Journal: Cell Reports Medicine

Article Title: iPSC-derived hypoimmunogenic tissue resident memory T cells mediate robust anti-tumor activity against cervical cancer

doi: 10.1016/j.xcrm.2023.101327

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Article Snippet: VEGF , Miltenyi Biotec , Cat# 130-109-386.

Techniques: Recombinant, DNA Extraction, Sequencing, Staining, Flow Cytometry, Cell Stimulation, Software

Journal: Cell Communication and Signaling : CCS

Article Title: Endomucin selectively regulates vascular endothelial growth factor receptor-2 endocytosis through its interaction with AP2

doi: 10.1186/s12964-024-01606-w

Figure Lengend Snippet: Essential role of EMCN in facilitating VEGFR2 and AP2 interaction and clathrin recruitment. A Colocalization of clathrin HC (heavy chain) (green) and VEGFR2 (red) were visualized in control HRECs with or without VEGF165 (10 ng/ml). Examples of colocalization of VEGFR2 and clathrin HC (white arrowhead), and clathrin (white arrow) were shown in magnified view. Bar = 10 µm B Fraction of VEGFR2 that colocalized with clathrin was quantified by Image J CoJAP plugin. Student t-test was used for the comparison. * P < 0.05, n = 3. C Colocalization of clathrin HC (heavy chain) (green) and VEGFR2 (red) were visualized in siNT or siEMCN HRECs with VEGF165 (10 ng/ml) stimulation. Examples of colocalization of VEGFR2 and clathrin HC (white arrowheads), and VEGFR2 (white arrow) were shown in magnified view. Bar = 10 µm D Fraction of VEGFR2 that colocalized with clathrin in siNT and siEMCN HRECs was quantified. Student t-test was used for the comparison. ** P < 0.01, n = 3. E Colocalization of clathrin HC (heavy chain) (green) and AP2 (red) were visualized in control in siNT and siEMCN HRECs with VEGF(10 ng/ml) stimulation. Examples of colocalization of clathrin HC and AP2 (white arrowhead) were shown in magnified view. Bar = 10 µm ( F ) Fraction of clathrin that colocalized with AP2 in siNT and siEMCN HRECs with VEGF stimulation were quantified. Student t-test was used for the comparison. P > 0.05, n = 3. G EMCN is required for interaction between VEGFR2 and AP2A2. HRECs were lysed and VEGFR2 that co-immunoprecipitated with AP2A2 in the presence and absence of EMCN was observed. n = 3

Article Snippet: Recombinant human VEGF165 (#293-VE), PlGF-2 (#6837-PL), and FGF2 (#233-FB) were purchased from R&D Systems.

Techniques: Control, Comparison, Immunoprecipitation

Journal: Cell Communication and Signaling : CCS

Article Title: Endomucin selectively regulates vascular endothelial growth factor receptor-2 endocytosis through its interaction with AP2

doi: 10.1186/s12964-024-01606-w

Figure Lengend Snippet: EMCN does not modulate VEGF165 or PIGF-induced endothelial migration or VEGFR1 internalization. A EMCN knockdown did not affect PlGF-2-induced HREC migration. HRECs were transfected with either siNT or siEMCN, mechanically scratched, stimulated with PlGF-2 (10 ng/ml) or VEGF165 (10 ng/ml), and the resulting cell migration was quantified by image analysis (left). ** P < 0.01, n = 6 or 9. Representative images of each group at time zero (white dashed line) and 15 h time (yellow dashed line) points (right). The scale bar represents 500 µm. B Illustration of the cell surface receptor internalization assay. Growth factors bind to its cell surface receptors and induce receptor internalization. Cell surface proteins are biotinylated, the cell surface fraction is separated using avidin resin, and western blot analysis were used to analyze the fraction of receptors remaining at the cell surface. C HRECs incubated in serum-free media were stimulated with VEGF165 (10 ng/ml) for 30 min with and without EMCN knockdown, and cell surface membrane-bound VEGFR1 (mVEGFR1) levels were analyzed by western blot analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, n = 6. One-way ANOVA was used for statistical analysis

Article Snippet: Recombinant human VEGF165 (#293-VE), PlGF-2 (#6837-PL), and FGF2 (#233-FB) were purchased from R&D Systems.

Techniques: Migration, Knockdown, Transfection, Cell Surface Receptor Assay, Avidin-Biotin Assay, Western Blot, Incubation, Membrane

Journal: Cell Communication and Signaling : CCS

Article Title: Endomucin selectively regulates vascular endothelial growth factor receptor-2 endocytosis through its interaction with AP2

doi: 10.1186/s12964-024-01606-w

Figure Lengend Snippet: EMCN is not required for FGF2-induced HREC cell migration or FGFR1 internalization. A EMCN knockdown did not affect FGF2-induced HREC migration. HRECs were transfected with either siNT or siEMCN, incubated in serum-free media for 8 h, mechanically scratched, and stimulated with FGF2 (10 ng/ml) or VEGF165 (10 ng/ml). Quantification of cell migration for all the treatment groups based on image analysis (left). Student t-test was used for comparisons within groups. * P < 0.05, n = 8. Representative images of each group at time zero (white dashed line) and 15 h time (yellow dashed line) points (right). The scale bar represents 500 µm. B EMCN knockdown did not affect FGF2-induced FGFR1 internalization in HREC. Serum-starved HRECs were stimulated with FGF2 for 45 min, and then the cell surface proteins were isolated and visualized by western blot. Quantification of FGFR1 at the cell surface from all treatment groups by western blot analysis (left). Student-t test was used for statistical analysis. * P < 0.05, n = 7. A representative western blot for all treatment groups (right). C EMCN does not interact with VEGFR1 or FGFR1 in HRECs. HRECs overexpressing myc-tagged EMCN were lysed, and cell surface receptors that co-immunoprecipitated with EMCN were observed. n = 3. Note that the IgG and Myc groups were overexposed together for the better detection of the different receptors because of the low protein levels, while the input groups were kept at a lower exposure

Article Snippet: Recombinant human VEGF165 (#293-VE), PlGF-2 (#6837-PL), and FGF2 (#233-FB) were purchased from R&D Systems.

Techniques: Migration, Knockdown, Transfection, Incubation, Isolation, Western Blot, Immunoprecipitation

Journal: Cell Communication and Signaling : CCS

Article Title: Endomucin selectively regulates vascular endothelial growth factor receptor-2 endocytosis through its interaction with AP2

doi: 10.1186/s12964-024-01606-w

Figure Lengend Snippet: EMCN knockdown inhibits VEGF121 induced VEGFR2 internalization and HRECs migration similar to VEGF165. A Both VEGF165 (10 ng/ml)- and VEGF121 (7.29 ng/ml)-induced migration were inhibited with EMCN knockdown. Quantification of cell migration by image analysis is shown (left). Student-t test was used for comparisons between groups. * P < 0.05, ** P < 0.01, n = 10. Representative images of each group at time zero (white dashed line) and 15 h time (yellow dashed line) points. The scale bar represents 500 µm. B Schematic representation of VEGFA isoforms, VEGF165 and VEGF121. C HRECs were treated with siEMCN stimulated with VEGF121 (7.29 ng/ml) for a time course of up to 120 min. VEGF121 induced significant VEGFR2 endocytosis after 60 min of stimulation, except when EMCN was knockdown. One-way ANOVA was used for comparation within group. Student-t test was used for comparation between siNT and siEMCN at the same time point. # P < 0.05, * P < 0.05, *** P < 0.001, **** P < 0.0001, n = 3. D Representative image of the western blot for VEGFR2 internalization in both siNT and siEMCN groups. CD31 was blotted as cell surface fraction loading control

Article Snippet: Recombinant human VEGF165 (#293-VE), PlGF-2 (#6837-PL), and FGF2 (#233-FB) were purchased from R&D Systems.

Techniques: Knockdown, Migration, Western Blot, Control

Journal: PLoS Computational Biology

Article Title: Optimizing antibody affinity and stability by the automated design of the variable light-heavy chain interfaces

doi: 10.1371/journal.pcbi.1007207

Figure Lengend Snippet: ( a ) G6 and 18 low-energy designs, each encoding 4–10 mutations relative to G6 (number of mutations is indicated above the bars) were tested for binding using yeast display at 8 nM VEGF concentration, resulting in seven designs that showed comparable or higher binding signal compared to G6. G6 des1 and G6 des13 were chosen for further characterization (colored in blue and orange, respectively). ( b ) SPR kinetic analysis of VEGF binding with twofold dilutions from a maximal concentration of 100 nM by G6, G6 des1 , and G6 des13 Fabs demonstrated faster binding on-rate in the designs ( k a = 2.3 * 10 5 M -1 s -1 , 3.27 * 10 5 M -1 s -1 and 5.3 * 10 5 M -1 s -1 , respectively). G6 des13 also improved binding off-rate ( k d = 3.2 * 10 −5 s -1 compared to 6 * 10 −5 s -1 in G6), resulting in an improved dissociation constant ( K D = 60 pM compared to 270 pM in G6). ( c & d ) Thermal denaturation and temperature of aggregation onset experiments, respectively, using microscale thermophoresis indicated substantially higher apparent stability in the designs. ( e ) Computational mutation-tolerance mapping indicated 11 positions at the vL-vH interface of the anti-VEGF antibody G6 (spheres) with potentially tolerated mutations. Thumbnails indicate selected mutations in a model structure of G6 des13 relative to G6 (gray). ( f ) Expression levels in HEK293 cells of G6 and the designs formatted as IgG were measured using Western blot analysis showing approximately an order of magnitude improvement in IgG expression levels for the designs. (g) Native mass-spectrometry analysis exhibited higher tolerance to applied collision energy in G6 des13 compared to G6, both formatted as IgG. The error bars represent standard deviations inferred from three repeats.

Article Snippet: The wild-type and designed antibodies were tested for binding by flow cytometry with 8 nM biotinylated VEGF (Recombinant Human VEGF 165, Biotinylated Protein R&D systems).

Techniques: Binding Assay, Concentration Assay, Microscale Thermophoresis, Mutagenesis, Expressing, Western Blot, Mass Spectrometry