huvec cells  (Lonza)


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    Structured Review

    Lonza huvec cells
    KSHV colocalizes with macropinocytic marker dextran and not with transferrin and caveolin. (A and D) Colocalization of KSHV with dextran. Uninfected and infected <t>HMVEC-d</t> (A) and <t>HUVEC</t> (D) cells were incubated at 37°C with Texas Red-labeled dextran and KSHV for 5, 10, and 15 min. Cells were washed in HBSS, fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked in 5% BSA, and then incubated with anti-gpK8.1A mAb followed by Alexa Fluor 488-labeled goat anti-mouse secondary antibody. Magnification, ×40. White arrows indicate colocalization of KSHV with dextran. (B and E) KSHV does not colocalize with transferrin. Uninfected cells and cells infected with KSHV and Alexa Fluor 594-conjugated transferrin were examined with anti-gpK8.1A antibody. Magnification, ×40. Yellow arrows indicate virus particles not colocalized with transferrin. (C) KSHV does not colocalize with caveolin. Uninfected and infected cells were incubated with anti-gpK8.1A and anticaveolin antibodies for 1 h at room temperature. The staining was visualized by incubation with Alexa Fluor 488-labeled antibody for gpK8.1A and Alexa Fluor 594-labeled secondary antibody for caveolin. Magnification, ×40. Yellow arrows indicate virus particles not colocalized with caveolin. Boxed areas are shown enlarged in the right-hand panels. ', min; UN, uninfected.
    Huvec Cells, supplied by Lonza, used in various techniques. Bioz Stars score: 97/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus Utilizes an Actin Polymerization-Dependent Macropinocytic Pathway To Enter Human Dermal Microvascular Endothelial and Human Umbilical Vein Endothelial Cells ▿"

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Utilizes an Actin Polymerization-Dependent Macropinocytic Pathway To Enter Human Dermal Microvascular Endothelial and Human Umbilical Vein Endothelial Cells ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02498-08

    KSHV colocalizes with macropinocytic marker dextran and not with transferrin and caveolin. (A and D) Colocalization of KSHV with dextran. Uninfected and infected HMVEC-d (A) and HUVEC (D) cells were incubated at 37°C with Texas Red-labeled dextran and KSHV for 5, 10, and 15 min. Cells were washed in HBSS, fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked in 5% BSA, and then incubated with anti-gpK8.1A mAb followed by Alexa Fluor 488-labeled goat anti-mouse secondary antibody. Magnification, ×40. White arrows indicate colocalization of KSHV with dextran. (B and E) KSHV does not colocalize with transferrin. Uninfected cells and cells infected with KSHV and Alexa Fluor 594-conjugated transferrin were examined with anti-gpK8.1A antibody. Magnification, ×40. Yellow arrows indicate virus particles not colocalized with transferrin. (C) KSHV does not colocalize with caveolin. Uninfected and infected cells were incubated with anti-gpK8.1A and anticaveolin antibodies for 1 h at room temperature. The staining was visualized by incubation with Alexa Fluor 488-labeled antibody for gpK8.1A and Alexa Fluor 594-labeled secondary antibody for caveolin. Magnification, ×40. Yellow arrows indicate virus particles not colocalized with caveolin. Boxed areas are shown enlarged in the right-hand panels. ', min; UN, uninfected.
    Figure Legend Snippet: KSHV colocalizes with macropinocytic marker dextran and not with transferrin and caveolin. (A and D) Colocalization of KSHV with dextran. Uninfected and infected HMVEC-d (A) and HUVEC (D) cells were incubated at 37°C with Texas Red-labeled dextran and KSHV for 5, 10, and 15 min. Cells were washed in HBSS, fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked in 5% BSA, and then incubated with anti-gpK8.1A mAb followed by Alexa Fluor 488-labeled goat anti-mouse secondary antibody. Magnification, ×40. White arrows indicate colocalization of KSHV with dextran. (B and E) KSHV does not colocalize with transferrin. Uninfected cells and cells infected with KSHV and Alexa Fluor 594-conjugated transferrin were examined with anti-gpK8.1A antibody. Magnification, ×40. Yellow arrows indicate virus particles not colocalized with transferrin. (C) KSHV does not colocalize with caveolin. Uninfected and infected cells were incubated with anti-gpK8.1A and anticaveolin antibodies for 1 h at room temperature. The staining was visualized by incubation with Alexa Fluor 488-labeled antibody for gpK8.1A and Alexa Fluor 594-labeled secondary antibody for caveolin. Magnification, ×40. Yellow arrows indicate virus particles not colocalized with caveolin. Boxed areas are shown enlarged in the right-hand panels. ', min; UN, uninfected.

    Techniques Used: Marker, Infection, Incubation, Labeling, Staining

    KSHV induces actin polymerization in HMVEC-d (A) and HUVEC (B) cells. HMVEC-d and HUVEC cells were left uninfected or infected with KSHV (MOI of 10) at 37°C for different times (', min), fixed, permeabilized, and stained for polymerized actin by using Alexa Fluor 488-labeled phalloidin for 30 min at room temperature. Red arrowheads indicate the accumulated actin stress fibers, white arrows indicate hair-like membrane filopodial extensions, and white arrowheads indicate sites of membrane ruffling. Magnification, ×40.
    Figure Legend Snippet: KSHV induces actin polymerization in HMVEC-d (A) and HUVEC (B) cells. HMVEC-d and HUVEC cells were left uninfected or infected with KSHV (MOI of 10) at 37°C for different times (', min), fixed, permeabilized, and stained for polymerized actin by using Alexa Fluor 488-labeled phalloidin for 30 min at room temperature. Red arrowheads indicate the accumulated actin stress fibers, white arrows indicate hair-like membrane filopodial extensions, and white arrowheads indicate sites of membrane ruffling. Magnification, ×40.

    Techniques Used: Infection, Staining, Labeling

    KSHV entry in HMVEC-d and HUVEC cells is dynamin independent. (A) HMVEC-d cells showing transfection with WT dynamin. Cells were transfected with 1 μg of GFP-WT dynamin plasmid or 1 μg of GFP-K44A dynamin plasmid and observed under a microscope after 24 h. a and c, GFP; b and d, phase-contrast microscopy. Magnification, ×10. (B) HMVEC-d cells were transfected with 1 μg of GFP-WT or GFP-K44A dynamin plasmid. After 24 h, cells were infected with KSHV at an MOI of 10 for different times and KSHV internalization was measured by real-time DNA PCR. Histogram shows internalized copy numbers of KSHV in HMVEC-d cells transfected with WT and K44A dynamin plasmids. (C and D) Dynasore did not affect internalization in HUVEC (C) and HMVEC-d (D) cells. HUVEC cells and HMVEC-d cells grown in six-well plates were treated with 80 μM dynasore for 1 h at 37°C and infected with KSHV at an MOI of 10 for different times, and KSHV internalization measured by real-time DNA PCR for ORF73 gene. (E) Dynasore treatment affects KSHV internalization in HFF cells. Cells grown in six-well plates were treated with 100 μM dynasore and infected with KSHV at an MOI of 10 for different times, and KSHV internalization measured by real-time DNA PCR. Histogram shows the internalized copy numbers of KSHV DNA with and without dynasore treatment. Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three experiments.
    Figure Legend Snippet: KSHV entry in HMVEC-d and HUVEC cells is dynamin independent. (A) HMVEC-d cells showing transfection with WT dynamin. Cells were transfected with 1 μg of GFP-WT dynamin plasmid or 1 μg of GFP-K44A dynamin plasmid and observed under a microscope after 24 h. a and c, GFP; b and d, phase-contrast microscopy. Magnification, ×10. (B) HMVEC-d cells were transfected with 1 μg of GFP-WT or GFP-K44A dynamin plasmid. After 24 h, cells were infected with KSHV at an MOI of 10 for different times and KSHV internalization was measured by real-time DNA PCR. Histogram shows internalized copy numbers of KSHV in HMVEC-d cells transfected with WT and K44A dynamin plasmids. (C and D) Dynasore did not affect internalization in HUVEC (C) and HMVEC-d (D) cells. HUVEC cells and HMVEC-d cells grown in six-well plates were treated with 80 μM dynasore for 1 h at 37°C and infected with KSHV at an MOI of 10 for different times, and KSHV internalization measured by real-time DNA PCR for ORF73 gene. (E) Dynasore treatment affects KSHV internalization in HFF cells. Cells grown in six-well plates were treated with 100 μM dynasore and infected with KSHV at an MOI of 10 for different times, and KSHV internalization measured by real-time DNA PCR. Histogram shows the internalized copy numbers of KSHV DNA with and without dynasore treatment. Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three experiments.

    Techniques Used: Transfection, Plasmid Preparation, Microscopy, Infection, Polymerase Chain Reaction, Standard Deviation

    2) Product Images from "Antifungal benzimidazoles disrupt vasculature by targeting one of nine β-tubulins"

    Article Title: Antifungal benzimidazoles disrupt vasculature by targeting one of nine β-tubulins

    Journal: bioRxiv

    doi: 10.1101/2020.09.15.298828

    TBZ specifically inhibits the human β-tubulin TUBB8, not TUBB4, in humanized yeast and HUVEC cell culture. ( A ) Overview. TBZ’s isotype specificity was identified in 2 ways. (Left) Recombinant human β-tubulins TUBB4 and TUBB8 were individually overexpressed in HUVEC cell culture to monitor comet lengths in the presence of TBZ. (Right) Using humanized yeast wherein yeast TUB2 was singly humanized by either of 2 replaceable human β-tubulins TUBB4 or TUBB8 to screen for differential sensitivity towards TBZ. ( B ) Reduced EB3 comet length after 1% DMSO, 250 µM TBZ treatment compared to 1% DMSO treated control. A . Comet length is similar in EB3, TUBB8 transfected HUVECs compared to EB3 transfected controls expressing native tubulins, but comets are longer in most EB3, TUBB4 transfected cells. B . Comet length is statistically similar between cells treated with 1% DMSO; however, following 30 minutes of 1% DMSO, 250 µM TBZ treatment TUBB4 transfected cells have significantly longer EB3 comets than HUVECs with TUBB8 or expressing native tubulins. ( C ) Growth profiles of humanized yeast strains show TBZ’s isotype specificity to TUBB8. When grown in the presence of TBZ, Strains carrying the wild-type TUB2 (blue) and human TUBB8 (green) genes are sensitive to TBZ while humanized TUBB4 strains (orange) are resistant. Mean +/-standard deviation indicated by solid lines and shaded boundaries, respectively.
    Figure Legend Snippet: TBZ specifically inhibits the human β-tubulin TUBB8, not TUBB4, in humanized yeast and HUVEC cell culture. ( A ) Overview. TBZ’s isotype specificity was identified in 2 ways. (Left) Recombinant human β-tubulins TUBB4 and TUBB8 were individually overexpressed in HUVEC cell culture to monitor comet lengths in the presence of TBZ. (Right) Using humanized yeast wherein yeast TUB2 was singly humanized by either of 2 replaceable human β-tubulins TUBB4 or TUBB8 to screen for differential sensitivity towards TBZ. ( B ) Reduced EB3 comet length after 1% DMSO, 250 µM TBZ treatment compared to 1% DMSO treated control. A . Comet length is similar in EB3, TUBB8 transfected HUVECs compared to EB3 transfected controls expressing native tubulins, but comets are longer in most EB3, TUBB4 transfected cells. B . Comet length is statistically similar between cells treated with 1% DMSO; however, following 30 minutes of 1% DMSO, 250 µM TBZ treatment TUBB4 transfected cells have significantly longer EB3 comets than HUVECs with TUBB8 or expressing native tubulins. ( C ) Growth profiles of humanized yeast strains show TBZ’s isotype specificity to TUBB8. When grown in the presence of TBZ, Strains carrying the wild-type TUB2 (blue) and human TUBB8 (green) genes are sensitive to TBZ while humanized TUBB4 strains (orange) are resistant. Mean +/-standard deviation indicated by solid lines and shaded boundaries, respectively.

    Techniques Used: Cell Culture, Recombinant, Transfection, Expressing, Standard Deviation

    3) Product Images from "Kalkitoxin Inhibits Angiogenesis, Disrupts Cellular Hypoxic Signaling, and Blocks Mitochondrial Electron Transport in Tumor Cells"

    Article Title: Kalkitoxin Inhibits Angiogenesis, Disrupts Cellular Hypoxic Signaling, and Blocks Mitochondrial Electron Transport in Tumor Cells

    Journal: Marine Drugs

    doi: 10.3390/md13031552

    Kalkitoxin inhibits hypoxia-stimulated tumor angiogenesis by blocking the induction of angiogenic factor VEGF. ( A ) T47D cells were exposed to hypoxic conditions (1% O 2 , 16 h) in the presence and absence of kalkitoxin at the specified concentrations. The levels of secreted VEGF protein in the conditioned media samples were determined by ELISA and normalized to the amount of total cellular proteins (average + standard deviation, n = 3). “C”—media control. The p values of statistically significant differences when compared to the media controls are shown; ( B ) Representative HUVEC tube formation assay results. T47D cell-conditioned media samples were prepared as described in (A). Both negative (Basal Media, e ) and positive (VEGF, f ) controls are included at the bottom. A 100 µm scale bar is included in each panel; ( C ) Average + standard deviation of tube length, quantified from three randomly selected fields for each specified condition. The p -values are shown for statistically significant differences; ( D ) Branching points, data determined and presented as described in (C).
    Figure Legend Snippet: Kalkitoxin inhibits hypoxia-stimulated tumor angiogenesis by blocking the induction of angiogenic factor VEGF. ( A ) T47D cells were exposed to hypoxic conditions (1% O 2 , 16 h) in the presence and absence of kalkitoxin at the specified concentrations. The levels of secreted VEGF protein in the conditioned media samples were determined by ELISA and normalized to the amount of total cellular proteins (average + standard deviation, n = 3). “C”—media control. The p values of statistically significant differences when compared to the media controls are shown; ( B ) Representative HUVEC tube formation assay results. T47D cell-conditioned media samples were prepared as described in (A). Both negative (Basal Media, e ) and positive (VEGF, f ) controls are included at the bottom. A 100 µm scale bar is included in each panel; ( C ) Average + standard deviation of tube length, quantified from three randomly selected fields for each specified condition. The p -values are shown for statistically significant differences; ( D ) Branching points, data determined and presented as described in (C).

    Techniques Used: Blocking Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, HUVEC Tube Formation Assay

    4) Product Images from "VEGF regulates local inhibitory complement proteins in the eye and kidney"

    Article Title: VEGF regulates local inhibitory complement proteins in the eye and kidney

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI86418

    VEGF regulates CFH expression in the glomerulus. Adult mice with an induced deletion of podocyte Vegfa showed reduced CFH staining in their glomeruli compared with control mice ( A , CFH red, podocin green, DAPI blue) and showed glomerular C3 staining ( B , C3 green, podocin red, DAPI blue). Human GEnC and podocytes stained positively for CFH (red, DAPI blue) under normal cell culture conditions ( C , media). This was removed by 0.1 M acetic acid treatment ( C , after AA). CFH staining recurred after 24 hours in serum-free media (SFM), and this was significantly increased by VEGF treatment in a dose-dependent manner. HUVECs also showed CFH staining, but they showed a different response to VEGF treatment ( C ). HEK293 cells did not show CFH staining, and there was no change with VEGF treatment ( C ). Immunofluorescent studies were validated using Western blotting of cell lysates ( D , n = 4), condition media ( E , representative, n = 4), and qPCR ( F , n = 4) shown for GEnC and podocytes ( D and F , n = 4, 1-way ANOVA). Twenty-four hours of VEGF treatment also reduced GEnC and podocyte C3d ( G ) (green, DAPI blue) deposits after cell-surface complement activation. ( A and B ) n = 8–10/group. 20 glomeruli/animal imaged for each antibody tested and averaged. Unpaired, 2-tailed t test. ( C and G ) Representative images shown from 4 independent experiments. Ten images obtained for each condition. MFI was calculated for CFH/C3d and corrected for cell number determined by DAPI-stained nuclei to semi-quantitatively compare expression. One-way ANOVA with Bonferroni’s post hoc analysis. Scale bars: 10 μm ( A ); 50 μm ( B , GEnC); 25 μm ( B , podocytes); 50 μm ( B , HUVEC); 50 μm ( B , HEK293); 50 μm ( C ); 50 μm ( G ). * P
    Figure Legend Snippet: VEGF regulates CFH expression in the glomerulus. Adult mice with an induced deletion of podocyte Vegfa showed reduced CFH staining in their glomeruli compared with control mice ( A , CFH red, podocin green, DAPI blue) and showed glomerular C3 staining ( B , C3 green, podocin red, DAPI blue). Human GEnC and podocytes stained positively for CFH (red, DAPI blue) under normal cell culture conditions ( C , media). This was removed by 0.1 M acetic acid treatment ( C , after AA). CFH staining recurred after 24 hours in serum-free media (SFM), and this was significantly increased by VEGF treatment in a dose-dependent manner. HUVECs also showed CFH staining, but they showed a different response to VEGF treatment ( C ). HEK293 cells did not show CFH staining, and there was no change with VEGF treatment ( C ). Immunofluorescent studies were validated using Western blotting of cell lysates ( D , n = 4), condition media ( E , representative, n = 4), and qPCR ( F , n = 4) shown for GEnC and podocytes ( D and F , n = 4, 1-way ANOVA). Twenty-four hours of VEGF treatment also reduced GEnC and podocyte C3d ( G ) (green, DAPI blue) deposits after cell-surface complement activation. ( A and B ) n = 8–10/group. 20 glomeruli/animal imaged for each antibody tested and averaged. Unpaired, 2-tailed t test. ( C and G ) Representative images shown from 4 independent experiments. Ten images obtained for each condition. MFI was calculated for CFH/C3d and corrected for cell number determined by DAPI-stained nuclei to semi-quantitatively compare expression. One-way ANOVA with Bonferroni’s post hoc analysis. Scale bars: 10 μm ( A ); 50 μm ( B , GEnC); 25 μm ( B , podocytes); 50 μm ( B , HUVEC); 50 μm ( B , HEK293); 50 μm ( C ); 50 μm ( G ). * P

    Techniques Used: Expressing, Mouse Assay, Staining, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, Activation Assay

    5) Product Images from "Interactions between endocarditis-derived Streptococcus gallolyticus subsp. gallolyticus isolates and human endothelial cells"

    Article Title: Interactions between endocarditis-derived Streptococcus gallolyticus subsp. gallolyticus isolates and human endothelial cells

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-10-78

    Influence of cell type (EA.hy926/HUVEC) and cell condition (stressed/non-stressed) on the adherence and invasion characteristics of S. gallolyticus . (A) Adhesion to and invasion of endothelial cell lines EA.hy926 and HUVECs after infection with 1 - 9 × 10 5 CFU/mL of different S. gallolyticus strains. (B) S. gallolyticus strain's adhesion to and invasion of EA.hy926 with and without mechanical stretch 24 h prior to infection with 1 - 9 × 10 5 CFU/mL bacteria. Results were determined after a 2 h exposure followed by additional 2 h incubation in the presence of antibiotics. n.d.: not detectable.
    Figure Legend Snippet: Influence of cell type (EA.hy926/HUVEC) and cell condition (stressed/non-stressed) on the adherence and invasion characteristics of S. gallolyticus . (A) Adhesion to and invasion of endothelial cell lines EA.hy926 and HUVECs after infection with 1 - 9 × 10 5 CFU/mL of different S. gallolyticus strains. (B) S. gallolyticus strain's adhesion to and invasion of EA.hy926 with and without mechanical stretch 24 h prior to infection with 1 - 9 × 10 5 CFU/mL bacteria. Results were determined after a 2 h exposure followed by additional 2 h incubation in the presence of antibiotics. n.d.: not detectable.

    Techniques Used: Infection, Incubation

    6) Product Images from "A novel xenograft model reveals invasive mesenchymal transition and ineffective angiogenic response during anti-angiogenic therapy resistance"

    Article Title: A novel xenograft model reveals invasive mesenchymal transition and ineffective angiogenic response during anti-angiogenic therapy resistance

    Journal: bioRxiv

    doi: 10.1101/272328

    Increased but ineffective angiogenic gene expression in a multi-generational xenograft model of bevacizumab resistance. ( a ) The top 30 upregulated genes from the significantly enriched angiogenesis gene ontology were selected for validation by qPCR. ( b ) qPCR validation of 29 of the top 30 angiogenesis genes revealed significant overexpression of angiogenesis related genes by generation 1 of U87-Bev R , peaking by generation 4. ( c ) Dot plots separating the PCR results for the 29 pro-angiogenic genes into 3 categories: those that were upregulated with each U87-Bev R generation, peaking at generation 9 (left); those that peaked in U87-Bev R generation 4 (middle); and those with no trend from generation to generation of U87-Bev R (right). ( d ) Multiple secreted VEGF-independent pro-angiogenic factors were upregulated across U87-Bev R generations by qPCR. ( e ) HUVEC cells on matrigel were exposed to CM from U87-Bev S and U87-Bev R of multiple generations in the absence and presence of bevacizumab. U87-Bev R CM drove increased endothelial segment and mesh formation compared to U87-Bev S conditioned media (P=0.0007-0.01). While bevacizumab blocked U87-Bev S effects on HUVEC cells (P=0.0004), by generation nine, bevacizumab failed to block the increased U87-Bev R effects on HUVEC cells (P > 0.05). *, p
    Figure Legend Snippet: Increased but ineffective angiogenic gene expression in a multi-generational xenograft model of bevacizumab resistance. ( a ) The top 30 upregulated genes from the significantly enriched angiogenesis gene ontology were selected for validation by qPCR. ( b ) qPCR validation of 29 of the top 30 angiogenesis genes revealed significant overexpression of angiogenesis related genes by generation 1 of U87-Bev R , peaking by generation 4. ( c ) Dot plots separating the PCR results for the 29 pro-angiogenic genes into 3 categories: those that were upregulated with each U87-Bev R generation, peaking at generation 9 (left); those that peaked in U87-Bev R generation 4 (middle); and those with no trend from generation to generation of U87-Bev R (right). ( d ) Multiple secreted VEGF-independent pro-angiogenic factors were upregulated across U87-Bev R generations by qPCR. ( e ) HUVEC cells on matrigel were exposed to CM from U87-Bev S and U87-Bev R of multiple generations in the absence and presence of bevacizumab. U87-Bev R CM drove increased endothelial segment and mesh formation compared to U87-Bev S conditioned media (P=0.0007-0.01). While bevacizumab blocked U87-Bev S effects on HUVEC cells (P=0.0004), by generation nine, bevacizumab failed to block the increased U87-Bev R effects on HUVEC cells (P > 0.05). *, p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Over Expression, Polymerase Chain Reaction, Blocking Assay

    7) Product Images from "VE-statin/egfl7 regulates vascular elastogenesis by interacting with lysyl oxidases"

    Article Title: VE-statin/egfl7 regulates vascular elastogenesis by interacting with lysyl oxidases

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2008.103

    Endogenous VE-statin/egfl7 secreted by HUVEC endothelial cells colocalizes with LoxL2. ( A ) Endogenous VE-statin/egfl7 is detected mainly in insoluble extracts from HUVEC cells (soluble compared with urea extractions) as evidenced by western blot. Anti-VE-statin/egfl7 antibody specificity is established by complete signal loss in cell extracts from HUVEC cells transfected with siRNA targeting VE-statin/egfl7 messenger. ns, nonspecific signal. ( B ) Endogenous VE-statin/egfl7 is detected by immunofluorescence in HUVEC cells cultured to high density but not in normal human dermal fibroblasts from neonates (NHDF). ( C ) Mature elastic fibres are undetectable in HUVEC cells but are actively deposited by NHDF in similar culture conditions. ( D ) RT–PCR analysis of endogenous VE-statin/egfl7, elastin and lysyl oxidases mRNA levels in HUVEC and NHDF cells cultured at confluence. ( E ) Colocalization of endogenous VE-statin/egfl7 (top) and LoxL2 (middle) in HUVEC cells by immunofluorescence. Bottom: merged image of VE-statin/egfl7 and LoxL2.
    Figure Legend Snippet: Endogenous VE-statin/egfl7 secreted by HUVEC endothelial cells colocalizes with LoxL2. ( A ) Endogenous VE-statin/egfl7 is detected mainly in insoluble extracts from HUVEC cells (soluble compared with urea extractions) as evidenced by western blot. Anti-VE-statin/egfl7 antibody specificity is established by complete signal loss in cell extracts from HUVEC cells transfected with siRNA targeting VE-statin/egfl7 messenger. ns, nonspecific signal. ( B ) Endogenous VE-statin/egfl7 is detected by immunofluorescence in HUVEC cells cultured to high density but not in normal human dermal fibroblasts from neonates (NHDF). ( C ) Mature elastic fibres are undetectable in HUVEC cells but are actively deposited by NHDF in similar culture conditions. ( D ) RT–PCR analysis of endogenous VE-statin/egfl7, elastin and lysyl oxidases mRNA levels in HUVEC and NHDF cells cultured at confluence. ( E ) Colocalization of endogenous VE-statin/egfl7 (top) and LoxL2 (middle) in HUVEC cells by immunofluorescence. Bottom: merged image of VE-statin/egfl7 and LoxL2.

    Techniques Used: Western Blot, Transfection, Immunofluorescence, Cell Culture, Reverse Transcription Polymerase Chain Reaction

    8) Product Images from "Anti-angiogenic SPARC peptides inhibit progression of neuroblastoma tumors"

    Article Title: Anti-angiogenic SPARC peptides inhibit progression of neuroblastoma tumors

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-9-138

    SPARC peptides FSEN and FSEC inhibit endothelial cell migration . HUVEC cells were treated with serial dilutions of SPARC peptides with or without 10 ng/ml bFGF in a modified Boyden chamber. Relative stimulation was calculated as percentage of bFGF-induced migration. (A) Peptide FSEN inhibited bFGF-stimulated endothelial migration with an EC 50 of ~2 nM. (B) Peptide FSEC displayed a strong dose-dependent inhibition of bFGF-stimulated endothelial migration with an EC 50 ~1 pM. Dark circles represent bFGF-stimulated migration; light circles represent basal migration in the absence of an activator.
    Figure Legend Snippet: SPARC peptides FSEN and FSEC inhibit endothelial cell migration . HUVEC cells were treated with serial dilutions of SPARC peptides with or without 10 ng/ml bFGF in a modified Boyden chamber. Relative stimulation was calculated as percentage of bFGF-induced migration. (A) Peptide FSEN inhibited bFGF-stimulated endothelial migration with an EC 50 of ~2 nM. (B) Peptide FSEC displayed a strong dose-dependent inhibition of bFGF-stimulated endothelial migration with an EC 50 ~1 pM. Dark circles represent bFGF-stimulated migration; light circles represent basal migration in the absence of an activator.

    Techniques Used: Migration, Modification, Inhibition

    9) Product Images from "Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing"

    Article Title: Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04525-w

    Laminin HBD peptides promote cell adhesion in vitro. a , b A total of 3000 cells/well human lung fibroblasts were cultured a without or b with 5 mM EDTA in FGM-2 culture media containing 1% FBS. c , d A total of 3000 cells/well HUVEC were cultured c without or d with 5 mM EDTA in EBM-2 culture media containing 100 ng/mL VEGF-A165 and 1% FBS. Cells were plated on 1 μg/mL laminin peptide pre-coated non-tissue culture-treated plates and incubated for 30 min at 37 °C. After plate washes, cell numbers were quantified using a CyQUANT assay ( n = 10, mean ± SEM). The signals obtained from BSA-coated wells are normalized to 1, and relative fold increases of cell numbers were calculated. Statistical analyses were performed using ANOVA with Tukey’s test. Kruskal–Wallis test followed by Dunn’s multiple comparison was used in b , c . * p
    Figure Legend Snippet: Laminin HBD peptides promote cell adhesion in vitro. a , b A total of 3000 cells/well human lung fibroblasts were cultured a without or b with 5 mM EDTA in FGM-2 culture media containing 1% FBS. c , d A total of 3000 cells/well HUVEC were cultured c without or d with 5 mM EDTA in EBM-2 culture media containing 100 ng/mL VEGF-A165 and 1% FBS. Cells were plated on 1 μg/mL laminin peptide pre-coated non-tissue culture-treated plates and incubated for 30 min at 37 °C. After plate washes, cell numbers were quantified using a CyQUANT assay ( n = 10, mean ± SEM). The signals obtained from BSA-coated wells are normalized to 1, and relative fold increases of cell numbers were calculated. Statistical analyses were performed using ANOVA with Tukey’s test. Kruskal–Wallis test followed by Dunn’s multiple comparison was used in b , c . * p

    Techniques Used: In Vitro, Cell Culture, Incubation, CyQUANT Assay

    10) Product Images from "Cavin-2 regulates the activity and stability of endothelial nitric-oxide synthase (eNOS) in angiogenesis"

    Article Title: Cavin-2 regulates the activity and stability of endothelial nitric-oxide synthase (eNOS) in angiogenesis

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.794743

    Validation of the specificity of Cavin-2 knockdown in HUVECs. HUVEC cells were knocked down with individual siRNAs (numbers 5, 6, 7, and 8) against Cavin-2 ( siCavin-2 ) and non-targeting control ( siControl ). A , WCLs were analyzed in a Western blot for Cavin-2 and β-Actin. Cavin-2 protein expression was severely lost in siRNA numbers 6 and 7. β-Actin serves as a loading control. B , in vitro angiogenesis assay on control and two individual Cavin-2 siRNAs (numbers 6 and 7) in HUVECs show a defective tube formation upon loss of cavin-2. C , graphs represent the quantitation of mean ± S.D. of the number of branches and the total network length in HUVECs computed using ImageJ from three independent experiments. *, indicates p
    Figure Legend Snippet: Validation of the specificity of Cavin-2 knockdown in HUVECs. HUVEC cells were knocked down with individual siRNAs (numbers 5, 6, 7, and 8) against Cavin-2 ( siCavin-2 ) and non-targeting control ( siControl ). A , WCLs were analyzed in a Western blot for Cavin-2 and β-Actin. Cavin-2 protein expression was severely lost in siRNA numbers 6 and 7. β-Actin serves as a loading control. B , in vitro angiogenesis assay on control and two individual Cavin-2 siRNAs (numbers 6 and 7) in HUVECs show a defective tube formation upon loss of cavin-2. C , graphs represent the quantitation of mean ± S.D. of the number of branches and the total network length in HUVECs computed using ImageJ from three independent experiments. *, indicates p

    Techniques Used: Western Blot, Expressing, In Vitro, Angiogenesis Assay, Quantitation Assay

    Cavin-2 is required for cell proliferation, migration, and invasion in HUVEC cells. A, Western blot analysis of the WCLs from HUVECs after knockdown of non-targeting control ( siControl ) or cavin-2 ( siCavin-2 ) using siRNAs shows a severe loss of Cavin-2 expression. β-Actin serves as a loading control. B , siControl and siCavin-2 HUVECs were analyzed for cell proliferation at the mentioned time points using MTT assay showing a defective cell proliferation of siCavin-2 HUVECs. C , control and Cavin-2 knockdown HUVEC cells were analyzed for cell migration using a wound healing assay. The cells shown are pictured post-scratch at 0 and 16 h showing a defective cell migration after Cavin-2 loss. D , areas of the wound closure were quantified at 16 h after scratch using ImageJ. E , cell migration analyzed using control and Cavin-2 knockdown HUVECs using a transwell migration chamber (8-micron pore diameter) after being incubated for 12 h show a defective cell migration of sicavin-2 HUEVCs. F , quantification of the migrated crystal violet-stained endothelial cells after 12 h of migration using ImageJ. G , Matrigel invasion of control and Cavin-2 knockdown HUVECs after 48 h using a Boyden chamber assay signify that cavin-2 loss severely affects invasive potential of HUVECs. The cells were fixed in 4% paraformaldehyde and stained with crystal violet. H , quantification of the cells invasion through the Matrigel-coated filter was performed using ImageJ. Results in the graph presented are mean ± S.D. of three independent experiments. * indicates p
    Figure Legend Snippet: Cavin-2 is required for cell proliferation, migration, and invasion in HUVEC cells. A, Western blot analysis of the WCLs from HUVECs after knockdown of non-targeting control ( siControl ) or cavin-2 ( siCavin-2 ) using siRNAs shows a severe loss of Cavin-2 expression. β-Actin serves as a loading control. B , siControl and siCavin-2 HUVECs were analyzed for cell proliferation at the mentioned time points using MTT assay showing a defective cell proliferation of siCavin-2 HUVECs. C , control and Cavin-2 knockdown HUVEC cells were analyzed for cell migration using a wound healing assay. The cells shown are pictured post-scratch at 0 and 16 h showing a defective cell migration after Cavin-2 loss. D , areas of the wound closure were quantified at 16 h after scratch using ImageJ. E , cell migration analyzed using control and Cavin-2 knockdown HUVECs using a transwell migration chamber (8-micron pore diameter) after being incubated for 12 h show a defective cell migration of sicavin-2 HUEVCs. F , quantification of the migrated crystal violet-stained endothelial cells after 12 h of migration using ImageJ. G , Matrigel invasion of control and Cavin-2 knockdown HUVECs after 48 h using a Boyden chamber assay signify that cavin-2 loss severely affects invasive potential of HUVECs. The cells were fixed in 4% paraformaldehyde and stained with crystal violet. H , quantification of the cells invasion through the Matrigel-coated filter was performed using ImageJ. Results in the graph presented are mean ± S.D. of three independent experiments. * indicates p

    Techniques Used: Migration, Western Blot, Expressing, MTT Assay, Wound Healing Assay, Incubation, Staining, Boyden Chamber Assay

    11) Product Images from "A Novel Human Cytomegalovirus Locus Modulates Cell Type-Specific Outcomes of Infection"

    Article Title: A Novel Human Cytomegalovirus Locus Modulates Cell Type-Specific Outcomes of Infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002444

    Expression and function of UL133-UL138 locus in primary endothelial cells. (A) Four endothelial cell types (HMVEC, HUVEC, HUAEC or HAEC) and one epithelial cell type (HRCE) were infected with TB40E-WT or TB40E- UL133-UL138 NULL at MOI 0.1. At 10 dpi, virus titers in lysates were determined by TCID 50 on MRC5 cells. (B) HMVEC-L cells infected with TB40E-WT, TB40E- UL133-UL138 NULL or TB40E-UL138 Stop at MOI 0.1 were harvested at indicated times post infection to analyze multi-step replication. Virus titers were determined by TCID 50 on MRC5 cells. (C) HMVEC cells infected with TB40E-WT or TB40E-UL136 myc at MOI 2.0 were harvested at 2 and 5 days post infection (dpi) and lysates prepared from equal numbers of cells were analyzed by immunoblotting using either mouse anti-myc antibody to detect pUL136 or rabbit polyclonal antibodies directed against each UL133-UL138 locus protein.
    Figure Legend Snippet: Expression and function of UL133-UL138 locus in primary endothelial cells. (A) Four endothelial cell types (HMVEC, HUVEC, HUAEC or HAEC) and one epithelial cell type (HRCE) were infected with TB40E-WT or TB40E- UL133-UL138 NULL at MOI 0.1. At 10 dpi, virus titers in lysates were determined by TCID 50 on MRC5 cells. (B) HMVEC-L cells infected with TB40E-WT, TB40E- UL133-UL138 NULL or TB40E-UL138 Stop at MOI 0.1 were harvested at indicated times post infection to analyze multi-step replication. Virus titers were determined by TCID 50 on MRC5 cells. (C) HMVEC cells infected with TB40E-WT or TB40E-UL136 myc at MOI 2.0 were harvested at 2 and 5 days post infection (dpi) and lysates prepared from equal numbers of cells were analyzed by immunoblotting using either mouse anti-myc antibody to detect pUL136 or rabbit polyclonal antibodies directed against each UL133-UL138 locus protein.

    Techniques Used: Expressing, Infection

    12) Product Images from "Identification and characterization of highly versatile peptide-vectors that bind non-competitively to the low-density lipoprotein receptor for in vivo targeting and delivery of small molecules and protein cargos"

    Article Title: Identification and characterization of highly versatile peptide-vectors that bind non-competitively to the low-density lipoprotein receptor for in vivo targeting and delivery of small molecules and protein cargos

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0191052

    In vitro validation of VH411 phage binding. (A) Representative epifluorescence photomicrographs of CHO-hTfR-EGFP and CHO-hLDLR-EGFP cells (green) incubated with 1.10 10 VH411 fd phage immunodetected with an anti-PVIII antibody diluted at 1/3000 (red). Cell nuclei are labeled with Hoechst#33258 at 0.5μg/mL(blue). Co-labeling appears in yellow/orange in the merged pictures. fd phage do not bind on cells that express the hTfR-EGFP. (B) LDLR expression and binding of phage with affinity for LDLR in the absence or presence of LDL at 0.5 mg/mL was evaluated by FACS on HUVEC for 20 min at 4°C in order to avoid endocytosis. In the present example, the cells were incubated with the anti-PVIII antibody (1/1000) detected with an allophycocyanine (APC) secondary antibody (1/800) (vertical axis) (B1, B3, B4, B5, B6) and with the anti-LDLR antibody (1/50) detected with a secondary FITC-labeled antibody (1/800) (horizontal axis) (B2, B3, B4, B5, B6). Phage VH549 (linear) and VH411 (cyclic) were added to cells in the absence (B3, B5 respectively) or presence of LDL (B4, B6 respectively). The number of positive cells was standardized with 5,000 events for each test. The results were expressed in arbitrary units of fluorescence. LDL strongly decreases the binding of VH549 but has no impact on VH411 binding. (C) Quantification of the fluorescence signal in zone Q2 of the graphs in B. No shift is measured in Q2 for the VH411 phage in the presence of LDL while a 56% reduction in Q2 signal is measured for VH549 phage with LDL indicating competition for the LDL binding site. Statistical analysis was performed using an analysis of variance, followed by Student’s test. Values represent the mean of 3 independent experiments; ***p ≤ 0.001.
    Figure Legend Snippet: In vitro validation of VH411 phage binding. (A) Representative epifluorescence photomicrographs of CHO-hTfR-EGFP and CHO-hLDLR-EGFP cells (green) incubated with 1.10 10 VH411 fd phage immunodetected with an anti-PVIII antibody diluted at 1/3000 (red). Cell nuclei are labeled with Hoechst#33258 at 0.5μg/mL(blue). Co-labeling appears in yellow/orange in the merged pictures. fd phage do not bind on cells that express the hTfR-EGFP. (B) LDLR expression and binding of phage with affinity for LDLR in the absence or presence of LDL at 0.5 mg/mL was evaluated by FACS on HUVEC for 20 min at 4°C in order to avoid endocytosis. In the present example, the cells were incubated with the anti-PVIII antibody (1/1000) detected with an allophycocyanine (APC) secondary antibody (1/800) (vertical axis) (B1, B3, B4, B5, B6) and with the anti-LDLR antibody (1/50) detected with a secondary FITC-labeled antibody (1/800) (horizontal axis) (B2, B3, B4, B5, B6). Phage VH549 (linear) and VH411 (cyclic) were added to cells in the absence (B3, B5 respectively) or presence of LDL (B4, B6 respectively). The number of positive cells was standardized with 5,000 events for each test. The results were expressed in arbitrary units of fluorescence. LDL strongly decreases the binding of VH549 but has no impact on VH411 binding. (C) Quantification of the fluorescence signal in zone Q2 of the graphs in B. No shift is measured in Q2 for the VH411 phage in the presence of LDL while a 56% reduction in Q2 signal is measured for VH549 phage with LDL indicating competition for the LDL binding site. Statistical analysis was performed using an analysis of variance, followed by Student’s test. Values represent the mean of 3 independent experiments; ***p ≤ 0.001.

    Techniques Used: In Vitro, Binding Assay, Incubation, Labeling, Expressing, FACS, Fluorescence

    13) Product Images from "Regulation of TLR7/9 responses in plasmacytoid dendritic cells by BST2 and ILT7 receptor interaction"

    Article Title: Regulation of TLR7/9 responses in plasmacytoid dendritic cells by BST2 and ILT7 receptor interaction

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20090547

    Analysis of BST2 expression and potential BST2–ILT7 cis-interaction. (A) HEK293, NHDF, HUVEC, and HaCat cells, cultured in the absence or presence of 500 units/ml of IFN-α for 48 h, were stained with anti-BST2 mAb 26F8. Staining with isotype-matched control Ig is shown in the gray shaded area. Data are representative of three independent experiments. (B) Fresh isolated pDCs from peripheral blood or cells activated with various stimuli for 48 h were analyzed for surface BST2 expression by flow cytometry. Data are representative of two independent experiments. (C) Human Burkitt’s lymphoma Namalwa cells transduced with different pDC receptor complexes, i.e., BDCA2/FcϵRIγ or ILT7/FcϵRIγ, were analyzed for surface BST2 and ILT7 expression. Data are representative of three independent experiments.
    Figure Legend Snippet: Analysis of BST2 expression and potential BST2–ILT7 cis-interaction. (A) HEK293, NHDF, HUVEC, and HaCat cells, cultured in the absence or presence of 500 units/ml of IFN-α for 48 h, were stained with anti-BST2 mAb 26F8. Staining with isotype-matched control Ig is shown in the gray shaded area. Data are representative of three independent experiments. (B) Fresh isolated pDCs from peripheral blood or cells activated with various stimuli for 48 h were analyzed for surface BST2 expression by flow cytometry. Data are representative of two independent experiments. (C) Human Burkitt’s lymphoma Namalwa cells transduced with different pDC receptor complexes, i.e., BDCA2/FcϵRIγ or ILT7/FcϵRIγ, were analyzed for surface BST2 and ILT7 expression. Data are representative of three independent experiments.

    Techniques Used: Expressing, Cell Culture, Staining, Isolation, Flow Cytometry, Transduction

    14) Product Images from "Identification and characterization of highly versatile peptide-vectors that bind non-competitively to the low-density lipoprotein receptor for in vivo targeting and delivery of small molecules and protein cargos"

    Article Title: Identification and characterization of highly versatile peptide-vectors that bind non-competitively to the low-density lipoprotein receptor for in vivo targeting and delivery of small molecules and protein cargos

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0191052

    In vitro validation of VH411 phage binding. (A) Representative epifluorescence photomicrographs of CHO-hTfR-EGFP and CHO-hLDLR-EGFP cells (green) incubated with 1.10 10 VH411 fd phage immunodetected with an anti-PVIII antibody diluted at 1/3000 (red). Cell nuclei are labeled with Hoechst#33258 at 0.5μg/mL(blue). Co-labeling appears in yellow/orange in the merged pictures. fd phage do not bind on cells that express the hTfR-EGFP. (B) LDLR expression and binding of phage with affinity for LDLR in the absence or presence of LDL at 0.5 mg/mL was evaluated by FACS on HUVEC for 20 min at 4°C in order to avoid endocytosis. In the present example, the cells were incubated with the anti-PVIII antibody (1/1000) detected with an allophycocyanine (APC) secondary antibody (1/800) (vertical axis) (B1, B3, B4, B5, B6) and with the anti-LDLR antibody (1/50) detected with a secondary FITC-labeled antibody (1/800) (horizontal axis) (B2, B3, B4, B5, B6). Phage VH549 (linear) and VH411 (cyclic) were added to cells in the absence (B3, B5 respectively) or presence of LDL (B4, B6 respectively). The number of positive cells was standardized with 5,000 events for each test. The results were expressed in arbitrary units of fluorescence. LDL strongly decreases the binding of VH549 but has no impact on VH411 binding. (C) Quantification of the fluorescence signal in zone Q2 of the graphs in B. No shift is measured in Q2 for the VH411 phage in the presence of LDL while a 56% reduction in Q2 signal is measured for VH549 phage with LDL indicating competition for the LDL binding site. Statistical analysis was performed using an analysis of variance, followed by Student’s test. Values represent the mean of 3 independent experiments; ***p ≤ 0.001.
    Figure Legend Snippet: In vitro validation of VH411 phage binding. (A) Representative epifluorescence photomicrographs of CHO-hTfR-EGFP and CHO-hLDLR-EGFP cells (green) incubated with 1.10 10 VH411 fd phage immunodetected with an anti-PVIII antibody diluted at 1/3000 (red). Cell nuclei are labeled with Hoechst#33258 at 0.5μg/mL(blue). Co-labeling appears in yellow/orange in the merged pictures. fd phage do not bind on cells that express the hTfR-EGFP. (B) LDLR expression and binding of phage with affinity for LDLR in the absence or presence of LDL at 0.5 mg/mL was evaluated by FACS on HUVEC for 20 min at 4°C in order to avoid endocytosis. In the present example, the cells were incubated with the anti-PVIII antibody (1/1000) detected with an allophycocyanine (APC) secondary antibody (1/800) (vertical axis) (B1, B3, B4, B5, B6) and with the anti-LDLR antibody (1/50) detected with a secondary FITC-labeled antibody (1/800) (horizontal axis) (B2, B3, B4, B5, B6). Phage VH549 (linear) and VH411 (cyclic) were added to cells in the absence (B3, B5 respectively) or presence of LDL (B4, B6 respectively). The number of positive cells was standardized with 5,000 events for each test. The results were expressed in arbitrary units of fluorescence. LDL strongly decreases the binding of VH549 but has no impact on VH411 binding. (C) Quantification of the fluorescence signal in zone Q2 of the graphs in B. No shift is measured in Q2 for the VH411 phage in the presence of LDL while a 56% reduction in Q2 signal is measured for VH549 phage with LDL indicating competition for the LDL binding site. Statistical analysis was performed using an analysis of variance, followed by Student’s test. Values represent the mean of 3 independent experiments; ***p ≤ 0.001.

    Techniques Used: In Vitro, Binding Assay, Incubation, Labeling, Expressing, FACS, Fluorescence

    15) Product Images from "Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing"

    Article Title: Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04525-w

    Laminin HBD peptides promote cell adhesion in vitro. a , b A total of 3000 cells/well human lung fibroblasts were cultured a without or b with 5 mM EDTA in FGM-2 culture media containing 1% FBS. c , d A total of 3000 cells/well HUVEC were cultured c without or d with 5 mM EDTA in EBM-2 culture media containing 100 ng/mL VEGF-A165 and 1% FBS. Cells were plated on 1 μg/mL laminin peptide pre-coated non-tissue culture-treated plates and incubated for 30 min at 37 °C. After plate washes, cell numbers were quantified using a CyQUANT assay ( n = 10, mean ± SEM). The signals obtained from BSA-coated wells are normalized to 1, and relative fold increases of cell numbers were calculated. Statistical analyses were performed using ANOVA with Tukey’s test. Kruskal–Wallis test followed by Dunn’s multiple comparison was used in b , c . * p
    Figure Legend Snippet: Laminin HBD peptides promote cell adhesion in vitro. a , b A total of 3000 cells/well human lung fibroblasts were cultured a without or b with 5 mM EDTA in FGM-2 culture media containing 1% FBS. c , d A total of 3000 cells/well HUVEC were cultured c without or d with 5 mM EDTA in EBM-2 culture media containing 100 ng/mL VEGF-A165 and 1% FBS. Cells were plated on 1 μg/mL laminin peptide pre-coated non-tissue culture-treated plates and incubated for 30 min at 37 °C. After plate washes, cell numbers were quantified using a CyQUANT assay ( n = 10, mean ± SEM). The signals obtained from BSA-coated wells are normalized to 1, and relative fold increases of cell numbers were calculated. Statistical analyses were performed using ANOVA with Tukey’s test. Kruskal–Wallis test followed by Dunn’s multiple comparison was used in b , c . * p

    Techniques Used: In Vitro, Cell Culture, Incubation, CyQUANT Assay

    16) Product Images from "Identification and characterization of highly versatile peptide-vectors that bind non-competitively to the low-density lipoprotein receptor for in vivo targeting and delivery of small molecules and protein cargos"

    Article Title: Identification and characterization of highly versatile peptide-vectors that bind non-competitively to the low-density lipoprotein receptor for in vivo targeting and delivery of small molecules and protein cargos

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0191052

    In vitro validation of VH411 phage binding. (A) Representative epifluorescence photomicrographs of CHO-hTfR-EGFP and CHO-hLDLR-EGFP cells (green) incubated with 1.10 10 VH411 fd phage immunodetected with an anti-PVIII antibody diluted at 1/3000 (red). Cell nuclei are labeled with Hoechst#33258 at 0.5μg/mL(blue). Co-labeling appears in yellow/orange in the merged pictures. fd phage do not bind on cells that express the hTfR-EGFP. (B) LDLR expression and binding of phage with affinity for LDLR in the absence or presence of LDL at 0.5 mg/mL was evaluated by FACS on HUVEC for 20 min at 4°C in order to avoid endocytosis. In the present example, the cells were incubated with the anti-PVIII antibody (1/1000) detected with an allophycocyanine (APC) secondary antibody (1/800) (vertical axis) (B1, B3, B4, B5, B6) and with the anti-LDLR antibody (1/50) detected with a secondary FITC-labeled antibody (1/800) (horizontal axis) (B2, B3, B4, B5, B6). Phage VH549 (linear) and VH411 (cyclic) were added to cells in the absence (B3, B5 respectively) or presence of LDL (B4, B6 respectively). The number of positive cells was standardized with 5,000 events for each test. The results were expressed in arbitrary units of fluorescence. LDL strongly decreases the binding of VH549 but has no impact on VH411 binding. (C) Quantification of the fluorescence signal in zone Q2 of the graphs in B. No shift is measured in Q2 for the VH411 phage in the presence of LDL while a 56% reduction in Q2 signal is measured for VH549 phage with LDL indicating competition for the LDL binding site. Statistical analysis was performed using an analysis of variance, followed by Student’s test. Values represent the mean of 3 independent experiments; ***p ≤ 0.001.
    Figure Legend Snippet: In vitro validation of VH411 phage binding. (A) Representative epifluorescence photomicrographs of CHO-hTfR-EGFP and CHO-hLDLR-EGFP cells (green) incubated with 1.10 10 VH411 fd phage immunodetected with an anti-PVIII antibody diluted at 1/3000 (red). Cell nuclei are labeled with Hoechst#33258 at 0.5μg/mL(blue). Co-labeling appears in yellow/orange in the merged pictures. fd phage do not bind on cells that express the hTfR-EGFP. (B) LDLR expression and binding of phage with affinity for LDLR in the absence or presence of LDL at 0.5 mg/mL was evaluated by FACS on HUVEC for 20 min at 4°C in order to avoid endocytosis. In the present example, the cells were incubated with the anti-PVIII antibody (1/1000) detected with an allophycocyanine (APC) secondary antibody (1/800) (vertical axis) (B1, B3, B4, B5, B6) and with the anti-LDLR antibody (1/50) detected with a secondary FITC-labeled antibody (1/800) (horizontal axis) (B2, B3, B4, B5, B6). Phage VH549 (linear) and VH411 (cyclic) were added to cells in the absence (B3, B5 respectively) or presence of LDL (B4, B6 respectively). The number of positive cells was standardized with 5,000 events for each test. The results were expressed in arbitrary units of fluorescence. LDL strongly decreases the binding of VH549 but has no impact on VH411 binding. (C) Quantification of the fluorescence signal in zone Q2 of the graphs in B. No shift is measured in Q2 for the VH411 phage in the presence of LDL while a 56% reduction in Q2 signal is measured for VH549 phage with LDL indicating competition for the LDL binding site. Statistical analysis was performed using an analysis of variance, followed by Student’s test. Values represent the mean of 3 independent experiments; ***p ≤ 0.001.

    Techniques Used: In Vitro, Binding Assay, Incubation, Labeling, Expressing, FACS, Fluorescence

    17) Product Images from "Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling"

    Article Title: Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201703206

    The GPCR downstream effector SNAP23 regulates MVB–PM fusion. (a) Network of proteins of interest together with direct interactors with altered phosphorylation levels upon histamine (100 µM) stimulation as identified by phosphoproteomics in HeLa and HUVEC cells. Proteins of interest are depicted with a blue rim. FC, fold change. (b) Graph showing the signal intensity values of phosphorylated peptides from proteins of interest before and after stimulation with 100 µM histamine. Data represent means ± SD of two technical replicates per condition. *, P
    Figure Legend Snippet: The GPCR downstream effector SNAP23 regulates MVB–PM fusion. (a) Network of proteins of interest together with direct interactors with altered phosphorylation levels upon histamine (100 µM) stimulation as identified by phosphoproteomics in HeLa and HUVEC cells. Proteins of interest are depicted with a blue rim. FC, fold change. (b) Graph showing the signal intensity values of phosphorylated peptides from proteins of interest before and after stimulation with 100 µM histamine. Data represent means ± SD of two technical replicates per condition. *, P

    Techniques Used:

    GPCR activation triggers MVB–PM fusion in single cells in a calcium-independent manner. (a) Schematic model of imaging setup. (b) Fusion activity of HeLa cells stimulated with KCl (70 mM), caffeine (20 mM), or histamine (100 µM). n ≥ 8 cells per condition. (c) Total projection of fusion events over a 60-s time course onto cells before (top) and after (bottom) stimulation with histamine (100 µM). Pseudocolored as in Fig. 1 j . (d) Measurement of individual HeLa cells ( n = 14) before and during stimulation with histamine (100 µM). (e) Mean fusion kinetics of CD63-pHluorin HeLa cells ( n = 6) showing the distribution of fusion events over time (dark blue line; SD is in light blue) and the calcium levels (red) during histamine stimulation (gray-shaded block). (f) Heat maps revealing calcium responses (measured by Fluo-4) upon histamine stimulation obtained by subtracting the fluorescent intensity values before stimulation from those after 8-s stimulation. Cells were nontreated or incubated with a buffer with fast (BAPTA) or slow (EGTA) calcium-binding kinetics. Bars, 10 µm. (g) Quantification of fusion activity of histamine-stimulated HeLa cells in the presence of EGTA (top) or BAPTA (bottom) buffers. n ≥ 10 cells per condition. (h) Measurement of individual HUVEC cells ( n = 30) before and after stimulation with histamine (100 µM). *, P
    Figure Legend Snippet: GPCR activation triggers MVB–PM fusion in single cells in a calcium-independent manner. (a) Schematic model of imaging setup. (b) Fusion activity of HeLa cells stimulated with KCl (70 mM), caffeine (20 mM), or histamine (100 µM). n ≥ 8 cells per condition. (c) Total projection of fusion events over a 60-s time course onto cells before (top) and after (bottom) stimulation with histamine (100 µM). Pseudocolored as in Fig. 1 j . (d) Measurement of individual HeLa cells ( n = 14) before and during stimulation with histamine (100 µM). (e) Mean fusion kinetics of CD63-pHluorin HeLa cells ( n = 6) showing the distribution of fusion events over time (dark blue line; SD is in light blue) and the calcium levels (red) during histamine stimulation (gray-shaded block). (f) Heat maps revealing calcium responses (measured by Fluo-4) upon histamine stimulation obtained by subtracting the fluorescent intensity values before stimulation from those after 8-s stimulation. Cells were nontreated or incubated with a buffer with fast (BAPTA) or slow (EGTA) calcium-binding kinetics. Bars, 10 µm. (g) Quantification of fusion activity of histamine-stimulated HeLa cells in the presence of EGTA (top) or BAPTA (bottom) buffers. n ≥ 10 cells per condition. (h) Measurement of individual HUVEC cells ( n = 30) before and after stimulation with histamine (100 µM). *, P

    Techniques Used: Activation Assay, Imaging, Activity Assay, Blocking Assay, Incubation, Binding Assay

    18) Product Images from "Cellular nanoscale stiffness patterns governed by intracellular forces"

    Article Title: Cellular nanoscale stiffness patterns governed by intracellular forces

    Journal: Nature materials

    doi: 10.1038/s41563-019-0391-7

    Imaging nanoscale stiffness of cells. a, Force vs. distance curves acquired while scanning a living fibroblast show low imaging forces and depth of indentation. Inset shows illustration of a T-shaped cantilever probing an adherent cell. b-f , Effective elastic modulus maps acquired from the periphery of living melanoma ( b ), HUVEC ( c ), fibroblast ( d,f ), and CHO cells ( e ). Effective elastic modulus values in logarithmic scale are color-coded and superimposed to the surface topography. The elastic modulus profile (inset) across the dashed line in ( f ) shows clearly resolved features separated by 30 nm, which might be single actin filaments, due to their diameter and linearity. Color bars are 9 to 140 kPa ( b ), 0.3 to 2.8 MPa ( c ), 12 to 370 kPa ( d ), 74 to 738 kPa ( e ), and 28 to 880 kPa ( f ).
    Figure Legend Snippet: Imaging nanoscale stiffness of cells. a, Force vs. distance curves acquired while scanning a living fibroblast show low imaging forces and depth of indentation. Inset shows illustration of a T-shaped cantilever probing an adherent cell. b-f , Effective elastic modulus maps acquired from the periphery of living melanoma ( b ), HUVEC ( c ), fibroblast ( d,f ), and CHO cells ( e ). Effective elastic modulus values in logarithmic scale are color-coded and superimposed to the surface topography. The elastic modulus profile (inset) across the dashed line in ( f ) shows clearly resolved features separated by 30 nm, which might be single actin filaments, due to their diameter and linearity. Color bars are 9 to 140 kPa ( b ), 0.3 to 2.8 MPa ( c ), 12 to 370 kPa ( d ), 74 to 738 kPa ( e ), and 28 to 880 kPa ( f ).

    Techniques Used: Imaging

    19) Product Images from "Anti-Cancer Effects of YAP Inhibitor (CA3) in Combination with Sorafenib against Hepatocellular Carcinoma (HCC) in Patient-Derived Multicellular Tumor Spheroid Models (MCTS)"

    Article Title: Anti-Cancer Effects of YAP Inhibitor (CA3) in Combination with Sorafenib against Hepatocellular Carcinoma (HCC) in Patient-Derived Multicellular Tumor Spheroid Models (MCTS)

    Journal: Cancers

    doi: 10.3390/cancers14112733

    Establishment of MCTS model using patient-derived HCCs and stromal cells. ( a ) Hematoxylin and eosin staining of MCTS; ( b ) Immunohistochemical analysis of α-SMA (marker for hepatic stellate cells, LX2), fibronectin (marker for fibroblasts, WI38), and CD34 (marker for endothelial cells, HUVEC) of consecutive sections of the MCTS model generated from patient-derived primary HCC cells cocultured with human stromal cells (LX2, WI38, and HUVEC). Representative images taken under a microscope (×200). Scale bar, 50 µm.
    Figure Legend Snippet: Establishment of MCTS model using patient-derived HCCs and stromal cells. ( a ) Hematoxylin and eosin staining of MCTS; ( b ) Immunohistochemical analysis of α-SMA (marker for hepatic stellate cells, LX2), fibronectin (marker for fibroblasts, WI38), and CD34 (marker for endothelial cells, HUVEC) of consecutive sections of the MCTS model generated from patient-derived primary HCC cells cocultured with human stromal cells (LX2, WI38, and HUVEC). Representative images taken under a microscope (×200). Scale bar, 50 µm.

    Techniques Used: Derivative Assay, Staining, Immunohistochemistry, Marker, Generated, Microscopy

    20) Product Images from "Molecular Cytogenetics Reveals Mosaicism in Human Umbilical Vein Endothelial Cells"

    Article Title: Molecular Cytogenetics Reveals Mosaicism in Human Umbilical Vein Endothelial Cells

    Journal: Genes

    doi: 10.3390/genes13061012

    Composite karyogram of HUVEC cells at the 425-band level of resolution. Chromosomes 1–22 showed no consistent variation of banding pattern, while several cells displayed abnormal morphology in the terminal region of the long arm of chromosome X (Xq Ab as indicated by arrow) and the rest of the cells exhibit normal Xq region (Xq N). Images were captured under 100× magnification.
    Figure Legend Snippet: Composite karyogram of HUVEC cells at the 425-band level of resolution. Chromosomes 1–22 showed no consistent variation of banding pattern, while several cells displayed abnormal morphology in the terminal region of the long arm of chromosome X (Xq Ab as indicated by arrow) and the rest of the cells exhibit normal Xq region (Xq N). Images were captured under 100× magnification.

    Techniques Used:

    21) Product Images from "Differential responses of MET activations to MET kinase inhibitor and neutralizing antibody"

    Article Title: Differential responses of MET activations to MET kinase inhibitor and neutralizing antibody

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-018-1628-y

    INC280 and MetMab inhibit HGF induced tube formation in HUVEC cells. a Representative images of HUVEC cell tube formation treated with either INC280 (1 µM) or MetMab (1 µM). HUVEC cells were cultured in a 24-well plate coated with 50 μl Matrigel for the treatments as indicated. Triplicates were used for each treatment. After 16 h, cells were imaged by phase-contrast microscopy. Five images were taken randomly from each well. Tube formation before and after treatment are shown in upper and lower panel, respectively. b Quantification of tube-like structures as described above in a . The number of tubes is calculated as mean ± SD of 15 images that are measured by Image J. Fold change refers to the average number of tubes of each treatment group as compared to HGF stimulation alone group. Vertical bar refers to standard deviation. *Compared with the HGF stimulation control group, inhibition was found to be statistically significant (student t test, p
    Figure Legend Snippet: INC280 and MetMab inhibit HGF induced tube formation in HUVEC cells. a Representative images of HUVEC cell tube formation treated with either INC280 (1 µM) or MetMab (1 µM). HUVEC cells were cultured in a 24-well plate coated with 50 μl Matrigel for the treatments as indicated. Triplicates were used for each treatment. After 16 h, cells were imaged by phase-contrast microscopy. Five images were taken randomly from each well. Tube formation before and after treatment are shown in upper and lower panel, respectively. b Quantification of tube-like structures as described above in a . The number of tubes is calculated as mean ± SD of 15 images that are measured by Image J. Fold change refers to the average number of tubes of each treatment group as compared to HGF stimulation alone group. Vertical bar refers to standard deviation. *Compared with the HGF stimulation control group, inhibition was found to be statistically significant (student t test, p

    Techniques Used: Cell Culture, Microscopy, Standard Deviation, Inhibition

    22) Product Images from "Chronic Exposure to HIV-Derived Protein Tat Impairs Endothelial Function via Indirect Alteration in Fat Mass and Nox1-Mediated Mechanisms in Mice"

    Article Title: Chronic Exposure to HIV-Derived Protein Tat Impairs Endothelial Function via Indirect Alteration in Fat Mass and Nox1-Mediated Mechanisms in Mice

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms222010977

    Tat does not have direct effect on endothelial function. Body weight ( A ), subcutaneous fat depot (SQF/BW; B ), visceral fat depot (VAT/BW; C ), concentration response curves (CRC) to acetylcholine (Ach; D ) and sodium nitroprusside (SNP; E ) in aortic rings from control (vehicle-treated) and TAT-treated mice (TAT, 3.2 µg/kg per day for 3 days, i.p.). CRC to acetylcholine (Ach; F ) and sodium nitroprusside (SNP; G ) in the presence of vehicle or Tat protein (20 ng/mL) in aortic rings from control mice. Gene expression of Nox1 ( H ) in vehicle (control) and Tat-treated (20 ng/mL for 24 h) HAEC and HUVEC cells. Data are presented as mean ± SEM. n = 5–6.
    Figure Legend Snippet: Tat does not have direct effect on endothelial function. Body weight ( A ), subcutaneous fat depot (SQF/BW; B ), visceral fat depot (VAT/BW; C ), concentration response curves (CRC) to acetylcholine (Ach; D ) and sodium nitroprusside (SNP; E ) in aortic rings from control (vehicle-treated) and TAT-treated mice (TAT, 3.2 µg/kg per day for 3 days, i.p.). CRC to acetylcholine (Ach; F ) and sodium nitroprusside (SNP; G ) in the presence of vehicle or Tat protein (20 ng/mL) in aortic rings from control mice. Gene expression of Nox1 ( H ) in vehicle (control) and Tat-treated (20 ng/mL for 24 h) HAEC and HUVEC cells. Data are presented as mean ± SEM. n = 5–6.

    Techniques Used: Concentration Assay, Mouse Assay, Expressing

    23) Product Images from "Direct phosphorylation and stabilization of HIF-1α by PIM1 kinase drives angiogenesis in solid tumors"

    Article Title: Direct phosphorylation and stabilization of HIF-1α by PIM1 kinase drives angiogenesis in solid tumors

    Journal: bioRxiv

    doi: 10.1101/2021.03.17.435865

    PIM1 induces angiogenesis in vivo and in vitro. A) Dox-PIM1 PC3 cells were transfected with siHIF-1/2α prior to treatment with dox for 24 h, and lysates were collected for immunoblotting and conditioned media (CM) was harvested for in vitro angiogenesis assay. B) Representative images of tube formation at 1 and 6 hours after plating HUVEC cells in CM. C) Quantification of mean tube length and D) total branch points as measured with Image J Angiogenesis Analyzer plug-in. E) VEGF-A levels in CM from the indicated conditions were measured by ELISA. F) Twelve mice (n=3/group) were injected with the indicated RKO cell lines, and tumor volume was measured over time. G) Mice were injected with 2 nmol of Angiosense 750EX 24 hours prior to imaging for fluorescence intensity. Vascular index was calculated by normalizing the bioluminescence signal to tumor volume. H) Tumors derived from each cell line were harvested and immunostained with CD31, PIM1, HIF-1α, and CC3. I) Quantification of IHC. Scale bar = 50 μm. *p
    Figure Legend Snippet: PIM1 induces angiogenesis in vivo and in vitro. A) Dox-PIM1 PC3 cells were transfected with siHIF-1/2α prior to treatment with dox for 24 h, and lysates were collected for immunoblotting and conditioned media (CM) was harvested for in vitro angiogenesis assay. B) Representative images of tube formation at 1 and 6 hours after plating HUVEC cells in CM. C) Quantification of mean tube length and D) total branch points as measured with Image J Angiogenesis Analyzer plug-in. E) VEGF-A levels in CM from the indicated conditions were measured by ELISA. F) Twelve mice (n=3/group) were injected with the indicated RKO cell lines, and tumor volume was measured over time. G) Mice were injected with 2 nmol of Angiosense 750EX 24 hours prior to imaging for fluorescence intensity. Vascular index was calculated by normalizing the bioluminescence signal to tumor volume. H) Tumors derived from each cell line were harvested and immunostained with CD31, PIM1, HIF-1α, and CC3. I) Quantification of IHC. Scale bar = 50 μm. *p

    Techniques Used: In Vivo, In Vitro, Transfection, Angiogenesis Assay, Enzyme-linked Immunosorbent Assay, Mouse Assay, Injection, Imaging, Fluorescence, Derivative Assay, Immunohistochemistry

    24) Product Images from "Hemodynamic forces tune the arrest, adhesion and extravasation of circulating tumor cells"

    Article Title: Hemodynamic forces tune the arrest, adhesion and extravasation of circulating tumor cells

    Journal: bioRxiv

    doi: 10.1101/183046

    Laminar flow stimulates the protrusive and remodeling activity of endothelial cells (A) Experimental workflow and representative image of transfected HUVECs. Representative time projection images of pCAG-EGFP expressing HUVECs in flow and no flow conditions, and quantification of the number of protrusion over time. (B) SEM images and quantification showing the number of protrusion in flow vs no flow condition (16h treatment). (C) 100-nm fluorescent beads perfusion over arrested tumor cells in the microfluidic channels. Single particle tracking analysis from high-speed confocal acquisition at different height (Z-step) (right) and quantifications (left). (D) Immunostaining and SEM representative images of TCs (LifeAct, red) arrested on a monolayer of HUVECs (PECAM, green) is shown, in two flow conditions (0 and 400/sec). (E) Quantification of the number of transmigrated TCs and of transmigrated TCs with remodeled HUVECs.
    Figure Legend Snippet: Laminar flow stimulates the protrusive and remodeling activity of endothelial cells (A) Experimental workflow and representative image of transfected HUVECs. Representative time projection images of pCAG-EGFP expressing HUVECs in flow and no flow conditions, and quantification of the number of protrusion over time. (B) SEM images and quantification showing the number of protrusion in flow vs no flow condition (16h treatment). (C) 100-nm fluorescent beads perfusion over arrested tumor cells in the microfluidic channels. Single particle tracking analysis from high-speed confocal acquisition at different height (Z-step) (right) and quantifications (left). (D) Immunostaining and SEM representative images of TCs (LifeAct, red) arrested on a monolayer of HUVECs (PECAM, green) is shown, in two flow conditions (0 and 400/sec). (E) Quantification of the number of transmigrated TCs and of transmigrated TCs with remodeled HUVECs.

    Techniques Used: Activity Assay, Transfection, Expressing, Single-particle Tracking, Immunostaining

    25) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus Glycoproteins B and K8.1 Regulate Virion Egress and Synthesis of Vascular Endothelial Growth Factor and Viral Interleukin-6 in BCBL-1 Cells ▿"

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Glycoproteins B and K8.1 Regulate Virion Egress and Synthesis of Vascular Endothelial Growth Factor and Viral Interleukin-6 in BCBL-1 Cells ▿

    Journal:

    doi: 10.1128/JVI.01889-09

    Effect of K8.1 and gB inhibition on HUVEC microtubule formation. (A) Photomicrographs of HUVEC monolayers treated with conditioned medium obtained from BCBL-1 cells. BCBL-1 cell supernatants were collected as described in Materials and Methods. HUVECs
    Figure Legend Snippet: Effect of K8.1 and gB inhibition on HUVEC microtubule formation. (A) Photomicrographs of HUVEC monolayers treated with conditioned medium obtained from BCBL-1 cells. BCBL-1 cell supernatants were collected as described in Materials and Methods. HUVECs

    Techniques Used: Inhibition

    Effect of K8.1 and gB inhibition on HUVEC migration. (A) Photomicrographs of endothelial cell migration toward conditioned medium obtained from BCBL-1 cells. Conditioned medium was collected from BCBL-1 cells treated with either siRNAs or anti-gB and
    Figure Legend Snippet: Effect of K8.1 and gB inhibition on HUVEC migration. (A) Photomicrographs of endothelial cell migration toward conditioned medium obtained from BCBL-1 cells. Conditioned medium was collected from BCBL-1 cells treated with either siRNAs or anti-gB and

    Techniques Used: Inhibition, Migration

    26) Product Images from "Direct phosphorylation and stabilization of HIF-1α by PIM1 kinase drives angiogenesis in solid tumors"

    Article Title: Direct phosphorylation and stabilization of HIF-1α by PIM1 kinase drives angiogenesis in solid tumors

    Journal: bioRxiv

    doi: 10.1101/2021.03.17.435865

    PIM1 induces angiogenesis in vivo and in vitro. A) Dox-PIM1 PC3 cells were transfected with siHIF-1/2α prior to treatment with dox for 24 h, and lysates were collected for immunoblotting and conditioned media (CM) was harvested for in vitro angiogenesis assay. B) Representative images of tube formation at 1 and 6 hours after plating HUVEC cells in CM. C) Quantification of mean tube length and D) total branch points as measured with Image J Angiogenesis Analyzer plug-in. E) VEGF-A levels in CM from the indicated conditions were measured by ELISA. F) Twelve mice (n=3/group) were injected with the indicated RKO cell lines, and tumor volume was measured over time. G) Mice were injected with 2 nmol of Angiosense 750EX 24 hours prior to imaging for fluorescence intensity. Vascular index was calculated by normalizing the bioluminescence signal to tumor volume. H) Tumors derived from each cell line were harvested and immunostained with CD31, PIM1, HIF-1α, and CC3. I) Quantification of IHC. Scale bar = 50 μm. *p
    Figure Legend Snippet: PIM1 induces angiogenesis in vivo and in vitro. A) Dox-PIM1 PC3 cells were transfected with siHIF-1/2α prior to treatment with dox for 24 h, and lysates were collected for immunoblotting and conditioned media (CM) was harvested for in vitro angiogenesis assay. B) Representative images of tube formation at 1 and 6 hours after plating HUVEC cells in CM. C) Quantification of mean tube length and D) total branch points as measured with Image J Angiogenesis Analyzer plug-in. E) VEGF-A levels in CM from the indicated conditions were measured by ELISA. F) Twelve mice (n=3/group) were injected with the indicated RKO cell lines, and tumor volume was measured over time. G) Mice were injected with 2 nmol of Angiosense 750EX 24 hours prior to imaging for fluorescence intensity. Vascular index was calculated by normalizing the bioluminescence signal to tumor volume. H) Tumors derived from each cell line were harvested and immunostained with CD31, PIM1, HIF-1α, and CC3. I) Quantification of IHC. Scale bar = 50 μm. *p

    Techniques Used: In Vivo, In Vitro, Transfection, Angiogenesis Assay, Enzyme-linked Immunosorbent Assay, Mouse Assay, Injection, Imaging, Fluorescence, Derivative Assay, Immunohistochemistry

    27) Product Images from "An Autoimmune Basis for Raynaud’s Phenomenon: Murine Model and Human Disease"

    Article Title: An Autoimmune Basis for Raynaud’s Phenomenon: Murine Model and Human Disease

    Journal: Arthritis & rheumatology (Hoboken, N.J.)

    doi: 10.1002/art.40505

    K10 as a cold-inducible target of Raynaud’s-associated antibodies. Cultured cells were kept at 37°C in complete medium (see Methods). Where indicated for cold exposure, cells were exposed to 4°C medium for 1 minute 24 hours before cell harvesting, then returned to 37°C. A . Anti-endothelial activity by immunofluorescence of HUVEC (see Methods) with an RNP+ serum from a Raynaud’s patient. B . Reverse immunophenotyping immunoprecipitation (see Methods) of HUVEC lysates with an anti-RNP+ human Raynaud’s Phenomenon serum identified a ~56kD band (arrow) that was not pre-cleared by an RNP+ serum from a patient without Raynaud’s Phenomenon (RP-), for MSMS analysis. C . Expression and cold-induced upregulation of mRNA levels by real time PCR were quantitated for K10 and control cytokeratin 18 (K18), normalized to expression of 18S ribosomal RNA. The endothelial cell line HUVEC expressed much higher levels of K10 message than epithelial HEK293 cells or lymphoid Jurkat cells at 37°C, and further dramatically upregulated K10 but not K18 expression levels after cold exposure. D . Expression and upregulation of K10 protein in HUVEC. While low levels of K10 protein were present in HUVEC cells at 37°C, the quantity of protein dramatically increased in cold-exposed cells (compared to GAPDH loading control).
    Figure Legend Snippet: K10 as a cold-inducible target of Raynaud’s-associated antibodies. Cultured cells were kept at 37°C in complete medium (see Methods). Where indicated for cold exposure, cells were exposed to 4°C medium for 1 minute 24 hours before cell harvesting, then returned to 37°C. A . Anti-endothelial activity by immunofluorescence of HUVEC (see Methods) with an RNP+ serum from a Raynaud’s patient. B . Reverse immunophenotyping immunoprecipitation (see Methods) of HUVEC lysates with an anti-RNP+ human Raynaud’s Phenomenon serum identified a ~56kD band (arrow) that was not pre-cleared by an RNP+ serum from a patient without Raynaud’s Phenomenon (RP-), for MSMS analysis. C . Expression and cold-induced upregulation of mRNA levels by real time PCR were quantitated for K10 and control cytokeratin 18 (K18), normalized to expression of 18S ribosomal RNA. The endothelial cell line HUVEC expressed much higher levels of K10 message than epithelial HEK293 cells or lymphoid Jurkat cells at 37°C, and further dramatically upregulated K10 but not K18 expression levels after cold exposure. D . Expression and upregulation of K10 protein in HUVEC. While low levels of K10 protein were present in HUVEC cells at 37°C, the quantity of protein dramatically increased in cold-exposed cells (compared to GAPDH loading control).

    Techniques Used: Cell Culture, Cell Harvesting, Activity Assay, Immunofluorescence, Immunoprecipitation, Expressing, Real-time Polymerase Chain Reaction

    28) Product Images from "Adipose-derived mesenchymal stem cells (ASCs) may favour breast cancer recurrence via HGF/c-Met signaling"

    Article Title: Adipose-derived mesenchymal stem cells (ASCs) may favour breast cancer recurrence via HGF/c-Met signaling

    Journal: Oncotarget

    doi:

    ASCs sustain tumor angiogenesis A) ASCs migratory activity. B) Angiogenic potential of co-cultured ASCs. HUVEC cells as control. Evaluation of VEGFR2, CD31 expression and CD34+ subpopulation in co-cultured ASCs versus ASCs grown alone. C) GFP expression in ASC1 trans-infected cells. D) Murine CD31 (red) in xenografts from co-transplanted MDA-MB-231 and GFP-expressing ASC1 (green). Nuclei counterstained with DAPI. D) VEGFR expression in breast cancer cells co-cultured versus grown alone.
    Figure Legend Snippet: ASCs sustain tumor angiogenesis A) ASCs migratory activity. B) Angiogenic potential of co-cultured ASCs. HUVEC cells as control. Evaluation of VEGFR2, CD31 expression and CD34+ subpopulation in co-cultured ASCs versus ASCs grown alone. C) GFP expression in ASC1 trans-infected cells. D) Murine CD31 (red) in xenografts from co-transplanted MDA-MB-231 and GFP-expressing ASC1 (green). Nuclei counterstained with DAPI. D) VEGFR expression in breast cancer cells co-cultured versus grown alone.

    Techniques Used: Activity Assay, Cell Culture, Expressing, Infection, Multiple Displacement Amplification

    29) Product Images from "Engineered natural killer cells impede the immunometabolic CD73-adenosine axis in solid tumors"

    Article Title: Engineered natural killer cells impede the immunometabolic CD73-adenosine axis in solid tumors

    Journal: eLife

    doi: 10.7554/eLife.73699

    CD73.CAR NK cells rescue NK cell dysfunction under hypoxia. ( A ) Cytotoxicity of human primary NK cells against lung adenocarcinoma targets under normoxic and chemically-induced hypoxic conditions. ( B ) Cytotoxicity of CD73.mRNACAR-NK cells and untransfected primary human NK cells against lung adenocarcinoma targets under chemically-induced hypoxic conditions. ( C ) Killing of lung adenocarcinoma cells by CD73.mRNACAR-NK cells and anti-CD73 antibody (7G2) for two individual donors. ( D ) Killing of HUVEC cells by CD73.mRNACAR-NK cells and primary human NK cells for three individual donors. ( E ) Production of adenosine by lung adenocarcinoma cells after treatment with CD73.CAR-NK cells and untransduced primary human NK cells for three individual donors. Unless otherwise stated, data are representative of three donors. *p
    Figure Legend Snippet: CD73.CAR NK cells rescue NK cell dysfunction under hypoxia. ( A ) Cytotoxicity of human primary NK cells against lung adenocarcinoma targets under normoxic and chemically-induced hypoxic conditions. ( B ) Cytotoxicity of CD73.mRNACAR-NK cells and untransfected primary human NK cells against lung adenocarcinoma targets under chemically-induced hypoxic conditions. ( C ) Killing of lung adenocarcinoma cells by CD73.mRNACAR-NK cells and anti-CD73 antibody (7G2) for two individual donors. ( D ) Killing of HUVEC cells by CD73.mRNACAR-NK cells and primary human NK cells for three individual donors. ( E ) Production of adenosine by lung adenocarcinoma cells after treatment with CD73.CAR-NK cells and untransduced primary human NK cells for three individual donors. Unless otherwise stated, data are representative of three donors. *p

    Techniques Used:

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    Lonza cell lines huvec
    Performance of CEC detection and identification of microvascular cells. The graph in (A) shows the recovery rate of <t>HUVEC</t> and <t>L‐HMVEC</t> spiked in a healthy whole blood sample at 100, 1000 and 10 000 cells mL −1 . The graph in (B) shows the percentage of CD36 positive cells detected on HMVEC and HUVEC at increasing spiking concentrations as in (A). The results are expressed as mean ± SD of duplicate quantification. CECs, circulating endothelial cells
    Cell Lines Huvec, supplied by Lonza, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Lonza human umbilical vein endothelial cells huvec
    Cell viability levels of <t>HUVEC</t> after exposure to proteinoid NPs, measured by <t>XTT</t> assay. Cells (3 × 10 5 ) were incubated for 48 h with proteinoid NPs dispersed in PBS (1 mg/mL) according to the experimental section. Untreated cells (positive control) were similarly incubated, as well as free doxorubicin, (100 nmol/ml, negative control). Each bar represents mean ± standard deviations of six separate samples.
    Human Umbilical Vein Endothelial Cells Huvec, supplied by Lonza, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    Depletion of ERRα increases angiogenesis in HUVEC. (A) Quantification of DAPI-stained nuclei in control and ERRα knockdown HUVEC grown in EGM2. ***, P

    Journal: Molecular and Cellular Biology

    Article Title: Loss of Estrogen-Related Receptor Alpha Facilitates Angiogenesis in Endothelial Cells

    doi: 10.1128/MCB.00411-18

    Figure Lengend Snippet: Depletion of ERRα increases angiogenesis in HUVEC. (A) Quantification of DAPI-stained nuclei in control and ERRα knockdown HUVEC grown in EGM2. ***, P

    Article Snippet: HUVEC (Lonza) were grown in EGM2 medium (PromoCell) in a humidified incubator at 37°C and 5% CO2 .

    Techniques: Staining

    Gene expression comparison of iECs to primary endothelial cells. (A) PCA relating gene expression profiles of vehicle-treated fibroblasts, blue squares; iPSCs, black triangles; HAEC, orange circles; HCAEC, yellow circles; HUVEC, red circles; and two iEC lines, green diamonds. Three biological replicates represent each cell line. (B) Scatter plot comparing baseline gene expression profiles of the mean expression level for the three primary cell beds and iEC lines. Squares represent genes that deviate by 3 standard deviations from the least squares regression line (gray).

    Journal: Chemical research in toxicology

    Article Title: A Stem Cell-Derived Endothelial Cell Model that Responds to Tobacco Smoke Like Primary Endothelial Cells

    doi: 10.1021/acs.chemrestox.9b00363

    Figure Lengend Snippet: Gene expression comparison of iECs to primary endothelial cells. (A) PCA relating gene expression profiles of vehicle-treated fibroblasts, blue squares; iPSCs, black triangles; HAEC, orange circles; HCAEC, yellow circles; HUVEC, red circles; and two iEC lines, green diamonds. Three biological replicates represent each cell line. (B) Scatter plot comparing baseline gene expression profiles of the mean expression level for the three primary cell beds and iEC lines. Squares represent genes that deviate by 3 standard deviations from the least squares regression line (gray).

    Article Snippet: Primary endothelial cells (human aorta (HAEC), coronary artery (HCAEC), and umbilicus (HUVEC)) were purchased from Lonza and cultured in EGM™-2.

    Techniques: Expressing

    Genes expressed selectively in EC from human aorta, coronary artery, or umbilicus. RNAseq data are shown for genes expressed at ten-fold higher counts in HAEC (orange) or HUVEC (red), or twenty-fold higher in HCAEC (yellow) relative to the average of the other two EC beds. For each gene the median number of transcripts per 22 million is plotted +/− the standard error for each EC bed. Zero transcript counts were plotted as 1 transcript to fit the logarithmic scale.

    Journal: Chemical research in toxicology

    Article Title: A Stem Cell-Derived Endothelial Cell Model that Responds to Tobacco Smoke Like Primary Endothelial Cells

    doi: 10.1021/acs.chemrestox.9b00363

    Figure Lengend Snippet: Genes expressed selectively in EC from human aorta, coronary artery, or umbilicus. RNAseq data are shown for genes expressed at ten-fold higher counts in HAEC (orange) or HUVEC (red), or twenty-fold higher in HCAEC (yellow) relative to the average of the other two EC beds. For each gene the median number of transcripts per 22 million is plotted +/− the standard error for each EC bed. Zero transcript counts were plotted as 1 transcript to fit the logarithmic scale.

    Article Snippet: Primary endothelial cells (human aorta (HAEC), coronary artery (HCAEC), and umbilicus (HUVEC)) were purchased from Lonza and cultured in EGM™-2.

    Techniques: