human vegf standard  (R&D Systems)

Journal: Molecular Oncology

Article Title: A group of novel VEGF splice variants as alternative therapeutic targets in renal cell carcinoma

doi: 10.1002/1878-0261.13401

Figure Lengend Snippet: VEGF XXX/NF isoforms are expressed in normal tissues and cancer cells. (A) VEGF XXX/NF splice variants. Primers used for RT‐(q)PCR analyses are indicated by the arrows. The names of the different VEGF XXX isoforms and the corresponding VEGF XXX/NF isoforms are indicated. (B) RT‐qPCR analyses of the expression of the different VEGF and VEGF XXX/NF in normal tissues; *** P < 0.001 vs VEGF in TIME cells; ### P < 0.001 vs VEGF XXX/NF in TIME cells. (C) RT‐qPCR analyses of VEGF and VEGF XXX/NF expression in RCC cell lines (RCC4, ACHN, CAKI‐2, RCC10, 786‐VHL, 786‐O, A498). Results are expressed as percent (%) of VEGF in TIME cells. *** P < 0.001 vs VEGF in TIME cells; ### P < 0.001 vs VEGF XXX/NF in TIME cells. (D) Summary table of VEGF and VEGF XXX/NF expression levels in normal tissue and RCC cells. (E) ELISA of VEGF (Peprotech) and VEGF XXX/NF (rabbit anti‐VEGF XXX/NF clone # 2) expression in supernatant of RCC cells *** P < 0.001 vs VEGF in TIME cells; ### P < 0.001 vs VEGF XXX/NF in RCC10 (two‐way ANOVA). Data are presented as the mean ± standard error of the mean (SEM). All experiments were performed with at least three biological duplicates ( n = 3) for each group in triplicate.

Article Snippet: RCC cell lines were seeded in 6‐well plates (500 000) and grown in DMEM medium containing 0.5% FBS for 48 h. VEGF assays were performed using the human VEGF standard development kit TMB ELISA (Peprotech ® , Cranbury, NJ, USA; Human VEGF 165 Standard TMB ELISA Development Kit, 900‐T10) according to the manufacturer's recommendations.

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Molecular Oncology

Article Title: A group of novel VEGF splice variants as alternative therapeutic targets in renal cell carcinoma

doi: 10.1002/1878-0261.13401

Figure Lengend Snippet: VEGF 222/NF binds VEGF‐receptors and co‐receptors and stimulates angiogenesis. (A, B) Specific binding of VEGF 222/NF to VEGFRs (VEGFR1, VEGFR2, VEGFR3) (A) and to NRPs (NRP1, NRP2) (B). (C) VEGF 222/NF induces phosphorylation of VEGFR2 and activation of downstream signalling pathways AKT and ERK. Confluent monolayers of TIME/endothelial cells were kept serum starved for 2 h and then treated with VEGF 165 (Va, 100 ng·mL −1 ) or VEGF 222/NF (NF, 100 ng/mL) for the indicated times. Figure shows representative images from three independent experiments. (D) Quantification of VEGFR2, ERK and AKT activity measure as a ratio between the amounts of phosphorylated and unmodified protein. The untreated (NT) condition at 5 min was used as reference, n = 3, * P < 0.05; ** P < 0.01; *** P < 0.001. (E) Cell proliferation assay of serum‐starved endothelial cells (TIME) treated with VEGF 222/NF (100 ng·mL −1 ) or not. Cells were counted for 7 days. (F–G) Wound scratch assays performed with serum‐starved TIME cells treated with VEGF 222/NF (100 ng·mL −1 ). Wound closure was determined at 3‐, 6‐, 9‐ and 12‐h after treatment. (H) In vitro permeability assay. Monolayers of serum‐depleted TIME cells were treated with PBS or VEGF 222/NF (100 ng·mL −1 ) for 30 min. Permeability was assessed by streptavidin‐HRP and TMB substrate staining. (I) In vivo permeability assay. Mice were injected intravenously with Evans blue, followed by PBS or VEGF 222/NF (500 ng·mL −1 ). The amount of Evans blue was determined colorimetrically in mouse ears (upper panel). Representative photos of vascular leakage induced by PBS or VEGF 222/NF (bottom panel). (J) In vivo plug assay. Mice were injected with a low concentration of Matrigel® containing PBS or VEGF 222/NF (1 μg·mL −1 ) and the level of haemoglobin content was measured 15 days after implantation. Representative photos of the Matrigel® plug 15 days after implantation (lower panel). Data were expressed as mean ± SEM. ** P 0.01, *** P < 0.001 vs PBS (two‐way ANOVA was used to assess statistical difference for cell proliferation and a Mann–Whitney test for the permeability and plug assays). Results of in vitro experiments are presented as the mean ± SEM. All experiments were performed with at least three biological duplicates ( n = 3) for each group in triplicate.

Article Snippet: RCC cell lines were seeded in 6‐well plates (500 000) and grown in DMEM medium containing 0.5% FBS for 48 h. VEGF assays were performed using the human VEGF standard development kit TMB ELISA (Peprotech ® , Cranbury, NJ, USA; Human VEGF 165 Standard TMB ELISA Development Kit, 900‐T10) according to the manufacturer's recommendations.

Techniques: Binding Assay, Phospho-proteomics, Activation Assay, Activity Assay, Proliferation Assay, In Vitro, Permeability, Staining, In Vivo, Injection, Plug Assay, Concentration Assay, MANN-WHITNEY

Journal: Molecular Oncology

Article Title: A group of novel VEGF splice variants as alternative therapeutic targets in renal cell carcinoma

doi: 10.1002/1878-0261.13401

Figure Lengend Snippet: Inhibition of VEGF XXX/NF delays the growth of experimental RCC tumours. (A) The affinity of bevacizumab for VEGF 165 and VEGF 222/NF was determined by ELISA, n = 3. (B) The growth curve of experimental tumours generated with 786‐O cells after treatment with anti‐KLH ( n = 6), anti‐VEGF XXX/NF ( n = 5) and bevacizumab ( n = 5). (C) Weight of 786‐O tumours at the end of the experiment. (D–E) Quantification of Ki67‐positive cells in 786‐O tumours. Cell proliferation was detected by Ki67 immunofluorescence labelling (green) and Hoechst33342 nuclear DNA counterstaining (blue). Scale bar: 50 μm. (F) Immunofluorescence detection of CD31, α‐SMA (upper panel) and LYVE1 (lower panel) in 786‐O tumours treated with control, anti‐VEGF XXX/NF antibodies or bevacizumab. Scale bar: 200 μm. (G) Number of mature (CD31 + , α‐SMA + ) vessels in the different tumour sections. (H) Number of lymphatic vessels (LYVE1 + ) in the tumour sections. * P < 0.05, *** P < 0.001 vs control, # P < 0.01, ## P < 0.01, ### P < 0.001 vs bevacizumab (one‐way ANOVA). (I) Plasma levels of VEGF XXX/NF and VEGF increased in the bevacizumab‐treated group. ELISA of plasma levels of VEGF and VEGF XXX/NF in mice with 786‐O tumours treated with bevacizumab or KLH or anti‐mouse VEGF XXX/NF antibodies. * P < 0.05, ** P < 0.01 vs KLH, # P < 0.05 vs bevacizumab (one‐way ANOVA).

Article Snippet: RCC cell lines were seeded in 6‐well plates (500 000) and grown in DMEM medium containing 0.5% FBS for 48 h. VEGF assays were performed using the human VEGF standard development kit TMB ELISA (Peprotech ® , Cranbury, NJ, USA; Human VEGF 165 Standard TMB ELISA Development Kit, 900‐T10) according to the manufacturer's recommendations.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Generated, Immunofluorescence, Control, Clinical Proteomics