huvecs  (Thermo Fisher)


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    Structured Review

    Thermo Fisher huvecs
    Gap junction-mediated cell-cell communication. (A) Dye transfer assay between <t>calcein</t> AM-labeled <t>HUVECs</t> (green) and RFP-HUVECs (red) after 24 h co-culture on gelatin and hFDM, respectively; the images in the left side show only the RFP-HUVECs, and the merged images of RFP-HUVECs and calcein AM-labeled HUVECs are on the right side. Positive signals (white triangles) are identified via the RFP-HUVECs (red) with green dye (calcein AM) uptake. (B) Expression of Cx43 protein (green) with HUVECs cultured on either gelatin or hFDM for 3 days, along with F-actin staining (red). (C) Percentage of calcein dye-positive RFP-HUVECs as calculated via image analysis after the dye transfer assay; the percentage was averaged by counting the number of dye-positive RFP-HUVECs against total RFP-HUVECs in a given image. (D) Quantitative evaluation of average Cx43 area per cell area. All scale bars are 50 μm. Statistically significant difference: * p
    Huvecs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Novel skin patch combining human fibroblast-derived matrix and ciprofloxacin for infected wound healing"

    Article Title: Novel skin patch combining human fibroblast-derived matrix and ciprofloxacin for infected wound healing

    Journal: Theranostics

    doi: 10.7150/thno.26837

    Gap junction-mediated cell-cell communication. (A) Dye transfer assay between calcein AM-labeled HUVECs (green) and RFP-HUVECs (red) after 24 h co-culture on gelatin and hFDM, respectively; the images in the left side show only the RFP-HUVECs, and the merged images of RFP-HUVECs and calcein AM-labeled HUVECs are on the right side. Positive signals (white triangles) are identified via the RFP-HUVECs (red) with green dye (calcein AM) uptake. (B) Expression of Cx43 protein (green) with HUVECs cultured on either gelatin or hFDM for 3 days, along with F-actin staining (red). (C) Percentage of calcein dye-positive RFP-HUVECs as calculated via image analysis after the dye transfer assay; the percentage was averaged by counting the number of dye-positive RFP-HUVECs against total RFP-HUVECs in a given image. (D) Quantitative evaluation of average Cx43 area per cell area. All scale bars are 50 μm. Statistically significant difference: * p
    Figure Legend Snippet: Gap junction-mediated cell-cell communication. (A) Dye transfer assay between calcein AM-labeled HUVECs (green) and RFP-HUVECs (red) after 24 h co-culture on gelatin and hFDM, respectively; the images in the left side show only the RFP-HUVECs, and the merged images of RFP-HUVECs and calcein AM-labeled HUVECs are on the right side. Positive signals (white triangles) are identified via the RFP-HUVECs (red) with green dye (calcein AM) uptake. (B) Expression of Cx43 protein (green) with HUVECs cultured on either gelatin or hFDM for 3 days, along with F-actin staining (red). (C) Percentage of calcein dye-positive RFP-HUVECs as calculated via image analysis after the dye transfer assay; the percentage was averaged by counting the number of dye-positive RFP-HUVECs against total RFP-HUVECs in a given image. (D) Quantitative evaluation of average Cx43 area per cell area. All scale bars are 50 μm. Statistically significant difference: * p

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

    2) Product Images from "Metastasis-associated miR-23a from nasopharyngeal carcinoma-derived exosomes mediates angiogenesis by repressing a novel target gene TSGA10"

    Article Title: Metastasis-associated miR-23a from nasopharyngeal carcinoma-derived exosomes mediates angiogenesis by repressing a novel target gene TSGA10

    Journal: Oncogene

    doi: 10.1038/s41388-018-0183-6

    MiR-23a is highly expressed in NPC-exo. a Scheme of exosome isolation by differential ultracentrifugation. b Representative electron microscopy image of NPC-exo. c Nanoparticle tracking analysis displayed the size distribution of exosomes isolated from NPC. d Western blot analysis of exosomal markers. CNE2 cell line was used as controls for exosomes characterized. Flotillin-1 was used as a loading control. e Uptake of exosomes in HUVECs by confocal microscopy. Blue: Hoechst staining; green: PKH67-labeled exosomes. f , g qRT-PCR of miRNA level in exosomes isolated from serum (grouping based on the clinical features of patients at the blood-drawing time) or NPC cells. T test. h qRT-PCR of miRNA expression in exosomes isolated from miR-23a-treated CM. One-way ANOVA
    Figure Legend Snippet: MiR-23a is highly expressed in NPC-exo. a Scheme of exosome isolation by differential ultracentrifugation. b Representative electron microscopy image of NPC-exo. c Nanoparticle tracking analysis displayed the size distribution of exosomes isolated from NPC. d Western blot analysis of exosomal markers. CNE2 cell line was used as controls for exosomes characterized. Flotillin-1 was used as a loading control. e Uptake of exosomes in HUVECs by confocal microscopy. Blue: Hoechst staining; green: PKH67-labeled exosomes. f , g qRT-PCR of miRNA level in exosomes isolated from serum (grouping based on the clinical features of patients at the blood-drawing time) or NPC cells. T test. h qRT-PCR of miRNA expression in exosomes isolated from miR-23a-treated CM. One-way ANOVA

    Techniques Used: Isolation, Electron Microscopy, Western Blot, Confocal Microscopy, Staining, Labeling, Quantitative RT-PCR, Expressing

    MiR-23a enhances HUVEC proliferation, migration, and tube formation. a Transfection efficiency was measured by qRT-PCR. One-way ANOVA. b The cell growth of transfected HUVECs was measured by CCK8 assay. Two-way ANOVA. c , d Flow cytometry analysis of the cell cycle was performed at 36 h after transfection. The graph summarizes the results of three independent experiments. One-way ANOVA. e , f Wound-healing assay showed cell migration in transfected HUVECs. Two-way ANOVA. g , h Transwell migration assays were performed to measure cell migration. Cell numbers were calculated as the average of 10 randomly picked fields. One-way ANOVA. i HUVECs were inoculated in Matrigel and the indicated images were captured. j , k Analyses of p-ERK expression in transfected cells
    Figure Legend Snippet: MiR-23a enhances HUVEC proliferation, migration, and tube formation. a Transfection efficiency was measured by qRT-PCR. One-way ANOVA. b The cell growth of transfected HUVECs was measured by CCK8 assay. Two-way ANOVA. c , d Flow cytometry analysis of the cell cycle was performed at 36 h after transfection. The graph summarizes the results of three independent experiments. One-way ANOVA. e , f Wound-healing assay showed cell migration in transfected HUVECs. Two-way ANOVA. g , h Transwell migration assays were performed to measure cell migration. Cell numbers were calculated as the average of 10 randomly picked fields. One-way ANOVA. i HUVECs were inoculated in Matrigel and the indicated images were captured. j , k Analyses of p-ERK expression in transfected cells

    Techniques Used: Migration, Transfection, Quantitative RT-PCR, CCK-8 Assay, Flow Cytometry, Cytometry, Wound Healing Assay, Expressing

    3) Product Images from "Control of endothelial quiescence by FOXO-regulated metabolites"

    Article Title: Control of endothelial quiescence by FOXO-regulated metabolites

    Journal: Nature Cell Biology

    doi: 10.1038/s41556-021-00637-6

    FOXO1 induces S -2HG generation by regulating BCAA catabolism. a , Expression of canonical FOXO1 targets and OGDH complex subunits in HUVECs transduced with a control-encoding (AdCtrl) and FOXO1 A3 -encoding (AdFOXO1 A3 ) adenovirus. Cells were collected 24 h after transduction and analysed by RNA-seq ( n = 3 independent samples). b , Volcano plot showing increased levels of BCAA catabolites in FOXO1 A3 -expressing HUVECs ( n = 4 independent experiments). c , KMV metabolite levels in HUVECs transduced with a doxycycline-inducible control-encoding (iLentiCtrl) or FOXO1 A3 -encoding lentivirus (iLentiFOXO1 A3 ) ( n = 6 (iLentiCtrl) and 10 (iLentiFOXO1 A3 ) independent samples). d , Changes in BCAA metabolites in AdCtrl versus AdFOXO1 A3 expressing HUVECs. Data represent the fold-change relative to control ( n = 4 independent samples). e , OGDH activity assay in control (PBS, Ctrl) or KMV-treated HUVECs ( n = 3 independent experiments). f , Decreased basal and maximal (FCCP) OCRs in HUVECs treated with KMV for 48 h compared to control ( n = 5 (control) or 8 (KMV) independent samples). g , 2HG metabolite levels in control and KMV-treated HUVECs measured by LC–MS ( n = 9 independent samples). h , 2HG chiral derivatization and enantioselective MS measurement of R - and S -2HG levels in control or KMV-treated HUVECs ( n = 9 independent samples). For a – h , the data represent the mean ± s.e.m.; two-tailed unpaired t -test, * P
    Figure Legend Snippet: FOXO1 induces S -2HG generation by regulating BCAA catabolism. a , Expression of canonical FOXO1 targets and OGDH complex subunits in HUVECs transduced with a control-encoding (AdCtrl) and FOXO1 A3 -encoding (AdFOXO1 A3 ) adenovirus. Cells were collected 24 h after transduction and analysed by RNA-seq ( n = 3 independent samples). b , Volcano plot showing increased levels of BCAA catabolites in FOXO1 A3 -expressing HUVECs ( n = 4 independent experiments). c , KMV metabolite levels in HUVECs transduced with a doxycycline-inducible control-encoding (iLentiCtrl) or FOXO1 A3 -encoding lentivirus (iLentiFOXO1 A3 ) ( n = 6 (iLentiCtrl) and 10 (iLentiFOXO1 A3 ) independent samples). d , Changes in BCAA metabolites in AdCtrl versus AdFOXO1 A3 expressing HUVECs. Data represent the fold-change relative to control ( n = 4 independent samples). e , OGDH activity assay in control (PBS, Ctrl) or KMV-treated HUVECs ( n = 3 independent experiments). f , Decreased basal and maximal (FCCP) OCRs in HUVECs treated with KMV for 48 h compared to control ( n = 5 (control) or 8 (KMV) independent samples). g , 2HG metabolite levels in control and KMV-treated HUVECs measured by LC–MS ( n = 9 independent samples). h , 2HG chiral derivatization and enantioselective MS measurement of R - and S -2HG levels in control or KMV-treated HUVECs ( n = 9 independent samples). For a – h , the data represent the mean ± s.e.m.; two-tailed unpaired t -test, * P

    Techniques Used: Expressing, Transduction, RNA Sequencing Assay, Activity Assay, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test

    4) Product Images from "MPDZ promotes DLL4-induced Notch signaling during angiogenesis"

    Article Title: MPDZ promotes DLL4-induced Notch signaling during angiogenesis

    Journal: eLife

    doi: 10.7554/eLife.32860

    MPDZ promotes Notch signaling activity. ( A ) HUVECs were either transduced with lentivirus expressing GFP (sh-ctrl) or with lentivirus expressing shRNA against MPDZ (sh-MPDZ). Expression level of Notch target genes HEY1 , HEY2 and HES1 were analyzed by qPCR 48 hr after transduction. Data are presented as mean ±SD. n ≥ 3; *, p
    Figure Legend Snippet: MPDZ promotes Notch signaling activity. ( A ) HUVECs were either transduced with lentivirus expressing GFP (sh-ctrl) or with lentivirus expressing shRNA against MPDZ (sh-MPDZ). Expression level of Notch target genes HEY1 , HEY2 and HES1 were analyzed by qPCR 48 hr after transduction. Data are presented as mean ±SD. n ≥ 3; *, p

    Techniques Used: Activity Assay, Transduction, Expressing, shRNA, Real-time Polymerase Chain Reaction

    MPDZ inhibits sprouting angiogenesis in vitro. ( A ) HUVEC were transduced with lentivirus-expressing shRNA against MPDZ (sh-MPDZ) or expressing GFP (sh-ctrl). Sprouting angiogenesis of collagen-embedded spheroids was analyzed 72 hr after transduction. Spheroids were cultured under basal conditions or stimulated with VEGF-A (25 ng/ml). Quantification shows length of all sprouts of each spheroid. n = 4 experiments with 10 spheroids per condition. **, p
    Figure Legend Snippet: MPDZ inhibits sprouting angiogenesis in vitro. ( A ) HUVEC were transduced with lentivirus-expressing shRNA against MPDZ (sh-MPDZ) or expressing GFP (sh-ctrl). Sprouting angiogenesis of collagen-embedded spheroids was analyzed 72 hr after transduction. Spheroids were cultured under basal conditions or stimulated with VEGF-A (25 ng/ml). Quantification shows length of all sprouts of each spheroid. n = 4 experiments with 10 spheroids per condition. **, p

    Techniques Used: In Vitro, Transduction, Expressing, shRNA, Cell Culture

    Mpdz does not affect cell cell junction assembly. ( A, B ) HUVECs were either transduced with lentivirus expressing GFP (sh-ctrl) or with lentivirus expressing shRNA against MPDZ (sh-MPDZ). Cells were cultured under sparse conditions ( A ) or confluent conditions ( B ). After PFA fixation cells were stained for DLL1 and Nectin-2 or DLL4 and Nectin-2 and counterstained with DAPI. Images were acquired with the confocal microscope LSM 700. Arrow indicates co-localization of DLL1/4 with Nectin-2 at the cell membrane. Arrow head indicates diminished co-localization at the cell membrane. Scale bar: 10 µm. ( C ) HUVECs were either transfected with control siRNA (si-ctrl) or with siRNA against Nectin-2 (si-Nectin-2). After PFA fixation cells were stained for DLL1 and Nectin-2 or DLL4 and Nectin-2. Images were acquired with the confocal microscope LSM 700. Arrow indicates localization of DLL1/4 at the cell membrane.Scale bar: 10 µm.
    Figure Legend Snippet: Mpdz does not affect cell cell junction assembly. ( A, B ) HUVECs were either transduced with lentivirus expressing GFP (sh-ctrl) or with lentivirus expressing shRNA against MPDZ (sh-MPDZ). Cells were cultured under sparse conditions ( A ) or confluent conditions ( B ). After PFA fixation cells were stained for DLL1 and Nectin-2 or DLL4 and Nectin-2 and counterstained with DAPI. Images were acquired with the confocal microscope LSM 700. Arrow indicates co-localization of DLL1/4 with Nectin-2 at the cell membrane. Arrow head indicates diminished co-localization at the cell membrane. Scale bar: 10 µm. ( C ) HUVECs were either transfected with control siRNA (si-ctrl) or with siRNA against Nectin-2 (si-Nectin-2). After PFA fixation cells were stained for DLL1 and Nectin-2 or DLL4 and Nectin-2. Images were acquired with the confocal microscope LSM 700. Arrow indicates localization of DLL1/4 at the cell membrane.Scale bar: 10 µm.

    Techniques Used: Transduction, Expressing, shRNA, Cell Culture, Staining, Microscopy, Transfection

    5) Product Images from "Matrix metalloproteinase (MMP)-degradable tissue engineered periosteum coordinates allograft healing via early stage recruitment and support of host neurovasculature"

    Article Title: Matrix metalloproteinase (MMP)-degradable tissue engineered periosteum coordinates allograft healing via early stage recruitment and support of host neurovasculature

    Journal: Biomaterials

    doi: 10.1016/j.biomaterials.2020.120535

    Representative images of HUVEC/hMSC spheroids within (A) hydrolytically degradable hydrogels (Hydro-Gel), (B, C and D) MMP-degradable hydrogels (MMP-Gel) with and without MMP inhibitor respectively after 5 days. Confocal microscopy (D) illustrates that HUVECs and hMSCs concomitantly sprouted in MMP-Gel. (E) Cell sprouting in different gels was quantified as average sprouting length after 1, 3, and 5 days using one-way ANOVA with Dunnett’s post-hoc analysis, where n =3, scale bar = 200 μm, p
    Figure Legend Snippet: Representative images of HUVEC/hMSC spheroids within (A) hydrolytically degradable hydrogels (Hydro-Gel), (B, C and D) MMP-degradable hydrogels (MMP-Gel) with and without MMP inhibitor respectively after 5 days. Confocal microscopy (D) illustrates that HUVECs and hMSCs concomitantly sprouted in MMP-Gel. (E) Cell sprouting in different gels was quantified as average sprouting length after 1, 3, and 5 days using one-way ANOVA with Dunnett’s post-hoc analysis, where n =3, scale bar = 200 μm, p

    Techniques Used: Confocal Microscopy

    6) Product Images from "Uremic Advanced Glycation End Products and Protein‐Bound Solutes Induce Endothelial Dysfunction Through Suppression of Krüppel‐Like Factor 2"

    Article Title: Uremic Advanced Glycation End Products and Protein‐Bound Solutes Induce Endothelial Dysfunction Through Suppression of Krüppel‐Like Factor 2

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    doi: 10.1161/JAHA.117.007566

    Uremic advanced glycation end products ( AGE s) induce endothelial reactive oxygen species ( ROS ) and leukocyte adhesion through suppression of Krüppel‐like factor 2 ( KLF 2). Human umbilical vein endothelial cells ( HUVEC s) were transfected with KLF 2 overexpression adenovirus (recombinant adenovirus expressing KLF2 and green fluorescent protein [GFP] [KLF2‐Ad]) or empty vector (recombinant adenovirus expressing GFP [Ctrl‐Ad]) and exposed to the AGE compound carboxymethyllysine (CML)‐modified BSA at 5.4 mg/L, unmodified BSA , nonuremic porcine serum ( NS ), or uremic porcine serum ( US ) for 24 hours. ROS production was measured in AGE ‐ or serum‐treated cells using the Cell ROX Orange Reagent fluorescent probe with 100 nmol angiotensin II ( AT II ) as a positive control. Representative images from AGE (A) and serum (B) treated cells are shown. C and D, Cellular ROS production was quantified by measuring individual cell fluorescence intensity in 16 high‐power fields from AGE (C) or serum (D) treated cells (n=18 per group; 2‐way ANOVA with the Holm‐Sidak post hoc test; repeated 3 separate times). E and F, Monocyte adhesion to HUVEC monolayers was measured in AGE (E) or serum (F) treated cells using Calcein red‐orange labeled THP ‐1 cells, with tumor necrosis factor α ( TNF α) as a positive control (Ctrl; n=18 per group; 2‐way ANOVA with the Holm‐Sidak post hoc test; repeated 3 separate times). Data are presented as mean± SEM . AU indicates arbitrary unit; and NS, nonsignificant. **** P
    Figure Legend Snippet: Uremic advanced glycation end products ( AGE s) induce endothelial reactive oxygen species ( ROS ) and leukocyte adhesion through suppression of Krüppel‐like factor 2 ( KLF 2). Human umbilical vein endothelial cells ( HUVEC s) were transfected with KLF 2 overexpression adenovirus (recombinant adenovirus expressing KLF2 and green fluorescent protein [GFP] [KLF2‐Ad]) or empty vector (recombinant adenovirus expressing GFP [Ctrl‐Ad]) and exposed to the AGE compound carboxymethyllysine (CML)‐modified BSA at 5.4 mg/L, unmodified BSA , nonuremic porcine serum ( NS ), or uremic porcine serum ( US ) for 24 hours. ROS production was measured in AGE ‐ or serum‐treated cells using the Cell ROX Orange Reagent fluorescent probe with 100 nmol angiotensin II ( AT II ) as a positive control. Representative images from AGE (A) and serum (B) treated cells are shown. C and D, Cellular ROS production was quantified by measuring individual cell fluorescence intensity in 16 high‐power fields from AGE (C) or serum (D) treated cells (n=18 per group; 2‐way ANOVA with the Holm‐Sidak post hoc test; repeated 3 separate times). E and F, Monocyte adhesion to HUVEC monolayers was measured in AGE (E) or serum (F) treated cells using Calcein red‐orange labeled THP ‐1 cells, with tumor necrosis factor α ( TNF α) as a positive control (Ctrl; n=18 per group; 2‐way ANOVA with the Holm‐Sidak post hoc test; repeated 3 separate times). Data are presented as mean± SEM . AU indicates arbitrary unit; and NS, nonsignificant. **** P

    Techniques Used: Transfection, Over Expression, Recombinant, Expressing, Plasmid Preparation, Modification, Positive Control, Fluorescence, Labeling

    Uremic porcine serum suppresses endothelial Krüppel‐like factor 2 ( KLF 2) expression, which is attenuated by hemodynamic shear stress and dialysis. A, Changes in KLF 2 mRNA expression were assessed over a 24‐hour time course by quantitative real‐time PCR in human umbilical vein endothelial cells ( HUVEC s) incubated with 10% uremic porcine serum. One‐way ANOVA with the Dunn post hoc test was used to calculate statistical significance between treated cells at each time point (n=4 per group; repeated 3 separate times). B, Western blot analysis and quantification of KLF 2 from HUVEC s incubated with increasing concentrations of normal porcine serum ( NS ) or uremic porcine serum ( US ) for 24 hours; basal medium was used as a control (n=4 per group; repeated 3 separate times). C, Western blot analysis and quantification of KLF 2 from HUVEC s cultured under periodic unidirectional flow using orbital shear rings. Cells were grown to confluence in shear rings and preconditioned for 72 hours under shear stress of 11 dyne/cm 2 on an orbital shaker (200 rpm) or static culture. Cells were then treated as in B and compared to untreated cells (NT) (n=4 per group; repeated 3 separate times). D, Western blot analysis and quantification of KLF 2 from HUVEC s incubated with 10% uremic porcine and then switched to dialyzed porcine serum ( DS ) after 48 hours (n=4 per group; repeated 3 separate times). Arrow marks time of switch from uremic to dialyzed serum. Quantification of protein expression above each blot is relative to baseline or untreated cells and normalized to β‐tubulin expression. Two‐way ANOVA with the Dunn post hoc test was used to calculate statistical significance between serum type and concentrations. Data presented as mean± SEM . NS indicates nonsignificant. * P
    Figure Legend Snippet: Uremic porcine serum suppresses endothelial Krüppel‐like factor 2 ( KLF 2) expression, which is attenuated by hemodynamic shear stress and dialysis. A, Changes in KLF 2 mRNA expression were assessed over a 24‐hour time course by quantitative real‐time PCR in human umbilical vein endothelial cells ( HUVEC s) incubated with 10% uremic porcine serum. One‐way ANOVA with the Dunn post hoc test was used to calculate statistical significance between treated cells at each time point (n=4 per group; repeated 3 separate times). B, Western blot analysis and quantification of KLF 2 from HUVEC s incubated with increasing concentrations of normal porcine serum ( NS ) or uremic porcine serum ( US ) for 24 hours; basal medium was used as a control (n=4 per group; repeated 3 separate times). C, Western blot analysis and quantification of KLF 2 from HUVEC s cultured under periodic unidirectional flow using orbital shear rings. Cells were grown to confluence in shear rings and preconditioned for 72 hours under shear stress of 11 dyne/cm 2 on an orbital shaker (200 rpm) or static culture. Cells were then treated as in B and compared to untreated cells (NT) (n=4 per group; repeated 3 separate times). D, Western blot analysis and quantification of KLF 2 from HUVEC s incubated with 10% uremic porcine and then switched to dialyzed porcine serum ( DS ) after 48 hours (n=4 per group; repeated 3 separate times). Arrow marks time of switch from uremic to dialyzed serum. Quantification of protein expression above each blot is relative to baseline or untreated cells and normalized to β‐tubulin expression. Two‐way ANOVA with the Dunn post hoc test was used to calculate statistical significance between serum type and concentrations. Data presented as mean± SEM . NS indicates nonsignificant. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Incubation, Western Blot, Cell Culture, Flow Cytometry

    Uremic advanced glycation end products ( AGE s) suppress endothelial Krüppel‐like factor 2 ( KLF 2) through receptor for AGE ( RAGE ) signaling. A, Western blot analysis and quantification of KLF 2 from human umbilical vein endothelial cells ( HUVEC s) exposed to individual protein‐bound uremic toxins, including carboxymethyllysine (CML)‐modified BSA, p‐cresol sulfate ( PCS ), indoxyl sulfate ( IS ), or vehicle BSA for 24 hours (n=5 per group; 1‐way ANOVA with the Dunn post hoc test; repeated 3 separate times). B, In‐cell Western analysis of KLF 2 expression from HUVEC s treated with increasing concentrations of CML ‐modified BSA (n=8 per group; repeated 3 separate times). Quantification of KLF 2 protein expression is relative to untreated cells and normalized to β‐tubulin expression. A sigmoidal fit of the densitometry data was used to determine the IC 50 of CML . C, Changes in KLF 2 mRNA expression were assessed over a 24‐hour time course by quantitative real‐time PCR (qPCR) in HUVEC s incubated for 24 hours with CML (5.4 mg/L). One‐way ANOVA with the Dunn post hoc test was used to calculate statistical significance between time points (n=6 per group; repeated 3 separate times). D and E, Knockdown of RAGE expression abolished the suppression of endothelial KLF 2 after CML treatment. RAGE or scrambled (scr) small interfering RNA was transfected before treatment with CML (5.4 mg/L) for 24 hours. Expression of KLF 2 (D) and RAGE (E) mRNA was assessed by qPCR (n=4 per group; 2‐way ANOVA with the Dunn post hoc test; repeated 3 separate times). Data are presented as mean± SEM . NS indicates nonsignificant. * P
    Figure Legend Snippet: Uremic advanced glycation end products ( AGE s) suppress endothelial Krüppel‐like factor 2 ( KLF 2) through receptor for AGE ( RAGE ) signaling. A, Western blot analysis and quantification of KLF 2 from human umbilical vein endothelial cells ( HUVEC s) exposed to individual protein‐bound uremic toxins, including carboxymethyllysine (CML)‐modified BSA, p‐cresol sulfate ( PCS ), indoxyl sulfate ( IS ), or vehicle BSA for 24 hours (n=5 per group; 1‐way ANOVA with the Dunn post hoc test; repeated 3 separate times). B, In‐cell Western analysis of KLF 2 expression from HUVEC s treated with increasing concentrations of CML ‐modified BSA (n=8 per group; repeated 3 separate times). Quantification of KLF 2 protein expression is relative to untreated cells and normalized to β‐tubulin expression. A sigmoidal fit of the densitometry data was used to determine the IC 50 of CML . C, Changes in KLF 2 mRNA expression were assessed over a 24‐hour time course by quantitative real‐time PCR (qPCR) in HUVEC s incubated for 24 hours with CML (5.4 mg/L). One‐way ANOVA with the Dunn post hoc test was used to calculate statistical significance between time points (n=6 per group; repeated 3 separate times). D and E, Knockdown of RAGE expression abolished the suppression of endothelial KLF 2 after CML treatment. RAGE or scrambled (scr) small interfering RNA was transfected before treatment with CML (5.4 mg/L) for 24 hours. Expression of KLF 2 (D) and RAGE (E) mRNA was assessed by qPCR (n=4 per group; 2‐way ANOVA with the Dunn post hoc test; repeated 3 separate times). Data are presented as mean± SEM . NS indicates nonsignificant. * P

    Techniques Used: Western Blot, Modification, In-Cell ELISA, Expressing, Real-time Polymerase Chain Reaction, Incubation, Small Interfering RNA, Transfection

    Uremic suppression of Krüppel‐like factor 2 (KLF2) is not blocked by the receptor for advanced glycation end product (RAGE) antagonist azeliragon (TTP488). A, Human umbilical vein endothelial cells (HUVECs) were treated with 1 to 5 μmol/L of the RAGE antagonist TTP 488 for 4 hours, followed by exposure to 5.4 mg/L carboxymethyllysine ( CML )‐modified BSA for 12 hours. KLF 2 transcript levels were determined by real‐time quantitative (qPCR). B through E, HUVEC s were pretreated with 5 μmol/L TTP 488, followed by exposure to 10% uremic ( UPS ) or nonuremic (NPS) porcine serum for 12 hours. Expression levels of KLF 2 (B) and regulated genes, including endothelial NO synthase ( eNOS; C), thrombomodulin ( TBMD ; D), and vascular cell adhesion molecule 1 ( VCAM 1; E), were assessed by qPCR (n=4 per group; repeated 3 separate times). Data are presented as mean± SEM . NS indicates nonsignificant. * P
    Figure Legend Snippet: Uremic suppression of Krüppel‐like factor 2 (KLF2) is not blocked by the receptor for advanced glycation end product (RAGE) antagonist azeliragon (TTP488). A, Human umbilical vein endothelial cells (HUVECs) were treated with 1 to 5 μmol/L of the RAGE antagonist TTP 488 for 4 hours, followed by exposure to 5.4 mg/L carboxymethyllysine ( CML )‐modified BSA for 12 hours. KLF 2 transcript levels were determined by real‐time quantitative (qPCR). B through E, HUVEC s were pretreated with 5 μmol/L TTP 488, followed by exposure to 10% uremic ( UPS ) or nonuremic (NPS) porcine serum for 12 hours. Expression levels of KLF 2 (B) and regulated genes, including endothelial NO synthase ( eNOS; C), thrombomodulin ( TBMD ; D), and vascular cell adhesion molecule 1 ( VCAM 1; E), were assessed by qPCR (n=4 per group; repeated 3 separate times). Data are presented as mean± SEM . NS indicates nonsignificant. * P

    Techniques Used: Modification, Real-time Polymerase Chain Reaction, Expressing

    7) Product Images from "Human umbilical cord multipotent mesenchymal stromal cells alleviate acute ischemia-reperfusion injury of spermatogenic cells via reducing inflammatory response and oxidative stress"

    Article Title: Human umbilical cord multipotent mesenchymal stromal cells alleviate acute ischemia-reperfusion injury of spermatogenic cells via reducing inflammatory response and oxidative stress

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-020-01813-5

    hUC-MSC-CM inhibits inflammatory factors production and inflammatory pathway in HUVECs. HUVECs stimulated by 10 ng/ml TNF-α were cultured with or without hUC-MSC-CM. Twenty-four hours later, the cells were harvested for RNA extract. a Real-time PCR analysis of mRNA levels of TNF-α, IL-1β, and Selectin-E of HUVECs. The level of mRNA in HUVECs was set as 1. Data was collected from at least 3 separated experiments. b ELISA analysis of IL-1β concentration in the supernatant of HUVECs. c Western blot analysis of Selectin-E expression of HUVECs with Selectin-E and β-actin antibodies. d Western blot analysis of P65 and P38 activation of HUVECs with antibodies against P-P65, P65, P-P38, P38, and β-actin. CM represents hUC-MSC-CM
    Figure Legend Snippet: hUC-MSC-CM inhibits inflammatory factors production and inflammatory pathway in HUVECs. HUVECs stimulated by 10 ng/ml TNF-α were cultured with or without hUC-MSC-CM. Twenty-four hours later, the cells were harvested for RNA extract. a Real-time PCR analysis of mRNA levels of TNF-α, IL-1β, and Selectin-E of HUVECs. The level of mRNA in HUVECs was set as 1. Data was collected from at least 3 separated experiments. b ELISA analysis of IL-1β concentration in the supernatant of HUVECs. c Western blot analysis of Selectin-E expression of HUVECs with Selectin-E and β-actin antibodies. d Western blot analysis of P65 and P38 activation of HUVECs with antibodies against P-P65, P65, P-P38, P38, and β-actin. CM represents hUC-MSC-CM

    Techniques Used: Cell Culture, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot, Expressing, Activation Assay

    8) Product Images from "DIP2A Functions as a FSTL1 Receptor *"

    Article Title: DIP2A Functions as a FSTL1 Receptor *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.069468

    Involvement of DIP2A in FSTL1 binding to endothelial cells. A , detection of DIP2A on the cell surface of HUVECs. HUVECs were incubated with anti-DIP2A antibody ( red ; mouse IgG, 5 μg/ml) or control mouse IgG ( black ) for 60 min. Cells were stained with Alexa Fluor® 488-conjugated secondary antibody and analyzed using a FACScan. B , immunocytochemical analysis of HUVECs with anti-DIP2A antibody. HUVECs were incubated with anti-DIP2A antibody, followed by staining with Alexa Fluor® 594-conjugated secondary antibody ( red ). Nuclei were stained with 4′,6-diamidino-2-phenylindole ( blue ). Representative pictures are shown. C , reduction of DIP2A mRNA expression in HUVECs following transfection with siRNA against DIP2A. At 48 h after transfection of HUVECs with siRNA against DIP2A or control siRNA, DIP2A mRNA levels were determined by quantitative real-time PCR analysis and expressed relative to 36B4 levels ( n = 3). D , effect of knockdown of DIP2A on the binding of FSTL1 to HUVECs. HUVECs were transfected with siRNA targeting DIP2A (○) or unrelated siRNA (●), incubated with increasing concentrations of biotinylated recombinant FSTL1 for 60 min, and treated with streptavidin-conjugated horseradish peroxidase, followed by incubation with QuantaBlu fluorogenic peroxidase substrate. The binding was assessed with a fluorescent microplate reader ( n = 6–7). The data were analyzed with Microsoft Excel to generate a logarithmic trend line.
    Figure Legend Snippet: Involvement of DIP2A in FSTL1 binding to endothelial cells. A , detection of DIP2A on the cell surface of HUVECs. HUVECs were incubated with anti-DIP2A antibody ( red ; mouse IgG, 5 μg/ml) or control mouse IgG ( black ) for 60 min. Cells were stained with Alexa Fluor® 488-conjugated secondary antibody and analyzed using a FACScan. B , immunocytochemical analysis of HUVECs with anti-DIP2A antibody. HUVECs were incubated with anti-DIP2A antibody, followed by staining with Alexa Fluor® 594-conjugated secondary antibody ( red ). Nuclei were stained with 4′,6-diamidino-2-phenylindole ( blue ). Representative pictures are shown. C , reduction of DIP2A mRNA expression in HUVECs following transfection with siRNA against DIP2A. At 48 h after transfection of HUVECs with siRNA against DIP2A or control siRNA, DIP2A mRNA levels were determined by quantitative real-time PCR analysis and expressed relative to 36B4 levels ( n = 3). D , effect of knockdown of DIP2A on the binding of FSTL1 to HUVECs. HUVECs were transfected with siRNA targeting DIP2A (○) or unrelated siRNA (●), incubated with increasing concentrations of biotinylated recombinant FSTL1 for 60 min, and treated with streptavidin-conjugated horseradish peroxidase, followed by incubation with QuantaBlu fluorogenic peroxidase substrate. The binding was assessed with a fluorescent microplate reader ( n = 6–7). The data were analyzed with Microsoft Excel to generate a logarithmic trend line.

    Techniques Used: Binding Assay, Incubation, Staining, Expressing, Transfection, Real-time Polymerase Chain Reaction, Recombinant

    Interaction of FSTL1 with a novel binding protein, DIP2A. A , detection of FSTL1-binding protein in membrane fractions of HUVECs. Membrane fractions were incubated in the presence or absence of FLAG-FSTL1 protein (2 μg/ml) and then immunoprecipitated with anti-FLAG affinity gel, followed by SDS-PAGE. Proteins were stained with carrier-complexed silver. The arrow indicates possible binding protein partners of FSTL1. B , DIP2A is immunoprecipitated by FLAG-FSTL1 from HUVEC membranes. Immunoprecipitated material was subjected to SDS-PAGE, followed by Western blot ( WB ) analysis with anti-DIP2A and anti-FSTL1 antibodies. C , FSTL1 is immunoprecipitated with nickel resin when DIP2A-His is present in the cell lysates. COS-7 cells were transfected with DIP2A-His or mock-transfected. Cell lysates were incubated in the presence or absence of recombinant FSTL1 protein ( rFstl1 ; 400 ng) for 1 h and then subjected to immunoprecipitation with nickel resin. Immunoprecipitated material was subjected to SDS-PAGE, and Western blot analysis was performed with anti-DIP2A and anti-FSTL1 antibodies.
    Figure Legend Snippet: Interaction of FSTL1 with a novel binding protein, DIP2A. A , detection of FSTL1-binding protein in membrane fractions of HUVECs. Membrane fractions were incubated in the presence or absence of FLAG-FSTL1 protein (2 μg/ml) and then immunoprecipitated with anti-FLAG affinity gel, followed by SDS-PAGE. Proteins were stained with carrier-complexed silver. The arrow indicates possible binding protein partners of FSTL1. B , DIP2A is immunoprecipitated by FLAG-FSTL1 from HUVEC membranes. Immunoprecipitated material was subjected to SDS-PAGE, followed by Western blot ( WB ) analysis with anti-DIP2A and anti-FSTL1 antibodies. C , FSTL1 is immunoprecipitated with nickel resin when DIP2A-His is present in the cell lysates. COS-7 cells were transfected with DIP2A-His or mock-transfected. Cell lysates were incubated in the presence or absence of recombinant FSTL1 protein ( rFstl1 ; 400 ng) for 1 h and then subjected to immunoprecipitation with nickel resin. Immunoprecipitated material was subjected to SDS-PAGE, and Western blot analysis was performed with anti-DIP2A and anti-FSTL1 antibodies.

    Techniques Used: Binding Assay, Incubation, Immunoprecipitation, SDS Page, Staining, Western Blot, Transfection, Recombinant

    Involvement of DIP2A in FSTL1-mediated Akt signaling. HUVECs were transfected with siRNA against DIP2A or control siRNA. A and B , DIP2A mediates the stimulatory actions of FSTL1 on Akt phosphorylation in HUVECs. A , after 16 h of incubation in serum-free medium, siRNA-transfected HUVECs were treated with recombinant FSTL1 ( rFstl1 ; 100 ng/ml) or vehicle for 30 min. B , after transfection with siRNA, HUVECs were transduced with Ad-FSTL1 and Ad-β-galactosidase for 8 h and incubated in serum-free medium for 24 h. Akt phosphorylation ( P-Akt ) levels were determined by Western blot analysis. Representative blots are shown from one of three independent experiments.
    Figure Legend Snippet: Involvement of DIP2A in FSTL1-mediated Akt signaling. HUVECs were transfected with siRNA against DIP2A or control siRNA. A and B , DIP2A mediates the stimulatory actions of FSTL1 on Akt phosphorylation in HUVECs. A , after 16 h of incubation in serum-free medium, siRNA-transfected HUVECs were treated with recombinant FSTL1 ( rFstl1 ; 100 ng/ml) or vehicle for 30 min. B , after transfection with siRNA, HUVECs were transduced with Ad-FSTL1 and Ad-β-galactosidase for 8 h and incubated in serum-free medium for 24 h. Akt phosphorylation ( P-Akt ) levels were determined by Western blot analysis. Representative blots are shown from one of three independent experiments.

    Techniques Used: Transfection, Incubation, Recombinant, Transduction, Western Blot

    9) Product Images from "High Glucose-treated Macrophages Augment E-Selectin Expression in Endothelial Cells *"

    Article Title: High Glucose-treated Macrophages Augment E-Selectin Expression in Endothelial Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.230540

    Luciferase reporter analysis of the E-selectin promoter and the roles of NF-κB and AP-1 in HG-MCM-induction of E-selectin promoter activity and transcription in HUVECs. A , HUVECs were cotransfected with 5′-deletion constructs and stimulated with HG-MCM for 4 h. E-selectin promoter activity was measured by luciferase assay normalized to β-galactosidase activity and is shown relative to that of HUVECs transfected with p540-Luc (set at 100%). B , NF-κB p65 and AP-1 activation levels were determined by TF ELISA. All bar graphs represent fold increases over the levels in control ECs ( CL ), calculated as the mean ± S.E. from three independent experiments. *, p
    Figure Legend Snippet: Luciferase reporter analysis of the E-selectin promoter and the roles of NF-κB and AP-1 in HG-MCM-induction of E-selectin promoter activity and transcription in HUVECs. A , HUVECs were cotransfected with 5′-deletion constructs and stimulated with HG-MCM for 4 h. E-selectin promoter activity was measured by luciferase assay normalized to β-galactosidase activity and is shown relative to that of HUVECs transfected with p540-Luc (set at 100%). B , NF-κB p65 and AP-1 activation levels were determined by TF ELISA. All bar graphs represent fold increases over the levels in control ECs ( CL ), calculated as the mean ± S.E. from three independent experiments. *, p

    Techniques Used: Luciferase, Activity Assay, Construct, Transfection, Activation Assay, Enzyme-linked Immunosorbent Assay

    10) Product Images from "High Glucose Activates YAP Signaling to Promote Vascular Inflammation"

    Article Title: High Glucose Activates YAP Signaling to Promote Vascular Inflammation

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2021.665994

    Effects of high glucose and shear stress on YAP activation in endothelial cells. (A) Representative protein expression blots and bar graphs of phospho and total YAP, phospho and total TAZ in HUVECs cultured under either normal or high glucose and subjected to either laminar flow (LF, 12 dyn/cm 2 ) or oscillatory flow (OF, 0.5 ± 6 dyn/cm 2 ; 1 Hz) for 72 h. (B) Representative protein expression blots of VCAM-1 and CYR61 and bar graphs of VCAM-1 in HUVECs cultured under either normal or high glucose and subjected to LF or OF for 72 h. (C) Representative microphotographs and bar graphs showing THP-1 monocyte attachments to HUVECs cultured under either normal or high glucose, subjected to LF for 72 h and additionally treated with the YAP/TEAD inhibitor K-975 (200 nM). DMSO was used as control for K-975. (D) Representative microphotographs and bar graphs showing THP-1 monocyte attachments to HUVECs cultured under either normal or high glucose and subjected to either LF or OF for 72 h. THP-1 monocytes are GFP positive (green fluorescence). Nuclei are stained by DAPI (blue fluorescence). Data are presented as the mean ± SEM. N = 4–11/group for panel A; N = 6–15/group for panel B; N = 4–5/group for panel C and N = 5–6/group for panel D. ∗ p
    Figure Legend Snippet: Effects of high glucose and shear stress on YAP activation in endothelial cells. (A) Representative protein expression blots and bar graphs of phospho and total YAP, phospho and total TAZ in HUVECs cultured under either normal or high glucose and subjected to either laminar flow (LF, 12 dyn/cm 2 ) or oscillatory flow (OF, 0.5 ± 6 dyn/cm 2 ; 1 Hz) for 72 h. (B) Representative protein expression blots of VCAM-1 and CYR61 and bar graphs of VCAM-1 in HUVECs cultured under either normal or high glucose and subjected to LF or OF for 72 h. (C) Representative microphotographs and bar graphs showing THP-1 monocyte attachments to HUVECs cultured under either normal or high glucose, subjected to LF for 72 h and additionally treated with the YAP/TEAD inhibitor K-975 (200 nM). DMSO was used as control for K-975. (D) Representative microphotographs and bar graphs showing THP-1 monocyte attachments to HUVECs cultured under either normal or high glucose and subjected to either LF or OF for 72 h. THP-1 monocytes are GFP positive (green fluorescence). Nuclei are stained by DAPI (blue fluorescence). Data are presented as the mean ± SEM. N = 4–11/group for panel A; N = 6–15/group for panel B; N = 4–5/group for panel C and N = 5–6/group for panel D. ∗ p

    Techniques Used: Activation Assay, Expressing, Cell Culture, Fluorescence, Staining

    11) Product Images from "Ghrelin suppresses inflammation in HUVECs by inhibiting ubiquitin-mediated uncoupling protein 2 degradation"

    Article Title: Ghrelin suppresses inflammation in HUVECs by inhibiting ubiquitin-mediated uncoupling protein 2 degradation

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2017.2977

    Ghrelin inhibits the oxidized low-density lipoprotein (oxLDL)-induced inflammatory response in human umbilical vein endothelial cells (HUVECs). (A) HUVECs were either pre-treated with ghrelin or left untreated, followed by oxLDL stimulation. RT-qPCR was conducted to determine the mRNA levels of IL-6, CCL-2, ICAM-1 and VCAM-1. (B) HUVECs were pre-treated with gradiatint concentrations of ghrelin followed by oxLDL stimulation. RT-qPCR was conducted to monitor the mRNA levels of IL-6, CCL-2, ICAM-1 and VCAM-1. Quantitative experiments were repeated at least 3 times. * P
    Figure Legend Snippet: Ghrelin inhibits the oxidized low-density lipoprotein (oxLDL)-induced inflammatory response in human umbilical vein endothelial cells (HUVECs). (A) HUVECs were either pre-treated with ghrelin or left untreated, followed by oxLDL stimulation. RT-qPCR was conducted to determine the mRNA levels of IL-6, CCL-2, ICAM-1 and VCAM-1. (B) HUVECs were pre-treated with gradiatint concentrations of ghrelin followed by oxLDL stimulation. RT-qPCR was conducted to monitor the mRNA levels of IL-6, CCL-2, ICAM-1 and VCAM-1. Quantitative experiments were repeated at least 3 times. * P

    Techniques Used: Quantitative RT-PCR

    12) Product Images from "Integrated bioinformatics analysis identifies microRNA-376a-3p as a new microRNA biomarker in patient with coronary artery disease"

    Article Title: Integrated bioinformatics analysis identifies microRNA-376a-3p as a new microRNA biomarker in patient with coronary artery disease

    Journal: American Journal of Translational Research

    doi:

    Downregulation of miR-376a-3p inhibited proliferation of HUVECs. A. HUVECs were transfected with 10 nM miR-376a-3p mimics or inhibitor for 72 h. RT-qPCR was used to detect the level of miR-376a-3p in HUVECs. **P
    Figure Legend Snippet: Downregulation of miR-376a-3p inhibited proliferation of HUVECs. A. HUVECs were transfected with 10 nM miR-376a-3p mimics or inhibitor for 72 h. RT-qPCR was used to detect the level of miR-376a-3p in HUVECs. **P

    Techniques Used: Transfection, Quantitative RT-PCR

    Overexpression of miR-376a-3p increased growth of HUVECs via downregulating NRIP1. A. Sequence alignment of miR-376a-3p with the putative binding sites within the WT regions of NRIP1. B. Dual luciferase reporter assay was used to detect the luciferase activity in HUVECs following co-transfecting with NRIP1-WT/MUT 3’-UTR plasmid and miR-376-3p mimics. **P
    Figure Legend Snippet: Overexpression of miR-376a-3p increased growth of HUVECs via downregulating NRIP1. A. Sequence alignment of miR-376a-3p with the putative binding sites within the WT regions of NRIP1. B. Dual luciferase reporter assay was used to detect the luciferase activity in HUVECs following co-transfecting with NRIP1-WT/MUT 3’-UTR plasmid and miR-376-3p mimics. **P

    Techniques Used: Over Expression, Sequencing, Binding Assay, Luciferase, Reporter Assay, Activity Assay, Plasmid Preparation

    Downregulation of miR-376a-3p induced apoptosis of HUVECs. A, B. HUVECs were transfected with 10 nM miR-376a-3p inhibitor for 72 h. Apoptotic cells were detected with Annexin V and PI double staining. Apoptosis cell rates were calculated. **P
    Figure Legend Snippet: Downregulation of miR-376a-3p induced apoptosis of HUVECs. A, B. HUVECs were transfected with 10 nM miR-376a-3p inhibitor for 72 h. Apoptotic cells were detected with Annexin V and PI double staining. Apoptosis cell rates were calculated. **P

    Techniques Used: Transfection, Double Staining

    13) Product Images from "Oxidized Low Density Lipoprotein-Induced Atherogenic Response of Human Umbilical Vascular Endothelial Cells (HUVECs) was Protected by Atorvastatin by Regulating miR-26a-5p/Phosphatase and Tensin Homolog (PTEN)"

    Article Title: Oxidized Low Density Lipoprotein-Induced Atherogenic Response of Human Umbilical Vascular Endothelial Cells (HUVECs) was Protected by Atorvastatin by Regulating miR-26a-5p/Phosphatase and Tensin Homolog (PTEN)

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.918405

    MiR-26a-5p inhibits ox-LDL-induced migration and the release of adhesion-related molecules by regulating PTEN. ( A ) Predicted consequential pairing of PTEN and miR-26a-5p. ( B ) qRT-PCR was applied to detect the expression of PTEN in HUVECs after transfection with miR-26a-5p mimic, miR-26a-5p inhibitor and NC; n=6. ** P
    Figure Legend Snippet: MiR-26a-5p inhibits ox-LDL-induced migration and the release of adhesion-related molecules by regulating PTEN. ( A ) Predicted consequential pairing of PTEN and miR-26a-5p. ( B ) qRT-PCR was applied to detect the expression of PTEN in HUVECs after transfection with miR-26a-5p mimic, miR-26a-5p inhibitor and NC; n=6. ** P

    Techniques Used: Migration, Quantitative RT-PCR, Expressing, Transfection

    ( A–D ) The expressions of miR-26a-5p, miR-29b, miR-214, and miR-363-3p were downregulated by ATV treatment. RNA levels of miR-26a-5p, miR-29b, miR-214, and miR-363-3p in HUVECs after being treated with 100 mg/L ox-LDL and 10 μM ATV for 24 hours. ### P
    Figure Legend Snippet: ( A–D ) The expressions of miR-26a-5p, miR-29b, miR-214, and miR-363-3p were downregulated by ATV treatment. RNA levels of miR-26a-5p, miR-29b, miR-214, and miR-363-3p in HUVECs after being treated with 100 mg/L ox-LDL and 10 μM ATV for 24 hours. ### P

    Techniques Used:

    ATV inhibits ox-LDL-induced apoptosis and migration of HUVECs by downregulating the expression of miR-26a-5p. ( A, B ) qRT-PCR was applied to detect the expression of miR-26a-5p in HUVECs after transfection with ( A ) miR-26a-5p mimic, ( B ) miR-26a-5p inhibitor and negative control (NC). ( C ) TUNEL staining was used to observe the percentage of apoptotic cells. ( D–F ) After being treated by ATV and ox-LDL for 24 hours, HUVECs were transfected with miR-26a-5p mimic, miR-26a-5p inhibitor, or NC to detect the ( D ) relative migration ratio, mRNA levels of ( E ) MCP-1 and ( F ) ICAM-1. Scale bar: 20 μm. ### P
    Figure Legend Snippet: ATV inhibits ox-LDL-induced apoptosis and migration of HUVECs by downregulating the expression of miR-26a-5p. ( A, B ) qRT-PCR was applied to detect the expression of miR-26a-5p in HUVECs after transfection with ( A ) miR-26a-5p mimic, ( B ) miR-26a-5p inhibitor and negative control (NC). ( C ) TUNEL staining was used to observe the percentage of apoptotic cells. ( D–F ) After being treated by ATV and ox-LDL for 24 hours, HUVECs were transfected with miR-26a-5p mimic, miR-26a-5p inhibitor, or NC to detect the ( D ) relative migration ratio, mRNA levels of ( E ) MCP-1 and ( F ) ICAM-1. Scale bar: 20 μm. ### P

    Techniques Used: Migration, Expressing, Quantitative RT-PCR, Transfection, Negative Control, TUNEL Assay, Staining

    14) Product Images from "The Anti-Angiogenic Activity of a Cystatin F Homologue from the Buccal Glands of Lampetra morii"

    Article Title: The Anti-Angiogenic Activity of a Cystatin F Homologue from the Buccal Glands of Lampetra morii

    Journal: Marine Drugs

    doi: 10.3390/md16120477

    MTT assay showed the inhibitory effects of rLm-cystatin F on the HUVEC’s proliferation. PBS was used as a negative control. The same volume of rLm-cystatin F (0, 1.9, 3.8, 5.7, 7.5, 9.4, and 11.3 μM, final concentration) was added into the HUVECs in the 96-well plates at 37 °C for 24 h. Relative to the PBS group, * p
    Figure Legend Snippet: MTT assay showed the inhibitory effects of rLm-cystatin F on the HUVEC’s proliferation. PBS was used as a negative control. The same volume of rLm-cystatin F (0, 1.9, 3.8, 5.7, 7.5, 9.4, and 11.3 μM, final concentration) was added into the HUVECs in the 96-well plates at 37 °C for 24 h. Relative to the PBS group, * p

    Techniques Used: MTT Assay, Negative Control, Concentration Assay

    Transwell assays showed the inhibitory effects of rLm-cystatin F on the HUVEC’s migration ( a ) and invasion ( b ). PBS was used as a negative control. Relative to the PBS group, * p
    Figure Legend Snippet: Transwell assays showed the inhibitory effects of rLm-cystatin F on the HUVEC’s migration ( a ) and invasion ( b ). PBS was used as a negative control. Relative to the PBS group, * p

    Techniques Used: Migration, Negative Control

    MTT assay showed that rLm-cystatin F thwarted HUVECs adhered to fibronectin, laminin, and collagen IV. PBS (0 µM rLm-cystatin F) was used as a negative control. Relative to the PBS group, * p
    Figure Legend Snippet: MTT assay showed that rLm-cystatin F thwarted HUVECs adhered to fibronectin, laminin, and collagen IV. PBS (0 µM rLm-cystatin F) was used as a negative control. Relative to the PBS group, * p

    Techniques Used: MTT Assay, Negative Control

    The inhibitory effects of rLm-cystatin F on the tube formation from HUVECs in vitro. PBS was used as a negative control. Relative to the PBS group, ** p
    Figure Legend Snippet: The inhibitory effects of rLm-cystatin F on the tube formation from HUVECs in vitro. PBS was used as a negative control. Relative to the PBS group, ** p

    Techniques Used: In Vitro, Negative Control

    15) Product Images from "Elevated miR-195-5p expression in deep vein thrombosis and mechanism of action in the regulation of vascular endothelial cell physiology"

    Article Title: Elevated miR-195-5p expression in deep vein thrombosis and mechanism of action in the regulation of vascular endothelial cell physiology

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2019.8166

    Effect of miR-195-5p downregulation on HUVECs. (A) After transfection with inhibitor control or miR-195-5p inhibitor for 48 h, the level of miR-195-5p in HUVECs was detected using RT-qPCR. (B) After transfection with inhibitor control or miR-195-5p inhibitor for 48 h, the cell viability of HUVECs was detected using MTT assay. (C) After transfection with inhibitor control or miR-195-5p inhibitor for 48 h, cell apoptosis of HUVECs was detected using flow cytometry, and the cell apoptosis rate (Q2 + Q4) was calculated and presented. After transfection with inhibitor control or miR-195-5p inhibitor for 48 h, the mRNA level of (D) Bcl-2 and (E) Bax in HUVECs was detected using RT-qPCR. (F) After transfection with inhibitor control or miR-195-5p inhibitor for 48 h, the protein level of Bcl-2 and Bax in HUVECs was detected using western blotting. Control, HUVECs without any treatment; inhibitor control, HUVECs transfected with inhibitor control for 48 h; inhibitor, HUVECs were transfected with miR-195-5p inhibitor for 48 h. Data are presented as the mean ± SD. *P
    Figure Legend Snippet: Effect of miR-195-5p downregulation on HUVECs. (A) After transfection with inhibitor control or miR-195-5p inhibitor for 48 h, the level of miR-195-5p in HUVECs was detected using RT-qPCR. (B) After transfection with inhibitor control or miR-195-5p inhibitor for 48 h, the cell viability of HUVECs was detected using MTT assay. (C) After transfection with inhibitor control or miR-195-5p inhibitor for 48 h, cell apoptosis of HUVECs was detected using flow cytometry, and the cell apoptosis rate (Q2 + Q4) was calculated and presented. After transfection with inhibitor control or miR-195-5p inhibitor for 48 h, the mRNA level of (D) Bcl-2 and (E) Bax in HUVECs was detected using RT-qPCR. (F) After transfection with inhibitor control or miR-195-5p inhibitor for 48 h, the protein level of Bcl-2 and Bax in HUVECs was detected using western blotting. Control, HUVECs without any treatment; inhibitor control, HUVECs transfected with inhibitor control for 48 h; inhibitor, HUVECs were transfected with miR-195-5p inhibitor for 48 h. Data are presented as the mean ± SD. *P

    Techniques Used: Transfection, Quantitative RT-PCR, MTT Assay, Flow Cytometry, Cytometry, Western Blot

    Effect of miR-195-5p upregulation on HUVECs. (A) After transfection with mimic control or miR-195-5p mimic for 48 h, the level of miR-195-5p in HUVECs was detected using RT-qPCR. After transfection with control-plasmid or Bcl-2-plasmid for 48 h, the mRNA and protein level of Bcl-2 in HUVECs was detected using (B) RT-qPCR and (C) western blotting. (D) After transfection with mimic control, miR-195-5p mimic or miR-195-5p mimic + Bcl-2-plasmid for 48 h, the cell viability of HUVECs was detected using MTT assay. (E) After transfection with mimic control, miR-195-5p mimic or miR-195-5p mimic + Bcl-2-plasmid for 48 h, the cell apoptosis of HUVECs was detected using flow cytometry, and the cell apoptosis rate (Q2 + Q4) was calculated and presented. (F) After transfection with mimic control, miR-195-5p mimic or miR-195-5p mimic + Bcl-2-plasmid for 48 h, the protein level of Bcl-2 and Bax in HUVECs was detected using western blotting. After transfection with mimic control, miR-195-5p mimic or miR-195-5p mimic+Bcl-2-plasmid for 48 h, the mRNA level of (G) Bcl-2 and (H) Bax in HUVECs was detected using RT-qPCR. Control, HUVECs without any treatment; mimic control, HUVECs transfected with mimic control for 48 h; miR-195-5p mimic, HUVECs transfected with miR-195-5p mimic for 48 h; control-plasmid, HUVECs transfected with control-plasmid for 48 h; Bcl-2-plasmid, HUVECs transfected with Bcl-2-plasmid for 48 h; miR-195-5p mimic+Bcl-2-plasmid, HUVECs co-transfected with miR-195-5p mimic + Bcl-2-plasmid for 48 h. Data are presented as the mean ± SD. **P
    Figure Legend Snippet: Effect of miR-195-5p upregulation on HUVECs. (A) After transfection with mimic control or miR-195-5p mimic for 48 h, the level of miR-195-5p in HUVECs was detected using RT-qPCR. After transfection with control-plasmid or Bcl-2-plasmid for 48 h, the mRNA and protein level of Bcl-2 in HUVECs was detected using (B) RT-qPCR and (C) western blotting. (D) After transfection with mimic control, miR-195-5p mimic or miR-195-5p mimic + Bcl-2-plasmid for 48 h, the cell viability of HUVECs was detected using MTT assay. (E) After transfection with mimic control, miR-195-5p mimic or miR-195-5p mimic + Bcl-2-plasmid for 48 h, the cell apoptosis of HUVECs was detected using flow cytometry, and the cell apoptosis rate (Q2 + Q4) was calculated and presented. (F) After transfection with mimic control, miR-195-5p mimic or miR-195-5p mimic + Bcl-2-plasmid for 48 h, the protein level of Bcl-2 and Bax in HUVECs was detected using western blotting. After transfection with mimic control, miR-195-5p mimic or miR-195-5p mimic+Bcl-2-plasmid for 48 h, the mRNA level of (G) Bcl-2 and (H) Bax in HUVECs was detected using RT-qPCR. Control, HUVECs without any treatment; mimic control, HUVECs transfected with mimic control for 48 h; miR-195-5p mimic, HUVECs transfected with miR-195-5p mimic for 48 h; control-plasmid, HUVECs transfected with control-plasmid for 48 h; Bcl-2-plasmid, HUVECs transfected with Bcl-2-plasmid for 48 h; miR-195-5p mimic+Bcl-2-plasmid, HUVECs co-transfected with miR-195-5p mimic + Bcl-2-plasmid for 48 h. Data are presented as the mean ± SD. **P

    Techniques Used: Transfection, Quantitative RT-PCR, Plasmid Preparation, Western Blot, MTT Assay, Flow Cytometry, Cytometry

    Bcl-2 is a direct target gene of miR-195-5p. (A) TargetScan software was used to predict a binding site for miR-195-5p in the 3′UTR of Bcl-2. (B) Luciferase activity of a dual-luciferase reporter vector containing wild-type 3′UTR-Bcl-2 or a mutant 3′UTR-Bcl-2. Data are presented as the mean ± SD of three independent experiments. (C) The mRNA expression levels of Bcl-2 in the peripheral blood from 15 healthy volunteers (Control) and in the peripheral blood from 15 DVT patients using reverse transcription-quantitative PCR. (D) Representative western blotting of Bcl-2 in the peripheral blood from 2 healthy volunteers (Control) and in the peripheral blood from 2 DVT patients. (E) The ratio of Bcl-2/β-actin was calculated in the different groups. WT, wild-type; MUT, mutant-type; WT-Bcl-2, HUVECs co-transfected with WT 3′UTR-Bcl-2 and either mimic control or miR-195-5p; MUT-Bcl-2, HUVECs transfected with MUT 3′UTR-Bcl-2. Data are presented as the mean ± SD. **P
    Figure Legend Snippet: Bcl-2 is a direct target gene of miR-195-5p. (A) TargetScan software was used to predict a binding site for miR-195-5p in the 3′UTR of Bcl-2. (B) Luciferase activity of a dual-luciferase reporter vector containing wild-type 3′UTR-Bcl-2 or a mutant 3′UTR-Bcl-2. Data are presented as the mean ± SD of three independent experiments. (C) The mRNA expression levels of Bcl-2 in the peripheral blood from 15 healthy volunteers (Control) and in the peripheral blood from 15 DVT patients using reverse transcription-quantitative PCR. (D) Representative western blotting of Bcl-2 in the peripheral blood from 2 healthy volunteers (Control) and in the peripheral blood from 2 DVT patients. (E) The ratio of Bcl-2/β-actin was calculated in the different groups. WT, wild-type; MUT, mutant-type; WT-Bcl-2, HUVECs co-transfected with WT 3′UTR-Bcl-2 and either mimic control or miR-195-5p; MUT-Bcl-2, HUVECs transfected with MUT 3′UTR-Bcl-2. Data are presented as the mean ± SD. **P

    Techniques Used: Software, Binding Assay, Luciferase, Activity Assay, Plasmid Preparation, Mutagenesis, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection

    16) Product Images from "Elucidation of Stannin Function Using Microarray Analysis: Implications for Cell Cycle Control"

    Article Title: Elucidation of Stannin Function Using Microarray Analysis: Implications for Cell Cycle Control

    Journal: Gene Expression

    doi:

    Microarray experimental design. Four groups of HUVECs were plated (two biological replicates per group). The groups were: a control group grown in EGM complete media, a group that was grown as control but that received a 200 ng/ml dose of TNF-α 60 h after plating, a group grown as control that received 20 nM Snn siRNA 24 h after plating, and a “Combo” group that was both transfected with Snn siRNA and dosed with TNF-α at the appropriate time points. Before any treatments, HUVECs were allowed 24 h of undisturbed growth subsequent to plating. The RNA isolated from each plate of HUVECs at the “Harvest” time point was split into three aliquots, each used on a separate microarray for a total of six technical replicates per group (one array each from the control and combo groups did not process correctly and so these groups have n = 5 rather than 6).
    Figure Legend Snippet: Microarray experimental design. Four groups of HUVECs were plated (two biological replicates per group). The groups were: a control group grown in EGM complete media, a group that was grown as control but that received a 200 ng/ml dose of TNF-α 60 h after plating, a group grown as control that received 20 nM Snn siRNA 24 h after plating, and a “Combo” group that was both transfected with Snn siRNA and dosed with TNF-α at the appropriate time points. Before any treatments, HUVECs were allowed 24 h of undisturbed growth subsequent to plating. The RNA isolated from each plate of HUVECs at the “Harvest” time point was split into three aliquots, each used on a separate microarray for a total of six technical replicates per group (one array each from the control and combo groups did not process correctly and so these groups have n = 5 rather than 6).

    Techniques Used: Microarray, Transfection, Isolation

    Cotreatment with TNF-α and Snn siRNA alters HUVEC progression through the cell cycle. The proportion of cells in each phase of the cell cycle was analyzed via flow cytometry after propidium iodide staining. HUVECs exposed to both TNF-α and Snn siRNA showed significant changes in their cell cycle compared to those treated with either TNF-α or Snn siRNA alone. HUVECs were transfected with Snn siRNA (20 nM for 24 h) and were then treated with TNF-α (200 ng/ml) and allowed to incubate for an additional 24 h. Neither TNF-α nor Snn siRNA alone had any effect on the progression of HUVECs through the cell cycle. * p
    Figure Legend Snippet: Cotreatment with TNF-α and Snn siRNA alters HUVEC progression through the cell cycle. The proportion of cells in each phase of the cell cycle was analyzed via flow cytometry after propidium iodide staining. HUVECs exposed to both TNF-α and Snn siRNA showed significant changes in their cell cycle compared to those treated with either TNF-α or Snn siRNA alone. HUVECs were transfected with Snn siRNA (20 nM for 24 h) and were then treated with TNF-α (200 ng/ml) and allowed to incubate for an additional 24 h. Neither TNF-α nor Snn siRNA alone had any effect on the progression of HUVECs through the cell cycle. * p

    Techniques Used: Flow Cytometry, Cytometry, Staining, Transfection

    Alteration of HUVEC cell growth by treatment with TNF-α and/or Snn siRNA. Cell numbers were determined using a hemacytometer after treatment with TNF-α (200 ng/ml) for up to 48 h with/without exposure to Snn siRNA (20 nM for up to 72 h). Growth is presented as a percent of the plating density of 2.0 ± 10 5 cells. Treatment with TNF-α alone resulted in significant inhibition of growth at both 24 and 48 h of exposure (72 and 96 h of growth). Treatment of TNF-α-stimulated HUVECs with Snn siRNA resulted in significant inhibition of cell growth compared with TNF-α alone or siRNA alone and both 24 and 48 h of TNF-α exposure. Treatment of Snn siRNA alone resulted in a significant inhibition of cell growth compared to vehicle-treated controls by 72 h of exposure. * p
    Figure Legend Snippet: Alteration of HUVEC cell growth by treatment with TNF-α and/or Snn siRNA. Cell numbers were determined using a hemacytometer after treatment with TNF-α (200 ng/ml) for up to 48 h with/without exposure to Snn siRNA (20 nM for up to 72 h). Growth is presented as a percent of the plating density of 2.0 ± 10 5 cells. Treatment with TNF-α alone resulted in significant inhibition of growth at both 24 and 48 h of exposure (72 and 96 h of growth). Treatment of TNF-α-stimulated HUVECs with Snn siRNA resulted in significant inhibition of cell growth compared with TNF-α alone or siRNA alone and both 24 and 48 h of TNF-α exposure. Treatment of Snn siRNA alone resulted in a significant inhibition of cell growth compared to vehicle-treated controls by 72 h of exposure. * p

    Techniques Used: Inhibition

    17) Product Images from "Effects of substrate stiffness on the biological behavior of human umbilical vein endothelial cells"

    Article Title: Effects of substrate stiffness on the biological behavior of human umbilical vein endothelial cells

    Journal: bioRxiv

    doi: 10.1101/803312

    Total GAG content in HUVECs cultured under different substrate stiffness/rigidity. All experiments were repeated independently in triplicate. Bars are standard deviation (SD) based on three replicate samples. Different letters indicate significant differences in t- test ( p
    Figure Legend Snippet: Total GAG content in HUVECs cultured under different substrate stiffness/rigidity. All experiments were repeated independently in triplicate. Bars are standard deviation (SD) based on three replicate samples. Different letters indicate significant differences in t- test ( p

    Techniques Used: Cell Culture, Standard Deviation

    Effects of different substrate stiffness/rigidity on CX40 protein expression in HUVECs. All experiments were repeated independently in triplicate. Bars are standard deviation (SD) based on three replicate samples. Different letters indicate significant differences in t- test ( p
    Figure Legend Snippet: Effects of different substrate stiffness/rigidity on CX40 protein expression in HUVECs. All experiments were repeated independently in triplicate. Bars are standard deviation (SD) based on three replicate samples. Different letters indicate significant differences in t- test ( p

    Techniques Used: Expressing, Standard Deviation

    Effects of substrate stiffness/rigidity on CX40 mRNA expression in HUVECs. All experiments were repeated independently in triplicate. Bars are standard deviation (SD) based on three replicate samples. Different letters indicate significant differences in t- test ( p
    Figure Legend Snippet: Effects of substrate stiffness/rigidity on CX40 mRNA expression in HUVECs. All experiments were repeated independently in triplicate. Bars are standard deviation (SD) based on three replicate samples. Different letters indicate significant differences in t- test ( p

    Techniques Used: Expressing, Standard Deviation

    HUVEC staining with FITC phalloidin. All experiments were repeated independently in triplicate. Different letters indicate significant differences in t- test ( p
    Figure Legend Snippet: HUVEC staining with FITC phalloidin. All experiments were repeated independently in triplicate. Different letters indicate significant differences in t- test ( p

    Techniques Used: Staining

    Effects of substrate stiffness/rigidity on proliferation of HUVECs based on bromodeoxyuridine (BrdU) assay. All experiments were repeated independently in triplicate. Different letters indicate significant differences in t- test ( p
    Figure Legend Snippet: Effects of substrate stiffness/rigidity on proliferation of HUVECs based on bromodeoxyuridine (BrdU) assay. All experiments were repeated independently in triplicate. Different letters indicate significant differences in t- test ( p

    Techniques Used: BrdU Staining

    18) Product Images from "A steroid like phytochemical Antcin M is an anti-aging reagent that eliminates hyperglycemia-accelerated premature senescence in dermal fibroblasts by direct activation of Nrf2 and SIRT-1"

    Article Title: A steroid like phytochemical Antcin M is an anti-aging reagent that eliminates hyperglycemia-accelerated premature senescence in dermal fibroblasts by direct activation of Nrf2 and SIRT-1

    Journal: Oncotarget

    doi: 10.18632/oncotarget.11229

    Schematic representation of antcin M-mediated protection against HG-accelarated stress-induced premature senescence in HNDFs and HUVECs Hyperglycemia induces intracellular ROS, which triggers p38 MAPK and JNK/SAMP activation. The activated p38 MAPK and JNK/SAPK promotes transcriptional activation of p53 and FoxO1 by acetylation. P53 and FoxO1-mediated up-regulation of p16 INK4A and p21 CIP1 distrubs cyclins and CDKs, which increase protein stability of pRB and allow to G 0 /G 1 cell-cycle arrest and senescence. Conversely, activated p38 MAPK and JNK/SAPK reduce SIRT-1 level by phosphorylating Ser47, eventually losing deacetylation activity. However, treatment with antcin M activates Nrf2-dependent anti-oxidant genes such as HO-1 and NQO-1 followed by activation of PI3K/AKT and ER1/2 kinases, which facilitates ROS inhibition and upregulates SIRT-1 expression in HNDFs and HUVECs. Results expressed as mean ± SEM of three indipendent expriments. Statistical significance at Ф P
    Figure Legend Snippet: Schematic representation of antcin M-mediated protection against HG-accelarated stress-induced premature senescence in HNDFs and HUVECs Hyperglycemia induces intracellular ROS, which triggers p38 MAPK and JNK/SAMP activation. The activated p38 MAPK and JNK/SAPK promotes transcriptional activation of p53 and FoxO1 by acetylation. P53 and FoxO1-mediated up-regulation of p16 INK4A and p21 CIP1 distrubs cyclins and CDKs, which increase protein stability of pRB and allow to G 0 /G 1 cell-cycle arrest and senescence. Conversely, activated p38 MAPK and JNK/SAPK reduce SIRT-1 level by phosphorylating Ser47, eventually losing deacetylation activity. However, treatment with antcin M activates Nrf2-dependent anti-oxidant genes such as HO-1 and NQO-1 followed by activation of PI3K/AKT and ER1/2 kinases, which facilitates ROS inhibition and upregulates SIRT-1 expression in HNDFs and HUVECs. Results expressed as mean ± SEM of three indipendent expriments. Statistical significance at Ф P

    Techniques Used: Activation Assay, Activity Assay, Inhibition, Expressing

    19) Product Images from "Administered circulating microparticles derived from lung cancer patients markedly improved angiogenesis, blood flow and ischemic recovery in rat critical limb ischemia"

    Article Title: Administered circulating microparticles derived from lung cancer patients markedly improved angiogenesis, blood flow and ischemic recovery in rat critical limb ischemia

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-015-0381-8

    Upper Panel ) Matrigel Assay for angiogenesis with and without Patient ’ s Lung cancer- derived microparticles (Lc- MPs) treatment (n = 6). A to C) After 5-hour cell culture [1.0 × 10 4 human umbilical vein endothelial cells (HUVECs)], Matrigel-assay angiogenesis was observed by microscopic findings (100×) in without (A) and with Lc-MPs treatment [3.0 × 10 5 (B) and 6.0 × 10 5 (C) MPs, respectively]. D) Analytical results of number of tubules (white arrows), * vs. other groups with different symbols (*, †, ‡), p
    Figure Legend Snippet: Upper Panel ) Matrigel Assay for angiogenesis with and without Patient ’ s Lung cancer- derived microparticles (Lc- MPs) treatment (n = 6). A to C) After 5-hour cell culture [1.0 × 10 4 human umbilical vein endothelial cells (HUVECs)], Matrigel-assay angiogenesis was observed by microscopic findings (100×) in without (A) and with Lc-MPs treatment [3.0 × 10 5 (B) and 6.0 × 10 5 (C) MPs, respectively]. D) Analytical results of number of tubules (white arrows), * vs. other groups with different symbols (*, †, ‡), p

    Techniques Used: Matrigel Assay, Derivative Assay, Cell Culture

    20) Product Images from "Shear Stress Responses of Adult Blood Outgrowth Endothelial Cells Seeded on Bioartificial Tissue"

    Article Title: Shear Stress Responses of Adult Blood Outgrowth Endothelial Cells Seeded on Bioartificial Tissue

    Journal: Tissue Engineering. Part A

    doi: 10.1089/ten.tea.2011.0055

    eNOS expression and NOx production by HBOECs and HUVECs seeded on bioartificial tissue and cultured under static and flow conditions for 24 h. eNOS staining of HBOEC cross sections after (A) static culture or (B) flow exposure. (C) NO production
    Figure Legend Snippet: eNOS expression and NOx production by HBOECs and HUVECs seeded on bioartificial tissue and cultured under static and flow conditions for 24 h. eNOS staining of HBOEC cross sections after (A) static culture or (B) flow exposure. (C) NO production

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

    VCAM-1 and ICAM-1 expression by HBOECs and HUVECs seeded on bioartificial tissue and cultured under static or flow conditions for 24 h, in the presence or absence of TNF-α. (A) VCAM-1 expression. (B) ICAM-1 expression. Plots show mean±SE
    Figure Legend Snippet: VCAM-1 and ICAM-1 expression by HBOECs and HUVECs seeded on bioartificial tissue and cultured under static or flow conditions for 24 h, in the presence or absence of TNF-α. (A) VCAM-1 expression. (B) ICAM-1 expression. Plots show mean±SE

    Techniques Used: Expressing, Cell Culture, Flow Cytometry

    Retention, elongation, and alignment of HBOECs and HUVECs seeded on bioartificial tissue and exposed to shear stress. VE-Cadherin staining of (A, B) HBOECs and (C, D) HUVECs. (A, C) Statically cultured cells. (B, D) Cells cultured under 15 dyn/cm
    Figure Legend Snippet: Retention, elongation, and alignment of HBOECs and HUVECs seeded on bioartificial tissue and exposed to shear stress. VE-Cadherin staining of (A, B) HBOECs and (C, D) HUVECs. (A, C) Statically cultured cells. (B, D) Cells cultured under 15 dyn/cm

    Techniques Used: Staining, Cell Culture

    21) Product Images from "Featured Article: Hypoxia-inducible factor-1α dependent nuclear entry of factor inhibiting HIF-1"

    Article Title: Featured Article: Hypoxia-inducible factor-1α dependent nuclear entry of factor inhibiting HIF-1

    Journal: Experimental Biology and Medicine

    doi: 10.1177/1535370215570821

    Effects of HIF-1α gene silencing on HIF-1α protein and mRNA levels. The HUVECs treated with DMOG only for 4 h as control group (D), Control), or pretreated with 50 nM mismatched siRNA (mmsiRNA), 50 nM, or 100 nM
    Figure Legend Snippet: Effects of HIF-1α gene silencing on HIF-1α protein and mRNA levels. The HUVECs treated with DMOG only for 4 h as control group (D), Control), or pretreated with 50 nM mismatched siRNA (mmsiRNA), 50 nM, or 100 nM

    Techniques Used:

    22) Product Images from "Coculturing Human Islets with Proangiogenic Support Cells to Improve Islet Revascularization at the Subcutaneous Transplantation Site"

    Article Title: Coculturing Human Islets with Proangiogenic Support Cells to Improve Islet Revascularization at the Subcutaneous Transplantation Site

    Journal: Tissue Engineering. Part A

    doi: 10.1089/ten.tea.2015.0317

    Sprout formation in Matrigel. (A–D) Representative phase-contrast microscopy images of control islets (A) and CIs (B–D) taken 48 h after Matrigel embedding. (E) Representative fluorescent image of human umbilical vein endothelial cell/human mesenchymal stromal cell (HUVEC/hMSC)-CI 48 h after Matrigel embedding with HUVECs labeled with DiO ( green ) and hMSCs with DiI ( red ). Both HUVECs and hMSCs contribute to sprout formation ( insert ; scale bar: 50 μm). (F) Quantification of sprout formation 24, 48, and 96 h after Matrigel embedding. Results are presented as mean ± SEM of five independent experiments. **
    Figure Legend Snippet: Sprout formation in Matrigel. (A–D) Representative phase-contrast microscopy images of control islets (A) and CIs (B–D) taken 48 h after Matrigel embedding. (E) Representative fluorescent image of human umbilical vein endothelial cell/human mesenchymal stromal cell (HUVEC/hMSC)-CI 48 h after Matrigel embedding with HUVECs labeled with DiO ( green ) and hMSCs with DiI ( red ). Both HUVECs and hMSCs contribute to sprout formation ( insert ; scale bar: 50 μm). (F) Quantification of sprout formation 24, 48, and 96 h after Matrigel embedding. Results are presented as mean ± SEM of five independent experiments. **

    Techniques Used: Microscopy, Labeling

    23) Product Images from "Activation of autophagy during farnesyl pyrophosphate synthase inhibition is mediated through PI3K/AKT/mTOR signaling"

    Article Title: Activation of autophagy during farnesyl pyrophosphate synthase inhibition is mediated through PI3K/AKT/mTOR signaling

    Journal: The Journal of International Medical Research

    doi: 10.1177/0300060519875371

    FPPS inhibition induced autophagy in HUVECs. (a) HUVECs treated with the FPPS inhibitor IBAN (100 µM, 12 hours) were co-transfected with RFP-LC3 and GFP-LC3 and observed under confocal microscopy (scale bar: 10 µm). Red dots represent autolysosomes and yellow dots represent autophagosomes. ***P
    Figure Legend Snippet: FPPS inhibition induced autophagy in HUVECs. (a) HUVECs treated with the FPPS inhibitor IBAN (100 µM, 12 hours) were co-transfected with RFP-LC3 and GFP-LC3 and observed under confocal microscopy (scale bar: 10 µm). Red dots represent autolysosomes and yellow dots represent autophagosomes. ***P

    Techniques Used: Inhibition, Transfection, Confocal Microscopy

    FPPS inhibition potentiates cell growth inhibition via impairment of autophagy. HUVECs were treated with IBAN (100 µM) with or without chloroquine (25 µM) for 24 hours. (a,b) Morphological changes of HUVECs following treatments were visualized using an inverted microscope (scale bar: 20 µm). (c) Cell proliferation was assessed using an MTS assay. The formazan dye produced by viable cells was quantified by measuring absorbance at 490 nm. (d) HUVECs were transiently transfected with an Atg7-specific siRNA. HUVECs were subsequently treated with IBAN (100 µM) for 24 hours, and cell proliferation was assessed using an MTS assay. *P
    Figure Legend Snippet: FPPS inhibition potentiates cell growth inhibition via impairment of autophagy. HUVECs were treated with IBAN (100 µM) with or without chloroquine (25 µM) for 24 hours. (a,b) Morphological changes of HUVECs following treatments were visualized using an inverted microscope (scale bar: 20 µm). (c) Cell proliferation was assessed using an MTS assay. The formazan dye produced by viable cells was quantified by measuring absorbance at 490 nm. (d) HUVECs were transiently transfected with an Atg7-specific siRNA. HUVECs were subsequently treated with IBAN (100 µM) for 24 hours, and cell proliferation was assessed using an MTS assay. *P

    Techniques Used: Inhibition, Inverted Microscopy, MTS Assay, Produced, Transfection

    24) Product Images from "Biomimetic hydrogels with pro-angiogenic properties"

    Article Title: Biomimetic hydrogels with pro-angiogenic properties

    Journal: Biomaterials

    doi: 10.1016/j.biomaterials.2010.01.104

    Time-lapse confocal videomicroscopy shows HUVECs and 10T1/2 undergoing tubule formation in hydrogels
    Figure Legend Snippet: Time-lapse confocal videomicroscopy shows HUVECs and 10T1/2 undergoing tubule formation in hydrogels

    Techniques Used:

    25) Product Images from "Activation of PPAR-δ induces microRNA-100 and decreases the uptake of very low-density lipoprotein in endothelial cells"

    Article Title: Activation of PPAR-δ induces microRNA-100 and decreases the uptake of very low-density lipoprotein in endothelial cells

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.13160

    miR-100 suppresses VLDL receptor (VLDLR) expression and VLDL uptake. (A) Quantitative PCR for validation of the efficiency of HUVECs transfected with miR-100 mimic (5 nM). (B) Quantitative PCR analysing VLDL receptor mRNA level after 24 h
    Figure Legend Snippet: miR-100 suppresses VLDL receptor (VLDLR) expression and VLDL uptake. (A) Quantitative PCR for validation of the efficiency of HUVECs transfected with miR-100 mimic (5 nM). (B) Quantitative PCR analysing VLDL receptor mRNA level after 24 h

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

    26) Product Images from "LncRNA Xist Contributes to Endogenous Neurological Repair After Chronic Compressive Spinal Cord Injury by Promoting Angiogenesis Through the miR-32-5p/Notch-1 Axis"

    Article Title: LncRNA Xist Contributes to Endogenous Neurological Repair After Chronic Compressive Spinal Cord Injury by Promoting Angiogenesis Through the miR-32-5p/Notch-1 Axis

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2020.00744

    Effect of the Xist/miR-32-5p axis on angiogenesis in HUVECs. (A) Representative images of the tube formation assay. HUVECs were exposed to different treatments for 48 h and subsequently subjected to hypoxia for a further 48 h. Tube formation was measured using a CCK8 kit, and sprouting and wound healing assays were performed. (B) Quantitation of tube formation was performed by RT-qPCR. (C) Representative images of the sprouting assay. (D) Quantitation of sprouting was performed by RT-qPCR. (E) Representative images of wound healing. (F) Quantitation of the migration rate was performed by RT-qPCR. Three independent experiments were carried out. * p
    Figure Legend Snippet: Effect of the Xist/miR-32-5p axis on angiogenesis in HUVECs. (A) Representative images of the tube formation assay. HUVECs were exposed to different treatments for 48 h and subsequently subjected to hypoxia for a further 48 h. Tube formation was measured using a CCK8 kit, and sprouting and wound healing assays were performed. (B) Quantitation of tube formation was performed by RT-qPCR. (C) Representative images of the sprouting assay. (D) Quantitation of sprouting was performed by RT-qPCR. (E) Representative images of wound healing. (F) Quantitation of the migration rate was performed by RT-qPCR. Three independent experiments were carried out. * p

    Techniques Used: Tube Formation Assay, Quantitation Assay, Quantitative RT-PCR, Migration

    The Xist/miR-32-5p axis exerts its role in angiogenesis by regulating Notch-1 expression. (A) The expression of Xist in HUVECs under hypoxic condition was quantitated by RT-qPCR. (B) HUVECs were exposed to different treatments. The expression of Notch-1 in HUVECs following transfection was quantitated by RT-qPCR. (C) The expression of Hes-1 following transfection was quantitated by RT-qPCR. Three independent experiments were carried out. *** p
    Figure Legend Snippet: The Xist/miR-32-5p axis exerts its role in angiogenesis by regulating Notch-1 expression. (A) The expression of Xist in HUVECs under hypoxic condition was quantitated by RT-qPCR. (B) HUVECs were exposed to different treatments. The expression of Notch-1 in HUVECs following transfection was quantitated by RT-qPCR. (C) The expression of Hes-1 following transfection was quantitated by RT-qPCR. Three independent experiments were carried out. *** p

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection

    27) Product Images from "Advanced PLGA hybrid scaffold with a bioactive PDRN/BMP2 nanocomplex for angiogenesis and bone regeneration using human fetal MSCs"

    Article Title: Advanced PLGA hybrid scaffold with a bioactive PDRN/BMP2 nanocomplex for angiogenesis and bone regeneration using human fetal MSCs

    Journal: Science Advances

    doi: 10.1126/sciadv.abj1083

    Biological ability conformation of each component of the NC. ( A ) Cell migration assay: optical images (scale bars:,200 μm) and quantification for 24 and 48 hours. ( B ) Tubule-forming assay: calcein AM–stained images (scale bars, 200 μm) and quantification of HUVECs. ( C ) Immunofluorescence staining labeled with VEGF (scale bars, 50 μm). ( D ) Gene expression onto the scaffolds related to angiogenesis, VEGF, and ANG2 using hfMSCs. ns, not significant. ( E ) Schematic illustration of angiogenesis through A2a receptor stimulation induced by PDRN and NC. # P
    Figure Legend Snippet: Biological ability conformation of each component of the NC. ( A ) Cell migration assay: optical images (scale bars:,200 μm) and quantification for 24 and 48 hours. ( B ) Tubule-forming assay: calcein AM–stained images (scale bars, 200 μm) and quantification of HUVECs. ( C ) Immunofluorescence staining labeled with VEGF (scale bars, 50 μm). ( D ) Gene expression onto the scaffolds related to angiogenesis, VEGF, and ANG2 using hfMSCs. ns, not significant. ( E ) Schematic illustration of angiogenesis through A2a receptor stimulation induced by PDRN and NC. # P

    Techniques Used: Cell Migration Assay, Staining, Immunofluorescence, Labeling, Expressing

    28) Product Images from "Propranolol Suppresses Proliferation and Migration of HUVECs through Regulation of the miR-206/VEGFA Axis"

    Article Title: Propranolol Suppresses Proliferation and Migration of HUVECs through Regulation of the miR-206/VEGFA Axis

    Journal: BioMed Research International

    doi: 10.1155/2021/7629176

    Downregulation of miR-206 abolishes the antioxidant capacity of propranolol in HUVECs. Cells were treated with propranolol or propranolol+miR-206 antagomir, the changes of (a) SOD activity, (b) GSH level, and (c) MDA level in HUVECs were measured, respectively. ∗∗ P
    Figure Legend Snippet: Downregulation of miR-206 abolishes the antioxidant capacity of propranolol in HUVECs. Cells were treated with propranolol or propranolol+miR-206 antagomir, the changes of (a) SOD activity, (b) GSH level, and (c) MDA level in HUVECs were measured, respectively. ∗∗ P

    Techniques Used: Activity Assay, Multiple Displacement Amplification

    Propranolol inhibits survival of HUVECs through demethylation of pre-miR-206. (a) Top-ten upregulated miRNAs in propranolol-treated (80 μ M) HUVECs comparing with control cells. (b) HUVECs were treated with 80 μ M propranolol for 48 h, and the mRNA level of miR-206 was determined by RT-qPCR. (c) Methylation and unmethylation status of the pre-miR-206 gene were measured by MS-PCR. (d) The mRNA level of miR-206 in cells was determined by RT-qPCR after miR-206 antagomir transfection. (e) Cell viability was detected by CCK-8 kit. (f) Cell apoptosis was analyzed on flow cytometry after Annexin V/PI double staining. ∗∗ P
    Figure Legend Snippet: Propranolol inhibits survival of HUVECs through demethylation of pre-miR-206. (a) Top-ten upregulated miRNAs in propranolol-treated (80 μ M) HUVECs comparing with control cells. (b) HUVECs were treated with 80 μ M propranolol for 48 h, and the mRNA level of miR-206 was determined by RT-qPCR. (c) Methylation and unmethylation status of the pre-miR-206 gene were measured by MS-PCR. (d) The mRNA level of miR-206 in cells was determined by RT-qPCR after miR-206 antagomir transfection. (e) Cell viability was detected by CCK-8 kit. (f) Cell apoptosis was analyzed on flow cytometry after Annexin V/PI double staining. ∗∗ P

    Techniques Used: Quantitative RT-PCR, Methylation, Polymerase Chain Reaction, Transfection, CCK-8 Assay, Flow Cytometry, Double Staining

    Downregulation of miR-206 reverses propranolol-induced inhibition of cell migration in HUVECs. HUVECs were divided into three groups: control, 80 μ M propranolol, 80 μ M propranolol + miR-206 antagomir. (a) and (b) Representative scratching area of cells and the calculated wound healing rate were indicated. (c) and (d) Migrated cells were stained with 0.2% crystal violet and counted at 3 random fields. (e) Representative images of tube formation were captured by microscopy at 24 h. (f) and (g) Branch points counts and capillary length were calculated at 3 random fields. ∗∗ P
    Figure Legend Snippet: Downregulation of miR-206 reverses propranolol-induced inhibition of cell migration in HUVECs. HUVECs were divided into three groups: control, 80 μ M propranolol, 80 μ M propranolol + miR-206 antagomir. (a) and (b) Representative scratching area of cells and the calculated wound healing rate were indicated. (c) and (d) Migrated cells were stained with 0.2% crystal violet and counted at 3 random fields. (e) Representative images of tube formation were captured by microscopy at 24 h. (f) and (g) Branch points counts and capillary length were calculated at 3 random fields. ∗∗ P

    Techniques Used: Inhibition, Migration, Staining, Microscopy

    Downregulation of miR-206 eliminates propranolol-induced cell cycle arrest in HUVECs. Cells were treated with propranolol or propranolol+miR-206 antagomir. (a) The protein levels of CDK4, Cyclin D1, and cleaved caspase 3 were detected by western blotting. (b)–(d) The relative expression levels of CDK4, Cyclin D1, and cleaved caspase 3 were quantified by normalizing to β -actin. (e) Cell cycle distribution was analyzed by flow cytometry, and the result was quantified by flowjo software. ∗∗ P
    Figure Legend Snippet: Downregulation of miR-206 eliminates propranolol-induced cell cycle arrest in HUVECs. Cells were treated with propranolol or propranolol+miR-206 antagomir. (a) The protein levels of CDK4, Cyclin D1, and cleaved caspase 3 were detected by western blotting. (b)–(d) The relative expression levels of CDK4, Cyclin D1, and cleaved caspase 3 were quantified by normalizing to β -actin. (e) Cell cycle distribution was analyzed by flow cytometry, and the result was quantified by flowjo software. ∗∗ P

    Techniques Used: Western Blot, Expressing, Flow Cytometry, Software

    29) Product Images from "Lactate induces vascular permeability via disruption of VE-cadherin in endothelial cells during sepsis"

    Article Title: Lactate induces vascular permeability via disruption of VE-cadherin in endothelial cells during sepsis

    Journal: Science Advances

    doi: 10.1126/sciadv.abm8965

    Calpain activation is required for lactate-induced disruption of VE-cadherin in ECs. Lactate (0.5 g/kg body weight) was administrated through intraperitoneal injection 6 hours after CLP or sham surgery. ( A and B ) Representative immunofluorescent staining images of calpain1 (red, A) and calpain2 (red, B) in ECs of the lung tissues. ECs were stained with CD31 (green), and nuclei were stained with DAPI (blue) ( n = 5). ( C ) Western blot analysis of calpain1 and calpain2 expressions following lactate treatment in ECs ( n = 3). ( D ) Calpain enzyme activity in ECs treated with lactic acid or sodium lactate ( n = 3). ( E ) Representative immunofluorescent staining images of VE-cadherin (red), calpain1 (green), and nuclei (DAPI, blue) in HUVECs treated with or without lactate ( n = 3). Scale bar, 20 μm. ( F and G ) HUVECs were treated with lactate for 6 hours. Protein lysates (200 μg) were precipitated with anti–VE-cadherin antibody followed by immunoblotting with anti-calpain1 (F) or anti-calpain2 (G) antibodies. LacH, lactic acid. LacNa, sodium lactate. Two-way ANOVA with Tukey’s test (A and B). One-way ANOVA with Tukey’s test (C and D). Student’s two-tailed unpaired t test (E). * P
    Figure Legend Snippet: Calpain activation is required for lactate-induced disruption of VE-cadherin in ECs. Lactate (0.5 g/kg body weight) was administrated through intraperitoneal injection 6 hours after CLP or sham surgery. ( A and B ) Representative immunofluorescent staining images of calpain1 (red, A) and calpain2 (red, B) in ECs of the lung tissues. ECs were stained with CD31 (green), and nuclei were stained with DAPI (blue) ( n = 5). ( C ) Western blot analysis of calpain1 and calpain2 expressions following lactate treatment in ECs ( n = 3). ( D ) Calpain enzyme activity in ECs treated with lactic acid or sodium lactate ( n = 3). ( E ) Representative immunofluorescent staining images of VE-cadherin (red), calpain1 (green), and nuclei (DAPI, blue) in HUVECs treated with or without lactate ( n = 3). Scale bar, 20 μm. ( F and G ) HUVECs were treated with lactate for 6 hours. Protein lysates (200 μg) were precipitated with anti–VE-cadherin antibody followed by immunoblotting with anti-calpain1 (F) or anti-calpain2 (G) antibodies. LacH, lactic acid. LacNa, sodium lactate. Two-way ANOVA with Tukey’s test (A and B). One-way ANOVA with Tukey’s test (C and D). Student’s two-tailed unpaired t test (E). * P

    Techniques Used: Activation Assay, Injection, Staining, Western Blot, Activity Assay, Two Tailed Test

    Lactate-suppressed adenylyl cyclase activity contributes to VE-cadherin down-regulation by activation of RAF1/MEK/ERK signaling. ( A ) A scheme depicting lactate mode of action in regulating VE-cadherin stability via GPR81/cAMP signaling. ( B ) HUVECs were treated with adenylyl cyclase activator (forskolin, 10 μM) before lactate stimulation for 6 hours. Representative immunofluorescent staining images of VE-cadherin (red) and nuclei (DAPI, blue) in HUVECs. ( C ) Western blot analysis of VE-cadherin in HUVECs pretreated with forskolin followed by lactate stimulation for 6 hours ( n = 4). ( D ) Western blot analysis of RAF1/MEK/ERK signaling in HUVECs pretreated with forskolin followed by lactate stimulation ( n = 4). ( E ) HUVECs were transfected with siRNAs for MEK1/MEK2 and scramble control siRNA for 24 hours before lactate stimulation for 6 hours. Expression of VE-cadherin, p-ERK1/2, and MEK1/2 were assessed by Western blot ( n = 4). ( F ) HUVECs were treated with EPAC agonist (8-CPT-2Me-cAMP, 100 μM) before lactate stimulation for 6 hours. Representative immunofluorescent staining images of VE-cadherin (red) and nuclei (DAPI, blue) in HUVECs. ( G ) Western blot analysis of VE-cadherin in HUVECs pretreated with EPAC agonist followed by lactate stimulation for 6 hours ( n = 4). ( H ) Western blot analysis of RAF1/MEK/ERK signaling in HUVECs pretreated with forskolin followed by lactate stimulation ( n = 4). EPAC, exchange protein directly activated by cAMP. Con, control. Two-way ANOVA with Tukey’s test. * P
    Figure Legend Snippet: Lactate-suppressed adenylyl cyclase activity contributes to VE-cadherin down-regulation by activation of RAF1/MEK/ERK signaling. ( A ) A scheme depicting lactate mode of action in regulating VE-cadherin stability via GPR81/cAMP signaling. ( B ) HUVECs were treated with adenylyl cyclase activator (forskolin, 10 μM) before lactate stimulation for 6 hours. Representative immunofluorescent staining images of VE-cadherin (red) and nuclei (DAPI, blue) in HUVECs. ( C ) Western blot analysis of VE-cadherin in HUVECs pretreated with forskolin followed by lactate stimulation for 6 hours ( n = 4). ( D ) Western blot analysis of RAF1/MEK/ERK signaling in HUVECs pretreated with forskolin followed by lactate stimulation ( n = 4). ( E ) HUVECs were transfected with siRNAs for MEK1/MEK2 and scramble control siRNA for 24 hours before lactate stimulation for 6 hours. Expression of VE-cadherin, p-ERK1/2, and MEK1/2 were assessed by Western blot ( n = 4). ( F ) HUVECs were treated with EPAC agonist (8-CPT-2Me-cAMP, 100 μM) before lactate stimulation for 6 hours. Representative immunofluorescent staining images of VE-cadherin (red) and nuclei (DAPI, blue) in HUVECs. ( G ) Western blot analysis of VE-cadherin in HUVECs pretreated with EPAC agonist followed by lactate stimulation for 6 hours ( n = 4). ( H ) Western blot analysis of RAF1/MEK/ERK signaling in HUVECs pretreated with forskolin followed by lactate stimulation ( n = 4). EPAC, exchange protein directly activated by cAMP. Con, control. Two-way ANOVA with Tukey’s test. * P

    Techniques Used: Activity Assay, Activation Assay, Staining, Western Blot, Transfection, Expressing

    Lactate-induced VE-cadherin disorganization is mediated by GPR81 signaling. ( A and B ) GPR81 was silenced by transfection with specific siRNA for 24 hours before lactate stimulation. Cells transfected with scramble siRNAs were used as controls. VE-cadherin expression was assessed by Western blot (A), and VE-cadherin localization was examined by immunofluorescent staining (B). Scale bar, 20 μm. ( C ) Levels of FITC-dextran penetration through GPR81-silenced HUVEC monolayer upon lactate stimulation ( n = 4). Cells transfected with scramble siRNAs were used as controls. ( D and E ) HUVECs were treated with GPR81 antagonist 3-OBA (5 mM) for 1 hour before lactate stimulation. Expression of VE-cadherin was assessed by Western blot (D) ( n = 3). Permeability was examined by levels of FITC-dextran penetration through endothelium (E) ( n = 4). ( F ) HUVECs were treated with GPR81 antagonist 3-OBA (5 mM) for 1 hour before lactate stimulation. Expression of ERK, p-ERK, and calpain1 was assessed by Western blot ( n = 3). ( G ) HUVECs were treated with an MCT inhibitor (CHC, 3 mM) for 1 hour before lactate stimulation. Expression of ERK, p-ERK, and calpain1 was assessed by Western blot ( n = 3). Two-way ANOVA with Tukey’s test. 3-OBA, 3-hydroxy-butyrate acid. CHC, 2-Cyano-3-(4-hydroxyphenyl)-2-propenoic acid. siR, siRNA. * P
    Figure Legend Snippet: Lactate-induced VE-cadherin disorganization is mediated by GPR81 signaling. ( A and B ) GPR81 was silenced by transfection with specific siRNA for 24 hours before lactate stimulation. Cells transfected with scramble siRNAs were used as controls. VE-cadherin expression was assessed by Western blot (A), and VE-cadherin localization was examined by immunofluorescent staining (B). Scale bar, 20 μm. ( C ) Levels of FITC-dextran penetration through GPR81-silenced HUVEC monolayer upon lactate stimulation ( n = 4). Cells transfected with scramble siRNAs were used as controls. ( D and E ) HUVECs were treated with GPR81 antagonist 3-OBA (5 mM) for 1 hour before lactate stimulation. Expression of VE-cadherin was assessed by Western blot (D) ( n = 3). Permeability was examined by levels of FITC-dextran penetration through endothelium (E) ( n = 4). ( F ) HUVECs were treated with GPR81 antagonist 3-OBA (5 mM) for 1 hour before lactate stimulation. Expression of ERK, p-ERK, and calpain1 was assessed by Western blot ( n = 3). ( G ) HUVECs were treated with an MCT inhibitor (CHC, 3 mM) for 1 hour before lactate stimulation. Expression of ERK, p-ERK, and calpain1 was assessed by Western blot ( n = 3). Two-way ANOVA with Tukey’s test. 3-OBA, 3-hydroxy-butyrate acid. CHC, 2-Cyano-3-(4-hydroxyphenyl)-2-propenoic acid. siR, siRNA. * P

    Techniques Used: Transfection, Expressing, Western Blot, Staining, Permeability

    30) Product Images from "Knockdown of long non-coding RNA plasmacytoma variant translocation 1 relieves ox-LDL-induced endothelial cell injury through regulating microRNA-30c-5p in atherosclerosis"

    Article Title: Knockdown of long non-coding RNA plasmacytoma variant translocation 1 relieves ox-LDL-induced endothelial cell injury through regulating microRNA-30c-5p in atherosclerosis

    Journal: Bioengineered

    doi: 10.1080/21655979.2021.2019878

    Knockdown of PVT1 facilitated proliferation in ox-LDL-induced HUVECs. HUVECs were transfected with control-siRNA or lncRNA PVT1-siRNA for 24 h, then exposed to ox-LDL for 24 h. (a and b) PVT1 expression levels were measured via RT-qPCR. (c) The viability of HUVECs was assessed via CCK-8 assays after 24 h, 48 h, and 72 h. (d and e) Apoptotic cells were detected by FCM. (f) Western blot analysis of Bax and Bcl-2 expression. (g and h) RT-qPCR analysis of Bax and Bcl-2 mRNA levels. **P
    Figure Legend Snippet: Knockdown of PVT1 facilitated proliferation in ox-LDL-induced HUVECs. HUVECs were transfected with control-siRNA or lncRNA PVT1-siRNA for 24 h, then exposed to ox-LDL for 24 h. (a and b) PVT1 expression levels were measured via RT-qPCR. (c) The viability of HUVECs was assessed via CCK-8 assays after 24 h, 48 h, and 72 h. (d and e) Apoptotic cells were detected by FCM. (f) Western blot analysis of Bax and Bcl-2 expression. (g and h) RT-qPCR analysis of Bax and Bcl-2 mRNA levels. **P

    Techniques Used: Transfection, Expressing, Quantitative RT-PCR, CCK-8 Assay, Western Blot

    miR-30 c-5p relieved the negative effects on proliferation, apoptosis of ox-LDL-treated HUVECs by lncRNA PVT1-knockdown. HUVECs were transfected with control-siRNA, lncRNA PVT1-siRNA, inhibitor control, or miR-30 c-5p inhibitor for 24 h, then induced by ox-LDL for 24 h. (a) miR-30 c-5p expression in different groups. (b) lncRNA PVT1 expression in different groups. (c) Proliferation of HUVECs was assessed via CCK-8 assays after 24 h, 48 h, and 72 h. (d and e) Apoptotic cells were enumerated using FCM. (f) Western blot analysis of Bax and Bcl-2 expression. (g and h) RT-qPCR analysis of Bax and Bcl-2 mRNA levels. **P
    Figure Legend Snippet: miR-30 c-5p relieved the negative effects on proliferation, apoptosis of ox-LDL-treated HUVECs by lncRNA PVT1-knockdown. HUVECs were transfected with control-siRNA, lncRNA PVT1-siRNA, inhibitor control, or miR-30 c-5p inhibitor for 24 h, then induced by ox-LDL for 24 h. (a) miR-30 c-5p expression in different groups. (b) lncRNA PVT1 expression in different groups. (c) Proliferation of HUVECs was assessed via CCK-8 assays after 24 h, 48 h, and 72 h. (d and e) Apoptotic cells were enumerated using FCM. (f) Western blot analysis of Bax and Bcl-2 expression. (g and h) RT-qPCR analysis of Bax and Bcl-2 mRNA levels. **P

    Techniques Used: Transfection, Expressing, CCK-8 Assay, Western Blot, Quantitative RT-PCR

    miR-30 c-5p relieved negative effects on inflammatory responses of ox-LDL-stimulated HUVECs by lncRNA PVT1 knockdown. (a-c) TNF-α, IL-1β, and IL-6 expression was assessed using ELISA. **P
    Figure Legend Snippet: miR-30 c-5p relieved negative effects on inflammatory responses of ox-LDL-stimulated HUVECs by lncRNA PVT1 knockdown. (a-c) TNF-α, IL-1β, and IL-6 expression was assessed using ELISA. **P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay

    Expression of PVT1 in serum samples and HUVECs treated with ox-LDL by qRT-PCR. (a) High expression of PVT1 in serum samples. (b) Significantly high expression of PVT1 in HUVECs. **P
    Figure Legend Snippet: Expression of PVT1 in serum samples and HUVECs treated with ox-LDL by qRT-PCR. (a) High expression of PVT1 in serum samples. (b) Significantly high expression of PVT1 in HUVECs. **P

    Techniques Used: Expressing, Quantitative RT-PCR

    Inhibition of PVT1 suppressed inflammatory responses in ox-LDL-stimulated HUVECs. (a-c) TNF-α, IL-1β, and IL-6 expression was determined using ELISA. **P
    Figure Legend Snippet: Inhibition of PVT1 suppressed inflammatory responses in ox-LDL-stimulated HUVECs. (a-c) TNF-α, IL-1β, and IL-6 expression was determined using ELISA. **P

    Techniques Used: Inhibition, Expressing, Enzyme-linked Immunosorbent Assay

    miR-30 c-5p expression in AS patient serum and ox-LDL-induced HUVECs. (a) Low expression of miR-30 c-5p in serum samples. (b) Significantly low expression of miR-30 c-5p in ox-LDL-induced HUVECs. (c) miR-30 c-5p expression in HUVECs transfected with inhibitor control or miR-30 c-5p inhibitor. (d) miR-30 c-5p expression in HUVECs transfected with control-siRNA, lncRNA PVT1-siRNA, lncRNA PVT1-siRNA+inhibitor control, or lncRNA PVT1-siRNA+miR-30 c-5p inhibitor.**P
    Figure Legend Snippet: miR-30 c-5p expression in AS patient serum and ox-LDL-induced HUVECs. (a) Low expression of miR-30 c-5p in serum samples. (b) Significantly low expression of miR-30 c-5p in ox-LDL-induced HUVECs. (c) miR-30 c-5p expression in HUVECs transfected with inhibitor control or miR-30 c-5p inhibitor. (d) miR-30 c-5p expression in HUVECs transfected with control-siRNA, lncRNA PVT1-siRNA, lncRNA PVT1-siRNA+inhibitor control, or lncRNA PVT1-siRNA+miR-30 c-5p inhibitor.**P

    Techniques Used: Expressing, Transfection

    31) Product Images from "ECM-derived biophysical cues mediate interstitial flow-induced sprouting angiogenesis"

    Article Title: ECM-derived biophysical cues mediate interstitial flow-induced sprouting angiogenesis

    Journal: bioRxiv

    doi: 10.1101/2022.06.04.494804

    Sprouting area of HUVECs cultured in microvessel analogues of both collagen-only and collagen/HA matrices in the responses of various pharmaceutical drugs treatments (i.e. CD44 blocking, MMP inhibition and enzymatic matrix degradation) under interstitial flow on day 1. The data were expressed as mean ± standard error (n ≥ 3 for all experimental conditions). One-way ANOVA followed by post-hoc unpaired, two-tailed Student t test was performed to evaluate the statistical significance. * indicates p-value
    Figure Legend Snippet: Sprouting area of HUVECs cultured in microvessel analogues of both collagen-only and collagen/HA matrices in the responses of various pharmaceutical drugs treatments (i.e. CD44 blocking, MMP inhibition and enzymatic matrix degradation) under interstitial flow on day 1. The data were expressed as mean ± standard error (n ≥ 3 for all experimental conditions). One-way ANOVA followed by post-hoc unpaired, two-tailed Student t test was performed to evaluate the statistical significance. * indicates p-value

    Techniques Used: Cell Culture, Blocking Assay, Inhibition, Two Tailed Test

    32) Product Images from "ZNF354C Mediated by DNMT1 Ameliorates Lung Ischemia-Reperfusion Oxidative Stress Injury by Reducing TFPI Promoter Methylation to Upregulate TFPI"

    Article Title: ZNF354C Mediated by DNMT1 Ameliorates Lung Ischemia-Reperfusion Oxidative Stress Injury by Reducing TFPI Promoter Methylation to Upregulate TFPI

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2022/7288729

    ZNF354C overexpression induced proliferation and migration through upregulating TFPI in OGD/R-induced HUVECs. OGD/R-treated HUVECs were transfected with ZNF354C overexpression plasmids or shZNF354C, respectively. ZNF354C and TFPI expressions were assessed via (a) RT-qPCR and (b) western blot. (c) TFPI level was analyzed with ELISA kit. (d) IF staining of TFPI in the processed HUVECs. (e) Cell viability was evaluated by CCK-8. (f) HUVECs in each group were subjected to EdU staining. (g) The images of cell migration were obtained using Transwell. (h) EdU-positive cells were quantitated in line with EdU staining results. (i) Quantitative analysis of migratory cells. (j) The binding of ZNF354C and TFPI promoter was confirmed through ChIP in each group. (k) The binding of RNA polymerase II on TFPI 5′UTR was examined via ChIP. The experiments were repeated three times independently.
    Figure Legend Snippet: ZNF354C overexpression induced proliferation and migration through upregulating TFPI in OGD/R-induced HUVECs. OGD/R-treated HUVECs were transfected with ZNF354C overexpression plasmids or shZNF354C, respectively. ZNF354C and TFPI expressions were assessed via (a) RT-qPCR and (b) western blot. (c) TFPI level was analyzed with ELISA kit. (d) IF staining of TFPI in the processed HUVECs. (e) Cell viability was evaluated by CCK-8. (f) HUVECs in each group were subjected to EdU staining. (g) The images of cell migration were obtained using Transwell. (h) EdU-positive cells were quantitated in line with EdU staining results. (i) Quantitative analysis of migratory cells. (j) The binding of ZNF354C and TFPI promoter was confirmed through ChIP in each group. (k) The binding of RNA polymerase II on TFPI 5′UTR was examined via ChIP. The experiments were repeated three times independently.

    Techniques Used: Over Expression, Migration, Transfection, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, CCK-8 Assay, Binding Assay, Chromatin Immunoprecipitation

    5-Aza decreased TFPI methylation, increased TFPI expression, accelerated proliferation and migration, and enhanced the combination of TFPI and ZNF354C in OGD/R-treated HUVECs. OGD/R-mediated HUVECs were treated with 5-Aza (low: 0.1 μ M, mid: 1 μ M, high: 10 μ M) for 24 h. (a) MSP was utilized to identify the methylation level of TFPI promoter. TFPI level was certified by applying (b) RT-qPCR, (c) western blot, (d) ELISA, and (e) IF staining in the processed HUVECs. Cell proliferation was determined with (f) CCK-8 and (g) EdU staining. (i) Migration in HUVECs was tested with Transwell. (h) EdU-positive cells and (j) migrated cells were counted. (k) The impact of 5-Aza on the binding of ZNF354C and TFPI promoter was confirmed by ChIP. (l) ChIP was adopted to analyze the effect of 5-Aza on the binding of RNA polymerase II on TFPI 5′UTR. The experiments were repeated three times independently.
    Figure Legend Snippet: 5-Aza decreased TFPI methylation, increased TFPI expression, accelerated proliferation and migration, and enhanced the combination of TFPI and ZNF354C in OGD/R-treated HUVECs. OGD/R-mediated HUVECs were treated with 5-Aza (low: 0.1 μ M, mid: 1 μ M, high: 10 μ M) for 24 h. (a) MSP was utilized to identify the methylation level of TFPI promoter. TFPI level was certified by applying (b) RT-qPCR, (c) western blot, (d) ELISA, and (e) IF staining in the processed HUVECs. Cell proliferation was determined with (f) CCK-8 and (g) EdU staining. (i) Migration in HUVECs was tested with Transwell. (h) EdU-positive cells and (j) migrated cells were counted. (k) The impact of 5-Aza on the binding of ZNF354C and TFPI promoter was confirmed by ChIP. (l) ChIP was adopted to analyze the effect of 5-Aza on the binding of RNA polymerase II on TFPI 5′UTR. The experiments were repeated three times independently.

    Techniques Used: Methylation, Expressing, Migration, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, CCK-8 Assay, Binding Assay, Chromatin Immunoprecipitation

    OGD/R induced hypermethylation of TFPI in HUVECs, and TFPI could interact with ZNF354C. (a) Methylation level of TFPI promoter was tested with MSP in OGD/R-treated HUVECs. (b) The methylation level was quantified based on MSP results. (c) The predicated binding sites between TFPI and ZNF354C. (d) Luciferase activity of TFPI promoter in ZNF354C-overexpressed HUVECs was determined by applying dual luciferase reporter gene. (e) EMSA was utilized to test the binding of TFPI promoter and ZNF354C. (f) The interaction between ZNF354C antibody and TFPI promoter was confirmed with ChIP in HUVECs under OGD/R. (g) HUVECs were incubated with Poly II antibody and beads, and the binding of RNA polymerase II on TFPI 5′UTR was examined using ChIP in HUVECs under OGD/R. The experiments were repeated three times independently.
    Figure Legend Snippet: OGD/R induced hypermethylation of TFPI in HUVECs, and TFPI could interact with ZNF354C. (a) Methylation level of TFPI promoter was tested with MSP in OGD/R-treated HUVECs. (b) The methylation level was quantified based on MSP results. (c) The predicated binding sites between TFPI and ZNF354C. (d) Luciferase activity of TFPI promoter in ZNF354C-overexpressed HUVECs was determined by applying dual luciferase reporter gene. (e) EMSA was utilized to test the binding of TFPI promoter and ZNF354C. (f) The interaction between ZNF354C antibody and TFPI promoter was confirmed with ChIP in HUVECs under OGD/R. (g) HUVECs were incubated with Poly II antibody and beads, and the binding of RNA polymerase II on TFPI 5′UTR was examined using ChIP in HUVECs under OGD/R. The experiments were repeated three times independently.

    Techniques Used: Methylation, Binding Assay, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Incubation

    DNMT1 suppressed proliferation and migration and reduced the interaction of TFPI and ZNF354C in OGD/R-induced HUVECs. HUVECs under OGD/R were transfected with DNMT1 overexpression plasmids or shDNMT1, respectively. (a) MSP displayed the change in the methylation level of TFPI promoter. The level of TFPI was tested through (b) RT-qPCR and (c) western blot, (d) ELISA, and (e) IF staining. (f) EdU staining was utilized to confirm the change of proliferation. (g) Transwell exhibited the change of cell migration. (h) CCK-8 presented the change in cell viability. (i) Based on the EdU staining data, EdU-positive cells were quantitated. (j) Migratory cells were quantitated in line with Transwell data. (k) ChIP demonstrated the impact of DNMT1 on the binding of ZNF354C and TFPI promoter. (l) ChIP showed the influence of DNMT1 on the binding of RNA polymerase II on TFPI 5′UTR. The experiments were repeated three times independently.
    Figure Legend Snippet: DNMT1 suppressed proliferation and migration and reduced the interaction of TFPI and ZNF354C in OGD/R-induced HUVECs. HUVECs under OGD/R were transfected with DNMT1 overexpression plasmids or shDNMT1, respectively. (a) MSP displayed the change in the methylation level of TFPI promoter. The level of TFPI was tested through (b) RT-qPCR and (c) western blot, (d) ELISA, and (e) IF staining. (f) EdU staining was utilized to confirm the change of proliferation. (g) Transwell exhibited the change of cell migration. (h) CCK-8 presented the change in cell viability. (i) Based on the EdU staining data, EdU-positive cells were quantitated. (j) Migratory cells were quantitated in line with Transwell data. (k) ChIP demonstrated the impact of DNMT1 on the binding of ZNF354C and TFPI promoter. (l) ChIP showed the influence of DNMT1 on the binding of RNA polymerase II on TFPI 5′UTR. The experiments were repeated three times independently.

    Techniques Used: Migration, Transfection, Over Expression, Methylation, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, CCK-8 Assay, Chromatin Immunoprecipitation, Binding Assay

    33) Product Images from "Vascular Regulation by Super Enhancer-Derived LINC00607"

    Article Title: Vascular Regulation by Super Enhancer-Derived LINC00607

    Journal: Frontiers in Cardiovascular Medicine

    doi: 10.3389/fcvm.2022.881916

    LINC00607 regulates basal EC and VSMC function. (A) Volcano plot indicating the DEGs upon 607-KD in HUVECs. Red and blue dots represent significantly up-regulated and down-regulated genes (with P -value cut-off of 0.01). Vertical dotted lines correspond to 2-fold differences. (B) Pathway enrichment analyses of DEGs upon 607-KD in HUVECs. Top 15 GO terms ranked by fold enrichment score for both the down- and up-regulated DEGs. (C) Expression of select down-regulated DEGs involved in angiogenesis due to LINC00607 knockdown (KD), as compared to scramble LNA (Scr). Heatmap is plotted based on z-scaled gene expression levels. (D) Representative images of tube formation of HUVEC transfected with either scramble or 607-KD at the indicated final concentration. The images were taken after incubating in Matrigel for 4 h. Scale bar = 100 μm. (E) Quantitative data of tube formation represented by numbers of tubes in randomly selected views. (F) DEGs upon 607-KD in VSMCs were plotted as in (A) . (G) Pathway enrichment analyses of DEGs upon 607-KD in VSMCs. Top 15 GO terms are plotted as in (B) . (H) Venn diagram showing the number of common DEGs upon 607-KD in HUVECs and VSMCs (in green). Numbers of unique DEGs in each cell type (pink for HUVECs and blue for VSMCs) are indicated. (I) Polygon radar chart showing the top 5 enriched pathways of the common DEGs in HUVECs and VSMCs. Data values for each vertex represent the number of DEGs classified in the indicated gene pathway in blue and the P -value (in -log 10 ) in orange. (J) Expression heatmap of representative DEGs commonly affected by 607-KD in ECs and VSMCs.
    Figure Legend Snippet: LINC00607 regulates basal EC and VSMC function. (A) Volcano plot indicating the DEGs upon 607-KD in HUVECs. Red and blue dots represent significantly up-regulated and down-regulated genes (with P -value cut-off of 0.01). Vertical dotted lines correspond to 2-fold differences. (B) Pathway enrichment analyses of DEGs upon 607-KD in HUVECs. Top 15 GO terms ranked by fold enrichment score for both the down- and up-regulated DEGs. (C) Expression of select down-regulated DEGs involved in angiogenesis due to LINC00607 knockdown (KD), as compared to scramble LNA (Scr). Heatmap is plotted based on z-scaled gene expression levels. (D) Representative images of tube formation of HUVEC transfected with either scramble or 607-KD at the indicated final concentration. The images were taken after incubating in Matrigel for 4 h. Scale bar = 100 μm. (E) Quantitative data of tube formation represented by numbers of tubes in randomly selected views. (F) DEGs upon 607-KD in VSMCs were plotted as in (A) . (G) Pathway enrichment analyses of DEGs upon 607-KD in VSMCs. Top 15 GO terms are plotted as in (B) . (H) Venn diagram showing the number of common DEGs upon 607-KD in HUVECs and VSMCs (in green). Numbers of unique DEGs in each cell type (pink for HUVECs and blue for VSMCs) are indicated. (I) Polygon radar chart showing the top 5 enriched pathways of the common DEGs in HUVECs and VSMCs. Data values for each vertex represent the number of DEGs classified in the indicated gene pathway in blue and the P -value (in -log 10 ) in orange. (J) Expression heatmap of representative DEGs commonly affected by 607-KD in ECs and VSMCs.

    Techniques Used: Expressing, Transfection, Concentration Assay

    Stimulus-dependent regulatory function of LINC00607 in ECs. (A) Representative images of smFISH detecting LINC00607 (green) in HUVEC treated with normal glucose (5.5 mM D-glucose) and osmolarity control (NM) or 25 mM D-glucose and 5 ng/ml TNFα (HT) for 3 days. DAPI staining indicates the nuclei. Scale bar = 20 μm. (B) Quantitative data of LINC00607 signal per nucleus. LINC00607 signal from 18 nuclei for NM and 24 nuclei for HT was quantified using ImageJ. (C) Venn diagram showing the number of common DEGs in HUVECs upon 607-KD under basal and HT conditions (green). Numbers of unique DEGs in each condition (pink for NM and blue for HT) are indicated. (D) Expression pattern of select DEGs commonly regulated by 607-KD in HUVECs under both baseline and HT. Heatmap is plotted based on z-scaled average gene expression levels. (E) Putative sequence of c-Myc binding site at the LINC00607 promoters (Chr2: 215826423-215837423, Chr2: 215830609-215841609, and Chr2: 215842628-215853628) predicted by TRANSFAC. The HUVEC c-Myc ChIP-seq tracks (ENCODE data ENCFF000RUU) at LINC00607 (green), SERPINE1 (red), and APEX1 (purple) genomic loci were plotted using Epigenome Browser. CAGE tracks indicating the putative alternative TSS for LINC00607 is shown in blue (F) Network map showing MYC as a top candidate TF for LINC00607-regulated DEGs. The relationship between MYC and downstream targets are displayed as edges between nodes. The color intensity of each node represents fold change expression, red (upregulated), and green (downregulated). The edges denote predicted relationships with orange indicating activation, blue indicating inhibition and gray representing an unpredicted effect. (G,H) qPCR analysis of indicated transcripts in HUVECs transfected with respective siRNAs under basal condition (G) or HT (H) . The respective scramble control was set as 1. Data represents mean ± SEM from 3 independent experiments. * P
    Figure Legend Snippet: Stimulus-dependent regulatory function of LINC00607 in ECs. (A) Representative images of smFISH detecting LINC00607 (green) in HUVEC treated with normal glucose (5.5 mM D-glucose) and osmolarity control (NM) or 25 mM D-glucose and 5 ng/ml TNFα (HT) for 3 days. DAPI staining indicates the nuclei. Scale bar = 20 μm. (B) Quantitative data of LINC00607 signal per nucleus. LINC00607 signal from 18 nuclei for NM and 24 nuclei for HT was quantified using ImageJ. (C) Venn diagram showing the number of common DEGs in HUVECs upon 607-KD under basal and HT conditions (green). Numbers of unique DEGs in each condition (pink for NM and blue for HT) are indicated. (D) Expression pattern of select DEGs commonly regulated by 607-KD in HUVECs under both baseline and HT. Heatmap is plotted based on z-scaled average gene expression levels. (E) Putative sequence of c-Myc binding site at the LINC00607 promoters (Chr2: 215826423-215837423, Chr2: 215830609-215841609, and Chr2: 215842628-215853628) predicted by TRANSFAC. The HUVEC c-Myc ChIP-seq tracks (ENCODE data ENCFF000RUU) at LINC00607 (green), SERPINE1 (red), and APEX1 (purple) genomic loci were plotted using Epigenome Browser. CAGE tracks indicating the putative alternative TSS for LINC00607 is shown in blue (F) Network map showing MYC as a top candidate TF for LINC00607-regulated DEGs. The relationship between MYC and downstream targets are displayed as edges between nodes. The color intensity of each node represents fold change expression, red (upregulated), and green (downregulated). The edges denote predicted relationships with orange indicating activation, blue indicating inhibition and gray representing an unpredicted effect. (G,H) qPCR analysis of indicated transcripts in HUVECs transfected with respective siRNAs under basal condition (G) or HT (H) . The respective scramble control was set as 1. Data represents mean ± SEM from 3 independent experiments. * P

    Techniques Used: Staining, Expressing, Sequencing, Binding Assay, Chromatin Immunoprecipitation, Activation Assay, Inhibition, Real-time Polymerase Chain Reaction, Transfection

    LINC00607 inhibition reverses the effect of HT in ECs. (A) Experimental design of HT to NM switch experiment. HUVECs were treated with NM or HT for 3 days, and then switched to NM medium for another 1 (H3N1) or 3 days (H3N3) before cell harvest. (B) qPCR detection of FN1 mRNA levels from experiment shown as in (A) . (C) Experimental design of the “reverse” experiment. HUVECs in biological replicates were treated with NM or HT for 7 days, or HT for 4 days before LINC00607 knockdown using LNA GapmeR (607-KD). (D) LINC00607 expression quantified by scRNA-seq of three groups of ECs. (E–H) Representative reversible ( FN1 and SERPINE1 ) and irreversible ( CCL2 and SMAD3 ) DEGs. (I) UMAP of HT-KD scRNA-seq data. (J) LINC00607 expression in HT-KD samples shown on UMAP separated by 7 clusters. Note that Cluster 0, 1, and 6 show high LINC00607 levels (expression level > 1, i.e., 607-Hi cells) and Cluster 4 shows the lowest LINC00607 level (i.e., 607-Lo cells). (K) LINC00607 expression in 7 clusters of cells in HT-KD samples plotted by UMAP. (L–N) Representative DEGs in HT-KD samples plotted on UMAP. (O) Heatmap showing the expression of indicated DEGs in LINC00607-Lo vs. -Hi ECs.
    Figure Legend Snippet: LINC00607 inhibition reverses the effect of HT in ECs. (A) Experimental design of HT to NM switch experiment. HUVECs were treated with NM or HT for 3 days, and then switched to NM medium for another 1 (H3N1) or 3 days (H3N3) before cell harvest. (B) qPCR detection of FN1 mRNA levels from experiment shown as in (A) . (C) Experimental design of the “reverse” experiment. HUVECs in biological replicates were treated with NM or HT for 7 days, or HT for 4 days before LINC00607 knockdown using LNA GapmeR (607-KD). (D) LINC00607 expression quantified by scRNA-seq of three groups of ECs. (E–H) Representative reversible ( FN1 and SERPINE1 ) and irreversible ( CCL2 and SMAD3 ) DEGs. (I) UMAP of HT-KD scRNA-seq data. (J) LINC00607 expression in HT-KD samples shown on UMAP separated by 7 clusters. Note that Cluster 0, 1, and 6 show high LINC00607 levels (expression level > 1, i.e., 607-Hi cells) and Cluster 4 shows the lowest LINC00607 level (i.e., 607-Lo cells). (K) LINC00607 expression in 7 clusters of cells in HT-KD samples plotted by UMAP. (L–N) Representative DEGs in HT-KD samples plotted on UMAP. (O) Heatmap showing the expression of indicated DEGs in LINC00607-Lo vs. -Hi ECs.

    Techniques Used: Inhibition, Real-time Polymerase Chain Reaction, Expressing

    34) Product Images from "Exosomes Derived from Bone Mesenchymal Stem Cells with the Stimulation of Fe3O4 Nanoparticles and Static Magnetic Field Enhance Wound Healing Through Upregulated miR-21-5p"

    Article Title: Exosomes Derived from Bone Mesenchymal Stem Cells with the Stimulation of Fe3O4 Nanoparticles and Static Magnetic Field Enhance Wound Healing Through Upregulated miR-21-5p

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S275650

    Upregulated miR-21-5p in mag-BMSC-Exos can be transferred to HUVECs and HSFs. (***) p
    Figure Legend Snippet: Upregulated miR-21-5p in mag-BMSC-Exos can be transferred to HUVECs and HSFs. (***) p

    Techniques Used:

    35) Product Images from "Selective inhibition of JNK located on mitochondria protects against mitochondrial dysfunction and cell death caused by endoplasmic reticulum stress in mice with LPS-induced ALI/ARDS"

    Article Title: Selective inhibition of JNK located on mitochondria protects against mitochondrial dysfunction and cell death caused by endoplasmic reticulum stress in mice with LPS-induced ALI/ARDS

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2022.5141

    ER stress can induce mitochondrial dysfunction and cell death. (A) Representative western blots of cyto c leakage to the cytosol, p-Bcl-2, cleaved caspase3 and Bax in HUVECs and A549 cells treated with the ER stress activator tunicamycin. (B) Changes in superoxide anion content and mitochondrial membrane permeability (ΔΨ) after HUVECs and A549 cells were treated with the ER stress activator tunicamycin; magnification, ×200. (C) Cell apoptosis detected by double labelling of HUVECs and A549 cells treated with the ER stress activator tunicamycin with annexin-V-FITC and PI. The data are expressed as the mean ± standard error of the mean, n=4/group, * P
    Figure Legend Snippet: ER stress can induce mitochondrial dysfunction and cell death. (A) Representative western blots of cyto c leakage to the cytosol, p-Bcl-2, cleaved caspase3 and Bax in HUVECs and A549 cells treated with the ER stress activator tunicamycin. (B) Changes in superoxide anion content and mitochondrial membrane permeability (ΔΨ) after HUVECs and A549 cells were treated with the ER stress activator tunicamycin; magnification, ×200. (C) Cell apoptosis detected by double labelling of HUVECs and A549 cells treated with the ER stress activator tunicamycin with annexin-V-FITC and PI. The data are expressed as the mean ± standard error of the mean, n=4/group, * P

    Techniques Used: Western Blot, Permeability

    ER stress can induce activation of JNK and mitochondrial localization of JNK in A549 cells and HUVECs. (A) Representative western blots of p-c-Jun, c-Jun, p-JNK and JNK in the total proteins of HUVECs and A549 cells treated with the ER stress activator tunicamycin. (B) Normalized ratio of p-c-Jun/c-Jun in the total proteins. (C) Relative content of JNK in the total proteins. (D) Normalized ratio of p-JNK/JNK in the total proteins. (E) Representative western blots of p-JNK and JNK in the mitochondrial proteins. (F) Relative content of p-JNK in the mitochondrial proteins. (G) Relative content of JNK in the mitochondrial proteins. (H) Normalized ratio of p-JNK/JNK in the mitochondrial proteins. (I) Representative western blots of p-JNK and JNK in the cytosolic/nuclear proteins. (J) Relative content of p-JNK in the cytosolic/nuclear proteins. (K) Relative content of JNK in the cytosolic/nuclear proteins. (L) Normalized ratio of p-JNK/JNK in the cytosolic/nuclear proteins. The data are expressed as the mean ± standard error of the mean. n=4/group. * P
    Figure Legend Snippet: ER stress can induce activation of JNK and mitochondrial localization of JNK in A549 cells and HUVECs. (A) Representative western blots of p-c-Jun, c-Jun, p-JNK and JNK in the total proteins of HUVECs and A549 cells treated with the ER stress activator tunicamycin. (B) Normalized ratio of p-c-Jun/c-Jun in the total proteins. (C) Relative content of JNK in the total proteins. (D) Normalized ratio of p-JNK/JNK in the total proteins. (E) Representative western blots of p-JNK and JNK in the mitochondrial proteins. (F) Relative content of p-JNK in the mitochondrial proteins. (G) Relative content of JNK in the mitochondrial proteins. (H) Normalized ratio of p-JNK/JNK in the mitochondrial proteins. (I) Representative western blots of p-JNK and JNK in the cytosolic/nuclear proteins. (J) Relative content of p-JNK in the cytosolic/nuclear proteins. (K) Relative content of JNK in the cytosolic/nuclear proteins. (L) Normalized ratio of p-JNK/JNK in the cytosolic/nuclear proteins. The data are expressed as the mean ± standard error of the mean. n=4/group. * P

    Techniques Used: Activation Assay, Western Blot

    36) Product Images from "The CREB/KMT5A complex regulates PTP1B to modulate high glucose-induced endothelial inflammatory factor levels in diabetic nephropathy"

    Article Title: The CREB/KMT5A complex regulates PTP1B to modulate high glucose-induced endothelial inflammatory factor levels in diabetic nephropathy

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-021-03629-4

    KMT5A suppression participates in high glucose-mediated endothelial inflammation by augmenting PTP1B expression in endothelial cells. A Western blot analysis of KMT5A, PTP1B and p-p65 levels in HUVECs. B – F qPCR analysis of mRNA expression of KMT5A, PTP1B, IL-1β, TNFα, and IL-6. G Western blot analysis of KMT5A, PTP1B and p-p65 levels in HUVECs. H – L qPCR analysis of mRNA expression of KMT5A, PTP1B, IL-1β, TNFα, and IL-6. (* p
    Figure Legend Snippet: KMT5A suppression participates in high glucose-mediated endothelial inflammation by augmenting PTP1B expression in endothelial cells. A Western blot analysis of KMT5A, PTP1B and p-p65 levels in HUVECs. B – F qPCR analysis of mRNA expression of KMT5A, PTP1B, IL-1β, TNFα, and IL-6. G Western blot analysis of KMT5A, PTP1B and p-p65 levels in HUVECs. H – L qPCR analysis of mRNA expression of KMT5A, PTP1B, IL-1β, TNFα, and IL-6. (* p

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

    CREB interacts with KMT5A to regulate PTP1B transcriptional activity in endothelial cells. A CREB and H4K20me1 were enriched at the PTP1B promoter region. B The putative CREB binding site of PTP1B. C PTP1B promoter activity was determined by luciferase reporter assays. D western blot analysis of KMT5A and CREB levels in HUVECs. E , F qPCR analysis of mRNA expression of KMT5A and CREB. G western blot analysis of KMT5A and CREB levels in HUVECs. H , I qPCR analysis of mRNA expression of KMT5A and CREB. J western blot analysis of KMT5A and PTP1B levels in HUVECs. K , L qPCR analysis of mRNA expression of KMT5A and PTP1B. (* p
    Figure Legend Snippet: CREB interacts with KMT5A to regulate PTP1B transcriptional activity in endothelial cells. A CREB and H4K20me1 were enriched at the PTP1B promoter region. B The putative CREB binding site of PTP1B. C PTP1B promoter activity was determined by luciferase reporter assays. D western blot analysis of KMT5A and CREB levels in HUVECs. E , F qPCR analysis of mRNA expression of KMT5A and CREB. G western blot analysis of KMT5A and CREB levels in HUVECs. H , I qPCR analysis of mRNA expression of KMT5A and CREB. J western blot analysis of KMT5A and PTP1B levels in HUVECs. K , L qPCR analysis of mRNA expression of KMT5A and PTP1B. (* p

    Techniques Used: Activity Assay, Binding Assay, Luciferase, Western Blot, Real-time Polymerase Chain Reaction, Expressing

    CREB interacts with KMT5A. A Several proteins that interact with CREB are shown ( https://inbio-discover.intomics.com/map.html#search ). B The association between CREB and KMT5A in HUVECs was confirmed by CoIP. C Colocalization of CREB and KMT5A in HUVECs was assessed by confocal microscopy. D western blot analysis of KMT5A and H4K20me1 levels in HUVECs. E qPCR analysis of mRNA expression of KMT5A (* p
    Figure Legend Snippet: CREB interacts with KMT5A. A Several proteins that interact with CREB are shown ( https://inbio-discover.intomics.com/map.html#search ). B The association between CREB and KMT5A in HUVECs was confirmed by CoIP. C Colocalization of CREB and KMT5A in HUVECs was assessed by confocal microscopy. D western blot analysis of KMT5A and H4K20me1 levels in HUVECs. E qPCR analysis of mRNA expression of KMT5A (* p

    Techniques Used: Co-Immunoprecipitation Assay, Confocal Microscopy, Western Blot, Real-time Polymerase Chain Reaction, Expressing

    CREB participates in high glucose-induced p65 phosphorylation and inflammatory factor levels via negative regulation of PTP1B expression in endothelial cells. A Western blot analysis of CREB, PTP1B and p-p65 levels in HUVECs. B – F qPCR analysis of mRNA expression of CREB, PTP1B, IL-1β, TNFα and IL-6. G western blot analysis of CREB, PTP1B and p-p65 levels in HUVECs. H – L qPCR analysis of mRNA expression of CREB, PTP1B, IL-1β, TNFα and IL-6. (* p
    Figure Legend Snippet: CREB participates in high glucose-induced p65 phosphorylation and inflammatory factor levels via negative regulation of PTP1B expression in endothelial cells. A Western blot analysis of CREB, PTP1B and p-p65 levels in HUVECs. B – F qPCR analysis of mRNA expression of CREB, PTP1B, IL-1β, TNFα and IL-6. G western blot analysis of CREB, PTP1B and p-p65 levels in HUVECs. H – L qPCR analysis of mRNA expression of CREB, PTP1B, IL-1β, TNFα and IL-6. (* p

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

    37) Product Images from "Cyclin G2 Is Involved in the Proliferation of Placental Trophoblast Cells and Their Interactions with Endothelial Cells"

    Article Title: Cyclin G2 Is Involved in the Proliferation of Placental Trophoblast Cells and Their Interactions with Endothelial Cells

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.926414

    CCNG2 decreases the capacity of HTR8/SVneo cells to integrate into HUVECs. ( A, C ) Fluorescent images showing the 24-h co-culture of HUVECs with HTR8/SVneo cells overexpressing CCNG2 ( A ) and HTR8/SVneo cells with CCNG2 silencing ( C ). The left images show the cellular networks formed by green-stained HTR8/SVneo cells. The middle images show the cellular networks formed by red-stained HUVECs. The right images show the merged images. Original magnification×100. ( B, D ) Statistical analyses of the integration into HUVECs of HTR8/SVneo cells overexpressing CCNG2 ( B ) and HTR8/SVneo cells with CCNG2 silencing ( D ), as determined using ImageJ software. Each experiment was independently performed 3 times.* P
    Figure Legend Snippet: CCNG2 decreases the capacity of HTR8/SVneo cells to integrate into HUVECs. ( A, C ) Fluorescent images showing the 24-h co-culture of HUVECs with HTR8/SVneo cells overexpressing CCNG2 ( A ) and HTR8/SVneo cells with CCNG2 silencing ( C ). The left images show the cellular networks formed by green-stained HTR8/SVneo cells. The middle images show the cellular networks formed by red-stained HUVECs. The right images show the merged images. Original magnification×100. ( B, D ) Statistical analyses of the integration into HUVECs of HTR8/SVneo cells overexpressing CCNG2 ( B ) and HTR8/SVneo cells with CCNG2 silencing ( D ), as determined using ImageJ software. Each experiment was independently performed 3 times.* P

    Techniques Used: Co-Culture Assay, Staining, Software

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    CCN family member 1 (CCN1) siRNA inhibits human umbilical vein endothelial cell (HUVEC) proliferation and induces HUVEC apoptosis under hypoxic conditions. <t>HUVECs</t> were divided into the normoxia group and the hypoxia group (HUVECs exposed to hypoxia); the hypoxia group was further subdivided into the hypoxia-scrambled siRNA group (HUVECs <t>transfected</t> with scrambled siRNA plasmid under hypoxic conditions) and the hypoxia-CCN1 siRNA group (HUVECs transfected with the CCN1 siRNA plasmid under hypoxic conditions). (A) Cell proliferation was evaluated by CCK-8 assay, each day for 4 days following transfection. Day 1 was the day of transfection. (B) Cell apoptosis was determined by flow cytometry using Annexin V/propidium iodide (PI) staining 2 days following transfection. Annexin V was set as the horizontal axis and PI was set as the vertical axis. Upper right (UR) quadrant, late apoptotic or necrotic cells; lower left (LL) quadrant, dual-negative/normal cells; lower right (LR) quadrant, early apoptotic cells; and upper left (UL) quadrant, mechanically damaged cells. The optical densities (at 450 nm) and the total percentages of apoptotic cells are presented as the means ± standard deviation (SD) of 3 independent experiments. ** P
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    CCN family member 1 (CCN1) siRNA inhibits human umbilical vein endothelial cell (HUVEC) proliferation and induces HUVEC apoptosis under hypoxic conditions. HUVECs were divided into the normoxia group and the hypoxia group (HUVECs exposed to hypoxia); the hypoxia group was further subdivided into the hypoxia-scrambled siRNA group (HUVECs transfected with scrambled siRNA plasmid under hypoxic conditions) and the hypoxia-CCN1 siRNA group (HUVECs transfected with the CCN1 siRNA plasmid under hypoxic conditions). (A) Cell proliferation was evaluated by CCK-8 assay, each day for 4 days following transfection. Day 1 was the day of transfection. (B) Cell apoptosis was determined by flow cytometry using Annexin V/propidium iodide (PI) staining 2 days following transfection. Annexin V was set as the horizontal axis and PI was set as the vertical axis. Upper right (UR) quadrant, late apoptotic or necrotic cells; lower left (LL) quadrant, dual-negative/normal cells; lower right (LR) quadrant, early apoptotic cells; and upper left (UL) quadrant, mechanically damaged cells. The optical densities (at 450 nm) and the total percentages of apoptotic cells are presented as the means ± standard deviation (SD) of 3 independent experiments. ** P

    Journal: International Journal of Molecular Medicine

    Article Title: CCN1/Cyr61-PI3K/AKT signaling promotes retinal neovascularization in oxygen-induced retinopathy

    doi: 10.3892/ijmm.2015.2371

    Figure Lengend Snippet: CCN family member 1 (CCN1) siRNA inhibits human umbilical vein endothelial cell (HUVEC) proliferation and induces HUVEC apoptosis under hypoxic conditions. HUVECs were divided into the normoxia group and the hypoxia group (HUVECs exposed to hypoxia); the hypoxia group was further subdivided into the hypoxia-scrambled siRNA group (HUVECs transfected with scrambled siRNA plasmid under hypoxic conditions) and the hypoxia-CCN1 siRNA group (HUVECs transfected with the CCN1 siRNA plasmid under hypoxic conditions). (A) Cell proliferation was evaluated by CCK-8 assay, each day for 4 days following transfection. Day 1 was the day of transfection. (B) Cell apoptosis was determined by flow cytometry using Annexin V/propidium iodide (PI) staining 2 days following transfection. Annexin V was set as the horizontal axis and PI was set as the vertical axis. Upper right (UR) quadrant, late apoptotic or necrotic cells; lower left (LL) quadrant, dual-negative/normal cells; lower right (LR) quadrant, early apoptotic cells; and upper left (UL) quadrant, mechanically damaged cells. The optical densities (at 450 nm) and the total percentages of apoptotic cells are presented as the means ± standard deviation (SD) of 3 independent experiments. ** P

    Article Snippet: The plasmids (500 ng/µ l) were transiently transfected into the HUVECs using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

    Techniques: Transfection, Plasmid Preparation, CCK-8 Assay, Flow Cytometry, Cytometry, Staining, Standard Deviation

    Silencing of Stab2 or Stab1 expression in endothelial cells inhibits endothelial sprouting and migration. (A,B) siRNA mediated silencing of Stab2 (A) or Stab1 (B) inhibited basal, VEGF-A and VEGF-C driven in-gel sprouting in HUVECs. (C,D) Representative spheroids from the sprouting assay shown in (A,B). (E,F) Silencing of Stab2 (E) or Stab1 (F) in HUVECs inhibited basal and VEGF-A driven endothelial migration in the modified Boyden chamber assay. HUVECs were transfected with two different Stab2 or Stab1 siRNAs and allowed to migrate through a membrane for 3 h using 25 ng/ml VEGF-A. Migrated cells were stained and counted under the microscope. n = 3 per group. *p

    Journal: PLoS ONE

    Article Title: HOXC9 Regulates Formation of Parachordal Lymphangioplasts and the Thoracic Duct in Zebrafish via Stabilin 2

    doi: 10.1371/journal.pone.0058311

    Figure Lengend Snippet: Silencing of Stab2 or Stab1 expression in endothelial cells inhibits endothelial sprouting and migration. (A,B) siRNA mediated silencing of Stab2 (A) or Stab1 (B) inhibited basal, VEGF-A and VEGF-C driven in-gel sprouting in HUVECs. (C,D) Representative spheroids from the sprouting assay shown in (A,B). (E,F) Silencing of Stab2 (E) or Stab1 (F) in HUVECs inhibited basal and VEGF-A driven endothelial migration in the modified Boyden chamber assay. HUVECs were transfected with two different Stab2 or Stab1 siRNAs and allowed to migrate through a membrane for 3 h using 25 ng/ml VEGF-A. Migrated cells were stained and counted under the microscope. n = 3 per group. *p

    Article Snippet: HUVECs were transfected with the indicated siRNA (final concentration: 200 nM) using Oligofectamine (Invitrogen).

    Techniques: Expressing, Migration, Modification, Boyden Chamber Assay, Transfection, Staining, Microscopy

    Regulation of TRAF6 expression and the related signaling pathway by TAM-derived exosomes. a , b TRAF6 gene silencing efficiency of EC cells by siTRAF6-1 and siTRAF6-2. c , d TRAF6 depletion by siTRAF6-1 and siTRAF6-2 reduced the migration of HUVECs. The mean ± SD of three independent experiments is shown, and statistical significance is indicated by *(P

    Journal: Cancer Cell International

    Article Title: Suppression of endothelial cell migration by tumor associated macrophage-derived exosomes is reversed by epithelial ovarian cancer exosomal lncRNA

    doi: 10.1186/s12935-017-0430-x

    Figure Lengend Snippet: Regulation of TRAF6 expression and the related signaling pathway by TAM-derived exosomes. a , b TRAF6 gene silencing efficiency of EC cells by siTRAF6-1 and siTRAF6-2. c , d TRAF6 depletion by siTRAF6-1 and siTRAF6-2 reduced the migration of HUVECs. The mean ± SD of three independent experiments is shown, and statistical significance is indicated by *(P

    Article Snippet: RNA extraction and MicroRNA profiling by RT-PCR RNA from exosomes was isolated and enriched with a Total Exosome RNA and Protein Isolation Kit (Invitrogen, CA, USA) according to the user’s guide, and the total RNA of HUVECs stimulated with the exosomes (60 µg/ml) after 48 h was extracted using TRIzol (Invitrogen, CA, USA).

    Techniques: Expressing, Derivative Assay, Migration

    EOC-derived exosomes reverse the suppression of HUVEC migration by TAMs. a , b TAM-derived exosomes and EOCSKOV3 cell exosomes stimulate HUVECs. The inhibition of endothelial cell migration by TAM-derived exosomes was reversed, however, by the direct effect of SKOV3 exosomes in promoting endothelial cell migration. c NF-κB phosphorylation was inhibited after incubation with exosomes derived from SKOV3 cells. d 2 lncRNAs identified as potential NF-κB pathway-associated genes in EOCSKOV3 cell exosomes. e A representative immunoblot of phosphorylated(p-)NF-κB and total NF-κB in HUVECs overexpressing the two lncRNAs

    Journal: Cancer Cell International

    Article Title: Suppression of endothelial cell migration by tumor associated macrophage-derived exosomes is reversed by epithelial ovarian cancer exosomal lncRNA

    doi: 10.1186/s12935-017-0430-x

    Figure Lengend Snippet: EOC-derived exosomes reverse the suppression of HUVEC migration by TAMs. a , b TAM-derived exosomes and EOCSKOV3 cell exosomes stimulate HUVECs. The inhibition of endothelial cell migration by TAM-derived exosomes was reversed, however, by the direct effect of SKOV3 exosomes in promoting endothelial cell migration. c NF-κB phosphorylation was inhibited after incubation with exosomes derived from SKOV3 cells. d 2 lncRNAs identified as potential NF-κB pathway-associated genes in EOCSKOV3 cell exosomes. e A representative immunoblot of phosphorylated(p-)NF-κB and total NF-κB in HUVECs overexpressing the two lncRNAs

    Article Snippet: RNA extraction and MicroRNA profiling by RT-PCR RNA from exosomes was isolated and enriched with a Total Exosome RNA and Protein Isolation Kit (Invitrogen, CA, USA) according to the user’s guide, and the total RNA of HUVECs stimulated with the exosomes (60 µg/ml) after 48 h was extracted using TRIzol (Invitrogen, CA, USA).

    Techniques: Derivative Assay, Migration, Inhibition, Incubation

    Internalization of the exosomes into recipient cells. HUVECs in culture were incubated with TAM-derived exosomes labeled with PKH67 ( green ). HUVECs were fixed with cold methanol and mounted with DAPI. High magnification images of HUVECs incubated with exosomes ( a – c ) or low magnification images of HUVECs incubated with exosomes ( d – f )

    Journal: Cancer Cell International

    Article Title: Suppression of endothelial cell migration by tumor associated macrophage-derived exosomes is reversed by epithelial ovarian cancer exosomal lncRNA

    doi: 10.1186/s12935-017-0430-x

    Figure Lengend Snippet: Internalization of the exosomes into recipient cells. HUVECs in culture were incubated with TAM-derived exosomes labeled with PKH67 ( green ). HUVECs were fixed with cold methanol and mounted with DAPI. High magnification images of HUVECs incubated with exosomes ( a – c ) or low magnification images of HUVECs incubated with exosomes ( d – f )

    Article Snippet: RNA extraction and MicroRNA profiling by RT-PCR RNA from exosomes was isolated and enriched with a Total Exosome RNA and Protein Isolation Kit (Invitrogen, CA, USA) according to the user’s guide, and the total RNA of HUVECs stimulated with the exosomes (60 µg/ml) after 48 h was extracted using TRIzol (Invitrogen, CA, USA).

    Techniques: Incubation, Derivative Assay, Labeling

    The expression of endothelial cell miRNA after co-culture with exosomes and miR-146b-5p can inhibit the migration of endothelial cells. a Quantification of individual miRNAs in HUVECs with or without exosomes derived from tumor-associated macrophages. The y-axis represents the relative miRNA expression level. The results are presented as the mean ± SD. miRNA expression in HUVECs with or without exosomes derived from tumor-associated macrophages. When HUVECs were co-cultured with exosomes derived from tumor-associated macrophages, a modest but statistically significant increase of miRNA expression was observed for miR-146b (*P

    Journal: Cancer Cell International

    Article Title: Suppression of endothelial cell migration by tumor associated macrophage-derived exosomes is reversed by epithelial ovarian cancer exosomal lncRNA

    doi: 10.1186/s12935-017-0430-x

    Figure Lengend Snippet: The expression of endothelial cell miRNA after co-culture with exosomes and miR-146b-5p can inhibit the migration of endothelial cells. a Quantification of individual miRNAs in HUVECs with or without exosomes derived from tumor-associated macrophages. The y-axis represents the relative miRNA expression level. The results are presented as the mean ± SD. miRNA expression in HUVECs with or without exosomes derived from tumor-associated macrophages. When HUVECs were co-cultured with exosomes derived from tumor-associated macrophages, a modest but statistically significant increase of miRNA expression was observed for miR-146b (*P

    Article Snippet: RNA extraction and MicroRNA profiling by RT-PCR RNA from exosomes was isolated and enriched with a Total Exosome RNA and Protein Isolation Kit (Invitrogen, CA, USA) according to the user’s guide, and the total RNA of HUVECs stimulated with the exosomes (60 µg/ml) after 48 h was extracted using TRIzol (Invitrogen, CA, USA).

    Techniques: Expressing, Co-Culture Assay, Migration, Derivative Assay, Cell Culture

    DFO effect on HIF-1α protein in senescent HUVECs. ( A ) qPCR analysis of HIF-1α mRNA in control and DFO-treated senescent HUVECs using the ΔCt method; HPRT1 mRNA was used for normalization. ( B ) Representative HIF-1α and ( D ) Hsp90 western blots in control and DFO-treated (100 µM, 8 hours) senescent HUVECs. Equal protein loading was confirmed probing with GAPDH. ( C , E ) The graphs present densitometric band analysis normalized to GAPDH in arbitrary units (AU). The data represent means ± SD. Control vs . DFO-treated senescent HUVECs cells. ***p

    Journal: Scientific Reports

    Article Title: MicroRNA-126 regulates Hypoxia-Inducible Factor-1α which inhibited migration, proliferation, and angiogenesis in replicative endothelial senescence

    doi: 10.1038/s41598-019-43689-3

    Figure Lengend Snippet: DFO effect on HIF-1α protein in senescent HUVECs. ( A ) qPCR analysis of HIF-1α mRNA in control and DFO-treated senescent HUVECs using the ΔCt method; HPRT1 mRNA was used for normalization. ( B ) Representative HIF-1α and ( D ) Hsp90 western blots in control and DFO-treated (100 µM, 8 hours) senescent HUVECs. Equal protein loading was confirmed probing with GAPDH. ( C , E ) The graphs present densitometric band analysis normalized to GAPDH in arbitrary units (AU). The data represent means ± SD. Control vs . DFO-treated senescent HUVECs cells. ***p

    Article Snippet: Real-time PCRTotal RNA was extracted from early passage and senescent HUVECs, as well as control and DFO-treated (100 µM, 8 hours) senescent HUVECs using the mirVana PARIS RNA and Native Protein Purification Kit (Ambion), according to the manufacturer’s instructions. cDNA was synthesized using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, California, USA) with 2 µg of total RNA primed with random hexamer primers, following the manufacturer’s instructions.

    Techniques: Real-time Polymerase Chain Reaction, Western Blot

    DFO effect on wound healing in senescent HUVECs. ( A ) Representative photomicrographs of senescent and DFO-treated senescent HUVECs cells 8 hours after wounding. Red lines indicate the edge of the wound repopulating cells. Magnification 100x. ( B ) Time course of changes in the size of the remaining wound. The data points represent the % open area means ± SD. n = 4 in duplicate.

    Journal: Scientific Reports

    Article Title: MicroRNA-126 regulates Hypoxia-Inducible Factor-1α which inhibited migration, proliferation, and angiogenesis in replicative endothelial senescence

    doi: 10.1038/s41598-019-43689-3

    Figure Lengend Snippet: DFO effect on wound healing in senescent HUVECs. ( A ) Representative photomicrographs of senescent and DFO-treated senescent HUVECs cells 8 hours after wounding. Red lines indicate the edge of the wound repopulating cells. Magnification 100x. ( B ) Time course of changes in the size of the remaining wound. The data points represent the % open area means ± SD. n = 4 in duplicate.

    Article Snippet: Real-time PCRTotal RNA was extracted from early passage and senescent HUVECs, as well as control and DFO-treated (100 µM, 8 hours) senescent HUVECs using the mirVana PARIS RNA and Native Protein Purification Kit (Ambion), according to the manufacturer’s instructions. cDNA was synthesized using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, California, USA) with 2 µg of total RNA primed with random hexamer primers, following the manufacturer’s instructions.

    Techniques: