Journal: Aging (Albany NY)

Article Title: Histone methyltransferase Smyd2 drives vascular aging by its enhancer-dependent activity

doi: 10.18632/aging.204449

Figure Lengend Snippet: Smyd2 is upregulated in vascular aging both in vivo and in vitro . ( A , B ) The in vivo aging model was established by Ang II infusion in mice for 28 days and harvested the aortas. The protein expressions of Smyd2, VCAM-1, and p21 were detected by immunoblots ( A ). The immunohistochemistry assay was performed with Smyd2 antibody. Scale bars, 50 μm ( B ). ( C – E ) Ang II (100 nM, 48 h)-induced RAECs was used as the in vitro aging model. Smyd2 and the senescence-related phenotype markers were increased in a dose-dependent manner in Ang II-induced RAECs ( C ). The protein expression of Smyd2 was increased in a time-dependent manner in Ang II-induced RAECs ( D ). RT-qPCR analysis of Smyd2 and Cdkn1a genes in Ang II-induced RAECs ( E ). Data are presented as the mean ± SEMs, * p < 0.05, ** p < 0.01, *** p < 0.001, each acquired from three individual experiments ( n = 3).

Article Snippet: Reagents and antibodies used in this study were obtained as follows: angiotension II (Ang II) was purchased from Meilunbio (Meilun Biotechnology, Dalian, China); the Smyd2 specific inhibitor LLY-507 was purchased from MedChem Express (MCE, USA); Smyd2, p53, p21, p16 and γ-H2AX antibodies were obtained from Cell Signaling Biotechnology (Danvers, MA, USA); H3K4me3, H3K4me1 and H3K27ac antibodies were purchased from Abcam (Cambridge, MA, USA); COX-2, VCAM-1 and GAPDH were purchased from Proteintech (Rosemont, IL, USA); p-ATM and p-Chk2 antibodies were purchased from Absci (MD, USA); IL-6 and iNOS antibodies were purchased from ABclonal (Wuhan, China).

Techniques: In Vivo, In Vitro, Western Blot, Immunohistochemistry, Expressing, Quantitative RT-PCR

Journal: Aging (Albany NY)

Article Title: Histone methyltransferase Smyd2 drives vascular aging by its enhancer-dependent activity

doi: 10.18632/aging.204449

Figure Lengend Snippet: Knockdown of Smyd2 attenuates Ang II-induced RAECs senescence. Smyd2 was knocked down using si Smyd2 in Ang II-induced RAECs, si CTL was used as the negative control. ( A , B ) The SA-β-gal staining and EdU incorporation assay. Representative staining images are shown in the figure left, and the statistical analysis of positive cells is shown on the right. Scale bars, 200 μm. ( C ) Western blot analysis of Smyd2 and the senescence markers (p53, p21 and p16), the proinflammatory mediators (iNOS, VCAM-1, COX-2 and IL-6) and the DNA damage marker (p-ATM) protein. Representative western blot images are shown in the figure above, and the statistical analysis of relative protein expression is shown below. GAPDH was used as the loading control. ( D ) The mRNA expression of Il6 gene by RT-qPCR analysis. ( E , F ) Immunofluorescence double staining of Smyd2 and the senescence markers (p53 and p16). Scale bars, 100 μm. Data are presented as the mean ± SEMs, * p < 0.05, ** p < 0.01, *** p < 0.001, each acquired from three individual experiments ( n = 3).

Article Snippet: Reagents and antibodies used in this study were obtained as follows: angiotension II (Ang II) was purchased from Meilunbio (Meilun Biotechnology, Dalian, China); the Smyd2 specific inhibitor LLY-507 was purchased from MedChem Express (MCE, USA); Smyd2, p53, p21, p16 and γ-H2AX antibodies were obtained from Cell Signaling Biotechnology (Danvers, MA, USA); H3K4me3, H3K4me1 and H3K27ac antibodies were purchased from Abcam (Cambridge, MA, USA); COX-2, VCAM-1 and GAPDH were purchased from Proteintech (Rosemont, IL, USA); p-ATM and p-Chk2 antibodies were purchased from Absci (MD, USA); IL-6 and iNOS antibodies were purchased from ABclonal (Wuhan, China).

Techniques: Negative Control, Staining, Western Blot, Marker, Expressing, Quantitative RT-PCR, Immunofluorescence, Double Staining

Journal: Aging (Albany NY)

Article Title: Histone methyltransferase Smyd2 drives vascular aging by its enhancer-dependent activity

doi: 10.18632/aging.204449

Figure Lengend Snippet: Inhibition of Smyd2 alleviates Ang II-induced senescence associated phenotypes in vitro . ( A , B ) SA-β-gal staining ( A ) and EdU incorporation ( B ) assays in Ang II-induced RAECs with LLY-507 (3 μM) pretreatment. Representative staining images are shown in the figure left, and the statistical analysis of positive cells is shown in the figure right. Scale bars, 100 μm. ( C ) Expressions of senescence markers (p53, p21 and p16), pro-inflammatory molecules (VCAM-1 and COX-2) and DNA damage markers (p-Chk2 and γH2AX) were detected by western blot analysis upon LLY-507 pretreatment in Ang II-induced RAECs. Representative western blot images are shown on the left, and the statistical analysis of relative protein expressions is shown on the right. GAPDH was used as the loading control. ( D ) The mRNA levels of Nos2 (encoding the iNOS protein) in Ang II-induced RAECs with LLY-507 pretreatment was examined by RT-qPCR. ( E , F ) Immunofluorescence double staining of Smyd2 and the senescence markers (p53 and p16) upon LLY-507 pretreatment in Ang II-induced RAECs. Scale bars, 100 μm or 50 μm. Data are presented as the mean ± SEMs, * p < 0.05, ** p < 0.01, *** p < 0.001, each acquired from three individual experiments ( n = 3).

Article Snippet: Reagents and antibodies used in this study were obtained as follows: angiotension II (Ang II) was purchased from Meilunbio (Meilun Biotechnology, Dalian, China); the Smyd2 specific inhibitor LLY-507 was purchased from MedChem Express (MCE, USA); Smyd2, p53, p21, p16 and γ-H2AX antibodies were obtained from Cell Signaling Biotechnology (Danvers, MA, USA); H3K4me3, H3K4me1 and H3K27ac antibodies were purchased from Abcam (Cambridge, MA, USA); COX-2, VCAM-1 and GAPDH were purchased from Proteintech (Rosemont, IL, USA); p-ATM and p-Chk2 antibodies were purchased from Absci (MD, USA); IL-6 and iNOS antibodies were purchased from ABclonal (Wuhan, China).

Techniques: Inhibition, In Vitro, Staining, Western Blot, Quantitative RT-PCR, Immunofluorescence, Double Staining

Journal: Aging (Albany NY)

Article Title: Histone methyltransferase Smyd2 drives vascular aging by its enhancer-dependent activity

doi: 10.18632/aging.204449

Figure Lengend Snippet: Smyd2 heterozygous knockout mice ameliorates senescence-associated phenotypes upon Ang II infusion. Smyd2 +/− mice and their wild-type littermates (WT) were infused with Ang II for 4 weeks. ( A ) Immunoblots of the proinflammatory mediators (COX-2 and VCAM-1). ( B , C ) The immunofluorescence analysis of Smyd2 with p53 or VCAM-1 in the cross-sectional area of blood vessels. Scale bars, 100 μm. Data are presented as the mean ± SEMs, *** p < 0.001, each acquired from three individual experiments ( n = 3).

Article Snippet: Reagents and antibodies used in this study were obtained as follows: angiotension II (Ang II) was purchased from Meilunbio (Meilun Biotechnology, Dalian, China); the Smyd2 specific inhibitor LLY-507 was purchased from MedChem Express (MCE, USA); Smyd2, p53, p21, p16 and γ-H2AX antibodies were obtained from Cell Signaling Biotechnology (Danvers, MA, USA); H3K4me3, H3K4me1 and H3K27ac antibodies were purchased from Abcam (Cambridge, MA, USA); COX-2, VCAM-1 and GAPDH were purchased from Proteintech (Rosemont, IL, USA); p-ATM and p-Chk2 antibodies were purchased from Absci (MD, USA); IL-6 and iNOS antibodies were purchased from ABclonal (Wuhan, China).

Techniques: Knock-Out, Western Blot, Immunofluorescence

Journal: Aging (Albany NY)

Article Title: Histone methyltransferase Smyd2 drives vascular aging by its enhancer-dependent activity

doi: 10.18632/aging.204449

Figure Lengend Snippet: Smyd2 is associated with other vascular aging-related diseases. ( A ) The protein expressions of Smyd2, the senescence marker (p21) and the proinflammatory mediators (VCAM-1, COX-2 and IL-6) were examined in a later passage of RAECs by Western blot. GAPDH was used as the loading control. ( B ) Western blot analysis of Smyd2 and the senescence markers (p21, VCAM-1 and IL-6) in arteries of two-kidney one-clip renovascular hypertensive (2K1C) mice. The statistical analysis of relative protein expression is shown in the figure below. ( C ) Western blot analysis of Smyd2 in arteries of db/db mice and its control db/m mice. ( D ) The mRNA levels of Smyd2 and the senescence marker genes ( Ptgs2, Cdkn1a and Trp53 ) in arteries of db/db mice and its control db/m mice. ( E ) The expression of Smyd2 in mice atherosclerotic artery samples was evaluated by western blot. ( F ) The protein level of Smyd2 in thoracic aorta in healthy (control) and atherosclerotic (AS) patients evaluated by western blot. Data are presented as the mean ± SEMs, n = 3 or 6. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Reagents and antibodies used in this study were obtained as follows: angiotension II (Ang II) was purchased from Meilunbio (Meilun Biotechnology, Dalian, China); the Smyd2 specific inhibitor LLY-507 was purchased from MedChem Express (MCE, USA); Smyd2, p53, p21, p16 and γ-H2AX antibodies were obtained from Cell Signaling Biotechnology (Danvers, MA, USA); H3K4me3, H3K4me1 and H3K27ac antibodies were purchased from Abcam (Cambridge, MA, USA); COX-2, VCAM-1 and GAPDH were purchased from Proteintech (Rosemont, IL, USA); p-ATM and p-Chk2 antibodies were purchased from Absci (MD, USA); IL-6 and iNOS antibodies were purchased from ABclonal (Wuhan, China).

Techniques: Marker, Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: Blocking VCAM-1 ameliorates hypertensive cardiac remodeling by impeding macrophage infiltration

doi: 10.3389/fphar.2022.1058268

Figure Lengend Snippet: VCAM-1 is upregulated in HF patients and Ang II-infused mice. (A) Analysis of serum VCAM-1 levels in HF patients and control subjects by ELISA ( n = 30) (B) Analysis of serum BNP levels in HF patients and control subjects by ELISA ( n = 30) (C) WT mice were infused with saline or Ang II (1,000 ng/kg/min) for 14 days qPCR analysis of VCAM-1 mRNA levels in heart tissues ( n = 6) (D) Immunoblot analysis of VCAM-1 protein levels in heart tissues (top) and quantification of relative protein levels (bottom, n = 4) (E) Immunohistochemical staining of VCAM-1 in heart sections (left). Scale bar: 50 μm. Quantification of the VCAM-1-positive area (right, n = 6). The data are expressed as the M ± SD, and n represents the number of human subjects or animal samples. * p < 0.05 vs saline group and *** p < 0.001 vs saline group or control group.

Article Snippet: The membranes were incubated with appropriate antibodies against VCAM-1 (1:200, Proteintech), calcineurin A (1:800, CST), phosphor (p)-STAT3 (1:500, CST), STAT3 (1:500, Arigo), TGF-β1 (1:800, CST), p-Smad2/3 (1:500, CST), Smad2/3 (1:1,000, CST), p-IKKα (1:500, CST), IKKα (1:1,000, CST), p-p65 (1:500, CST), p65 (1:1,000, CST), NOX1 (1:800, CST), NOX4 (1:800, CST) and GAPDH (1:2000, HUABIO).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Immunohistochemical staining, Staining

Journal: Frontiers in Pharmacology

Article Title: Blocking VCAM-1 ameliorates hypertensive cardiac remodeling by impeding macrophage infiltration

doi: 10.3389/fphar.2022.1058268

Figure Lengend Snippet: Inhibition of VCAM-1 attenuates Ang II-induced hypertension, cardiac hypertrophy and cardiac dysfunction. (A) WT mice were infused with saline or Ang II (1,000 ng/kg/min) for 14 days and cotreated with a VCAM-1 neutralizing antibody at a dose of 0.1 and 0.2 mg/mouse or IgG control every 2 days (B) Systolic blood pressure (SBP) in each group ( n = 6) (C) Echocardiographic analysis (left) and quantification of the ejection fraction (EF%) (right, n = 6) (D) H&E staining of heart sections (left). Scale bar: 0.5 cm. The ratio of heart weight to body weight (HW/BW) (right, n = 6) (E) WGA staining of heart sections (left). Scale bar: 50 μm. Analysis of the cross-sectional area of myocytes (right, n = 6) (F) qPCR analysis of ANF, BNP and MYH7 levels in the heart ( n = 6) (G) Immunoblot analysis of cardiac calcineurin A (CaNA) and p-STAT3 (left) and analysis of the level of these proteins (right, n = 4). The data are expressed as the M ± SD, and n represents the number of animals. *** p < 0.001 vs saline + IgG group. # p < 0.05, ## p < 0.01 and ### p < 0.001 vs Ang II + IgG group.

Article Snippet: The membranes were incubated with appropriate antibodies against VCAM-1 (1:200, Proteintech), calcineurin A (1:800, CST), phosphor (p)-STAT3 (1:500, CST), STAT3 (1:500, Arigo), TGF-β1 (1:800, CST), p-Smad2/3 (1:500, CST), Smad2/3 (1:1,000, CST), p-IKKα (1:500, CST), IKKα (1:1,000, CST), p-p65 (1:500, CST), p65 (1:1,000, CST), NOX1 (1:800, CST), NOX4 (1:800, CST) and GAPDH (1:2000, HUABIO).

Techniques: Inhibition, Staining, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Blocking VCAM-1 ameliorates hypertensive cardiac remodeling by impeding macrophage infiltration

doi: 10.3389/fphar.2022.1058268

Figure Lengend Snippet: Blockade of VCAM-1 suppresses Ang II induced cardiac fibrosis. (A) Masson’s trichrome staining of heart sections (left). Scale bar: 50 μm. Analysis of the fibrotic areas (right, n = 6) (B) Immunofluorescence staining of heart sections with an anti-collagen III antibody (left). Scale bar: 50 μm. Analysis of collagen III positive areas (right, n = 6) (C) Immunohistochemical staining of heart sections with an anti-α-SMA antibody (left). Scale bar: 50 μm. Analysis of α-SMA-positive areas (right, n = 6) (D) qPCR analysis of collagen I, collagen III and α-SMA expression in the heart ( n = 6) (E) Immunoblot analysis of TGF-β1 and p-Smad2/3 (left) and analysis of the levels of these proteins (right, n = 4). The data are expressed as the M ± SD, and n represents the number of animals. ** p < 0.01 and *** p < 0.001 vs saline + IgG group. # p < 0.05, ## p < 0.01 and ### p < 0.001 vs Ang II + IgG group.

Article Snippet: The membranes were incubated with appropriate antibodies against VCAM-1 (1:200, Proteintech), calcineurin A (1:800, CST), phosphor (p)-STAT3 (1:500, CST), STAT3 (1:500, Arigo), TGF-β1 (1:800, CST), p-Smad2/3 (1:500, CST), Smad2/3 (1:1,000, CST), p-IKKα (1:500, CST), IKKα (1:1,000, CST), p-p65 (1:500, CST), p65 (1:1,000, CST), NOX1 (1:800, CST), NOX4 (1:800, CST) and GAPDH (1:2000, HUABIO).

Techniques: Staining, Immunofluorescence, Immunohistochemical staining, Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Blocking VCAM-1 ameliorates hypertensive cardiac remodeling by impeding macrophage infiltration

doi: 10.3389/fphar.2022.1058268

Figure Lengend Snippet: Blocking VCAM-1 inhibits Ang II-induced VLA-4 + macrophage infiltration and the expression of proinflammatory cytokines. (A) H&E staining of heart sections. Scale bar: 100 μm (B) Immunohistochemical staining of heart sections with an anti-CD68 antibody (left). Scale bar: 50 μm. Analysis of CD68-positive macrophages (right, n = 6) (C) Immunofluorescence staining of heart sections with an anti-CD68 and anti-VLA-4 antibody (left). Scale bar: 50 μm. Analysis of CD68 and VLA-4-positive areas (right, n = 6) (D) qPCR analysis of IL-1β, IL-6 and TNF-α expression in the heart ( n = 6) (E) Immunoblot analysis of VLA-4, p-IKKα and p-p65 (left) and analysis of the levels of these proteins (right, n = 4). The data are expressed as the M ± SD, and n represents the number of animals. * p < 0.05 and *** p < 0.001 vs saline + IgG group. # p < 0.05 and ### p < 0.001 vs Ang II + IgG group.

Article Snippet: The membranes were incubated with appropriate antibodies against VCAM-1 (1:200, Proteintech), calcineurin A (1:800, CST), phosphor (p)-STAT3 (1:500, CST), STAT3 (1:500, Arigo), TGF-β1 (1:800, CST), p-Smad2/3 (1:500, CST), Smad2/3 (1:1,000, CST), p-IKKα (1:500, CST), IKKα (1:1,000, CST), p-p65 (1:500, CST), p65 (1:1,000, CST), NOX1 (1:800, CST), NOX4 (1:800, CST) and GAPDH (1:2000, HUABIO).

Techniques: Blocking Assay, Expressing, Staining, Immunohistochemical staining, Immunofluorescence, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Blocking VCAM-1 ameliorates hypertensive cardiac remodeling by impeding macrophage infiltration

doi: 10.3389/fphar.2022.1058268

Figure Lengend Snippet: Blockade of VCAM-1 reduces Ang II-induced oxidative stress and DNA damage. (A) Immunofluorescence staining of heart sections (left). Scale bar: 50 μm. Analysis of the DHE fluorescence intensity (right, n = 6) (B) Immunofluorescence staining of heart sections with an anti-γ-H2AX antibody (left). Scale bar: 20 μm. Analysis of γ-H2AX-positive nuclei (right, n = 6) (C) Immunohistochemical staining of heart sections with an anti-nitrotyrosine antibody (left). Scale bar: 50 μm. Analysis of nitrotyrosine-positive areas (right, n = 6) (D) qPCR analysis of NOX1, NOX2 and NOX4 expression in the heart ( n = 6) (E) Immunoblot analysis of NOX1 and NOX4 (left) and analysis the levels of these proteins (right, n = 4). The data are expressed as the M ± SD, and n represents the number of animals. *** p < 0.001 vs saline + IgG group. # p < 0.05, ## p < 0.01 and ### p < 0.001 vs Ang II + IgG group.

Article Snippet: The membranes were incubated with appropriate antibodies against VCAM-1 (1:200, Proteintech), calcineurin A (1:800, CST), phosphor (p)-STAT3 (1:500, CST), STAT3 (1:500, Arigo), TGF-β1 (1:800, CST), p-Smad2/3 (1:500, CST), Smad2/3 (1:1,000, CST), p-IKKα (1:500, CST), IKKα (1:1,000, CST), p-p65 (1:500, CST), p65 (1:1,000, CST), NOX1 (1:800, CST), NOX4 (1:800, CST) and GAPDH (1:2000, HUABIO).

Techniques: Immunofluorescence, Staining, Fluorescence, Immunohistochemical staining, Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Blocking VCAM-1 ameliorates hypertensive cardiac remodeling by impeding macrophage infiltration

doi: 10.3389/fphar.2022.1058268

Figure Lengend Snippet: Blocking VCAM-1 inhibits macrophage adhesion and migration and reduces cardiomyocyte hypertrophy and myofibroblast activation in vitro . (A) The bone marrow-derived macrophages (BMMs) were stimulated with Ang II (100 nM) or saline for 24 h. ELISA analysis of the IL-1β, IL-6 and TNF-α levels ( n = 6) (B) HUVECs were pretreated an anti-VCAM-1 antibody (5 μg/ml) or IgG control for 2 h and then stimulated with Ang II (100 nM) or saline for an additional 24 h. Adhesion of PKH26-labeled BMMs (red, left, scale bar: 100 μm) and analysis of adherent cells (right, n = 6 fields) (C) Migration of BMMs (left). Scale bar: 100 μm. Analysis of migrated cells (right, n = 6 fields) (D) HUVECs and BMMs were co-cultured and treated with anti-VCAM-1 (5 μg/ml) and Ang II (100 nM) for 24 h. The supernatants were collected and added to CMs or CFs for 24 h. Cardiomyocyte size was determined using immunostaining with anti-α-actinin antibody. (E) The mRNA levels of ANF and BNP in CMs were analyzed by qPCR analysis ( n = 6). (F) The protein levels of CaNA and p-STAT3 in lysates from CMs were determined by immunoblotting analysis (left), and quantification of relative protein level (right, n = 3 independent experiments). (G) The mRNA levels of collagen I and collagen III in CFs were analyzed by qPCR analysis ( n = 6). (H) The protein levels of TGF-β1 and p-Smad2/3 in lysates from CFs were evaluated by immunoblotting analysis (left), and quantification of relative protein level (right, n = 3 independent experiments).

Article Snippet: The membranes were incubated with appropriate antibodies against VCAM-1 (1:200, Proteintech), calcineurin A (1:800, CST), phosphor (p)-STAT3 (1:500, CST), STAT3 (1:500, Arigo), TGF-β1 (1:800, CST), p-Smad2/3 (1:500, CST), Smad2/3 (1:1,000, CST), p-IKKα (1:500, CST), IKKα (1:1,000, CST), p-p65 (1:500, CST), p65 (1:1,000, CST), NOX1 (1:800, CST), NOX4 (1:800, CST) and GAPDH (1:2000, HUABIO).

Techniques: Blocking Assay, Migration, Activation Assay, In Vitro, Derivative Assay, Enzyme-linked Immunosorbent Assay, Labeling, Cell Culture, Immunostaining, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Blocking VCAM-1 ameliorates hypertensive cardiac remodeling by impeding macrophage infiltration

doi: 10.3389/fphar.2022.1058268

Figure Lengend Snippet: A working model of the mechanism by which VCAM-1 regulates Ang II-induced cardiac remodeling. Ang II infusion upregulates VCAM-1 expression, which promotes VLA-4 + macrophage adhesion to the endothelium and infiltration into the heart, inducing the release of abundant proinflammatory cytokines and ROS to activate multiple signaling pathways, thereby leading to cardiomyocyte hypertrophy, myofibroblast activation, and subsequent cardiac remodeling. Conversely, selective blockade of VCAM-1 effectively prevents these effects.

Article Snippet: The membranes were incubated with appropriate antibodies against VCAM-1 (1:200, Proteintech), calcineurin A (1:800, CST), phosphor (p)-STAT3 (1:500, CST), STAT3 (1:500, Arigo), TGF-β1 (1:800, CST), p-Smad2/3 (1:500, CST), Smad2/3 (1:1,000, CST), p-IKKα (1:500, CST), IKKα (1:1,000, CST), p-p65 (1:500, CST), p65 (1:1,000, CST), NOX1 (1:800, CST), NOX4 (1:800, CST) and GAPDH (1:2000, HUABIO).

Techniques: Expressing, Activation Assay

Journal: Cell & Bioscience

Article Title: Neutrophil extracellular traps promote thrombogenicity in cerebral venous sinus thrombosis

doi: 10.1186/s13578-022-00845-z

Figure Lengend Snippet: NETs induce thrombogenicity in HBMECs. HBMECs were incubated with isolated NETs in the presence or absence of DNase I, APC and sivelestat. a , b Representative images showing ICAM-1 (red) and VCAM-1 (green) in treated ECs as observed by confocal microscopy. c The expression of ICAM-1 (red) and VCAM-1 (green) was measured by western blotting. d , e Representative images showing TF (red) and CD31 (green) expression on ECs. f The expression of TF on ECs was measured by western blotting. g , h Representative images showing VWF (red) and CD31 (green) expression on ECs incubated with NETs. i The expression of VWF in the supernatant in each group. (j) Fibrin formation in ECs incubated with NETs (0.5 μg/mL) in the presence of DNase I, APC and sivelestat. Statistics: Ordinary one-way ANOVA (i, j). The inset scale bars in a , b , d , e , g and h are 20 μm. ***P < 0.001.****P < 0.0001

Article Snippet: ECs were cultured on fibronectin-coated slide flasks and stained with anti-CD31 (ab9498, 1 μg/mL), anti-von Willebrand factor (vWF, Proteintech, 1 μg/mL), anti-ICAM-1 (Proteintech, 1 μg/mL), anti-VCAM-1 (Proteintech, 1 μg/mL), and anti-TF antibodies (ab48647, 1 μg/mL).

Techniques: Incubation, Isolation, Confocal Microscopy, Expressing, Western Blot