system ecis 1600 applied biophysics troy ny  (Applied BioPhysics)


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    Applied BioPhysics system ecis 1600 applied biophysics troy ny
    System Ecis 1600 Applied Biophysics Troy Ny, supplied by Applied BioPhysics, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ecis  (Applied BioPhysics)


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    Applied BioPhysics ecis
    Ecis, supplied by Applied BioPhysics, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96w20idf pet 96 well array ecis plates  (Applied BioPhysics)


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    Applied BioPhysics 96w20idf pet 96 well array ecis plates
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    ecis cultureware electrode arrays 8w10e  (Applied BioPhysics)


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    Applied BioPhysics ecis cultureware electrode arrays 8w10e
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    ecis software  (Applied BioPhysics)


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    Applied BioPhysics ecis software
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    ecis 1600r applied biophysics tiruppathi  (Applied BioPhysics)


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    Applied BioPhysics ecis 1600r applied biophysics tiruppathi
    (A) Confluent HUVECs were exposed or not to 1.5 μg/ml CN03 for 3 h, lysed and the indicated active GTPases were detected by pull-down assays. (B and C) Confluent HUVECs (B) or HDMVEC (C) were exposed to 1.5 μg/ml or 5 μg/ml CN03, respectively, for 3 h, fixed and stained for VE-cadherin and PECAM-1. Right images show two-fold enlargements of the squared areas highlighting the morphology of linear (top) and reticular (bottom) AJs. Bottom plots show quantification of junctional VE-cadherin and PECAM-1 levels and the percentage of reticular AJs area. Scale bars, 50 μm. (D and E) Protein expression changes of PECAM-1 and VE-cadherin in HUVECs (D) and HDMVEC (E) exposed to CN03 as in B and C. (F) CN03 induces surface localization of PECAM-1 and VE-cadherin Surface-biotinylated proteins were isolated by pull-down assay with NeutrAvidin-agarose in HUVECs exposed or not to CN03 as in A. VE-cadherin and PECAM-1 protein levels from the pull-down fraction were compared with total protein levels from the lysates by western blot analysis. Non-biotinylated cells were analyzed in parallel as a control of the pulldown assay. Immunoblots of tubulin are shown as loading controls. Plots show the mean□±□SEM from at least three independent experiments. (G) TEER analysis with an <t>ECIS</t> system of HDMVEC exposed to 5 μg/ml CN03. *, P< 0.05; **, P< 0.01; *** P<0.001.
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    1) Product Images from "Harnessing homeostatically active RhoC at cell junctions preserves human endothelial barrier function during inflammation"

    Article Title: Harnessing homeostatically active RhoC at cell junctions preserves human endothelial barrier function during inflammation

    Journal: bioRxiv

    doi: 10.1101/2024.05.17.594667

    (A) Confluent HUVECs were exposed or not to 1.5 μg/ml CN03 for 3 h, lysed and the indicated active GTPases were detected by pull-down assays. (B and C) Confluent HUVECs (B) or HDMVEC (C) were exposed to 1.5 μg/ml or 5 μg/ml CN03, respectively, for 3 h, fixed and stained for VE-cadherin and PECAM-1. Right images show two-fold enlargements of the squared areas highlighting the morphology of linear (top) and reticular (bottom) AJs. Bottom plots show quantification of junctional VE-cadherin and PECAM-1 levels and the percentage of reticular AJs area. Scale bars, 50 μm. (D and E) Protein expression changes of PECAM-1 and VE-cadherin in HUVECs (D) and HDMVEC (E) exposed to CN03 as in B and C. (F) CN03 induces surface localization of PECAM-1 and VE-cadherin Surface-biotinylated proteins were isolated by pull-down assay with NeutrAvidin-agarose in HUVECs exposed or not to CN03 as in A. VE-cadherin and PECAM-1 protein levels from the pull-down fraction were compared with total protein levels from the lysates by western blot analysis. Non-biotinylated cells were analyzed in parallel as a control of the pulldown assay. Immunoblots of tubulin are shown as loading controls. Plots show the mean□±□SEM from at least three independent experiments. (G) TEER analysis with an ECIS system of HDMVEC exposed to 5 μg/ml CN03. *, P< 0.05; **, P< 0.01; *** P<0.001.
    Figure Legend Snippet: (A) Confluent HUVECs were exposed or not to 1.5 μg/ml CN03 for 3 h, lysed and the indicated active GTPases were detected by pull-down assays. (B and C) Confluent HUVECs (B) or HDMVEC (C) were exposed to 1.5 μg/ml or 5 μg/ml CN03, respectively, for 3 h, fixed and stained for VE-cadherin and PECAM-1. Right images show two-fold enlargements of the squared areas highlighting the morphology of linear (top) and reticular (bottom) AJs. Bottom plots show quantification of junctional VE-cadherin and PECAM-1 levels and the percentage of reticular AJs area. Scale bars, 50 μm. (D and E) Protein expression changes of PECAM-1 and VE-cadherin in HUVECs (D) and HDMVEC (E) exposed to CN03 as in B and C. (F) CN03 induces surface localization of PECAM-1 and VE-cadherin Surface-biotinylated proteins were isolated by pull-down assay with NeutrAvidin-agarose in HUVECs exposed or not to CN03 as in A. VE-cadherin and PECAM-1 protein levels from the pull-down fraction were compared with total protein levels from the lysates by western blot analysis. Non-biotinylated cells were analyzed in parallel as a control of the pulldown assay. Immunoblots of tubulin are shown as loading controls. Plots show the mean□±□SEM from at least three independent experiments. (G) TEER analysis with an ECIS system of HDMVEC exposed to 5 μg/ml CN03. *, P< 0.05; **, P< 0.01; *** P<0.001.

    Techniques Used: Staining, Expressing, Isolation, Pull Down Assay, Western Blot

    (A) TEER and Rb measurements of confluent HDMVECs exposed to bacterial LPS and 3 h later treated or not with 5 μg/ml CN03. Bottom plots show the quantification of TEER decrease after 10 h of LPS stimulation. (B) Confluent HDMVEC were incubated with LPS and CN03 as in A, fixed and stained for VE-cadherin, PECAM-1 and F-actin. Bottom left and central plots show the quantification of junctional VE-cadherin and PECAM-1 levels. Bottom right plot shows the quantification of intercellular gaps identified by automatic image processing with Fiji (C) Confluent HLMVEC were exposed to 30% sera from healthy donors or sera from patients suffering severe sepsis and TEER was measured with an ECIS system for 48 h. 30% FBS was used as control. (D and E) . Endothelial cells were exposed or not to septic sera with or without CN03 for 24 h, fixed and stained for VE-cadherin, PECAM-1 and F-actin. Intercellular gaps were identified by automatic image processing with Fiji and quantified (E). Plots show the mean□±□SEM of cell quantifications from at least three independent experiments. Scale bars, 50 μm. *, P< 0.05; *** P<0.001.
    Figure Legend Snippet: (A) TEER and Rb measurements of confluent HDMVECs exposed to bacterial LPS and 3 h later treated or not with 5 μg/ml CN03. Bottom plots show the quantification of TEER decrease after 10 h of LPS stimulation. (B) Confluent HDMVEC were incubated with LPS and CN03 as in A, fixed and stained for VE-cadherin, PECAM-1 and F-actin. Bottom left and central plots show the quantification of junctional VE-cadherin and PECAM-1 levels. Bottom right plot shows the quantification of intercellular gaps identified by automatic image processing with Fiji (C) Confluent HLMVEC were exposed to 30% sera from healthy donors or sera from patients suffering severe sepsis and TEER was measured with an ECIS system for 48 h. 30% FBS was used as control. (D and E) . Endothelial cells were exposed or not to septic sera with or without CN03 for 24 h, fixed and stained for VE-cadherin, PECAM-1 and F-actin. Intercellular gaps were identified by automatic image processing with Fiji and quantified (E). Plots show the mean□±□SEM of cell quantifications from at least three independent experiments. Scale bars, 50 μm. *, P< 0.05; *** P<0.001.

    Techniques Used: Incubation, Staining

    (A) Cartoon showing chimeric constructs of GFP conjugated to the RhoA and RhoC hypervariable regions (GFP-Rho-hyper). RhoA contains a S residue two positions upstream the prenylation site, whereas RhoC contains a R residue. GFP-RhoA-hyper and GFP-RhoC-hyper containing the wild type hypervariable regions, as well as mutated sequences in which the RhoA S residue was substituted by R (GFP-RhoA-hyper S188R ) and the RhoC R residue replaced by S (GFP-RhoC-hyper R188S ) were expressed in HUVECs for 24 h, cells fixed and stained for VE-cadherin. Bottom left plots show the Manders’ coefficients for GFP-hyper chimeras and VE-cadherin. (B) The RhoC R residue was substituted by S or the uncharged amino acid A in GFP-RhoC constructs. Expression plasmids containing the indicated chimeras were ectopically expressed for 24 h, cells were fixed, stained for VE-cadherin and junctional co-localization quantified with Manders’ coefficient. (C) HUVECs expressing the indicated GFP-Rho proteins for 48 h were lysed and subjected to pull-down assays with GST-RTK to detect GTP-loaded RhoA and RhoC. Total and active fractions were immunoblotted with the indicated antibodies. Right graph shows the relative quantification of GFP-Rho activation with respect to GFP-RhoC activity. (D) HUVECs transiently expressing the indicated GFP-Rho proteins were plated on ECIS arrays for 48 h and TEER and Rb analyzed. Plots show the mean□±□SEM from at least three independent experiments. *, P< 0.05; **, P< 0.01; *** P<0.001.
    Figure Legend Snippet: (A) Cartoon showing chimeric constructs of GFP conjugated to the RhoA and RhoC hypervariable regions (GFP-Rho-hyper). RhoA contains a S residue two positions upstream the prenylation site, whereas RhoC contains a R residue. GFP-RhoA-hyper and GFP-RhoC-hyper containing the wild type hypervariable regions, as well as mutated sequences in which the RhoA S residue was substituted by R (GFP-RhoA-hyper S188R ) and the RhoC R residue replaced by S (GFP-RhoC-hyper R188S ) were expressed in HUVECs for 24 h, cells fixed and stained for VE-cadherin. Bottom left plots show the Manders’ coefficients for GFP-hyper chimeras and VE-cadherin. (B) The RhoC R residue was substituted by S or the uncharged amino acid A in GFP-RhoC constructs. Expression plasmids containing the indicated chimeras were ectopically expressed for 24 h, cells were fixed, stained for VE-cadherin and junctional co-localization quantified with Manders’ coefficient. (C) HUVECs expressing the indicated GFP-Rho proteins for 48 h were lysed and subjected to pull-down assays with GST-RTK to detect GTP-loaded RhoA and RhoC. Total and active fractions were immunoblotted with the indicated antibodies. Right graph shows the relative quantification of GFP-Rho activation with respect to GFP-RhoC activity. (D) HUVECs transiently expressing the indicated GFP-Rho proteins were plated on ECIS arrays for 48 h and TEER and Rb analyzed. Plots show the mean□±□SEM from at least three independent experiments. *, P< 0.05; **, P< 0.01; *** P<0.001.

    Techniques Used: Construct, Residue, Staining, Expressing, Activation Assay, Activity Assay

    ecis 8w10e pet culture ware  (Applied BioPhysics)


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    Applied BioPhysics ecis 8w10e pet culture ware
    Confluent monolayers of primary human umbilical vein endothelial cells (HUVEC) were infected with adenoviral vectors to express MARCH2-GFP, MARCH2 LD-GFP, MARCH4-GFP or GFP. After 24 hours cells were fixed for immunofluorescence or lysed for western blot analysis. Monolayers were co-labeled for (A) VE-cadherin and F-actin or (B) N-cadherin and p120-catenin (Scale bar = 100 μm). Expression of GFP or GFP tagged MARCH ligases were also visualized by fluorescence in these monolayers. (C) Western blot analysis for VE-cad, N-cad, VEGR-2, GFP and actin. Band density for VE-cad, N-cad, VEGFR-2, and actin were quantified by Multi Gauge (V3.0) and presented as mean± SEM, n = 4 (D) Confluent monolayers grown on <t>ECIS</t> chamber slides were infected with adenoviral vectors, and trans-endothelial electrical resistance was continuously measured by ECIS for 24 hrs.
    Ecis 8w10e Pet Culture Ware, supplied by Applied BioPhysics, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "MARCH family E3 ubiquitin ligases selectively target and degrade cadherin family proteins"

    Article Title: MARCH family E3 ubiquitin ligases selectively target and degrade cadherin family proteins

    Journal: PLOS ONE

    doi: 10.1371/journal.pone.0290485

    Confluent monolayers of primary human umbilical vein endothelial cells (HUVEC) were infected with adenoviral vectors to express MARCH2-GFP, MARCH2 LD-GFP, MARCH4-GFP or GFP. After 24 hours cells were fixed for immunofluorescence or lysed for western blot analysis. Monolayers were co-labeled for (A) VE-cadherin and F-actin or (B) N-cadherin and p120-catenin (Scale bar = 100 μm). Expression of GFP or GFP tagged MARCH ligases were also visualized by fluorescence in these monolayers. (C) Western blot analysis for VE-cad, N-cad, VEGR-2, GFP and actin. Band density for VE-cad, N-cad, VEGFR-2, and actin were quantified by Multi Gauge (V3.0) and presented as mean± SEM, n = 4 (D) Confluent monolayers grown on ECIS chamber slides were infected with adenoviral vectors, and trans-endothelial electrical resistance was continuously measured by ECIS for 24 hrs.
    Figure Legend Snippet: Confluent monolayers of primary human umbilical vein endothelial cells (HUVEC) were infected with adenoviral vectors to express MARCH2-GFP, MARCH2 LD-GFP, MARCH4-GFP or GFP. After 24 hours cells were fixed for immunofluorescence or lysed for western blot analysis. Monolayers were co-labeled for (A) VE-cadherin and F-actin or (B) N-cadherin and p120-catenin (Scale bar = 100 μm). Expression of GFP or GFP tagged MARCH ligases were also visualized by fluorescence in these monolayers. (C) Western blot analysis for VE-cad, N-cad, VEGR-2, GFP and actin. Band density for VE-cad, N-cad, VEGFR-2, and actin were quantified by Multi Gauge (V3.0) and presented as mean± SEM, n = 4 (D) Confluent monolayers grown on ECIS chamber slides were infected with adenoviral vectors, and trans-endothelial electrical resistance was continuously measured by ECIS for 24 hrs.

    Techniques Used: Infection, Immunofluorescence, Western Blot, Labeling, Expressing, Fluorescence

    ecis zθ  (Applied BioPhysics)


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    Applied BioPhysics ecis zθ
    Ecis Zθ, supplied by Applied BioPhysics, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cell substrate impedance sensing ecis 91  (Applied BioPhysics)


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    Applied BioPhysics cell substrate impedance sensing ecis 91
    Cell Substrate Impedance Sensing Ecis 91, supplied by Applied BioPhysics, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ecis zθ  (Applied BioPhysics)


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    Applied BioPhysics ecis zθ
    a – h HUVECs were treated with siRNA (si) targeting PNUTS or a control sequence. a-b) HUVEC barrier resistance was assessed by <t>ECIS</t> at 4000 Hz for 48 h, presented as average resistance ± SEM of n = 3 experiments, 8 biological replicates per group and experiment. c HUVECs were seeded in transwells and HRP passage through <t>the</t> <t>endothelial</t> monolayer was assessed by absorption measurements (450 nm) and shown as percentage of total HRP ( n = 5). d , e Cell-cell interaction was assessed by modeling of data obtained by ECIS measured as R b (Ω*cm 2 ); presented as average resistance ± SEM of n = 3 experiments, 8 biological replicates per group and experiment. f Cell surface presence of PECAM1 and VE-cadherin was assessed by flow cytometry 48 h after siRNA transfection ( n = 3). g Total PECAM1 and VE-cadherin levels were assessed by Western Blotting (WB). Tubulin and β-actin expression was used as loading control. h Cells were grown into confluence and immuno-stained for VE-cadherin, PECAM1 and F-actin. DAPI was used to stain nuclei. The presence of intercellular gaps in endothelial monolayers was measured by quantifying the intercellular areas versus the total area in 4 fields per image, 3 images per experiment, n = 4. i Schematic representation of the PNUTS lentiviral vectors used for the barrier rescue experiments. Both vectors included silent mutations in the seed sequence of siPNUTS. HUVECs were transduced with the indicated constructs for 8–10 days and later transfected with siControl or siPNUTS to silence endogenous expression of PNUTS, before subjecting them to ECIS and cell counting (4–6 independent experiments, 4 biological replicates per group and experiment). j PNUTS expression in total cell lysates of HUVECs treated with indicated vectors and siRNAs was analyzed by WB, Tubulin was used as a loading control. Representative WB is shown. k Change in cell-cell interaction measurement from ECIS 48 h after cell seeding, measured as variation of R b of siPNUTS- vs siControl-treated cells. l Cell proliferation of ECIS-assayed HUVECs, measured as % number of cells relative to control. * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars depict the standard error of the mean (SEM).
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    Images

    1) Product Images from "Aging-regulated PNUTS maintains endothelial barrier function via SEMA3B suppression"

    Article Title: Aging-regulated PNUTS maintains endothelial barrier function via SEMA3B suppression

    Journal: Communications Biology

    doi: 10.1038/s42003-024-06230-5

    a – h HUVECs were treated with siRNA (si) targeting PNUTS or a control sequence. a-b) HUVEC barrier resistance was assessed by ECIS at 4000 Hz for 48 h, presented as average resistance ± SEM of n = 3 experiments, 8 biological replicates per group and experiment. c HUVECs were seeded in transwells and HRP passage through the endothelial monolayer was assessed by absorption measurements (450 nm) and shown as percentage of total HRP ( n = 5). d , e Cell-cell interaction was assessed by modeling of data obtained by ECIS measured as R b (Ω*cm 2 ); presented as average resistance ± SEM of n = 3 experiments, 8 biological replicates per group and experiment. f Cell surface presence of PECAM1 and VE-cadherin was assessed by flow cytometry 48 h after siRNA transfection ( n = 3). g Total PECAM1 and VE-cadherin levels were assessed by Western Blotting (WB). Tubulin and β-actin expression was used as loading control. h Cells were grown into confluence and immuno-stained for VE-cadherin, PECAM1 and F-actin. DAPI was used to stain nuclei. The presence of intercellular gaps in endothelial monolayers was measured by quantifying the intercellular areas versus the total area in 4 fields per image, 3 images per experiment, n = 4. i Schematic representation of the PNUTS lentiviral vectors used for the barrier rescue experiments. Both vectors included silent mutations in the seed sequence of siPNUTS. HUVECs were transduced with the indicated constructs for 8–10 days and later transfected with siControl or siPNUTS to silence endogenous expression of PNUTS, before subjecting them to ECIS and cell counting (4–6 independent experiments, 4 biological replicates per group and experiment). j PNUTS expression in total cell lysates of HUVECs treated with indicated vectors and siRNAs was analyzed by WB, Tubulin was used as a loading control. Representative WB is shown. k Change in cell-cell interaction measurement from ECIS 48 h after cell seeding, measured as variation of R b of siPNUTS- vs siControl-treated cells. l Cell proliferation of ECIS-assayed HUVECs, measured as % number of cells relative to control. * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars depict the standard error of the mean (SEM).
    Figure Legend Snippet: a – h HUVECs were treated with siRNA (si) targeting PNUTS or a control sequence. a-b) HUVEC barrier resistance was assessed by ECIS at 4000 Hz for 48 h, presented as average resistance ± SEM of n = 3 experiments, 8 biological replicates per group and experiment. c HUVECs were seeded in transwells and HRP passage through the endothelial monolayer was assessed by absorption measurements (450 nm) and shown as percentage of total HRP ( n = 5). d , e Cell-cell interaction was assessed by modeling of data obtained by ECIS measured as R b (Ω*cm 2 ); presented as average resistance ± SEM of n = 3 experiments, 8 biological replicates per group and experiment. f Cell surface presence of PECAM1 and VE-cadherin was assessed by flow cytometry 48 h after siRNA transfection ( n = 3). g Total PECAM1 and VE-cadherin levels were assessed by Western Blotting (WB). Tubulin and β-actin expression was used as loading control. h Cells were grown into confluence and immuno-stained for VE-cadherin, PECAM1 and F-actin. DAPI was used to stain nuclei. The presence of intercellular gaps in endothelial monolayers was measured by quantifying the intercellular areas versus the total area in 4 fields per image, 3 images per experiment, n = 4. i Schematic representation of the PNUTS lentiviral vectors used for the barrier rescue experiments. Both vectors included silent mutations in the seed sequence of siPNUTS. HUVECs were transduced with the indicated constructs for 8–10 days and later transfected with siControl or siPNUTS to silence endogenous expression of PNUTS, before subjecting them to ECIS and cell counting (4–6 independent experiments, 4 biological replicates per group and experiment). j PNUTS expression in total cell lysates of HUVECs treated with indicated vectors and siRNAs was analyzed by WB, Tubulin was used as a loading control. Representative WB is shown. k Change in cell-cell interaction measurement from ECIS 48 h after cell seeding, measured as variation of R b of siPNUTS- vs siControl-treated cells. l Cell proliferation of ECIS-assayed HUVECs, measured as % number of cells relative to control. * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars depict the standard error of the mean (SEM).

    Techniques Used: Sequencing, Flow Cytometry, Transfection, Western Blot, Expressing, Staining, Transduction, Construct, Cell Counting

    a The two subsets of RNAseq data we obtained, from endothelial-depleted PNUTS mouse lungs and PNUTS knockdown (KD) HUVECs, were compared to find common targets of PNUTS depletion. The Venn diagram depicts the finding of 277 common transcripts, which were functionally analysed using KEGG pathways analysis, shown in the table below. b Changes in axon guidance gene expression after PNUTS silencing was assessed by RT-qPCR. Expression values are relative siControl-treated HUVECs and normalized to RPSA mRNA ( n = 6). c Volcano plot showing the distribution of gene expression in PNUTS KD versus control HUVECs. SEMA3B is marked in red. d Supernatant SEMA3B concentration was determined by ELISA 72 h after PNUTS silencing ( n = 4). e Expression levels of Sema3b mRNA in intima samples of PNUTS EC-KO mice was assessed by RT-qPCR, relative to WT samples and normalized to Rplp0 mRNA. f HUVECs were treated with vehicle or Tautomycetin (166 nM) for 48 h and mRNA was analyzed by RT-qPCR for expression of SEMA3B, normalized to RPSA mRNA ( n = 3). g Expression of SEMA3B was measured in mRNA samples of HUVECs assayed in Fig. , relative to control cells and normalized to RPSA mRNA. h - i HUVECs were co-transfected with siPNUTS and/or siSEMA3B were subjected to ECIS for 48 h ( n = 4 independent experiments, 4 biological replicates per group and experiment). h Cell-cell interaction was modeled. i Endothelial resistance was measured at 4000 Hz. j Top panel: siSEMA3B rescues the effect of PNUTS silencing on adherence junctions (shown as PECAM1 IF staining). Bottom panel: PECAM1 IF staining shows time course of change in adherens junctions upon stimulation with recombinant human SEMA3B. k Cell-cell interaction was modeled using ECIS. The arrow indicates the 48 h time point at which recombinant SEMA3B was added to the medium (or not; untreated). Quantification was performed at 60 h ( n = 4 biological replicates and 3–4 technical replicates). l Graphic summary of the proposed mechanism. In young individuals, PNUTS interacts and promotes activity of PP1, which represses the expression of SEMA3B. Endothelial cells are in homeostasis and maintain their barrier function. During aging, PNUTS is repressed in endothelial cells. The absence of PNUTS inhibits PP1 function at the SEMA3B promoter activating SEMA3B expression. SEMA3B exerts repulsive signals between endothelial cells, promoting intercellular gaps and disrupting the barrier. This provokes a series of critical changes in the cells leading to cellular senescence. * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars depict the standard error of the mean (SEM).
    Figure Legend Snippet: a The two subsets of RNAseq data we obtained, from endothelial-depleted PNUTS mouse lungs and PNUTS knockdown (KD) HUVECs, were compared to find common targets of PNUTS depletion. The Venn diagram depicts the finding of 277 common transcripts, which were functionally analysed using KEGG pathways analysis, shown in the table below. b Changes in axon guidance gene expression after PNUTS silencing was assessed by RT-qPCR. Expression values are relative siControl-treated HUVECs and normalized to RPSA mRNA ( n = 6). c Volcano plot showing the distribution of gene expression in PNUTS KD versus control HUVECs. SEMA3B is marked in red. d Supernatant SEMA3B concentration was determined by ELISA 72 h after PNUTS silencing ( n = 4). e Expression levels of Sema3b mRNA in intima samples of PNUTS EC-KO mice was assessed by RT-qPCR, relative to WT samples and normalized to Rplp0 mRNA. f HUVECs were treated with vehicle or Tautomycetin (166 nM) for 48 h and mRNA was analyzed by RT-qPCR for expression of SEMA3B, normalized to RPSA mRNA ( n = 3). g Expression of SEMA3B was measured in mRNA samples of HUVECs assayed in Fig. , relative to control cells and normalized to RPSA mRNA. h - i HUVECs were co-transfected with siPNUTS and/or siSEMA3B were subjected to ECIS for 48 h ( n = 4 independent experiments, 4 biological replicates per group and experiment). h Cell-cell interaction was modeled. i Endothelial resistance was measured at 4000 Hz. j Top panel: siSEMA3B rescues the effect of PNUTS silencing on adherence junctions (shown as PECAM1 IF staining). Bottom panel: PECAM1 IF staining shows time course of change in adherens junctions upon stimulation with recombinant human SEMA3B. k Cell-cell interaction was modeled using ECIS. The arrow indicates the 48 h time point at which recombinant SEMA3B was added to the medium (or not; untreated). Quantification was performed at 60 h ( n = 4 biological replicates and 3–4 technical replicates). l Graphic summary of the proposed mechanism. In young individuals, PNUTS interacts and promotes activity of PP1, which represses the expression of SEMA3B. Endothelial cells are in homeostasis and maintain their barrier function. During aging, PNUTS is repressed in endothelial cells. The absence of PNUTS inhibits PP1 function at the SEMA3B promoter activating SEMA3B expression. SEMA3B exerts repulsive signals between endothelial cells, promoting intercellular gaps and disrupting the barrier. This provokes a series of critical changes in the cells leading to cellular senescence. * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars depict the standard error of the mean (SEM).

    Techniques Used: Expressing, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transfection, Staining, Recombinant, Activity Assay

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    (A) Confluent HUVECs were exposed or not to 1.5 μg/ml CN03 for 3 h, lysed and the indicated active GTPases were detected by pull-down assays. (B and C) Confluent HUVECs (B) or HDMVEC (C) were exposed to 1.5 μg/ml or 5 μg/ml CN03, respectively, for 3 h, fixed and stained for VE-cadherin and PECAM-1. Right images show two-fold enlargements of the squared areas highlighting the morphology of linear (top) and reticular (bottom) AJs. Bottom plots show quantification of junctional VE-cadherin and PECAM-1 levels and the percentage of reticular AJs area. Scale bars, 50 μm. (D and E) Protein expression changes of PECAM-1 and VE-cadherin in HUVECs (D) and HDMVEC (E) exposed to CN03 as in B and C. (F) CN03 induces surface localization of PECAM-1 and VE-cadherin Surface-biotinylated proteins were isolated by pull-down assay with NeutrAvidin-agarose in HUVECs exposed or not to CN03 as in A. VE-cadherin and PECAM-1 protein levels from the pull-down fraction were compared with total protein levels from the lysates by western blot analysis. Non-biotinylated cells were analyzed in parallel as a control of the pulldown assay. Immunoblots of tubulin are shown as loading controls. Plots show the mean□±□SEM from at least three independent experiments. (G) TEER analysis with an ECIS system of HDMVEC exposed to 5 μg/ml CN03. *, P< 0.05; **, P< 0.01; *** P<0.001.

    Journal: bioRxiv

    Article Title: Harnessing homeostatically active RhoC at cell junctions preserves human endothelial barrier function during inflammation

    doi: 10.1101/2024.05.17.594667

    Figure Lengend Snippet: (A) Confluent HUVECs were exposed or not to 1.5 μg/ml CN03 for 3 h, lysed and the indicated active GTPases were detected by pull-down assays. (B and C) Confluent HUVECs (B) or HDMVEC (C) were exposed to 1.5 μg/ml or 5 μg/ml CN03, respectively, for 3 h, fixed and stained for VE-cadherin and PECAM-1. Right images show two-fold enlargements of the squared areas highlighting the morphology of linear (top) and reticular (bottom) AJs. Bottom plots show quantification of junctional VE-cadherin and PECAM-1 levels and the percentage of reticular AJs area. Scale bars, 50 μm. (D and E) Protein expression changes of PECAM-1 and VE-cadherin in HUVECs (D) and HDMVEC (E) exposed to CN03 as in B and C. (F) CN03 induces surface localization of PECAM-1 and VE-cadherin Surface-biotinylated proteins were isolated by pull-down assay with NeutrAvidin-agarose in HUVECs exposed or not to CN03 as in A. VE-cadherin and PECAM-1 protein levels from the pull-down fraction were compared with total protein levels from the lysates by western blot analysis. Non-biotinylated cells were analyzed in parallel as a control of the pulldown assay. Immunoblots of tubulin are shown as loading controls. Plots show the mean□±□SEM from at least three independent experiments. (G) TEER analysis with an ECIS system of HDMVEC exposed to 5 μg/ml CN03. *, P< 0.05; **, P< 0.01; *** P<0.001.

    Article Snippet: Trans-endothelial electric resistance (TEER) assays with an electric cell-substrate impedance sensing system (ECIS 1600R; Applied Biophysics)(Tiruppathi et al., 1992) were performed as described (Colas-Algora et al., 2019; ).

    Techniques: Staining, Expressing, Isolation, Pull Down Assay, Western Blot

    (A) TEER and Rb measurements of confluent HDMVECs exposed to bacterial LPS and 3 h later treated or not with 5 μg/ml CN03. Bottom plots show the quantification of TEER decrease after 10 h of LPS stimulation. (B) Confluent HDMVEC were incubated with LPS and CN03 as in A, fixed and stained for VE-cadherin, PECAM-1 and F-actin. Bottom left and central plots show the quantification of junctional VE-cadherin and PECAM-1 levels. Bottom right plot shows the quantification of intercellular gaps identified by automatic image processing with Fiji (C) Confluent HLMVEC were exposed to 30% sera from healthy donors or sera from patients suffering severe sepsis and TEER was measured with an ECIS system for 48 h. 30% FBS was used as control. (D and E) . Endothelial cells were exposed or not to septic sera with or without CN03 for 24 h, fixed and stained for VE-cadherin, PECAM-1 and F-actin. Intercellular gaps were identified by automatic image processing with Fiji and quantified (E). Plots show the mean□±□SEM of cell quantifications from at least three independent experiments. Scale bars, 50 μm. *, P< 0.05; *** P<0.001.

    Journal: bioRxiv

    Article Title: Harnessing homeostatically active RhoC at cell junctions preserves human endothelial barrier function during inflammation

    doi: 10.1101/2024.05.17.594667

    Figure Lengend Snippet: (A) TEER and Rb measurements of confluent HDMVECs exposed to bacterial LPS and 3 h later treated or not with 5 μg/ml CN03. Bottom plots show the quantification of TEER decrease after 10 h of LPS stimulation. (B) Confluent HDMVEC were incubated with LPS and CN03 as in A, fixed and stained for VE-cadherin, PECAM-1 and F-actin. Bottom left and central plots show the quantification of junctional VE-cadherin and PECAM-1 levels. Bottom right plot shows the quantification of intercellular gaps identified by automatic image processing with Fiji (C) Confluent HLMVEC were exposed to 30% sera from healthy donors or sera from patients suffering severe sepsis and TEER was measured with an ECIS system for 48 h. 30% FBS was used as control. (D and E) . Endothelial cells were exposed or not to septic sera with or without CN03 for 24 h, fixed and stained for VE-cadherin, PECAM-1 and F-actin. Intercellular gaps were identified by automatic image processing with Fiji and quantified (E). Plots show the mean□±□SEM of cell quantifications from at least three independent experiments. Scale bars, 50 μm. *, P< 0.05; *** P<0.001.

    Article Snippet: Trans-endothelial electric resistance (TEER) assays with an electric cell-substrate impedance sensing system (ECIS 1600R; Applied Biophysics)(Tiruppathi et al., 1992) were performed as described (Colas-Algora et al., 2019; ).

    Techniques: Incubation, Staining

    (A) Cartoon showing chimeric constructs of GFP conjugated to the RhoA and RhoC hypervariable regions (GFP-Rho-hyper). RhoA contains a S residue two positions upstream the prenylation site, whereas RhoC contains a R residue. GFP-RhoA-hyper and GFP-RhoC-hyper containing the wild type hypervariable regions, as well as mutated sequences in which the RhoA S residue was substituted by R (GFP-RhoA-hyper S188R ) and the RhoC R residue replaced by S (GFP-RhoC-hyper R188S ) were expressed in HUVECs for 24 h, cells fixed and stained for VE-cadherin. Bottom left plots show the Manders’ coefficients for GFP-hyper chimeras and VE-cadherin. (B) The RhoC R residue was substituted by S or the uncharged amino acid A in GFP-RhoC constructs. Expression plasmids containing the indicated chimeras were ectopically expressed for 24 h, cells were fixed, stained for VE-cadherin and junctional co-localization quantified with Manders’ coefficient. (C) HUVECs expressing the indicated GFP-Rho proteins for 48 h were lysed and subjected to pull-down assays with GST-RTK to detect GTP-loaded RhoA and RhoC. Total and active fractions were immunoblotted with the indicated antibodies. Right graph shows the relative quantification of GFP-Rho activation with respect to GFP-RhoC activity. (D) HUVECs transiently expressing the indicated GFP-Rho proteins were plated on ECIS arrays for 48 h and TEER and Rb analyzed. Plots show the mean□±□SEM from at least three independent experiments. *, P< 0.05; **, P< 0.01; *** P<0.001.

    Journal: bioRxiv

    Article Title: Harnessing homeostatically active RhoC at cell junctions preserves human endothelial barrier function during inflammation

    doi: 10.1101/2024.05.17.594667

    Figure Lengend Snippet: (A) Cartoon showing chimeric constructs of GFP conjugated to the RhoA and RhoC hypervariable regions (GFP-Rho-hyper). RhoA contains a S residue two positions upstream the prenylation site, whereas RhoC contains a R residue. GFP-RhoA-hyper and GFP-RhoC-hyper containing the wild type hypervariable regions, as well as mutated sequences in which the RhoA S residue was substituted by R (GFP-RhoA-hyper S188R ) and the RhoC R residue replaced by S (GFP-RhoC-hyper R188S ) were expressed in HUVECs for 24 h, cells fixed and stained for VE-cadherin. Bottom left plots show the Manders’ coefficients for GFP-hyper chimeras and VE-cadherin. (B) The RhoC R residue was substituted by S or the uncharged amino acid A in GFP-RhoC constructs. Expression plasmids containing the indicated chimeras were ectopically expressed for 24 h, cells were fixed, stained for VE-cadherin and junctional co-localization quantified with Manders’ coefficient. (C) HUVECs expressing the indicated GFP-Rho proteins for 48 h were lysed and subjected to pull-down assays with GST-RTK to detect GTP-loaded RhoA and RhoC. Total and active fractions were immunoblotted with the indicated antibodies. Right graph shows the relative quantification of GFP-Rho activation with respect to GFP-RhoC activity. (D) HUVECs transiently expressing the indicated GFP-Rho proteins were plated on ECIS arrays for 48 h and TEER and Rb analyzed. Plots show the mean□±□SEM from at least three independent experiments. *, P< 0.05; **, P< 0.01; *** P<0.001.

    Article Snippet: Trans-endothelial electric resistance (TEER) assays with an electric cell-substrate impedance sensing system (ECIS 1600R; Applied Biophysics)(Tiruppathi et al., 1992) were performed as described (Colas-Algora et al., 2019; ).

    Techniques: Construct, Residue, Staining, Expressing, Activation Assay, Activity Assay

    Confluent monolayers of primary human umbilical vein endothelial cells (HUVEC) were infected with adenoviral vectors to express MARCH2-GFP, MARCH2 LD-GFP, MARCH4-GFP or GFP. After 24 hours cells were fixed for immunofluorescence or lysed for western blot analysis. Monolayers were co-labeled for (A) VE-cadherin and F-actin or (B) N-cadherin and p120-catenin (Scale bar = 100 μm). Expression of GFP or GFP tagged MARCH ligases were also visualized by fluorescence in these monolayers. (C) Western blot analysis for VE-cad, N-cad, VEGR-2, GFP and actin. Band density for VE-cad, N-cad, VEGFR-2, and actin were quantified by Multi Gauge (V3.0) and presented as mean± SEM, n = 4 (D) Confluent monolayers grown on ECIS chamber slides were infected with adenoviral vectors, and trans-endothelial electrical resistance was continuously measured by ECIS for 24 hrs.

    Journal: PLOS ONE

    Article Title: MARCH family E3 ubiquitin ligases selectively target and degrade cadherin family proteins

    doi: 10.1371/journal.pone.0290485

    Figure Lengend Snippet: Confluent monolayers of primary human umbilical vein endothelial cells (HUVEC) were infected with adenoviral vectors to express MARCH2-GFP, MARCH2 LD-GFP, MARCH4-GFP or GFP. After 24 hours cells were fixed for immunofluorescence or lysed for western blot analysis. Monolayers were co-labeled for (A) VE-cadherin and F-actin or (B) N-cadherin and p120-catenin (Scale bar = 100 μm). Expression of GFP or GFP tagged MARCH ligases were also visualized by fluorescence in these monolayers. (C) Western blot analysis for VE-cad, N-cad, VEGR-2, GFP and actin. Band density for VE-cad, N-cad, VEGFR-2, and actin were quantified by Multi Gauge (V3.0) and presented as mean± SEM, n = 4 (D) Confluent monolayers grown on ECIS chamber slides were infected with adenoviral vectors, and trans-endothelial electrical resistance was continuously measured by ECIS for 24 hrs.

    Article Snippet: For ECIS experiments, HUVEC were seeded onto ECIS 8W10E PET culture ware (Applied Biophysics), and readings were taken every 5 minutes at single frequency 4000Hz.

    Techniques: Infection, Immunofluorescence, Western Blot, Labeling, Expressing, Fluorescence