Journal: Frontiers in Endocrinology

Article Title: A novel link between chronic inflammation and humanin regulation in children

doi: 10.3389/fendo.2023.1142310

Figure Lengend Snippet: Local effects of TNF on humanin, PCNA, SOX9 and TRAF2 expressions in growth plates tissue specimens (n=3) in patient 1 and patient 2 analyzed separately. Quantitative analysis of (A, E) humanin staining, (B, F) PCNA staining, (C, G) SOX9 staining, and (D, H) TRAF2 staining. Error bars indicate mean ± SE. Students t-test was used to analyze differences between groups.

Article Snippet: Next, slides were incubated with anti-humanin antibody (NB100-56877; Novus Biologicals, Littleton, Colorado, USA), anti-proliferating cell nuclear antigen (PCNA) antibody (ab-18197; Abcam, Cambridge, United Kingdom), anti-SOX9 antibody (ab-5355; Sigma-Aldrich, Burlington, MA, USA), and anti-TRAF2 antibody (NB100-56173SS; Novus Biologicals, Littleton, Colorado, USA) overnight at 4°C, 1:300 diluted for all antibodies.

Techniques: Staining

Journal: Frontiers in Endocrinology

Article Title: A novel link between chronic inflammation and humanin regulation in children

doi: 10.3389/fendo.2023.1142310

Figure Lengend Snippet: TNF suppressed SOX9, PCNA and TRAF2 expressions in human growth plate tissue specimens (n=6) obtained from 2 children or human chondrocytes. (A, B) Quantitative analysis of SOX9 staining (yellow arrows), calculated as number of positive cells per mm². (C) Relative expression of SOX9 assessed by qPCR in HCS-2/8 cells treated for 72 hours with TNF at 10, 30, 100, 300 ng/ml concentrations (n=3). (D, E) Quantitative analysis of PCNA staining (yellow arrows), calculated as number of positive cells per mm². (F) Relative expression of PCNA assessed by qPCR in HCS-2/8 cells treated for 72 hours with TNF at 10, 30, 100, 300 ng/ml concentrations (n=3). (G) Western blot analysis of SOX9 and PCNA expressions in HCS-2/8 cells treated with TNF (100 ng/ml). (H, I) Quantification of SOX9 and PCNA expressions by three independent Western blot experiments. (J, K) Quantitative analysis of TRAF2 staining (yellow arrows), calculated as number of positive cells per mm². Error bars indicate mean ± SE. Students t-test was used to analyze differences between groups.

Article Snippet: Next, slides were incubated with anti-humanin antibody (NB100-56877; Novus Biologicals, Littleton, Colorado, USA), anti-proliferating cell nuclear antigen (PCNA) antibody (ab-18197; Abcam, Cambridge, United Kingdom), anti-SOX9 antibody (ab-5355; Sigma-Aldrich, Burlington, MA, USA), and anti-TRAF2 antibody (NB100-56173SS; Novus Biologicals, Littleton, Colorado, USA) overnight at 4°C, 1:300 diluted for all antibodies.

Techniques: Staining, Expressing, Western Blot

Journal: Angiogenesis

Article Title: Aflibercept exhibits VEGF binding stoichiometry distinct from bevacizumab and does not support formation of immune-like complexes

doi: 10.1007/s10456-016-9515-8

Figure Lengend Snippet: Aflibercept forms 1:1 complexes with VEGF 165 . The molar masses of aflibercept:VEGF 165 and bevacizumab:VEGF 165 complexes were analyzed by multi-angle laser light scattering detection coupled to SEC. The differential refractive index ( right y axis) and the measured molar mass ( left y axis) of peaks are indicated as a function of elution volume for each sample. The experimentally determined molar masses are indicated by horizontal lines. Cartoons of free VEGF 165 and complexes of aflibercept or bevacizumab bound to VEGF 165 are shown. Complexes of VEGF 165 with bevacizumab ( a ) or aflibercept ( b ) at various molar ratios were incubated for 12 h at ambient temperature. Following incubation, the samples were kept at 4 °C in the autosampler prior to injection (~100–200 µg per sample) onto a Superose 12 column pre-equilibrated in 10 mM phosphate containing 500 mM NaCl buffer (pH 7.0) with a flow rate of 0.3 mL/min. Chromatograms of VEGF 165 and bevacizumab ( a ) or aflibercept ( b ) are superimposed to indicate the elution profiles of the unbound proteins. The 1:1 molar ratio complexes yielded similar elution profiles and are not shown for the purposes of clarity

Article Snippet: Immune complexes were preassembled by mixing antibody + antigen at a 1:1 molar ratio (126 μg M90 [Florida Hospital] + 45 μg CD154 [Peprotech], or 35 μg VEGF [Peprotech, produced in E. coli ] + either 126 μg bevacizumab [Genentech] or 92 μg aflibercept [Regeneron] ± heparin [Sigma-Aldrich] in PBS).

Techniques: Refractive Index, Incubation, Injection

Journal: Angiogenesis

Article Title: Aflibercept exhibits VEGF binding stoichiometry distinct from bevacizumab and does not support formation of immune-like complexes

doi: 10.1007/s10456-016-9515-8

Figure Lengend Snippet: In vitro binding affinity of bevacizumab and aflibercept for Fcγ receptors in the presence and absence of VEGF 121 and VEGF 165 determined by SPR (Biacore)

Article Snippet: Immune complexes were preassembled by mixing antibody + antigen at a 1:1 molar ratio (126 μg M90 [Florida Hospital] + 45 μg CD154 [Peprotech], or 35 μg VEGF [Peprotech, produced in E. coli ] + either 126 μg bevacizumab [Genentech] or 92 μg aflibercept [Regeneron] ± heparin [Sigma-Aldrich] in PBS).

Techniques: In Vitro, Binding Assay, Injection

Journal: Angiogenesis

Article Title: Aflibercept exhibits VEGF binding stoichiometry distinct from bevacizumab and does not support formation of immune-like complexes

doi: 10.1007/s10456-016-9515-8

Figure Lengend Snippet: Aflibercept:VEGF 165 complexes do not activate platelets in vitro. a Preformed equal molar (200 nM) aflibercept:VEGF 165 or bevacizumab:VEGF 165 complexes were added to primed (1 µM epinephrine), washed platelets containing 200 nM UFH unfractionated heparin and percent light transmittance monitored at 600 nm. Thrombin (1 nM, Chrono-PAR) acted as the positive control. b A range (400–50 nM) of preformed equal molar bevacizumab:VEGF 165 complexes were added to primed, washed platelets containing UFH unfractionated heparin and percent light transmittance monitored. A similar experiment using aflibercept:VEGF 165 complexes did not activate platelets (data not shown), and thus, only data for the 200 nM complex ( a ) are shown. Serotonin release was measured from platelets stimulated in the presence of a range of concentrations (0.1, 0.2, 0.5 or 1.0 µM) of UFH with aflibercept:VEGF 165 complex ( c ) or bevacizumab:VEGF 165 complex ( d ). Inhibitor:ligand complex concentration was 500 nM

Article Snippet: Immune complexes were preassembled by mixing antibody + antigen at a 1:1 molar ratio (126 μg M90 [Florida Hospital] + 45 μg CD154 [Peprotech], or 35 μg VEGF [Peprotech, produced in E. coli ] + either 126 μg bevacizumab [Genentech] or 92 μg aflibercept [Regeneron] ± heparin [Sigma-Aldrich] in PBS).

Techniques: In Vitro, Positive Control, Concentration Assay

Journal: Angiogenesis

Article Title: Aflibercept exhibits VEGF binding stoichiometry distinct from bevacizumab and does not support formation of immune-like complexes

doi: 10.1007/s10456-016-9515-8

Figure Lengend Snippet: Aflibercept + VEGF 165 + unfractionated heparin (UFC) complexes do not activate platelets in vivo. B6;SJL-Tg (FcγRIIa)11Mkz (FCGR2A) mice were injected with PBS ( n = 5) or preformed immune complexes ( n = 10 per group) via the tail vein. Ten minutes after reagent injection, mice were anesthetized, blood was collected by cardiac puncture, and platelet counts were measured using an automated cell counter. Following blood draws, animals were killed and lungs were dissected, rinsed, and embedded in paraffin. Paraffin blocks were sliced, and cut sections (2 µm thick) were deparaffinized, rehydrated, and stained. Shown are platelet counts per individual FcγRIIa animal by treatment group ( a ) along with representative H&E microscopy sections for PBS ( b ) bevacizumab (Bev) + VEGF 165 + heparin ( c ) and aflibercept + VEGF 165 + heparin ( d ). The horizontal line in ( a ) represents approximately 60 % of reduction from baseline mean platelet count. Microscopy images were captured at ×200 magnification. Insets represent ×700 magnification

Article Snippet: Immune complexes were preassembled by mixing antibody + antigen at a 1:1 molar ratio (126 μg M90 [Florida Hospital] + 45 μg CD154 [Peprotech], or 35 μg VEGF [Peprotech, produced in E. coli ] + either 126 μg bevacizumab [Genentech] or 92 μg aflibercept [Regeneron] ± heparin [Sigma-Aldrich] in PBS).

Techniques: In Vivo, Injection, Staining, Microscopy

Journal: Angiogenesis

Article Title: Aflibercept exhibits VEGF binding stoichiometry distinct from bevacizumab and does not support formation of immune-like complexes

doi: 10.1007/s10456-016-9515-8

Figure Lengend Snippet: Aflibercept does not exhibit significant cell surface binding to ARPE-19 and HUVEC. Cell surface binding of aflibercept and bevacizumab was evaluated using ARPE-19 ( a ) and HUVEC ( b ). Cells were pre-seeded on collagen-coated 96-well plates. ARPE-19 cells were incubated with 5 nM bevacizumab or aflibercept alone or in the presence of 10 nM VEGF 165 or 10 nM VEGF 121 at 37 °C for 1 h. HUVEC were incubated with 15 nM bevacizumab or aflibercept alone or in the presence of 10 nM VEGF 165 or 10 nM VEGF 121 at 37 °C for 1 h. Surface-bound inhibitor was detected by incubation with A488-anti-hIgG (Fc-specific) at 4 °C. Cells were washed, fixed with 4 % paraformaldehyde, and counterstained with a nucleic acid counterstain (DAPI for ARPE-19 or DRAQ5 for HUVEC, red fluorescence) prior to analysis. Cell surface binding was evaluated with secondary antibody alone ( left column ), bevacizumab ( middle column ) or aflibercept ( right column ) in the presence of VEGF 121 ( a – c ), VEGF 165 ( d – f ) or no ligand ( g – i ). Scale bar = 50 μm in ( a ) and 100 μm in ( b )

Article Snippet: Immune complexes were preassembled by mixing antibody + antigen at a 1:1 molar ratio (126 μg M90 [Florida Hospital] + 45 μg CD154 [Peprotech], or 35 μg VEGF [Peprotech, produced in E. coli ] + either 126 μg bevacizumab [Genentech] or 92 μg aflibercept [Regeneron] ± heparin [Sigma-Aldrich] in PBS).

Techniques: Binding Assay, Incubation, Fluorescence

Journal: Angiogenesis

Article Title: Aflibercept exhibits VEGF binding stoichiometry distinct from bevacizumab and does not support formation of immune-like complexes

doi: 10.1007/s10456-016-9515-8

Figure Lengend Snippet: In vitro binding of bevacizumab and aflibercept to NRP1.mFc and heparin–biotin by SPR (Biacore) in the presence of VEGF 165 . a Human NRP1.mFc (155 RU) was captured on an anti-mouse Fc-coupled chip surface. The histogram represents bevacizumab and aflibercept at concentrations of 500, 100, 10, 5, and 1 nM either alone or pre-complexed at 1:1 molar ratio with human VEGF 121 or VEGF 165 . b Heparin–biotin (35 RU) was captured on a neutravidin-coupled chip surface. The histogram represents bevacizumab and aflibercept at concentrations of 500, 100, 10, 5, and 1 nM either alone or pre-complexed at 1:1 molar ratio with human VEGF 121 or VEGF 165 . Representative sensograms of bevacizumab and aflibercept binding to NRP1.mFc and heparin at 5 nM are shown in Supplementary Figure 7

Article Snippet: Immune complexes were preassembled by mixing antibody + antigen at a 1:1 molar ratio (126 μg M90 [Florida Hospital] + 45 μg CD154 [Peprotech], or 35 μg VEGF [Peprotech, produced in E. coli ] + either 126 μg bevacizumab [Genentech] or 92 μg aflibercept [Regeneron] ± heparin [Sigma-Aldrich] in PBS).

Techniques: In Vitro, Binding Assay

Journal: Angiogenesis

Article Title: Aflibercept exhibits VEGF binding stoichiometry distinct from bevacizumab and does not support formation of immune-like complexes

doi: 10.1007/s10456-016-9515-8

Figure Lengend Snippet: Cell surface binding of bevacizumab is directly proportional to endogenous VEGF 165 concentration. ARPE-19 cells, cultured on human fibronectin-coated coverslips in six-well plates, were treated with equimolar concentration (1.68 μM) of bevacizumab, aflibercept, or hFc for 3 days starting from different time points (Day 2, 4, 9, and 14) followed by immunofluorescence staining of cell surface-bound inhibitor ( red fluorescence, detected with mouse antihuman IgG Fc-specific, and secondary Ab, goat anti-mouse IgG-Alexa flour 594. Nuclei were counterstained with DAPI in blue). Cells and culture media at various culture times (Days 2, 6, 9, and 28) were collected for next-generation sequencing and ELISA of VEGF expression levels. Cells treated with bevacizumab ( a – d ) showed an increased cell surface binding in confluent ARPE-19 cell culture, coincident with the upregulation of VEGF expression ( m , n ). Cells treated with aflibercept ( e – h ) or control protein hFc ( i – l ) showed minimal binding at all time points. Scale Bar = 50 μm

Article Snippet: Immune complexes were preassembled by mixing antibody + antigen at a 1:1 molar ratio (126 μg M90 [Florida Hospital] + 45 μg CD154 [Peprotech], or 35 μg VEGF [Peprotech, produced in E. coli ] + either 126 μg bevacizumab [Genentech] or 92 μg aflibercept [Regeneron] ± heparin [Sigma-Aldrich] in PBS).

Techniques: Binding Assay, Concentration Assay, Cell Culture, Immunofluorescence, Staining, Fluorescence, Next-Generation Sequencing, Enzyme-linked Immunosorbent Assay, Expressing, Control