Journal: BMB Reports

Article Title: MMPP is a novel VEGFR2 inhibitor that suppresses angiogenesis via VEGFR2/AKT/ERK/NF-κB pathway

doi: 10.5483/BMBRep.2023-0150

Figure Lengend Snippet: Effects on migration, invasion, and tube formation in HUVECs and new vessel sprouting in mouse aortic ring. (A) A wound healing assay was conducted to identify migratory ability. The area of the scratch was captured at 0 h (top) and 24 h (bottom) in every group. Quantification of wound area normalized to the scramble control. (B) Transwell invasion assay. The cells penetrating the BME-coated membrane were fixed, and stained, and images were captured using a microscope. (C) Tube formation assay. The results of tube formation were capture by microscopy. Quantification of capillary network formation for 16 h using Image J. (D) Ex-vivo mouse aortic ring assay. Aortic ring was incubated in BME gel with MMPP and/or VEGFA for 6 days. Quantification of branching length using Image J. Data are represented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA.

Article Snippet: Human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Migration, Wound Healing Assay, Control, Transwell Invasion Assay, Membrane, Staining, Microscopy, Tube Formation Assay, Ex Vivo, Aortic Ring Assay, Incubation

Journal: BMB Reports

Article Title: MMPP is a novel VEGFR2 inhibitor that suppresses angiogenesis via VEGFR2/AKT/ERK/NF-κB pathway

doi: 10.5483/BMBRep.2023-0150

Figure Lengend Snippet: Docking studies with MMPP and VEGFR2 and effects of VEGFR2 downstream signaling. (A) 3D diagram showing the docking complex of MMPP (CID: 122517441) with the VEGFR2 ABD (PBD code: 4asd). (B) CETSA analyzed the thermal stabilization of VEGFR2 protein at different temperatures (40, 45, 50, 55, and 60°C). HUVECs were starved in serum-free medium for 24 h, and then treated with MMPP (10 μg/ml) for 2 h. (C) Cell-based ELISA assessed the VEGFR2 inhibitory effect to measure VEGFR2 kinase activity. HUVECs were treated with MMPP for 30 min, and phosphorylation levels of VEGFR2 (Tyr 1175) were measured at 450 nm. (D) Immunoblotting analyses of phosphorylation levels of VEGFR2, AKT, and ERK. HUVECs were starved in serum-free medium for 24 h. MMPP (10 μg/ml) was treated for 1 h, subsequently VEGFA (40 ng/ml) for 30 min. Bar graphs were represented the band intensities of phosphorylated form normalized with normal form. (E) p65 was examined using immunoblotting of the nucleic and cytosolic fraction. p65 was normalized with PARP in nucleic fraction. MMPP (10 μg/ml) was treated for 1 h, subsequently VEGFA (40 ng/ml) for 1 h. (F) Translocation of p65 was observed using immunofluorescence staining. MMPP (10 μg/ml) was treated for 1 h, subsequently VEGFA (40 ng/ml) for 1 h. Data are represented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA.

Article Snippet: Human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: In-Cell ELISA, Activity Assay, Phospho-proteomics, Western Blot, Translocation Assay, Immunofluorescence, Staining

Journal: BMB Reports

Article Title: MMPP is a novel VEGFR2 inhibitor that suppresses angiogenesis via VEGFR2/AKT/ERK/NF-κB pathway

doi: 10.5483/BMBRep.2023-0150

Figure Lengend Snippet: Effects on mRNA levels of angiogenesis-related factor. mRNA levels of (A) VEGFA , (B) VEGFR2 , (C) MMP2 , and (D) MMP9 were determined using RT-PCR. HUVECs were starved in serum-free medium for 24 h. The cells were treated with MMPP (10 μg/ml) for 1 h, subsequently VEGFA (40 ng/ml) for 24 h. Data are represented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA.

Article Snippet: Human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: BMB Reports

Article Title: MMPP is a novel VEGFR2 inhibitor that suppresses angiogenesis via VEGFR2/AKT/ERK/NF-κB pathway

doi: 10.5483/BMBRep.2023-0150

Figure Lengend Snippet: Effects on tube formation, migration, and aortic ring in BCCM. (A) Wound healing assay was performed on HUVECs treated with CCM, BCCM and MMPP + BCCM to analyze their migratory ability. (B) The images of HUVECs tubes formation in CCM, BCCM and MMPP + BCCM were captured, and then the number of junctions was quantified by Image J. (C) The microvessels sprouts of mouse aortic rings were captured in the treatment of CCM, BCCM and MMPP + BCCM. Data are represented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA. (D) Schematic diagram of anti-angiogenic effects of MMPP in endothelial cells. The figure was created using the Motfolio PPT Drawing Toolkits (motfolio.com).

Article Snippet: Human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Migration, Wound Healing Assay

Journal: Bio-protocol

Article Title: Western Blotting and Immunoprecipitation of Native Human PIEZO1 Channels

doi: 10.21769/BioProtoc.5385

Figure Lengend Snippet: (A) Representative western blot probing PIEZO1 and α-ACTININ from various cell lines. For protein lanes: 1, sonicated HCF; 2, HCF; 3, 95 °C boiled HCF; 4, HUVEC; 5, PAOEC; 6, HCF (high passage number); 7, BJ-5ta; 8, HEK293T; 9, MCF-7; 10, HeLa; 11, LnCaP; 12, HEK293T- Piezo1-/- . (B) Example of the quantified intensity level of PIEZO1 signals and α-ACTININ signals from multiple cell lines.

Article Snippet: Porcine aortic endothelial cells (PAOEC) (Cell Applications, Inc., catalog number: P304-05, isolated from normal healthy porcine aorta) 9.

Techniques: Western Blot, Sonication