itgav rabbit pab  (Bioss)

Journal: Frontiers in Pharmacology

Article Title: Histidine-rich glycoprotein inhibits TNF-α–induced tube formation in human vascular endothelial cells

doi: 10.3389/fphar.2025.1561628

Figure Lengend Snippet: Primer Sequences Used for qPCR Analysis.

Article Snippet: The integrin alpha V antibody was obtained from R&D Systems (AF1219-SP, Minneapolis, MN, United States), and anti-integrin beta eight antibodies were sourced from Thermo Fisher Scientific (PA5-100843, Waltham, MA, United States).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Histidine-rich glycoprotein inhibits TNF-α–induced tube formation in human vascular endothelial cells

doi: 10.3389/fphar.2025.1561628

Figure Lengend Snippet: TNF-α induces integrin αV and β8 expression in EA. hy926 cells. (A) Relative mRNA expression of integrins αV, β3, and β8 in EA. hy926 cells treated with 100 ng/mL TNF-α for 4.5 h. mRNA levels were measured using qRT-PCR and normalized against GAPDH levels. The results are expressed as mean ± SD based on data from three independent experiments ( n = 3). ** p < 0.01 when compared to the control group. To assess the differences among the groups, an independent samples t-test was employed for statistical analysis. (B) Western blots show an increase in integrin αV and β8 protein levels. Data are presented as mean ± SD of values from three independent experiments ( n = 3). To determine statistical significance, one-way ANOVA was performed, followed by Tukey’s post hoc analysis.* p < 0.05, ** p < 0.01 compared to the control.

Article Snippet: The integrin alpha V antibody was obtained from R&D Systems (AF1219-SP, Minneapolis, MN, United States), and anti-integrin beta eight antibodies were sourced from Thermo Fisher Scientific (PA5-100843, Waltham, MA, United States).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Histidine-rich glycoprotein inhibits TNF-α–induced tube formation in human vascular endothelial cells

doi: 10.3389/fphar.2025.1561628

Figure Lengend Snippet: Effect of integrin αV and β8 on TNF-α-dependent tube formation in EA. hy926 cells. Cells were transfected with siRNAs targeting integrins αV and β8. (A) qRT-PCR analysis of the extent of suppression of target genes integrin αV and β8 by RNAi. After 24 h, αV siRNA suppressed the expression of the αV gene by 81% and β8 siRNA suppressed the expression of the β8 gene by 94%. Tukey’s post hoc test was used for statistical analysis (** p < 0.01, †† p < 0.01), and the data are presented as mean ± SD from three independent experiments ( n = 3). (B) FACS analysis of integrin αV and β8 protein expression. Mean fluorescence intensity (MFI) of integrin αV, β8, and αVβ8 was measured in EA. hy926 cells transfected with control siRNA, αV siRNA, β8 siRNA, or both. Knockdown of αV and β8 significantly reduced the protein levels of these integrins (p < 0.01), further confirming the efficiency of RNAi-mediated suppression. (C) Tube formation assays involved EA. hy926 cells treated with control, control siRNA, αV siRNA, β8 siRNA, or αV + β8 siRNA, followed by TNF-α (100 ng/mL) stimulation. Scale bar: 200 μm. (D) TNF-α treatment significantly enhanced the tube length compared with that in the control siRNA treatment, but this increase was notably mitigated by treatment with αV and β8 siRNAs. Results are expressed as mean ± SD from three independent experiments ( n = 3). (** p < 0.01, †† p < 0.01; Tukey’s post hoc test).

Article Snippet: The integrin alpha V antibody was obtained from R&D Systems (AF1219-SP, Minneapolis, MN, United States), and anti-integrin beta eight antibodies were sourced from Thermo Fisher Scientific (PA5-100843, Waltham, MA, United States).

Techniques: Transfection, Quantitative RT-PCR, Expressing, Fluorescence, Control, Knockdown

Journal: Frontiers in Pharmacology

Article Title: Histidine-rich glycoprotein inhibits TNF-α–induced tube formation in human vascular endothelial cells

doi: 10.3389/fphar.2025.1561628

Figure Lengend Snippet: The effect of HRG on TNF-α-induced integrin expression and tube formation in EA. hy926 cells. (A) Representative images of tube formation in EA. hy926 cells treated with TNF-α (100 ng/mL) and various concentrations of HRG (10, 30, and 75 μg/mL). Scale bar: 200 μm. (B) Treatment with HRG at 10, 30, and 75 μg/mL progressively reduced TNF-α-induced tube formation, as evidenced by a decrease in number of branches, number of branch points, and total tube length. Data are shown as mean ± SD from three independent experiments ( n = 3). (** p < 0.01, † p < 0.05, †† p < 0.01; Tukey’s post hoc test). (C) qRT-PCR analysis showing the relative mRNA levels of integrin αV and β8 in EA. hy926 cells treated with TNF-α and HRG. TNF-α treatment significantly upregulated the mRNA levels of integrin αV and β8, whereas cotreatment with HRG markedly suppressed this upregulation (** p < 0.01, †† p < 0.01; Tukey’s post hoc test). (D) FACS analysis of the cell surface expression of integrin αVβ8 in EA.hy926 cells treated with TNF-α and HRG. The mean fluorescence intensity (MFI) values indicate that TNF-α significantly increased the expression of integrin αVβ8, which was significantly reduced by cotreatment with HRG. Data represent mean ± SD from three independent experiments ( n = 3) (** p < 0.01, † p < 0.05, †† p < 0.01; Tukey’s post hoc test). (E) Immunofluorescence images illustrating the impact of HRG on TNF-α-induced integrin αVβ8 expression in EA. hy926 cells. Cells were treated with TNF-α (100 ng/mL) with or without HRG. Immunostaining was performed for integrin αVβ8 (green), F-actin using phalloidin (red), and nuclei with DAPI (blue). Merged images show the colocalization of these markers. HRG treatment reduced the TNF-α–induced increase in the expression of integrin αVβ8. Scale bars: 200 μm.

Article Snippet: The integrin alpha V antibody was obtained from R&D Systems (AF1219-SP, Minneapolis, MN, United States), and anti-integrin beta eight antibodies were sourced from Thermo Fisher Scientific (PA5-100843, Waltham, MA, United States).

Techniques: Expressing, Quantitative RT-PCR, Fluorescence, Immunofluorescence, Immunostaining

Journal: Frontiers in Pharmacology

Article Title: Histidine-rich glycoprotein inhibits TNF-α–induced tube formation in human vascular endothelial cells

doi: 10.3389/fphar.2025.1561628

Figure Lengend Snippet: HRG-induced nuclear translocation of NRF2 and its inhibitory effects on TNF-α-induced angiogenic processes in EA.hy926 cells. (A) Analysis of NRF2 expression via Western blotting in the nuclear and cytoplasmic fractions of EA. hy926 cells treated with HRG (75 μg/mL) over different time points (1, 2, 4, 6, and 9 h). Loading controls for nuclear and cytoplasmic proteins were histone H3 and β-actin, respectively. HRG treatment induced translocation of NRF2 to the nucleus in a time-dependent manner; unt: untreated. (B) Representative images of tube formation in EA. hy926 cells treated with TNF-α (100 ng/mL) and various concentrations of SML0959, an NRF2 activator (1, 3, and 10 μM). Scale bar: 200 μm. (C) Number of branches, number of branch points, and total tube length under each condition. Treatment with NRF2 activator significantly reduced TNF-α–induced tube formation in a dose-dependent manner. Data are presented as mean ± SD of values from three independent experiments ( n = 3) (** p < 0.01, † p < 0.05, †† p < 0.01; Tukey’s post hoc test). (D) qRT-PCR analysis showing the relative mRNA levels of integrin αV and β8 in EA. hy926 cells treated with TNF-α and NRF2 activator. Treatment with an NRF2 activator significantly reduced the TNF-α–induced mRNA levels of integrin αV and β8. Data are presented as mean ± SD of values from three independent experiments ( n = 3) (** p < 0.01, †† p < 0.01; Tukey’s post hoc test). (E) FACS analysis of the cell surface expression of integrin αVβ8 in EA. hy926 cells treated with TNF-α and NRF2 activator. The MFI values indicate that TNF-α significantly increased the expression of integrin αVβ8, which was significantly reduced by cotreatment with NRF2 activator. Data are presented as mean ± SD of values from three independent experiments ( n = 3). (** p < 0.01, † p < 0.05, †† p < 0.01; Tukey’s post hoc test).

Article Snippet: The integrin alpha V antibody was obtained from R&D Systems (AF1219-SP, Minneapolis, MN, United States), and anti-integrin beta eight antibodies were sourced from Thermo Fisher Scientific (PA5-100843, Waltham, MA, United States).

Techniques: Translocation Assay, Expressing, Western Blot, Quantitative RT-PCR

Journal: Frontiers in Pharmacology

Article Title: Histidine-rich glycoprotein inhibits TNF-α–induced tube formation in human vascular endothelial cells

doi: 10.3389/fphar.2025.1561628

Figure Lengend Snippet: The effect of TNF-α and HRG on integrin αVβ8 expression and tube formation in EA. hy926 endothelial cells. Immunostaining was performed for integrin αVβ8 (green), actin filaments (stained with phalloidin, red), and nuclei (stained with DAPI, blue). The images show untreated cells (control, left) and cells treated with TNF-α (right). Representative images of the tube formation assay showing green fluorescence from tube-like structures formed by endothelial cells. Scale bars: 20 μm for immunostaining images and 200 μm for tube formation images. TNF-α treatment led to a significant increase in integrin αVβ8 expression and subsequent tube formation, whereas HRG/NRF2 signaling modulates this process by inhibiting integrin αVβ8 expression and angiogenesis.

Article Snippet: The integrin alpha V antibody was obtained from R&D Systems (AF1219-SP, Minneapolis, MN, United States), and anti-integrin beta eight antibodies were sourced from Thermo Fisher Scientific (PA5-100843, Waltham, MA, United States).

Techniques: Expressing, Immunostaining, Staining, Control, Tube Formation Assay, Fluorescence

Journal: bioRxiv

Article Title: Overcoming Immune Checkpoint Inhibitor Resistance via Potent and Selective Dual αvβ6/8 Inhibitors Based on Engineered Lasso Peptides

doi: 10.1101/2025.01.28.635346

Figure Lengend Snippet:

Article Snippet: For the integrins αvβ1, αvβ3, αvβ5, αvβ6 and αvβ8, binding was visualized using a mouse anti-human integrin αv (CD51) monoclonal antibody targeting the αv subunit (cat. no. MAB1219 was purchased from R&D Systems).

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay