Journal: Pharmaceutics

Article Title: Elovanoids Counteract Inflammatory Signaling, Autophagy, Endoplasmic Reticulum Stress, and Senescence Gene Programming in Human Nasal Epithelial Cells Exposed to Allergens

doi: 10.3390/pharmaceutics14010113

Figure Lengend Snippet: ELVN-34 reduce the expression of pro-inflammatory cytokines, chemokines and cell adhesion molecule in HNEpC challenged with LPS or poly(I:C). HNEpC challenged with LPS or poly(I:C) (30 μg/mL) display enhanced production of pro-inflammatory cytokines and chemokines IL-6, IL-1β, IL-8/CXCL8, CCL2/MCP-1, CXCL1/KC/GRO, VEGF, and cell adhesion molecule ICAM1(CD54) compared to non-treated cells. This increase in the production of pro-inflammatory molecules is abrogated by the addition of ELVN-34 (500 nM) 30 min post-challenge. LPS and poly(I:C) induce a reduction of anti-inflammatory IL-10 expression in HNEpC that is restored to normal levels after treatment with ELVN-34 (500 nM). The results showed the averages of three independent experiments. (**** p < 0.0001, NS—not significant).

Article Snippet: In addition, the expression of the following cytokines was analyzed in the supernatant of HNEpC exposed to different allergic inductors: IL-6 (Catalog# 6802, Chondrex) range of detection: (9–600 pg/mL), IL-1β (Catalog# 6805, Chondrex) range of detection: (4–250 pg/mL) IL-8/CXCL8 (Quantikine ELISA Kit, Catalog# D8000C, R&D Systems) range of detection: (31–2000 pg/mL), vascular endothelial growth factor (VEGF) (Catalog# 6810, Chondrex) range of detection: (31–2000 pg/mL), ICAM1(CD54) (ELISA Kit, Catalog# ab100640, Abcam) range of detection: (16–1000 pg/mL), CXCL1/KC/GRO (Catalog# 6825, Chondrex) range of detection: CCL2/MCP-1 (Catalog# 6821, Chondrex) range of detection: (16–1000 pg/mL), and IL-10 (Catalog# 6806, Chondrex) range of detection: (8–500 pg/mL).

Techniques: Expressing

Journal: BioMed Research International

Article Title: Rapid Production of Human VEGF-A following Intradermal Injection of Modified VEGF-A mRNA Demonstrated by Cutaneous Microdialysis in the Rabbit and Pig In Vivo

doi: 10.1155/2019/3915851

Figure Lengend Snippet: Placement of VEGF-A mRNA injections in relation to the microdialysis probe in the rabbit study. For further details, see methods.

Article Snippet: An Alexa-labelled detection antibody against human VEGF-A (R&D systems) was then flowed through the column and the florescence intensity was used for quantification of the ligand.

Techniques:

Journal: BioMed Research International

Article Title: Rapid Production of Human VEGF-A following Intradermal Injection of Modified VEGF-A mRNA Demonstrated by Cutaneous Microdialysis in the Rabbit and Pig In Vivo

doi: 10.1155/2019/3915851

Figure Lengend Snippet: Placement of VEGF-A mRNA injections in relation to the microdialysis probe in the pig study. For further details, see methods.

Article Snippet: An Alexa-labelled detection antibody against human VEGF-A (R&D systems) was then flowed through the column and the florescence intensity was used for quantification of the ligand.

Techniques:

Journal: BioMed Research International

Article Title: Rapid Production of Human VEGF-A following Intradermal Injection of Modified VEGF-A mRNA Demonstrated by Cutaneous Microdialysis in the Rabbit and Pig In Vivo

doi: 10.1155/2019/3915851

Figure Lengend Snippet: Concentrations of human VEGF-A in eluates from 100 kDa microdialysis probes intradermally inserted in the rabbit hind leg. Microdialysis was started at t=0 h and four id injections of VEGF-A mRNA injections (50 μ g each) were given at t=1 h. Dotted line indicates Lower Limit of Quantification (LLOQ, 33.4 pg/mL). Two probes were inserted in each rabbit. Values shown are mean ± SEM (n=4).

Article Snippet: An Alexa-labelled detection antibody against human VEGF-A (R&D systems) was then flowed through the column and the florescence intensity was used for quantification of the ligand.

Techniques:

Journal: BioMed Research International

Article Title: Rapid Production of Human VEGF-A following Intradermal Injection of Modified VEGF-A mRNA Demonstrated by Cutaneous Microdialysis in the Rabbit and Pig In Vivo

doi: 10.1155/2019/3915851

Figure Lengend Snippet: Concentrations of human VEGF-A in microdialysis eluates from 100 kDa microdialysis probes intradermally inserted on the pig (n=3) abdomen. Values presented as individual values in eluate from each probe. Six intradermal VEGF-A mRNA injections (50 μ L each, total 24, 120, or 600 μ g VEGF-A mRNA) were given at t=0 h. At t=1.5 h after the injections, microdialysis was started. Eluates were collected during the time intervals from 1.5 h to 3.5 h, from 3.5 h to 5.5 h, and from 5.5 h to 7.5 h, respectively. Dotted vertical line indicates Lower Limit of Quantification (33.4 pg/mL); eluate samples below LLOQ are depicted as 0.5 ∗ LLOQ=16.7 pg/mL. Six probes were inserted in each pig, two probes per dose except the 120 μ g dose, where only four probes were used to assess human VEGF-A production following injection of 24 or 600 μ g VEGF-A mRNA. The remaining two probes were used to study effects following injection of citrate/saline vehicle.

Article Snippet: An Alexa-labelled detection antibody against human VEGF-A (R&D systems) was then flowed through the column and the florescence intensity was used for quantification of the ligand.

Techniques: Injection, Saline

Journal: BioMed Research International

Article Title: Rapid Production of Human VEGF-A following Intradermal Injection of Modified VEGF-A mRNA Demonstrated by Cutaneous Microdialysis in the Rabbit and Pig In Vivo

doi: 10.1155/2019/3915851

Figure Lengend Snippet: Amounts (pg/mg) of human VEGF-A in skin biopsies excised approximately 8 hours after intradermal injection of VEGF-A mRNA in 3 pigs. Each dose was administered as 6 (24 and 600 μ g dose) or 4 (120 μ g dose) separate injections at two different sites in each pig. Hatched data point in the 24 μ g dose, depicted at 0.1 pg/mg, indicating amount below Lower Limit of Quantification (0.28 pg/mg).

Article Snippet: An Alexa-labelled detection antibody against human VEGF-A (R&D systems) was then flowed through the column and the florescence intensity was used for quantification of the ligand.

Techniques: Injection

Journal: Circulation

Article Title: Collagen-Targeting Vascular Endothelial Growth Factor Improves Cardiac Performance After Myocardial Infarction

doi: 10.1161/circulationaha.108.800565

Figure Lengend Snippet: Figure 1. Collagen-binding ability of VEGF and CBD-VEGF in vitro. A, The amount of VEGF or CBD-VEGF bound to collagen was mea- sured by ELISA assay (n6 in each group/dose). Data are presented as meanSEM. *P0.05; **P0.01. B, Kd values for binding of VEGF or CBD-VEGF to collagen were calculated by Scatchard analysis. Slope of each line1/Kd.

Article Snippet: The remaining proteins on collagen were detected with an anti-VEGF monoclonal antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, Calif).

Techniques: Binding Assay, In Vitro, Enzyme-linked Immunosorbent Assay

Journal: Circulation

Article Title: Collagen-Targeting Vascular Endothelial Growth Factor Improves Cardiac Performance After Myocardial Infarction

doi: 10.1161/circulationaha.108.800565

Figure Lengend Snippet: Figure 2. Biological activity of growth factors on collagen. A, HUVEC growth–promoting activity was detected by MTT assay, which resulted in dose-response curves (n6 in each group/dose). B, Collagen-coated plates were washed extensively after incubation with VEGF or CBD-VEGF. Next, the mitogenic activities of the proteins that remained on HUVECs were determined by MTT assay (n6 in each group/dose). Data are presented as meanSEM. **P0.01.

Article Snippet: The remaining proteins on collagen were detected with an anti-VEGF monoclonal antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, Calif).

Techniques: Activity Assay, MTT Assay, Incubation

Journal: Circulation

Article Title: Collagen-Targeting Vascular Endothelial Growth Factor Improves Cardiac Performance After Myocardial Infarction

doi: 10.1161/circulationaha.108.800565

Figure Lengend Snippet: Figure 3. VEGF and CBD-VEGF induced blood vessel formation in collagen mem- branes implanted subcutaneously. A, Macroscopic view of the implants retrieved 2 weeks later. Scale bar5 mm. B through D, Immunohistochemical stain- ing of von Willebrand factor on collagen membranes loaded with PBS (B), VEGF (C), or CBD-VEGF (D). Arrows point to typical blood vessels. E, Blood vessel density is expressed as the number of blood vessels per field (n5 in each group). Data are presented as meanSEM *P0.05, **P0.01. Scale bar50 m.

Article Snippet: The remaining proteins on collagen were detected with an anti-VEGF monoclonal antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, Calif).

Techniques: Immunohistochemical staining, Staining

Journal: Circulation

Article Title: Collagen-Targeting Vascular Endothelial Growth Factor Improves Cardiac Performance After Myocardial Infarction

doi: 10.1161/circulationaha.108.800565

Figure Lengend Snippet: Figure 4. Binding ability of VEGF and CBD-VEGF to the infarcted heart. A, After ligation, saline, VEGF, or CBD-VEGF was injected into 5 regions in the border zone surrounding the infarct. Black dots indicate injection sites. B, Three hours after injection, exogenous pro- tein levels were detected by Western blot with anti-polyhistidine antibody. -Actin was used as internal control. C, Quantitation of pro- tein bands (n4 in each group). D, Exogenous protein levels in serum were detected 3 and 6 hours after injection (n6 in each group). Data are presented as meanSEM. *P0.05, **P0.01.

Article Snippet: The remaining proteins on collagen were detected with an anti-VEGF monoclonal antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, Calif).

Techniques: Binding Assay, Ligation, Saline, Injection, Western Blot, Control, Quantitation Assay

Journal: Circulation

Article Title: Collagen-Targeting Vascular Endothelial Growth Factor Improves Cardiac Performance After Myocardial Infarction

doi: 10.1161/circulationaha.108.800565

Figure Lengend Snippet: Figure 5. Histological analysis of hearts 4 weeks after myocardial infarction. Heart sections were stained with Masson trichrome. A, Control group. B, VEGF group. C, CBD-VEGF group. D, Percentage of infarct area in the whole LV area. E, Wall thickness of infarct area. n7 in each group. Data are presented as meanSEM. *P0.05. Scale bar1 mm.

Article Snippet: The remaining proteins on collagen were detected with an anti-VEGF monoclonal antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, Calif).

Techniques: Staining, Control

Journal: Circulation

Article Title: Collagen-Targeting Vascular Endothelial Growth Factor Improves Cardiac Performance After Myocardial Infarction

doi: 10.1161/circulationaha.108.800565

Figure Lengend Snippet: Figure 6. Capillary density in the infarct border zone. Von Wille- brand factor antibody was used in immunohistochemistry of blood vessels. A, control group. B, VEGF group. C, CBD-VEGF group. D, Quantitative analysis of capillary density. Capillary density is expressed as the number of capillary vessels per field. n7 in each group. Data are presented as meanSEM. *P0.05, **P0.01. Scale bar50 m.

Article Snippet: The remaining proteins on collagen were detected with an anti-VEGF monoclonal antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, Calif).

Techniques: Immunohistochemistry, Control

Journal: Nature Communications

Article Title: Selective targeting of ligand-dependent and -independent signaling by GPCR conformation-specific anti-US28 intrabodies

doi: 10.1038/s41467-021-24574-y

Figure Lengend Snippet: a Overview of biotinylation of proximal proteins by US28-Bio ID2. b US28-mediated inositol phosphate (IP) accumulation in HEK293T cells expressing HA-US28 wildtype (US28 WT) or HA-US28-Bio ID2(No Nb) alone or co-expressed with non-US28 targeting intrabody-mVenus (Irr Nb mV) or VUN103-mVenus (VUN103 mV). IP levels were normalized to US28 WT No Nbt ( n = 3 independent experiments). c Detection of biotinylated proteins in lysates from HEK293T cells with and without expression of HA-US28-Bio ID2 alone (No Nb) or withIrr Nb mV or VUN103 mV by Western blot using streptavidin-HRP. Representative blot shown from three independent biological replicates. d Detection of pyruvate carboxylase (PC), intrabody-mVenus (mVenus), Gα q and actin in lysates and biotinylated protein pulldown samples of HEK293T cells with or without HA-US28-Bio ID2 alone (No Nb) or withVUN103 mV or Irr Nb mV. Representative blot shown from three independent biological replicates. Samples are derived from the same experiment and blots were processed in parallel. e Quantification of biotinylated Gα q protein levels of biological triplicate samples. Data from three independent experiments using independent biological replicates. f BRET between US28-Nluc and Venus-mini-Gα q upon inducible expression of non-US28 targeting intrabody (Irr Nb-FLAG) VUN103 (VUN103-FLAG)(Intrabody). BRET signals were normalized to that in non-induced HEK293T cells (No intrabody) ( n = 3 independent experiments). g Detection of PC, mVenus, β-arrestin1/2 (β−Arr 1/2), and 14-3-3 in lysates and biotinylated protein pulldown samples of HEK293T cells with or without HA-US28-Bio ID2 alone (No Nb) or with VUN103 mV or Irr Nb mV. Representative blot shown from three independent biological replicates. Samples are derived from the same experiment and blots were processed in parallel. h Quantification of biotinylated βArr-1/2 protein levels in biological triplicate samples. Data from three independent experiments using independent biological replicates. i BRET between US28-Nluc and β-arrestin2-mVenus upon inducible expression of Irr Nb-FLAG or VUN103-FLAG(Intrabody). BRET signals were normalized to that in non-induced HEK293T cells (No intrabody) ( n = 3 independent experiments). All data are plotted as mean ± SD. Statistical analyses were performed using unpaired two-tailed t -test. ns, p > 0.05. Source data are provided as a Source Data file.

Article Snippet: Wells were subsequently washed with wash buffer and were incubated with biotinylated Rabbit Anti-Human VEGF detection antibody (1:800 in dilution buffer, #500-P10BT, Peprotech) for 2 h at RT.

Techniques: Expressing, Western Blot, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Selective targeting of ligand-dependent and -independent signaling by GPCR conformation-specific anti-US28 intrabodies

doi: 10.1038/s41467-021-24574-y

Figure Lengend Snippet: a U251 with inducible US28 expression (No Nb) were transduced with lentiviral vector expressing inducible non-US28 targeting intrabody-mVenus (Irr Nb mV) or VUN103-mVenus (VUN103 mV). Cells were (induced) or were not (Non-induced) induced with doxycycline. US28 was detected using an anti-US28 antibody (US28, red). Nanobody binding was detected using the mVenus-tag (Nb mV, green). Representative data of three independent experiments. b Cells were seeded in hanging droplet plates and expression of US28 (and intrabodies) were or were not induced with doxycycline. Representative data of three independent experiments. c Spheroid areas were quantified and normalized to the spheroid area of non-induced US28 negative (US28 neg) spheroid ( n = 6 spheroids for non-induced and induced No Nb samples; n = 8 spheroids for non-induced Irr Nb mV samples; n = 12 spheroids for induced Irr Nb mV samples; n = 7 spheroids for non-induced VUN103 mV samples; n = 11 spheroids for induced VUN103 mV samples). d Western blot of phospho-STAT3 (p-STAT3), total STAT3, immediate early proteins (IE), intrabody-mVenus (mVenus) and actin of U251 cells (No Nb), U251cells transduced with a lentiviral vector expressing inducible non-US28 targeting intrabody-mVenus (Irr Nb mV) or VUN103-mVenus (VUN103 mV). Cells were not infected or infected with HCMV wild-type virus (WT). Representative blot shown from three independent experiments using independent biological replicates. Samples are derived from the same experiment and blots were processed in parallel. e U251 cells (No Nb), U251cells transduced with a lentiviral vector expressing inducible non-US28 targeting intrabody-mVenus (Irr Nb mV) or VUN103-mVenus (VUN103 mV)were uninfected or infected with HCMV wild-type virus (WT) or HCMV virus lacking US28 (HCMV ΔUS28). Conditioned serum of infected cells was harvested and VEGF levels were determined using ELISA ( n = 3 independent experiments). All data are plotted as mean ± SD. Statistical analyses were performed using an unpaired two-tailed t -test. ns, p > 0.05. Source data are provided as a Source Data file.

Article Snippet: Wells were subsequently washed with wash buffer and were incubated with biotinylated Rabbit Anti-Human VEGF detection antibody (1:800 in dilution buffer, #500-P10BT, Peprotech) for 2 h at RT.

Techniques: Expressing, Transduction, Plasmid Preparation, Binding Assay, Western Blot, Infection, Virus, Derivative Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Neuropilin 1 and Neuropilin 2 gene invalidation or pharmacological inhibition reveals their relevance for the treatment of metastatic renal cell carcinoma

doi: 10.1186/s13046-021-01832-x

Figure Lengend Snippet: NRP1 or NRP2 gene invalidation results in inhibition of cell proliferation and migration. a The locus of the NRP1 gene was sequenced in control (NRP1 Ctrl) and in two independent clones (#NRP1 2.2 and #NRP1 2.7) KO for NRP1 . b The locus of NRP2 was sequenced in control (NRP2 Ctrl) and in two independent clones (#NRP2 2.3 and #NRP1 2.28) KO for NRP2 . c NRP1 and NRP2 mRNA levels were tested by qPCR in control (786O), in two independent clones (#NRP1 2.2 and 2.7) KO for NRP1 , and in two independent clones (#NRP2 2.3 and 2.28) KO for NRP2. d NRP1 and NRP2 protein levels were evaluated by flow cytometry in control (786O), in two independent clones (#NRP1 2.2 and 2.7) KO for NRP1 , and in two independent clones (#NRP2 2.3 and 2.28) KO for NRP2. e The proliferation of NRP1 and NRP2 KO cells were tested by counting the cells at the indicated time points. f The migration of NRPs KO cells was determined in scratch assays by measuring the time of wound closure. g VEGFA and VEGFC expression was tested in control (Ctrl) and KO clones by ELISA. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: CXCL8 cytokines and VEGFA were detected by using PeproTech ELISA kits according to the manufacturer’s indication as already described [ ].

Techniques: Inhibition, Migration, Clone Assay, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Neuropilin 1 and Neuropilin 2 gene invalidation or pharmacological inhibition reveals their relevance for the treatment of metastatic renal cell carcinoma

doi: 10.1186/s13046-021-01832-x

Figure Lengend Snippet: High NRPa-308 concentration stimulates the production of NRPs ligands and of pro-angiogenic/pro-inflammatory cytokines. The effects of NRPa-308 and sunitinib on the production of different cytokines were evaluated by ELISA; a VEGFA, b VEGFC, production ( c ) CXCL8, d CXCL5. * p < 0.05; **p < 0.01; *** p < 0.001

Article Snippet: CXCL8 cytokines and VEGFA were detected by using PeproTech ELISA kits according to the manufacturer’s indication as already described [ ].

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Clinical and Molecular Allergy : CMA

Article Title: Hypoxia modulates human eosinophil function

doi: 10.1186/1476-7961-8-10

Figure Lengend Snippet: The effect of hypoxia on VEGF and IL-8 release from eosinophils . Eosinophils were cultured in medium in normoxia or hypoxia. After o.n. incubation VEGF (A) and IL-8 (B) concentrations were measured in supernatants by specific ELISAs. Data are the mean ± SEM of three experiments. (* p ≤ 0.05; ** p ≤ 0.005).

Article Snippet: VEGF was detected by Human VEGF ELISA Development kit (PeproTech Inc.) and IL-8 by DuoSet kit (R&D system) according to the manufacturer's instructions.

Techniques: Cell Culture, Incubation

Journal: Clinical and Molecular Allergy : CMA

Article Title: Hypoxia modulates human eosinophil function

doi: 10.1186/1476-7961-8-10

Figure Lengend Snippet: Effect of GM-CSF signaling on hypoxia mediated HIF-1α and VEGF regulation . Eosinophils were incubated in normoxia/hypoxia in the absence or presence of GM-CSF. Western blot analysis was performed for phospho ERK1/2 and total ERK1/2 (A) or pP38 (B). The results are from one representative experiment out of three. (C) Eosinophils were cultured in medium with or without GM-CSF and with or without PD98059 (left panel; n = 5) or SB203580 (right panel; n = 3). MAPK inhibitors were added at the starting of the culture. VEGF levels were analyzed by ELISA. Data are the mean ± SEM of three experiments (* p ≤ 0.05). (D) Eosinophils were cultured for 3 h in medium alone (lanes 1 and 5), with GM-CSF alone (lanes 2 and 6) or with GM-CSF with PD98059 (lanes 3 and 7) or SB203580 (lanes 4 and 8). HIF-1α levels were analyzed by Western blot (left panel). Densitometry analysis is shown as a column chart (right panel). Data are representative of four experiments.

Article Snippet: VEGF was detected by Human VEGF ELISA Development kit (PeproTech Inc.) and IL-8 by DuoSet kit (R&D system) according to the manufacturer's instructions.

Techniques: Incubation, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epithelial Membrane Protein-2 (EMP2) Antibody Blockade Reduces Corneal Neovascularization in an In Vivo Model

doi: 10.1167/iovs.18-24345

Figure Lengend Snippet: Anti-EMP2–treated animals have reduced central cornea thickness and decreased CD34, VEGF, and CD31 staining in cornea. ([A], top row) H&E-stained sections were obtained, and the average of three measurements in the central region of each burned cornea were taken. ([A], second row) Immunohistochemical staining of CD34 expression in the treated and untreated corneas show an increase in CD34-positive staining in the saline and control antibody–treated animals, with markedly reduced CD34 staining in anti-EMP2 treated animals. Little to no CD34 expression is observed in the control unburned cornea. (A, third row) Immunohistochemical staining of VEGF expression shows a similar increase in VEGF expression of saline and control antibody treated animals, with markedly reduced VEGF staining in anti-EMP2 treated animals. (A, bottom row) Staining via immunofluorescence with anti-CD31, an endothelial cell specific marker, shows increased expression, as predicted, in the control burns (burn + saline and burn + control IgG), as compared to the unburned cornea. However, concordant with the CD34 histochemical staining, the animals who received the burn and anti-EMP2 IgG exhibited reduced new vessel formation as compared with the control burns. (B) The overall thickness of the central cornea in the anti-EMP2 antibody treated group is significantly reduced compared to the saline treated group (*P < 0.05). (C) The average of three measurements per eye of epithelial thickness from central cornea were taken. Although all burned eyes have significantly reduced epithelial thickness compared to unburned control, the overall thickness of the epithelial layer of the cornea is unchanged and unaffected regardless of type of treatment (saline, control IgG, or anti-EMP2 IgG). (D) Quantitation of CD34 and (E) CD31 positively stained corneal sections demonstrate similar trends with immunohistochemical and immunofluorescent staining (*P < 0.05). Scale bar: 50 μm.

Article Snippet: Secreted VEGF from conditioned media was detected and analyzed per manufacturer's instructions (Human VEGF Quantikine ELISA kit; R&D Systems, Minneapolis, MN, USA).

Techniques: Staining, Immunohistochemical staining, Expressing, Saline, Control, Immunofluorescence, Marker, Quantitation Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epithelial Membrane Protein-2 (EMP2) Antibody Blockade Reduces Corneal Neovascularization in an In Vivo Model

doi: 10.1167/iovs.18-24345

Figure Lengend Snippet: Incubation with anti-EMP2 antibody results in decreased EMP2 expression and decreased VEGF expression in vitro in HCLE cells. (A) HCLE cells were incubated with the anti-EMP2 antibody for up to 48 hours, and expression of EMP2 and VEGF quantified by Western blot. (B) Reduction of EMP2 expression decreased to a nadir at 12 to 24 hours, and (C) reduction in VEGF expression reached a nadir at 12 hours.

Article Snippet: Secreted VEGF from conditioned media was detected and analyzed per manufacturer's instructions (Human VEGF Quantikine ELISA kit; R&D Systems, Minneapolis, MN, USA).

Techniques: Incubation, Expressing, In Vitro, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epithelial Membrane Protein-2 (EMP2) Antibody Blockade Reduces Corneal Neovascularization in an In Vivo Model

doi: 10.1167/iovs.18-24345

Figure Lengend Snippet: Supernatants from HCLE cells previously incubated with anti-EMP2 antibody show reduction in secreted VEGF and reduced cell migration in a transwell migration assay. (A) Secreted VEGF-A was measured by ELISA in the cell culture supernatants of HCLE cells incubated with anti-EMP2 antibody for 0 to 48 hours. There is a time-dependent reduction in VEGF secretion with EMP2 blockade. (B) Supernatants of HCLE cells incubated with anti-EMP2 for 0 to 48 hours were used to test function in an HUVEC transwell migration assay. Of note, there is a statistically significant reduction in the number of cells that have migrated after 12 and 24 hours (*P < 0.05).

Article Snippet: Secreted VEGF from conditioned media was detected and analyzed per manufacturer's instructions (Human VEGF Quantikine ELISA kit; R&D Systems, Minneapolis, MN, USA).

Techniques: Incubation, Migration, Transwell Migration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cancer gene therapy

Article Title: MicroRNA-140-5p inhibits invasion and angiogenesis through targeting VEGF-A in breast cancer.

doi: 10.1038/cgt.2017.30

Figure Lengend Snippet: Figure 2. VEGF-A is the direct target of miR-140-5p. (a) A putative miR-140-5p-binding site exists in the 3′-UTR of VEGF-A mRNA, and three point mutations were generated in the binding site. (b) Luciferase reporter assay for HEK-293T WT and Mut 3′-UTRs. (c) miR-140-5p re-expression reduced VEGF-A mRNA levels as compared with the negative control in MCF-7 and MDA-MB-231 cells. (d) The expression of miR-140-5p in MCF-7 and MDA-MB-231 cells decreased the intracellular VEGF-A protein levels, as shown by western blot analysis. (e) The expression of miR-140-5p in MCF-7 and MDA-MB-231 cells decreased secreted VEGF-A protein levels, as shown by ELISA.

Article Snippet: MCF-7 and MDA-MB-231 cells (5 × 103, 6 × 103 cells per well, respectively) in 96-well plates were transfected with miR-140-5p mimics and N-Control for 48 h. Culture medium was collected and secreted VEGF-A was detected with the human VEGF-A ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Binding Assay, Generated, Luciferase, Reporter Assay, Expressing, Negative Control, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: European radiology

Article Title: Magnetic resonance imaging for monitoring the effects of thalidomide on experimental human breast cancers

doi: 10.1007/s00330-008-1111-x

Figure Lengend Snippet: Representative immunohistochemically stained human breast cancer sections (MDA-MB-435) showing leakage of macromolecular contrast medium (streptavidin-biotin reaction), vascular richness (lectin and RECA-1 antibody), and VEGF in tumor cells. Thalidomide treatment reduces the extravasation of albumin-(Gd-DTPA)27-(biotin)11 without reducing the abundance of tumor blood vessels. a Tumor section from the saline-control group administered albumin-(Gd-DTPA)27-(biotin)11. The strong red signal indicates extravascular 1-h accumulation of biotin-labeled contrast medium surrounding the yellow-green tumor microvessels. b After 7 days of treatment with thalidomide, the density of red-fluorescent, biotin-labeled contrast agent extravasated over 1 h is strongly reduced compared with a, indicating a reduction in leakage and extravascular accumulation of the macromolecules. Confocal microscopic images of tumor vessels in MDA-MB-435 tumors after treatment with saline (c, d) or thalidomide (e, f) for 7 days show no noticeable change in the area density of perfused blood vessels (green lectin-stained) or total blood vessels (red, RECA-1 stained). No difference is observable (c–f) between saline-control and thalidomide-treated groups with regard to tumor vascularity. (g, h). Representative MDA-MB-435 tumor sections after immunohistochemical staining for human VEGF after a 7-day, three-injection treatment protocol with saline (g) or thalidomide (h). No difference in amount VEGF immunoreactivity was detected in the two groups. Scale bar 115 μm in c–f and 120 μm in a, b

Article Snippet: VEGF immunoreactivity of the human tumor cells was detected using goat polyclonal antibody anti-human VEGF (1:500, R&D Systems, Minneapolis, Minn.).

Techniques: Staining, Saline, Control, Labeling, Immunohistochemical staining, Injection

Journal: European radiology

Article Title: Magnetic resonance imaging for monitoring the effects of thalidomide on experimental human breast cancers

doi: 10.1007/s00330-008-1111-x

Figure Lengend Snippet: Measurement of RECA-1 and VEGF area density in tumors treated with thalidomide or saline control. Values obtained after 7 days of therapy

Article Snippet: VEGF immunoreactivity of the human tumor cells was detected using goat polyclonal antibody anti-human VEGF (1:500, R&D Systems, Minneapolis, Minn.).

Techniques: Saline, Control