Journal: Journal of Pharmaceutical Analysis

Article Title: Development of a novel NanoBRET high-throughput drug screening assay for human GnRH receptor using sulfo-cyanine 5 fluorophore

doi: 10.1016/j.jpha.2025.101532

Figure Lengend Snippet: Receptor binding assays of sulfo-cyanine 5-D-Lys 6 -gonadotropin-releasing hormone (sCy 5 -D-Lys 6 -GnRH) in SCL60 cells (a human embryonic kidney 293 cell line stably expressing rat gonadotropin-releasing hormone (GnRH) receptor). (A) Saturation binding assay . SCL60 cells were incubated with various concentrations (0, 0.1–100 nM) of sCy 5 -D-Lys 6 -GnRH for 4 h at 4 °C. Following incubation, unbound ligands were removed by washing the cells with ice-cold Dulbecco's phosphate-buffered saline (DPBS), and relative fluorescence intensity/units (RFU) were measured at emission 680 nm (Em680) under excitation of 640 nm (Ex640). Total binding (TB) was assessed in the absence of unlabeled GnRH I, while non-specific binding (NSB) was determined in the presence of a 100-fold excess of unlabeled GnRH I. Specific binding (SB) was calculated by subtracting NSB from TB. (B) Inhibition binding assay. SCL60 cells were incubated with 10 nM sCy 5 -D-Lys 6 -GnRH and increasing concentrations of unlabeled GnRH I (0–10 μM) for 4 h at 4 °C. After incubation, unbound ligands were washed away with ice-cold DPBS, followed by measurement of RFU. Representative results from three independent experiments, each performed in quadruplicate, are shown. Data are presented as mean ± standard deviations (SD).

Article Snippet: Human embryonic kidney 293 (HEK293, ECACC: 85120602) and African green monkey kidney fibroblast (COS-7, ECACC: 87021302) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Binding Assay, Stable Transfection, Expressing, Saturation Assay, Incubation, Saline, Fluorescence, Inhibition

Journal: Journal of Pharmaceutical Analysis

Article Title: Development of a novel NanoBRET high-throughput drug screening assay for human GnRH receptor using sulfo-cyanine 5 fluorophore

doi: 10.1016/j.jpha.2025.101532

Figure Lengend Snippet: Phosphorylation of extracellular signal-regulated kinase 1/2 (p-ERK1/2) induced by sulfo-cyanine 5-D-Lys 6 -gonadotropin-releasing hormone (sCy 5 -D-Lys 6 -GnRH) , gonadotropin-releasing hormone (GnRH) I, and sCy 5 -kisspeptin 10 (sCy 5 -KP10) in human embryonic kidney 293 (HEK293) cell lines stably expressing rat GnRH receptor (SCL60) or human GnRH receptor (hGnRHR). (A) Time-dependent p-ERK1/2. SCL60 cells were treated with 10 nM sCy 5 -D-Lys 6 -GnRH or GnRH I for the indicated times. (B) Dose-dependent p-ERK1/2. SCL60 cells were treated with vehicle (–∞) or increasing concentrations of sCy 5 -D-Lys 6 -GnRH or GnRH I for 5 min. (C) Ligand-induced ERK1/2 phosphorylation and its inhibition by GnRH antagonist Cetrorelix. HEK 293 cells stably expressing hGnRHR were treated with 10 nM of sCy 5 -D-Lys 6 -GnRH, GnRH I, or 1 μM of sCy 5 -KP10, in the absence or presence of 1 μM of Cetrorelix, for 5 min. β-actin was used as a loading control. Representative blots from three independent experiments are presented. ∗∗ P < 0.01, ∗∗∗ P < 0.001 compared with vehicle-treated controls.

Article Snippet: Human embryonic kidney 293 (HEK293, ECACC: 85120602) and African green monkey kidney fibroblast (COS-7, ECACC: 87021302) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Phospho-proteomics, Stable Transfection, Expressing, Inhibition, Control

Journal: Journal of Pharmaceutical Analysis

Article Title: Development of a novel NanoBRET high-throughput drug screening assay for human GnRH receptor using sulfo-cyanine 5 fluorophore

doi: 10.1016/j.jpha.2025.101532

Figure Lengend Snippet: Protein expression levels of N-terminal secretory signal peptide-NanoLuciferase-human gonadotropin-releasing hormone receptor (N-secNluc-hGnRHR) and its mutants. (A) The protein expression levels of N-secNluc-hGnRHR-K191E and N-secNluc-hGnRHR-K191 deletion (K191Δ) mutants relative to the N-secNluc-hGnRHR were determined by the measurement of relative luminescence intensity/units (RLU) in human embryonic kidney 293 (HEK293) cells transiently expressing N-secNluc-hGnRHRs. (B) The cell surface receptor expression levels of N-secNLuc-hGnRHR and N-secNluc-hGnRHR-K191Δ, transiently expressed in African green monkey kidney fibroblast (COS-7) cells, were measured by relative fluorescence intensity/units (RFU) at emission 680 (Em680) nm under excitation of 640 (Ex640) nm through receptor binding assays using 10, 50, and 100 nM, respectively, of sulfo-cyanine 5-D-Lys 6 -gonadotropin-releasing hormone (sCy 5 -D-Lys 6 -GnRH). The N-secNluc-hGnRHR-K191Δ (solid/black bars) construct gave a 4.5 to 5-fold higher receptor expression than that of N-secNluc-hGnRHR (open/white bars) on the cell membrane ( ∗∗∗ P < 0.001, n = 3).

Article Snippet: Human embryonic kidney 293 (HEK293, ECACC: 85120602) and African green monkey kidney fibroblast (COS-7, ECACC: 87021302) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Cell Surface Receptor Assay, Fluorescence, Binding Assay, Construct, Membrane

Journal: Journal of Pharmaceutical Analysis

Article Title: Development of a novel NanoBRET high-throughput drug screening assay for human GnRH receptor using sulfo-cyanine 5 fluorophore

doi: 10.1016/j.jpha.2025.101532

Figure Lengend Snippet: Confocal microscopy image analysis of sulfo-cyanine 5-D-Lys 6 -gonadotropin-releasing hormone (sCy 5 -D-Lys 6 -GnRH) binding to human embryonic kidney 293 (HEK293) cells expressing gonadotropin-releasing hormone (GnRH) receptor. (A) Fluorescence of sCy 5 -D-Lys 6 -GnRH bound HEK293 cells expressing N-terminal secretory signal peptide-NanoLuciferase-human gonadotropin-releasing hormone receptor with K191 deletion (N-secNluc-hGnRHR-K191Δ). (B) Overlay of both fluorescence and brightfield/differential interference contrast (DIC) images of the cells in sCy 5 -D-Lys 6 -GnRH bound HEK293 cells expressing N-secNluc-hGnRHR-K191Δ. (C) Abolishment of fluorescence of sCy 5 -D-Lys 6 -GnRH bound HEK293 cells expressing N-secNluc-hGnRHR-K191Δ by co-treatment of the cells with 1 μM of GnRH I. (D) Overlay of both fluorescence and brightfield/DIC images of the HEK293 cells expressing N-secNluc-hGnRHR-K191Δ treated with both sCy 5 -D-Lys 6 -GnRH and 1 μM of GnRH I. (E) The cell membrane localisation of fluorescence of sCy 5 -D-Lys 6 -GnRH bound HEK293 cells expressing GnRHR and the monomeric teal fluorescent protein-H-Ras membrane biomarker (mTFP-H-Ras). (F) Cell membrane localisation of mTFP-H-Ras membrane biomarker of the sCy 5 -D-Lys 6 -GnRH incubated HEK293 cells expressing GnRHR and mTFP-H-Ras.

Article Snippet: Human embryonic kidney 293 (HEK293, ECACC: 85120602) and African green monkey kidney fibroblast (COS-7, ECACC: 87021302) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Confocal Microscopy, Binding Assay, Expressing, Fluorescence, Membrane, Biomarker Discovery, Incubation

Journal: Journal of Pharmaceutical Analysis

Article Title: Development of a novel NanoBRET high-throughput drug screening assay for human GnRH receptor using sulfo-cyanine 5 fluorophore

doi: 10.1016/j.jpha.2025.101532

Figure Lengend Snippet: NanoLuciferase (Nluc) bioluminescence resonance energy transfer (NanoBRET)-based saturation and inhibition binding assays between sulfo-cyanine 5-D-Lys 6 -gonadotropin-releasing hormone (sCy 5 -D-Lys 6 -GnRH) andN-terminal secretory signal peptide-NanoLuciferase-human gonadotropin-releasing hormone receptor and its K191 deletion (N-secNluc-hGnRHR and N-secNluc-hGnRHR-K191Δ) transiently expressed in human embryonic kidney 293 (HEK293) cells. For saturation binding assays, transfected HEK293 cells were incubated with various concentrations (0, 1 nM–100 nM) of sCy 5 -D-Lys 6 -GnRH in the absence of (total binding, TB) or presence (non-specific binding (NSB)) of a 100-fold excess of unlabeled gonadotropin-releasing hormone I (GnRH I) for 30 min at 37 °C, followed by the immediate addition of the substrate furimazine (1:1000) before measurement of emissions. For inhibition assays, transfected HEK293 cells were incubated with 10 nM sCy 5 -D-Lys 6 -GnRH and increasing concentrations of unlabeled GnRH I (0, 0.1 nM to 1 μM) for 4 h at 4 °C. The plates were then brought to room temperature, and furimazine (1:1000) was added immediately before measurement. (A) Saturation binding of sCy 5 -D-Lys 6 -GnRH to N-secNluc-hGnRHR. (B) Saturation binding of sCy 5 -D-Lys 6 -GnRH to N-secNluc-hGnRHR-K191Δ. (C) Inhibition binding of N-secNluc-hGnRHR by GnRH I. (D) Inhibition binding of N-secNluc-hGnRHR-K191Δ by GnRH I. Raw bioluminescence resonance energy transfer (BRET) ratios were calculated by the division of the emission at 610 nm longpass (610LP) by the emission at 460 nm with a bandpass of 80 (460BP80). Representative results from at least three independent experiments, each performed in triplicate, are shown.

Article Snippet: Human embryonic kidney 293 (HEK293, ECACC: 85120602) and African green monkey kidney fibroblast (COS-7, ECACC: 87021302) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Bioluminescence Resonance Energy Transfer, Inhibition, Binding Assay, Transfection, Incubation

Journal: Journal of Pharmaceutical Analysis

Article Title: Development of a novel NanoBRET high-throughput drug screening assay for human GnRH receptor using sulfo-cyanine 5 fluorophore

doi: 10.1016/j.jpha.2025.101532

Figure Lengend Snippet: Kinetics of sulfo-cyanine 5-D-Lys 6 -gonadotropin-releasing hormone (sCy 5 -D-Lys 6 -GnRH) binding toN-terminal secretory signal peptide-NanoLuciferase-human gonadotropin-releasing hormone receptor with K191 deletion (N-secNluc-hGnRHR-K191Δ) stably expressed in human embryonic kidney 293 (HEK293) cells were examined by measurement of bioluminescence resonance energy transfer (BRET) within a continuous time. The cells were seeded into a 96-well plate at a density of 5 × 10 5 cells per well and incubated with furimazine (1:1000 dilution) for 3 min. After the incubation, three different concentrations of sCy 5 -D-Lys 6 -GnRH, approximating the dissociation equilibrium constant ( K D ) value, were added. Emissions were simultaneously measured at 610 nm longpass (610LP) and 460 nm with a bandpass of 80 nm (460BP80) over 40 min at 37 °C. A representative result from three independent experiments, each performed in triplicate, is shown. Data are presented as mean ± standard deviations (SD).

Article Snippet: Human embryonic kidney 293 (HEK293, ECACC: 85120602) and African green monkey kidney fibroblast (COS-7, ECACC: 87021302) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Binding Assay, Stable Transfection, Bioluminescence Resonance Energy Transfer, Incubation

Journal: Journal of Pharmaceutical Analysis

Article Title: Development of a novel NanoBRET high-throughput drug screening assay for human GnRH receptor using sulfo-cyanine 5 fluorophore

doi: 10.1016/j.jpha.2025.101532

Figure Lengend Snippet: NanoLuciferase (Nluc) bioluminescence resonance energy transfer (NanoBRET)-based ligand binding and the Z′ factor of ligand displacements of sulfo-cyanine 5-D-Lys 6 -gonadotropin-releasing hormone (sCy 5 -D-Lys 6 -GnRH) at(N-terminal secretory signal peptide-NanoLuciferase-human gonadotropin-releasing hormone receptor with K191 deletion (N-secNluc-hGnRHR-K191Δ) stably expressed in human embryonic kidney 293 (HEK293) cells. (A) Saturation binding of sCy 5 -D-Lys 6 -GnRH at HEK293 cells stably expressing N-secNluc-hGnRHR-K191Δ. The cells were incubated with the indicated concentrations of sCy 5 -D-Lys 6 -GnRH in the absence of (total binding, TB) or presence (non-specific binding (NSB)) of a 100-fold excess of unlabeled gonadotropin-releasing hormone (GnRH) I for 30 min at 37 °C. The emissions at 610 nm longpass (610LP) and 460 nm with a bandpass of 80 nm (460BP80) were measured following the addition of the substrate furimazine. (B) Competitive inhibition binding of HEK293 cells stably expressing N-secNluc-hGnRHR-K191Δ. The cells were incubated with 10 nM of sCy 5 -D-Lys 6 -GnRH and increasing concentrations of unlabeled Cetrorelix, GnRH I, or GnRH II, ranging from 0 (B 0 , the maximum binding) to 1 μM, and the emissions were then measured as above. (C) Z′ factor values were calculated for the competitive inhibition bindings of sCy 5 -D-Lys 6 -GnRH to N-secNluc-hGnRHR-K191Δ. Data are presented as mean ± standard deviations (SD) from three independent assays performed in triplicate.

Article Snippet: Human embryonic kidney 293 (HEK293, ECACC: 85120602) and African green monkey kidney fibroblast (COS-7, ECACC: 87021302) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Bioluminescence Resonance Energy Transfer, Ligand Binding Assay, Stable Transfection, Binding Assay, Expressing, Incubation, Inhibition

Journal: Journal of Pharmaceutical Analysis

Article Title: Development of a novel NanoBRET high-throughput drug screening assay for human GnRH receptor using sulfo-cyanine 5 fluorophore

doi: 10.1016/j.jpha.2025.101532

Figure Lengend Snippet: Validation of high-throughput screening (HTS) using a small-scale library. Human embryonic kidney 293 (HEK293) cells stably expressing N-terminal secretory signal peptide-NanoLuciferase-human gonadotropin-releasing hormone receptor with K191 deletion (N-secNluc-hGnRHR-K191Δ ) were plated at densities of 5 × 10 5 cells per well in 96-well plates and were then incubated with 10 nM of sulfo-cyanine 5-D-Lys 6 -gonadotropin-releasing hormone (sCy 5 -D-Lys 6 -GnRH) and increasing concentrations of various ligands, such as gonadotropin-releasing hormone (GnRH) I and II, N-lactoyl-phenylalanine, respectively, ranging from 0 (B 0 , the maximum binding) to 10 μM at 4 °C for 4 h. The plates were then brought to room temperature, and furimazine (1:2000, 5 μM) was added and incubated for 5 min. Emissions were simultaneously measured at 610 nm longpass and 460 nm with a bandpass of 80 nm. Raw bioluminescence resonance energy transfer (BRET) ratios were calculated by the division of the emission at 610 nm by the emission at 460 nm. (A) Inhibition of specific BRET signal between sCy 5 -D-Lys 6 -GnRH and N-secNluc-hGnRHR-K191Δ by endogenous GnRHs and their analogues, peptide and non-peptide antagonists. (B) Effect of various GnRH-unrelated molecules on the BRET ratio. Representative results from three independent experiments, each performed in quadruplicate, are shown.

Article Snippet: Human embryonic kidney 293 (HEK293, ECACC: 85120602) and African green monkey kidney fibroblast (COS-7, ECACC: 87021302) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Biomarker Discovery, High Throughput Screening Assay, Stable Transfection, Expressing, Incubation, Binding Assay, Bioluminescence Resonance Energy Transfer, Inhibition, Analogues

Journal: Journal of Pharmaceutical Analysis

Article Title: Development of a novel NanoBRET high-throughput drug screening assay for human GnRH receptor using sulfo-cyanine 5 fluorophore

doi: 10.1016/j.jpha.2025.101532

Figure Lengend Snippet: NanoLuciferase (Nluc) bioluminescence resonance energy transfer (NanoBRET)-based saturation and inhibition binding of Boron-dipyrromethene 630/650-D-Lys 6 -gonadotropin-releasing hormone (BODIPY630/650-D-Lys 6 -GnRH) atN-terminal secretory signal peptide-NanoLuciferase-human gonadotropin-releasing hormone receptor with K191 deletion (N-secNluc-hGnRHR-K191Δ) stably expressed in human embryonic kidney 293 (HEK293) cells. (A) Saturation binding assay. HEK293 cells stably expressing S-N-Nluc-GnRHR-K191Δ were incubated with various concentrations of BODIPY630/650-D-Lys 6 -GnRH in the absence of (total binding, TB) or presence (non-specific binding (NSB)) of a 100-fold excess of unlabeled gonadotropin-releasing hormone (GnRH) I for 30 min at 37 °C. Emissions at 610 nm longpass (610LP) and 460 nm with a bandpass of 80 nm (460BP80) were measured simultaneously following the addition of the substrate furimazine. (B) Inhibition binding assay. HEK293 cells stably expressing S-N-Nluc-GnRHR-K191Δ were incubated with 10 nM BODIPY630/650-D-Lys 6 -GnRH and increasing concentrations of unlabeled GnRH I (0–1 μM) for 4 h at 4 °C. The plate was then brought to room temperature, and furimazine was added immediately before measurement of emissions. The raw bioluminescence resonance energy transfer (BRET) ratios were then calculated. Representative results from at least three independent experiments, each performed in triplicate, are shown.

Article Snippet: Human embryonic kidney 293 (HEK293, ECACC: 85120602) and African green monkey kidney fibroblast (COS-7, ECACC: 87021302) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Bioluminescence Resonance Energy Transfer, Inhibition, Binding Assay, Stable Transfection, Saturation Assay, Expressing, Incubation

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: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) The extracellular domain of mouse VEGFR-3 was immobilized on microtiter wells and incubated with the X6 phage display library. Bar graph shows enrichment in the number of phage recovered [in transducing units (TU)] after consecutive rounds of selection (I, II, and III). (*) Round I was not quantified to prevent the loss of phage displaying unique peptides. ( B ) Peptide identified by sequencing phage bound to VEGFR-3 (round III) ( n , number of phages sequenced). ( C and D ) Binding of control phage Fd (white bars) and phage PCAIWF (B, black bars) and WVCSGG (C, black bars) to VEGF receptors and co-receptors immobilized on microtiter wells. ( E and F ) Inhibition of phage PCAIWF (E) or WVCSGG (F) binding to immobilized VEGFR-3 by synthetic peptide PCAIWF or control peptide (CARAC). The minus sign indicates that no synthetic peptide was added to the assay. ( G ) Dose-response assay. Phage PCAIWF was incubated with immobilized VEGFR-3 in the presence of synthetic peptides PCAIWF, PSAIWF, or CARAC (control). Percentage relative to phage binding in the absence of competing peptide. In all cases, bars represent means ± SEM from triplicate plating. Statistics, Student’s t test (** P ≤ 0.01 and *** P ≤ 0.001).

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Incubation, Selection, Sequencing, Binding Assay, Control, Inhibition

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Binding of phage PCAIWF to immobilized VEGFR-3 in the presence or absence of VEGF-A or VEGF-C (10 ng/ml). ( B ) Binding of phage PCAIWF to immobilized VEGFR-3 in the presence of increasing concentrations of VEGF-C. Percentage relative to phage binding in the absence of VEGF-C. ( C ) Cartoon showing the three-dimensional structure of the complex VEGF-C (red) bound to VEGFR-2 IgD2-3 (shown in orange and green, respectively) (Protein Data Bank #2X1W). ( D ) Analysis by SDS–polyacrylamide gel electrophoresis of purified recombinant IgD2 and IgD2-3 proteins containing the ligand-binding domain of VEGFR-3. ( E ) Binding of phage PCAIWF to VEGFR-3 and its recombinant Ig domains immobilized on microtiter wells in the presence or absence of the synthetic peptide PCAIWF or its scramble version, IFCAPW (100 μg/ml). Phage binding was quantified by FLISA using an anti-bacteriophage sera. ( F ) Binding of VEGF-C to microtiter wells coated with immobilized recombinant ligand binding domains IgD2 and IgD2-3 of VEGFR-3 in the presence or absence of synthetic peptides PCAIWF and WVCSGG or the scramble control peptide (IFCAPW). For phage experiments (A and B), bars represent mean ± SEM from triplicate plating; for FLISA assays ( E to G ), bars represent means ± SEM from duplicate wells. Statistics, Student’s t test [not significant (N.S.), P > 0.05; * P ≤ 0.05 and *** P ≤ 0.001].

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Binding Assay, Polyacrylamide Gel Electrophoresis, Purification, Recombinant, Ligand Binding Assay, Fluorophore-linked Immunoabsorbent Assay, Control

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Representation of the VEGF family, their receptors, and pattern of interaction. ( B to F ) Recombinant proteins for the human VEGFR-3 (B), VEGFR-2 (C and E), and VEGFR-1 (D and F) extracellular domains were immobilized on microtiter wells and incubated with the human ligands VEGF-C (B and C), PlGF (D), and VEGF-A (E and F) in the presence or absence of synthetic peptides PCAIWF and PSAIWF or the scramble control peptide (IFCAPW). Growth factors bound to the wells were quantified by FLISA using immunospecific antibodies and fluorescent detection. Bars represent means ± SEM from duplicate wells. Statistics, analysis of variance (ANOVA) (Tukey’s multiple comparison test) (* P ≤ 0.05; ** P ≤ 0.01 and *** P ≤ 0.001).

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Recombinant, Incubation, Control, Fluorophore-linked Immunoabsorbent Assay, Comparison

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Immunoblot analysis of phosphorylated and total forms of ERK1/2 in LECs incubated with VEGF-A, VEGF-C, or FGF (100 ng/ml) in the presence or absence of peptide PCAIWF or scramble (IFCAPW) (30 μg/ml). ( B ) Ratio of fluorescent intensity for phosphorylated and total ERK1/2. Bars represent means ± SEM from three independent measurements of the immunoblot membrane. Two independent experiments were performed with similar results. Bars represent means ± SEM from triplicate readings. Statistics, ANOVA (Tukey’s multiple comparison test) (* P ≤ 0.05).

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Western Blot, Incubation, Membrane, Comparison

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Tube formation by HUVECs in Matrigel induced by VEGF or VEGF-C in the presence or absence of peptide PCAIWF or scramble (500 μg/ml, embedded in the Matrigel layer). ( B ) Number of tubes formed between endothelial cells. Bars represent means ± SEM from triplicate wells. Statistics, Student’s t test (* P ≤ 0.05). Two independent experiments were performed with similar results.

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques:

Journal: Cell reports

Article Title: Immune gene diversity and STING1 variants in shaping cancer immunity across different genetic ancestry populations

doi: 10.1016/j.celrep.2025.116882

Figure Lengend Snippet: (A) The K-means clustering algorithm was used to group the cancer cell lines based on their similarity of IFN-β and ISG expression in response to cGAMP treatment (10 μg/mL, 30 min). The STING1 haplotypes of each cell line are indicated using color coding. (B) Heatmap plot illustrates the changes in expression levels of IFN-β and ISGs following cGAMP treatment. The K-means group and STING1 haplotypes of each cancer cell line are represented by color codes at the top of the heatmap. (C) Illustration of the dual gRNA/CRISPR strategy used to knock out an entire STING1 gene allele in the STING1 heterozygous cancer cell lines. (D) rhPCR-based allelic discrimination was used for genotyping the STING1 gene in the collected clones. The normalized reporter signals (Rn) for allele 1 (FAM) and allele 2 (VIC) were plotted on the x and y axes, respectively. The allelic discrimination plot shows three distinct genotype clusters, including individuals homozygous (depicted in red) and heterozygous (shown in green) for the reference allele and those homozygous for the alternate allele (represented in blue). The homogeneous HAQ/HAQ line HARA and WT/WT line H1373 served as genotyping controls. (E) Western blot analysis of p-TBK1 and p-STAT1 was conducted in isogenic SKOV3 cells carrying either WT or HAQ STING1 , following treatment with 10 μg/mL cGAMP. (F) Bubble plot illustrates the results of gene set enrichment analysis (GSEA) for the upregulated genes identified by RNA-seq following treatment with 10 μg/mL cGAMP for 30 min in isogenic SKOV3 cells carrying WT/HAQ, WT, or HAQ STING1 . The IFN gene sets were obtained from Reactome, and the transcription-factor-regulated gene sets were sourced from TRANSFAC. The bubble size corresponds to the −log (adjusted p value), and the color intensity indicates the number of genes within each gene set. (G) Violin plot shows the fold changes of the commonly upregulated genes identified by RNA-seq after treatment with 10 μg/mL cGAMP for 30 min in isogenic SKOV3 cells carrying WT/HAQ, WT, or HAQ STING1 . (H) The fold changes in expression levels of IFN-β and ISGs, as measured by RT-qPCR, following treatment with 10 μg/mL cGAMP for 30 min in isogenic SKOV3 and TOV21G cells carrying WT/HAQ, WT, or HAQ STING1 . (I) The numbers of T cells (Jurkat-CXCR3, top) and NK cells (NK92, bottom) that migrated to the lower compartment, where isogenic SKOV3 cells were pretreated with 5 μg/mL poly(dA:dT) for 4 h. (J) WT or HAQ STING1 cDNA was transduced into SKOV3 or TOV21G cells in which the endogenous STING1 had been knocked out using CRISPR. The expression of STING1 protein was subsequently detected through western blots. (K) The levels of IFN-β expression measured by ELISA following treatment with 10 μg/mL cGAMP for 30 min in SKOV3 and TOV21G cells transduced with either WT or HAQ STING1 . (L) WT or HAQ STING1 cDNA was transduced into HEK293 reporter cells, which lacked endogenous STING1 expression and contained dual reporters. The expression of STING1 protein was subsequently detected through western blots. (M) The activities of ISRE and hIFN-β reporters after treatment with 10 μg/mL cGAMP for 30 min in 293-Dual Null reporter transduced with either WT or HAQ STING1 . Data are represented as means ± SD. * p < 0.05 and ** p < 0.01.

Article Snippet: 293-Dual TM Null Cells , InvivoGen , Cat #293d-null.

Techniques: Expressing, CRISPR, Knock-Out, RNase H-dependent PCR, Clone Assay, Western Blot, RNA Sequencing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transduction