Journal: International Journal of Molecular Sciences

Article Title: E7050 Suppresses the Growth of Multidrug-Resistant Human Uterine Sarcoma by Inhibiting Angiogenesis via Targeting of VEGFR2-Mediated Signaling Pathways

doi: 10.3390/ijms24119606

Figure Lengend Snippet: Effects of E7050 on VEGFR2-mediated signaling pathways in VEGF-stimulated HUVECs. Cells were serum-starved for 6 h and then pretreated with E7050 (5 and 10 μM) or vehicle for 1 h, followed by stimulation with VEGF (100 ng/mL) for another 10 min (VEGFR2, PLCγ1, FAK, and Src) or 30 min (Akt, JNK, and p38 MAPK) before proteins were collected. The expression and phosphorylation status of VEGFR2 and its downstream effectors, including PLCγ1, FAK, Src, Akt, JNK, and p38 MAPK, were detected by Western blotting using respective antibodies. ( A ) E7050 inhibited the phosphorylation (Tyr1175) of VEGFR2 induced by VEGF in HUVECs. ( B ) The quantified results show that the ratio of p-VEGFR2 protein normalized to the total amount of VEGFR2 protein, which was measured by densitometry. ( C ) E7050 inhibited the phosphorylation of PLCγ1, FAK, and Src in VEGF-stimulated HUVECs. Calculated ratios of ( D ) p-PLCγ1, ( E ) p-FAK, and ( F ) p-Src normalized to the relative total protein levels are shown. ( G ) E7050 inhibited the phosphorylation of Akt, JNK, and p38 MAPK in VEGF-stimulated HUVECs. The compiled results of the ratios of ( H ) p-Akt, ( I ) p-JNK, and ( J ) p-p38 MAPK normalized to the relative total protein levels are shown. The data are presented as mean ± SEM of three independent experiments. # p < 0.05 compared with the untreated cells. * p < 0.05 compared with the vehicle-treated cells.

Article Snippet: The concentrations of VEGF in the culture supernatants of E7050-treated MES-SA/Dx5 cells were measured using the commercial human VEGF ELISA Kit PicoKine TM according to the instructions of the manufacturer (Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Protein-Protein interactions, Expressing, Phospho-proteomics, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: E7050 Suppresses the Growth of Multidrug-Resistant Human Uterine Sarcoma by Inhibiting Angiogenesis via Targeting of VEGFR2-Mediated Signaling Pathways

doi: 10.3390/ijms24119606

Figure Lengend Snippet: Effect of E7050 on the expression level of HGF in MES-SA/Dx5 cells. ( A ) Cells were treated with various concentrations (5–25 μM) of E7050 for 24 h. Whole cell extracts were prepared and subjected to Western blotting using antibodies against HGF and β-actin. β-actin was used as an internal loading control. ( B ) Densitometric analysis of blots relative to HGF protein after normalization with β-actin. ( C ) Secreted HGF in cell culture media was determined by ELISA. Data are presented as the mean ± SEM of three independent experiments. * p < 0.05 versus vehicle-treated control cells.

Article Snippet: The concentrations of VEGF in the culture supernatants of E7050-treated MES-SA/Dx5 cells were measured using the commercial human VEGF ELISA Kit PicoKine TM according to the instructions of the manufacturer (Boster Biological Technology, Pleasanton, CA, USA).

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

Journal: International Journal of Molecular Sciences

Article Title: E7050 Suppresses the Growth of Multidrug-Resistant Human Uterine Sarcoma by Inhibiting Angiogenesis via Targeting of VEGFR2-Mediated Signaling Pathways

doi: 10.3390/ijms24119606

Figure Lengend Snippet: Effects of E7050 on VEGFR2-mediated signaling pathways in cultured MES-SA/Dx5 cells-derived conditioned medium (CM)-treated HUVECs. Cells were serum-starved for 6 h and pretreated with E7050 (5 and 10 μM) or vehicle for 1 h, followed by the addition of VEGF (100 ng/mL) or CM (from cultured MES-SA/Dx5 cells) for another 10 min (VEGFR2, FAK, and Src) or 30 min (Akt, JNK, and p38 MAPK) before protein extraction. The expression and phosphorylation status of VEGFR2 and its downstream effectors, including FAK, Src, Akt, JNK, and p38 MAPK, were detected by Western blotting using specific antibodies. ( A ) E7050 inhibited the phosphorylation of VEGFR2 and Src in MES-SA/Dx5 CM-induced HUVECs. ( B ) The relative band density of p-VEGFR2 protein was normalized to total VEGFR2 protein, which was measured by densitometry. Calculated ratios of ( C ) p-FAK and ( D ) p-Src normalized to the relative total protein levels are shown. ( E ) E7050 inhibited the phosphorylation of Akt, JNK, and p38 MAPK in MES-SA/Dx5 CM-induced HUVECs. The compiled results of the ratios of ( F ) p-Akt, ( G ) p-JNK, and ( H ) p-p38 MAPK normalized to relative total protein levels are shown. The data are presented as mean ± SEM of three independent experiments. # p < 0.05 compared with the untreated cells. * p < 0.05 compared with the vehicle-treated cells.

Article Snippet: The concentrations of VEGF in the culture supernatants of E7050-treated MES-SA/Dx5 cells were measured using the commercial human VEGF ELISA Kit PicoKine TM according to the instructions of the manufacturer (Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Protein-Protein interactions, Cell Culture, Derivative Assay, Protein Extraction, Expressing, Phospho-proteomics, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: E7050 Suppresses the Growth of Multidrug-Resistant Human Uterine Sarcoma by Inhibiting Angiogenesis via Targeting of VEGFR2-Mediated Signaling Pathways

doi: 10.3390/ijms24119606

Figure Lengend Snippet: Effects of E7050 on tumor growth and angiogenesis in the MES-SA/Dx5 cell line-derived xenograft mouse model. Histological characteristics in the tumor tissue sections of MES-SA/Dx5 xenografts obtained from vehicle- and E7050-treated nude mice on day 28 were measured by H&E staining. The expression levels of CD31, VEGF, and p-VEGFR2 (Tyr1175) in tumor tissue sections were also examined by immunohistochemical analyses. Representative photomicrographs of H&E and immunohistochemical staining in tumor tissue sections from vehicle control and E7050-treated groups of mice are shown. Scale bar: 100 μm.

Article Snippet: The concentrations of VEGF in the culture supernatants of E7050-treated MES-SA/Dx5 cells were measured using the commercial human VEGF ELISA Kit PicoKine TM according to the instructions of the manufacturer (Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Derivative Assay, Staining, Expressing, Immunohistochemical staining, Control

Journal: International Journal of Molecular Sciences

Article Title: E7050 Suppresses the Growth of Multidrug-Resistant Human Uterine Sarcoma by Inhibiting Angiogenesis via Targeting of VEGFR2-Mediated Signaling Pathways

doi: 10.3390/ijms24119606

Figure Lengend Snippet: Schematic diagram of a proposed mechanism of E7050-induced anti-angiogenic activity. E7050 exerts anti-angiogenic effects in VEGF-stimulated endothelial cells by downregulating the phosphorylation of VEGFR2 and its downstream mediators, including PLCγ1, FAK, Src, Akt, JNK, and p38 MAPK. The blockage of VEGFR2-mediated signaling cascade pathways by E7050 contributes to the inhibition of proliferation, migration, and tube formation in endothelial cells.

Article Snippet: The concentrations of VEGF in the culture supernatants of E7050-treated MES-SA/Dx5 cells were measured using the commercial human VEGF ELISA Kit PicoKine TM according to the instructions of the manufacturer (Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Activity Assay, Phospho-proteomics, Inhibition, Migration

Journal: Journal of Neuroinflammation

Article Title: Succinate-induced macrophage polarization and RBP4 secretion promote vascular sprouting in ocular neovascularization

doi: 10.1186/s12974-023-02998-1

Figure Lengend Snippet: Effect of RBP4 on sprouting in vivo and in vitro. HUVECs were treated with 10 ng/ml RBP4 for 48 h, with or without 1 μM Ki8751 pretreatment for 12 h. A Relative mRNA expression of ANGPT2, TIE1, HEY1 and DLL4 in RBP4-treated HUVECs ( n = 3). B Relative mRNA expression of ANGPT2, TIE1, HEY1 and DLL4 in the control, RBP4 and RBP4 + Ki8751 groups ( n = 3). C , D Protein levels of VEGFR2 in the three groups ( n = 3). E Immunofluorescence staining of DAPI (blue) and VEGFR2 (red) in HUVECs. Scale bar: 50 μm. F Co-IP examination of VEGFR2-RBP4 interaction. H , J , K Retinal IB4 staining of P14 in OIR and relative number of tip cells as well as filopodia. Scale bar: 200 μm ( n = 3). G , I Choroidal sprouting analysis in three groups ( n = 3). Scale bar: 500 μm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Administration of the VEGFR2 inhibitor Ki8751 (1 μM for 12 h, #HY-12038, MCE) reduced VEGFR2 expression and attenuated RBP4 promotion of ANGPT2 and TIE1 as well as inhibition of HEY1 and DLL4 (Fig. B).

Techniques: In Vivo, In Vitro, Expressing, Control, Immunofluorescence, Staining, Co-Immunoprecipitation Assay