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 nanobiotechnology

Article Title: ALPL regulates pro-angiogenic capacity of mesenchymal stem cells through ATP-P2X7 axis controlled exosomes secretion.

doi: 10.1186/s12951-024-02396-6

Figure Lengend Snippet: Fig. 5 ALPL mutation increases hBMMSCs exosomes secretion through ATP axis. (A) HPP hBMMSCs was treated with 2 U/mL ATP-apyrase (HPP + Apy) and the exosomes markers were analyzed. Exosomes proteins from equal volumes of culture supernatant of Nor, HPP and HPP + Apy hBMMSCs were loaded for Western Blot. (B) Exosomes volumes derived from each groups were detected by exosomes ELISA complete kit. (C-D) The intracellular expression of CD9 and CD81 were analyzed by Western Blot. (E) Immunofluorescent staining showed CD9 and CD81-positive labeled exosomal proteins localized in Nor, HPP and HPP + Apy hBMMSCs. Scale bar, 10 μm. (F) Nor hBMMSCs was treated with 10µmol/L ATP and the exosomes markers were analyzed. Exo somal proteins from equal volumes of culture supernatant of Nor and Nor + ATP hBMMSCs were loaded for Western Blot. (G) Exosomes volumes derived from Nor and Nor + ATP groups were detected by exosomes ELSA complete kit. (H-I) The intracellular expression of CD9 and CD81 were analyzed by Western Blot. (J) CD9 and CD81-positive labeled exosomal proteins localized in Nor and Nor + ATP hBMMSCs, as assessed by immunofluorescent staining. Scale bar, 10 μm. All results were generated in three independent experiments. Data were shown as mean ± standard deviation (SD); *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: The secreted VEGF, PDGFBB, Angiostatin and Endostatin in the samples were quantified using human VEGF ELISA Kit (Proteintech, KE00216, China), human PDGFBB ELISA Kit (Proteintech, KE00161, China), human Angiostatin ELISA kit (Neobioscience Technology, E-EL-H6165, China) and human Endostatin ELISA Kit (Proteintech, KE00259, China) according to manufacture’s instruction.

Techniques: Mutagenesis, Western Blot, Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing, Staining, Labeling, Generated, Standard Deviation

Journal: Cancers

Article Title: Endothelial-Specific Molecule 1 Inhibition Lessens Productive Angiogenesis and Tumor Metastasis to Overcome Bevacizumab Resistance

doi: 10.3390/cancers14225681

Figure Lengend Snippet: ESM1 promoted the proliferation as well as increased migration of HUVEC in vitro. ( a ) Immunofluorescence analysis of MDA-MB-231-S or MDA-MB-231-S ovESM1 tumor sections from mice treated with PBS or bevacizumab. *, p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001, by t test. ( b ) HUVEC cells were incubated by conditioned medium of MDA-MB-231-S, MDA-MB-231-S ovESM1 or MDA-MB-231-R for 24 h and the survival was measured by MTT assay, * p < 0.05; ** p < 0.01; *** p < 0.001, by t test. ( c ) Dot blotting of ESM1 in MDA-MB-231-S, MDA-MB-231-S ovESM1 , MDA-MB-231-R or MDA-MB-231-R shESM1#1–3 tumor cells supernatant. ( d ) HUVEC cells were incubated by conditioned medium of MDA-MB-231-S, MDA-MB-231-R or MDA-MB-231-R shESM1#1–3 for 24 h and the survival was measured by MTT assay, *** p < 0.001, **** p < 0.0001 by t test. ( e,f ) HUVEC cells were incubated by conditioned medium of MDA-MB-231-S, MDA-MB-231-S ovESM1 , MDA-MB-231-R or MDA-MB-231-R shESM1#1–3 for 24 or 36 h and the migration was measured by wound healing assay, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, by t test. ( g ) qRT-PCR of VEGFA mRNA levels in MDA-MB-231-S, MDA-MB-231-R, MDA-MB-231-S ovESM1 cells. n =3/group. ( h ) ELISA for analyzing the level of secreted VEGFA in MDA-MB-231-S and MDA-MB-231-S ovESM1 cells. n =3. **** p < 0.0001 by t test. ( i ) qRT-PCR of VEGFA mRNA levels in HUVEC with or without human ESM1 added. * p < 0.05, n = 3/group. ( j ) ELISA for analyzing the level of secreted VEGFA in HUVEC cells with or without human ESM1 added. **** p < 0.0001 by t test. ( k ) Serum concentration of VEGFA in MDA-MB-231-S and MDA-MB-231-S ovESM1 bearing mice treated with bevacizumab was detected by ELISA. n = 4/group. **** p < 0.0001 by t test. ( l ) Serum concentration-time curve of VEGFA in MDA-MB-231-S/R bearing mice treated with bevacizumab was detected by ELISA. n = 3/group. ** p < 0.01; *** p < 0.001; **** p < 0.0001, by t test. ( m ) HUVEC cells were incubated by conditioned medium of MDA-MB-231-S, MDA-MB-231-S ovESM1 or MDA-MB-231-R with or without bevacizumab (0.1 mg/mL) for 24 h and the survival was measured by MTT assay, * p < 0.05; ** p < 0.01; *** p < 0.001, by t test. ( n ) HUVEC cells were incubated by conditioned medium that added bevacizumab (0.1 mg/mL) of MDA-MB-231-S, MDA-MB-231-S ovESM1 or MDA-MB-231-R for 24 and the migration was measured by wound healing assay, * p < 0.05; ** p < 0.01, by t test.

Article Snippet: Serum and cellular supernatant VEGFA were measured using enzyme linked immunosorbent assay (ELISA) with a Human VEGFA ELISA Kit (CHE0043, 4A Biotech, Beijing, China), according to the manufacturer’s instructions.

Techniques: Migration, In Vitro, Immunofluorescence, Incubation, MTT Assay, Wound Healing Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Medicina

Article Title: VEGF Profile in Early Undifferentiated Arthritis Cohort

doi: 10.3390/medicina58060833

Figure Lengend Snippet: VEGF level distribution at the onset of undifferentiated arthritis between disease outcomes after 12 months of follow-up. SpA: spondyloarthropathies (reactive arthritis, axial or peripheral spondylarthritis, psoriatic arthritis); RA: rheumatoid arthritis; other: other autoimmune inflammatory diseases (systemic lupus erythematosus, undifferentiated connective tissue disease, IgG 4 related disease); UA: undifferentiated arthritis; VEGF: vascular endothelial growth factor.

Article Snippet: ELISA kit for human VEGF-A was from IBL International, Germany (catalog no. BE55101).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Cell-free regenerative medicine: identifying the best source of mesenchymal stem cells for skin therapy in Systemic Sclerosis

doi: 10.3389/fcell.2025.1518412

Figure Lengend Snippet: Comparative analysis of cytokine content between AT-MSC-CM, BM-MSC-CM, WJ-MSC-CM, and CB-MSC-CM. AT-MSCs, BM-MSCs, WJ-MSCs, and CB-MSCs were cultured in basal medium. After 24 h, CM from these cells were collected and analyzed for pro-inflammatory (A) , anti-inflammatory (B) , and immune-regulatory (C) cytokine content by using multiplex human cytokine approach or ELISA kits (see Methods). Data were reported as mean of three separate samples. For multiple comparisons, one-way ANOVA was assessed. $ denotes statistically significant values compared with AT-MSC-CM; # denotes statistically significant values compared with BM-MSC-CM; § denotes statistically significant values compared with WJ-MSC-CM; ¥ denotes statistically significant values compared with CB-MSC-CM.

Article Snippet: VEGF-A Human ELISA Kit (Elabscience, Catalog No E-EL-HO111, Wuhan, China) is an in vitro enzyme-linked immunosorbent assay, which was performed for the quantitative measurement of human VEGF-A in cell lysates and conditioned media.

Techniques: Cell Culture, Multiplex Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Cell-free regenerative medicine: identifying the best source of mesenchymal stem cells for skin therapy in Systemic Sclerosis

doi: 10.3389/fcell.2025.1518412

Figure Lengend Snippet: Comparative analysis of cytokine content in AT-MSC-CM, BM-MSC-CM, WJ-MSC-CM, and CB-MSC-CM. AT-MSCs, BM-MSCs, WJ-MSCs, and CB-MSCs were cultured in basal medium. After 24 h, CM from these cells were collected and analyzed for chemokine (A) and growth factor (B) content by using multiplex human cytokine approach or ELISA kits (see Methods). Values are mean ± SEM of three independent samples. For multiple comparisons, one-way ANOVA was assessed. $ denotes statistically significant values compared with AT-MSC-CM; # denotes statistically significant values compared with BM-MSC-CM; § denotes statistically significant values compared with WJ-MSC-CM; ¥ denotes statistically significant values compared with CB-MSC-CM.

Article Snippet: VEGF-A Human ELISA Kit (Elabscience, Catalog No E-EL-HO111, Wuhan, China) is an in vitro enzyme-linked immunosorbent assay, which was performed for the quantitative measurement of human VEGF-A in cell lysates and conditioned media.

Techniques: Cell Culture, Multiplex Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Cell-free regenerative medicine: identifying the best source of mesenchymal stem cells for skin therapy in Systemic Sclerosis

doi: 10.3389/fcell.2025.1518412

Figure Lengend Snippet: Effects of BM-MSC-CM and WJ-MSC-CM on SSc fibroblasts. (A) VEGF-A mRNA expression, (B) VEGF-B mRNA expression, (C) VEGF-C mRNA expression, (D) VEGF-D mRNA expression in SSc fibroblasts treated or not with BM-MSC-CM and WJ-MSC-CM for 24 h at 37°C in a humidified (5% CO 2 ) incubator. mRNA expression levels expressed as fold change (%) over control (untreated cells). (E) Cellular lysate and (F) conditioned medium from SSc fibroblasts, treated or not with BM-MSC-CM and WJ-MSC-CM for 24 h at 37°C in a humidified (5% CO 2 ) incubator, were analyzed for protein expression of VEGF-A by ELISA. Values are mean ± SEM of three SSc patients. (G) Primary dermal fibroblasts isolated from digital ulcers of SSc patient were treated with basal medium and WJ-MSC-CM for 24 h and analyzed for their capacity to migrate into wounds created by using a sterile pipette tip. Scratch images were acquired using inverted microscope and ×4 magnification. Data were plotted and expressed as a percentage of the length of wound size over T0 (assumed as 100%). Error bars represent standard deviation of the mean of triplicate measurements within one experiment. * p < 0.05; ** p < 0.001. (H) Masson’s trichrome staining to qualitatively compare wound healing performance and collagen production between control and treated group of primary skin fibroblasts isolated from 3 SSc patients. The microscopic images were observed under an inverted microscope (Leica DMi1) equipped with camera (FLEXACAM C1, Leica) at ×4 magnification.

Article Snippet: VEGF-A Human ELISA Kit (Elabscience, Catalog No E-EL-HO111, Wuhan, China) is an in vitro enzyme-linked immunosorbent assay, which was performed for the quantitative measurement of human VEGF-A in cell lysates and conditioned media.

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Isolation, Sterility, Transferring, Inverted Microscopy, Standard Deviation, Staining