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human dermal blood microvascular endothelial cells  (Cell Applications Inc)


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    Structured Review

    Cell Applications Inc human dermal blood microvascular endothelial cells
    a shPIM3 or control (shScr) silenced human umbilical vein <t>endothelial</t> cells (HUVECs) and dermal <t>microvascular</t> endothelial cells <t>(BECs)</t> were stained for vascular endothelial cadherin (CDH5) and F-actin. Nuclei were stained using DAPI. Relative CDH5 signal intensity ( b ) and area ( c ) (normalized to number of nuclei) in HUVEC ( n = 3 independent experiments for shScr, n = 6 independent experiments for shPIM3) and in BEC ( n = 3 for shScr, n = 5 for shPIM3). Western blot ( d ) and quantification ( e ) of CDH5 in HUVECs treated as in ( a ). n = 3 independent experiments for shScr, n = 6 for shPIM3. f shPIM3 or shScr silenced BECs were stained for α- and β-catenin (CTNNA1 and CTNNB1) and HUVECs for δ-catenin (CTNND1) and F-actin. Nuclei were stained using DAPI. Relative α-catenin ( g , h ), β-catenin ( i , j ) and δ-catenin ( k , l ) signal intensities (per field) and area (normalized to number of nuclei) relative to control (shScr). n = 3 for shScr, n = 6 for shPIM3 ( g – j ). n = 3 for shScr, n = 5 for shPIM3 ( k , l ). m Schematic representation of CDH5 and α-, β- and δ-catenin in adherens junctions created in Biorender.com. Two-tailed unpaired t -test ( b , c , e , g – l ). Data are presented as mean values + /- SD. Data is pooled from independent experiments using two shPIM3 clones in b , c , e , g – l . Scale bars 50 μm ( a , f ); 25 μm in close-up images ( a , f ). Source data are provided as a Source Data file.
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    Images

    1) Product Images from "Endothelial Pim3 kinase protects the vascular barrier during lung metastasis"

    Article Title: Endothelial Pim3 kinase protects the vascular barrier during lung metastasis

    Journal: Nature Communications

    doi: 10.1038/s41467-024-54445-1

    a shPIM3 or control (shScr) silenced human umbilical vein endothelial cells (HUVECs) and dermal microvascular endothelial cells (BECs) were stained for vascular endothelial cadherin (CDH5) and F-actin. Nuclei were stained using DAPI. Relative CDH5 signal intensity ( b ) and area ( c ) (normalized to number of nuclei) in HUVEC ( n = 3 independent experiments for shScr, n = 6 independent experiments for shPIM3) and in BEC ( n = 3 for shScr, n = 5 for shPIM3). Western blot ( d ) and quantification ( e ) of CDH5 in HUVECs treated as in ( a ). n = 3 independent experiments for shScr, n = 6 for shPIM3. f shPIM3 or shScr silenced BECs were stained for α- and β-catenin (CTNNA1 and CTNNB1) and HUVECs for δ-catenin (CTNND1) and F-actin. Nuclei were stained using DAPI. Relative α-catenin ( g , h ), β-catenin ( i , j ) and δ-catenin ( k , l ) signal intensities (per field) and area (normalized to number of nuclei) relative to control (shScr). n = 3 for shScr, n = 6 for shPIM3 ( g – j ). n = 3 for shScr, n = 5 for shPIM3 ( k , l ). m Schematic representation of CDH5 and α-, β- and δ-catenin in adherens junctions created in Biorender.com. Two-tailed unpaired t -test ( b , c , e , g – l ). Data are presented as mean values + /- SD. Data is pooled from independent experiments using two shPIM3 clones in b , c , e , g – l . Scale bars 50 μm ( a , f ); 25 μm in close-up images ( a , f ). Source data are provided as a Source Data file.
    Figure Legend Snippet: a shPIM3 or control (shScr) silenced human umbilical vein endothelial cells (HUVECs) and dermal microvascular endothelial cells (BECs) were stained for vascular endothelial cadherin (CDH5) and F-actin. Nuclei were stained using DAPI. Relative CDH5 signal intensity ( b ) and area ( c ) (normalized to number of nuclei) in HUVEC ( n = 3 independent experiments for shScr, n = 6 independent experiments for shPIM3) and in BEC ( n = 3 for shScr, n = 5 for shPIM3). Western blot ( d ) and quantification ( e ) of CDH5 in HUVECs treated as in ( a ). n = 3 independent experiments for shScr, n = 6 for shPIM3. f shPIM3 or shScr silenced BECs were stained for α- and β-catenin (CTNNA1 and CTNNB1) and HUVECs for δ-catenin (CTNND1) and F-actin. Nuclei were stained using DAPI. Relative α-catenin ( g , h ), β-catenin ( i , j ) and δ-catenin ( k , l ) signal intensities (per field) and area (normalized to number of nuclei) relative to control (shScr). n = 3 for shScr, n = 6 for shPIM3 ( g – j ). n = 3 for shScr, n = 5 for shPIM3 ( k , l ). m Schematic representation of CDH5 and α-, β- and δ-catenin in adherens junctions created in Biorender.com. Two-tailed unpaired t -test ( b , c , e , g – l ). Data are presented as mean values + /- SD. Data is pooled from independent experiments using two shPIM3 clones in b , c , e , g – l . Scale bars 50 μm ( a , f ); 25 μm in close-up images ( a , f ). Source data are provided as a Source Data file.

    Techniques Used: Control, Staining, Western Blot, Two Tailed Test, Clone Assay

    Control or AZD-1208 treated human umbilical vein endothelial cells (HUVEC) ( a ) and human dermal microvascular blood endothelial cells (BEC) ( b ) were analyzed using ECIS electrical cell impedance sensing. Arrows indicate initiation of serum starvation (arrow 1) and initiation of treatment (arrow 2). Shown are representative experiments with triplicate samples with SEM. Significant differences based on three independent experiments ( n = 3, Ctrl vs AZD-1208 at indicated times after treatment initiation) for HUVEC at 24 h 1 µM ( p = 0.0372), 10 µM ( p = 0.0026), at 48 h 1 µM ( p = 0.0456), 10 µM ( p < 0.0001) and at 72 h 10 µM ( p = 0.0053) and for BEC at 48 h 1 µM ( p = 0.0086), 10 µM ( p = 0.0012). c Representative images of HUVEC and BEC treated with AZD-1208 (1 μM) or 0.1% DMSO (Ctrl) for 24 h in reduced 2.5% serum, and stained for CDH5, F-actin and nuclei (DAPI). Relative CDH5 signal intensity (per field) ( d ) and area (normalized to number of nuclei) ( e ). n = 3 independent experiments. f , g CDH5 Western blot and quantification of HUVEC treated as in ( c ). n = 3 independent experiments. h AZD-1208 (30 mg kg −1 ) or vehicle (Ctrl) was orally administered daily for 5 days. CDH5 and collagen IV (Col IV) were stained in thick lung sections. Shown are maximum intensity projections of confocal z-stacks. i Magnification of maximum intensity projection of CDH5 stained lung sections (top) with surface masking (below). Arrows indicate gaps in CDH5 staining. Quantification of CDH5 intensity ( j ) and area ( k ), and number of gaps in CDH5 staining ( l ) as explained in materials and methods. n = 4 independent experiments. Mixed-effects analysis ( a ) and two-way ANOVA ( b ) both with multiple comparisons, two-tailed unpaired t -test ( d , e , g , j , l ), two-sided Mann-Whitney U test ( k ). Data are presented as mean values + /- SD ( d , e , g , j – l ,) or + /- SEM ( a , b ). Scale bars 50 µm ( c ); 25 µm ( h , close-up images in c ) and 10 µm ( i ). Source data are provided as a Source Data file.
    Figure Legend Snippet: Control or AZD-1208 treated human umbilical vein endothelial cells (HUVEC) ( a ) and human dermal microvascular blood endothelial cells (BEC) ( b ) were analyzed using ECIS electrical cell impedance sensing. Arrows indicate initiation of serum starvation (arrow 1) and initiation of treatment (arrow 2). Shown are representative experiments with triplicate samples with SEM. Significant differences based on three independent experiments ( n = 3, Ctrl vs AZD-1208 at indicated times after treatment initiation) for HUVEC at 24 h 1 µM ( p = 0.0372), 10 µM ( p = 0.0026), at 48 h 1 µM ( p = 0.0456), 10 µM ( p < 0.0001) and at 72 h 10 µM ( p = 0.0053) and for BEC at 48 h 1 µM ( p = 0.0086), 10 µM ( p = 0.0012). c Representative images of HUVEC and BEC treated with AZD-1208 (1 μM) or 0.1% DMSO (Ctrl) for 24 h in reduced 2.5% serum, and stained for CDH5, F-actin and nuclei (DAPI). Relative CDH5 signal intensity (per field) ( d ) and area (normalized to number of nuclei) ( e ). n = 3 independent experiments. f , g CDH5 Western blot and quantification of HUVEC treated as in ( c ). n = 3 independent experiments. h AZD-1208 (30 mg kg −1 ) or vehicle (Ctrl) was orally administered daily for 5 days. CDH5 and collagen IV (Col IV) were stained in thick lung sections. Shown are maximum intensity projections of confocal z-stacks. i Magnification of maximum intensity projection of CDH5 stained lung sections (top) with surface masking (below). Arrows indicate gaps in CDH5 staining. Quantification of CDH5 intensity ( j ) and area ( k ), and number of gaps in CDH5 staining ( l ) as explained in materials and methods. n = 4 independent experiments. Mixed-effects analysis ( a ) and two-way ANOVA ( b ) both with multiple comparisons, two-tailed unpaired t -test ( d , e , g , j , l ), two-sided Mann-Whitney U test ( k ). Data are presented as mean values + /- SD ( d , e , g , j – l ,) or + /- SEM ( a , b ). Scale bars 50 µm ( c ); 25 µm ( h , close-up images in c ) and 10 µm ( i ). Source data are provided as a Source Data file.

    Techniques Used: Control, Staining, Western Blot, Two Tailed Test, MANN-WHITNEY



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    a. HMVBECs were used to test the induction of arterial and Notch genes under the conditions of static (ST), ST with VEGF (20ng/ml), FSS (12dyn/cm 2 ) and FSS with VEGF. Gene expression was examined by quantitative RT-PCR followed by quantification relative to ST. b. FSS-induced expression of GJA4, GJA5 and Hey1 in HMVBECs with a gradient of VEGF. Gene expression was examined by quantitative RT-PCR followed by quantification relative to ST. FSS experiments were conducted using Vitroflo system. All conditions under FSS were in the same Vitroflo plate. c. HUVECs treated with siRNA were used to test FSS-induced expression of arterial genes and the effect of VEGF. d. A working model demonstrating VEGF signals through VEGFR2-PLCγ1 to counteract FSS-induced arterial specification. Student t-test was used for statistics. p<0.05 was determined as statistically significant. ns: not significant.

    Journal: bioRxiv

    Article Title: VEGF counteracts shear stress-determined arterial fate specification during capillary remodeling

    doi: 10.1101/2024.01.23.576920

    Figure Lengend Snippet: a. HMVBECs were used to test the induction of arterial and Notch genes under the conditions of static (ST), ST with VEGF (20ng/ml), FSS (12dyn/cm 2 ) and FSS with VEGF. Gene expression was examined by quantitative RT-PCR followed by quantification relative to ST. b. FSS-induced expression of GJA4, GJA5 and Hey1 in HMVBECs with a gradient of VEGF. Gene expression was examined by quantitative RT-PCR followed by quantification relative to ST. FSS experiments were conducted using Vitroflo system. All conditions under FSS were in the same Vitroflo plate. c. HUVECs treated with siRNA were used to test FSS-induced expression of arterial genes and the effect of VEGF. d. A working model demonstrating VEGF signals through VEGFR2-PLCγ1 to counteract FSS-induced arterial specification. Student t-test was used for statistics. p<0.05 was determined as statistically significant. ns: not significant.

    Article Snippet: Human microvascular blood endothelial cells (HMVBECs) (HDBECs, Promocell, #C-12211) were maintained in EGM2-MV full growth medium (Lonza).

    Techniques: Expressing, Quantitative RT-PCR

    a. HMVBECs were subjected to VEGF (20ng/ml) or FSS (12 dyn/cm 2 ) to test the activation of Notch signaling in a time course, with DMSO or γ-secretase inhibitor DAPT treatments. b. HMVBECs treated with siCTRL or siDll4 were subjected to VEGF and FSS to test the activation of Notch signaling. c-d. Working models demonstrating the mechanisms by which VEGF and FSS use to activate Notch signaling. VEGF activates the expression of Dll4, which consequently serves as a ligand to activate Notch signaling in a neighboring cell. By contrast, FSS does not rely on increasing Dll4 expression to activate Notch. Increase of Dll4 expression is a result rather than a cause of FSS-induced Notch activation. e. FSS-induced Notch signaling and expression of arterial genes. HMVBECs were subjected to FSS in a time course in the absence or presence of VEGF.

    Journal: bioRxiv

    Article Title: VEGF counteracts shear stress-determined arterial fate specification during capillary remodeling

    doi: 10.1101/2024.01.23.576920

    Figure Lengend Snippet: a. HMVBECs were subjected to VEGF (20ng/ml) or FSS (12 dyn/cm 2 ) to test the activation of Notch signaling in a time course, with DMSO or γ-secretase inhibitor DAPT treatments. b. HMVBECs treated with siCTRL or siDll4 were subjected to VEGF and FSS to test the activation of Notch signaling. c-d. Working models demonstrating the mechanisms by which VEGF and FSS use to activate Notch signaling. VEGF activates the expression of Dll4, which consequently serves as a ligand to activate Notch signaling in a neighboring cell. By contrast, FSS does not rely on increasing Dll4 expression to activate Notch. Increase of Dll4 expression is a result rather than a cause of FSS-induced Notch activation. e. FSS-induced Notch signaling and expression of arterial genes. HMVBECs were subjected to FSS in a time course in the absence or presence of VEGF.

    Article Snippet: Human microvascular blood endothelial cells (HMVBECs) (HDBECs, Promocell, #C-12211) were maintained in EGM2-MV full growth medium (Lonza).

    Techniques: Activation Assay, Expressing

    a. Pretreated with DMSO or 5uM DAPT for 3h, HMVBECs were subjected to FSS to induce arterial differentiation for 16h. Samples were analyzed by western blot. b. Representative retinal artery and branching arterioles at p5 from the Notch reporter mouse line CBF:H2B-Venus. Arterial ECs were labeled by immunostaining of Sox17 and colocalized with endogenous Venus signal in the nuclei. Arrow heads indicate Sox17 high /CBF-arterial ECs. Scale bars: 25 µm. c. In the background of DAPT-induced Notch inhibition (P1-3), arterial morphogenesis in P4 retina from control and PLCg1 iECKO mice. White arrow heads indicate arterial morphogenesis marked by Sox17 high ECs; yellow arrow heads indicate smooth muscle coverage. Abbreviations: A: artery; V: vein. Scale bars: 100 µm.

    Journal: bioRxiv

    Article Title: VEGF counteracts shear stress-determined arterial fate specification during capillary remodeling

    doi: 10.1101/2024.01.23.576920

    Figure Lengend Snippet: a. Pretreated with DMSO or 5uM DAPT for 3h, HMVBECs were subjected to FSS to induce arterial differentiation for 16h. Samples were analyzed by western blot. b. Representative retinal artery and branching arterioles at p5 from the Notch reporter mouse line CBF:H2B-Venus. Arterial ECs were labeled by immunostaining of Sox17 and colocalized with endogenous Venus signal in the nuclei. Arrow heads indicate Sox17 high /CBF-arterial ECs. Scale bars: 25 µm. c. In the background of DAPT-induced Notch inhibition (P1-3), arterial morphogenesis in P4 retina from control and PLCg1 iECKO mice. White arrow heads indicate arterial morphogenesis marked by Sox17 high ECs; yellow arrow heads indicate smooth muscle coverage. Abbreviations: A: artery; V: vein. Scale bars: 100 µm.

    Article Snippet: Human microvascular blood endothelial cells (HMVBECs) (HDBECs, Promocell, #C-12211) were maintained in EGM2-MV full growth medium (Lonza).

    Techniques: Western Blot, Labeling, Immunostaining, Inhibition, Control

    a shPIM3 or control (shScr) silenced human umbilical vein endothelial cells (HUVECs) and dermal microvascular endothelial cells (BECs) were stained for vascular endothelial cadherin (CDH5) and F-actin. Nuclei were stained using DAPI. Relative CDH5 signal intensity ( b ) and area ( c ) (normalized to number of nuclei) in HUVEC ( n = 3 independent experiments for shScr, n = 6 independent experiments for shPIM3) and in BEC ( n = 3 for shScr, n = 5 for shPIM3). Western blot ( d ) and quantification ( e ) of CDH5 in HUVECs treated as in ( a ). n = 3 independent experiments for shScr, n = 6 for shPIM3. f shPIM3 or shScr silenced BECs were stained for α- and β-catenin (CTNNA1 and CTNNB1) and HUVECs for δ-catenin (CTNND1) and F-actin. Nuclei were stained using DAPI. Relative α-catenin ( g , h ), β-catenin ( i , j ) and δ-catenin ( k , l ) signal intensities (per field) and area (normalized to number of nuclei) relative to control (shScr). n = 3 for shScr, n = 6 for shPIM3 ( g – j ). n = 3 for shScr, n = 5 for shPIM3 ( k , l ). m Schematic representation of CDH5 and α-, β- and δ-catenin in adherens junctions created in Biorender.com. Two-tailed unpaired t -test ( b , c , e , g – l ). Data are presented as mean values + /- SD. Data is pooled from independent experiments using two shPIM3 clones in b , c , e , g – l . Scale bars 50 μm ( a , f ); 25 μm in close-up images ( a , f ). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Endothelial Pim3 kinase protects the vascular barrier during lung metastasis

    doi: 10.1038/s41467-024-54445-1

    Figure Lengend Snippet: a shPIM3 or control (shScr) silenced human umbilical vein endothelial cells (HUVECs) and dermal microvascular endothelial cells (BECs) were stained for vascular endothelial cadherin (CDH5) and F-actin. Nuclei were stained using DAPI. Relative CDH5 signal intensity ( b ) and area ( c ) (normalized to number of nuclei) in HUVEC ( n = 3 independent experiments for shScr, n = 6 independent experiments for shPIM3) and in BEC ( n = 3 for shScr, n = 5 for shPIM3). Western blot ( d ) and quantification ( e ) of CDH5 in HUVECs treated as in ( a ). n = 3 independent experiments for shScr, n = 6 for shPIM3. f shPIM3 or shScr silenced BECs were stained for α- and β-catenin (CTNNA1 and CTNNB1) and HUVECs for δ-catenin (CTNND1) and F-actin. Nuclei were stained using DAPI. Relative α-catenin ( g , h ), β-catenin ( i , j ) and δ-catenin ( k , l ) signal intensities (per field) and area (normalized to number of nuclei) relative to control (shScr). n = 3 for shScr, n = 6 for shPIM3 ( g – j ). n = 3 for shScr, n = 5 for shPIM3 ( k , l ). m Schematic representation of CDH5 and α-, β- and δ-catenin in adherens junctions created in Biorender.com. Two-tailed unpaired t -test ( b , c , e , g – l ). Data are presented as mean values + /- SD. Data is pooled from independent experiments using two shPIM3 clones in b , c , e , g – l . Scale bars 50 μm ( a , f ); 25 μm in close-up images ( a , f ). Source data are provided as a Source Data file.

    Article Snippet: Human Umbilical Vein ECs (HUVEC) (Cell Applications, Inc., San Diego, CA, USA) and Human Dermal Blood Microvascular Endothelial Cells (a.k.a.

    Techniques: Control, Staining, Western Blot, Two Tailed Test, Clone Assay

    Control or AZD-1208 treated human umbilical vein endothelial cells (HUVEC) ( a ) and human dermal microvascular blood endothelial cells (BEC) ( b ) were analyzed using ECIS electrical cell impedance sensing. Arrows indicate initiation of serum starvation (arrow 1) and initiation of treatment (arrow 2). Shown are representative experiments with triplicate samples with SEM. Significant differences based on three independent experiments ( n = 3, Ctrl vs AZD-1208 at indicated times after treatment initiation) for HUVEC at 24 h 1 µM ( p = 0.0372), 10 µM ( p = 0.0026), at 48 h 1 µM ( p = 0.0456), 10 µM ( p < 0.0001) and at 72 h 10 µM ( p = 0.0053) and for BEC at 48 h 1 µM ( p = 0.0086), 10 µM ( p = 0.0012). c Representative images of HUVEC and BEC treated with AZD-1208 (1 μM) or 0.1% DMSO (Ctrl) for 24 h in reduced 2.5% serum, and stained for CDH5, F-actin and nuclei (DAPI). Relative CDH5 signal intensity (per field) ( d ) and area (normalized to number of nuclei) ( e ). n = 3 independent experiments. f , g CDH5 Western blot and quantification of HUVEC treated as in ( c ). n = 3 independent experiments. h AZD-1208 (30 mg kg −1 ) or vehicle (Ctrl) was orally administered daily for 5 days. CDH5 and collagen IV (Col IV) were stained in thick lung sections. Shown are maximum intensity projections of confocal z-stacks. i Magnification of maximum intensity projection of CDH5 stained lung sections (top) with surface masking (below). Arrows indicate gaps in CDH5 staining. Quantification of CDH5 intensity ( j ) and area ( k ), and number of gaps in CDH5 staining ( l ) as explained in materials and methods. n = 4 independent experiments. Mixed-effects analysis ( a ) and two-way ANOVA ( b ) both with multiple comparisons, two-tailed unpaired t -test ( d , e , g , j , l ), two-sided Mann-Whitney U test ( k ). Data are presented as mean values + /- SD ( d , e , g , j – l ,) or + /- SEM ( a , b ). Scale bars 50 µm ( c ); 25 µm ( h , close-up images in c ) and 10 µm ( i ). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Endothelial Pim3 kinase protects the vascular barrier during lung metastasis

    doi: 10.1038/s41467-024-54445-1

    Figure Lengend Snippet: Control or AZD-1208 treated human umbilical vein endothelial cells (HUVEC) ( a ) and human dermal microvascular blood endothelial cells (BEC) ( b ) were analyzed using ECIS electrical cell impedance sensing. Arrows indicate initiation of serum starvation (arrow 1) and initiation of treatment (arrow 2). Shown are representative experiments with triplicate samples with SEM. Significant differences based on three independent experiments ( n = 3, Ctrl vs AZD-1208 at indicated times after treatment initiation) for HUVEC at 24 h 1 µM ( p = 0.0372), 10 µM ( p = 0.0026), at 48 h 1 µM ( p = 0.0456), 10 µM ( p < 0.0001) and at 72 h 10 µM ( p = 0.0053) and for BEC at 48 h 1 µM ( p = 0.0086), 10 µM ( p = 0.0012). c Representative images of HUVEC and BEC treated with AZD-1208 (1 μM) or 0.1% DMSO (Ctrl) for 24 h in reduced 2.5% serum, and stained for CDH5, F-actin and nuclei (DAPI). Relative CDH5 signal intensity (per field) ( d ) and area (normalized to number of nuclei) ( e ). n = 3 independent experiments. f , g CDH5 Western blot and quantification of HUVEC treated as in ( c ). n = 3 independent experiments. h AZD-1208 (30 mg kg −1 ) or vehicle (Ctrl) was orally administered daily for 5 days. CDH5 and collagen IV (Col IV) were stained in thick lung sections. Shown are maximum intensity projections of confocal z-stacks. i Magnification of maximum intensity projection of CDH5 stained lung sections (top) with surface masking (below). Arrows indicate gaps in CDH5 staining. Quantification of CDH5 intensity ( j ) and area ( k ), and number of gaps in CDH5 staining ( l ) as explained in materials and methods. n = 4 independent experiments. Mixed-effects analysis ( a ) and two-way ANOVA ( b ) both with multiple comparisons, two-tailed unpaired t -test ( d , e , g , j , l ), two-sided Mann-Whitney U test ( k ). Data are presented as mean values + /- SD ( d , e , g , j – l ,) or + /- SEM ( a , b ). Scale bars 50 µm ( c ); 25 µm ( h , close-up images in c ) and 10 µm ( i ). Source data are provided as a Source Data file.

    Article Snippet: Human Umbilical Vein ECs (HUVEC) (Cell Applications, Inc., San Diego, CA, USA) and Human Dermal Blood Microvascular Endothelial Cells (a.k.a.

    Techniques: Control, Staining, Western Blot, Two Tailed Test, MANN-WHITNEY

    Establishment of the Experimental System. (A) . Demonstration of Dermal Microvascular Endothelial Cell (DMEC) labeling. Fluorescent image of nuclear-localized mCherry overlaid on a phase contrast image of confluent DMECs. (B) . The proliferation of DMECs in full growth medium (dotted line) is exponential (solid line is a fitted exponential curve). (C) . Cell cycle analysis of relative DNA content determined by labeling live cells with Hoescht and imaged with a microscope. Full growth medium (left) produces a familiar distribution of cells while 24 h in Basal Proliferation Medium (BPM) (right) produces far fewer cells in S- and G2/M-phases. (D) . Demonstration of YOYO-1 dye to detect dead cells. (E) .) Example of nuclei (closed circles) and dead cells (open circles) over time in BPM. (F) . Cell death in BPM is countered by inhibition of caspase activity via 10 µM Q-VD-OPh.

    Journal: Frontiers in Pharmacology

    Article Title: Identification of kinases activated by multiple pro-angiogenic growth factors

    doi: 10.3389/fphar.2022.1022722

    Figure Lengend Snippet: Establishment of the Experimental System. (A) . Demonstration of Dermal Microvascular Endothelial Cell (DMEC) labeling. Fluorescent image of nuclear-localized mCherry overlaid on a phase contrast image of confluent DMECs. (B) . The proliferation of DMECs in full growth medium (dotted line) is exponential (solid line is a fitted exponential curve). (C) . Cell cycle analysis of relative DNA content determined by labeling live cells with Hoescht and imaged with a microscope. Full growth medium (left) produces a familiar distribution of cells while 24 h in Basal Proliferation Medium (BPM) (right) produces far fewer cells in S- and G2/M-phases. (D) . Demonstration of YOYO-1 dye to detect dead cells. (E) .) Example of nuclei (closed circles) and dead cells (open circles) over time in BPM. (F) . Cell death in BPM is countered by inhibition of caspase activity via 10 µM Q-VD-OPh.

    Article Snippet: Human Dermal blood microvascular endothelial cells (DMECs) isolated from a single donor were purchased from Lonza (CC-2183).

    Techniques: Labeling, Cell Cycle Assay, Microscopy, Inhibition, Activity Assay

    Concentration-Dependent Population Dynamics of Dermal Microvascular Endothelial Cells (DMECs) in the Presence of Pro-Angiogenic Growth Factors. (A) . The number of cells ( N ) over time for four concentrations of FGF2. (B) . The number of dead cells ( M ) over time for the experiment shown in panel (A) . (C) . The proliferation rate, p , for the same experiment as in panels A,B. (D) . The birth rate, B , as a function of time. (E) . The death rate, D , as a function of time.

    Journal: Frontiers in Pharmacology

    Article Title: Identification of kinases activated by multiple pro-angiogenic growth factors

    doi: 10.3389/fphar.2022.1022722

    Figure Lengend Snippet: Concentration-Dependent Population Dynamics of Dermal Microvascular Endothelial Cells (DMECs) in the Presence of Pro-Angiogenic Growth Factors. (A) . The number of cells ( N ) over time for four concentrations of FGF2. (B) . The number of dead cells ( M ) over time for the experiment shown in panel (A) . (C) . The proliferation rate, p , for the same experiment as in panels A,B. (D) . The birth rate, B , as a function of time. (E) . The death rate, D , as a function of time.

    Article Snippet: Human Dermal blood microvascular endothelial cells (DMECs) isolated from a single donor were purchased from Lonza (CC-2183).

    Techniques: Concentration Assay

    The dose-response proliferative behavior of Dermal Microvascular Endothelial Cells (DMECs) to Three Pro-Angiogenic Growth Factors. The proliferation rate (A) , the birth rate (B) , and the death rate (C) for FGF2, VEGFA, and HGF over a range of concentrations. Note that the y -axes share the same scale to facilitate comparison of the relative magnitude of each.

    Journal: Frontiers in Pharmacology

    Article Title: Identification of kinases activated by multiple pro-angiogenic growth factors

    doi: 10.3389/fphar.2022.1022722

    Figure Lengend Snippet: The dose-response proliferative behavior of Dermal Microvascular Endothelial Cells (DMECs) to Three Pro-Angiogenic Growth Factors. The proliferation rate (A) , the birth rate (B) , and the death rate (C) for FGF2, VEGFA, and HGF over a range of concentrations. Note that the y -axes share the same scale to facilitate comparison of the relative magnitude of each.

    Article Snippet: Human Dermal blood microvascular endothelial cells (DMECs) isolated from a single donor were purchased from Lonza (CC-2183).

    Techniques:

    Identification of kinases important for the proliferation rate of Dermal Microvascular Endothelial Cells (DMECs) in each pro-angiogenic growth factor studied. (A) . Magnitudes of the Kinome Regression (KIR) coefficients (i.e., influential kinases) for each identified growth factor. (B) . Venn diagram revealing the exclusivity and commonalities in the set of kinases identified for each growth factor. (C) . The list of the intersection of kinases implicated in all three growth factors. Kinases are ordered according to a rank obtained by summing each kinase’s ranks across all growth factors.

    Journal: Frontiers in Pharmacology

    Article Title: Identification of kinases activated by multiple pro-angiogenic growth factors

    doi: 10.3389/fphar.2022.1022722

    Figure Lengend Snippet: Identification of kinases important for the proliferation rate of Dermal Microvascular Endothelial Cells (DMECs) in each pro-angiogenic growth factor studied. (A) . Magnitudes of the Kinome Regression (KIR) coefficients (i.e., influential kinases) for each identified growth factor. (B) . Venn diagram revealing the exclusivity and commonalities in the set of kinases identified for each growth factor. (C) . The list of the intersection of kinases implicated in all three growth factors. Kinases are ordered according to a rank obtained by summing each kinase’s ranks across all growth factors.

    Article Snippet: Human Dermal blood microvascular endothelial cells (DMECs) isolated from a single donor were purchased from Lonza (CC-2183).

    Techniques:

    Orthogonal evidence supporting the role of kinases identified by Kinome Regression (KIR) Analysis. (A) . Targeting growth factor receptors with 5 nM of siRNA reduces the proliferation rate of Dermal Microvascular Endothelial Cells (DMECs) in the presence of cognate growth factors while minimizing reduction in proliferation in other growth factors. (B) . Targeting kinases with siRNA largely agrees with kinases implicated by KIR. Here, the effect is defined as the average over all three siRNAs used for each kinase and normalized to the no-siRNA control. Error bars are s. e.m. The asterisks indicate growth factors context in which the siRNA targeting a given kinase produced a statistically significant result ( p < 0.05 from multiple comparison testing).

    Journal: Frontiers in Pharmacology

    Article Title: Identification of kinases activated by multiple pro-angiogenic growth factors

    doi: 10.3389/fphar.2022.1022722

    Figure Lengend Snippet: Orthogonal evidence supporting the role of kinases identified by Kinome Regression (KIR) Analysis. (A) . Targeting growth factor receptors with 5 nM of siRNA reduces the proliferation rate of Dermal Microvascular Endothelial Cells (DMECs) in the presence of cognate growth factors while minimizing reduction in proliferation in other growth factors. (B) . Targeting kinases with siRNA largely agrees with kinases implicated by KIR. Here, the effect is defined as the average over all three siRNAs used for each kinase and normalized to the no-siRNA control. Error bars are s. e.m. The asterisks indicate growth factors context in which the siRNA targeting a given kinase produced a statistically significant result ( p < 0.05 from multiple comparison testing).

    Article Snippet: Human Dermal blood microvascular endothelial cells (DMECs) isolated from a single donor were purchased from Lonza (CC-2183).

    Techniques: Produced