouting from 3D spheroids. Substantial differences among cell populations were found in culturing spheroids on Matrigel®. A front of gel invasion was rapidly (24 h) detected in MPC-derived spheroids while spheroids from P2-MSCs and HUVECs showed more compact structures with sharper edges ( a ). After 1 week, MPC spheroids showed loose network structures and sprouting over 250 μm from the edge. Sprouting from P2-MSC spheroids was definitely reduced with only few cells detected within a shorter (35 μm) distance from the edge. Conversely, HUVECs showed no invading capacity ( b ). HUVEC human umbilical vein endothelial cell, MPC mesangiogenic progenitor cell, MSC mesenchymal stromal cell, N.D. not detected

    Techniques Used: Derivative Assay

    Histological examination of chicken embryo CAM grafts. Application of “no cell” grafts did not alter CAM morphology, allowing clear identification of the three-layer structure formed by endoderm ( ED ), mesoderm ( M ), and ectoderm ( ET ). MPC grafts showed extended areas of Geltrex™ degradation and intense tissue remodeling, with an increased number of newly formed microvessels ( arrows , exploded frame). P2-MSC implants maintained their compact structure and did not interact with CAM; no increment of vascularization was detected. HUVEC grafts revealed Geltrex™ degradation, CAM remodeling, and increased vascularization. Two representative samples for each cell type are displayed. HUVEC human umbilical vein endothelial cell, MPC mesangiogenic progenitor cell, MSC mesenchymal stromal cell
    Figure Legend Snippet: Histological examination of chicken embryo CAM grafts. Application of “no cell” grafts did not alter CAM morphology, allowing clear identification of the three-layer structure formed by endoderm ( ED ), mesoderm ( M ), and ectoderm ( ET ). MPC grafts showed extended areas of Geltrex™ degradation and intense tissue remodeling, with an increased number of newly formed microvessels ( arrows , exploded frame). P2-MSC implants maintained their compact structure and did not interact with CAM; no increment of vascularization was detected. HUVEC grafts revealed Geltrex™ degradation, CAM remodeling, and increased vascularization. Two representative samples for each cell type are displayed. HUVEC human umbilical vein endothelial cell, MPC mesangiogenic progenitor cell, MSC mesenchymal stromal cell

    Techniques Used: Chick Chorioallantoic Membrane Assay

    Ex-ovo CAM assay. Alterations of the vascular network were analyzed in 8-day chicken embryo CAM, 72 h after grafting Geltrex™ droplets containing MPCs, P2-MSCs, and HUVECs. MPC and HUVEC grafts stimulated the formation of a more complex vessel network as revealed by numerous afferent capillaries, while P2-MSCs did not show any significant effect with respect to “no cell” negative controls. Two representative samples for each cell type are displayed ( a ). White crosses , implant graft sites. Quantification of the capillary network surrounding the on-plants confirmed the increased complexity in the tests applying MPCs ( green ) or HUVECs ( pale blue ), in terms of total network length, branching points, number of segments, and mean segment length, with respect to “no cell” control ( black ) or MSCs ( red ) ( b ). * p
    Figure Legend Snippet: Ex-ovo CAM assay. Alterations of the vascular network were analyzed in 8-day chicken embryo CAM, 72 h after grafting Geltrex™ droplets containing MPCs, P2-MSCs, and HUVECs. MPC and HUVEC grafts stimulated the formation of a more complex vessel network as revealed by numerous afferent capillaries, while P2-MSCs did not show any significant effect with respect to “no cell” negative controls. Two representative samples for each cell type are displayed ( a ). White crosses , implant graft sites. Quantification of the capillary network surrounding the on-plants confirmed the increased complexity in the tests applying MPCs ( green ) or HUVECs ( pale blue ), in terms of total network length, branching points, number of segments, and mean segment length, with respect to “no cell” control ( black ) or MSCs ( red ) ( b ). * p

    Techniques Used: Chick Chorioallantoic Membrane Assay

    Cell culture characterization. Phase contrast microscopy of 6-day BM-MNCs in selective culture conditions showed adherent cells characterized by distinctive MPC morphology with round fried egg-shape and sporadic polar elongation ( a ). Flow cytometry showed homogeneous expression of MPC-related markers CD31, CD18, and CD11c and lack of MSC markers STRO-1, CD73, and CD90 ( b ). Immunofluorescence revealed F-actin typical podosome-like distribution ( red ) and intense positive stain for nestin ( green ) ( c ). Nuclei visualization was performed by DAPI staining ( blue ). No difference in nestin expression ( red ) was found between MPCs ( d.1 ) and HUVECs ( d.2 ) while von Willebrand factor ( vWF ) was detected in HUVECs only ( green ) ( d ). One-week MPC mesengenic differentiation (P1-MSCs) produced mixed cultures of flattened elongated multibranched cells with residual highly rifrangent MPCs. Subculturing P1-MSCs for a further week in MesenPRO® RS Medium led to monomorphic cultures of confluent fibroblastoid spindle-shaped cells (P2-MSCs) ( e ). Flow cytometry revealed a standard MSC phenotype for P2-MSCs ( f ). Immunofluorescence showed F-actin reorganization in stress fibers ( red ) and loss of nestin, occasionally expressed by few residual multibranched cells ( green ) ( g ). P2-MSCs were also able to differentiate selectively into adipocytes, as revealed by intracellular lipid droplet accumulation ( red in h.1 ) or osteocytes featuring intense extracellular calcium deposition ( green in h.2 ) ( h ). HUVEC human umbilical vein endothelial cell, MPC mesangiogenic progenitor cell, MSC mesenchymal stromal cell (Color figure online)
    Figure Legend Snippet: Cell culture characterization. Phase contrast microscopy of 6-day BM-MNCs in selective culture conditions showed adherent cells characterized by distinctive MPC morphology with round fried egg-shape and sporadic polar elongation ( a ). Flow cytometry showed homogeneous expression of MPC-related markers CD31, CD18, and CD11c and lack of MSC markers STRO-1, CD73, and CD90 ( b ). Immunofluorescence revealed F-actin typical podosome-like distribution ( red ) and intense positive stain for nestin ( green ) ( c ). Nuclei visualization was performed by DAPI staining ( blue ). No difference in nestin expression ( red ) was found between MPCs ( d.1 ) and HUVECs ( d.2 ) while von Willebrand factor ( vWF ) was detected in HUVECs only ( green ) ( d ). One-week MPC mesengenic differentiation (P1-MSCs) produced mixed cultures of flattened elongated multibranched cells with residual highly rifrangent MPCs. Subculturing P1-MSCs for a further week in MesenPRO® RS Medium led to monomorphic cultures of confluent fibroblastoid spindle-shaped cells (P2-MSCs) ( e ). Flow cytometry revealed a standard MSC phenotype for P2-MSCs ( f ). Immunofluorescence showed F-actin reorganization in stress fibers ( red ) and loss of nestin, occasionally expressed by few residual multibranched cells ( green ) ( g ). P2-MSCs were also able to differentiate selectively into adipocytes, as revealed by intracellular lipid droplet accumulation ( red in h.1 ) or osteocytes featuring intense extracellular calcium deposition ( green in h.2 ) ( h ). HUVEC human umbilical vein endothelial cell, MPC mesangiogenic progenitor cell, MSC mesenchymal stromal cell (Color figure online)

    Techniques Used: Cell Culture, Microscopy, Flow Cytometry, Cytometry, Expressing, Immunofluorescence, Staining, Produced, Subculturing Assay

    Hierarchical clustering analysis. Expression of the selected 29 genes reveals five major clusters and three of them were specific for each cell population. HUVECs exclusively expressed angiogenic-associated cluster ( pale blue ). A cluster of three genes ( CSF1R , SPP1 , and OCT-4A ; green ) was specific for MPCs and silenced after mesengenic differentiation in P2-MSCs, which upregulated the MSC/pericyte-associated cluster ( red ). As expected, MPCs were more hierarchically closely associated with P2-MSCs. HUVEC human umbilical vein endothelial cell, MPC mesangiogenic progenitor cell, MSC mesenchymal stromal cell (Color figure online)
    Figure Legend Snippet: Hierarchical clustering analysis. Expression of the selected 29 genes reveals five major clusters and three of them were specific for each cell population. HUVECs exclusively expressed angiogenic-associated cluster ( pale blue ). A cluster of three genes ( CSF1R , SPP1 , and OCT-4A ; green ) was specific for MPCs and silenced after mesengenic differentiation in P2-MSCs, which upregulated the MSC/pericyte-associated cluster ( red ). As expected, MPCs were more hierarchically closely associated with P2-MSCs. HUVEC human umbilical vein endothelial cell, MPC mesangiogenic progenitor cell, MSC mesenchymal stromal cell (Color figure online)

    Techniques Used: Expressing

    In-vitro angiogenesis-related assays. Freshly isolated MPCs as well as HUVECs revealed substantial Ac-LDL uptake, showing green fluorescence in most adherent cells. P2-MSCs showed no fluorescence ( a ). Quantitative evaluation (percentage of fluorescent areas) confirmed the observation ( b ). MPC transendothelial migration ability was reported in the presence of either FBS gradient or SDF-1β chemoattraction with a number of migrated cells on the outer surface of culture inserts ( c , blue–violet ). Conversely, few migrating P2-MSCs were reported exclusively under FBS gradient and only in part of the samples, compromising statistical significance ( d ) (“n.s.”, p > 0.05). Neither MPCs nor P2-MSCs were able to form CLS in capillary-like tube formation assay ( e , f , g ). Ac-LDL acetylated-low density lipoprotein, FBS fetal bovine serum, HUVEC human umbilical vein endothelial cell, MPC mesangiogenic progenitor cell, MSC mesenchymal stromal cell, N.D. not detected, SDF-1β stromal cell-derived factor 1 beta (Color figure online)
    Figure Legend Snippet: In-vitro angiogenesis-related assays. Freshly isolated MPCs as well as HUVECs revealed substantial Ac-LDL uptake, showing green fluorescence in most adherent cells. P2-MSCs showed no fluorescence ( a ). Quantitative evaluation (percentage of fluorescent areas) confirmed the observation ( b ). MPC transendothelial migration ability was reported in the presence of either FBS gradient or SDF-1β chemoattraction with a number of migrated cells on the outer surface of culture inserts ( c , blue–violet ). Conversely, few migrating P2-MSCs were reported exclusively under FBS gradient and only in part of the samples, compromising statistical significance ( d ) (“n.s.”, p > 0.05). Neither MPCs nor P2-MSCs were able to form CLS in capillary-like tube formation assay ( e , f , g ). Ac-LDL acetylated-low density lipoprotein, FBS fetal bovine serum, HUVEC human umbilical vein endothelial cell, MPC mesangiogenic progenitor cell, MSC mesenchymal stromal cell, N.D. not detected, SDF-1β stromal cell-derived factor 1 beta (Color figure online)

    Techniques Used: In Vitro, Isolation, Fluorescence, Migration, Capillary Tube Formation Assay, Derivative Assay

    Gene expression analysis. Endothelial-associated genes ( a ) were constitutively expressed by HUVECs ( blue ). PECAM and TIE1 were highly expressed in MPCs ( green ) and silenced during mesengenic differentiation. Conversely, P2-MSCs ( red ) activated TEK expression. MPC mesengenic differentiation was accompanied by mesenchymal/pericyte-associated gene ( b ) upregulation and significant reduction of MPC-related genes NES , OCT-4A , and SPP1 ( c ). Cytokine gene expression was not significantly modified following mesengenic induction ( d ). Differences over 1 log were evidenced for ANGPT2 and RANKL when comparing MPCs/P2-MSCs with HUVECs. ** p
    Figure Legend Snippet: Gene expression analysis. Endothelial-associated genes ( a ) were constitutively expressed by HUVECs ( blue ). PECAM and TIE1 were highly expressed in MPCs ( green ) and silenced during mesengenic differentiation. Conversely, P2-MSCs ( red ) activated TEK expression. MPC mesengenic differentiation was accompanied by mesenchymal/pericyte-associated gene ( b ) upregulation and significant reduction of MPC-related genes NES , OCT-4A , and SPP1 ( c ). Cytokine gene expression was not significantly modified following mesengenic induction ( d ). Differences over 1 log were evidenced for ANGPT2 and RANKL when comparing MPCs/P2-MSCs with HUVECs. ** p

    Techniques Used: Expressing, Modification

    24) Product Images from "Endothelial properties of third-trimester amniotic fluid stem cells cultured in hypoxia"

    Article Title: Endothelial properties of third-trimester amniotic fluid stem cells cultured in hypoxia

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-015-0204-0

    In vitro and in vivo endothelial differentiation and miR expression of AFS cells. a AFS cells were seeded over Matrigel-coated wells, and phase-contrast images showed network formation after 24 hours (scale bar = 100 μm). Image analysis of those network structures ( b ) produced values that were compared against standard positive control cells (HUVECs, left white bars ), and statistical significance is shown among AFS cells and control with asterisks, also between different concentrations of oxygen for the same trimester with hashes (n = 10). Immunofluorescence stainings of endothelial network ( c ) showed vWF (green signal) expression (scale bar = 100 μm). d Ability of endothelial cells to intake AcLDL: green vesicles are indicated by white arrows , phase contrast ( left ) and fluorescence detection ( right ) are shown for better appreciation (scale bar = 200 μm). * P
    Figure Legend Snippet: In vitro and in vivo endothelial differentiation and miR expression of AFS cells. a AFS cells were seeded over Matrigel-coated wells, and phase-contrast images showed network formation after 24 hours (scale bar = 100 μm). Image analysis of those network structures ( b ) produced values that were compared against standard positive control cells (HUVECs, left white bars ), and statistical significance is shown among AFS cells and control with asterisks, also between different concentrations of oxygen for the same trimester with hashes (n = 10). Immunofluorescence stainings of endothelial network ( c ) showed vWF (green signal) expression (scale bar = 100 μm). d Ability of endothelial cells to intake AcLDL: green vesicles are indicated by white arrows , phase contrast ( left ) and fluorescence detection ( right ) are shown for better appreciation (scale bar = 200 μm). * P

    Techniques Used: In Vitro, In Vivo, Expressing, Produced, Positive Control, Immunofluorescence, Fluorescence

    25) Product Images from "Nucleoside/nucleotide reverse transcriptase inhibitors attenuate angiogenesis and lymphangiogenesis by impairing receptor tyrosine kinases signalling in endothelial cells) Nucleoside/nucleotide reverse transcriptase inhibitors attenuate angiogenesis and lymphangiogenesis by impairing receptor tyrosine kinases signalling in endothelial cells"

    Article Title: Nucleoside/nucleotide reverse transcriptase inhibitors attenuate angiogenesis and lymphangiogenesis by impairing receptor tyrosine kinases signalling in endothelial cells) Nucleoside/nucleotide reverse transcriptase inhibitors attenuate angiogenesis and lymphangiogenesis by impairing receptor tyrosine kinases signalling in endothelial cells

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.14036

    Effects of NRTI treatment on the proliferation, migration and tube formation of microvascular ECs and microlymphatic ECs. (A) After TDF, AZT or 3TC treatment for 48 h, HMVECs, HUVECs and HDLECs were stained with PI and annexin‐V‐FITC, followed by detection on a flow cytometer. The populations of early apoptotic cells (annexin‐V positive/PI negative) and late apoptotic cells (PI positive) were evaluated as a % of total cells. Data represent the means ± SEM based on five independent experiments. ns, not significant. (B–E) After TDF, AZT or 3TC treatment for 48 h, confluent monolayers of ECs were subjected to a wound healing assay. Representative images of HMVECs and HDLECs are shown in (B) and (D) respectively. Wound closure was quantified as % in (C) and (E). Data represent the means ± SEM based on five independent experiments. Scale bar, 200 μm. * P
    Figure Legend Snippet: Effects of NRTI treatment on the proliferation, migration and tube formation of microvascular ECs and microlymphatic ECs. (A) After TDF, AZT or 3TC treatment for 48 h, HMVECs, HUVECs and HDLECs were stained with PI and annexin‐V‐FITC, followed by detection on a flow cytometer. The populations of early apoptotic cells (annexin‐V positive/PI negative) and late apoptotic cells (PI positive) were evaluated as a % of total cells. Data represent the means ± SEM based on five independent experiments. ns, not significant. (B–E) After TDF, AZT or 3TC treatment for 48 h, confluent monolayers of ECs were subjected to a wound healing assay. Representative images of HMVECs and HDLECs are shown in (B) and (D) respectively. Wound closure was quantified as % in (C) and (E). Data represent the means ± SEM based on five independent experiments. Scale bar, 200 μm. * P

    Techniques Used: Migration, Staining, Flow Cytometry, Cytometry, Wound Healing Assay

    26) Product Images from "Factor VIIa bound to endothelial cell protein C receptor activates protease activated receptor-1 and mediates cell signaling and barrier protection"

    Article Title: Factor VIIa bound to endothelial cell protein C receptor activates protease activated receptor-1 and mediates cell signaling and barrier protection

    Journal: Blood

    doi: 10.1182/blood-2010-09-310706

    FVIIa activation of p44/42 MAPK in endothelial cells . (A-B) Confluent monolayers of HUVECs were treated with FVIIa (10nM) or control vehicle for varying times at 37°C. Cell lysates were subjected to immunoblot analysis with phospho-specific or
    Figure Legend Snippet: FVIIa activation of p44/42 MAPK in endothelial cells . (A-B) Confluent monolayers of HUVECs were treated with FVIIa (10nM) or control vehicle for varying times at 37°C. Cell lysates were subjected to immunoblot analysis with phospho-specific or

    Techniques Used: Activation Assay

    FVIIa cleaves PAR1 on endothelial cells . Confluent monolayers of HUVECs were incubated at 37°C with (A) control buffer (○) or FVIIa (10nM; ●) for varying time periods, (B) varying concentrations of FVIIa for 1 hour, or (C) FVIIa
    Figure Legend Snippet: FVIIa cleaves PAR1 on endothelial cells . Confluent monolayers of HUVECs were incubated at 37°C with (A) control buffer (○) or FVIIa (10nM; ●) for varying time periods, (B) varying concentrations of FVIIa for 1 hour, or (C) FVIIa

    Techniques Used: Incubation

    27) Product Images from "MicroRNA-494 Regulates Endoplasmic Reticulum Stress in Endothelial Cells"

    Article Title: MicroRNA-494 Regulates Endoplasmic Reticulum Stress in Endothelial Cells

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2021.671461

    miR-494 is a negative regulator of ER stress in vitro . Relative mRNA expression of ER stress responsive genes as measured by qRT-PCR. (A,B) DDIT3 (CHOP), (C,D) spliced XBP1 in HUVECs treated with 10 μg/mL TCN 48h after transfection with (A,C) miR-494 mimic or (B,D) miR-494 inhibitor. Gene expression is normalized to GAPDH and mean fold changes compared to control treatments are shown. (E,F) Simple Western blot analysis of HUVECs transfected with miR-494 mimic or control (24 h) (E) or miR-494 inhibitor or control (F) followed by TCN (10 μg/mL) for 24 h. (G) Cell viability in HUVECs as treated in (A) Vertical dotted red line indicates non-adjacent lanes. Graphs are mean + SEM fold changes of biological replicates from n = 3 independent experiments. ∗ P
    Figure Legend Snippet: miR-494 is a negative regulator of ER stress in vitro . Relative mRNA expression of ER stress responsive genes as measured by qRT-PCR. (A,B) DDIT3 (CHOP), (C,D) spliced XBP1 in HUVECs treated with 10 μg/mL TCN 48h after transfection with (A,C) miR-494 mimic or (B,D) miR-494 inhibitor. Gene expression is normalized to GAPDH and mean fold changes compared to control treatments are shown. (E,F) Simple Western blot analysis of HUVECs transfected with miR-494 mimic or control (24 h) (E) or miR-494 inhibitor or control (F) followed by TCN (10 μg/mL) for 24 h. (G) Cell viability in HUVECs as treated in (A) Vertical dotted red line indicates non-adjacent lanes. Graphs are mean + SEM fold changes of biological replicates from n = 3 independent experiments. ∗ P

    Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, Western Blot

    miR-494 regulates target genes in cell survival and DNA replication. (A) Venn diagram showing the number of downregulated target proteins in a Tandem Mass Tag labeled Mass Spectrometry profile from HUVECs treated with TCN or transfected with miR-494 compared to vehicle treatment or control miR respectively. (B) Fold-change (compared to respective controls) of protein or mRNA levels as assessed by Mass Spectrometry or qRT-PCR respectively for the six targets that were downregulated in both TCN and miR-494 groups. All six targets harbor miR-494 binding sites in their 3′UTRs. (C) Representative Simple Western blot showing survivin ( BIRC5 ) and GINS4 levels in HUVECs 24 h after miR-494 transfection followed by TCN treatment (24 h). Right panels show quantitation of biological replicates. ∗ P
    Figure Legend Snippet: miR-494 regulates target genes in cell survival and DNA replication. (A) Venn diagram showing the number of downregulated target proteins in a Tandem Mass Tag labeled Mass Spectrometry profile from HUVECs treated with TCN or transfected with miR-494 compared to vehicle treatment or control miR respectively. (B) Fold-change (compared to respective controls) of protein or mRNA levels as assessed by Mass Spectrometry or qRT-PCR respectively for the six targets that were downregulated in both TCN and miR-494 groups. All six targets harbor miR-494 binding sites in their 3′UTRs. (C) Representative Simple Western blot showing survivin ( BIRC5 ) and GINS4 levels in HUVECs 24 h after miR-494 transfection followed by TCN treatment (24 h). Right panels show quantitation of biological replicates. ∗ P

    Techniques Used: Labeling, Mass Spectrometry, Transfection, Quantitative RT-PCR, Binding Assay, Western Blot, Quantitation Assay

    28) Product Images from "Evaluation of late outgrowth endothelial progenitor cell and umbilical vein endothelial cell responses to thromboresistant collagen-mimetic hydrogels"

    Article Title: Evaluation of late outgrowth endothelial progenitor cell and umbilical vein endothelial cell responses to thromboresistant collagen-mimetic hydrogels

    Journal: Journal of biomedical materials research. Part A

    doi: 10.1002/jbm.a.36045

    Quantitative assessment of the surface cell density of (A) EOCs and (B) HUVECs at 24 h and 72 h. Measurements are expressed as mean ± standard error of mean. Results were obtained from n = 6 independent samples per formulation with 10 images per sample. All values at 24 h for a given cell type were significantly different from the corresponding 72 h values. * significantly different from the 4 mg/mL PEG-Scl2-2 hydrogel at the corresponding time point, p
    Figure Legend Snippet: Quantitative assessment of the surface cell density of (A) EOCs and (B) HUVECs at 24 h and 72 h. Measurements are expressed as mean ± standard error of mean. Results were obtained from n = 6 independent samples per formulation with 10 images per sample. All values at 24 h for a given cell type were significantly different from the corresponding 72 h values. * significantly different from the 4 mg/mL PEG-Scl2-2 hydrogel at the corresponding time point, p

    Techniques Used:

    Representative images of EOCs and HUVECs stained with phalloidin and counterstained with DAPI on PEG-Scl2-2 and PEG-coll hydrogels at 24 h and 72 h. The scale bar in each image series equals 200 μm and applies to all images in the series.
    Figure Legend Snippet: Representative images of EOCs and HUVECs stained with phalloidin and counterstained with DAPI on PEG-Scl2-2 and PEG-coll hydrogels at 24 h and 72 h. The scale bar in each image series equals 200 μm and applies to all images in the series.

    Techniques Used: Staining

    Relative protein expression of the endothelial markers PECAM-1, Col IV, TM, and E-selectin by confluent EOCs and HUVECs cultured on PEG-Scl2-2 hydrogels and PEG-coll controls. For each cell type, n = 3–6 independent samples were analyzed for each hydrogel formulation. Measurements are expressed as mean ± standard error of the mean. * significantly different from the 8 mg/mL PEG-Scl2-2 hydrogel, p
    Figure Legend Snippet: Relative protein expression of the endothelial markers PECAM-1, Col IV, TM, and E-selectin by confluent EOCs and HUVECs cultured on PEG-Scl2-2 hydrogels and PEG-coll controls. For each cell type, n = 3–6 independent samples were analyzed for each hydrogel formulation. Measurements are expressed as mean ± standard error of the mean. * significantly different from the 8 mg/mL PEG-Scl2-2 hydrogel, p

    Techniques Used: Expressing, Cell Culture

    Influence of Scl2-2 concentration on the migration speed of (A) EOCs and (B) HUVECs. For each formulation, n = 110–290 cells were analyzed per cell type. Measurements are expressed as mean ± standard error of mean. * significantly different from the 4 mg/mL PEG-Scl2-2 hydrogel, p
    Figure Legend Snippet: Influence of Scl2-2 concentration on the migration speed of (A) EOCs and (B) HUVECs. For each formulation, n = 110–290 cells were analyzed per cell type. Measurements are expressed as mean ± standard error of mean. * significantly different from the 4 mg/mL PEG-Scl2-2 hydrogel, p

    Techniques Used: Concentration Assay, Migration

    Quantitative assessment of the fraction of surface covered by (A) EOCs and (B) HUVECs at 24 h and 72 h of culture. Measurements are expressed as mean ± standard error of the mean. For each hydrogel type, n = 5–6 independent samples were analyzed. All values at 24 h for a given cell type were significantly different from the corresponding 72 h values. * significantly different from the 4 mg/mL PEG-Scl2-2 hydrogel at the corresponding time point, p
    Figure Legend Snippet: Quantitative assessment of the fraction of surface covered by (A) EOCs and (B) HUVECs at 24 h and 72 h of culture. Measurements are expressed as mean ± standard error of the mean. For each hydrogel type, n = 5–6 independent samples were analyzed. All values at 24 h for a given cell type were significantly different from the corresponding 72 h values. * significantly different from the 4 mg/mL PEG-Scl2-2 hydrogel at the corresponding time point, p

    Techniques Used:

    Relative gene expression of the endothelial markers (A) CD34, (B) VEGFR2, (C) EphrinB2:EphB4, (D) VE-Cadherin, (E) vWF, and (F) NOS3 of confluent EOCs and HUVECs cultured on PEG-Scl2-2 hydrogels and PEG-coll controls. For each cell type, n = 3–6 independent samples were analyzed for each hydrogel formulation. Measurements are expressed as mean ± standard error of the mean. * significantly different from the 8 mg/mL PEG-Scl2-2 hydrogel, p
    Figure Legend Snippet: Relative gene expression of the endothelial markers (A) CD34, (B) VEGFR2, (C) EphrinB2:EphB4, (D) VE-Cadherin, (E) vWF, and (F) NOS3 of confluent EOCs and HUVECs cultured on PEG-Scl2-2 hydrogels and PEG-coll controls. For each cell type, n = 3–6 independent samples were analyzed for each hydrogel formulation. Measurements are expressed as mean ± standard error of the mean. * significantly different from the 8 mg/mL PEG-Scl2-2 hydrogel, p

    Techniques Used: Expressing, Cell Culture

    Integrin α 1 and α 2 subunit assessment by flow cytometry for EOCs and HUVECs. Gray filled curves represent negative controls. Black lined curves represent test samples.
    Figure Legend Snippet: Integrin α 1 and α 2 subunit assessment by flow cytometry for EOCs and HUVECs. Gray filled curves represent negative controls. Black lined curves represent test samples.

    Techniques Used: Flow Cytometry

    29) Product Images from "Arhgef15 Promotes Retinal Angiogenesis by Mediating VEGF-Induced Cdc42 Activation and Potentiating RhoJ Inactivation in Endothelial Cells"

    Article Title: Arhgef15 Promotes Retinal Angiogenesis by Mediating VEGF-Induced Cdc42 Activation and Potentiating RhoJ Inactivation in Endothelial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0045858

    Arhgef15 facilitates actin polymerization and cell motility in ECs. (A) Phase-contrast (PhC) microscopy in cultured HUVECs at 72 h after siRNA transfection or 30 min after Sema3E stimulation. (B) The ratio of collapsing cells in cultured HUVECs. All experiments were repeated at least four times. (C) Confocal microscopy for phalloidin in cultured HUVECs. Note the actin depolymerization after transfection of siRNAs for Arhgef15 and Cdc42, and after Sema3E stimulation. (D) Quantification of the cell surface area in cultured HUVECs. The number of cells analyzed were: si-Ctrl, 151; si-GEF15, 170; si-CDC42, 151; Sema3E, 168. (E) Confocal microscopy for phalloidin in cultured HUVECs. Note the thickening of actin fibers at 24 h after transfection of plasmid vectors expressing Arhgef15 and Cdc42-CA, in contrast to the disruption of actin fibers by RhoJ-WT overexpression. (F) Scratch-wound assay in HUVECs transfected with siRNAs. Note the reduced EC motility by knockdown of Arhgef15 and Cdc42. (G) Quantification of the wound closure. n = 3 per group. (H) Tube formation assay in HUVECs transfected with siRNAs. Note the inhibition of capillary-like network formation by knockdown of Arhgef15 and Cdc42. (I) Quantification of the total tube length. n = 7 per group. Scale bar: 50 µm (A); 10 µm (C and E); 100 µm (F and H). Error bars represent SEM; ** P
    Figure Legend Snippet: Arhgef15 facilitates actin polymerization and cell motility in ECs. (A) Phase-contrast (PhC) microscopy in cultured HUVECs at 72 h after siRNA transfection or 30 min after Sema3E stimulation. (B) The ratio of collapsing cells in cultured HUVECs. All experiments were repeated at least four times. (C) Confocal microscopy for phalloidin in cultured HUVECs. Note the actin depolymerization after transfection of siRNAs for Arhgef15 and Cdc42, and after Sema3E stimulation. (D) Quantification of the cell surface area in cultured HUVECs. The number of cells analyzed were: si-Ctrl, 151; si-GEF15, 170; si-CDC42, 151; Sema3E, 168. (E) Confocal microscopy for phalloidin in cultured HUVECs. Note the thickening of actin fibers at 24 h after transfection of plasmid vectors expressing Arhgef15 and Cdc42-CA, in contrast to the disruption of actin fibers by RhoJ-WT overexpression. (F) Scratch-wound assay in HUVECs transfected with siRNAs. Note the reduced EC motility by knockdown of Arhgef15 and Cdc42. (G) Quantification of the wound closure. n = 3 per group. (H) Tube formation assay in HUVECs transfected with siRNAs. Note the inhibition of capillary-like network formation by knockdown of Arhgef15 and Cdc42. (I) Quantification of the total tube length. n = 7 per group. Scale bar: 50 µm (A); 10 µm (C and E); 100 µm (F and H). Error bars represent SEM; ** P

    Techniques Used: Microscopy, Cell Culture, Transfection, Confocal Microscopy, Plasmid Preparation, Expressing, Over Expression, Scratch Wound Assay Assay, Tube Formation Assay, Inhibition

    30) Product Images from "Mechanical loading of intraluminal pressure mediates wound angiogenesis by regulating the TOCA family of F-BAR proteins"

    Article Title: Mechanical loading of intraluminal pressure mediates wound angiogenesis by regulating the TOCA family of F-BAR proteins

    Journal: Nature Communications

    doi: 10.1038/s41467-022-30197-8

    IP load-induced EC stretching causes detachment of CIP4 and TOCA1 from the leading edge of ECs to inhibit branch elongation. a – d Effects of biaxial stretching on colocalization of Arp2/3 complexes ( a , b ) and CIP4 ( c , d ) with F-actin at leading edges of HUVECs directionally migrating on stretching chambers. Confocal fluorescence images of HUVECs exposed to continuous biaxial stretch for 3 min after being stretched to 10% over 8 min (Stretch) or kept under static conditions (Control). Left upper, F-actin (red); left lower, ARPC2 (Arp2/3, green) ( a ) or CIP4 (green) ( c ). F-actin (upper), ARPC2 or CIP4 (middle), and merged (lower) images of boxed areas are enlarged on the right. In a , c , yellow arrows indicate the direction of cell migration. b , d Quantification of Arp2/3 complexes ( b ) and CIP4 ( d ) colocalized with F-actin at leading edges of HUVECs, as in a , c , respectively. Each dot represents an individual confocal image (blue, Control; red Stretch). Data are means ± s.e.m ( n = 8 regions examined over 2 independent experiments for each). ** p
    Figure Legend Snippet: IP load-induced EC stretching causes detachment of CIP4 and TOCA1 from the leading edge of ECs to inhibit branch elongation. a – d Effects of biaxial stretching on colocalization of Arp2/3 complexes ( a , b ) and CIP4 ( c , d ) with F-actin at leading edges of HUVECs directionally migrating on stretching chambers. Confocal fluorescence images of HUVECs exposed to continuous biaxial stretch for 3 min after being stretched to 10% over 8 min (Stretch) or kept under static conditions (Control). Left upper, F-actin (red); left lower, ARPC2 (Arp2/3, green) ( a ) or CIP4 (green) ( c ). F-actin (upper), ARPC2 or CIP4 (middle), and merged (lower) images of boxed areas are enlarged on the right. In a , c , yellow arrows indicate the direction of cell migration. b , d Quantification of Arp2/3 complexes ( b ) and CIP4 ( d ) colocalized with F-actin at leading edges of HUVECs, as in a , c , respectively. Each dot represents an individual confocal image (blue, Control; red Stretch). Data are means ± s.e.m ( n = 8 regions examined over 2 independent experiments for each). ** p

    Techniques Used: Fluorescence, Migration

    31) Product Images from "Innate immune cell response to host-parasite interaction in a human intestinal tissue microphysiological system"

    Article Title: Innate immune cell response to host-parasite interaction in a human intestinal tissue microphysiological system

    Journal: Science Advances

    doi: 10.1126/sciadv.abm8012

    Integrating primary intestinal epithelial cells in the human intestinal tissue MPS. ( A ) Schematic showing spatial distribution of the intestinal epithelium and endothelium in the intestinal tissue MPS. ( B ) Optimized culture protocol and timeline to set up the intestinal tissue MPS. ( C ) Tubular HUVEC endothelium and primary intestinal epithelium cells as shown by immunostaining of F-actin. ( D ) Differentiated tubular epithelium shows retention of phenotypic characteristics including expression of epithelial cell adhesion protein (i, E-cadherin), enterocyte-specific marker [ii, fatty acid–binding protein 1 (FABP1)], marker for mucin-producing cells (iii, MUC2A), and marker for protein involved in formation of microvilli (villin 1, iv). ( E ) Schematic representing the culture and differentiation of human primary intestinal epithelial cells in Transwells and in the intestinal tissue MPS. ( F and G ) Bar graphs showing differential gene expression in cells isolated from intestinal crypts and in epithelium cultured in the intestinal tissue MPS and Transwells. Genes analyzed include markers associated with the crypt and villus compartment of the intestinal tissue. Values are presented as means ± SD from four independent experiments involving tubular or monolayer epithelium generated from human intestinal organoids (* = Transwell versus lumen, **** P ≤ 0.0001, *** P ≤ 0.001, and ** P ≤ 0.01; # = isolated crypts versus lumen, #### P ≤ 0.0001 and # P ≤ 0.05; = isolated crypts versus Transwell, P ≤ 0.0001, P ≤ 0.001, P ≤ 0.01, and P ≤ 0.05). ns, not significant.
    Figure Legend Snippet: Integrating primary intestinal epithelial cells in the human intestinal tissue MPS. ( A ) Schematic showing spatial distribution of the intestinal epithelium and endothelium in the intestinal tissue MPS. ( B ) Optimized culture protocol and timeline to set up the intestinal tissue MPS. ( C ) Tubular HUVEC endothelium and primary intestinal epithelium cells as shown by immunostaining of F-actin. ( D ) Differentiated tubular epithelium shows retention of phenotypic characteristics including expression of epithelial cell adhesion protein (i, E-cadherin), enterocyte-specific marker [ii, fatty acid–binding protein 1 (FABP1)], marker for mucin-producing cells (iii, MUC2A), and marker for protein involved in formation of microvilli (villin 1, iv). ( E ) Schematic representing the culture and differentiation of human primary intestinal epithelial cells in Transwells and in the intestinal tissue MPS. ( F and G ) Bar graphs showing differential gene expression in cells isolated from intestinal crypts and in epithelium cultured in the intestinal tissue MPS and Transwells. Genes analyzed include markers associated with the crypt and villus compartment of the intestinal tissue. Values are presented as means ± SD from four independent experiments involving tubular or monolayer epithelium generated from human intestinal organoids (* = Transwell versus lumen, **** P ≤ 0.0001, *** P ≤ 0.001, and ** P ≤ 0.01; # = isolated crypts versus lumen, #### P ≤ 0.0001 and # P ≤ 0.05; = isolated crypts versus Transwell, P ≤ 0.0001, P ≤ 0.001, P ≤ 0.01, and P ≤ 0.05). ns, not significant.

    Techniques Used: Immunostaining, Expressing, Marker, Binding Assay, Isolation, Cell Culture, Generated

    Human intestinal tissue MPS for studying innate immune responses to parasitic infection. ( A ) Schematic representation of the design rationale for modeling parasite infection, in the intestinal epithelium, and innate immune cell responses. ( B ) Three-dimensional rendered illustration of the intestinal tissue MPS, within a polydimethylsiloxane (PDMS) device that includes tubular intestinal epithelium and endothelium within an ECM gel. Immune cells are introduced into the lumen of the endothelium for modeling and elucidating innate immune responses to parasite infection of the epithelium. ( C ) Schematic representation describing the experimental approach to set up each component (i to iv) of the MPS used in this study. ( D ) Fluorescence image showing the formation of a confluent Caco-2 intestinal epithelium (magenta) and HUVEC endothelium (yellow). ( E ) The model retains phenotypic characteristics of tight junction markers (i, ZO-1), microvilli markers (ii, villin), and markers of mucus producing goblet cells (iii, MUC2A) for the epithelium. ( F ) HUVEC endothelium in coculture with the epithelium retains expression of endothelial marker (CD31).
    Figure Legend Snippet: Human intestinal tissue MPS for studying innate immune responses to parasitic infection. ( A ) Schematic representation of the design rationale for modeling parasite infection, in the intestinal epithelium, and innate immune cell responses. ( B ) Three-dimensional rendered illustration of the intestinal tissue MPS, within a polydimethylsiloxane (PDMS) device that includes tubular intestinal epithelium and endothelium within an ECM gel. Immune cells are introduced into the lumen of the endothelium for modeling and elucidating innate immune responses to parasite infection of the epithelium. ( C ) Schematic representation describing the experimental approach to set up each component (i to iv) of the MPS used in this study. ( D ) Fluorescence image showing the formation of a confluent Caco-2 intestinal epithelium (magenta) and HUVEC endothelium (yellow). ( E ) The model retains phenotypic characteristics of tight junction markers (i, ZO-1), microvilli markers (ii, villin), and markers of mucus producing goblet cells (iii, MUC2A) for the epithelium. ( F ) HUVEC endothelium in coculture with the epithelium retains expression of endothelial marker (CD31).

    Techniques Used: Infection, Fluorescence, Expressing, Marker

    32) Product Images from "Antisense Oligonucleotides Targeting Y-Box Binding Protein-1 Inhibit Tumor Angiogenesis by Downregulating Bcl-xL-VEGFR2/-Tie Axes"

    Article Title: Antisense Oligonucleotides Targeting Y-Box Binding Protein-1 Inhibit Tumor Angiogenesis by Downregulating Bcl-xL-VEGFR2/-Tie Axes

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2017.09.004

    Involvement of YB-1 in Proliferation, Apoptosis, and Tube Formation in Vascular Endothelial Cells HUVECs and HPAECs were transfected with YB-1 ASO A or control ASO A (5 nM) and subjected to the following analyses. (A) Cell proliferation analysis with the WST-8 assay. *p
    Figure Legend Snippet: Involvement of YB-1 in Proliferation, Apoptosis, and Tube Formation in Vascular Endothelial Cells HUVECs and HPAECs were transfected with YB-1 ASO A or control ASO A (5 nM) and subjected to the following analyses. (A) Cell proliferation analysis with the WST-8 assay. *p

    Techniques Used: Transfection, Allele-specific Oligonucleotide

    YB-1 ASO A -Induced Apoptosis and Inhibition of Tube Formation Are Mediated by Bcl-xL Reduction in Vascular Endothelial Cells (A) Heatmap analysis of cDNA microarray data showing highly downregulated Bcl-xL expression, among Bcl-2 family genes, in HUVECs and HPAECs transfected with YB-1 ASO A (5 nM). (B) Downregulation of Bcl-xL expression was confirmed by qRT-PCR analysis (normalized to values for 18S ). *p
    Figure Legend Snippet: YB-1 ASO A -Induced Apoptosis and Inhibition of Tube Formation Are Mediated by Bcl-xL Reduction in Vascular Endothelial Cells (A) Heatmap analysis of cDNA microarray data showing highly downregulated Bcl-xL expression, among Bcl-2 family genes, in HUVECs and HPAECs transfected with YB-1 ASO A (5 nM). (B) Downregulation of Bcl-xL expression was confirmed by qRT-PCR analysis (normalized to values for 18S ). *p

    Techniques Used: Allele-specific Oligonucleotide, Inhibition, Microarray, Expressing, Transfection, Quantitative RT-PCR

    Construction of YB-1 ASOs and Their Effects on YB-1 mRNA and Protein Expression In Vitro (A) Schematic structures of several types of YB-1 ASOs. (B) T m values of YB-1 ASO duplexes with DNA and RNA target strands. (C) qRT-PCR of human YBX1 mRNA in HUVECs, HPAECs, and MIA PaCa-2 cells transfected with YB-1 ASOs or control ASO (normalized to B2M ). n = 3. (D) Immunoblots for YB-1 protein in HUVECs, HPAECs, and MIA PaCa-2 cells transfected with YB-1 ASOs or control ASO (5 nM). β-actin is the loading control. (E) Effects of i.v. administered YB-1 ASO A , YB-1 ASO L , or control ASO (once per week for 3 weeks at 10 mg/kg body weight) on liver function, evaluated by alanine aminotransferase (ALT) and T. Bil values. *p
    Figure Legend Snippet: Construction of YB-1 ASOs and Their Effects on YB-1 mRNA and Protein Expression In Vitro (A) Schematic structures of several types of YB-1 ASOs. (B) T m values of YB-1 ASO duplexes with DNA and RNA target strands. (C) qRT-PCR of human YBX1 mRNA in HUVECs, HPAECs, and MIA PaCa-2 cells transfected with YB-1 ASOs or control ASO (normalized to B2M ). n = 3. (D) Immunoblots for YB-1 protein in HUVECs, HPAECs, and MIA PaCa-2 cells transfected with YB-1 ASOs or control ASO (5 nM). β-actin is the loading control. (E) Effects of i.v. administered YB-1 ASO A , YB-1 ASO L , or control ASO (once per week for 3 weeks at 10 mg/kg body weight) on liver function, evaluated by alanine aminotransferase (ALT) and T. Bil values. *p

    Techniques Used: Expressing, In Vitro, Allele-specific Oligonucleotide, Quantitative RT-PCR, Transfection, Western Blot

    Knockdown of YB-1/Bcl-xL Downregulates VEGFR2 and Tie-1/-2 Expression in Angiogenic Endothelial Cells in Tumor Microenvironments (A and B) Western blot analysis showing inhibition of VEGFR2 and Tie-1/-2 expression in HUVECs or HPAECs transfected with YB-1 ASO A (5 nM) (A) or Bcl-xL siRNA (20 nM) (B). β-actin is the loading control. (C) Antitumor efficacy of YB-1 ASO A treatment (once per week for 4 weeks at 10 mg/kg body weight) was superior to that of bevacizumab (twice per week for 4 weeks at 10 mg/kg body weight) in mice bearing Suit2-GR xenografts. *p
    Figure Legend Snippet: Knockdown of YB-1/Bcl-xL Downregulates VEGFR2 and Tie-1/-2 Expression in Angiogenic Endothelial Cells in Tumor Microenvironments (A and B) Western blot analysis showing inhibition of VEGFR2 and Tie-1/-2 expression in HUVECs or HPAECs transfected with YB-1 ASO A (5 nM) (A) or Bcl-xL siRNA (20 nM) (B). β-actin is the loading control. (C) Antitumor efficacy of YB-1 ASO A treatment (once per week for 4 weeks at 10 mg/kg body weight) was superior to that of bevacizumab (twice per week for 4 weeks at 10 mg/kg body weight) in mice bearing Suit2-GR xenografts. *p

    Techniques Used: Expressing, Western Blot, Inhibition, Transfection, Allele-specific Oligonucleotide, Mouse Assay

    33) Product Images from "Two faces of bivalent domain regulate VEGFA responsiveness and angiogenesis"

    Article Title: Two faces of bivalent domain regulate VEGFA responsiveness and angiogenesis

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-020-2228-3

    EZH1-induced RNAPII pausing release was required for the activation of bdDEG. a Metagene plot showing RNAPII occupancy of upregulated bdDEG at 0, 1, 4, or 12 h of VEGFA treatment. VEGFA treatment stimulated release of paused RNAPII at 1 h. b The RNAPII PI of downregulated bdDEG (red) and upregulated bdDEG (blue) at hour 0, 1, 4, and 12. c Effect of inhibition of RNAPII pausing release or PRC2 methyltransferase activity on VEGFA activation of bdDEGs, as measured by RT-qPCR. HUVECs were treated with VEGFA and/or small molecule inhibitors (JQ1, flavopiridol, and DZNep) or vehicle (DMSO). Inhibition of RNAPII pause release (JQ1 and flavopiridol) but not PRC2 methyltransferase activity (DZNep) suppressed VEGFA-driven transcriptional activation of the six tested bdDEGs. Plots show mean ± SD; n = 4, two-tailed Student’s t -test: * P
    Figure Legend Snippet: EZH1-induced RNAPII pausing release was required for the activation of bdDEG. a Metagene plot showing RNAPII occupancy of upregulated bdDEG at 0, 1, 4, or 12 h of VEGFA treatment. VEGFA treatment stimulated release of paused RNAPII at 1 h. b The RNAPII PI of downregulated bdDEG (red) and upregulated bdDEG (blue) at hour 0, 1, 4, and 12. c Effect of inhibition of RNAPII pausing release or PRC2 methyltransferase activity on VEGFA activation of bdDEGs, as measured by RT-qPCR. HUVECs were treated with VEGFA and/or small molecule inhibitors (JQ1, flavopiridol, and DZNep) or vehicle (DMSO). Inhibition of RNAPII pause release (JQ1 and flavopiridol) but not PRC2 methyltransferase activity (DZNep) suppressed VEGFA-driven transcriptional activation of the six tested bdDEGs. Plots show mean ± SD; n = 4, two-tailed Student’s t -test: * P

    Techniques Used: Activation Assay, Inhibition, Activity Assay, Quantitative RT-PCR, Two Tailed Test

    34) Product Images from "? Opioids inhibit tumor angiogenesis by suppressing VEGF signaling"

    Article Title: ? Opioids inhibit tumor angiogenesis by suppressing VEGF signaling

    Journal: Scientific Reports

    doi: 10.1038/srep03213

    Inhibitory effects of KOR agonists, U50,488H and TRK820, on HUVEC migration and tube formation. (a) The boyden chamber assay. Inhibition of VEGF-induced chemotaxis was assessed after including DAMGO (10, 30 μM), SNC80 (10, 30 μM), U50,488H (10, 30 μM), or TRK820 (10, 30 μM) (n = 3, *p
    Figure Legend Snippet: Inhibitory effects of KOR agonists, U50,488H and TRK820, on HUVEC migration and tube formation. (a) The boyden chamber assay. Inhibition of VEGF-induced chemotaxis was assessed after including DAMGO (10, 30 μM), SNC80 (10, 30 μM), U50,488H (10, 30 μM), or TRK820 (10, 30 μM) (n = 3, *p

    Techniques Used: Migration, Boyden Chamber Assay, Inhibition, Chemotaxis Assay

    35) Product Images from "N-glycosylation controls the function of junctional adhesion molecule-A"

    Article Title: N-glycosylation controls the function of junctional adhesion molecule-A

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E14-12-1604

    JAM-A N-glycan content differs between epithelial and endothelial cells. Endogenous expression of JAM-A from MCF7, A549, Caco-2, HUVEC, and pulmonary microvascular endothelial cells (HPmvEC) (A) and exogenous expression of the protein in CHO and MDA-MB-231 (B) was analyzed for N-glycan content after lysis and incubation with agarose-bound lectins as described in the Materials and Methods . Bound proteins were subjected to Western blot analysis for detection of JAM-A. Data are representative of two to four experiments per cell type.
    Figure Legend Snippet: JAM-A N-glycan content differs between epithelial and endothelial cells. Endogenous expression of JAM-A from MCF7, A549, Caco-2, HUVEC, and pulmonary microvascular endothelial cells (HPmvEC) (A) and exogenous expression of the protein in CHO and MDA-MB-231 (B) was analyzed for N-glycan content after lysis and incubation with agarose-bound lectins as described in the Materials and Methods . Bound proteins were subjected to Western blot analysis for detection of JAM-A. Data are representative of two to four experiments per cell type.

    Techniques Used: Expressing, Multiple Displacement Amplification, Lysis, Incubation, Western Blot

    36) Product Images from "The microvascular niche instructs T cells in large vessel vasculitis via the VEGF-Jagged1-Notch pathway"

    Article Title: The microvascular niche instructs T cells in large vessel vasculitis via the VEGF-Jagged1-Notch pathway

    Journal: Science translational medicine

    doi: 10.1126/scitranslmed.aal3322

    Circulating VEGF in GCA patients up-regulates microvascular endothelial Jagged1 Plasma samples were collected from patients with GCA and age-matched healthy controls. Patients with RA served as disease controls. All data are mean ± SEM. (A and B) EC monolayers (HMVECs and HUVECs) were treated with 10% GCA plasma, RA plasma and control plasma. After 6 hours, mRNA transcripts for JAG1 , DLL1 and DLL4 were quantified by RT-PCR (A). After 24 hours, Jagged1 protein was measured by flow cytometry (B). Representative histograms and mean fluorescence intensities (MFIs) corrected by background subtraction from four to six independent experiments. (C) Plasma VEGF concentrations in healthy controls, RA patients and GCA patients. Each dot represents one individual. (D) Jagged1 protein expression on HMVECs and HUVECs treated with hVEGF (10 ng/ml) for 24 hours. Results from three to four experiments. (E) HMVECs were treated with GCA plasma ± VEGF receptor inhibitor axitinib (1 μM). Representative histogram of Jagged1 expression and MFIs are from four experiments. (F) HMVECs were treated with GCA plasma ± anti-VEGF blocking antibody (10 μg/ml) for six hours. JAG1 transcripts were measured by RT-PCR. Data from three experiments. (G and H) Slices of human medium-sized arteries were cultured for 5 days with hVEGF (100 ng/ml) or vehicle (G). Alternatively, the arteries were kept in medium containing 30% plasma from healthy donors or from GCA patients in the absence or presence of anti-VEGF antibody (100 μg/ml) (H). Jagged1 protein was visualized by immunohistochemical staining with anti-Jagged1 antibody. Isotype antibody was used as control for binding specificity. Representative images are from five experiments. Scale bars 20 μm.
    Figure Legend Snippet: Circulating VEGF in GCA patients up-regulates microvascular endothelial Jagged1 Plasma samples were collected from patients with GCA and age-matched healthy controls. Patients with RA served as disease controls. All data are mean ± SEM. (A and B) EC monolayers (HMVECs and HUVECs) were treated with 10% GCA plasma, RA plasma and control plasma. After 6 hours, mRNA transcripts for JAG1 , DLL1 and DLL4 were quantified by RT-PCR (A). After 24 hours, Jagged1 protein was measured by flow cytometry (B). Representative histograms and mean fluorescence intensities (MFIs) corrected by background subtraction from four to six independent experiments. (C) Plasma VEGF concentrations in healthy controls, RA patients and GCA patients. Each dot represents one individual. (D) Jagged1 protein expression on HMVECs and HUVECs treated with hVEGF (10 ng/ml) for 24 hours. Results from three to four experiments. (E) HMVECs were treated with GCA plasma ± VEGF receptor inhibitor axitinib (1 μM). Representative histogram of Jagged1 expression and MFIs are from four experiments. (F) HMVECs were treated with GCA plasma ± anti-VEGF blocking antibody (10 μg/ml) for six hours. JAG1 transcripts were measured by RT-PCR. Data from three experiments. (G and H) Slices of human medium-sized arteries were cultured for 5 days with hVEGF (100 ng/ml) or vehicle (G). Alternatively, the arteries were kept in medium containing 30% plasma from healthy donors or from GCA patients in the absence or presence of anti-VEGF antibody (100 μg/ml) (H). Jagged1 protein was visualized by immunohistochemical staining with anti-Jagged1 antibody. Isotype antibody was used as control for binding specificity. Representative images are from five experiments. Scale bars 20 μm.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Cytometry, Fluorescence, Expressing, Blocking Assay, Cell Culture, Immunohistochemistry, Staining, Binding Assay

    37) Product Images from "Establishment of a three-dimensional model to study human uterine angiogenesis"

    Article Title: Establishment of a three-dimensional model to study human uterine angiogenesis

    Journal: Molecular Human Reproduction

    doi: 10.1093/molehr/gax064

    Sphingosine 1-phosphate and GFs stimulate dose-dependent UtMVEC and HUVEC invasion. ( A ) UtMVECs and ( B ) HUVECs were seeded onto 2.5 mg/ml collagen matrices containing 0–1000 nM sphingosine 1-phosphate (S1P) in the presence of 40 ng/ml bFGF and VEGF. Cells were allowed to invade 24 h. Invasion density quantification was performed as described in the Materials and Methods section and is presented on the y -axis of panels (A) and (B), with manual and automated counts depicted to the left and right, respectively. At least six wells were averaged per treatment. Data in both panels show average values (±SD) from a representative experiment of three independent experiments performed. ( C ) Photographs of UtMVEC and HUVEC invasion responses (side view) at 24 h with indicated concentration of S1P. Scale bars represent 100 μm. Arrows indicate the cell monolayer. Statistical significance in panels (A) and (B) was determined using a one-way ANOVA with Tukey’s post hoc test where each letter represents a significant statistical difference at P
    Figure Legend Snippet: Sphingosine 1-phosphate and GFs stimulate dose-dependent UtMVEC and HUVEC invasion. ( A ) UtMVECs and ( B ) HUVECs were seeded onto 2.5 mg/ml collagen matrices containing 0–1000 nM sphingosine 1-phosphate (S1P) in the presence of 40 ng/ml bFGF and VEGF. Cells were allowed to invade 24 h. Invasion density quantification was performed as described in the Materials and Methods section and is presented on the y -axis of panels (A) and (B), with manual and automated counts depicted to the left and right, respectively. At least six wells were averaged per treatment. Data in both panels show average values (±SD) from a representative experiment of three independent experiments performed. ( C ) Photographs of UtMVEC and HUVEC invasion responses (side view) at 24 h with indicated concentration of S1P. Scale bars represent 100 μm. Arrows indicate the cell monolayer. Statistical significance in panels (A) and (B) was determined using a one-way ANOVA with Tukey’s post hoc test where each letter represents a significant statistical difference at P

    Techniques Used: Concentration Assay

    hCG increases UtMVEC invasion responses in a dose-dependent manner. ( A ) UtMVECs (Lonza Cat. CC-2564, Lot 2F1740) and HUVECs (Lonza Cat. C2517A, Lot 444770) derived from female donors, were seeded onto 2.5 mg/ml collagen matrices containing 0, 20, 100, 200 or 400 U/ml hCG in the presence of 40 ng/ml VEGF and bFGF. No S1P was added. Cells were allowed to invade for 24 h. Invasion density was quantified manually as the average number of invading cells in a 0.25 mm 2 field (±SD). At least three wells were counted and averaged per treatment. Results shown are from a representative experiment ( n = 3). Statistical significance was determined using a one-way ANOVA with Tukey’s post hoc test where each letter represents a significant statistical difference at P
    Figure Legend Snippet: hCG increases UtMVEC invasion responses in a dose-dependent manner. ( A ) UtMVECs (Lonza Cat. CC-2564, Lot 2F1740) and HUVECs (Lonza Cat. C2517A, Lot 444770) derived from female donors, were seeded onto 2.5 mg/ml collagen matrices containing 0, 20, 100, 200 or 400 U/ml hCG in the presence of 40 ng/ml VEGF and bFGF. No S1P was added. Cells were allowed to invade for 24 h. Invasion density was quantified manually as the average number of invading cells in a 0.25 mm 2 field (±SD). At least three wells were counted and averaged per treatment. Results shown are from a representative experiment ( n = 3). Statistical significance was determined using a one-way ANOVA with Tukey’s post hoc test where each letter represents a significant statistical difference at P

    Techniques Used: Derivative Assay

    Invading UtMVECs display increased signaling responses following treatment with S1P and GFs. UtMVECs and HUVEC controls were seeded onto 2.5 mg/ml type I collagen matrices in the presence (+) or the absence (−) of 1 μM S1P and 40 ng/ml VEGF and bFGF. Extracts of invading cells were made at the indicated times. Western blotting was performed with antibodies directed to activated Akt (pAkt, phosphorylated at Ser473), total Akt, activated ERK (pERK, phosphorylated at Thr202, Tyr204), total ERK, PECAM-1, VE-cadherin, membrane type 1-matrix metalloproteinase (MT1-MMP) and actin. Western blots from a representative experiment ( n = 3) are shown.
    Figure Legend Snippet: Invading UtMVECs display increased signaling responses following treatment with S1P and GFs. UtMVECs and HUVEC controls were seeded onto 2.5 mg/ml type I collagen matrices in the presence (+) or the absence (−) of 1 μM S1P and 40 ng/ml VEGF and bFGF. Extracts of invading cells were made at the indicated times. Western blotting was performed with antibodies directed to activated Akt (pAkt, phosphorylated at Ser473), total Akt, activated ERK (pERK, phosphorylated at Thr202, Tyr204), total ERK, PECAM-1, VE-cadherin, membrane type 1-matrix metalloproteinase (MT1-MMP) and actin. Western blots from a representative experiment ( n = 3) are shown.

    Techniques Used: Western Blot

    UtMVECs express EC markers. ( A ) RNA was isolated from HUVECs and UtMVECs (Passage 5) and reverse transcribed into cDNA. RT-PCR was performed using primers specific for Claudin-5, platelet endothelial cell adhesion molecule-1 (PECAM-1), vascular endothelial-cadherin (VE-cadherin), von Willebrand factor (vWF), Vimentin, Caveolin-1, human GAPDH, cytokeratin-8 and smooth muscle actin (α-SMA). ( B ) Protein extracts were analyzed by Western blotting using antibodies directed to Claudin-5, PECAM-1, VE-cadherin, vWF, Vimentin, Caveolin-1, GAPDH and α-SMA.
    Figure Legend Snippet: UtMVECs express EC markers. ( A ) RNA was isolated from HUVECs and UtMVECs (Passage 5) and reverse transcribed into cDNA. RT-PCR was performed using primers specific for Claudin-5, platelet endothelial cell adhesion molecule-1 (PECAM-1), vascular endothelial-cadherin (VE-cadherin), von Willebrand factor (vWF), Vimentin, Caveolin-1, human GAPDH, cytokeratin-8 and smooth muscle actin (α-SMA). ( B ) Protein extracts were analyzed by Western blotting using antibodies directed to Claudin-5, PECAM-1, VE-cadherin, vWF, Vimentin, Caveolin-1, GAPDH and α-SMA.

    Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Combined treatment with estrogen and progesterone selectively enhances UtMVEC invasion. ( A ) UtMVECs and HUVECs were seeded onto 2.5 mg/ml collagen matrices containing 300 nM S1P in the presence of 4 ng/ml VEGF and bFGF. Estrogen (E2) and progesterone (P4) were added at the following concentrations: 0 nM E2 and 0 nM P4, 0.1 nM E2 and 10 nM P4, 1 nM E2 and 100 nM P4, or 10 nM E2 and 1000 nM P4. Cells were allowed to invade for 24 h. At least three wells were counted per treatment and pooled from three independent experiments. The number of invading structures per well was normalized relative to the average control invasion (0 E2, 0 P4), (±SD). Statistical significance was determined using a one-way ANOVA with Tukey’s post hoc test where each letter represents a significant statistical difference at P
    Figure Legend Snippet: Combined treatment with estrogen and progesterone selectively enhances UtMVEC invasion. ( A ) UtMVECs and HUVECs were seeded onto 2.5 mg/ml collagen matrices containing 300 nM S1P in the presence of 4 ng/ml VEGF and bFGF. Estrogen (E2) and progesterone (P4) were added at the following concentrations: 0 nM E2 and 0 nM P4, 0.1 nM E2 and 10 nM P4, 1 nM E2 and 100 nM P4, or 10 nM E2 and 1000 nM P4. Cells were allowed to invade for 24 h. At least three wells were counted per treatment and pooled from three independent experiments. The number of invading structures per well was normalized relative to the average control invasion (0 E2, 0 P4), (±SD). Statistical significance was determined using a one-way ANOVA with Tukey’s post hoc test where each letter represents a significant statistical difference at P

    Techniques Used:

    Temporal UtMVEC angiogenic responses are comparable to HUVEC controls. UtMVECs and HUVECs were allowed to invade for 8, 12, 16 and 24 h. ( A ) Photographs of invasion responses (side view) at each time point. Scale bars represent 100 μm. Arrows point to cell monolayer, while arrowheads indicate lumens in invading cells. ( B ) Quantification of invasion density with time, recorded manually as the average number of invading cells in a 0.25 mm 2 field. Data shown represent average values (±SEM). ( C ) Invasion distance was quantified by measuring the distance from the cell monolayer to the tips of invading structures ( n > 100 structures per treatment group). Data shown represent average invasion distances (±SEM). Statistical significance in panels (B) and (C) was determined using a one-way ANOVA with Tukey’s post hoc test where each letter represents significant statistical difference at P
    Figure Legend Snippet: Temporal UtMVEC angiogenic responses are comparable to HUVEC controls. UtMVECs and HUVECs were allowed to invade for 8, 12, 16 and 24 h. ( A ) Photographs of invasion responses (side view) at each time point. Scale bars represent 100 μm. Arrows point to cell monolayer, while arrowheads indicate lumens in invading cells. ( B ) Quantification of invasion density with time, recorded manually as the average number of invading cells in a 0.25 mm 2 field. Data shown represent average values (±SEM). ( C ) Invasion distance was quantified by measuring the distance from the cell monolayer to the tips of invading structures ( n > 100 structures per treatment group). Data shown represent average invasion distances (±SEM). Statistical significance in panels (B) and (C) was determined using a one-way ANOVA with Tukey’s post hoc test where each letter represents significant statistical difference at P

    Techniques Used:

    Wall shear stress synergizes with S1P and GFs to stimulate UtMVEC invasion. UtMVECs and HUVEC controls were seeded onto 3D collagen matrices containing nothing (wall shear stress: WSS), 1 μM S1P (WSS+S1P), 4 ng/ml VEGF and bFGF (WSS+GF), or 1 μM S1P and 4 ng/ml VEGF and bFGF (WSS+S1P+GF). In all treatment groups, 5.3 dyn/cm 2 WSS was applied and cells were allowed to invade for 24 h. ( A ) Average invasion density (±SD) of UtMVECs and HUVEC controls after 24 h of invasion was quantified manually. Statistical significance was determined using a one-way ANOVA with Tukey’s post hoc test where each letter represents a significant statistical difference at P
    Figure Legend Snippet: Wall shear stress synergizes with S1P and GFs to stimulate UtMVEC invasion. UtMVECs and HUVEC controls were seeded onto 3D collagen matrices containing nothing (wall shear stress: WSS), 1 μM S1P (WSS+S1P), 4 ng/ml VEGF and bFGF (WSS+GF), or 1 μM S1P and 4 ng/ml VEGF and bFGF (WSS+S1P+GF). In all treatment groups, 5.3 dyn/cm 2 WSS was applied and cells were allowed to invade for 24 h. ( A ) Average invasion density (±SD) of UtMVECs and HUVEC controls after 24 h of invasion was quantified manually. Statistical significance was determined using a one-way ANOVA with Tukey’s post hoc test where each letter represents a significant statistical difference at P

    Techniques Used:

    38) Product Images from "Angiogenic Factor AGGF1 Activates Autophagy with an Essential Role in Therapeutic Angiogenesis for Heart Disease"

    Article Title: Angiogenic Factor AGGF1 Activates Autophagy with an Essential Role in Therapeutic Angiogenesis for Heart Disease

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1002529

    Molecular signaling pathway for AGGF-induced autophagy. (A) Western blot analysis for phosphorylation of JNK with protein extracts from HUVECs treated with different concentrations of AGGF1 for 4 h. (B) Activation of JNK in HUVECs treated with AGGF1 protein (500 ng/ ml) for different time points. (C) JNK inhibitors II and III inhibited AGGF1-induced autophagy (LC3-II levels) and activation of JNK. (D) Co-immunoprecipitation to assess the assembly of Beclin1-Vps34-Atg14L complex. Immunoprecipitation, beclin 1 antibody, or control IgG; Western blot, individual antibodies for Vsp34, Atg14L, or beclin 1. (E) Vps34 lipid kinase activity with ELISA assays ( n = 3/group). (F) Autophagic activity assay for membrane-associated PI(3)K (GFP-2xFYVE dots). HUVECs were transfected with GFP-2xFYVE for 24 h and then treated with JNK inhibitor II or control DMSO, followed by treatment with AGGF1 or IgG ( n = 6/group). Scale bar = 10 μm. (G) Autophagic activity assay for degradation of long-lived, damaged proteins by 3 H-leucine release in HUVECs ( n = 6/group). ( H ) JNK1 siRNA inhibited AGGF1-induced autophagy (LC3-II levels) and expression of JNK1. ( I ) Co-immunoprecipitation to assess the assembly of the Beclin1-Vps34-Atg14L complex by JNK1 siRNA. ( J ) Atg14L siRNA inhibited AGGF1-induced autophagy (LC3-II levels) and expression of Atg14L. ( K ) Co-immunoprecipitation to assess the assembly of the Beclin1-Vps34-Atg14L complex by Atg14L siRNA. Underlying data are shown in S1 Data .
    Figure Legend Snippet: Molecular signaling pathway for AGGF-induced autophagy. (A) Western blot analysis for phosphorylation of JNK with protein extracts from HUVECs treated with different concentrations of AGGF1 for 4 h. (B) Activation of JNK in HUVECs treated with AGGF1 protein (500 ng/ ml) for different time points. (C) JNK inhibitors II and III inhibited AGGF1-induced autophagy (LC3-II levels) and activation of JNK. (D) Co-immunoprecipitation to assess the assembly of Beclin1-Vps34-Atg14L complex. Immunoprecipitation, beclin 1 antibody, or control IgG; Western blot, individual antibodies for Vsp34, Atg14L, or beclin 1. (E) Vps34 lipid kinase activity with ELISA assays ( n = 3/group). (F) Autophagic activity assay for membrane-associated PI(3)K (GFP-2xFYVE dots). HUVECs were transfected with GFP-2xFYVE for 24 h and then treated with JNK inhibitor II or control DMSO, followed by treatment with AGGF1 or IgG ( n = 6/group). Scale bar = 10 μm. (G) Autophagic activity assay for degradation of long-lived, damaged proteins by 3 H-leucine release in HUVECs ( n = 6/group). ( H ) JNK1 siRNA inhibited AGGF1-induced autophagy (LC3-II levels) and expression of JNK1. ( I ) Co-immunoprecipitation to assess the assembly of the Beclin1-Vps34-Atg14L complex by JNK1 siRNA. ( J ) Atg14L siRNA inhibited AGGF1-induced autophagy (LC3-II levels) and expression of Atg14L. ( K ) Co-immunoprecipitation to assess the assembly of the Beclin1-Vps34-Atg14L complex by Atg14L siRNA. Underlying data are shown in S1 Data .

    Techniques Used: Western Blot, Activation Assay, Immunoprecipitation, Activity Assay, Enzyme-linked Immunosorbent Assay, Transfection, Expressing

    39) Product Images from "Photo-Targeted Nanoparticles"

    Article Title: Photo-Targeted Nanoparticles

    Journal: Nano letters

    doi: 10.1021/nl903411s

    Nanoparticle targeting. (A) Attachment of nanoparticles with caged YIGSR peptide to HUVECs in illuminated and non-illuminated cultures. The particles appear white. (B) Percentage of HUVECs and MSCs targeted by nanoparticles. The nanoparticles were labeled according the type of surface ligand: “Non-caged”: YIGSR, “Caged”: Y(DMNB)IGSR, “Non-ad” the non-adhering peptide (FHPDYRVI).
    Figure Legend Snippet: Nanoparticle targeting. (A) Attachment of nanoparticles with caged YIGSR peptide to HUVECs in illuminated and non-illuminated cultures. The particles appear white. (B) Percentage of HUVECs and MSCs targeted by nanoparticles. The nanoparticles were labeled according the type of surface ligand: “Non-caged”: YIGSR, “Caged”: Y(DMNB)IGSR, “Non-ad” the non-adhering peptide (FHPDYRVI).

    Techniques Used: Labeling

    40) Product Images from "Temporal Transition of Mechanical Characteristics of HUVEC/MSC Spheroids Using a Microfluidic Chip with Force Sensor Probes"

    Article Title: Temporal Transition of Mechanical Characteristics of HUVEC/MSC Spheroids Using a Microfluidic Chip with Force Sensor Probes

    Journal: Micromachines

    doi: 10.3390/mi7120221

    Transitions of measured SI and size of spheroids. ( A ) Maps of HUVEC/MSC spheroids; ( B ) maps of Rho-associated kinase (ROCK) 10 spheroids; ( C ) maps of ROCK100 spheroids.
    Figure Legend Snippet: Transitions of measured SI and size of spheroids. ( A ) Maps of HUVEC/MSC spheroids; ( B ) maps of Rho-associated kinase (ROCK) 10 spheroids; ( C ) maps of ROCK100 spheroids.

    Techniques Used:

    Comparison of relative gene expression of YAP1 in HUVEC/MSC, ROCK10 and ROCK100 spheroids.
    Figure Legend Snippet: Comparison of relative gene expression of YAP1 in HUVEC/MSC, ROCK10 and ROCK100 spheroids.

    Techniques Used: Expressing

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    Lonza huvecs
    Hif1a Is Positively Correlated with Tgfbs and Biliary Markers and Negatively Correlated with Hepatocyte Markers in Mouse Liver Development (A) Microarray gene expression analysis of mouse fetal liver from E9.5 to 17.5. (B) Immunofluorescence staining for HIF1A (red), HIF2A (red), DLK1 (green), and nuclei (blue, DAPI) in E10.5 mouse liver. Scale bar, 100 μm (upper) or 20 μm (lower). (C) Correlation analysis of hypoxia- ( Hif1a ), hepatocyte- ( Alb and Rbp4 ), and cholangiocyte- (others) associated markers in mouse livers from E9.5 to 8-week-old mice. (D) hiPSC-LBs cultured for 10 days (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: <t>KO1-HUVECs</t> [MSCV-KO1]; no label: <t>MSCs;</t> scale bar, 250 μm). (E) ELISA on protein secretion in hiPSC-LBs cultured for 10 days (mean ± SD; n = 15 independent experiments; ∗∗ p
    Huvecs, supplied by Lonza, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Lonza cell lines huvec
    Performance of CEC detection and identification of microvascular cells. The graph in (A) shows the recovery rate of <t>HUVEC</t> and <t>L‐HMVEC</t> spiked in a healthy whole blood sample at 100, 1000 and 10 000 cells mL −1 . The graph in (B) shows the percentage of CD36 positive cells detected on HMVEC and HUVEC at increasing spiking concentrations as in (A). The results are expressed as mean ± SD of duplicate quantification. CECs, circulating endothelial cells
    Cell Lines Huvec, supplied by Lonza, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Lonza human umbilical vein endothelial cells huvec
    Cell viability levels of <t>HUVEC</t> after exposure to proteinoid NPs, measured by <t>XTT</t> assay. Cells (3 × 10 5 ) were incubated for 48 h with proteinoid NPs dispersed in PBS (1 mg/mL) according to the experimental section. Untreated cells (positive control) were similarly incubated, as well as free doxorubicin, (100 nmol/ml, negative control). Each bar represents mean ± standard deviations of six separate samples.
    Human Umbilical Vein Endothelial Cells Huvec, supplied by Lonza, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Hif1a Is Positively Correlated with Tgfbs and Biliary Markers and Negatively Correlated with Hepatocyte Markers in Mouse Liver Development (A) Microarray gene expression analysis of mouse fetal liver from E9.5 to 17.5. (B) Immunofluorescence staining for HIF1A (red), HIF2A (red), DLK1 (green), and nuclei (blue, DAPI) in E10.5 mouse liver. Scale bar, 100 μm (upper) or 20 μm (lower). (C) Correlation analysis of hypoxia- ( Hif1a ), hepatocyte- ( Alb and Rbp4 ), and cholangiocyte- (others) associated markers in mouse livers from E9.5 to 8-week-old mice. (D) hiPSC-LBs cultured for 10 days (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar, 250 μm). (E) ELISA on protein secretion in hiPSC-LBs cultured for 10 days (mean ± SD; n = 15 independent experiments; ∗∗ p

    Journal: Stem Cell Reports

    Article Title: Optimal Hypoxia Regulates Human iPSC-Derived Liver Bud Differentiation through Intercellular TGFB Signaling

    doi: 10.1016/j.stemcr.2018.06.015

    Figure Lengend Snippet: Hif1a Is Positively Correlated with Tgfbs and Biliary Markers and Negatively Correlated with Hepatocyte Markers in Mouse Liver Development (A) Microarray gene expression analysis of mouse fetal liver from E9.5 to 17.5. (B) Immunofluorescence staining for HIF1A (red), HIF2A (red), DLK1 (green), and nuclei (blue, DAPI) in E10.5 mouse liver. Scale bar, 100 μm (upper) or 20 μm (lower). (C) Correlation analysis of hypoxia- ( Hif1a ), hepatocyte- ( Alb and Rbp4 ), and cholangiocyte- (others) associated markers in mouse livers from E9.5 to 8-week-old mice. (D) hiPSC-LBs cultured for 10 days (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar, 250 μm). (E) ELISA on protein secretion in hiPSC-LBs cultured for 10 days (mean ± SD; n = 15 independent experiments; ∗∗ p

    Article Snippet: HUVECs and MSCs were cultured in endothelial cell growth medium (EGM; Lonza) or MSC growth medium (MSCGM; Lonza).

    Techniques: Microarray, Expressing, Immunofluorescence, Staining, Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    TGFB Signal Inhibition Promotes Hepatocyte Differentiation in Liver Buds (A) Confocal imaging of hiPSC-LBs cultured with various concentrations of A83-01 for 15 days in Excess-hypoxia group (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar from left to right, 250, 100, and 100 μm). (B) Image analysis of HUVEC abundance in hiPSC-LBs cultured with various A83-01 concentrations for 15 days in Excess-hypoxia group. Fluorescence intensity of KO1 protein expression in HUVECs was evaluated as HUVEC abundance in hiPSC-LBs (left: mean ± SD; n = 9–17 independent experiments; ∗∗ p

    Journal: Stem Cell Reports

    Article Title: Optimal Hypoxia Regulates Human iPSC-Derived Liver Bud Differentiation through Intercellular TGFB Signaling

    doi: 10.1016/j.stemcr.2018.06.015

    Figure Lengend Snippet: TGFB Signal Inhibition Promotes Hepatocyte Differentiation in Liver Buds (A) Confocal imaging of hiPSC-LBs cultured with various concentrations of A83-01 for 15 days in Excess-hypoxia group (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar from left to right, 250, 100, and 100 μm). (B) Image analysis of HUVEC abundance in hiPSC-LBs cultured with various A83-01 concentrations for 15 days in Excess-hypoxia group. Fluorescence intensity of KO1 protein expression in HUVECs was evaluated as HUVEC abundance in hiPSC-LBs (left: mean ± SD; n = 9–17 independent experiments; ∗∗ p

    Article Snippet: HUVECs and MSCs were cultured in endothelial cell growth medium (EGM; Lonza) or MSC growth medium (MSCGM; Lonza).

    Techniques: Inhibition, Imaging, Cell Culture, Fluorescence, Expressing

    TGFB Signals from the Mesenchyme and Endothelium Are Candidate Regulators of O 2 -Dependent Hepatocyte Differentiation in Liver Buds (A) Phase-contrast and confocal images of hiPSC-LBs cultured for 1 (phase) or 5 (confocal) days (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar, 250 μm). (B) Boxplots of TGFB family gene expression in hiPSC-LBs cultured for 5 and 15 days. The error bars represent the maximum and minimum values; n = 9 (day 5) and 10 (day 15) independent experiments; ∗ p

    Journal: Stem Cell Reports

    Article Title: Optimal Hypoxia Regulates Human iPSC-Derived Liver Bud Differentiation through Intercellular TGFB Signaling

    doi: 10.1016/j.stemcr.2018.06.015

    Figure Lengend Snippet: TGFB Signals from the Mesenchyme and Endothelium Are Candidate Regulators of O 2 -Dependent Hepatocyte Differentiation in Liver Buds (A) Phase-contrast and confocal images of hiPSC-LBs cultured for 1 (phase) or 5 (confocal) days (green: eGFP-iPSC-DE cells [AAVS1:EGFP]; red: KO1-HUVECs [MSCV-KO1]; no label: MSCs; scale bar, 250 μm). (B) Boxplots of TGFB family gene expression in hiPSC-LBs cultured for 5 and 15 days. The error bars represent the maximum and minimum values; n = 9 (day 5) and 10 (day 15) independent experiments; ∗ p

    Article Snippet: HUVECs and MSCs were cultured in endothelial cell growth medium (EGM; Lonza) or MSC growth medium (MSCGM; Lonza).

    Techniques: Cell Culture, Expressing

    EphA1 regulates EPC proangiogenic potency in a paracrine fashion in vitro. a A1: WB assay showing the EphA1 protein expression in HCC cells. A2: EPC incorporation into HUVECs, EPC’s uptake of DiI-ac-LDL ( red ) together with HUVEC tube formation ( blue ). A3: EPC incorporation assay analysis data (** P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: EphA1 activation promotes the homing of endothelial progenitor cells to hepatocellular carcinoma for tumor neovascularization through the SDF-1/CXCR4 signaling pathway

    doi: 10.1186/s13046-016-0339-6

    Figure Lengend Snippet: EphA1 regulates EPC proangiogenic potency in a paracrine fashion in vitro. a A1: WB assay showing the EphA1 protein expression in HCC cells. A2: EPC incorporation into HUVECs, EPC’s uptake of DiI-ac-LDL ( red ) together with HUVEC tube formation ( blue ). A3: EPC incorporation assay analysis data (** P

    Article Snippet: EPCs were labeled with Dil-ac-LDL (Molecular Probes, 2 ug/mL) at 37 °C for 20 min. After washing with PBS, 1,000 of the Dil-ac-LDL labeled EPCs were mixed with 10,000 of HUVECs in 100 uL of 10 % FBS/EGM-2 MV medium (Lonza) in order to evaluate the contribution of EPCs to Endothelial Cells derived tube formation.

    Techniques: In Vitro, Western Blot, Expressing

    Performance of CEC detection and identification of microvascular cells. The graph in (A) shows the recovery rate of HUVEC and L‐HMVEC spiked in a healthy whole blood sample at 100, 1000 and 10 000 cells mL −1 . The graph in (B) shows the percentage of CD36 positive cells detected on HMVEC and HUVEC at increasing spiking concentrations as in (A). The results are expressed as mean ± SD of duplicate quantification. CECs, circulating endothelial cells

    Journal: Research and Practice in Thrombosis and Haemostasis

    Article Title: Circulating endothelial cells as biomarker for cardiovascular diseases, et al. Circulating endothelial cells as biomarker for cardiovascular diseases

    doi: 10.1002/rth2.12158

    Figure Lengend Snippet: Performance of CEC detection and identification of microvascular cells. The graph in (A) shows the recovery rate of HUVEC and L‐HMVEC spiked in a healthy whole blood sample at 100, 1000 and 10 000 cells mL −1 . The graph in (B) shows the percentage of CD36 positive cells detected on HMVEC and HUVEC at increasing spiking concentrations as in (A). The results are expressed as mean ± SD of duplicate quantification. CECs, circulating endothelial cells

    Article Snippet: 2.3 Cell culture and cell spiking The endothelial cell lines HUVEC (human umbilical vein endothelial cells), L‐HMVEC (lung human microvascular endothelial cells), HPAEC (human pulmonary arterial endothelial cells), and HAEC were obtained from Lonza (Basel, Switzerland).

    Techniques: Capillary Electrochromatography

    Stability of EPCs (A), CECs (B, D), and mvCECs (C, E) in blood samples collected with different anticoagulants. Analyses were performed on fresh blood samples, 0 h, and after 24 h, 48 h and 72 h of storage at 4°C. EPC (A) and CEC (B, C) were quantified on whole blood samples collected from heathy donors in Transfix, EDTA or Lithium Heparin. Recovery of CECs and mvCECs (D, E) in Transfix tubes was assessed on whole blood samples spiked with HUVEC or L‐HMVEC at 100 cells mL −1 and expressed in percentage of time=0 h. Results are expressed as mean ± SD of two or three independent experiments. TF, transfix; LH, Lithium Heparin; CECs, circulating endothelial cells; EPCs, endothelial progenitor cells

    Journal: Research and Practice in Thrombosis and Haemostasis

    Article Title: Circulating endothelial cells as biomarker for cardiovascular diseases, et al. Circulating endothelial cells as biomarker for cardiovascular diseases

    doi: 10.1002/rth2.12158

    Figure Lengend Snippet: Stability of EPCs (A), CECs (B, D), and mvCECs (C, E) in blood samples collected with different anticoagulants. Analyses were performed on fresh blood samples, 0 h, and after 24 h, 48 h and 72 h of storage at 4°C. EPC (A) and CEC (B, C) were quantified on whole blood samples collected from heathy donors in Transfix, EDTA or Lithium Heparin. Recovery of CECs and mvCECs (D, E) in Transfix tubes was assessed on whole blood samples spiked with HUVEC or L‐HMVEC at 100 cells mL −1 and expressed in percentage of time=0 h. Results are expressed as mean ± SD of two or three independent experiments. TF, transfix; LH, Lithium Heparin; CECs, circulating endothelial cells; EPCs, endothelial progenitor cells

    Article Snippet: 2.3 Cell culture and cell spiking The endothelial cell lines HUVEC (human umbilical vein endothelial cells), L‐HMVEC (lung human microvascular endothelial cells), HPAEC (human pulmonary arterial endothelial cells), and HAEC were obtained from Lonza (Basel, Switzerland).

    Techniques: Capillary Electrochromatography

    Cell viability levels of HUVEC after exposure to proteinoid NPs, measured by XTT assay. Cells (3 × 10 5 ) were incubated for 48 h with proteinoid NPs dispersed in PBS (1 mg/mL) according to the experimental section. Untreated cells (positive control) were similarly incubated, as well as free doxorubicin, (100 nmol/ml, negative control). Each bar represents mean ± standard deviations of six separate samples.

    Journal: Scientific Reports

    Article Title: Engineering and use of proteinoid polymers and nanocapsules containing agrochemicals

    doi: 10.1038/s41598-020-66172-w

    Figure Lengend Snippet: Cell viability levels of HUVEC after exposure to proteinoid NPs, measured by XTT assay. Cells (3 × 10 5 ) were incubated for 48 h with proteinoid NPs dispersed in PBS (1 mg/mL) according to the experimental section. Untreated cells (positive control) were similarly incubated, as well as free doxorubicin, (100 nmol/ml, negative control). Each bar represents mean ± standard deviations of six separate samples.

    Article Snippet: Materials and methodsThe following analytical-grade chemicals were purchased from commercial sources and used without further purification: L-glutamic acid (E), L-phenylalanine (F), L-histidine (H), L-lysine (K), L-tryptophan (W), glufosinate (Ef), poly(L-lactic acid) (PLLA, Mw of 2 kDa), dodecyl aldehyde (DA), sodium cyanoborohydride, poly(ethylene glycol) NHS ester, Cyanine3 NHS ester (Cy3 NHS), auxin (sodium salt), doxorubicin, human serum albumin (HSA), Triton-x-100, bovine plasma fibrinogen, Murashige and Skoog (MS) and plant agar from Sigma (Rehovot, Israel); phosphate buffered saline (PBS), minimum essential medium Eagle’s supplement (MEM), fetal bovine serum (FBS), glutamine, penicillin, streptomycin, sodium 3´-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) and mycoplasma detection kits from Biological Industries (Bet Haemek, Israel); human umbilical vein endothelial cells (HUVEC) and their culture medium EGM-2 from Lonza Israel; Water was purified by passing deionized water through an Elgastat Spectrum reverse osmosis system (Elga Ltd., High Wycombe, UK).

    Techniques: XTT Assay, Incubation, Positive Control, Negative Control