human umbilical vein endothelial cells huvecs  (Lonza)


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    Structured Review

    Lonza human umbilical vein endothelial cells huvecs
    Schematic detailing the collection of conditioned media. (A) Endothelial networks are formed in GelMA hydrogels by co-culturing <t>HUVECs</t> and <t>NHLFs</t> for 1 week. (B) Endothelial networks formed after a week in culture can be visualized by staining for CD31. Scale bar = 200 μm.
    Human Umbilical Vein Endothelial Cells Huvecs, supplied by Lonza, used in various techniques. Bioz Stars score: 98/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Multidimensional hydrogel models reveal endothelial network angiocrine signals increase glioblastoma cell number, invasion, and temozolomide resistance"

    Article Title: Multidimensional hydrogel models reveal endothelial network angiocrine signals increase glioblastoma cell number, invasion, and temozolomide resistance

    Journal: Integrative Biology

    doi: 10.1093/intbio/zyaa010

    Schematic detailing the collection of conditioned media. (A) Endothelial networks are formed in GelMA hydrogels by co-culturing HUVECs and NHLFs for 1 week. (B) Endothelial networks formed after a week in culture can be visualized by staining for CD31. Scale bar = 200 μm.
    Figure Legend Snippet: Schematic detailing the collection of conditioned media. (A) Endothelial networks are formed in GelMA hydrogels by co-culturing HUVECs and NHLFs for 1 week. (B) Endothelial networks formed after a week in culture can be visualized by staining for CD31. Scale bar = 200 μm.

    Techniques Used: Staining

    2) Product Images from "MicroRNA miR-27 Inhibits Adenovirus Infection by Suppressing the Expression of SNAP25 and TXN2"

    Article Title: MicroRNA miR-27 Inhibits Adenovirus Infection by Suppressing the Expression of SNAP25 and TXN2

    Journal: Journal of Virology

    doi: 10.1128/JVI.00159-17

    Inhibition of Ad infection by miR-27a/b. (A) The copy numbers of miR-27a/b in HeLa cells, H1299 cells, HUVECs, and NHLFs were determined by quantitative RT-PCR analysis. (B to D) HeLa cells were transfected with miR-27a/b mimics (B) or inhibitors (C) at the indicated doses, or H1299 cells, HUVECs, and NHLFs were transfected with miR-27a/b mimics or inhibitors at 20 nM (D). The cells were then infected with WT-Ad at 100 VP/cell. After 24 h of incubation, the copy numbers of WT-Ad genomic DNA in the cells were determined by quantitative PCR analysis. (E) HeLa cells were transfected with miR-27a/b mimics or inhibitors at 20 nM, followed by infection with WT-Ad at 100 VP/cell. After 24 h of incubation, IFU titers of the WT-Ad progeny in the cells were determined by infectious titer assay. (F) HeLa cells were transfected with a control plasmid (pHM5-U6) or a pre-miR-27a/b-expressing plasmid (pHM5-U6-pre-miR-27a or -b), followed by infection with WT-Ad at 100 VP/cell. After 24 h of incubation, the copy numbers of WT-Ad genomic DNA in the cells were determined. The data are expressed as means and SD ( n = 3 or 4). *, P
    Figure Legend Snippet: Inhibition of Ad infection by miR-27a/b. (A) The copy numbers of miR-27a/b in HeLa cells, H1299 cells, HUVECs, and NHLFs were determined by quantitative RT-PCR analysis. (B to D) HeLa cells were transfected with miR-27a/b mimics (B) or inhibitors (C) at the indicated doses, or H1299 cells, HUVECs, and NHLFs were transfected with miR-27a/b mimics or inhibitors at 20 nM (D). The cells were then infected with WT-Ad at 100 VP/cell. After 24 h of incubation, the copy numbers of WT-Ad genomic DNA in the cells were determined by quantitative PCR analysis. (E) HeLa cells were transfected with miR-27a/b mimics or inhibitors at 20 nM, followed by infection with WT-Ad at 100 VP/cell. After 24 h of incubation, IFU titers of the WT-Ad progeny in the cells were determined by infectious titer assay. (F) HeLa cells were transfected with a control plasmid (pHM5-U6) or a pre-miR-27a/b-expressing plasmid (pHM5-U6-pre-miR-27a or -b), followed by infection with WT-Ad at 100 VP/cell. After 24 h of incubation, the copy numbers of WT-Ad genomic DNA in the cells were determined. The data are expressed as means and SD ( n = 3 or 4). *, P

    Techniques Used: Inhibition, Infection, Quantitative RT-PCR, Transfection, Incubation, Real-time Polymerase Chain Reaction, Infectious Titer Assay, Plasmid Preparation, Expressing

    3) Product Images from "Conductive GelMA–Collagen–AgNW Blended Hydrogel for Smart Actuator"

    Article Title: Conductive GelMA–Collagen–AgNW Blended Hydrogel for Smart Actuator

    Journal: Polymers

    doi: 10.3390/polym13081217

    Biocompatibility analysis of the conductive GelMA–collagen–AgNW blended hydrogel. Live (green)/dead (red) fluorescence image of the human umbilical vein endothelial cells (HUVECs) cultured within the conductive GelMA–collagen–AgNW blended hydrogel (** p
    Figure Legend Snippet: Biocompatibility analysis of the conductive GelMA–collagen–AgNW blended hydrogel. Live (green)/dead (red) fluorescence image of the human umbilical vein endothelial cells (HUVECs) cultured within the conductive GelMA–collagen–AgNW blended hydrogel (** p

    Techniques Used: Fluorescence, Cell Culture

    4) Product Images from "Krüppel-like factor 14, a coronary artery disease associated transcription factor, inhibits endothelial inflammation via NF-κB signaling pathway"

    Article Title: Krüppel-like factor 14, a coronary artery disease associated transcription factor, inhibits endothelial inflammation via NF-κB signaling pathway

    Journal: Atherosclerosis

    doi: 10.1016/j.atherosclerosis.2018.09.018

    KLF14 inhibits pro-inflammatory cytokine-induced expression of inflammatory markers in endothelial cells. HCAECs (A-C) and HUVECs (D-F) were infected with 10 MOI of adenovirus encoding lacZ (AdLaZ) or human KLF14 (AdKLF14) for 48 hours and then stimulated with 5 ng/ml IL-1β or 2 ng/ml TNFα respectively for 4 hours. Endothelial inflammatory markers VCAM-1 and SELE (encoding E-selectin) mRNA abundance was determined by qRT-PCR (A and D). VCAM-1 and E-selectin protein levels were determined by western blot (B and E) and quantitatively analyzed (C and F). (G-I) Mouse pulmonary endothelial cells were isolated from Klf14 global KO mice (KO) and their wild type littermates (WT) and stimulated with 20 ng/ml IL-1β for 4 hours. (G) Vcam-1 and Sele mRNA abundance was determined by qRT-PCR. Vcam-1 protein levels (H) were determined by western blot and quantitatively analyzed (I). ** p
    Figure Legend Snippet: KLF14 inhibits pro-inflammatory cytokine-induced expression of inflammatory markers in endothelial cells. HCAECs (A-C) and HUVECs (D-F) were infected with 10 MOI of adenovirus encoding lacZ (AdLaZ) or human KLF14 (AdKLF14) for 48 hours and then stimulated with 5 ng/ml IL-1β or 2 ng/ml TNFα respectively for 4 hours. Endothelial inflammatory markers VCAM-1 and SELE (encoding E-selectin) mRNA abundance was determined by qRT-PCR (A and D). VCAM-1 and E-selectin protein levels were determined by western blot (B and E) and quantitatively analyzed (C and F). (G-I) Mouse pulmonary endothelial cells were isolated from Klf14 global KO mice (KO) and their wild type littermates (WT) and stimulated with 20 ng/ml IL-1β for 4 hours. (G) Vcam-1 and Sele mRNA abundance was determined by qRT-PCR. Vcam-1 protein levels (H) were determined by western blot and quantitatively analyzed (I). ** p

    Techniques Used: Expressing, Infection, Quantitative RT-PCR, Western Blot, Isolation, Mouse Assay

    5) Product Images from "The effect of cytokines produced by human adipose tissue on monocyte adhesion to the endothelium"

    Article Title: The effect of cytokines produced by human adipose tissue on monocyte adhesion to the endothelium

    Journal: Cell Adhesion & Migration

    doi: 10.1080/19336918.2019.1644856

    Relative gene expression of adhesion molecules with the addition of TNF-α, IL-1ß and MCP-1 inhibitor compared to negative control. Inhibition of selected cytokines decreased gene expression of adhesion molecules – VCAM-1, ICAM-1 and SelE compared to negative control (unstimulated endothelial cells). VCAM-1 – vascular cell adhesion molecule 1; ICAM-1 – intercellular adhesion molecule 1; SelE – selectin E; IL-1ß – interleukin 1ß; TNF α – tumor necrosis factor α; MCP-1 – monocyte chemoattractant protein-1 (CCL2), * p
    Figure Legend Snippet: Relative gene expression of adhesion molecules with the addition of TNF-α, IL-1ß and MCP-1 inhibitor compared to negative control. Inhibition of selected cytokines decreased gene expression of adhesion molecules – VCAM-1, ICAM-1 and SelE compared to negative control (unstimulated endothelial cells). VCAM-1 – vascular cell adhesion molecule 1; ICAM-1 – intercellular adhesion molecule 1; SelE – selectin E; IL-1ß – interleukin 1ß; TNF α – tumor necrosis factor α; MCP-1 – monocyte chemoattractant protein-1 (CCL2), * p

    Techniques Used: Expressing, Negative Control, Inhibition

    Effect of ATCM on relative gene expression in endothelial cells compared to the negative control. ** p
    Figure Legend Snippet: Effect of ATCM on relative gene expression in endothelial cells compared to the negative control. ** p

    Techniques Used: Expressing, Negative Control

    Stimulation of endothelial cells by IL-1ß and TNF-α at concentrations measured in ATCM. IL-1ß (200 pg/ml) and TNF-α (30 pg/ml). NC – negative control, IL-1ß – interleukin 1ß, TNF-α – tumor necrosis factor α, * p
    Figure Legend Snippet: Stimulation of endothelial cells by IL-1ß and TNF-α at concentrations measured in ATCM. IL-1ß (200 pg/ml) and TNF-α (30 pg/ml). NC – negative control, IL-1ß – interleukin 1ß, TNF-α – tumor necrosis factor α, * p

    Techniques Used: Negative Control

    Effect of inhibition of selected cytokines during stimulation of endothelial cells. NC – negative control, ATCM – adipose tissue-conditioned media, IL-1ß – interleukin 1ß, TNF-α – tumor necrosis factor α, MCP-1 – monocyte chemoattractant protein-1 (CCL2), RANTES – CCL5 – regulated the activation, normal T cell expression and secretion, *** p
    Figure Legend Snippet: Effect of inhibition of selected cytokines during stimulation of endothelial cells. NC – negative control, ATCM – adipose tissue-conditioned media, IL-1ß – interleukin 1ß, TNF-α – tumor necrosis factor α, MCP-1 – monocyte chemoattractant protein-1 (CCL2), RANTES – CCL5 – regulated the activation, normal T cell expression and secretion, *** p

    Techniques Used: Inhibition, Negative Control, Activation Assay, Expressing

    6) Product Images from "Cross-tissue, single-cell stromal atlas identifies shared pathological fibroblast phenotypes in four chronic inflammatory diseases"

    Article Title: Cross-tissue, single-cell stromal atlas identifies shared pathological fibroblast phenotypes in four chronic inflammatory diseases

    Journal: Med (New York, N.Y.)

    doi: 10.1016/j.medj.2022.05.002

    Convergence of fibroblasts from distinct tissues with in vitro activation (A) Study design with three conditions: fibroblasts cultured alone (control), with an equal mixture of endothelial cells (ECs), and in supernatant extracted from activated T cells (T cells). Each condition was repeated in three lung-derived and three synovium-derived fibroblast cell lines. (B) Total cell numbers per donor per condition in log scale. (C) scRNA-seq profiles of cultured cells were visualized in 3D with integrative analysis and UMAP projection. Each subpanel highlights the location of fibroblasts from the control, T cell, and EC conditions and colors fibroblasts by tissue. (D) Within each tissue, activation signatures were derived for the EC and T cell conditions and plotted in a heatmap of pseudobulk samples (rows) by genes (columns), colored by centered and scaled log 2 fold change (versus control). Three representative genes were selected for each activation signature. (E) For each condition, we plotted the per-gene changes for synovial fibroblasts (x axis) against lung fibroblasts (y axis) and highlighted the three representative genes from (D). (F) We compared the in vitro activation changes with cluster marker signatures from the cross-tissue atlas with correlation analysis. Error bars denote 99% CI for the Pearson correlation statistic. (G) Correlation analysis of fibroblasts cultured with ECs in a 3D culture system. (H) Magnification of the correlation of SPARC + COL3A1 + (C4) cluster markers (x axis) with the 3D EC synovial activation signature (y axis). Genes significantly ( p
    Figure Legend Snippet: Convergence of fibroblasts from distinct tissues with in vitro activation (A) Study design with three conditions: fibroblasts cultured alone (control), with an equal mixture of endothelial cells (ECs), and in supernatant extracted from activated T cells (T cells). Each condition was repeated in three lung-derived and three synovium-derived fibroblast cell lines. (B) Total cell numbers per donor per condition in log scale. (C) scRNA-seq profiles of cultured cells were visualized in 3D with integrative analysis and UMAP projection. Each subpanel highlights the location of fibroblasts from the control, T cell, and EC conditions and colors fibroblasts by tissue. (D) Within each tissue, activation signatures were derived for the EC and T cell conditions and plotted in a heatmap of pseudobulk samples (rows) by genes (columns), colored by centered and scaled log 2 fold change (versus control). Three representative genes were selected for each activation signature. (E) For each condition, we plotted the per-gene changes for synovial fibroblasts (x axis) against lung fibroblasts (y axis) and highlighted the three representative genes from (D). (F) We compared the in vitro activation changes with cluster marker signatures from the cross-tissue atlas with correlation analysis. Error bars denote 99% CI for the Pearson correlation statistic. (G) Correlation analysis of fibroblasts cultured with ECs in a 3D culture system. (H) Magnification of the correlation of SPARC + COL3A1 + (C4) cluster markers (x axis) with the 3D EC synovial activation signature (y axis). Genes significantly ( p

    Techniques Used: In Vitro, Activation Assay, Cell Culture, Derivative Assay, Marker

    7) Product Images from "The STRIPAK complex components FAM40A and FAM40B regulate endothelial cell contractility via ROCKs"

    Article Title: The STRIPAK complex components FAM40A and FAM40B regulate endothelial cell contractility via ROCKs

    Journal: BMC Cell Biology

    doi: 10.1186/s12860-018-0175-y

    Effects of FAM40A or FAM40B on RhoA activity and MLC2 phosphorylation. a HUVECs were transfected with siRNAs targeting FAM40A or FAM40B. After 72 h, cells were lysed and whole cell lysates used in a GST-RBD pulldown assay to determine levels of active GTP-loaded RhoA. A representative immunoblot for total and GTP-loaded RhoA is shown ( n = 3). b Immunoblot of phospho-MLC2 (pMLC2) and MLC2 in FAM40A and FAM40B-depleted HUVECs. Quantification of pMLC2 levels was performed by individually normalising pMLC2 and MLC2 levels to GAPDH levels. The ratio of the normalised pMLC2 level to the normalised MLC2 level was used as a measure of MLC2 phosphorylation. Data are means of 3 independent experiments ± SEM. n.s. not significant; Student’s t -test compared to siControl. c HUVECs were transfected with siRNAs targeting FAM40A or FAM40B and seeded onto fibronectin-coated glass coverslips to form confluent monolayers. 72 h after transfection cells were fixed and stained for F-actin and pMLC2. Images are compressed stacks of 10–15 z-sections. Asterisks indicate examples of regions where pMLC2 co-localizes with stress fibers. Images shown are compressed stacks of 10–15 z-sections and are representative of 2 independent experiments. Scale bar, 20 μm. Dual colour intensity profiles for F-actin and pMLC2 are indicated for representative cells with the region scanned indicated as a red line on each image
    Figure Legend Snippet: Effects of FAM40A or FAM40B on RhoA activity and MLC2 phosphorylation. a HUVECs were transfected with siRNAs targeting FAM40A or FAM40B. After 72 h, cells were lysed and whole cell lysates used in a GST-RBD pulldown assay to determine levels of active GTP-loaded RhoA. A representative immunoblot for total and GTP-loaded RhoA is shown ( n = 3). b Immunoblot of phospho-MLC2 (pMLC2) and MLC2 in FAM40A and FAM40B-depleted HUVECs. Quantification of pMLC2 levels was performed by individually normalising pMLC2 and MLC2 levels to GAPDH levels. The ratio of the normalised pMLC2 level to the normalised MLC2 level was used as a measure of MLC2 phosphorylation. Data are means of 3 independent experiments ± SEM. n.s. not significant; Student’s t -test compared to siControl. c HUVECs were transfected with siRNAs targeting FAM40A or FAM40B and seeded onto fibronectin-coated glass coverslips to form confluent monolayers. 72 h after transfection cells were fixed and stained for F-actin and pMLC2. Images are compressed stacks of 10–15 z-sections. Asterisks indicate examples of regions where pMLC2 co-localizes with stress fibers. Images shown are compressed stacks of 10–15 z-sections and are representative of 2 independent experiments. Scale bar, 20 μm. Dual colour intensity profiles for F-actin and pMLC2 are indicated for representative cells with the region scanned indicated as a red line on each image

    Techniques Used: Activity Assay, Transfection, Staining

    Effect of FAM40A and FAM40B depletion on endothelial junctions and permeability. a HUVECs were transfected with siRNAs targeting FAM40A or FAM40B and seeded onto fibronectin-coated glass coverslips to form monolayers. 72 h after transfection cells were fixed and stained for F-actin and VE-cadherin. b Immunofluorescence analysis of FAM40A and FAM40B-depleted HUVECs for F-actin and ZO-1. Images are compressed stacks of 10–15 z-sections and are representative of 3 independent experiments. Scale bars, 40 μm. c Magnified images highlighting anchoring of stress fibers at cell-cell junctions. d HUVECs were transfected with the indicated siRNAs and plated at confluency onto transwell inserts. 72 h after transfection, FITC-dextran was added to the upper chamber. After a further 80 min the fluorescence of media in the lower chamber was determined. Data show mean permeability ± SEM as % of siControl; n = 3 independent experiments for siFAM40A; n = 5 independent experiments for siFAM40B. * p
    Figure Legend Snippet: Effect of FAM40A and FAM40B depletion on endothelial junctions and permeability. a HUVECs were transfected with siRNAs targeting FAM40A or FAM40B and seeded onto fibronectin-coated glass coverslips to form monolayers. 72 h after transfection cells were fixed and stained for F-actin and VE-cadherin. b Immunofluorescence analysis of FAM40A and FAM40B-depleted HUVECs for F-actin and ZO-1. Images are compressed stacks of 10–15 z-sections and are representative of 3 independent experiments. Scale bars, 40 μm. c Magnified images highlighting anchoring of stress fibers at cell-cell junctions. d HUVECs were transfected with the indicated siRNAs and plated at confluency onto transwell inserts. 72 h after transfection, FITC-dextran was added to the upper chamber. After a further 80 min the fluorescence of media in the lower chamber was determined. Data show mean permeability ± SEM as % of siControl; n = 3 independent experiments for siFAM40A; n = 5 independent experiments for siFAM40B. * p

    Techniques Used: Permeability, Transfection, Staining, Immunofluorescence, Fluorescence

    FAM40A and FAM40B regulate endothelial loop formation. a HUVECs were transfected with siRNAs targeting FAM40A and FAM40B. 48 h after transfection cells were seeded onto a layer of Matrigel. Loops were allowed to form for 24 h. Scale bar, 200 μm. Graph (right panel) shows quantification of number of loops per field. At least 5 fields were scored per condition in each experiment. Data show mean ± SEM normalised to siControl; n = 4 for siFAM40A-1, siFAM40A-2 and siFAM40B-1; n = 3 for siFAM40B-2. b Arrows highlight deformation of the Matrigel substrate by FAM40-depleted cells. Scale bars, 200 μm. c HUVECs were allowed to form loops on polymerised Matrigel for 24 h, and were fixed and stained for F-actin, and with DAPI to visualise nuclei. Images are sections taken at a single z-position and are representative of 3 independent experiments. Boxed areas are shown at higher magnification (inset images) to highlight F-actin micro-spikes on cells. Scale bar, 40 μm. d FAM40A and FAM40B-depleted HUVECs were allowed to form loops on polymerised Martigel for 24 h. Cells were treated with 10 μM Y-27632 for 24 h. Scale bar, 200 μm. Graph (right panel) shows quantification of number of loops per field. At least 4 fields were scored per condition in each experiment. Data are mean ± SEM normalised to siControl, n = 3 independent experiments. * p
    Figure Legend Snippet: FAM40A and FAM40B regulate endothelial loop formation. a HUVECs were transfected with siRNAs targeting FAM40A and FAM40B. 48 h after transfection cells were seeded onto a layer of Matrigel. Loops were allowed to form for 24 h. Scale bar, 200 μm. Graph (right panel) shows quantification of number of loops per field. At least 5 fields were scored per condition in each experiment. Data show mean ± SEM normalised to siControl; n = 4 for siFAM40A-1, siFAM40A-2 and siFAM40B-1; n = 3 for siFAM40B-2. b Arrows highlight deformation of the Matrigel substrate by FAM40-depleted cells. Scale bars, 200 μm. c HUVECs were allowed to form loops on polymerised Matrigel for 24 h, and were fixed and stained for F-actin, and with DAPI to visualise nuclei. Images are sections taken at a single z-position and are representative of 3 independent experiments. Boxed areas are shown at higher magnification (inset images) to highlight F-actin micro-spikes on cells. Scale bar, 40 μm. d FAM40A and FAM40B-depleted HUVECs were allowed to form loops on polymerised Martigel for 24 h. Cells were treated with 10 μM Y-27632 for 24 h. Scale bar, 200 μm. Graph (right panel) shows quantification of number of loops per field. At least 4 fields were scored per condition in each experiment. Data are mean ± SEM normalised to siControl, n = 3 independent experiments. * p

    Techniques Used: Transfection, Staining

    CCM3 regulates stress fibers and angiogenic loop formation in endothelial cells. HUVECs were transfected with siRNAs targeting CCM3 or with a control siRNA. a After 72 h, the amount of CCM3 mRNA was determined by qPCR. Data are normalised to GAPDH mRNA levels and are the mean of 3 independent experiments ± SEM. b Left panels, HUVECs were seeded onto fibronectin-coated glass coverslips to form confluent monolayers. 72 h after transfection, cells were fixed and stained for F-actin and VE-cadherin. Images are compressed stacks of 10–15 confocal z-sections. Arrows indicate discontinuous junctions (VE-cadherin); Scale bar, 40 μm. Intensity profiles are indicated for representative cells with the region scanned indicated as a red line on each image. Right panel, stress fibers were quantified as described in Materials and Methods; data show mean ± SEM; n = 3 independent experiments. At least 150 cells were scored per condition in each experiment. c Left panels, 48 h after transfection, HUVECs were seeded onto a layer of Matrigel. Loops were allowed to form for 24 h, and then cells were fixed and stained for F-actin with phalloidin-Alexa546. Scale bar, 100 μm. Right, loop formation was quantified by scoring the number of loops per field using fluorescence images. 6 fields were scored per condition in each experiment. Results are shown as % of siControl. Data are mean ± SEM, n = 3 independent experiments; ** p
    Figure Legend Snippet: CCM3 regulates stress fibers and angiogenic loop formation in endothelial cells. HUVECs were transfected with siRNAs targeting CCM3 or with a control siRNA. a After 72 h, the amount of CCM3 mRNA was determined by qPCR. Data are normalised to GAPDH mRNA levels and are the mean of 3 independent experiments ± SEM. b Left panels, HUVECs were seeded onto fibronectin-coated glass coverslips to form confluent monolayers. 72 h after transfection, cells were fixed and stained for F-actin and VE-cadherin. Images are compressed stacks of 10–15 confocal z-sections. Arrows indicate discontinuous junctions (VE-cadherin); Scale bar, 40 μm. Intensity profiles are indicated for representative cells with the region scanned indicated as a red line on each image. Right panel, stress fibers were quantified as described in Materials and Methods; data show mean ± SEM; n = 3 independent experiments. At least 150 cells were scored per condition in each experiment. c Left panels, 48 h after transfection, HUVECs were seeded onto a layer of Matrigel. Loops were allowed to form for 24 h, and then cells were fixed and stained for F-actin with phalloidin-Alexa546. Scale bar, 100 μm. Right, loop formation was quantified by scoring the number of loops per field using fluorescence images. 6 fields were scored per condition in each experiment. Results are shown as % of siControl. Data are mean ± SEM, n = 3 independent experiments; ** p

    Techniques Used: Transfection, Real-time Polymerase Chain Reaction, Staining, Fluorescence

    Rho/ROCK signaling is required for stress fiber induction by FAM40 depletion. HUVECs were transfected with siRNAs targeting FAM40A, FAM40B or a control siRNA and seeded onto fibronectin-coated glass coverslips to form confluent monolayers. 72 h after transfection cells were treated with ( a ) 4 μg/ml C3 transferase for 2 h, or ( b ) 5 μM H1152 for 10 min after which they were fixed and stained for F-actin. Images are compressed stacks of 10–15 z-sections. Scale bars, 40 μm. Graphs show stress fiber content quantified from at least 150 cells per condition from 3 independent experiments. Error bars depict SEM values. *** (black) p
    Figure Legend Snippet: Rho/ROCK signaling is required for stress fiber induction by FAM40 depletion. HUVECs were transfected with siRNAs targeting FAM40A, FAM40B or a control siRNA and seeded onto fibronectin-coated glass coverslips to form confluent monolayers. 72 h after transfection cells were treated with ( a ) 4 μg/ml C3 transferase for 2 h, or ( b ) 5 μM H1152 for 10 min after which they were fixed and stained for F-actin. Images are compressed stacks of 10–15 z-sections. Scale bars, 40 μm. Graphs show stress fiber content quantified from at least 150 cells per condition from 3 independent experiments. Error bars depict SEM values. *** (black) p

    Techniques Used: Transfection, Staining

    FAM40A and FAM40B regulate stress fibers in endothelial cells. HUVECs were transfected with siRNAs targeting FAM40A, FAM40B or with a control siRNA, and analysed 72 after transfection. a The amount of FAM40A mRNA was determined by quantitative PCR. Data are normalised to GAPDH mRNA levels and are the mean ± SEM; n = 3. b Representative immunoblot of HUVEC lysates. Whole cell lysates were immunoblotted for FAM40B and GAPDH as a loading control; arrow indicates FAM40B protein. Lanes are from the same immunoblot. Quantification of FAM40B band intensities (to band) is shown above the blot, relative to siControl, and was carried out using ImageJ. c Cells were seeded onto fibronectin-coated glass coverslips to form confluent monolayers. Cells were fixed and stained for F-actin. Images are compressed stacks of 10–15 z-sections. Scale bar, 40 μm. Intensity profiles are indicated for representative cells with the region scanned indicated as a red line on each image. Graph (right panel) shows quantification of stress fibers from at least 150 cells per condition from 3 independent experiments ± SEM. d HUVECs were transfected with pHA-FAM40A, pHA-FAM40B or pmyc-CCM3, seeded onto fibronectin-coated glass coverslips and fixed after 24 h. Cells were stained for F-actin and the HA or myc epitope. Images are compressed stacks of 15–20 z-sections (FAM40A/B) or single images (CCM3) and are representative of 2 independent experiments. Scale bar, 20 μm. e HUVECs were transfected with control siRNA or siRNAs targeting FAM40A or FAM40B, followed after 48 h by transfection with pmyc-CCM3. After a further 24 h, stress fibers were quantified from at least 100 cells per condition from 2 independent experiments. * p
    Figure Legend Snippet: FAM40A and FAM40B regulate stress fibers in endothelial cells. HUVECs were transfected with siRNAs targeting FAM40A, FAM40B or with a control siRNA, and analysed 72 after transfection. a The amount of FAM40A mRNA was determined by quantitative PCR. Data are normalised to GAPDH mRNA levels and are the mean ± SEM; n = 3. b Representative immunoblot of HUVEC lysates. Whole cell lysates were immunoblotted for FAM40B and GAPDH as a loading control; arrow indicates FAM40B protein. Lanes are from the same immunoblot. Quantification of FAM40B band intensities (to band) is shown above the blot, relative to siControl, and was carried out using ImageJ. c Cells were seeded onto fibronectin-coated glass coverslips to form confluent monolayers. Cells were fixed and stained for F-actin. Images are compressed stacks of 10–15 z-sections. Scale bar, 40 μm. Intensity profiles are indicated for representative cells with the region scanned indicated as a red line on each image. Graph (right panel) shows quantification of stress fibers from at least 150 cells per condition from 3 independent experiments ± SEM. d HUVECs were transfected with pHA-FAM40A, pHA-FAM40B or pmyc-CCM3, seeded onto fibronectin-coated glass coverslips and fixed after 24 h. Cells were stained for F-actin and the HA or myc epitope. Images are compressed stacks of 15–20 z-sections (FAM40A/B) or single images (CCM3) and are representative of 2 independent experiments. Scale bar, 20 μm. e HUVECs were transfected with control siRNA or siRNAs targeting FAM40A or FAM40B, followed after 48 h by transfection with pmyc-CCM3. After a further 24 h, stress fibers were quantified from at least 100 cells per condition from 2 independent experiments. * p

    Techniques Used: Transfection, Real-time Polymerase Chain Reaction, Staining

    8) Product Images from "Orthogonal co-cultivation of smooth muscle cell and endothelial cell layers to construct in vivo-like vasculature"

    Article Title: Orthogonal co-cultivation of smooth muscle cell and endothelial cell layers to construct in vivo-like vasculature

    Journal: Biomicrofluidics

    doi: 10.1063/1.5068689

    The effects of shear flow on the orientation angle and spreading areas of co-cultured HUVECs. (a) Analysis of orientation angles of the VSMCs and the co-cultured HUVECs with respect to the shear flow. (b) Spreading areas of co-cultured HUVECs depending on shear flow. All error bars were measured by a standard deviation of means with n = 30. *** P
    Figure Legend Snippet: The effects of shear flow on the orientation angle and spreading areas of co-cultured HUVECs. (a) Analysis of orientation angles of the VSMCs and the co-cultured HUVECs with respect to the shear flow. (b) Spreading areas of co-cultured HUVECs depending on shear flow. All error bars were measured by a standard deviation of means with n = 30. *** P

    Techniques Used: Cell Culture, Standard Deviation

    Overall procedure for producing the in vivo like vasculature. (a) and (b) The microwrinkle patterned circular microchannel was first fabricated, and [(c) and (d)] the VSMCs were circumferentially aligned along the microwrinkles. (e) and (f) Then, HUVECs were cultured on the VSMCs and reoriented toward the flow direction by shear stress.
    Figure Legend Snippet: Overall procedure for producing the in vivo like vasculature. (a) and (b) The microwrinkle patterned circular microchannel was first fabricated, and [(c) and (d)] the VSMCs were circumferentially aligned along the microwrinkles. (e) and (f) Then, HUVECs were cultured on the VSMCs and reoriented toward the flow direction by shear stress.

    Techniques Used: In Vivo, Cell Culture

    Top, side, and front views of 3D z-stack images of 3D co-cultured VSMCs and HUVECs in the circular microfluidic channel. (a) A bottom half-circular channel and (b) a top half-circular channel were displayed by staining filamentous actin (green) and nuclei (blue) with Alexa Fluor 488 phalloidin and DAPI, respectively.
    Figure Legend Snippet: Top, side, and front views of 3D z-stack images of 3D co-cultured VSMCs and HUVECs in the circular microfluidic channel. (a) A bottom half-circular channel and (b) a top half-circular channel were displayed by staining filamentous actin (green) and nuclei (blue) with Alexa Fluor 488 phalloidin and DAPI, respectively.

    Techniques Used: Cell Culture, Staining

    The expression of VE-Cadherin of the HUVECs co-cultured on the aligned VSMC layers with a different flow rate. (a) Fluorescence images of the filamentous actin and nuclei of circumferentially aligned VSMCs on the microwrinkled fluidic channel. Fluorescence images of the VE-cadherin and nuclei of the HUVECs co-cultured on the aligned VSMC layers with a flow rate of (b) 0 μ l/h, (c) 100 μ l/h, and (d) 300 μ l/h. Scale bar: 100 μ m.
    Figure Legend Snippet: The expression of VE-Cadherin of the HUVECs co-cultured on the aligned VSMC layers with a different flow rate. (a) Fluorescence images of the filamentous actin and nuclei of circumferentially aligned VSMCs on the microwrinkled fluidic channel. Fluorescence images of the VE-cadherin and nuclei of the HUVECs co-cultured on the aligned VSMC layers with a flow rate of (b) 0 μ l/h, (c) 100 μ l/h, and (d) 300 μ l/h. Scale bar: 100 μ m.

    Techniques Used: Expressing, Cell Culture, Fluorescence

    9) Product Images from "Upregulation of C-Reactive Protein by Placenta-Derived Mesenchymal Stem Cells Promotes Angiogenesis in A Rat Model with Cirrhotic Liver"

    Article Title: Upregulation of C-Reactive Protein by Placenta-Derived Mesenchymal Stem Cells Promotes Angiogenesis in A Rat Model with Cirrhotic Liver

    Journal: International Journal of Stem Cells

    doi: 10.15283/ijsc20052

    PD-MSCs induce the expression of CRP and VEGF at the mRNA level. Schematic showing the experimental protocol (A). mRNA level of CRP (B), VEGF (D) was quantified in rat hepatocytes treated with CCl 4 and co-cultured with HUVECs and PD-MSCs. mRNA level of CRP (C), VEGF (E) was assayed in HUVECs after CCl 4 treatment and co-culture with rat hepatocytes and PD-MSCs. Duplicated data are represented as the mean±SD. *, p
    Figure Legend Snippet: PD-MSCs induce the expression of CRP and VEGF at the mRNA level. Schematic showing the experimental protocol (A). mRNA level of CRP (B), VEGF (D) was quantified in rat hepatocytes treated with CCl 4 and co-cultured with HUVECs and PD-MSCs. mRNA level of CRP (C), VEGF (E) was assayed in HUVECs after CCl 4 treatment and co-culture with rat hepatocytes and PD-MSCs. Duplicated data are represented as the mean±SD. *, p

    Techniques Used: Expressing, Cell Culture, Co-Culture Assay

    10) Product Images from "AECHL-1, a novel triterpenoid, targets tumor neo-vasculature and impairs the endothelial cell cytoskeleton"

    Article Title: AECHL-1, a novel triterpenoid, targets tumor neo-vasculature and impairs the endothelial cell cytoskeleton

    Journal: Angiogenesis

    doi: 10.1007/s10456-015-9466-5

    Effect of AECHL-1 on microfilament dynamics and motility-associated proteins. HUVECs were grown on coverslips till confluency and scratched with a pipette tip. Cells were pretreated with AECHL-1 for 4 h and then stimulated with VEGF and incubated for 9 h. Coverslips were then fixed and processed for immunostaining. Immunofluorescence analysis of HUVECs stained with phalloidin conjugated with Alexa Fluor 488 and a anti-IQGAP1 antibody b anti-WAVE-2 antibody. Membrane ruffles indicate active lamellipodial edge ( white arrow ). Images were taken by a confocal microscope at ×60 magnification. c Also at the indicated time (9 h), cells were harvested and subjected to Western blotting for detection of WAVE-2 and pRac/Cdc42. Membrane was stripped and reprobed with total Rac/Cdc42 and GAPDH to indicate equal loading
    Figure Legend Snippet: Effect of AECHL-1 on microfilament dynamics and motility-associated proteins. HUVECs were grown on coverslips till confluency and scratched with a pipette tip. Cells were pretreated with AECHL-1 for 4 h and then stimulated with VEGF and incubated for 9 h. Coverslips were then fixed and processed for immunostaining. Immunofluorescence analysis of HUVECs stained with phalloidin conjugated with Alexa Fluor 488 and a anti-IQGAP1 antibody b anti-WAVE-2 antibody. Membrane ruffles indicate active lamellipodial edge ( white arrow ). Images were taken by a confocal microscope at ×60 magnification. c Also at the indicated time (9 h), cells were harvested and subjected to Western blotting for detection of WAVE-2 and pRac/Cdc42. Membrane was stripped and reprobed with total Rac/Cdc42 and GAPDH to indicate equal loading

    Techniques Used: Transferring, Incubation, Immunostaining, Immunofluorescence, Staining, Microscopy, Western Blot

    Effect of AECHL-1 on survival and proliferation of HUVECs. a HUVECs treated with varying concentrations of AECHL-1 for 48 h, and cell viability was determined by MTT assay. b HUVECS treated with varying concentrations of AECHL-1 for 24 h and subjected to flow cytometry using Annexin V-FITC as a marker of apoptosis. c HUVECs treated with varying concentrations of AECHL-1 for 48 h, and cell proliferation was determined by tritiated thymidine assay. Columns, mean from three independent experiments performed in triplicate; bars , SE. * P
    Figure Legend Snippet: Effect of AECHL-1 on survival and proliferation of HUVECs. a HUVECs treated with varying concentrations of AECHL-1 for 48 h, and cell viability was determined by MTT assay. b HUVECS treated with varying concentrations of AECHL-1 for 24 h and subjected to flow cytometry using Annexin V-FITC as a marker of apoptosis. c HUVECs treated with varying concentrations of AECHL-1 for 48 h, and cell proliferation was determined by tritiated thymidine assay. Columns, mean from three independent experiments performed in triplicate; bars , SE. * P

    Techniques Used: MTT Assay, Flow Cytometry, Marker

    AECHL-1 inhibits VEGF-induced capillary structure formation, invasion and migration of endothelial cells. a AECHL-1 inhibited VEGF-induced tube formation of endothelial cells on Matrigel. b AECHL-1 inhibited HUVEC invasion. Migrated cells through the membrane were quantified. c AECHL-1 inhibited HUVEC migration. HUVEC monolayer was scratched by pipette and treated with or without 10 ng/mL VEGF. Migration is expressed as % gap closure of VEGF-treated well. Endothelial cells were photographed (magnification, ×10) using Image pro-plus and quantified using ImageJ software for all above-described experiments. Columns, mean from three independent experiments; bars, SE. * P
    Figure Legend Snippet: AECHL-1 inhibits VEGF-induced capillary structure formation, invasion and migration of endothelial cells. a AECHL-1 inhibited VEGF-induced tube formation of endothelial cells on Matrigel. b AECHL-1 inhibited HUVEC invasion. Migrated cells through the membrane were quantified. c AECHL-1 inhibited HUVEC migration. HUVEC monolayer was scratched by pipette and treated with or without 10 ng/mL VEGF. Migration is expressed as % gap closure of VEGF-treated well. Endothelial cells were photographed (magnification, ×10) using Image pro-plus and quantified using ImageJ software for all above-described experiments. Columns, mean from three independent experiments; bars, SE. * P

    Techniques Used: Migration, Transferring, Software

    11) Product Images from "Novel functions for 2‐phenylbenzimidazole‐5‐sulphonic acid: Inhibition of ovarian cancer cell responses and tumour angiogenesis, et al. Novel functions for 2‐phenylbenzimidazole‐5‐sulphonic acid: Inhibition of ovarian cancer cell responses and tumour angiogenesis"

    Article Title: Novel functions for 2‐phenylbenzimidazole‐5‐sulphonic acid: Inhibition of ovarian cancer cell responses and tumour angiogenesis, et al. Novel functions for 2‐phenylbenzimidazole‐5‐sulphonic acid: Inhibition of ovarian cancer cell responses and tumour angiogenesis

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14989

    PBSA inhibits endothelial tube formation through the down‐regulation of VEGF. A, Quiescent SKOV‐3 cells were pre‐treated with PBSA for 30 min, followed by 10% FBS stimulation for (left panel) 24 h or (right panel) 48 h. RT‐PCR and ELISA analyses were performed as described in Materials and methods. B, HUVEC tube formation assay was performed using conditioned media from SKOV‐3 cell culture treated as described above (A, left panel). Values represent the mean ± SD of at least three independent experiments. Statistical significance is indicated (* P
    Figure Legend Snippet: PBSA inhibits endothelial tube formation through the down‐regulation of VEGF. A, Quiescent SKOV‐3 cells were pre‐treated with PBSA for 30 min, followed by 10% FBS stimulation for (left panel) 24 h or (right panel) 48 h. RT‐PCR and ELISA analyses were performed as described in Materials and methods. B, HUVEC tube formation assay was performed using conditioned media from SKOV‐3 cell culture treated as described above (A, left panel). Values represent the mean ± SD of at least three independent experiments. Statistical significance is indicated (* P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, HUVEC Tube Formation Assay, Cell Culture

    12) Product Images from "Surface Engineering of FLT4-Targeted Nanocarriers Enhances Cell-Softening Glaucoma Therapy"

    Article Title: Surface Engineering of FLT4-Targeted Nanocarriers Enhances Cell-Softening Glaucoma Therapy

    Journal: ACS applied materials & interfaces

    doi: 10.1021/acsami.1c09294

    Normal and glaucomatous SC endothelial cells express FLT4/VEGFR3. (a) Illustration of the FLT4/VEGFR3 expression test in two normal SC endothelial cell strains (SC 78 and SC 84) and two SCg cell strains (SC 57g and SC 90g). (b) Flow cytometric analysis of FLT4 expression in SC and SCg cell strains. HUVECs are included as a control endothelial cell line that does not express FLT4 at high levels. FLT4 expression was detected by staining cells with antihuman FLT4-APC antibodies. Data are presented as mean ± s.e.m. ( n = 3). Significant differences between SC and HUVEC FLT4 MFI were determined by ANOVA with Dunnett’s multiple comparison test (5% significance level). * p
    Figure Legend Snippet: Normal and glaucomatous SC endothelial cells express FLT4/VEGFR3. (a) Illustration of the FLT4/VEGFR3 expression test in two normal SC endothelial cell strains (SC 78 and SC 84) and two SCg cell strains (SC 57g and SC 90g). (b) Flow cytometric analysis of FLT4 expression in SC and SCg cell strains. HUVECs are included as a control endothelial cell line that does not express FLT4 at high levels. FLT4 expression was detected by staining cells with antihuman FLT4-APC antibodies. Data are presented as mean ± s.e.m. ( n = 3). Significant differences between SC and HUVEC FLT4 MFI were determined by ANOVA with Dunnett’s multiple comparison test (5% significance level). * p

    Techniques Used: Expressing, Staining

    Differences in ligand biochemical accessibility modulate the rate of MC uptake by SC endothelial cells and vascular endothelial cells in vitro . (a,b) Determination of biochemical access to lipid-anchored targeting peptides displayed on polymeric MCs. (a) Illustration of the protease protection assay to evaluate peptide accessibility. (b) Trypsin proteolysis kinetics ( n = 5). Concentrations: [peptide] = 40 nM and [trypsin] = 800 nM. Pseudo-first-order association model fits are displayed for comparison, y = y 0 + ( y max – y 0 )*(1 – e − kx ), where k is the proteolysis rate (hours −1 ). In all cases, r 2 > 0.94. (c–f) The surface-displayed PG48 FLT4-targeting peptide significantly increases MC uptake by human SC cells and decreases uptake by HUVECs in vitro . (c) Illustration of PEG- b -PPS MC formulations and the cellular uptake study. (d,e) Cellular uptake by normal SC cells (d) or HUVECs (e). MFI determined by flow cytometry. The mean ± s.e.m. is displayed ( n = 3). (f) SC-targeting specificity defined here as SC MFI /HUVEC MFI . For (d–f), statistical significance was determined by ANOVA with post hoc Tukey’s multiple comparison test and a 5% significance level. **** p
    Figure Legend Snippet: Differences in ligand biochemical accessibility modulate the rate of MC uptake by SC endothelial cells and vascular endothelial cells in vitro . (a,b) Determination of biochemical access to lipid-anchored targeting peptides displayed on polymeric MCs. (a) Illustration of the protease protection assay to evaluate peptide accessibility. (b) Trypsin proteolysis kinetics ( n = 5). Concentrations: [peptide] = 40 nM and [trypsin] = 800 nM. Pseudo-first-order association model fits are displayed for comparison, y = y 0 + ( y max – y 0 )*(1 – e − kx ), where k is the proteolysis rate (hours −1 ). In all cases, r 2 > 0.94. (c–f) The surface-displayed PG48 FLT4-targeting peptide significantly increases MC uptake by human SC cells and decreases uptake by HUVECs in vitro . (c) Illustration of PEG- b -PPS MC formulations and the cellular uptake study. (d,e) Cellular uptake by normal SC cells (d) or HUVECs (e). MFI determined by flow cytometry. The mean ± s.e.m. is displayed ( n = 3). (f) SC-targeting specificity defined here as SC MFI /HUVEC MFI . For (d–f), statistical significance was determined by ANOVA with post hoc Tukey’s multiple comparison test and a 5% significance level. **** p

    Techniques Used: In Vitro, Flow Cytometry

    13) Product Images from "The impact of autophagy modulation on phenotype and survival of cardiac stromal cells under metabolic stress"

    Article Title: The impact of autophagy modulation on phenotype and survival of cardiac stromal cells under metabolic stress

    Journal: Cell Death Discovery

    doi: 10.1038/s41420-022-00924-7

    Autophagy induction enhances the pro-angiogenic paracrine capacity of CSCs. Tube-forming assay was performed with HUVECs exposed to conditioned media from ad-ATG7 or ad-LacZ transduced CSCs, in 5 or 50 mM glucose. Representative culture images are shown ( A , B ), together with the quantification of closed-loop number (C) and total tube length (D) ( n =4). Scale bars=200 µm. *P
    Figure Legend Snippet: Autophagy induction enhances the pro-angiogenic paracrine capacity of CSCs. Tube-forming assay was performed with HUVECs exposed to conditioned media from ad-ATG7 or ad-LacZ transduced CSCs, in 5 or 50 mM glucose. Representative culture images are shown ( A , B ), together with the quantification of closed-loop number (C) and total tube length (D) ( n =4). Scale bars=200 µm. *P

    Techniques Used:

    14) Product Images from "TNFα-senescence initiates a STAT-dependent positive feedback loop, leading to a sustained interferon signature, DNA damage, and cytokine secretion"

    Article Title: TNFα-senescence initiates a STAT-dependent positive feedback loop, leading to a sustained interferon signature, DNA damage, and cytokine secretion

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.101328

    Model of mechanisms involved in TNFα-induced senescence of HUVECs ( A ) TNFα activates JAK/STAT and p38 signaling pathways, which mediate increased expression of STAT1/3 phosphorylation. Activation of the JAK pathway leads to persistent phosphorylation of STAT1/3 signaling, which together with ROS, interferon genes, and other SASP components, drives a positive auto-regulatory loop, leading to sustained inflammation and stable senescence. ( B ) Inhibition of STAT1/3 with the JAK inhibitor AG490 decreased ROS and IL-6 production and decreased expression of interferon response genes. On the other hand, blockade of STAT1/3 expression decreased S phase entry of cells and increased p21 expression, leading to senescence.
    Figure Legend Snippet: Model of mechanisms involved in TNFα-induced senescence of HUVECs ( A ) TNFα activates JAK/STAT and p38 signaling pathways, which mediate increased expression of STAT1/3 phosphorylation. Activation of the JAK pathway leads to persistent phosphorylation of STAT1/3 signaling, which together with ROS, interferon genes, and other SASP components, drives a positive auto-regulatory loop, leading to sustained inflammation and stable senescence. ( B ) Inhibition of STAT1/3 with the JAK inhibitor AG490 decreased ROS and IL-6 production and decreased expression of interferon response genes. On the other hand, blockade of STAT1/3 expression decreased S phase entry of cells and increased p21 expression, leading to senescence.

    Techniques Used: Expressing, Activation Assay, Inhibition

    TNFα induces senescence and DNA damage in HUVECs ( A ) Long-term growth curve of cells exposed to recombinant human TNFα (5ng/ml). Untreated cells were used as controls. Population doubling and doubling times were calculated based on cell density at confluence. Data represent mean values from 3 independent experiments. ( B ) The percentage of BrdU-positive cells was determined by FACS analysis in cells untreated or chronically treated with TNFα at the concentration indicated. ( C ) Western blot analysis of p21, p16, and actin in cells treated with TNFα 5ng/ml for the indicated times. ( D ) SA-β-gal activity in TNFα (5ng/ml)-treated or control cells for the indicated number of days. ( E ) Percentages of SA-β-gal-positive cells in control or TNFα-treated cultures. The data represent 2 independent counts of 200 cells from 3 independent experiments. ( F ) Intracellular ROS levels were monitored by 2′,7′-dichlorodihydrofluorescein diacetate staining followed by flow cytometry. Bar graph represents percentage of DCFDA-positive cells treated with TNFα or medium alone. ( G ) Immunofluorescence detection of γH2AX foci in controls or cells treated with TNFα (5ng/ml) for indicated days. Data in A , B , E , and F represent mean value ± standard deviation (s.d.) from n=3, 2, 3, and 2 independent experiments, respectively.
    Figure Legend Snippet: TNFα induces senescence and DNA damage in HUVECs ( A ) Long-term growth curve of cells exposed to recombinant human TNFα (5ng/ml). Untreated cells were used as controls. Population doubling and doubling times were calculated based on cell density at confluence. Data represent mean values from 3 independent experiments. ( B ) The percentage of BrdU-positive cells was determined by FACS analysis in cells untreated or chronically treated with TNFα at the concentration indicated. ( C ) Western blot analysis of p21, p16, and actin in cells treated with TNFα 5ng/ml for the indicated times. ( D ) SA-β-gal activity in TNFα (5ng/ml)-treated or control cells for the indicated number of days. ( E ) Percentages of SA-β-gal-positive cells in control or TNFα-treated cultures. The data represent 2 independent counts of 200 cells from 3 independent experiments. ( F ) Intracellular ROS levels were monitored by 2′,7′-dichlorodihydrofluorescein diacetate staining followed by flow cytometry. Bar graph represents percentage of DCFDA-positive cells treated with TNFα or medium alone. ( G ) Immunofluorescence detection of γH2AX foci in controls or cells treated with TNFα (5ng/ml) for indicated days. Data in A , B , E , and F represent mean value ± standard deviation (s.d.) from n=3, 2, 3, and 2 independent experiments, respectively.

    Techniques Used: Recombinant, FACS, Concentration Assay, Western Blot, Activity Assay, Staining, Flow Cytometry, Immunofluorescence, Standard Deviation

    15) Product Images from "Preventive Effects of Quercetin against the Onset of Atherosclerosis-Related Acute Aortic Syndromes in Mice"

    Article Title: Preventive Effects of Quercetin against the Onset of Atherosclerosis-Related Acute Aortic Syndromes in Mice

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21197226

    The effects of quercetin on plasma tumor necrosis factor (TNF)-α concentration and the quercetin-3-O-β-D-glucuronide (Q3GA) effects in cultured human umbilical vein endothelial cells (HUVECs). ( a ) TNF-α concentration in plasma from AB group mice ( n = 5–8). Protein expression of VCAM-1 and extracellular signal-regulated kinase (ERK) 5 phosphorylation in HUVECs were analyzed by Western blotting. Representative bands (( b ) and upper panel of ( d )), and quantified intensity (( c ) and lower panel of ( d )) are shown. The mRNA expression of endothelial nitric oxide synthase (eNOS) in HUVECs are shown in ( e ). n = 4–6. Values are shown as fold increase to the average of control and are expressed as mean±SE. Statistical analyses were performed using two-way ANOVA for repeated measures and Bonferroni post hoc test. * p
    Figure Legend Snippet: The effects of quercetin on plasma tumor necrosis factor (TNF)-α concentration and the quercetin-3-O-β-D-glucuronide (Q3GA) effects in cultured human umbilical vein endothelial cells (HUVECs). ( a ) TNF-α concentration in plasma from AB group mice ( n = 5–8). Protein expression of VCAM-1 and extracellular signal-regulated kinase (ERK) 5 phosphorylation in HUVECs were analyzed by Western blotting. Representative bands (( b ) and upper panel of ( d )), and quantified intensity (( c ) and lower panel of ( d )) are shown. The mRNA expression of endothelial nitric oxide synthase (eNOS) in HUVECs are shown in ( e ). n = 4–6. Values are shown as fold increase to the average of control and are expressed as mean±SE. Statistical analyses were performed using two-way ANOVA for repeated measures and Bonferroni post hoc test. * p

    Techniques Used: Concentration Assay, Cell Culture, Mouse Assay, Expressing, Western Blot

    16) Product Images from "Endoglin regulates mural cell adhesion in the circulatory system"

    Article Title: Endoglin regulates mural cell adhesion in the circulatory system

    Journal: Cellular and Molecular Life Sciences

    doi: 10.1007/s00018-015-2099-4

    Schematic diagram showing the role of endoglin in integrin-mediated cell adhesion between ECs and mural cells/podocytes. a – c Blood vessels. a A normal blood vessel with an endothelial monolayer facing the lumen surrounded by vascular mural cells (VMCs) and ECM proteins. ECs and vascular mural cells share a common basal membrane (BM). During vascular development and stabilization, binding of the homeostatic chemokine CXCL12 to its receptor CXCR4 leads to activation of β1-integrins in VMCs. Then, endothelial endoglin binds to β1-integrins on VMCs. b In HHT1, endoglin haploinsufficiency leads to a decreased binding of endothelial endoglin to β1-integrins in VMCs. c In preeclampsia, soluble endoglin competes with membrane bound endoglin for the binding to β1-integrins in VMCs. d , e Kidney glomerulus. d A normal glomerulus showing pericytes bound to the glomerular basal membrane (GBM) through their surface integrins. e In preeclampsia, soluble endoglin competes with GBM for the binding to surface integrins in podocytes. The presence of endothelial endoglin in the lumen of the vessel and the existence of other adhesion molecules have been omitted for simplification
    Figure Legend Snippet: Schematic diagram showing the role of endoglin in integrin-mediated cell adhesion between ECs and mural cells/podocytes. a – c Blood vessels. a A normal blood vessel with an endothelial monolayer facing the lumen surrounded by vascular mural cells (VMCs) and ECM proteins. ECs and vascular mural cells share a common basal membrane (BM). During vascular development and stabilization, binding of the homeostatic chemokine CXCL12 to its receptor CXCR4 leads to activation of β1-integrins in VMCs. Then, endothelial endoglin binds to β1-integrins on VMCs. b In HHT1, endoglin haploinsufficiency leads to a decreased binding of endothelial endoglin to β1-integrins in VMCs. c In preeclampsia, soluble endoglin competes with membrane bound endoglin for the binding to β1-integrins in VMCs. d , e Kidney glomerulus. d A normal glomerulus showing pericytes bound to the glomerular basal membrane (GBM) through their surface integrins. e In preeclampsia, soluble endoglin competes with GBM for the binding to surface integrins in podocytes. The presence of endothelial endoglin in the lumen of the vessel and the existence of other adhesion molecules have been omitted for simplification

    Techniques Used: Binding Assay, Activation Assay

    Involvement of the endoglin RGD motif in adhesion between VSMCs and ECs. a HUVECs, labeled with CSFE ( green ), were incubated with the UASMC monolayers for 1 h at 37 °C with/without thalidomide, CXCL12 (CXC), SolEng (S.Eng), RGD peptide or DGR peptide, as indicated. Bound CSFE-labeled cells were lysed and quantified by Varioskan plate reader. The average of four different experiments in duplicate is shown. Statistical significances vs. control cells (** p
    Figure Legend Snippet: Involvement of the endoglin RGD motif in adhesion between VSMCs and ECs. a HUVECs, labeled with CSFE ( green ), were incubated with the UASMC monolayers for 1 h at 37 °C with/without thalidomide, CXCL12 (CXC), SolEng (S.Eng), RGD peptide or DGR peptide, as indicated. Bound CSFE-labeled cells were lysed and quantified by Varioskan plate reader. The average of four different experiments in duplicate is shown. Statistical significances vs. control cells (** p

    Techniques Used: Labeling, Incubation

    Endoglin silencing in endothelial and smooth muscle cells. a Primary cultures of HUVECs, HAECs and UASMCs (p4–8) were untreated (control), nucleofected with endoglin (Eng) specific siRNA (#s4677 and #s4679) and GFP ( green stain ), or nucleofected with scrambled siRNA (#AM4611 and #AM4613) and GFP (siRNA control). After 48 h, cells were observed by confocal microscopy. Cells cotransfected with GFP are visualized by their green fluorescence. b Flow cytometry analysis. Primary cultures of HUVECs, HAECs and UASMCs were untreated (control) or nucleofected with Eng siRNA or scrambled siRNA #AM4611 and #AM4613 (siRNA control). After 48 h, cells were analyzed by immunofluorescence flow cytometry with anti-endoglin mAb P4A4 ( green histograms) or a negative control mAb (X63; blue histograms ). The mean fluorescence intensity (MFI) of each sample is indicated. Endoglin expression is decreased upon transfection with specific siRNA in all cells. Cells nucleofected with scrambled siRNA showed the same endoglin expression levels as untreated cells (data not shown). c HUVECs and HAECs were incubated in matrigel to analyze tube formation. Confocal microscopy of untreated cells (control), nucleofected for 48 h with scrambled siRNA (AM4611, #1; AM4613, #2) or Eng siRNA (#s4677 and #s4679), or incubated with soluble endoglin (Sol.Eng) are shown. The histogram on the right indicates the percentage, respect to the control sample (100 %), of closing tubes under each experimental condition. Samples were in triplicates and the mean of the control condition was given the arbitrary value of 100. The average of five different experiments is shown. The statistical significance respect to control value (CTR) is indicated (*** p
    Figure Legend Snippet: Endoglin silencing in endothelial and smooth muscle cells. a Primary cultures of HUVECs, HAECs and UASMCs (p4–8) were untreated (control), nucleofected with endoglin (Eng) specific siRNA (#s4677 and #s4679) and GFP ( green stain ), or nucleofected with scrambled siRNA (#AM4611 and #AM4613) and GFP (siRNA control). After 48 h, cells were observed by confocal microscopy. Cells cotransfected with GFP are visualized by their green fluorescence. b Flow cytometry analysis. Primary cultures of HUVECs, HAECs and UASMCs were untreated (control) or nucleofected with Eng siRNA or scrambled siRNA #AM4611 and #AM4613 (siRNA control). After 48 h, cells were analyzed by immunofluorescence flow cytometry with anti-endoglin mAb P4A4 ( green histograms) or a negative control mAb (X63; blue histograms ). The mean fluorescence intensity (MFI) of each sample is indicated. Endoglin expression is decreased upon transfection with specific siRNA in all cells. Cells nucleofected with scrambled siRNA showed the same endoglin expression levels as untreated cells (data not shown). c HUVECs and HAECs were incubated in matrigel to analyze tube formation. Confocal microscopy of untreated cells (control), nucleofected for 48 h with scrambled siRNA (AM4611, #1; AM4613, #2) or Eng siRNA (#s4677 and #s4679), or incubated with soluble endoglin (Sol.Eng) are shown. The histogram on the right indicates the percentage, respect to the control sample (100 %), of closing tubes under each experimental condition. Samples were in triplicates and the mean of the control condition was given the arbitrary value of 100. The average of five different experiments is shown. The statistical significance respect to control value (CTR) is indicated (*** p

    Techniques Used: Staining, Confocal Microscopy, Fluorescence, Flow Cytometry, Immunofluorescence, Negative Control, Expressing, Transfection, Incubation

    Role of endoglin in adhesion between VSMCs and ECs. a , b Cell–cell adhesion in angiogenesis assays. a UASMCs were transfected with GFP and cocultured with unlabeled HUVECs at a 1:4 ratio in matrigel to analyze mural cell adhesion to ECs. Confocal microscopy of untreated cells (control) and cells incubated with soluble endoglin (Sol.Eng) or nucleofected with Eng siRNA are shown in representative photographs. The intensity of the staining according to the color scale (0–250) indicates mural cell adhesion to endothelial cells in 3D co-culture b Quantification of UASMCs binding to ECs was carried out by measuring the intensity profile using fluorescence confocal microscopy (SP5, Leica). The mean area in percentage, representing mural cell adhesion measured in different fields, is indicated. Samples were in triplicates and the mean of the control condition was given the arbitrary value of 100. The average of five different experiments is shown. c , d Cell adhesion assay. c HUVEC monolayers were incubated with UASMCs previously labeled with CSFE in the absence or in the presence of soluble endoglin. After 1 h incubation, wells were washed and the cells were visualized by confocal microscopy. d Binding of UASMCs to HUVECs in c was quantified by measuring the intensity profile using fluorescence confocal microscopy (SP5, Leica). The average of four independent experiments is shown. The statistical significance respect to control value (CTR) is indicated. * p
    Figure Legend Snippet: Role of endoglin in adhesion between VSMCs and ECs. a , b Cell–cell adhesion in angiogenesis assays. a UASMCs were transfected with GFP and cocultured with unlabeled HUVECs at a 1:4 ratio in matrigel to analyze mural cell adhesion to ECs. Confocal microscopy of untreated cells (control) and cells incubated with soluble endoglin (Sol.Eng) or nucleofected with Eng siRNA are shown in representative photographs. The intensity of the staining according to the color scale (0–250) indicates mural cell adhesion to endothelial cells in 3D co-culture b Quantification of UASMCs binding to ECs was carried out by measuring the intensity profile using fluorescence confocal microscopy (SP5, Leica). The mean area in percentage, representing mural cell adhesion measured in different fields, is indicated. Samples were in triplicates and the mean of the control condition was given the arbitrary value of 100. The average of five different experiments is shown. c , d Cell adhesion assay. c HUVEC monolayers were incubated with UASMCs previously labeled with CSFE in the absence or in the presence of soluble endoglin. After 1 h incubation, wells were washed and the cells were visualized by confocal microscopy. d Binding of UASMCs to HUVECs in c was quantified by measuring the intensity profile using fluorescence confocal microscopy (SP5, Leica). The average of four independent experiments is shown. The statistical significance respect to control value (CTR) is indicated. * p

    Techniques Used: Transfection, Confocal Microscopy, Incubation, Staining, Co-Culture Assay, Binding Assay, Fluorescence, Cell Adhesion Assay, Labeling

    Role of integrins in adhesion between VSMCs and ECs. UASMCs were labeled with CSFE ( green ) and HUVECs were labeled with CMTPX ( red ). Then, UASMCs were cocultured with HUVECs at a 1:4 ratio in matrigel to analyze mural cell adhesion to ECs. a , c Representative photographs of confocal microscopy analysis. a Cells treated with a control mAb IgG2b (CTR) and cells incubated with the inhibitory anti-β1 integrins mAb LIA1/2, the anti-β1 integrins mAb TS2/16 or the general integrins activator MnCl 2 . c Untreated cells (control) and cells incubated with the pericyte recruiter PDGF-BB or the integrins activator CXCL12 either in the absence or in the presence of the chemokine receptor (CXCR4) inhibitor AMD3100 (AMD). b , d Quantification of UASMCs binding to HUVECs from a and c , respectively. The intensity profile was measured using fluorescence confocal microscopy (SP5, Leica). The mean area in percentage, representing mural cell adhesion measured in different fields, is indicated. Histograms in b and d represent the mean of four and five independent experiments, respectively. The statistical significance respect to control value (CTR) is indicated. * p
    Figure Legend Snippet: Role of integrins in adhesion between VSMCs and ECs. UASMCs were labeled with CSFE ( green ) and HUVECs were labeled with CMTPX ( red ). Then, UASMCs were cocultured with HUVECs at a 1:4 ratio in matrigel to analyze mural cell adhesion to ECs. a , c Representative photographs of confocal microscopy analysis. a Cells treated with a control mAb IgG2b (CTR) and cells incubated with the inhibitory anti-β1 integrins mAb LIA1/2, the anti-β1 integrins mAb TS2/16 or the general integrins activator MnCl 2 . c Untreated cells (control) and cells incubated with the pericyte recruiter PDGF-BB or the integrins activator CXCL12 either in the absence or in the presence of the chemokine receptor (CXCR4) inhibitor AMD3100 (AMD). b , d Quantification of UASMCs binding to HUVECs from a and c , respectively. The intensity profile was measured using fluorescence confocal microscopy (SP5, Leica). The mean area in percentage, representing mural cell adhesion measured in different fields, is indicated. Histograms in b and d represent the mean of four and five independent experiments, respectively. The statistical significance respect to control value (CTR) is indicated. * p

    Techniques Used: Labeling, Confocal Microscopy, Incubation, Binding Assay, Fluorescence

    17) Product Images from "Tissue‐specific angiogenic and invasive properties of human neonatal thymus and bone MSCs: Role of SLIT3‐ROBO1, et al. Tissue‐specific angiogenic and invasive properties of human neonatal thymus and bone MSCs: Role of SLIT3‐ROBO1"

    Article Title: Tissue‐specific angiogenic and invasive properties of human neonatal thymus and bone MSCs: Role of SLIT3‐ROBO1, et al. Tissue‐specific angiogenic and invasive properties of human neonatal thymus and bone MSCs: Role of SLIT3‐ROBO1

    Journal: Stem Cells Translational Medicine

    doi: 10.1002/sctm.19-0448

    Neonatal thymus MSCs are more proangiogenic than subject‐matched nbMSCs. A, Conditioned medium from ntMSCs promoted greater HUVEC network formation as conditioned medium from nbMSCs and abMSCs. Scale bar = 200 μm. B, Quantification of HUVEC network formation in A. Each data point represents a randomly selected field of analysis (results are from n = 3 subjects). C, Neonatal thymus MSCs had greater transcript levels of ANGPT1 and HIF1A as compared to matched nbMSCs. Each data pair represents ntMSCs and nbMSCs that were isolated from a single patient and all data compared with a ratio t test. D, CD31 immunohistochemistry of MSC/HUVEC‐seeded collagen/fibronectin constructs implanted in NOD SCID mice for 14 days. Scale bars = 50 μm. E, Quantification of CD31 vascular density of constructs in D, showing that ntMSCs stimulated more angiogenesis in vivo (n = 5 animals with two constructs per animal; ntMSCs and nbMSCs isolated from the same subject). Each data point represents a randomly selected field of analysis
    Figure Legend Snippet: Neonatal thymus MSCs are more proangiogenic than subject‐matched nbMSCs. A, Conditioned medium from ntMSCs promoted greater HUVEC network formation as conditioned medium from nbMSCs and abMSCs. Scale bar = 200 μm. B, Quantification of HUVEC network formation in A. Each data point represents a randomly selected field of analysis (results are from n = 3 subjects). C, Neonatal thymus MSCs had greater transcript levels of ANGPT1 and HIF1A as compared to matched nbMSCs. Each data pair represents ntMSCs and nbMSCs that were isolated from a single patient and all data compared with a ratio t test. D, CD31 immunohistochemistry of MSC/HUVEC‐seeded collagen/fibronectin constructs implanted in NOD SCID mice for 14 days. Scale bars = 50 μm. E, Quantification of CD31 vascular density of constructs in D, showing that ntMSCs stimulated more angiogenesis in vivo (n = 5 animals with two constructs per animal; ntMSCs and nbMSCs isolated from the same subject). Each data point represents a randomly selected field of analysis

    Techniques Used: Isolation, Immunohistochemistry, Construct, Mouse Assay, In Vivo

    18) Product Images from "Collaborative Enhancement of Antibody Binding to Distinct PECAM-1 Epitopes Modulates Endothelial Targeting"

    Article Title: Collaborative Enhancement of Antibody Binding to Distinct PECAM-1 Epitopes Modulates Endothelial Targeting

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034958

    Binding parameters of anti-PECAM-1 [ 125 I]-mAbs to live cells expressing PECAM-1. Cell surface binding parameters (K d and B max ) of [ 125 I]-mAbs to PECAM-1 was determined by RIA-based method with ( A ) native huPECAM-1 on HUVECs, and ( B ) recombinant muPECAM-1 on REN-muP cells. Serial dilutions of [ 125 I]-mAbs were added to confluent cellular monolayers and incubated for 2 h at 4°C. The results shown are from a representative experiment, with the inset showing Scatchard plot of binding data. Note that total binding was corrected for NSB using 100−fold excess of unlabeled mAb for HUVECs or using parent REN cells for REN-muP binding. ( C – D ) K d and B max Binding parameters are for [ 125 I]-mAbs to huPECAM-1 and muPECAM-1 are listed. Results were determined by three independent RIA experiments performed in quadruplicate, with data expressed as mean ± SD.
    Figure Legend Snippet: Binding parameters of anti-PECAM-1 [ 125 I]-mAbs to live cells expressing PECAM-1. Cell surface binding parameters (K d and B max ) of [ 125 I]-mAbs to PECAM-1 was determined by RIA-based method with ( A ) native huPECAM-1 on HUVECs, and ( B ) recombinant muPECAM-1 on REN-muP cells. Serial dilutions of [ 125 I]-mAbs were added to confluent cellular monolayers and incubated for 2 h at 4°C. The results shown are from a representative experiment, with the inset showing Scatchard plot of binding data. Note that total binding was corrected for NSB using 100−fold excess of unlabeled mAb for HUVECs or using parent REN cells for REN-muP binding. ( C – D ) K d and B max Binding parameters are for [ 125 I]-mAbs to huPECAM-1 and muPECAM-1 are listed. Results were determined by three independent RIA experiments performed in quadruplicate, with data expressed as mean ± SD.

    Techniques Used: Binding Assay, Expressing, Recombinant, Incubation

    In vitro binding properties of mAb to live cells expressing PECAM-1. Cell surface binding of mAbs to PECAM-1 was determined by ELISA-based method with ( A ) HUVECs, ( B ) REN-muP cells. Proteins were added to confluent cellular monolayers at the indicated dilutions and incubated for 2 h at 4°C. The results shown are from a representative experiment. Non-targeted IgG or non-PECAM-1 expressing cells were used as negative control. Representative plots for mAb binding to MS1 cells are available in Figure S2 . ( C ) Analysis of the relative binding affinity of anti-PECAM-1 mAbs, when binding to cells is half-maximal (IC 50 ). Data points were fit as described under “ Methods .” The IC 50 is reported as the mean IC 50 value ± SD of three independent experiments performed in triplicate.
    Figure Legend Snippet: In vitro binding properties of mAb to live cells expressing PECAM-1. Cell surface binding of mAbs to PECAM-1 was determined by ELISA-based method with ( A ) HUVECs, ( B ) REN-muP cells. Proteins were added to confluent cellular monolayers at the indicated dilutions and incubated for 2 h at 4°C. The results shown are from a representative experiment. Non-targeted IgG or non-PECAM-1 expressing cells were used as negative control. Representative plots for mAb binding to MS1 cells are available in Figure S2 . ( C ) Analysis of the relative binding affinity of anti-PECAM-1 mAbs, when binding to cells is half-maximal (IC 50 ). Data points were fit as described under “ Methods .” The IC 50 is reported as the mean IC 50 value ± SD of three independent experiments performed in triplicate.

    Techniques Used: In Vitro, Binding Assay, Expressing, Enzyme-linked Immunosorbent Assay, Incubation, Negative Control

    Anti-PECAM-1 [ 125 I]-mAb binding in live cells is enhanced by paired mAb directed to adjacent PECAM-1 epitope. The modulation of PECAM-1 binding was determined by co-incubation of [ 125 I]-mAb with indicated concentrations of unlabeled self-paired mAb or paired mAb with cells for 2 h at 4°C. Binding data were plotted as [ 125 I]-mAb molecules bound per cell (mAb/cell) and data points were fit as described under “ Methods .” ( A and B ) Unlabeled mAb 62 competitively inhibits binding of [ 125 I]-mAb 62 to huPECAM-1 in HUVEC. However, mAb 37 enhances [ 125 I]-mAb 62 binding to huPECAM-1 in HUVEC by 1.5−fold over binding of [ 125 I]-mAb 62 alone. Interestingly, mAb 62 does not enhance the binding of [ 125 I]-mAb 37 ( Figure S3 ). ( C – D ) Collaborative binding studies of mAbs 390 and MEC13.3 with REN-muP cells as described in panel A. Unlabeled self-paired mAb 390 and mAb MEC13.3 competitively inhibit binding of [ 125 I]-mAb390 and [ 125 I]-mAb MEC13.3 to REN-muP cells, respectively. In contrast, mAb pairs [ 125 I]-mAb 390/MEC13.3 and [ 125 I]-mAb MEC13.3/390 enhance binding by ∼1.5−fold and ∼2.7−fold, respectively, over [ 125 I]-mAb alone (***, P
    Figure Legend Snippet: Anti-PECAM-1 [ 125 I]-mAb binding in live cells is enhanced by paired mAb directed to adjacent PECAM-1 epitope. The modulation of PECAM-1 binding was determined by co-incubation of [ 125 I]-mAb with indicated concentrations of unlabeled self-paired mAb or paired mAb with cells for 2 h at 4°C. Binding data were plotted as [ 125 I]-mAb molecules bound per cell (mAb/cell) and data points were fit as described under “ Methods .” ( A and B ) Unlabeled mAb 62 competitively inhibits binding of [ 125 I]-mAb 62 to huPECAM-1 in HUVEC. However, mAb 37 enhances [ 125 I]-mAb 62 binding to huPECAM-1 in HUVEC by 1.5−fold over binding of [ 125 I]-mAb 62 alone. Interestingly, mAb 62 does not enhance the binding of [ 125 I]-mAb 37 ( Figure S3 ). ( C – D ) Collaborative binding studies of mAbs 390 and MEC13.3 with REN-muP cells as described in panel A. Unlabeled self-paired mAb 390 and mAb MEC13.3 competitively inhibit binding of [ 125 I]-mAb390 and [ 125 I]-mAb MEC13.3 to REN-muP cells, respectively. In contrast, mAb pairs [ 125 I]-mAb 390/MEC13.3 and [ 125 I]-mAb MEC13.3/390 enhance binding by ∼1.5−fold and ∼2.7−fold, respectively, over [ 125 I]-mAb alone (***, P

    Techniques Used: Binding Assay, Incubation

    19) Product Images from "Early outgrowth cells versus endothelial colony forming cells functions in platelet aggregation"

    Article Title: Early outgrowth cells versus endothelial colony forming cells functions in platelet aggregation

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-015-0723-6

    Expression of cell surface markers by EOCs and ECFCs. Flow cytometric analysis of cell surface marker expression profile of EOCs ( gray ) and ECFCs ( white ) compared to PBMCs ( black ) and HUVECs ( dark gray ) using mouse anti-human PE-conjugated monoclonal antibodies against CD14, CD45, VEGFR2, CD34, CD144 and CD31. Histogram represents the mean data ± SEM of percent cell surface marker expression ( n ≥ 3)
    Figure Legend Snippet: Expression of cell surface markers by EOCs and ECFCs. Flow cytometric analysis of cell surface marker expression profile of EOCs ( gray ) and ECFCs ( white ) compared to PBMCs ( black ) and HUVECs ( dark gray ) using mouse anti-human PE-conjugated monoclonal antibodies against CD14, CD45, VEGFR2, CD34, CD144 and CD31. Histogram represents the mean data ± SEM of percent cell surface marker expression ( n ≥ 3)

    Techniques Used: Expressing, Flow Cytometry, Marker

    Expression of NOS and COX and release of NO and PGI 2 by EOCs and ECFCs. a Left : NOS and COX protein expression profile in PBMCs, EOCs, ECFCs and HUVECs. Representative blots of cell lysates analyzed for eNOS, iNOS, COX-1 and COX-2 by SDS-PAGE. Right : Histogram represents the mean of data ± SEM expressed as arbitrary units of optical density of blots on the left ( n ≥ 3); ( b , c ) supernatants from 4 × 10 6 /mL PBMCs, EOCs, ECFCs and HUVECs were assessed for PGI 2 release by radioimmunoassay of 6-keto-PGF 1α concentration (pg/mL) ( b ) and for NO release by nitrate/nitrite fluorometric assay for nitrate concentration (pmol) ( c ). Histograms represent the mean concentration ± SEM of nitrate and PGI 2 ( n ≥ 3, * p
    Figure Legend Snippet: Expression of NOS and COX and release of NO and PGI 2 by EOCs and ECFCs. a Left : NOS and COX protein expression profile in PBMCs, EOCs, ECFCs and HUVECs. Representative blots of cell lysates analyzed for eNOS, iNOS, COX-1 and COX-2 by SDS-PAGE. Right : Histogram represents the mean of data ± SEM expressed as arbitrary units of optical density of blots on the left ( n ≥ 3); ( b , c ) supernatants from 4 × 10 6 /mL PBMCs, EOCs, ECFCs and HUVECs were assessed for PGI 2 release by radioimmunoassay of 6-keto-PGF 1α concentration (pg/mL) ( b ) and for NO release by nitrate/nitrite fluorometric assay for nitrate concentration (pmol) ( c ). Histograms represent the mean concentration ± SEM of nitrate and PGI 2 ( n ≥ 3, * p

    Techniques Used: Expressing, SDS Page, RIA Assay, Concentration Assay

    Characterization of PBMC-derived EOCs and ECFCs. a Representative optical microscopy images of EOCs and ECFCs taken at 10× magnification using an inverted light microscope. EOCs display a heterogeneous population of round and elongated cells on fibronectin following 7 days of culture. ECFCs show a homogeneous population forming a cobblestone-like monolayer on collagen following 21 days of culture. b Representative confocal microscopy images showing EOCs and ECFCs triple staining for DiI-Ac-LDL uptake ( red ), Ulex-lectin binding ( green ) and TO-PRO-3 nuclear staining ( blue ) taken at 63× magnification. c Representative optical microscopy images showing the tube-like structure formation potential of EOCs and ECFCs compared to HUVECs on a Matrigel surface taken at 10× magnification using an inverted microscope
    Figure Legend Snippet: Characterization of PBMC-derived EOCs and ECFCs. a Representative optical microscopy images of EOCs and ECFCs taken at 10× magnification using an inverted light microscope. EOCs display a heterogeneous population of round and elongated cells on fibronectin following 7 days of culture. ECFCs show a homogeneous population forming a cobblestone-like monolayer on collagen following 21 days of culture. b Representative confocal microscopy images showing EOCs and ECFCs triple staining for DiI-Ac-LDL uptake ( red ), Ulex-lectin binding ( green ) and TO-PRO-3 nuclear staining ( blue ) taken at 63× magnification. c Representative optical microscopy images showing the tube-like structure formation potential of EOCs and ECFCs compared to HUVECs on a Matrigel surface taken at 10× magnification using an inverted microscope

    Techniques Used: Derivative Assay, Microscopy, Light Microscopy, Confocal Microscopy, Staining, Binding Assay, Inverted Microscopy

    20) Product Images from "Intracellular Delivery of Active Proteins by Polyphosphazene Polymers"

    Article Title: Intracellular Delivery of Active Proteins by Polyphosphazene Polymers

    Journal: Pharmaceutics

    doi: 10.3390/pharmaceutics13020249

    Uptake of PZ/protein complexes in different cell types. ( A ) HUVECs or ( B ) Cal27 cells incubated for 30 min at 37 °C with green FITC-avidin alone or complexed to PZ-PYR or PZ-PEG. Cells were washed, fixed, and incubated with Texas Red-labeled biotin-IgG to co-stain avidin on the cell surface in red (green + red = yellow) vs. internalized avidin (green alone). Blue = cell nuclei stained with DAPI. Dashed lines = cell borders as seen by bright field. Scale bar = 10 µm. Data are mean ± SEM. * p
    Figure Legend Snippet: Uptake of PZ/protein complexes in different cell types. ( A ) HUVECs or ( B ) Cal27 cells incubated for 30 min at 37 °C with green FITC-avidin alone or complexed to PZ-PYR or PZ-PEG. Cells were washed, fixed, and incubated with Texas Red-labeled biotin-IgG to co-stain avidin on the cell surface in red (green + red = yellow) vs. internalized avidin (green alone). Blue = cell nuclei stained with DAPI. Dashed lines = cell borders as seen by bright field. Scale bar = 10 µm. Data are mean ± SEM. * p

    Techniques Used: Incubation, Avidin-Biotin Assay, Labeling, Staining

    Binding of PZ/protein complexes to different cell types. ( A ) Human Umbilical Vein Endothelial Cells (HUVECs) or ( B ) Cal27 cells, incubated for 30 min at 37 °C with FITC-avidin alone or complexed to PZ-PYR or PZ-PEG. Fluorescence microcopy images (top) and image quantification of the mean intensity and sum intensity (bottom) are shown. Dashed lines = cell borders as seen by bright field. Scale bar = 10 µm. Data are mean ± SEM. * p
    Figure Legend Snippet: Binding of PZ/protein complexes to different cell types. ( A ) Human Umbilical Vein Endothelial Cells (HUVECs) or ( B ) Cal27 cells, incubated for 30 min at 37 °C with FITC-avidin alone or complexed to PZ-PYR or PZ-PEG. Fluorescence microcopy images (top) and image quantification of the mean intensity and sum intensity (bottom) are shown. Dashed lines = cell borders as seen by bright field. Scale bar = 10 µm. Data are mean ± SEM. * p

    Techniques Used: Binding Assay, Incubation, Avidin-Biotin Assay, Fluorescence

    21) Product Images from "The role of scaffold microarchitecture in engineering endothelial cell immunomodulation"

    Article Title: The role of scaffold microarchitecture in engineering endothelial cell immunomodulation

    Journal: Biomaterials

    doi: 10.1016/j.biomaterials.2012.06.052

    Experimental layout. A confluent endothelial monolayer (HUVECs) was activated by incubation with 10 ng/mL of TNF-α for 4 h. Top scheme : Thereafter, basal levels of THP-1 monocyte adhesion were analyzed by switching to EGM-2 media and incubate HUVEC monolayers with fluorescently labeled monocyte for 1 h before reading intensity of adherent cells. Two parallel sets of experiments were performed. Middle scheme : HUVECs were pre-incubated with conditioned media from flat 2D-ECs or contoured 3D-MEECs, then media changed to control EGM-2 and THP-1 added in suspension for 1 h before reading intensity of adherent cells. Bottom scheme : The same rational was pursued for monocytes, THP-1 cells were incubated with conditioned media from 2D-ECs or 3D-MEECs prior to use in the adhesion test.
    Figure Legend Snippet: Experimental layout. A confluent endothelial monolayer (HUVECs) was activated by incubation with 10 ng/mL of TNF-α for 4 h. Top scheme : Thereafter, basal levels of THP-1 monocyte adhesion were analyzed by switching to EGM-2 media and incubate HUVEC monolayers with fluorescently labeled monocyte for 1 h before reading intensity of adherent cells. Two parallel sets of experiments were performed. Middle scheme : HUVECs were pre-incubated with conditioned media from flat 2D-ECs or contoured 3D-MEECs, then media changed to control EGM-2 and THP-1 added in suspension for 1 h before reading intensity of adherent cells. Bottom scheme : The same rational was pursued for monocytes, THP-1 cells were incubated with conditioned media from 2D-ECs or 3D-MEECs prior to use in the adhesion test.

    Techniques Used: Incubation, Labeling

    22) Product Images from "Endothelial cells are not productively infected by SARS‐CoV‐2"

    Article Title: Endothelial cells are not productively infected by SARS‐CoV‐2

    Journal: Clinical & Translational Immunology

    doi: 10.1002/cti2.1350

    Endothelial cells can be infected with 2 × 10 6 PFU of SARS‐CoV‐2, but infection is abortive. (a) Viral replication shown as number of PFU mL −1 of supernatant from SARS‐CoV‐2 infected HUVEC, HMVEC‐L and Calu‐3 cells at 24 h, 48 h and 72 h after infection. n = 2 (HUVEC and HMVEC‐L), n = 3 (Calu‐3) independent experiments. (b) Representative immunofluorescent images of HMVEC‐L stained for nucleocapsid protein (NP) (shown as single channel in top panel) (green), Phalloidin (magenta) and DAPI (blue) with mock or SARS‐CoV‐2 infection from either the apical or basolateral side of the cells at 72 h after infection. Scale bar 50 µm. The white box is enlarged in Figure 3 . (c) Quantification of NP staining intensity in HMVEC‐L. n = 15 images from 3 independent experiments. (d) Western blot analysis showing NP protein levels in HUVEC, HMVEC‐L and Calu‐3 cells after 72 h of infection. (e) Quantification of NP protein levels in HUVEC, HMVEC‐L and Calu‐3. n = 3 independent experiments. (f) Representative immunofluorescent images of ACE2 overexpressing HMVEC‐L and control Calu‐3 cells stained for NP (shown as single channel in top panel) (green), ACE2 (shown as single channel in middle panel) (magenta), Phalloidin (grey) and DAPI (blue) with mock or SARS‐CoV‐2 infection from the apical side of the cells at 72 h after infection. Scale bar 50 µm. (g) Quantification of NP staining intensity in HMVEC‐L, HMVEC‐L + ACE2 and Calu‐3 with either low (6 × 10 4 ) or high (2 × 10 6 ) dose of SARS‐CoV‐2 infection. n = 15 images from 3 independent experiments. (h) Quantification of ACE2 staining intensity in HMVEC‐L, HMVEC‐L + ACE2 and Calu‐3. n = 10 (HMVEC‐L and Calu‐3), n = 20 (HMVEC‐L + ACE2) images from 2 independent experiments. (i) Viral replication shown as number of PFU mL −1 of supernatant from SARS‐CoV‐2 infected HMVEC‐L, HMVEC‐L + ACE2 and Calu‐3 cells at 72 h after infection. The grey dashed line indicates input level. n = 3 independent experiments. Data are presented as mean ± s.e.m. with individual data points indicated and colour coded per independent experimental replicate. Statistical significance was determined using the Kruskal–Wallis test between 24 h and other time points (a) , between HMVEC‐L and all other conditions (i) or the Mann–Whitney U ‐test between mock and + SARS‐CoV‐2 (c, e, g) . * P
    Figure Legend Snippet: Endothelial cells can be infected with 2 × 10 6 PFU of SARS‐CoV‐2, but infection is abortive. (a) Viral replication shown as number of PFU mL −1 of supernatant from SARS‐CoV‐2 infected HUVEC, HMVEC‐L and Calu‐3 cells at 24 h, 48 h and 72 h after infection. n = 2 (HUVEC and HMVEC‐L), n = 3 (Calu‐3) independent experiments. (b) Representative immunofluorescent images of HMVEC‐L stained for nucleocapsid protein (NP) (shown as single channel in top panel) (green), Phalloidin (magenta) and DAPI (blue) with mock or SARS‐CoV‐2 infection from either the apical or basolateral side of the cells at 72 h after infection. Scale bar 50 µm. The white box is enlarged in Figure 3 . (c) Quantification of NP staining intensity in HMVEC‐L. n = 15 images from 3 independent experiments. (d) Western blot analysis showing NP protein levels in HUVEC, HMVEC‐L and Calu‐3 cells after 72 h of infection. (e) Quantification of NP protein levels in HUVEC, HMVEC‐L and Calu‐3. n = 3 independent experiments. (f) Representative immunofluorescent images of ACE2 overexpressing HMVEC‐L and control Calu‐3 cells stained for NP (shown as single channel in top panel) (green), ACE2 (shown as single channel in middle panel) (magenta), Phalloidin (grey) and DAPI (blue) with mock or SARS‐CoV‐2 infection from the apical side of the cells at 72 h after infection. Scale bar 50 µm. (g) Quantification of NP staining intensity in HMVEC‐L, HMVEC‐L + ACE2 and Calu‐3 with either low (6 × 10 4 ) or high (2 × 10 6 ) dose of SARS‐CoV‐2 infection. n = 15 images from 3 independent experiments. (h) Quantification of ACE2 staining intensity in HMVEC‐L, HMVEC‐L + ACE2 and Calu‐3. n = 10 (HMVEC‐L and Calu‐3), n = 20 (HMVEC‐L + ACE2) images from 2 independent experiments. (i) Viral replication shown as number of PFU mL −1 of supernatant from SARS‐CoV‐2 infected HMVEC‐L, HMVEC‐L + ACE2 and Calu‐3 cells at 72 h after infection. The grey dashed line indicates input level. n = 3 independent experiments. Data are presented as mean ± s.e.m. with individual data points indicated and colour coded per independent experimental replicate. Statistical significance was determined using the Kruskal–Wallis test between 24 h and other time points (a) , between HMVEC‐L and all other conditions (i) or the Mann–Whitney U ‐test between mock and + SARS‐CoV‐2 (c, e, g) . * P

    Techniques Used: Infection, Staining, Western Blot, MANN-WHITNEY

    Endothelial cells express low levels of ACE2 and TMPRSS2 receptors. (a) Representative immunofluorescence images of BHK‐21, HUVEC, HMVEC‐L, Calu‐3 and HMVEC‐L + ACE2 overexpression cells stained for ACE2 (shown as single channel in top panel) (green), Phalloidin (magenta) and DAPI (blue). Scale bar 30 µm. (b) Quantification of ACE2 staining intensity, n = 15 images; 5 images per independent experiment, 3 independent experiments. (c) Western blot analysis showing both glycosylated (∼120 kDa) and deglycosylated (∼98 kDa) ACE2 protein. (d) Western blot analysis showing TMPRSS2 protein. (e) Quantification of protein levels for glycosylated ACE2 (∼120 kDa) and deglycosylated ACE2 (∼98 kDa) in HUVEC, HMVEC‐L, Calu‐3 and BHK‐21. n = 3 independent experiments. (f) Quantification of protein levels for TMPRSS2 in HUVEC, HMVEC‐L and Calu‐3. n = 4 (HUVEC and HMVEC‐L), n = 2 (Calu‐3) independent experiments. (g) qPCR shows presence of mRNA for ACE2, TMPRSS2 and NRP1 in HUVEC, HMVEC‐L and Calu‐3 cells. n = 3 independent experiments. Data are presented as mean ± s.e.m. with individual data points indicated and colour coded per independent experimental replicate. Statistical significance was determined using the Kruskal–Wallis test between Calu‐3 and HMVEC‐L +ACE2 and all others (b) or between Calu‐3 and all others (e, f, g) . * P
    Figure Legend Snippet: Endothelial cells express low levels of ACE2 and TMPRSS2 receptors. (a) Representative immunofluorescence images of BHK‐21, HUVEC, HMVEC‐L, Calu‐3 and HMVEC‐L + ACE2 overexpression cells stained for ACE2 (shown as single channel in top panel) (green), Phalloidin (magenta) and DAPI (blue). Scale bar 30 µm. (b) Quantification of ACE2 staining intensity, n = 15 images; 5 images per independent experiment, 3 independent experiments. (c) Western blot analysis showing both glycosylated (∼120 kDa) and deglycosylated (∼98 kDa) ACE2 protein. (d) Western blot analysis showing TMPRSS2 protein. (e) Quantification of protein levels for glycosylated ACE2 (∼120 kDa) and deglycosylated ACE2 (∼98 kDa) in HUVEC, HMVEC‐L, Calu‐3 and BHK‐21. n = 3 independent experiments. (f) Quantification of protein levels for TMPRSS2 in HUVEC, HMVEC‐L and Calu‐3. n = 4 (HUVEC and HMVEC‐L), n = 2 (Calu‐3) independent experiments. (g) qPCR shows presence of mRNA for ACE2, TMPRSS2 and NRP1 in HUVEC, HMVEC‐L and Calu‐3 cells. n = 3 independent experiments. Data are presented as mean ± s.e.m. with individual data points indicated and colour coded per independent experimental replicate. Statistical significance was determined using the Kruskal–Wallis test between Calu‐3 and HMVEC‐L +ACE2 and all others (b) or between Calu‐3 and all others (e, f, g) . * P

    Techniques Used: Immunofluorescence, Over Expression, Staining, Western Blot, Real-time Polymerase Chain Reaction

    23) Product Images from "The impact of patient co-morbidities on the regenerative capacity of cardiac explant-derived stem cells"

    Article Title: The impact of patient co-morbidities on the regenerative capacity of cardiac explant-derived stem cells

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-016-0321-4

    Relationship between the long-term risk of coronary heart disease events and the regenerative performance of EDCs. a Representative images of HUVEC tubules formed after exposure to media conditioned by EDCs sourced from patients with variable LTS scores. Correlation between patient LTS score and the ability of EDC conditioned media to stimulate HUVEC tubule formation ( n = 13). b Representative images of 4′,6-diamidino-2-phenylindole-stained circulating angiogenic ells (CACs) after 24 hours exposure to media conditioned by EDCs sourced from patients with variable LTS scores. Correlation between patient LTS score and the ability of EDC conditioned media to recruit CACs across a transwell membrane ( n = 11). c Correlation between patient LTS score improvements in echocardiographic left ventricular ejection fraction (LVEF) measured 2 and 3 weeks after intramyocardial injection into an immunodeficient mouse model of myocardial ischemia ( n = 37). d Correlation between LTS score and cytokine content within EDC conditioned media (IL-6, interleukin 6, n = 13; SDF-1α, stromal-derived factor 1 alpha; n = 15). e Representative screen capture images of media conditioned by EDCs from patients with high and low LTS score ( white arrows indicate exosomes). Correlation between LTS score and number of exosomes within EDC conditioned media ( n = 8). Also shown adjacent to each point is the mode ± SEM size distribution ( n = 3 technical repeats per sample). EDCs explant derived cells, HUVEC human umbilical vein endothelial cell, LTS long term stratification
    Figure Legend Snippet: Relationship between the long-term risk of coronary heart disease events and the regenerative performance of EDCs. a Representative images of HUVEC tubules formed after exposure to media conditioned by EDCs sourced from patients with variable LTS scores. Correlation between patient LTS score and the ability of EDC conditioned media to stimulate HUVEC tubule formation ( n = 13). b Representative images of 4′,6-diamidino-2-phenylindole-stained circulating angiogenic ells (CACs) after 24 hours exposure to media conditioned by EDCs sourced from patients with variable LTS scores. Correlation between patient LTS score and the ability of EDC conditioned media to recruit CACs across a transwell membrane ( n = 11). c Correlation between patient LTS score improvements in echocardiographic left ventricular ejection fraction (LVEF) measured 2 and 3 weeks after intramyocardial injection into an immunodeficient mouse model of myocardial ischemia ( n = 37). d Correlation between LTS score and cytokine content within EDC conditioned media (IL-6, interleukin 6, n = 13; SDF-1α, stromal-derived factor 1 alpha; n = 15). e Representative screen capture images of media conditioned by EDCs from patients with high and low LTS score ( white arrows indicate exosomes). Correlation between LTS score and number of exosomes within EDC conditioned media ( n = 8). Also shown adjacent to each point is the mode ± SEM size distribution ( n = 3 technical repeats per sample). EDCs explant derived cells, HUVEC human umbilical vein endothelial cell, LTS long term stratification

    Techniques Used: Staining, Injection, Derivative Assay

    24) Product Images from "Multidimensional hydrogel models reveal endothelial network angiocrine signals increase glioblastoma cell number, invasion, and temozolomide resistance"

    Article Title: Multidimensional hydrogel models reveal endothelial network angiocrine signals increase glioblastoma cell number, invasion, and temozolomide resistance

    Journal: bioRxiv

    doi: 10.1101/2020.01.18.911396

    Schematic detailing the collection of conditioned media. A) Endothelial networks are formed in GelMA hydrogels by co-culturing HUVECs and NHLFs for one week. B) Endothelial networks formed after a week in culture can be visualized by staining for CD31. Scale bar = 200 µm.
    Figure Legend Snippet: Schematic detailing the collection of conditioned media. A) Endothelial networks are formed in GelMA hydrogels by co-culturing HUVECs and NHLFs for one week. B) Endothelial networks formed after a week in culture can be visualized by staining for CD31. Scale bar = 200 µm.

    Techniques Used: Staining

    25) Product Images from "Exosomal HMGB1 derived from hypoxia‐conditioned bone marrow mesenchymal stem cells increases angiogenesis via the JNK/HIF‐1α pathway"

    Article Title: Exosomal HMGB1 derived from hypoxia‐conditioned bone marrow mesenchymal stem cells increases angiogenesis via the JNK/HIF‐1α pathway

    Journal: FEBS Open Bio

    doi: 10.1002/2211-5463.13142

    Characterization of BMSC‐derived exosomes. (A). Morphology of H‐MSC‐exos (hypoxia/exos) (left) and N‐MSC‐exos (normoxia/exos) (right) analyzed by transmission electron microscopy. Scale bars: 100 nm. (B) Western blot analysis of CD9 and CD81 expression in exosomes derived from hypoxia or normoxia‐conditioned BMSCs. (C). Uptake of exosomes in HUVECs by immunofluorescence staining. Scale bars: 100 μm.
    Figure Legend Snippet: Characterization of BMSC‐derived exosomes. (A). Morphology of H‐MSC‐exos (hypoxia/exos) (left) and N‐MSC‐exos (normoxia/exos) (right) analyzed by transmission electron microscopy. Scale bars: 100 nm. (B) Western blot analysis of CD9 and CD81 expression in exosomes derived from hypoxia or normoxia‐conditioned BMSCs. (C). Uptake of exosomes in HUVECs by immunofluorescence staining. Scale bars: 100 μm.

    Techniques Used: Derivative Assay, Transmission Assay, Electron Microscopy, Western Blot, Expressing, Immunofluorescence, Staining

    Hypoxic MSC‐derived exosomes enhance angiogenesis through HMGB1. (A) Western blot analysis of HMGB1 expression in HUVECs treated with H‐MSC‐exos (hypoxia/exos) or N‐MSC‐exos (normoxia/exos). (B) Quantitative PCR analysis of HMGB1 expression in HUVECs treated with siRNA. (C) Morphology of hypoxic MSC‐derived exosomes. (D) Western blot analysis of VEGF and CD31 expression in BMSC‐derived exosomes under different conditions. (E) Silencing of HMGB1 by siRNA reduced the viability of HUVECs induced by H‐MSC‐exos. (F, G) Silencing of HMGB1 reduced the tube formation of HUVECs enhanced by H‐MSC‐exos. (H, I) Knockdown of HMGB1 reduced H‐MSC‐exo‐induced VEGF/CD31 expression. * P
    Figure Legend Snippet: Hypoxic MSC‐derived exosomes enhance angiogenesis through HMGB1. (A) Western blot analysis of HMGB1 expression in HUVECs treated with H‐MSC‐exos (hypoxia/exos) or N‐MSC‐exos (normoxia/exos). (B) Quantitative PCR analysis of HMGB1 expression in HUVECs treated with siRNA. (C) Morphology of hypoxic MSC‐derived exosomes. (D) Western blot analysis of VEGF and CD31 expression in BMSC‐derived exosomes under different conditions. (E) Silencing of HMGB1 by siRNA reduced the viability of HUVECs induced by H‐MSC‐exos. (F, G) Silencing of HMGB1 reduced the tube formation of HUVECs enhanced by H‐MSC‐exos. (H, I) Knockdown of HMGB1 reduced H‐MSC‐exo‐induced VEGF/CD31 expression. * P

    Techniques Used: Derivative Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction

    Exosomal HMGB1 up‐regulates HIF‐1α expression. (A–C) Quantitative RT‐PCR and western blot analysis of HIF‐1α mRNA and protein expression in HUVECs under different conditions. (D, E) Treatment with BAY87‐2243 (40 μ m ) decreased the tube formation of HUVECs enhanced by H‐MSC‐exos. (F, G) Treatment with BAY87‐2243 (40 μ m ) decreased H‐MSC‐exo‐induced VEGF/CD31 expression. * P
    Figure Legend Snippet: Exosomal HMGB1 up‐regulates HIF‐1α expression. (A–C) Quantitative RT‐PCR and western blot analysis of HIF‐1α mRNA and protein expression in HUVECs under different conditions. (D, E) Treatment with BAY87‐2243 (40 μ m ) decreased the tube formation of HUVECs enhanced by H‐MSC‐exos. (F, G) Treatment with BAY87‐2243 (40 μ m ) decreased H‐MSC‐exo‐induced VEGF/CD31 expression. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot

    Exosomal HMGB1 promotes angiogenesis via the JNK/HIF‐1α cascade. (A, B) Western blot analysis of JNK expression and phosphorylation in HUVECs under different conditions. (C, D) Administration of SP600125 (60 μ m ) decreased the tube formation of HUVECs enhanced by H‐MSC‐exos. (E, F) Administration of SP600125 reduced H‐MSC‐exo‐induced VEGF/CD31 expression. * P
    Figure Legend Snippet: Exosomal HMGB1 promotes angiogenesis via the JNK/HIF‐1α cascade. (A, B) Western blot analysis of JNK expression and phosphorylation in HUVECs under different conditions. (C, D) Administration of SP600125 (60 μ m ) decreased the tube formation of HUVECs enhanced by H‐MSC‐exos. (E, F) Administration of SP600125 reduced H‐MSC‐exo‐induced VEGF/CD31 expression. * P

    Techniques Used: Western Blot, Expressing

    Effects of hypoxic MSC‐derived exosomes on the viability, migration and tube formation of HUVECs. (A). The viability of HUVECs was determined by CCK‐8 assay after treatment with H‐MSC‐exos (hypoxia/exos) or N‐MSC‐exos (normoxia/exos). (B, C) Transwell migration assay in HUVECs after exposure to H‐MSC‐exos or N‐MSC‐exos. (D, E) Tube formation assay in HUVECs treated with H‐MSC‐exos or N‐MSC‐exos. (F, G) Western blot analysis of VEGF and CD31 expression in HUVECs treated with H‐MSC‐exos or N‐MSC‐exos. * P
    Figure Legend Snippet: Effects of hypoxic MSC‐derived exosomes on the viability, migration and tube formation of HUVECs. (A). The viability of HUVECs was determined by CCK‐8 assay after treatment with H‐MSC‐exos (hypoxia/exos) or N‐MSC‐exos (normoxia/exos). (B, C) Transwell migration assay in HUVECs after exposure to H‐MSC‐exos or N‐MSC‐exos. (D, E) Tube formation assay in HUVECs treated with H‐MSC‐exos or N‐MSC‐exos. (F, G) Western blot analysis of VEGF and CD31 expression in HUVECs treated with H‐MSC‐exos or N‐MSC‐exos. * P

    Techniques Used: Derivative Assay, Migration, CCK-8 Assay, Transwell Migration Assay, Tube Formation Assay, Western Blot, Expressing

    26) Product Images from "In Vitro Microtumors Provide a Physiologically Predictive Tool for Breast Cancer Therapeutic Screening"

    Article Title: In Vitro Microtumors Provide a Physiologically Predictive Tool for Breast Cancer Therapeutic Screening

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123312

    hMSCs and endothelial tubules promote invasion and differentially effect proliferation of MDA-MB-231 and MCF-7 spheroids. A. Spheroids formed as described above under hypoxia. Then 96 well, flat bottom plates were coated with 50 μl of tubule formation matrix and incubated for one hour to polymerize the hydrogel. For wells with HUVECs, 12,500 cells were added to each well, and for remaining samples, EGM-2 was added. HUVECs were allowed to assemble into tubules for two hours. One spheroid was transferred to each of the wells in the plate. MCTS were allowed to settle for 1 hour. Then, 100 μl of medium was aspirated from each well. For wells with hMSCs, 10,000 cells were suspended in each ml of Invasion Matrix, and 50 μl was added to each well. For the remaining samples, 50 μl of tumor-aligned invasion matrix was added to each well. The plates were then incubated at 37°C, 5% CO 2 for 1 hour to polymerize the hydrogel, and 100 μl of TARPMI, 10% FBS was added to each well. Cultures were incubated under hypoxia for 96 hours. Images are provided for spheroids alone and for spheroids, HUVECs, and hMSCs for MCF-7 (A) and MDA-231 (B). Cultures were analyzed as described above for proliferation (C) and invasion (D), and samples were evaluated in quadruplicate. S = breast cancer MCTS, H = HUVEC network, and M = hMSCs. Scale bar = 500 μm. *P
    Figure Legend Snippet: hMSCs and endothelial tubules promote invasion and differentially effect proliferation of MDA-MB-231 and MCF-7 spheroids. A. Spheroids formed as described above under hypoxia. Then 96 well, flat bottom plates were coated with 50 μl of tubule formation matrix and incubated for one hour to polymerize the hydrogel. For wells with HUVECs, 12,500 cells were added to each well, and for remaining samples, EGM-2 was added. HUVECs were allowed to assemble into tubules for two hours. One spheroid was transferred to each of the wells in the plate. MCTS were allowed to settle for 1 hour. Then, 100 μl of medium was aspirated from each well. For wells with hMSCs, 10,000 cells were suspended in each ml of Invasion Matrix, and 50 μl was added to each well. For the remaining samples, 50 μl of tumor-aligned invasion matrix was added to each well. The plates were then incubated at 37°C, 5% CO 2 for 1 hour to polymerize the hydrogel, and 100 μl of TARPMI, 10% FBS was added to each well. Cultures were incubated under hypoxia for 96 hours. Images are provided for spheroids alone and for spheroids, HUVECs, and hMSCs for MCF-7 (A) and MDA-231 (B). Cultures were analyzed as described above for proliferation (C) and invasion (D), and samples were evaluated in quadruplicate. S = breast cancer MCTS, H = HUVEC network, and M = hMSCs. Scale bar = 500 μm. *P

    Techniques Used: Multiple Displacement Amplification, Incubation

    27) Product Images from "Overexpression of CCN3 Inhibits Inflammation and Progression of Atherosclerosis in Apolipoprotein E-Deficient Mice"

    Article Title: Overexpression of CCN3 Inhibits Inflammation and Progression of Atherosclerosis in Apolipoprotein E-Deficient Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0094912

    Effect of proinflammatory cytokines on CCN3 expression. HAECs (A) and HUVECs (B) were treated with TNF-α (10 ng/ml) or IL-1β (4 ng/ml) for 24 h. Total RNA and cell protein were harvested for qRT-PCR or Western blot analysis, respectively. mRNA expression was normalized to GAPDH ( N = 6, *p
    Figure Legend Snippet: Effect of proinflammatory cytokines on CCN3 expression. HAECs (A) and HUVECs (B) were treated with TNF-α (10 ng/ml) or IL-1β (4 ng/ml) for 24 h. Total RNA and cell protein were harvested for qRT-PCR or Western blot analysis, respectively. mRNA expression was normalized to GAPDH ( N = 6, *p

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot

    28) Product Images from "High Mobility Group Box 1 Inhibits Human Pulmonary Artery Endothelial Cell Migration via a Toll-like Receptor 4- and Interferon Response Factor 3-dependent Mechanism(s) *"

    Article Title: High Mobility Group Box 1 Inhibits Human Pulmonary Artery Endothelial Cell Migration via a Toll-like Receptor 4- and Interferon Response Factor 3-dependent Mechanism(s) *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.434142

    Differential gene induction by HMGB1 in HPAECs versus HUVECs. A and B , HPAECs ( A ) and HUVECs ( B ) were stimulated with HMGB1 (1 μg/ml) for 4 h and then assessed for the induction of ISG20, IFNβ, or iNOS mRNA by RT-PCR. C and D , the relative
    Figure Legend Snippet: Differential gene induction by HMGB1 in HPAECs versus HUVECs. A and B , HPAECs ( A ) and HUVECs ( B ) were stimulated with HMGB1 (1 μg/ml) for 4 h and then assessed for the induction of ISG20, IFNβ, or iNOS mRNA by RT-PCR. C and D , the relative

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    Differential signaling in HPAECs versus HUVECs. Western blot analysis for NUMA (150 kDa, nuclear marker), HSP90 (90 kDa, cytoplasmic marker), β-actin (42 kDa), NFκB-p65 (65 kDa), and IRF3 (45 kDa) in cytoplasmic and nuclear fractions from
    Figure Legend Snippet: Differential signaling in HPAECs versus HUVECs. Western blot analysis for NUMA (150 kDa, nuclear marker), HSP90 (90 kDa, cytoplasmic marker), β-actin (42 kDa), NFκB-p65 (65 kDa), and IRF3 (45 kDa) in cytoplasmic and nuclear fractions from

    Techniques Used: Western Blot, Marker

    29) Product Images from "GSK-3β regulates the endothelial-to-mesenchymal transition via reciprocal crosstalk between NSCLC cells and HUVECs in multicellular tumor spheroid models"

    Article Title: GSK-3β regulates the endothelial-to-mesenchymal transition via reciprocal crosstalk between NSCLC cells and HUVECs in multicellular tumor spheroid models

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-019-1050-1

    Interplay between NSCLC cells and HUVECs promotes chemoresistance in MCTSs. a and b NCI-H460 ( a ) and A549 ( b ) spheroids were co-cultured with or without stromal cells (WI38 or HUVECs) for 2 days, and then treated with 10 or 20 μM Gefitinib for 2 days. Chemoresistance was measured by staining with EthD-1, a cell death marker. The intensity of EthD-1 was analyzed and values were normalized to control (0.01% DMSO). c and d NCI-H460 ( c ) and A549 ( d ) spheroids were co-cultured with or without WI38 cells and HUVECs, and stained with EthD-1 to detect cell death, 2 days after treatment with 10 or 20 μM Cisplatin. Data are shown as mean ± SD from two independent experiments in triplicate. * p
    Figure Legend Snippet: Interplay between NSCLC cells and HUVECs promotes chemoresistance in MCTSs. a and b NCI-H460 ( a ) and A549 ( b ) spheroids were co-cultured with or without stromal cells (WI38 or HUVECs) for 2 days, and then treated with 10 or 20 μM Gefitinib for 2 days. Chemoresistance was measured by staining with EthD-1, a cell death marker. The intensity of EthD-1 was analyzed and values were normalized to control (0.01% DMSO). c and d NCI-H460 ( c ) and A549 ( d ) spheroids were co-cultured with or without WI38 cells and HUVECs, and stained with EthD-1 to detect cell death, 2 days after treatment with 10 or 20 μM Cisplatin. Data are shown as mean ± SD from two independent experiments in triplicate. * p

    Techniques Used: Cell Culture, Staining, Ethidium Homodimer Assay, Marker

    Secretomes from NSCLC spheroids induced endothelial-to-mesenchymal transition. a Experimental schematic of secretomes from NSCLC. NCI-H460 cells were cultured under 2D and 3D conditions using the same number of cells with the same amount of media. After 3 days, the conditioned medium (CM) was mixed with HUVEC original media at various ratios, and then used to treat HUVECs for 24 and 48 h. b Representative images of immunofluorescence staining for CD31 (green) and α-SMA (red) expression and nuclei (blue) in HUVECs treated with CM from 2D and 3D NCI-H460 cells at a ratio of 5:5. c HUVECs that were treated with CM from 2D and 3D NCI-H460 cells were harvested after 48 h incubation. HUVEC lysates were analyzed by western blotting using anti-CD31, anti-α-SMA, and anti-β-actin antibodies. d Quantification of relative expression levels of CD31 and α-SMA. Values were normalized to β-actin. Data are shown as mean ± SD from two independent experiments
    Figure Legend Snippet: Secretomes from NSCLC spheroids induced endothelial-to-mesenchymal transition. a Experimental schematic of secretomes from NSCLC. NCI-H460 cells were cultured under 2D and 3D conditions using the same number of cells with the same amount of media. After 3 days, the conditioned medium (CM) was mixed with HUVEC original media at various ratios, and then used to treat HUVECs for 24 and 48 h. b Representative images of immunofluorescence staining for CD31 (green) and α-SMA (red) expression and nuclei (blue) in HUVECs treated with CM from 2D and 3D NCI-H460 cells at a ratio of 5:5. c HUVECs that were treated with CM from 2D and 3D NCI-H460 cells were harvested after 48 h incubation. HUVEC lysates were analyzed by western blotting using anti-CD31, anti-α-SMA, and anti-β-actin antibodies. d Quantification of relative expression levels of CD31 and α-SMA. Values were normalized to β-actin. Data are shown as mean ± SD from two independent experiments

    Techniques Used: Cell Culture, Immunofluorescence, Staining, Expressing, Incubation, Western Blot

    Interaction between NSCLC cells and HUVECs in MCTSs affects the compactness of tumor spheroids. a and b NSCLC, which is stained by DiO, and HUVECs stained by DiD were cultured for 3 days in 2D or 3D culture systems and then the images were obtained. c NCI-H460 or A549 cells were co-cultured with HUVECs in 2D and 3D culture systems at a ratio of 5:5 for 48 h. Lysates were analyzed by immunoblotting using indicated antibodies. d NCI-H460, A549, and SK-MES-1 cells were co-cultured with stromal cells (WI38 cells and HUVECs) at a ratio of 5:5 for 1 day and 3 days. All bright-field images of spheroids were obtained using the Operetta® High Content Screening System with a 10× objective. e To calculate the volume of spheroids, their long and short diameters were measured. Data are shown as mean ± SD from two independent experiments in triplicate. * P
    Figure Legend Snippet: Interaction between NSCLC cells and HUVECs in MCTSs affects the compactness of tumor spheroids. a and b NSCLC, which is stained by DiO, and HUVECs stained by DiD were cultured for 3 days in 2D or 3D culture systems and then the images were obtained. c NCI-H460 or A549 cells were co-cultured with HUVECs in 2D and 3D culture systems at a ratio of 5:5 for 48 h. Lysates were analyzed by immunoblotting using indicated antibodies. d NCI-H460, A549, and SK-MES-1 cells were co-cultured with stromal cells (WI38 cells and HUVECs) at a ratio of 5:5 for 1 day and 3 days. All bright-field images of spheroids were obtained using the Operetta® High Content Screening System with a 10× objective. e To calculate the volume of spheroids, their long and short diameters were measured. Data are shown as mean ± SD from two independent experiments in triplicate. * P

    Techniques Used: Staining, Cell Culture, High Content Screening

    CHIR-99021, a GSK-3β inhibitor, suppresses the EndMT process. a NCI-H460 cells were co-cultured with HUVECs in 2D and 3D-culture systems at a ratio of 5:5, and then treated with the indicated drugs for 24 h. Cells were harvested and immunoblotted with anti-CD31, anti-VE-cadherin, anti-α-SMA, anti-vimentin, and anti-β-actin antibodies. b A549 were co-cultured with HUVEC under 2D and 3D condition at ratio of 5:5, and then treated with 2 μM CHIR-99021, 5 μM MSAB, 20 μM SB-216763, and 20 μM IWR-1. After 24 h, lysates were analyzed by immunoblotting using indicated antibodies. c NCI-H460 cells were co-cultured with HUVECs under 2D and 3D conditions and then treated with 2 μM CHIR-99021, 5 μM MSAB, and co-treated with both 2 μM CHIR-99021 and 5 μM MSAB. After 24 h, lysates were analyzed by immunoblotting using anti-CD31, anti-VE-cadherin, and anti-β-actin antibodies. d NCI-H460 cells were co-cultured with HUVECs under 3D conditions, and then treated with 2 μM CHIR-99021 for 24, 48, and 72 h. e NCI-H460 cells were co-cultured with HUVECs under 3D conditions, and then treated with 0.5, 1, and 2 μM CHIR-99021 for 24 h. Lysates were analyzed by immunoblotting. f Representative images of MCTSs containing NCI-H460 cells and HUVECs treated with CHIR-99021 for 1 day. Shown are spheroids stained with H E, and immunohistochemically stained for CD31. Scale bars = 50 μm. g NCI-H460 cells were co-cultured with HUVECs in a 3D culture system, and then treated with 1 μM CHIR-99021 for 24 h. Lysates were analyzed by immunoblotting using anti-phospho-GSK-3β (Tyr216), anti-phospho-GSK-3β (Ser9), anti-GSK-3β, anti-CD31, and anti-β-actin antibodies
    Figure Legend Snippet: CHIR-99021, a GSK-3β inhibitor, suppresses the EndMT process. a NCI-H460 cells were co-cultured with HUVECs in 2D and 3D-culture systems at a ratio of 5:5, and then treated with the indicated drugs for 24 h. Cells were harvested and immunoblotted with anti-CD31, anti-VE-cadherin, anti-α-SMA, anti-vimentin, and anti-β-actin antibodies. b A549 were co-cultured with HUVEC under 2D and 3D condition at ratio of 5:5, and then treated with 2 μM CHIR-99021, 5 μM MSAB, 20 μM SB-216763, and 20 μM IWR-1. After 24 h, lysates were analyzed by immunoblotting using indicated antibodies. c NCI-H460 cells were co-cultured with HUVECs under 2D and 3D conditions and then treated with 2 μM CHIR-99021, 5 μM MSAB, and co-treated with both 2 μM CHIR-99021 and 5 μM MSAB. After 24 h, lysates were analyzed by immunoblotting using anti-CD31, anti-VE-cadherin, and anti-β-actin antibodies. d NCI-H460 cells were co-cultured with HUVECs under 3D conditions, and then treated with 2 μM CHIR-99021 for 24, 48, and 72 h. e NCI-H460 cells were co-cultured with HUVECs under 3D conditions, and then treated with 0.5, 1, and 2 μM CHIR-99021 for 24 h. Lysates were analyzed by immunoblotting. f Representative images of MCTSs containing NCI-H460 cells and HUVECs treated with CHIR-99021 for 1 day. Shown are spheroids stained with H E, and immunohistochemically stained for CD31. Scale bars = 50 μm. g NCI-H460 cells were co-cultured with HUVECs in a 3D culture system, and then treated with 1 μM CHIR-99021 for 24 h. Lysates were analyzed by immunoblotting using anti-phospho-GSK-3β (Tyr216), anti-phospho-GSK-3β (Ser9), anti-GSK-3β, anti-CD31, and anti-β-actin antibodies

    Techniques Used: Cell Culture, Staining

    Reciprocal crosstalk between NSCLC cells and HUVECs induces activation of GSK-3β in multicellular tumor spheroid models. a NCI-H460 or A549 cells were cultured under 2D and 3D conditions for 2 days. The levels of TGF-β1 in culture supernatants were measured using ELISA. Results are presented as mean ± standard error of the means. * P
    Figure Legend Snippet: Reciprocal crosstalk between NSCLC cells and HUVECs induces activation of GSK-3β in multicellular tumor spheroid models. a NCI-H460 or A549 cells were cultured under 2D and 3D conditions for 2 days. The levels of TGF-β1 in culture supernatants were measured using ELISA. Results are presented as mean ± standard error of the means. * P

    Techniques Used: Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    30) Product Images from "Insulin Downregulates the Transcriptional Coregulator CITED2, an Inhibitor of Proangiogenic Function in Endothelial Cells"

    Article Title: Insulin Downregulates the Transcriptional Coregulator CITED2, an Inhibitor of Proangiogenic Function in Endothelial Cells

    Journal: Diabetes

    doi: 10.2337/db16-0001

    Proangiogneic endothelial cell function after reduced CITED2 expression. A and B : HUVECs were serum starved overnight and treated with insulin at 10 nmol/L for the times indicated. A : Western blot of HUVEC lysate. B : Quantitative analysis from three independent experiments. C – G : HUVECs were transfected with siRNA targeting CITED2 (siCITED2) or control siRNA (siCon). C : After transfection with siRNA, protein expression was measured in cell lysate by Western blotting. D : After siRNA transfection, HUVECs were plated on Matrigel and allowed to form tubes. Representative photos of phase-contrast microscopy are grayscale versions with adjusted contrast of the microphotos shown in Supplementary Fig. 5 . E : Quantitative analysis of tube formation from three independent experiments. For explanation of the terms master junctions, master segments, meshes, and length, see Supplementary Fig. 5 . F and G : HUVECs were transfected with siRNA 48 h before start of insulin treatment and serum starved 16 h before start of insulin treatment. Cultures were treated with 10 nmol/L insulin for 16 h and then labeled with EdU for an additional 4 h. EdU-labeled cells were stained with Alexa Fluor 647 (AF647) using Molecular Probes Click-iT procedure and analyzed by flow cytometry. F : Representative scatter plots of side scatter (SSC) versus AF647 fluorescence from flow cytometry. G : Quantitative analysis of EdU incorporation based on three independent experiments. * P
    Figure Legend Snippet: Proangiogneic endothelial cell function after reduced CITED2 expression. A and B : HUVECs were serum starved overnight and treated with insulin at 10 nmol/L for the times indicated. A : Western blot of HUVEC lysate. B : Quantitative analysis from three independent experiments. C – G : HUVECs were transfected with siRNA targeting CITED2 (siCITED2) or control siRNA (siCon). C : After transfection with siRNA, protein expression was measured in cell lysate by Western blotting. D : After siRNA transfection, HUVECs were plated on Matrigel and allowed to form tubes. Representative photos of phase-contrast microscopy are grayscale versions with adjusted contrast of the microphotos shown in Supplementary Fig. 5 . E : Quantitative analysis of tube formation from three independent experiments. For explanation of the terms master junctions, master segments, meshes, and length, see Supplementary Fig. 5 . F and G : HUVECs were transfected with siRNA 48 h before start of insulin treatment and serum starved 16 h before start of insulin treatment. Cultures were treated with 10 nmol/L insulin for 16 h and then labeled with EdU for an additional 4 h. EdU-labeled cells were stained with Alexa Fluor 647 (AF647) using Molecular Probes Click-iT procedure and analyzed by flow cytometry. F : Representative scatter plots of side scatter (SSC) versus AF647 fluorescence from flow cytometry. G : Quantitative analysis of EdU incorporation based on three independent experiments. * P

    Techniques Used: Cell Function Assay, Expressing, Western Blot, Transfection, Microscopy, Labeling, Staining, Flow Cytometry, Fluorescence

    CITED2 regulates HIF transactivation in endothelial cells. A : HUVECs were cotransfected with a luciferase reporter gene driven by a promoter containing HREs, which bind HIF, and a Renilla control plasmid to allow normalization for transfection efficiency and cell number. The cultures were cotransfected with a plasmid expressing CITED2 (pCITED2) or a control plasmid (pControl) and luciferase activity was measured in cell lysate. B and C : Lung endothelial cells were isolated from mice with deletion of the Cited2 gene targeted to endothelial cells [Tg(Cre) Cited2 L/L ] or their controls ( Cited2 L/L ). B : Western blot prepared from cell lysate. C : Quantitation based on densitometry in cultures from three pairs of animals. D : Expression of Edn1 mRNA in CD31 − and CD31 + cells in enzymatically dissociated gastrocnemius muscle. Cells were sorted by FACS after gating for single, viable, CD45 − cells. Tissue from three animals in each group was analyzed. E : Edn1 expression in gastrocnemius muscle 24 h after femoral artery ligation. mRNA in the ischemic muscle relative to the contralateral, nonischemic gastrocnemius muscle is shown. Tissue from 12 animals in each group was analyzed. * P
    Figure Legend Snippet: CITED2 regulates HIF transactivation in endothelial cells. A : HUVECs were cotransfected with a luciferase reporter gene driven by a promoter containing HREs, which bind HIF, and a Renilla control plasmid to allow normalization for transfection efficiency and cell number. The cultures were cotransfected with a plasmid expressing CITED2 (pCITED2) or a control plasmid (pControl) and luciferase activity was measured in cell lysate. B and C : Lung endothelial cells were isolated from mice with deletion of the Cited2 gene targeted to endothelial cells [Tg(Cre) Cited2 L/L ] or their controls ( Cited2 L/L ). B : Western blot prepared from cell lysate. C : Quantitation based on densitometry in cultures from three pairs of animals. D : Expression of Edn1 mRNA in CD31 − and CD31 + cells in enzymatically dissociated gastrocnemius muscle. Cells were sorted by FACS after gating for single, viable, CD45 − cells. Tissue from three animals in each group was analyzed. E : Edn1 expression in gastrocnemius muscle 24 h after femoral artery ligation. mRNA in the ischemic muscle relative to the contralateral, nonischemic gastrocnemius muscle is shown. Tissue from 12 animals in each group was analyzed. * P

    Techniques Used: Luciferase, Plasmid Preparation, Transfection, Expressing, Activity Assay, Isolation, Mouse Assay, Western Blot, Quantitation Assay, FACS, Ligation

    31) Product Images from "Regulation of Angiogenic Functions by Angiopoietins through Calcium-Dependent Signaling Pathways"

    Article Title: Regulation of Angiogenic Functions by Angiopoietins through Calcium-Dependent Signaling Pathways

    Journal: BioMed Research International

    doi: 10.1155/2015/965271

    Calcium-dependent AKT and MAPK activation upon Ang-1/Ang-2 stimulation. Ang-1 and Ang-2 induce Ca 2+ -dependent AKT, ERK1/2, and p38 phosphorylation. Confluent HUVECs were pretreated or not with 20 μ M BAPTA-AM for 1 h and stimulated with 100 ng/mL Ang-1 or 200 ng/mL Ang-2 for 30 min. Blots of total cell lysate were probed for the phosphorylation of downstream targets AKT, ERK1/2, and p38. To ensure equal loading membranes were reprobed for the total amount of the indicated proteins and for β -actin. Representative blots are shown from three to five independent experiments (a). Cells pretreated or not with 75 μ M 2-APB or 30 μ M 8Br-cADPR and stimulated with either Ang-1 or Ang-2. Blots of total cell lysate were probed for the phosphorylation of downstream target AKT. To ensure equal loading membranes were reprobed for the total amount of the indicated protein and for β -actin. Representative blots are shown from three independent experiments (b1, b2, c1, and c2).
    Figure Legend Snippet: Calcium-dependent AKT and MAPK activation upon Ang-1/Ang-2 stimulation. Ang-1 and Ang-2 induce Ca 2+ -dependent AKT, ERK1/2, and p38 phosphorylation. Confluent HUVECs were pretreated or not with 20 μ M BAPTA-AM for 1 h and stimulated with 100 ng/mL Ang-1 or 200 ng/mL Ang-2 for 30 min. Blots of total cell lysate were probed for the phosphorylation of downstream targets AKT, ERK1/2, and p38. To ensure equal loading membranes were reprobed for the total amount of the indicated proteins and for β -actin. Representative blots are shown from three to five independent experiments (a). Cells pretreated or not with 75 μ M 2-APB or 30 μ M 8Br-cADPR and stimulated with either Ang-1 or Ang-2. Blots of total cell lysate were probed for the phosphorylation of downstream target AKT. To ensure equal loading membranes were reprobed for the total amount of the indicated protein and for β -actin. Representative blots are shown from three independent experiments (b1, b2, c1, and c2).

    Techniques Used: Activation Assay

    2-APB and 8Br-cADPR impair Angs-dependent cell migration on HUVECs. Ang-1 and Ang-2 induce Ca 2+ -dependent FAK phosphorylation. Confluent HUVECs were pretreated or not with 20 μ M BAPTA-AM for 1 h and stimulated with 100 ng/mL Ang-1 or 200 ng/mL Ang-2 for 30 min. Blots of total cell lysate were probed for the phosphorylation of downstream target FAK. To ensure equal loading membranes were reprobed for the total amount of the indicated protein and for β -actin (a). Cells pretreated or not with 75 μ M 2-APB or 30 μ M 8Br-cADPR and stimulated with either Ang-1 or Ang-2 (b1, b2, c1, and c2). Representative blots are shown from three to five independent experiments. Ang-1-induced EC migration is affected by treatment with the second messengers inhibitor 2-APB while Ang-2-induced cell migration appears to be both IP 3 - and cADPR-dependent. Scratch assay to evaluate the cell migration capability in the indicated experimental conditions. Wounded monolayers at the time of manual damage ( t = 0 h upper panel) and after 24 h treatment (lower panel) with Ang-1 (d), Ang-2 (e), and inhibitors as indicated. Pictures are representative of three independent experiments.
    Figure Legend Snippet: 2-APB and 8Br-cADPR impair Angs-dependent cell migration on HUVECs. Ang-1 and Ang-2 induce Ca 2+ -dependent FAK phosphorylation. Confluent HUVECs were pretreated or not with 20 μ M BAPTA-AM for 1 h and stimulated with 100 ng/mL Ang-1 or 200 ng/mL Ang-2 for 30 min. Blots of total cell lysate were probed for the phosphorylation of downstream target FAK. To ensure equal loading membranes were reprobed for the total amount of the indicated protein and for β -actin (a). Cells pretreated or not with 75 μ M 2-APB or 30 μ M 8Br-cADPR and stimulated with either Ang-1 or Ang-2 (b1, b2, c1, and c2). Representative blots are shown from three to five independent experiments. Ang-1-induced EC migration is affected by treatment with the second messengers inhibitor 2-APB while Ang-2-induced cell migration appears to be both IP 3 - and cADPR-dependent. Scratch assay to evaluate the cell migration capability in the indicated experimental conditions. Wounded monolayers at the time of manual damage ( t = 0 h upper panel) and after 24 h treatment (lower panel) with Ang-1 (d), Ang-2 (e), and inhibitors as indicated. Pictures are representative of three independent experiments.

    Techniques Used: Migration, Wound Healing Assay

    32) Product Images from "Amine-modified nanoplastics promote the procoagulant activation of isolated human red blood cells and thrombus formation in rats"

    Article Title: Amine-modified nanoplastics promote the procoagulant activation of isolated human red blood cells and thrombus formation in rats

    Journal: Particle and Fibre Toxicology

    doi: 10.1186/s12989-022-00500-y

    Effects of PS-NPs on the adhesion of human RBCs to human umbilical vein endothelial cells (HUVECs) and thrombin generation. A Adhesion of RBCs incubated with PS-NPs to HUVECs was observed using fluorescence microscopy ( n = 4). The signal of endothelial cells is green fluorescence and that of RBCs is red fluorescence. The white circles indicate the aggregated RBCs. B Thrombin generation was determined at the various concentrations of PS-NPs at a wavelength of 405 nm ( n = 6). Data are presented as the mean ± SE. * p
    Figure Legend Snippet: Effects of PS-NPs on the adhesion of human RBCs to human umbilical vein endothelial cells (HUVECs) and thrombin generation. A Adhesion of RBCs incubated with PS-NPs to HUVECs was observed using fluorescence microscopy ( n = 4). The signal of endothelial cells is green fluorescence and that of RBCs is red fluorescence. The white circles indicate the aggregated RBCs. B Thrombin generation was determined at the various concentrations of PS-NPs at a wavelength of 405 nm ( n = 6). Data are presented as the mean ± SE. * p

    Techniques Used: Incubation, Fluorescence, Microscopy

    33) Product Images from "Resveratrol Promotes Diabetic Wound Healing via SIRT1-FOXO1-c-Myc Signaling Pathway-Mediated Angiogenesis"

    Article Title: Resveratrol Promotes Diabetic Wound Healing via SIRT1-FOXO1-c-Myc Signaling Pathway-Mediated Angiogenesis

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2019.00421

    The endothelial protective action of RES against HG is silent information regulator 1 (SIRT1) dependent, in vitro . Cell lysates of HUVECs were used to detect (A) the SIRT1 protein levels by immunoblotting. HUVECs were cultured either in NG or HG medium in the presence or absence of RES (10 μM) for 72 h, MAN was served as the osmotic control for the HG. (B) The quantitative analysis of immunoblot. (C) HUVECs capillary-like tube formation, scale bars = 300 μm (×25), (E) wound healing assay, scale bars = 300 μm (×25), (G) immunofluorescence with Ki67, scale bars = 100 μm (×200), (I) immunofluorescence with PCNA, scale bars = 100 μm (×200), (K) TUNEL assay, scale bars = 100 μm (×200), and (M) fluorescence with DHE, scale bars = 100 μm (×200), HUVECs were transduced with transfected with SIRT1 siRNA or control siRNA, respectively. After transduction, HUVECs were cultured either in NG or HG medium alone or with RES (10 μM) for 72 h. Quantification of the tube length (D) , the cell migration distance (F) , the Ki67 fluorescence intensity ratio (H) , the PCNA fluorescence intensity ratio (J) , the quantitative analysis of TUNEL + cells (L) , the DHE fluorescence intensity ratio (N) . All values displayed are means ± SEM of 8 independent experiments. # p
    Figure Legend Snippet: The endothelial protective action of RES against HG is silent information regulator 1 (SIRT1) dependent, in vitro . Cell lysates of HUVECs were used to detect (A) the SIRT1 protein levels by immunoblotting. HUVECs were cultured either in NG or HG medium in the presence or absence of RES (10 μM) for 72 h, MAN was served as the osmotic control for the HG. (B) The quantitative analysis of immunoblot. (C) HUVECs capillary-like tube formation, scale bars = 300 μm (×25), (E) wound healing assay, scale bars = 300 μm (×25), (G) immunofluorescence with Ki67, scale bars = 100 μm (×200), (I) immunofluorescence with PCNA, scale bars = 100 μm (×200), (K) TUNEL assay, scale bars = 100 μm (×200), and (M) fluorescence with DHE, scale bars = 100 μm (×200), HUVECs were transduced with transfected with SIRT1 siRNA or control siRNA, respectively. After transduction, HUVECs were cultured either in NG or HG medium alone or with RES (10 μM) for 72 h. Quantification of the tube length (D) , the cell migration distance (F) , the Ki67 fluorescence intensity ratio (H) , the PCNA fluorescence intensity ratio (J) , the quantitative analysis of TUNEL + cells (L) , the DHE fluorescence intensity ratio (N) . All values displayed are means ± SEM of 8 independent experiments. # p

    Techniques Used: In Vitro, Cell Culture, Wound Healing Assay, Immunofluorescence, TUNEL Assay, Fluorescence, Transduction, Transfection, Migration

    c-Myc participates in the endothelial protective action of RES against hyperglycemia in vitro . (A) Cell lysates of HUVECs were used to detect the FOXO1 and c-Myc protein levels by immunoblotting. HUVECs were transduced with Ad- FOXO1 and Ad- LacZ , respectively. After transduction, HUVECs were cultured either in NG or HG medium in the presence or absence of RES (10 μM) for 72 h. (B , C) The quantitative analysis of each immunoblots. (D) HUVECs capillary-like tube formation, scale bars = 300 μm (×25), (F) wound healing assay, scale bars = 300 μm (×25), (H) immunofluorescence with Ki67, scale bars = 100 μm (×200), (J) immunofluorescence with PCNA, scale bars = 100 μm (×200), (L) TUNEL assay, scale bars = 100 μm (×200), (N) fluorescence with DHE, scale bars = 100 μm (×200), HUVECs were transduced with transfected with c-Myc siRNA or control siRNA, respectively. After transduction, HUVECs were cultured either in NG, or HG medium alone or with RES (10 μM) for 72 h. Quantification of the tube length (E) , the cell migration distance (G) , the Ki67 fluorescence intensity ratio (I) , the PCNA fluorescence intensity ratio (K) , the quantitative analysis of TUNEL + cells (M) , the DHE fluorescence intensity ratio (O) . All values displayed are means ± SEM of 8 independent experiments. # p
    Figure Legend Snippet: c-Myc participates in the endothelial protective action of RES against hyperglycemia in vitro . (A) Cell lysates of HUVECs were used to detect the FOXO1 and c-Myc protein levels by immunoblotting. HUVECs were transduced with Ad- FOXO1 and Ad- LacZ , respectively. After transduction, HUVECs were cultured either in NG or HG medium in the presence or absence of RES (10 μM) for 72 h. (B , C) The quantitative analysis of each immunoblots. (D) HUVECs capillary-like tube formation, scale bars = 300 μm (×25), (F) wound healing assay, scale bars = 300 μm (×25), (H) immunofluorescence with Ki67, scale bars = 100 μm (×200), (J) immunofluorescence with PCNA, scale bars = 100 μm (×200), (L) TUNEL assay, scale bars = 100 μm (×200), (N) fluorescence with DHE, scale bars = 100 μm (×200), HUVECs were transduced with transfected with c-Myc siRNA or control siRNA, respectively. After transduction, HUVECs were cultured either in NG, or HG medium alone or with RES (10 μM) for 72 h. Quantification of the tube length (E) , the cell migration distance (G) , the Ki67 fluorescence intensity ratio (I) , the PCNA fluorescence intensity ratio (K) , the quantitative analysis of TUNEL + cells (M) , the DHE fluorescence intensity ratio (O) . All values displayed are means ± SEM of 8 independent experiments. # p

    Techniques Used: In Vitro, Transduction, Cell Culture, Western Blot, Wound Healing Assay, Immunofluorescence, TUNEL Assay, Fluorescence, Transfection, Migration

    Resveratrol attenuates hyperglycemia-induced endothelial dysfunction in vitro . (A) The presence of human umbilical vein endothelial cells (HUVECs) wound healing assay, scale bars = 300 μm (×25), (C) capillary-like tube formation, scale bars = 300 μm (×25), (E) immunofluorescence with Ki67, scale bars = 100 μm (×200), (G) immunofluorescence with PCNA, scale bars = 100 μm (×200), (I) TUNEL assay, scale bars = 100 μm (×200), and (K) fluorescence with DHE, scale bars = 100 μm (×200), HUVECs were cultured in different mediums containing NG (5.5 mM), HG (33 mM) alone, or with RES (10 μM) for 72 h, mannitol (MAN; 33 mM: 5.5 mM of glucose + 27.5 mM of D -mannitol) was served as the osmotic control for the HG. Quantification of the cell migration distance (B) , the tube length (D) , the Ki67 fluorescence intensity ratio (F) , the PCNA fluorescence intensity ratio (H) , the quantitative analysis of TUNEL + cells (J) , the DHE fluorescence intensity ratio (L) . All values displayed are means ± SEM of 8 independent experiments. # p
    Figure Legend Snippet: Resveratrol attenuates hyperglycemia-induced endothelial dysfunction in vitro . (A) The presence of human umbilical vein endothelial cells (HUVECs) wound healing assay, scale bars = 300 μm (×25), (C) capillary-like tube formation, scale bars = 300 μm (×25), (E) immunofluorescence with Ki67, scale bars = 100 μm (×200), (G) immunofluorescence with PCNA, scale bars = 100 μm (×200), (I) TUNEL assay, scale bars = 100 μm (×200), and (K) fluorescence with DHE, scale bars = 100 μm (×200), HUVECs were cultured in different mediums containing NG (5.5 mM), HG (33 mM) alone, or with RES (10 μM) for 72 h, mannitol (MAN; 33 mM: 5.5 mM of glucose + 27.5 mM of D -mannitol) was served as the osmotic control for the HG. Quantification of the cell migration distance (B) , the tube length (D) , the Ki67 fluorescence intensity ratio (F) , the PCNA fluorescence intensity ratio (H) , the quantitative analysis of TUNEL + cells (J) , the DHE fluorescence intensity ratio (L) . All values displayed are means ± SEM of 8 independent experiments. # p

    Techniques Used: In Vitro, Wound Healing Assay, Immunofluorescence, TUNEL Assay, Fluorescence, Cell Culture, Migration

    FOXO1 participates in the endothelial protective action of RES against hyperglycemia, in vitro . (A) Cell lysates of HUVECs were used to detect the SIRT1 and FOXO1 protein levels by immunoblotting. sqRT-PCR analysis of FOXO1 mRNA level in HUVECs. HUVECs were transduced with SIRT1 siRNA or control siRNA, respectively. After transduction, HUVECs were cultured either in NG or HG medium alone or with RES (10 μM) for 72 h, MAN was served as the osmotic control for the HG. (B – D) The quantitative analysis of (A) . (E) Cell lysates of HUVECs were used to detect the FOXO1 protein levels by immunoblotting. HUVECs were cultured either in NG or HG medium alone or with RES (10 μM) for 72 h, MAN was served as the osmotic control for the HG. For signaling pathway analysis, MG-132 (0.5 μM) was pretreated for 2 h before RES administration. (F) The quantitative analysis of (E) . (G) Nuclear and cytosolic extracts were isolated to detect the FOXO1 protein levels by immunoblotting. HUVECs treated as indicated in (A) . (H,I) The quantitative analysis of each immunoblots. (J) Representative immunofluorescence with FOXO1 in HUVECs, which treated as indicated in (A) . Scale bars = 5 μm (×400). (K) The quantitative analysis of nuclear/perinuclear FOXO1 fluorescence intensity ratio in (J) . (L) Cell lysates of HUVECs were used to detect the FOXO1 and c-Myc protein levels by immunoblotting. sqRT-PCR analysis of FOXO1 mRNA level in HUVECs. HUVECs were transduced with Ad- FOXO1 and Ad- LacZ , respectively. After transduction, HUVECs were cultured in NG. (M – O) The quantitative analysis of (L) . All values displayed are means ± SEM of 8 independent experiments. # p
    Figure Legend Snippet: FOXO1 participates in the endothelial protective action of RES against hyperglycemia, in vitro . (A) Cell lysates of HUVECs were used to detect the SIRT1 and FOXO1 protein levels by immunoblotting. sqRT-PCR analysis of FOXO1 mRNA level in HUVECs. HUVECs were transduced with SIRT1 siRNA or control siRNA, respectively. After transduction, HUVECs were cultured either in NG or HG medium alone or with RES (10 μM) for 72 h, MAN was served as the osmotic control for the HG. (B – D) The quantitative analysis of (A) . (E) Cell lysates of HUVECs were used to detect the FOXO1 protein levels by immunoblotting. HUVECs were cultured either in NG or HG medium alone or with RES (10 μM) for 72 h, MAN was served as the osmotic control for the HG. For signaling pathway analysis, MG-132 (0.5 μM) was pretreated for 2 h before RES administration. (F) The quantitative analysis of (E) . (G) Nuclear and cytosolic extracts were isolated to detect the FOXO1 protein levels by immunoblotting. HUVECs treated as indicated in (A) . (H,I) The quantitative analysis of each immunoblots. (J) Representative immunofluorescence with FOXO1 in HUVECs, which treated as indicated in (A) . Scale bars = 5 μm (×400). (K) The quantitative analysis of nuclear/perinuclear FOXO1 fluorescence intensity ratio in (J) . (L) Cell lysates of HUVECs were used to detect the FOXO1 and c-Myc protein levels by immunoblotting. sqRT-PCR analysis of FOXO1 mRNA level in HUVECs. HUVECs were transduced with Ad- FOXO1 and Ad- LacZ , respectively. After transduction, HUVECs were cultured in NG. (M – O) The quantitative analysis of (L) . All values displayed are means ± SEM of 8 independent experiments. # p

    Techniques Used: In Vitro, Polymerase Chain Reaction, Transduction, Cell Culture, Isolation, Western Blot, Immunofluorescence, Fluorescence

    FOXO1 participates in the endothelial protective action of RES against hyperglycemia, in vitro . (A) HUVECs capillary-like tube formation, scale bars = 300 μm (×25), (C) wound healing assay, scale bars = 300 μm (×25), (E) immunofluorescence with Ki67, scale bars = 100 μm (×200), (G) immunofluorescence with PCNA, scale bars = 100 μm (×200), (I) TUNEL assay, scale bars = 100 μm (×200), (K) fluorescence with DHE, scale bars = 100 μm (×200), HUVECs were transduced with Ad- FOXO1 and Ad- LacZ , respectively. After transduction, HUVECs were cultured either in NG or HG medium in the presence or absence of RES (10 μM) for 72 h, MAN was served as the osmotic control for the HG. Quantification of the tube length (B) , the cell migration distance (D) , the Ki67 fluorescence intensity ratio (F) , the PCNA fluorescence intensity ratio (H) , the quantitative analysis of TUNEL + cells (J) , the DHE fluorescence intensity ratio (L) . All values displayed are means ± SEM of 8 independent experiments. # p
    Figure Legend Snippet: FOXO1 participates in the endothelial protective action of RES against hyperglycemia, in vitro . (A) HUVECs capillary-like tube formation, scale bars = 300 μm (×25), (C) wound healing assay, scale bars = 300 μm (×25), (E) immunofluorescence with Ki67, scale bars = 100 μm (×200), (G) immunofluorescence with PCNA, scale bars = 100 μm (×200), (I) TUNEL assay, scale bars = 100 μm (×200), (K) fluorescence with DHE, scale bars = 100 μm (×200), HUVECs were transduced with Ad- FOXO1 and Ad- LacZ , respectively. After transduction, HUVECs were cultured either in NG or HG medium in the presence or absence of RES (10 μM) for 72 h, MAN was served as the osmotic control for the HG. Quantification of the tube length (B) , the cell migration distance (D) , the Ki67 fluorescence intensity ratio (F) , the PCNA fluorescence intensity ratio (H) , the quantitative analysis of TUNEL + cells (J) , the DHE fluorescence intensity ratio (L) . All values displayed are means ± SEM of 8 independent experiments. # p

    Techniques Used: In Vitro, Wound Healing Assay, Immunofluorescence, TUNEL Assay, Fluorescence, Transduction, Cell Culture, Migration

    34) Product Images from "Endoglin Modulates TGFβR2 Induced VEGF and Proinflammatory Cytokine Axis Mediated Angiogenesis in Prolonged DEHP-Exposed Breast Cancer Cells"

    Article Title: Endoglin Modulates TGFβR2 Induced VEGF and Proinflammatory Cytokine Axis Mediated Angiogenesis in Prolonged DEHP-Exposed Breast Cancer Cells

    Journal: Biomedicines

    doi: 10.3390/biomedicines10020417

    Representative and quantitative results of endoglin-mediated HUVEC tube formation. ( A ) Induced HUVEC tube formation in coculture with control and DEHP-exposed MDA-MB-231 cells at 8 h after seeding; endoglin knockdown reversed DEHP-induced HUVEC tube formation. Scale bar = 250 µm. ( B ) Quantitative evaluation of the number of tubes formed in coculture at 8 h after cell seeding. ( C ) Quantitative evaluation of average tube length in coculture at 8 h after cell seeding. ( D ) Quantitative evaluation of the number of nodes formed in coculture at 8 h after cell seeding. ( E ) Results of quantitative ELISA for VEGF levels in the cell culture medium of control and DEHP-exposed MDA-MB-231 cells (mock- and shENG-treated); ** p
    Figure Legend Snippet: Representative and quantitative results of endoglin-mediated HUVEC tube formation. ( A ) Induced HUVEC tube formation in coculture with control and DEHP-exposed MDA-MB-231 cells at 8 h after seeding; endoglin knockdown reversed DEHP-induced HUVEC tube formation. Scale bar = 250 µm. ( B ) Quantitative evaluation of the number of tubes formed in coculture at 8 h after cell seeding. ( C ) Quantitative evaluation of average tube length in coculture at 8 h after cell seeding. ( D ) Quantitative evaluation of the number of nodes formed in coculture at 8 h after cell seeding. ( E ) Results of quantitative ELISA for VEGF levels in the cell culture medium of control and DEHP-exposed MDA-MB-231 cells (mock- and shENG-treated); ** p

    Techniques Used: Multiple Displacement Amplification, Enzyme-linked Immunosorbent Assay, Cell Culture

    35) Product Images from "KLF4-Induced Connexin40 Expression Contributes to Arterial Endothelial Quiescence"

    Article Title: KLF4-Induced Connexin40 Expression Contributes to Arterial Endothelial Quiescence

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2019.00080

    Cx40 expression is gradually regulated by shear stress. (A) KLF4 expression in bEnd.3 cells under static conditions (St) or exposed to 24 h of LLSS and HLSS was assessed by qPCR. N = 3. (B) Cx40 expression in bEnd.3 cells under static conditions (St) or exposed to 24 h of LLSS and HLSS was assessed by qPCR. N = 3. (C) Representative images of Cx40 expression (green) in bEnd.3 cells under static conditions (St) or exposed to LLSS and HLSS for 24 h. Arrow indicates the direction of flow. Nuclei were stained with DAPI (blue). Scale bar represents 10 μm. (D) Schematic representation of flow regions in cell cultures induced by orbital rotation; green = HLSS and red = OSS. (E) Cx40 expression in bEnd.3 cells under static conditions (St) or exposed to 48 h of HLSS was assessed by Western blotting. N = 5. (F) Phase-contrast images of HUVECs under static conditions or exposed for 48 h of HLSS. (G,H) Cx40 expression in HUVECs under static conditions (St) or exposed to 48 h of HLSS was assessed by Western blotting (G) or qPCR ( H ; N = 6).
    Figure Legend Snippet: Cx40 expression is gradually regulated by shear stress. (A) KLF4 expression in bEnd.3 cells under static conditions (St) or exposed to 24 h of LLSS and HLSS was assessed by qPCR. N = 3. (B) Cx40 expression in bEnd.3 cells under static conditions (St) or exposed to 24 h of LLSS and HLSS was assessed by qPCR. N = 3. (C) Representative images of Cx40 expression (green) in bEnd.3 cells under static conditions (St) or exposed to LLSS and HLSS for 24 h. Arrow indicates the direction of flow. Nuclei were stained with DAPI (blue). Scale bar represents 10 μm. (D) Schematic representation of flow regions in cell cultures induced by orbital rotation; green = HLSS and red = OSS. (E) Cx40 expression in bEnd.3 cells under static conditions (St) or exposed to 48 h of HLSS was assessed by Western blotting. N = 5. (F) Phase-contrast images of HUVECs under static conditions or exposed for 48 h of HLSS. (G,H) Cx40 expression in HUVECs under static conditions (St) or exposed to 48 h of HLSS was assessed by Western blotting (G) or qPCR ( H ; N = 6).

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Staining, Western Blot

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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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    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
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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.
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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