Journal: Experimental and Therapeutic Medicine

Article Title: Expression levels of VEGF-C and VEGFR-3 in renal cell carcinoma and their association with lymph node metastasis

doi: 10.3892/etm.2021.9986

Figure Lengend Snippet: Immunohistochemical staining of VEGFR-3 expression. VEGFR-3 immunohistochemical staining in (A) normal renal tissues and (B) RCC tissues. (C) Lymphatic endothelial cells in RCC tissues were VEGFR-3 + (indicated by arrows). Scale bars, 100 µm. (D) Correlation between VEGF-C and VEGFR-3 expression in RCC tissues. Statistical significance was determined by Spearman's correlation test. RCC, renal cell carcinoma; VEGF-C, vascular endothelial growth factor-C; VEGFR-3, VEGF receptor-3.

Article Snippet: For antigen retrieval, the tissue sections were incubated with 0.01 M sodium citrate (pH 6) in a microwave oven at 95˚C for 10 min, followed by blocking with 5% normal goat serum (cat. no. ZLI-9021; OriGene Technologies, Inc.) for 10 min at room temperature, the tissue sections were incubated with rabbit anti-human VEGF-C monoclonal antibody (cat. no. BA0548; 1:200; Wuhan Boster Biological Technology Co., Ltd.), rabbit anti-human VEGFR-3 monoclonal antibody (cat. no. A01276-3; 1:200; Wuhan Boster Biological Technology Co., Ltd.) and mouse anti-human D2-40 monoclonal antibody (cat. no. ZM-0465; undiluted; OriGene Technologies, Inc.) for 12 h at 4˚C.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Molecular Cancer

Article Title: m 6 A methylation reader IGF2BP2 activates endothelial cells to promote angiogenesis and metastasis of lung adenocarcinoma

doi: 10.1186/s12943-023-01791-1

Figure Lengend Snippet: IGF2BP2 activates the PI3K-Akt signaling pathway through mediating the m 6 A modification of FLT4. A Fluorescent probe assay for detecting RNA levels of FLT4 in focal endothelial cells of LUAD patients with high or low CD34 expression. Bar, 50 μm. B Multiplex immunofluorescence assay for measuring protein levels of FLT4 in focal endothelial cells of LUAD patients with high or low CD34 expression. Bar, 50 μm. C Molecular docking for predicting the docking potential of IGF2BP2 protein with mRNA of FLT4. D Multiplex immunofluorescence assay for verifying protein levels of IGF2BP2 and RNA levels of FLT4 in focal endothelial cells of LUAD patients with high or low CD34 expression. Bar, 50 μm. E , F The m. 6 A levels of FLT4 in A549 and NCI-H1299 cells after transfection with si-NC or si-IGF2BP2. G , H IGF2BP2 protein levels in A549 and NCI-H1299 cells with si-NC or si-IGF2BP2 transfection. I - L FLT4, PI3K, and AKT expression levels in A549 and NCI-H1299 cells with si-NC or si-IGF2BP2 transfection. M The diagram for IGF2BP2-mediated FLT4-PI3K-Akt signaling pathway in regulating angiogenesis during LUAD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: The primary antibody against CD34, EPCAM, IGF2BP2, or FLT4 (Proteintech, Wuhan, China) was diluted with 1% BSA and incubated overnight at 4 °C.

Techniques: Modification, Expressing, Multiplex Assay, Immunofluorescence, Transfection

Journal: Molecular Cancer

Article Title: m 6 A methylation reader IGF2BP2 activates endothelial cells to promote angiogenesis and metastasis of lung adenocarcinoma

doi: 10.1186/s12943-023-01791-1

Figure Lengend Snippet: Development of the IGF2BP2-based prognostic scoring system for LUAD. A The global regulatory network of IGF2BP2-FLT4-PI3K-Akt signaling-angiogenesis. B Bubble plots demonstrating the correlation between the IGF2BP2-FLT4-PI3K-Akt signaling-angiogenesis key genes and clinical indicators in the TCGA-LUAD dataset. C , D Survival curves of OS and RFS outcomes for the low and high-score patients stratified by the IGF2BP2-based model in the TCGA-LUAD dataset. E Time-independent ROC curves for evaluating the performance of the IGF2BP2-based model in predicting OS and RFS outcomes in the TCGA-LUAD dataset. F , G External validation of OS analysis and time-independent ROC curves in the GSE72094 dataset. H , I Uni- and multivariate cox regression results on the IGF2BP2-based model and clinical parameters with patient survival in the TCGA-LUAD dataset. J Construction of the nomogram composed of the IGF2BP2-based model and stage. K - M Calibration curves for the nomogram-predicted and actual one-, three- and five-year OS outcomes

Article Snippet: The primary antibody against CD34, EPCAM, IGF2BP2, or FLT4 (Proteintech, Wuhan, China) was diluted with 1% BSA and incubated overnight at 4 °C.

Techniques: Biomarker Discovery

Journal: The Journal of Cell Biology

Article Title: Vascular endothelial growth factor can signal through platelet-derived growth factor receptors

doi: 10.1083/jcb.200608093

Figure Lengend Snippet: MSCs expressed no VEGFRs. The expression of VEGFR mRNA transcripts was examined by semiquantitative RT-PCR analysis and cell surface protein expression by single-color flow cytometry, using human MSCs, HUVECs as a VEGFR-positive control cell, and HDF as a VEGFR-negative control cell. (A) RNA isolated from MSCs, HUVECs, and HDFs were used to amplify VEGFR1-3, VEGF-A, NP-1, and NP-2 transcripts, with GAPDH as a control, and then resolved by agarose gel. Lane 1, VEGFR1 (99 bp); lane 2, VEGFR2 (81 bp); lane 3, VEGFR3 (87 bp); lane 4, VEGF-A (98 bp); lane 5, GAPDH (71 bp); lane 6, NP-1 (77 bp); lane 7, NP-2 (83 bp). Data are representative of six independent experiments, with two different pairs of primers for VEGFR1-3 used. (B) Flow cytometry using PE-conjugated antibodies. Analysis of VEGFR1-3 is represented by VEGFR1-, VEGFR2-, and VEGFR3-PE expression, respectively, with IgG 1 -PE expression as a control. A representative example of three independent experiments is shown.

Article Snippet: For single-color flow cytometry, MSCs, HUVECs, or HDFs (4×10 6 cells/ml) were incubated with either PE-conjugated anti–human VEGFR1-PE (FAB321P), VEGFR2-PE (FAB357P), or VEGFR3-PE (FAB3492P) antibodies, or control anti–IgG 1 -PE antibody (IC002P; R&D Systems).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Positive Control, Negative Control, Isolation, Control, Agarose Gel Electrophoresis

Journal: The Journal of Cell Biology

Article Title: Vascular endothelial growth factor can signal through platelet-derived growth factor receptors

doi: 10.1083/jcb.200608093

Figure Lengend Snippet: VEGF-A stimulated both PDGFRα and PDGFRβ tyrosine phosphorylation. Human phospho-RTK arrays were used to examine VEGF-A–induced RTK phosphorylation levels in MSC lysate samples. Arrays contain phosphotyrosine-positive control spots in each corner, having coordinates (A1, A2), (A23, A24), (F1, F2), (F23, F24), which were assigned a pixel density value of 100, which was used to normalize positive RTK spot pixel density values. Relevant RTK duplicate spot coordinates: PDGFRα = (C7, C8), PDGFRβ = (C9, C10), VEGFR1 = (D9, D10), VEGFR2 = (D11, D12), VEGFR3 = (D13, D14), EGFR = (B1, B2), FGFR3 = (B13, B14), Axl = (B21, B22), EphA7 = (E3, E4). (A) RTK array analysis of control MSC lysate, not stimulated with exogenous growth factor (basal). (B) RTK array analysis of lysates from MSCs transfected with 3 μg scrambled siRNA as a control, siRNA PDGFRα or siRNA PDGFRβ, stimulated using 20 ng/ml VEGF-A 165 in serum-free conditions for 10 min at 37°C. Each array was identically exposed to detection reagents and film. (C) Bar graph representing data from arrays (A and B). Mean pixel density ± the SD of duplicate spots, normalized against duplicate phosphotyrosine-positive control spots = 100. A representative example of two independent experiments is shown for each array analysis. *, P < 0.001 compared with the respective VEGF-A 165 –stimulated scrambled siRNA control.

Article Snippet: For single-color flow cytometry, MSCs, HUVECs, or HDFs (4×10 6 cells/ml) were incubated with either PE-conjugated anti–human VEGFR1-PE (FAB321P), VEGFR2-PE (FAB357P), or VEGFR3-PE (FAB3492P) antibodies, or control anti–IgG 1 -PE antibody (IC002P; R&D Systems).

Techniques: Phospho-proteomics, Positive Control, Control, Transfection