Journal: Oncogene

Article Title: Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer

doi: 10.1038/s41388-022-02382-y

Figure Lengend Snippet: Following ectopic expression of untagged NRP2B in C4–2B cells, nuclear and post-nuclear fractions were separated, and a pull-down assay was performed on the nuclear fraction with NRP2 antibody. Mass-Spectrometry was carried out with the pull-down samples. Using the genes detected in NRP2 Mass-Spectrometry. A. Sum of all the spectra associated with NRP2B pulldown sample is determined by the spectral graph. B. Venn-diagram represent the overlapping genes identified in two independent NRP2B mass-spectrometry assay. C. Group of nuclear pore proteins (Nups) identified through NRP2B Mass-Spectrometry. Schematic diagram indicating the relative positions of Nups around the nuclear pore. D. Validation of NRP2-Nup93 interactions in C4–2B. C4–2B cells were transfected with HA-tagged NRP2B. IP was carried out with HA-antibody and immunoblots were carried out with anti-Nup93. E. Endogenously, NRP2 and Nup93 interaction was validated in C4–2B and 22Rv1 cell lines. NRP2 was pull down with NRP2-specific antibody and IB with Nup93 antibody. F. NRP2B and AR interaction was carried out in C4–2B following ectopic interaction of HA-tagged NRP2B. Pulldown was carried out with HA-antibody and IP was carried out against NRP2 and AR. G. NRP2 and AR interaction was also monitored in PC3 and 22Rv1 following ectopic expression of HA-tagged NRP2B. IB was carried out against HA and AR. H. Co-IP for AR with NRP2 was carried out with pulldown of NRP2 by HA-antibody in C4–2B cells expressing wild type and mutant K892A NRP2B isoforms. NRP2B immunoblot was carried out with HA-antibody. I. IP with AR antibody was carried out to test the interaction among AR, Nup93 and NRP2 under the presence or absence of NRP2 and Nup 93 from the nuclear fraction of LNCaP C4–2B cells. An immunoblot was performed with anti-AR, anti-Nup93 and anti-NRP2 antibody. The Co-IP was carried out in the presence of 50ng/ml VEGF-C. J. PLA was carried out to validating the NRP2-AR interaction within the nucleus. Following ectopic expression of HA-tagged NRP2B, PLA was carried out with HA and endogenous AR antibodies under the presence or absence of NUP93. As a negative control, only HA-antibody was used for PLA reaction. Arrowhead indicates the immune-reactive PLA puncta. Nucleus was counter-stained with NUP98 to demarcate the nuclear periphery. DAPI used for nuclear staining. PLA quantification was shown in Bar diagram.

Article Snippet: NRP2B or NRP2A localization were chased with various experimental treatment condition for 1hrs under the following reagents VEGF-C (50ng/ml, R&D System, 752-VC-025), NRP2Fc (100ng/ml, R&D System, 2215-N2–025), SEMA3F (100ng/ml, R&D System, 9878-S3–025), Brefeldin A (0.5μM, Sigma, B6542).

Techniques: Expressing, Pull Down Assay, Mass Spectrometry, Biomarker Discovery, Transfection, Western Blot, Co-Immunoprecipitation Assay, Mutagenesis, Negative Control, Staining

Journal: World Journal of Gastroenterology

Article Title: Role of VEGF and CD44v6 in differentiating benign from malignant ascites

doi: 10.3748/wjg.v9.i11.2596

Figure Lengend Snippet: Comparison of VEGF concentrations in different kinds of ascites. Group 1: cirrhotic ascites, Group 2: tuberculous ascites, Group 3: malignant ascites.

Article Snippet: Immunoassay for human VEGF Concentrations of VEGF in ascites were determined with an ELISA kit (R&D Systems) following the manufacturer’s guidelines.

Techniques: Comparison

Journal: Drug Design, Development and Therapy

Article Title: Inhibition of lymphangiogenesis in vitro and in vivo by the multikinase inhibitor nintedanib

doi: 10.2147/DDDT.S130297

Figure Lengend Snippet: Effect of BIBF 1120 (nintedanib) on growth factor-induced proliferation of HLECs. Notes: ( A ) VEGF-C (50 ng/mL) enhanced proliferation of HLECs significantly. The enhanced effect was significantly reduced by 0.01 μM nintedanib ( P <0.05) and almost eliminated by 0.1 μM nintedanib ( P <0.01). ( B ) PDGF-BB (50 ng/mL) enhanced proliferation of HLECs significantly. The enhanced effect was significantly reduced by 0.01 μM nintedanib ( P <0.05) and almost eliminated by 0.1 μM nintedanib ( P <0.01). ( C ) A concentration of 50 ng/mL of bFGF enhanced proliferation of HLECs significantly. The enhanced effect was significantly reduced by 0.1 μM nintedanib ( P <0.05) and almost eliminated by 1 μM nintedanib ( P <0.01). * P -value <0.05. ** P -value <0.01. Abbreviations: HLECs, human lymphatic endothelial cells; VEGF-C, vascular endothelial growth factor-C; PDGF-BB, platelet-derived growth factor-BB; bFGF, basic fibroblast growth factor; OD, optical density.

Article Snippet: After incubation for 24 h, cells were treated with 50 ng/mL recombinant VEGF-C (PeproTech, Rocky Hill, NJ, USA), PDGF-BB (PeproTech), or bFGF (PeproTech) in the presence or absence of nintedanib at concentrations of 0.01, 0.1, and 1 μM.

Techniques: Concentration Assay, Derivative Assay

Journal: Drug Design, Development and Therapy

Article Title: Inhibition of lymphangiogenesis in vitro and in vivo by the multikinase inhibitor nintedanib

doi: 10.2147/DDDT.S130297

Figure Lengend Snippet: BIBF 1120 (nintedanib) suppressed growth factor-induced migration of HLECs. Notes: ( A ) Cells treated with 0.1% DMSO were used as controls. ( B1 and B2 ) VEGF-C (50 ng/mL) promoted migration of HLECs significantly, and the enhanced effect was significantly reduced by 0.1 μM nintedanib. ( C1 and C2 ) PDGF-BB (50 ng/mL) promoted migration of HLECs significantly, and the enhanced effect was significantly reduced by 0.1 μM nintedanib. ( D1 and D2 ) A concentration of 50 ng/mL of bFGF enhanced migration of HLECs significantly, and the enhanced effect was significantly reduced by 0.1 μM nintedanib. ( E ) The number of migrated cells stimulated by VEGF-C, PDGF-BB, or bFGF at a concentration of 50 ng/mL in the presence or absence of 0.1 μM nintedanib. A–D at 200× magnification; scale bar =50 μm. Abbreviations: HLECs, human lymphatic endothelial cells; DMSO, dimethyl sulfoxide; VEGF-C, vascular endothelial growth factor-C; PDGF-BB, platelet-derived growth factor-BB; bFGF, basic fibroblast growth factor.

Article Snippet: After incubation for 24 h, cells were treated with 50 ng/mL recombinant VEGF-C (PeproTech, Rocky Hill, NJ, USA), PDGF-BB (PeproTech), or bFGF (PeproTech) in the presence or absence of nintedanib at concentrations of 0.01, 0.1, and 1 μM.

Techniques: Migration, Concentration Assay, Derivative Assay

Journal: Drug Design, Development and Therapy

Article Title: Inhibition of lymphangiogenesis in vitro and in vivo by the multikinase inhibitor nintedanib

doi: 10.2147/DDDT.S130297

Figure Lengend Snippet: BIBF 1120 (nintedanib) inhibited growth factor-induced tube formation of HLECs. Notes: ( A ) Cells treated with 0.1% DMSO were used as controls. ( B1 and B2 ) VEGF-C (50 ng/mL) enhanced tube formation of HLECs significantly, and the enhanced effect was significantly reduced by 0.1 μM nintedanib. ( C1 and C2 ) PDGF-BB (50 ng/mL) enhanced tube formation of HLECs significantly, and the enhanced effect was significantly reduced by 0.1 μM nintedanib. ( D1 and D2 ) A concentration of 50 ng/mL of bFGF enhanced tube formation of HLECs significantly, and the enhanced effect was significantly reduced by 0.1 μM nintedanib. ( E ) The area of the tube structure after being stimulated by VEGF-C, PDGF-BB, and bFGF at a concentration of 50 ng/mL in the presence or absence of 0.1 μM nintedanib. ( F ) The length of the tube structure after being stimulated by VEGF-C, PDGF-BB, and bFGF at a concentration of 50 ng/mL in the presence or absence of 0.1 μM nintedanib. A–D at 100× magnification. Abbreviations: HLECs, human lymphatic endothelial cells; DMSO, dimethyl sulfoxide; VEGF-C, vascular endothelial growth factor-C; PDGF-BB, platelet-derived growth factor-BB; bFGF, basic fibroblast growth factor.

Article Snippet: After incubation for 24 h, cells were treated with 50 ng/mL recombinant VEGF-C (PeproTech, Rocky Hill, NJ, USA), PDGF-BB (PeproTech), or bFGF (PeproTech) in the presence or absence of nintedanib at concentrations of 0.01, 0.1, and 1 μM.

Techniques: Concentration Assay, Derivative Assay

Journal: The Journal of pathology

Article Title: Effect of VEGFC on lymph flow and inflammation-induced alveolar bone loss

doi: 10.1002/path.5456

Figure Lengend Snippet: AAV-VEGFC increases local lymphatic drainage and reduces alveolar bone loss in RA-associated periodontitis. Four-month-old TNF-Tg mice received local injection of AAV-GFP control virus (left maxillary) or AAV-VEGFC virus (right maxillary) into periodontal tissues of the left and right maxillary first molar, respectively. Two months after viral injection, mice were sacrificed and analyzed. (A) Expression of virus-encoded human VEGFC in periodontal tissues of TNF-Tg mice. (B) Paraffin sections of maxillae were subjected to IF for PDPN (red) and αSMA (green). Representative images of periodontal tissue showing the distribution of PDPN+/αSMA− lymphatic capillaries (red), PDPN+/αSMA+ collecting LVs (yellow), and PDPN−/αSMA+ blood vessels (green). Quantification of the PDPN+αSMA− area or PDPN+αSMA+ area to tissue area (%) was determined. The number of PDPN+αSMA+ collecting LVs per mm2 tissue areas (#/mm2) was calculated. (C) An example of in vivo washout of ICG in periodontal tissues reflecting lymph flow through all time points. ICG clearance (%) was quantified. Values shown are mean ± SEM. *p < 0.05 versus AAV-GFP control virus at the same time point. (D) Representative 3D scanned sections (upper panels) and reconstructed sections (lower panels) along the longitudinal direction of the maxillae. Bone mineral density (BMD, g/cm2) was analyzed. (E) Representative images of H&E-stained paraffin sections. Measurement of the bone levels by comparing the distance from the cemento-enamel junction (CEJ) to the alveolar bone crest (ABC) in μm was determined. (F) Representative images of TRAP-stained paraffin sections. The percentage of alveolar bone surface covered by osteoclasts (Oc.S/B.S) was determined. (G) Representative images of ALP-stained paraffin sections. The percentage of alveolar bone surface covered by osteoblasts (Ob.S/B.S) was determined. *p < 0.05 versus AAV-GFP control virus. N = 6–8. A two-tailed unpaired Student’s t-test was performed.

Article Snippet: A recombinant adeno-associated virus (AAV) encoding a full-length human VEGFC cDNA (NM_005429) was purchased from Genechem Biotech Inc (Shanghai, PR China).

Techniques: Injection, Virus, Expressing, In Vivo, Staining, Two Tailed Test

Journal: The Journal of pathology

Article Title: Effect of VEGFC on lymph flow and inflammation-induced alveolar bone loss

doi: 10.1002/path.5456

Figure Lengend Snippet: AAV-VEGFC increases local lymphatic drainage and reduces alveolar bone loss in ligature-induced periodontitis. Two-month-old WT mice were randomly divided into two groups (six mice per group). One group received local injection of AAV-GFP control virus in periodontal tissues of both the left and right maxillary first molar. Another group received AAV-VEGFC virus injection. Two weeks later, a 5–0 silk ligature was tied around the right maxillary first molar and the contralateral tooth was left unligated to serve as the baseline control. All the mice were sacrificed and analyzed 2 weeks after placement of the ligature. (A) Paraffin sections of maxillae were subjected to IF for PDPN (red) and αSMA (green). Representative images of periodontal tissue showing the distribution of PDPN+/αSMA− lymphatic capillaries (red), PDPN+/αSMA+ collecting LVs (yellow), and PDPN−/αSMA+ blood vessels (green). Quantification of the PDPN+αSMA− area or PDPN+αSMA+ area to tissue area (%) was determined. The number of PDPN+αSMA+ collecting LVs per mm2 tissue areas (#/mm2) was calculated. (B) An example of in vivo washout of ICG in periodontal tissues reflecting lymph flow through all time points. ICG clearance (%) was quantified. Values shown are mean ± SEM. *p < 0.05 versus control + AAV-GFP, #p < 0.05 versus periodontitis + AAV-VEGFC, at the same time point. (C) Representative 3D scanned sections (upper panels) and reconstructed sections (lower panels) along the longitudinal direction of the maxillae. Bone mineral density (BMD, g/cm2) was analyzed. (D) Representative images of H&E-stained paraffin sections. Measurement of the bone levels by comparing the distance from the cemento-enamel junction (CEJ) to the alveolar bone crest (ABC) in μm was determined. (E) Representative images of TRAP-stained paraffin sections. The percentage of alveolar bone surface covered by osteoclasts (Oc.S/B.S) was determined. (F) Representative images of ALP-stained paraffin sections. The percentage of alveolar bone surface covered by osteoblasts (Ob.S/B.S) was determined. *p < 0.05 in the indicated groups. N = 6. One-way ANOVA followed by Dunnett’s post hoc multiple comparisons test was performed.

Article Snippet: A recombinant adeno-associated virus (AAV) encoding a full-length human VEGFC cDNA (NM_005429) was purchased from Genechem Biotech Inc (Shanghai, PR China).

Techniques: Injection, Virus, In Vivo, Staining