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  • 97
    Name:
    Vascular Endothelial Growth Factor VEGF
    Description:
    The human VEGF165 coding cDNA was subcloned into an expression vector and expressed in yeast The recombinant human VEGF 165 homodimer was purified and stored in PBS buffer pH 7 4 containing 0 1 BSA
    Catalog Number:
    9943
    Price:
    None
    Category:
    Cytokines
    Source:
    Human Recombinant Protein
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc vegf a
    Proposed model by which CCR7 regulated <t>NF-κB/VEGF</t> pathway and induced angiogenesis in esophageal squamous carcinoma cells.
    The human VEGF165 coding cDNA was subcloned into an expression vector and expressed in yeast The recombinant human VEGF 165 homodimer was purified and stored in PBS buffer pH 7 4 containing 0 1 BSA
    https://www.bioz.com/result/vegf a/product/Cell Signaling Technology Inc
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    vegf a - by Bioz Stars, 2021-04
    97/100 stars

    Images

    1) Product Images from "CCR7 enhances the angiogenic capacity of esophageal squamous carcinoma cells in vitro via activation of the NF-κB/VEGF signaling pathway"

    Article Title: CCR7 enhances the angiogenic capacity of esophageal squamous carcinoma cells in vitro via activation of the NF-κB/VEGF signaling pathway

    Journal: American Journal of Translational Research

    doi:

    Proposed model by which CCR7 regulated NF-κB/VEGF pathway and induced angiogenesis in esophageal squamous carcinoma cells.
    Figure Legend Snippet: Proposed model by which CCR7 regulated NF-κB/VEGF pathway and induced angiogenesis in esophageal squamous carcinoma cells.

    Techniques Used:

    2) Product Images from "Generation of a functional and durable vascular niche by the adenoviral E4ORF1 gene"

    Article Title: Generation of a functional and durable vascular niche by the adenoviral E4ORF1 gene

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0805980105

    E4ORF1 tonically activates pAkt in PECs. ( A ) PECs (HUVECs) were infected with AdE4ORF1→7 vectors at 100 moi or AdE4ORF1 or AdE4ORF1 at 20 moi in serum/cytokine-free medium for 48 h. Immunoblot of lysates using polyclonal anti-phospho-ser473 Akt antibody ( pAkt ) ( Upper ) and total Akt antibody ( Akt ) ( Lower ) are shown. ( B ) PECs were transfected with 20 nM E4ORF1 siRNA, E4ORF6 siRNA or control GFP siRNA for 24 h and then exposed to AdE4ORF1→7 vectors (100 moi) in serum/cytokine-free medium for 2 days. Western blot analysis using anti-phospho-Akt antibody ( Upper ) or anti-total Akt antibody ( Lower ). ( C ) PECs (HUVECs) were infected with lenti-E4ORF1 or lenti-GFP for 3 days followed by Western blot for pAkt ( Upper ) or Akt ( Lower ). ( D ) Cell viability (MTT assay) of Lenti-E4ORF1 or control HUVECs incubated serum/cytokine-free for 5 days +/− wortmannin A (1 μM), rapamycin (8 nM), or NK-κB inhibitor CAPE (2 μM). ( E ) Lenti-E4ORF1 HUVECs incubated (serum/cytokine-free) in presence or absence of 10 μM PD98059 (PD) or SB203580 (SB) for 2 days before cell viability analysis ( P is not significant). ( F ) E4ORF1 + and E4ORF1 − ECs stimulated with or without 50 ng/ml of VEGF-A for 10 min followed by Western blot for phospho-MAPK ( Upper ) or level of β-actin ( Lower ).
    Figure Legend Snippet: E4ORF1 tonically activates pAkt in PECs. ( A ) PECs (HUVECs) were infected with AdE4ORF1→7 vectors at 100 moi or AdE4ORF1 or AdE4ORF1 at 20 moi in serum/cytokine-free medium for 48 h. Immunoblot of lysates using polyclonal anti-phospho-ser473 Akt antibody ( pAkt ) ( Upper ) and total Akt antibody ( Akt ) ( Lower ) are shown. ( B ) PECs were transfected with 20 nM E4ORF1 siRNA, E4ORF6 siRNA or control GFP siRNA for 24 h and then exposed to AdE4ORF1→7 vectors (100 moi) in serum/cytokine-free medium for 2 days. Western blot analysis using anti-phospho-Akt antibody ( Upper ) or anti-total Akt antibody ( Lower ). ( C ) PECs (HUVECs) were infected with lenti-E4ORF1 or lenti-GFP for 3 days followed by Western blot for pAkt ( Upper ) or Akt ( Lower ). ( D ) Cell viability (MTT assay) of Lenti-E4ORF1 or control HUVECs incubated serum/cytokine-free for 5 days +/− wortmannin A (1 μM), rapamycin (8 nM), or NK-κB inhibitor CAPE (2 μM). ( E ) Lenti-E4ORF1 HUVECs incubated (serum/cytokine-free) in presence or absence of 10 μM PD98059 (PD) or SB203580 (SB) for 2 days before cell viability analysis ( P is not significant). ( F ) E4ORF1 + and E4ORF1 − ECs stimulated with or without 50 ng/ml of VEGF-A for 10 min followed by Western blot for phospho-MAPK ( Upper ) or level of β-actin ( Lower ).

    Techniques Used: Infection, Transfection, Western Blot, MTT Assay, Incubation

    E4ORF1-mediated activation of FGF-2/FGF receptor pathway supports survival and proliferation of ECs. ( A ) HUVECs were infected with AdE4ORF1, AdE43,4,6,6/7 at 20 moi, or AdE4ORF1→7, or AdE4 Null (lacking all E4ORFs) at 100 moi in serum/cytokine-free medium for 48 h. Lysates were analyzed by immunoblot using polyclonal anti-FGF2 and anti-β-Actin antibodies. ( B–D ) Lenti-E4ORF1 HUVECs were transfected with siRNA against FGF-2 for 48 h. ( B ) Lysates were analyzed by immunoblot using polyclonal anti-FGF2, anti-FGF receptor-1, and anti-β-actin antibodies. ( C ) Quantification of ECs in serum/cytokine-free conditions by trypan blue exclusion. ( D ) Phase-contrast microscopy of E4ORF1 + ECs treated with control siRNA or FGF-2 siRNA, demonstrating detachment and death of PECs treated with siRNA against FGF-2 in serum/cytokine-free conditions. (Magnification: 200×.) ( E ) Lenti-E4ORF1 infected-HUVECs were treated with anti-FGF-R1 antibody or rabbit-IgG in serum/cytokine-free M199 with or without FGF-2 for 5 days and quantitated with the MTT assay. Data are mean ± SD of 3 experiments. ( F ) HUVECs were transduced with either lenti-GFP (control, E4ORF1 − ) or lenti-E4ORF1 (E4ORF1 + ) enumerated in serum/cytokine-free conditions in the presence and absence of FGF-2 (5 ng/ml) and/or VEGF-A at 10 ng/ml. Data represent mean ± SD ( n = 3).
    Figure Legend Snippet: E4ORF1-mediated activation of FGF-2/FGF receptor pathway supports survival and proliferation of ECs. ( A ) HUVECs were infected with AdE4ORF1, AdE43,4,6,6/7 at 20 moi, or AdE4ORF1→7, or AdE4 Null (lacking all E4ORFs) at 100 moi in serum/cytokine-free medium for 48 h. Lysates were analyzed by immunoblot using polyclonal anti-FGF2 and anti-β-Actin antibodies. ( B–D ) Lenti-E4ORF1 HUVECs were transfected with siRNA against FGF-2 for 48 h. ( B ) Lysates were analyzed by immunoblot using polyclonal anti-FGF2, anti-FGF receptor-1, and anti-β-actin antibodies. ( C ) Quantification of ECs in serum/cytokine-free conditions by trypan blue exclusion. ( D ) Phase-contrast microscopy of E4ORF1 + ECs treated with control siRNA or FGF-2 siRNA, demonstrating detachment and death of PECs treated with siRNA against FGF-2 in serum/cytokine-free conditions. (Magnification: 200×.) ( E ) Lenti-E4ORF1 infected-HUVECs were treated with anti-FGF-R1 antibody or rabbit-IgG in serum/cytokine-free M199 with or without FGF-2 for 5 days and quantitated with the MTT assay. Data are mean ± SD of 3 experiments. ( F ) HUVECs were transduced with either lenti-GFP (control, E4ORF1 − ) or lenti-E4ORF1 (E4ORF1 + ) enumerated in serum/cytokine-free conditions in the presence and absence of FGF-2 (5 ng/ml) and/or VEGF-A at 10 ng/ml. Data represent mean ± SD ( n = 3).

    Techniques Used: Activation Assay, Infection, Transfection, Microscopy, MTT Assay, Transduction

    3) Product Images from "Hypericin-photodynamic therapy induces human umbilical vein endothelial cell apoptosis"

    Article Title: Hypericin-photodynamic therapy induces human umbilical vein endothelial cell apoptosis

    Journal: Scientific Reports

    doi: 10.1038/srep18398

    Inhibition of VEGF-A-mediated PI3K/Akt pathway in HUVECs. The HUVECs were serum-starved for 24 h and stimulated in fresh medium with VEGF-A (25 ng/ml) and then treated with HY (0.062 μM) in combination with VEGF-A (25 ng/ml) for 24 h. The cells were exposed to a 585-nm LED light at a dose of 1.0 J/cm 2 and were incubation for 24 h. The cells were lysed and the proteins were harvested for western blot analysis of VEGF-A ( a ), p-Akt (Ser473) and Akt ( b ), and Bad ( c ). Densitometric measurements were analysed using AlphaEaseFC 4.0 software. The protein expression levels were normalized to those of the vehicle control (100%). Data are presented as means ± S.D. (n = 3); ** P
    Figure Legend Snippet: Inhibition of VEGF-A-mediated PI3K/Akt pathway in HUVECs. The HUVECs were serum-starved for 24 h and stimulated in fresh medium with VEGF-A (25 ng/ml) and then treated with HY (0.062 μM) in combination with VEGF-A (25 ng/ml) for 24 h. The cells were exposed to a 585-nm LED light at a dose of 1.0 J/cm 2 and were incubation for 24 h. The cells were lysed and the proteins were harvested for western blot analysis of VEGF-A ( a ), p-Akt (Ser473) and Akt ( b ), and Bad ( c ). Densitometric measurements were analysed using AlphaEaseFC 4.0 software. The protein expression levels were normalized to those of the vehicle control (100%). Data are presented as means ± S.D. (n = 3); ** P

    Techniques Used: Inhibition, Incubation, Western Blot, Software, Expressing

    4) Product Images from "Insulin-Containing Wound Dressing Promotes Diabetic Wound Healing Through Stabilizing HIF-1α"

    Article Title: Insulin-Containing Wound Dressing Promotes Diabetic Wound Healing Through Stabilizing HIF-1α

    Journal: Frontiers in Bioengineering and Biotechnology

    doi: 10.3389/fbioe.2020.592833

    Insulin-containing SF microparticles promoted diabetic wound healing through HIF-1α stabilization. (A,B) Insulin-containing dressing promoted HIF-1α stabilization and its downstream protein expression. The expressions of HIF-1α and downstream protein Col I, Col III, and VEGF-A in wounds on the fifth day were analyzed using western blot, and Photoshop was used in the quantification of immunoblots. Data are shown as mean ± SD. ** p
    Figure Legend Snippet: Insulin-containing SF microparticles promoted diabetic wound healing through HIF-1α stabilization. (A,B) Insulin-containing dressing promoted HIF-1α stabilization and its downstream protein expression. The expressions of HIF-1α and downstream protein Col I, Col III, and VEGF-A in wounds on the fifth day were analyzed using western blot, and Photoshop was used in the quantification of immunoblots. Data are shown as mean ± SD. ** p

    Techniques Used: Expressing, Western Blot

    5) Product Images from "Regulatory mechanisms of anthrax toxin receptor 1-dependent vascular and connective tissue homeostasis"

    Article Title: Regulatory mechanisms of anthrax toxin receptor 1-dependent vascular and connective tissue homeostasis

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    doi: 10.1016/j.matbio.2014.12.002

    Cell signaling changes in skin of mutant mice. (a) Real-time PCR shows reduced transcript levels for Vegfr1 and Itgb1 (left) and 3-fold increase in Vegfa transcripts (middle) in mutant skin extracts; ELISA shows 2-fold increase in VEGF plasma levels (right) in mutant mice (n = 6; *P
    Figure Legend Snippet: Cell signaling changes in skin of mutant mice. (a) Real-time PCR shows reduced transcript levels for Vegfr1 and Itgb1 (left) and 3-fold increase in Vegfa transcripts (middle) in mutant skin extracts; ELISA shows 2-fold increase in VEGF plasma levels (right) in mutant mice (n = 6; *P

    Techniques Used: Mutagenesis, Mouse Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Dysregulation of MMP2 activity in TEM8 mutant cells. (a) ELISA showing VEGF levels in lysates of stable endothelial cell lines with different degrees of Antxr1 knockdown (shTEM8-1/26%; shTEM8-2/60%; shTEM8-3/90%). (n = 3; *P
    Figure Legend Snippet: Dysregulation of MMP2 activity in TEM8 mutant cells. (a) ELISA showing VEGF levels in lysates of stable endothelial cell lines with different degrees of Antxr1 knockdown (shTEM8-1/26%; shTEM8-2/60%; shTEM8-3/90%). (n = 3; *P

    Techniques Used: Activity Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay

    6) Product Images from "6-Shogaol reduces progression of experimental endometriosis in vivo and in vitro via regulation of VGEF and inhibition of COX-2 and PGE2-mediated inflammatory responses"

    Article Title: 6-Shogaol reduces progression of experimental endometriosis in vivo and in vitro via regulation of VGEF and inhibition of COX-2 and PGE2-mediated inflammatory responses

    Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

    doi: 10.4196/kjpp.2018.22.6.627

    6-Shogaol regulates the expression of VEGF and Flk-1. (a) 6-Shogaol markedly down-regulates the expressions of VEGF and Flk-1 mRNA levels. (b) Relative mRNA expression levels. Values are represented as mean±SD, n=6. p
    Figure Legend Snippet: 6-Shogaol regulates the expression of VEGF and Flk-1. (a) 6-Shogaol markedly down-regulates the expressions of VEGF and Flk-1 mRNA levels. (b) Relative mRNA expression levels. Values are represented as mean±SD, n=6. p

    Techniques Used: Expressing

    Effect of 6-shogaol on VEGF and Flk-1 and COX-2 protein expressions. 6-Shogaol markedly regulates the expressions of VEGF and Flk-1 and COX-2 proteins (a). Representative immunoblot (b). Relative expressions of proteins. Values are represented as mean±SD, n=6. p
    Figure Legend Snippet: Effect of 6-shogaol on VEGF and Flk-1 and COX-2 protein expressions. 6-Shogaol markedly regulates the expressions of VEGF and Flk-1 and COX-2 proteins (a). Representative immunoblot (b). Relative expressions of proteins. Values are represented as mean±SD, n=6. p

    Techniques Used:

    7) Product Images from "Pharmacological Intervention of MKL/SRF signaling by CCG-1423 impedes Endothelial Cell Migration and Angiogenesis"

    Article Title: Pharmacological Intervention of MKL/SRF signaling by CCG-1423 impedes Endothelial Cell Migration and Angiogenesis

    Journal: Angiogenesis

    doi: 10.1007/s10456-017-9560-y

    CCG-1423 attenuates expression of several actin-binding proteins, suppresses membrane protrusion and motility of HmVEC A) Quantitative analyses of the effect of 5 M CCG-1423 treatment on VEGF-induced motility of HmVEC (these data are based on time-lapse analyses of randomly migrating HmVEC cells for 2 hrs) (**: p
    Figure Legend Snippet: CCG-1423 attenuates expression of several actin-binding proteins, suppresses membrane protrusion and motility of HmVEC A) Quantitative analyses of the effect of 5 M CCG-1423 treatment on VEGF-induced motility of HmVEC (these data are based on time-lapse analyses of randomly migrating HmVEC cells for 2 hrs) (**: p

    Techniques Used: Expressing, Binding Assay

    8) Product Images from "Different Sequences of Fractionated Low-Dose Proton and Single Iron-Radiation-Induced Divergent Biological Responses in the Heart"

    Article Title: Different Sequences of Fractionated Low-Dose Proton and Single Iron-Radiation-Induced Divergent Biological Responses in the Heart

    Journal: Radiation research

    doi: 10.1667/RR14667.1

    Protein analyses at 1 month postirradiation and 7 days after MI. Representative Western blot images of heart tissue homogenates from nonirradiated control mouse hearts and hearts of mice that were whole-body irradiated using different sequences at 1 month postirradiation in a radiation and aging plus MI model. Images represent scans of radiograms for phosphorylated (p), total (T) and loading control on day 7 for the following proteins: VEGF-A (38 kDa) and GAPDH (panel A); p-Erk1/2, T-Erk1/2 (42/44 kDa) and GAPDH (panel C); p-Akt (Ser473), T-Akt (62 kDa) and GAPDH (panel E). Quantification and graphic representation of total protein levels and phosphorylation using densitometric analysis of phospho-band intensities after adjusting for corresponding GAPDH (36 kDa) and total band intensities of heart tissue homogenates at 1 month postirradiation in a radiation and aging plus MI model for the following proteins: VEGF-A, p-Erk1/2 and p-Akt (Ser473) (panels B, D and F, respectively). Group 1: nonirradiated control (solid black bars); group 2: 1 H × 3 irradiation (dotted bars); group 3: 1 H × 3 + 56 Fe irradiation (diagonal line bars); and group 4: 56 Fe + 1 H × 3 irradiation (solid red bars). Results in all graphs are depicted as mean ± SEM (n = 6–8 animals per time point/group). Statistical significance was assigned when P
    Figure Legend Snippet: Protein analyses at 1 month postirradiation and 7 days after MI. Representative Western blot images of heart tissue homogenates from nonirradiated control mouse hearts and hearts of mice that were whole-body irradiated using different sequences at 1 month postirradiation in a radiation and aging plus MI model. Images represent scans of radiograms for phosphorylated (p), total (T) and loading control on day 7 for the following proteins: VEGF-A (38 kDa) and GAPDH (panel A); p-Erk1/2, T-Erk1/2 (42/44 kDa) and GAPDH (panel C); p-Akt (Ser473), T-Akt (62 kDa) and GAPDH (panel E). Quantification and graphic representation of total protein levels and phosphorylation using densitometric analysis of phospho-band intensities after adjusting for corresponding GAPDH (36 kDa) and total band intensities of heart tissue homogenates at 1 month postirradiation in a radiation and aging plus MI model for the following proteins: VEGF-A, p-Erk1/2 and p-Akt (Ser473) (panels B, D and F, respectively). Group 1: nonirradiated control (solid black bars); group 2: 1 H × 3 irradiation (dotted bars); group 3: 1 H × 3 + 56 Fe irradiation (diagonal line bars); and group 4: 56 Fe + 1 H × 3 irradiation (solid red bars). Results in all graphs are depicted as mean ± SEM (n = 6–8 animals per time point/group). Statistical significance was assigned when P

    Techniques Used: Western Blot, Mouse Assay, Irradiation

    9) Product Images from "Redox status of high-mobility group box 1 performs a dual role in angiogenesis of colorectal carcinoma"

    Article Title: Redox status of high-mobility group box 1 performs a dual role in angiogenesis of colorectal carcinoma

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.12577

    HMGB1, especially its disulfide-bonded form, could regulate the release of VEGF-A. Quantities of VEGF-A expressed after stimulation of HUVECs with increasing concentrations of rHMGB1 (A and C) or duration of stimulation (B and D). (E) At mRNA level, VEGF-A expression was significantly reduced via siRNA inhibition of HMGB1 and RAGE. (F) At protein level, siRNA targeting HMGB1, RAGE and TLR4 had an obvious influence on VEGF-A secretion. (G) at-HMGB1 (50 ng/ml) slightly up-regulated VEGF-A expression ( P > 0.05), which was reversed by effects of H 2 O 2 and si-RAGE. (H) Oxidation and inhibition by 2G7 mAb (1 μg/ml) or si-TLR4 significantly down-regulated the VEGF-A expression induced by ds-HMGB1 (500 ng/ml). For terminally oxidation, at-HMGB1 or ds-HMGB1 was previously exposed to H 2 O 2 (50mM) for 1 hr. (I) The similar VEGF-A reduction by H 2 O 2 , 2G7 mAb or si-TLR4 was determined with Western blot, whereas there displayed no effect of at-HMGB1 or its associated inhibition. *Indicates a P
    Figure Legend Snippet: HMGB1, especially its disulfide-bonded form, could regulate the release of VEGF-A. Quantities of VEGF-A expressed after stimulation of HUVECs with increasing concentrations of rHMGB1 (A and C) or duration of stimulation (B and D). (E) At mRNA level, VEGF-A expression was significantly reduced via siRNA inhibition of HMGB1 and RAGE. (F) At protein level, siRNA targeting HMGB1, RAGE and TLR4 had an obvious influence on VEGF-A secretion. (G) at-HMGB1 (50 ng/ml) slightly up-regulated VEGF-A expression ( P > 0.05), which was reversed by effects of H 2 O 2 and si-RAGE. (H) Oxidation and inhibition by 2G7 mAb (1 μg/ml) or si-TLR4 significantly down-regulated the VEGF-A expression induced by ds-HMGB1 (500 ng/ml). For terminally oxidation, at-HMGB1 or ds-HMGB1 was previously exposed to H 2 O 2 (50mM) for 1 hr. (I) The similar VEGF-A reduction by H 2 O 2 , 2G7 mAb or si-TLR4 was determined with Western blot, whereas there displayed no effect of at-HMGB1 or its associated inhibition. *Indicates a P

    Techniques Used: Expressing, Inhibition, Western Blot

    HMGB1 induced tumour angiogenesis. (A) HUVECs were co-cultured with HCT116 cells for 4 days in the absence or presence of the four HMGB1-associated antibodies, involving DPH1.1 mAb (0.5 μg/ml), 2G7 mAb (1 μg/ml), RAGE Ab (10 μg/ml) and TLR4 Ab (10 μg/ml). The supernatants were collected and analysed for VEGF-A secretion. (Ctrl indicates supernatants from HUVECs cultured alone; CC indicates supernatants co-cultured for 4 days without any inhibition). (B–E) Conditioned medium (CM) from HCT116 cells was applied to increase angiogenic properties of HUVECs. However, CM interfered with the four HMGB1-associated antibodies and VEGF-A Ab (10 μg/ml) had a counter effect in distinct conditions. Representative photos of three independent experiments are shown. *Compared with cells not treated by CM (Ctrl); P
    Figure Legend Snippet: HMGB1 induced tumour angiogenesis. (A) HUVECs were co-cultured with HCT116 cells for 4 days in the absence or presence of the four HMGB1-associated antibodies, involving DPH1.1 mAb (0.5 μg/ml), 2G7 mAb (1 μg/ml), RAGE Ab (10 μg/ml) and TLR4 Ab (10 μg/ml). The supernatants were collected and analysed for VEGF-A secretion. (Ctrl indicates supernatants from HUVECs cultured alone; CC indicates supernatants co-cultured for 4 days without any inhibition). (B–E) Conditioned medium (CM) from HCT116 cells was applied to increase angiogenic properties of HUVECs. However, CM interfered with the four HMGB1-associated antibodies and VEGF-A Ab (10 μg/ml) had a counter effect in distinct conditions. Representative photos of three independent experiments are shown. *Compared with cells not treated by CM (Ctrl); P

    Techniques Used: Cell Culture, Inhibition

    HMGB1 influenced angiogenic properties via different redox states. Exposure of HUVECs to 50 ng/ml at-HMGB1 promoted invasion of EC in Matrigel-coated chambers (A) as well as its migration in scratch wound assay (C). ds-HMGB1 (500 ng/ml) as well as its associated inhibition failed to influence EC to invade (B) or migrate (D). (E and F) Aortic rings were embedded in Matrigel containing at-HMGB1 (50 ng/ml) or ds-HMGB1 (500 ng/ml). Sprouts area from the rings was evaluated in the absence or presence of DPH1.1 mAb (0.5 μg/ml), 2G7 mAb (1 μg/ml), antibodies to RAGE, TLR4 and VEGF-A (all in concentration of 10 μg/ml). For terminally oxidation, at-HMGB1 or ds-HMGB1 was previously exposed to H 2 O 2 (50mM) for 1 hr. (G and H) Similar treatment was applied in a HUVEC tube formation assay. Representative photos of three independent experiments are shown. *Compared with cells not exposed to at-HMGB1 or ds-HMGB1 (Ctrl), P
    Figure Legend Snippet: HMGB1 influenced angiogenic properties via different redox states. Exposure of HUVECs to 50 ng/ml at-HMGB1 promoted invasion of EC in Matrigel-coated chambers (A) as well as its migration in scratch wound assay (C). ds-HMGB1 (500 ng/ml) as well as its associated inhibition failed to influence EC to invade (B) or migrate (D). (E and F) Aortic rings were embedded in Matrigel containing at-HMGB1 (50 ng/ml) or ds-HMGB1 (500 ng/ml). Sprouts area from the rings was evaluated in the absence or presence of DPH1.1 mAb (0.5 μg/ml), 2G7 mAb (1 μg/ml), antibodies to RAGE, TLR4 and VEGF-A (all in concentration of 10 μg/ml). For terminally oxidation, at-HMGB1 or ds-HMGB1 was previously exposed to H 2 O 2 (50mM) for 1 hr. (G and H) Similar treatment was applied in a HUVEC tube formation assay. Representative photos of three independent experiments are shown. *Compared with cells not exposed to at-HMGB1 or ds-HMGB1 (Ctrl), P

    Techniques Used: Migration, Scratch Wound Assay Assay, Inhibition, Concentration Assay, HUVEC Tube Formation Assay

    10) Product Images from "6-Shogaol reduces progression of experimental endometriosis in vivo and in vitro via regulation of VGEF and inhibition of COX-2 and PGE2-mediated inflammatory responses"

    Article Title: 6-Shogaol reduces progression of experimental endometriosis in vivo and in vitro via regulation of VGEF and inhibition of COX-2 and PGE2-mediated inflammatory responses

    Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

    doi: 10.4196/kjpp.2018.22.6.627

    Effect of 6-shogaol on VEGF and Flk-1 and COX-2 protein expressions. 6-Shogaol markedly regulates the expressions of VEGF and Flk-1 and COX-2 proteins (a). Representative immunoblot (b). Relative expressions of proteins. Values are represented as mean±SD, n=6. p
    Figure Legend Snippet: Effect of 6-shogaol on VEGF and Flk-1 and COX-2 protein expressions. 6-Shogaol markedly regulates the expressions of VEGF and Flk-1 and COX-2 proteins (a). Representative immunoblot (b). Relative expressions of proteins. Values are represented as mean±SD, n=6. p

    Techniques Used:

    6-Shogaol regulates the expression of VEGF and Flk-1. (a) 6-Shogaol markedly down-regulates the expressions of VEGF and Flk-1 mRNA levels. (b) Relative mRNA expression levels. Values are represented as mean±SD, n=6. p
    Figure Legend Snippet: 6-Shogaol regulates the expression of VEGF and Flk-1. (a) 6-Shogaol markedly down-regulates the expressions of VEGF and Flk-1 mRNA levels. (b) Relative mRNA expression levels. Values are represented as mean±SD, n=6. p

    Techniques Used: Expressing

    11) Product Images from "A novel bispecific c-MET/PD-1 antibody with therapeutic potential in solid cancer"

    Article Title: A novel bispecific c-MET/PD-1 antibody with therapeutic potential in solid cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.16173

    BsAb inhibits tumor development and chronic inflammation in vivo (A) Xenograft studies were performed using 5-6 week old male NOD/SCID mice (n=4/per group), by subcutaneous injection of MKN45 cells (5 × 10 6 cells). Tumor volumes measured on indicated days were plotted for the BsAb treatment and control groups. (B) MKN45 tumor xenografts were stripped 4 days after the last treatment. (C) Semi-quantification of IHC was expressed as percentage of positively stained cells. Representative images of tumors from 5 fields randomly were counted (n=4/per group), showing Ki-67, VEGF-A, and MMP-9 (magnification: 400×). (D) Serum levels of IL-6 and TNF-α were measured by ELISA. The assays were repeated 2 times and each sample has 3 holes, data represent the mean ± SD of 4 individual mice per group. **: P
    Figure Legend Snippet: BsAb inhibits tumor development and chronic inflammation in vivo (A) Xenograft studies were performed using 5-6 week old male NOD/SCID mice (n=4/per group), by subcutaneous injection of MKN45 cells (5 × 10 6 cells). Tumor volumes measured on indicated days were plotted for the BsAb treatment and control groups. (B) MKN45 tumor xenografts were stripped 4 days after the last treatment. (C) Semi-quantification of IHC was expressed as percentage of positively stained cells. Representative images of tumors from 5 fields randomly were counted (n=4/per group), showing Ki-67, VEGF-A, and MMP-9 (magnification: 400×). (D) Serum levels of IL-6 and TNF-α were measured by ELISA. The assays were repeated 2 times and each sample has 3 holes, data represent the mean ± SD of 4 individual mice per group. **: P

    Techniques Used: In Vivo, Mouse Assay, Injection, Immunohistochemistry, Staining, Enzyme-linked Immunosorbent Assay

    12) Product Images from "Spica prunellae promotes cancer cell apoptosis, inhibits cell proliferation and tumor angiogenesis in a mouse model of colorectal cancer via suppression of stat3 pathway"

    Article Title: Spica prunellae promotes cancer cell apoptosis, inhibits cell proliferation and tumor angiogenesis in a mouse model of colorectal cancer via suppression of stat3 pathway

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/1472-6882-13-144

    Effect of EESP on the expression of Bcl-2, Bax, Cyclin D1, CDK4, VEGF-A and VEGFR-2 in CRC mice. ( A ) The mRNA levels in tumor tissues from control and EESP-treated group were determined by RT-PCR. GAPDH was used as an internal control. The data of densitometric analysis were normalized to the mean mRNA expression of untreated control (100%). ( B-D ) The protein expression of Bcl-2, Bax, Cyclin D1, CDK4, VEGF-A and VEGFR-2 in tumor tissues was analyzed via immunohistochemical assay. The photographs are representative images taken at a magnification of 400 ×. Quantification of immunohistochemical assay was represented as percentage of positively-stained cells. Data shown are averages with S.D. (error bars) from 9 individual mouse in each group. *P
    Figure Legend Snippet: Effect of EESP on the expression of Bcl-2, Bax, Cyclin D1, CDK4, VEGF-A and VEGFR-2 in CRC mice. ( A ) The mRNA levels in tumor tissues from control and EESP-treated group were determined by RT-PCR. GAPDH was used as an internal control. The data of densitometric analysis were normalized to the mean mRNA expression of untreated control (100%). ( B-D ) The protein expression of Bcl-2, Bax, Cyclin D1, CDK4, VEGF-A and VEGFR-2 in tumor tissues was analyzed via immunohistochemical assay. The photographs are representative images taken at a magnification of 400 ×. Quantification of immunohistochemical assay was represented as percentage of positively-stained cells. Data shown are averages with S.D. (error bars) from 9 individual mouse in each group. *P

    Techniques Used: Expressing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry, Staining

    13) Product Images from "Annexin A12–26 Treatment Improves Skin Heterologous Transplantation by Modulating Inflammation and Angiogenesis Processes"

    Article Title: Annexin A12–26 Treatment Improves Skin Heterologous Transplantation by Modulating Inflammation and Angiogenesis Processes

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2018.01015

    AnxA1 2–26 increases angiogenesis in a dorsal chamber model in BALB/c mice. Mice were topically treated with Saline (10 μL), AnxA1 2–26 (1 mg/kg), and/or VEGF-A (10 ng/10 μL) in the dorsal skin. The treatments were administrated once per day, every 2 days, resulting three applications in each mouse. Representative images of the microcirculatory network of dorsal skin were obtained before (day 4) and after (day 9) treatments (A) . The images in the upper panel represent the stained normal tissue and in the lower panel, the same computational images obtained after inverting the colors are displayed (A) . The quantification of vessels is represented in B . The values express the mean ± SEM of five animals per group (ANOVA followed by Bonferroni’s Multiple Comparison Test). ∗ p
    Figure Legend Snippet: AnxA1 2–26 increases angiogenesis in a dorsal chamber model in BALB/c mice. Mice were topically treated with Saline (10 μL), AnxA1 2–26 (1 mg/kg), and/or VEGF-A (10 ng/10 μL) in the dorsal skin. The treatments were administrated once per day, every 2 days, resulting three applications in each mouse. Representative images of the microcirculatory network of dorsal skin were obtained before (day 4) and after (day 9) treatments (A) . The images in the upper panel represent the stained normal tissue and in the lower panel, the same computational images obtained after inverting the colors are displayed (A) . The quantification of vessels is represented in B . The values express the mean ± SEM of five animals per group (ANOVA followed by Bonferroni’s Multiple Comparison Test). ∗ p

    Techniques Used: Mouse Assay, Staining

    AnxA1 2–26 does not induce tube formation. HUVECs (2 × 10 4 cells/well) were incubated with PBS (control), AnxA1 2–26 (30 μM), and/or VEGF (50 ng/mL) for 6 h on Matrigel ® and the number of tube structures were quantified using an optical microscope (A,B) . PECAM-1 expression was evaluated by flow cytometry (C) . Scale bar = 10 μm. Results are expressed as the mean ± SEM of two independent experiments in triplicate (ANOVA followed by the Tukey’s multiple comparisons test). ∗ p
    Figure Legend Snippet: AnxA1 2–26 does not induce tube formation. HUVECs (2 × 10 4 cells/well) were incubated with PBS (control), AnxA1 2–26 (30 μM), and/or VEGF (50 ng/mL) for 6 h on Matrigel ® and the number of tube structures were quantified using an optical microscope (A,B) . PECAM-1 expression was evaluated by flow cytometry (C) . Scale bar = 10 μm. Results are expressed as the mean ± SEM of two independent experiments in triplicate (ANOVA followed by the Tukey’s multiple comparisons test). ∗ p

    Techniques Used: Incubation, Microscopy, Expressing, Flow Cytometry, Cytometry

    AnxA1 2–26 increases endothelial cell migration and actin polymerization. HUVECs (1 × 10 4 cells/well) were incubated with PBS (control), AnxA1 2–26 (30 μM), and/or VEGF-A (10 or 50 ng/mL) and cell proliferation was evaluated at 24, 48, and 72 h. Results are expressed as the mean ± SEM of cells of two independent experiments in triplicate (A) . HUVECs were incubated with different treatments for 48 h, later labeled with PI (50 μg/mL) and the cell cycle phases were evaluated (ANOVA followed by the Tukey’s multiple comparisons test) (B) . HUVEC migration was evaluated after 12 h of incubation with PBS (control), AnxA1 2–26 (1, 10, or 30 μM) and/or VEGF-A (50 ng/mL). Cell migration was monitored with images obtained before (0 h) and after (12 h) treatments (C,D) . HUVECs (1 × 10 4 cells/well) were incubated with different treatments for 2 h and later incubated with rhodamine-phalloidin to evaluate actin polymerization. The intensity of fluorescence was monitored using a fluorescent plate reader (E) and by confocal microscopy (F) . Scale bar = 10 μm. Results are expressed as the mean ± SEM of cells of two independent experiments in triplicate (ANOVA followed by the Bonferroni’s test). ∗ p
    Figure Legend Snippet: AnxA1 2–26 increases endothelial cell migration and actin polymerization. HUVECs (1 × 10 4 cells/well) were incubated with PBS (control), AnxA1 2–26 (30 μM), and/or VEGF-A (10 or 50 ng/mL) and cell proliferation was evaluated at 24, 48, and 72 h. Results are expressed as the mean ± SEM of cells of two independent experiments in triplicate (A) . HUVECs were incubated with different treatments for 48 h, later labeled with PI (50 μg/mL) and the cell cycle phases were evaluated (ANOVA followed by the Tukey’s multiple comparisons test) (B) . HUVEC migration was evaluated after 12 h of incubation with PBS (control), AnxA1 2–26 (1, 10, or 30 μM) and/or VEGF-A (50 ng/mL). Cell migration was monitored with images obtained before (0 h) and after (12 h) treatments (C,D) . HUVECs (1 × 10 4 cells/well) were incubated with different treatments for 2 h and later incubated with rhodamine-phalloidin to evaluate actin polymerization. The intensity of fluorescence was monitored using a fluorescent plate reader (E) and by confocal microscopy (F) . Scale bar = 10 μm. Results are expressed as the mean ± SEM of cells of two independent experiments in triplicate (ANOVA followed by the Bonferroni’s test). ∗ p

    Techniques Used: Migration, Incubation, Labeling, Fluorescence, Confocal Microscopy

    VEGF-A and AnxA1 2–26 decreases FPR1 expression on HUVECs. FPR1 was detected in the plasma membrane (arrows) and cytoplasm of cells under all experimental conditions (A) . Negative control (B) . Density of FPR1 immunogold particles in HUVECs (C) . Scale bar = 0.5 μm. Data are mean ± SEM of distinct cells analyzed ( n = 12–20/group) for each condition (ANOVA followed by the Bonferroni’s test). ∗ p
    Figure Legend Snippet: VEGF-A and AnxA1 2–26 decreases FPR1 expression on HUVECs. FPR1 was detected in the plasma membrane (arrows) and cytoplasm of cells under all experimental conditions (A) . Negative control (B) . Density of FPR1 immunogold particles in HUVECs (C) . Scale bar = 0.5 μm. Data are mean ± SEM of distinct cells analyzed ( n = 12–20/group) for each condition (ANOVA followed by the Bonferroni’s test). ∗ p

    Techniques Used: Expressing, Negative Control

    AnxA1 2–26 treatment improves the heterologous transplantation and induces angiogenesis. Histopathological analyses of skin transplanted fragments without (PBS) and with AnxA1 2–26 peptide treatment after 3 (A) , 10 (B) , 15 (C) , and 60 (D) days post-surgery. Cell infiltration (E) , TGF-β (F) , α-SMA (G) , FGF-b (H) , and VEGF-A (I) gene expression and VEGF-A protein (J) in the transplanted tissue. Host tissue (TH), transplanted scaffold (Sc), vessels ( ∗ ), fibroblasts (arrows). The inserts show high magnifications of the fibroblasts. The values express the mean ± SEM of five animals per group (ANOVA followed by the Bonferroni’s test). ∗ p
    Figure Legend Snippet: AnxA1 2–26 treatment improves the heterologous transplantation and induces angiogenesis. Histopathological analyses of skin transplanted fragments without (PBS) and with AnxA1 2–26 peptide treatment after 3 (A) , 10 (B) , 15 (C) , and 60 (D) days post-surgery. Cell infiltration (E) , TGF-β (F) , α-SMA (G) , FGF-b (H) , and VEGF-A (I) gene expression and VEGF-A protein (J) in the transplanted tissue. Host tissue (TH), transplanted scaffold (Sc), vessels ( ∗ ), fibroblasts (arrows). The inserts show high magnifications of the fibroblasts. The values express the mean ± SEM of five animals per group (ANOVA followed by the Bonferroni’s test). ∗ p

    Techniques Used: Transplantation Assay, Expressing

    14) Product Images from "Spatial regulation of VEGF receptor endocytosis in angiogenesis"

    Article Title: Spatial regulation of VEGF receptor endocytosis in angiogenesis

    Journal: Nature cell biology

    doi: 10.1038/ncb2679

    Endothelial Dab2 and PAR-3 regulate angiogenic vessel growth a , Overview of the P6 control and Dab2 iΔEC retinal vasculature. Anti-Dab2 (white/red) staining is shown in top panels. ECs, Isolectin B4 (IB4). Bottom panels show higher magnification of the angiogenic front. b , Defects in the P6 Pard3 iΔEC retinal vasculature. Anti-PAR-3 and IB4 (green) staining in middle panels show successful deletion of PAR-3 in Pard3 iΔEC vessels. Residual round signals correspond to autofluorescent blood cells. Bottom panels show higher magnification of the angiogenic front. c, d , Quantitation of filopodia number and length, tip cell number, EC-covered area, EC proliferation and vessels branch points in Dab2 mutant (iΔEC) ( c ) or Pard3 mutant ( d ) retinas compared to the corresponding control littermates (Ctrl). Dab2 mutant n=7, Pard3 mutant n=3. Percentage of reduction is indicated. Data represent the means±s.d. P values, two-tailed Student’s t-test. e , Reduced uptake of labelled VEGF-A (red) at the Dab2 iΔEC or Pard3 iΔEC angiogenic front compared to control littermates. Green arrowheads indicate VEGF-A spots, white arrowheads marks ECs with no or little uptake. f , Statistical analysis of internalised Alexa-coupled VEGF-A in the angiogenic front and central plexus. Data represent the means±s.d. of 6 independent experiments. P values, two-tailed Student’s t-test.
    Figure Legend Snippet: Endothelial Dab2 and PAR-3 regulate angiogenic vessel growth a , Overview of the P6 control and Dab2 iΔEC retinal vasculature. Anti-Dab2 (white/red) staining is shown in top panels. ECs, Isolectin B4 (IB4). Bottom panels show higher magnification of the angiogenic front. b , Defects in the P6 Pard3 iΔEC retinal vasculature. Anti-PAR-3 and IB4 (green) staining in middle panels show successful deletion of PAR-3 in Pard3 iΔEC vessels. Residual round signals correspond to autofluorescent blood cells. Bottom panels show higher magnification of the angiogenic front. c, d , Quantitation of filopodia number and length, tip cell number, EC-covered area, EC proliferation and vessels branch points in Dab2 mutant (iΔEC) ( c ) or Pard3 mutant ( d ) retinas compared to the corresponding control littermates (Ctrl). Dab2 mutant n=7, Pard3 mutant n=3. Percentage of reduction is indicated. Data represent the means±s.d. P values, two-tailed Student’s t-test. e , Reduced uptake of labelled VEGF-A (red) at the Dab2 iΔEC or Pard3 iΔEC angiogenic front compared to control littermates. Green arrowheads indicate VEGF-A spots, white arrowheads marks ECs with no or little uptake. f , Statistical analysis of internalised Alexa-coupled VEGF-A in the angiogenic front and central plexus. Data represent the means±s.d. of 6 independent experiments. P values, two-tailed Student’s t-test.

    Techniques Used: Staining, Quantitation Assay, Mutagenesis, Two Tailed Test

    Increased VEGF uptake and sprouting in the Prkci iΔEC central retina a, b , VEGF-A (red, a ) or VEGF-C ( b ) uptake in the Prkci iΔEC central plexus. Green arrowheads indicate ligand spots. Cell nuclei, DAPI (blue); ECs, Isolectin B4 (IB4, white). c , Statistical analysis of internalised VEGFs as shown in ( a ) and ( b ). Data represent the means±s.d. of 6 independent experiments. P values, two-tailed Student’s t-test. d , Phenotype of the Isolectin B4-stained (IB4) P6 Prkci iΔEC retinal vasculature. Higher magnification of the angiogenic front (middle) and central plexus (bottom) are shown. e–g , Quantitation of vessels branch points, EC area and proliferation ( e , n=3), the number and length of filopodia and the number of distal sprout tips at the angiogenic front ( f , n=6), and the number of ectopic sprouts and filopodia ( g , n=6) in the central retina of Prkci mutants (iΔEC) compared to control (Ctrl) retinas. Data represent the means±s.d. P values, two-tailed Student’s t-test
    Figure Legend Snippet: Increased VEGF uptake and sprouting in the Prkci iΔEC central retina a, b , VEGF-A (red, a ) or VEGF-C ( b ) uptake in the Prkci iΔEC central plexus. Green arrowheads indicate ligand spots. Cell nuclei, DAPI (blue); ECs, Isolectin B4 (IB4, white). c , Statistical analysis of internalised VEGFs as shown in ( a ) and ( b ). Data represent the means±s.d. of 6 independent experiments. P values, two-tailed Student’s t-test. d , Phenotype of the Isolectin B4-stained (IB4) P6 Prkci iΔEC retinal vasculature. Higher magnification of the angiogenic front (middle) and central plexus (bottom) are shown. e–g , Quantitation of vessels branch points, EC area and proliferation ( e , n=3), the number and length of filopodia and the number of distal sprout tips at the angiogenic front ( f , n=6), and the number of ectopic sprouts and filopodia ( g , n=6) in the central retina of Prkci mutants (iΔEC) compared to control (Ctrl) retinas. Data represent the means±s.d. P values, two-tailed Student’s t-test

    Techniques Used: Two Tailed Test, Staining, Quantitation Assay

    Spatial differences in VEGF uptake in the retina a , Quantitation of Alexa-labelled VEGF-A in retinal ECs at 10 and 45 min after injection. The formation of intracellular spots was blocked by MiTMAB. Data represent the means±s.d. of 6 independent experiments. P values, ANOVA. b , VEGF-A uptake in blood vessels of Flk iΔEC mice. Green arrowheads indicate internalised Alexa dye-coupled VEGF-A (red). Cell nuclei, DAPI (blue); ECs, Isolectin B4 (IB4, white). Data represent the means±s.d. of 6 independent experiments. P values, two-tailed student t test. c , Spatial distribution of VEGF-A or VEGF-C (red) uptake in retinal vessels. Green arrowheads indicate internalised label spots. Cell nuclei, DAPI (blue); ECs, Isolectin B4 (IB4, white).
    Figure Legend Snippet: Spatial differences in VEGF uptake in the retina a , Quantitation of Alexa-labelled VEGF-A in retinal ECs at 10 and 45 min after injection. The formation of intracellular spots was blocked by MiTMAB. Data represent the means±s.d. of 6 independent experiments. P values, ANOVA. b , VEGF-A uptake in blood vessels of Flk iΔEC mice. Green arrowheads indicate internalised Alexa dye-coupled VEGF-A (red). Cell nuclei, DAPI (blue); ECs, Isolectin B4 (IB4, white). Data represent the means±s.d. of 6 independent experiments. P values, two-tailed student t test. c , Spatial distribution of VEGF-A or VEGF-C (red) uptake in retinal vessels. Green arrowheads indicate internalised label spots. Cell nuclei, DAPI (blue); ECs, Isolectin B4 (IB4, white).

    Techniques Used: Quantitation Assay, Injection, Mouse Assay, Two Tailed Test

    Dab2 and PAR-3 control VEGF receptor internalisation a , Alexa546-labelled VEGF-A or VEGF-C (red) accumulated in the perinuclear region of control mouse ECs at 30 min after stimulation, which was strongly reduced after knockdown of Dab2 or Pard3 . Actin, Phalloidin (green); nuclei, DAPI (blue). b, c , Quantitation of Alexa546-positive peri-nuclear VEGF-A ( b ) or VEGF-C ( c ) spots. Two different siRNAs were used for Dab2 and Pard3 in ( b ). Data represent the means±s.d. of 6 independent experiments. P values, two-tailed Student’s t-test. At least 100 cells were scored in each experiment. d, e , Biochemical detection of biotinylated (surface) VEGFR2 and VEGFR3 in stimulated control and Dab2 ( d ) or Pard3 ( e ) KD cells. Antibodies used for immunoblotting and molecular weight marker are indicated. f , Activation of Rac1 in control and Dab2 or Pard3 KD mouse ECs stimulated with VEGF-A or VEGF-C for 5 min, as indicated.
    Figure Legend Snippet: Dab2 and PAR-3 control VEGF receptor internalisation a , Alexa546-labelled VEGF-A or VEGF-C (red) accumulated in the perinuclear region of control mouse ECs at 30 min after stimulation, which was strongly reduced after knockdown of Dab2 or Pard3 . Actin, Phalloidin (green); nuclei, DAPI (blue). b, c , Quantitation of Alexa546-positive peri-nuclear VEGF-A ( b ) or VEGF-C ( c ) spots. Two different siRNAs were used for Dab2 and Pard3 in ( b ). Data represent the means±s.d. of 6 independent experiments. P values, two-tailed Student’s t-test. At least 100 cells were scored in each experiment. d, e , Biochemical detection of biotinylated (surface) VEGFR2 and VEGFR3 in stimulated control and Dab2 ( d ) or Pard3 ( e ) KD cells. Antibodies used for immunoblotting and molecular weight marker are indicated. f , Activation of Rac1 in control and Dab2 or Pard3 KD mouse ECs stimulated with VEGF-A or VEGF-C for 5 min, as indicated.

    Techniques Used: Quantitation Assay, Two Tailed Test, Molecular Weight, Marker, Activation Assay

    Negative regulation of VEGF receptor internalisation by aPKC a , Phosphorylation of a GST-Dab2 fusion protein by recombinant PKCλ reduced its interaction with VEGFR3 and VEGFR2 in pull down assays. Densiometric (DM) values are shown below bands. ATP for the kinase activation was added (+) or absent (−), as indicated. CBB, Coomassie Brilliant Blue staining of GST fusion proteins. b , Western blot showing reduced Dab2 phoshorylation at Serine 24 (pS24) in cultured ECs after 30 min incubation with 5µM aPKC inhibitor. c , Association of Dab2 with immunoprecipitated VEGFR3 (anti-R3) was enhanced in Prkci iΔEC lung lysate compared to control littermates. No specific bands were immunoprecipitated with IgG. VEGFR3-associated Dab2 lacked detectable phosphorylation in Ser24 (pS24). d , Effect of aPKC inhibition on VEGF-A- or VEGF-C-induced VEGF receptor internalisation at indicated time points. Cells were preincubated with 5 µM of aPKC inhibitor for 30 min. Surface VEGFR2 (R2) and VEGFR3 (R3) were identified by biotinylation. e , Increased activation of MAP kinase (p-ERK1/2) by VEGF-A or VEGF-C after aPKC inhibition in cultured mouse ECs. Effects on AKT phosphorylation were comparably modest. Total ERK1/2 and AKT are shown as loading controls. Molecular weight marker (kD) is indicated. f , Anti-aPKC (total), anti-phospho-aPKC (p-aPKC, Thr560) and Isolectin B4 (IB4) staining of the P6 control and Prkci iΔEC retinal vasculature. g , Anti-aPKC immunostaining (red) labels ECs (IB4, green) at the angiogenic front and in the central retinal plexus. h , Phospho-aPKC (p-aPKC, Thr560) immunosignals were weak at the angiogenic front in comparison to vessels of the central plexus. Higher magnification of p-aPKC signals and merged channels in insets is shown on the right.
    Figure Legend Snippet: Negative regulation of VEGF receptor internalisation by aPKC a , Phosphorylation of a GST-Dab2 fusion protein by recombinant PKCλ reduced its interaction with VEGFR3 and VEGFR2 in pull down assays. Densiometric (DM) values are shown below bands. ATP for the kinase activation was added (+) or absent (−), as indicated. CBB, Coomassie Brilliant Blue staining of GST fusion proteins. b , Western blot showing reduced Dab2 phoshorylation at Serine 24 (pS24) in cultured ECs after 30 min incubation with 5µM aPKC inhibitor. c , Association of Dab2 with immunoprecipitated VEGFR3 (anti-R3) was enhanced in Prkci iΔEC lung lysate compared to control littermates. No specific bands were immunoprecipitated with IgG. VEGFR3-associated Dab2 lacked detectable phosphorylation in Ser24 (pS24). d , Effect of aPKC inhibition on VEGF-A- or VEGF-C-induced VEGF receptor internalisation at indicated time points. Cells were preincubated with 5 µM of aPKC inhibitor for 30 min. Surface VEGFR2 (R2) and VEGFR3 (R3) were identified by biotinylation. e , Increased activation of MAP kinase (p-ERK1/2) by VEGF-A or VEGF-C after aPKC inhibition in cultured mouse ECs. Effects on AKT phosphorylation were comparably modest. Total ERK1/2 and AKT are shown as loading controls. Molecular weight marker (kD) is indicated. f , Anti-aPKC (total), anti-phospho-aPKC (p-aPKC, Thr560) and Isolectin B4 (IB4) staining of the P6 control and Prkci iΔEC retinal vasculature. g , Anti-aPKC immunostaining (red) labels ECs (IB4, green) at the angiogenic front and in the central retinal plexus. h , Phospho-aPKC (p-aPKC, Thr560) immunosignals were weak at the angiogenic front in comparison to vessels of the central plexus. Higher magnification of p-aPKC signals and merged channels in insets is shown on the right.

    Techniques Used: Recombinant, Activation Assay, Staining, Western Blot, Cell Culture, Incubation, Immunoprecipitation, Inhibition, Molecular Weight, Marker, Immunostaining

    15) Product Images from "Curcumol attenuates liver sinusoidal endothelial cell angiogenesis via regulating Glis‐PROX1‐HIF‐1α in liver fibrosis, et al. Curcumol attenuates liver sinusoidal endothelial cell angiogenesis via regulating Glis‐PROX1‐HIF‐1α in liver fibrosis"

    Article Title: Curcumol attenuates liver sinusoidal endothelial cell angiogenesis via regulating Glis‐PROX1‐HIF‐1α in liver fibrosis, et al. Curcumol attenuates liver sinusoidal endothelial cell angiogenesis via regulating Glis‐PROX1‐HIF‐1α in liver fibrosis

    Journal: Cell Proliferation

    doi: 10.1111/cpr.12762

    Curcumol inhibits liver sinusoidal endothelial cell angiogenesis through regulating Hh signalling pathway in mice. A, Analysis of sinusoidal fenestrae (arrowhead) in liver tissues (n = 3). Scale bar = 1 μm. B, Immunohistochemical analyses of endothelial markers CD31, CD34 and vWF in liver tissues (n = 3). Scale bar = 100 μm. C, Western blot analysis of endothelial markers and angiogenic factors (n = 3). D, Immunofluorescence analysis of endothelial markers CD31 accompanied by HIF‐1α, VEGF‐A, Smo and PROX1 in liver tissues (n = 3). Scale bar = 20 μm. * P
    Figure Legend Snippet: Curcumol inhibits liver sinusoidal endothelial cell angiogenesis through regulating Hh signalling pathway in mice. A, Analysis of sinusoidal fenestrae (arrowhead) in liver tissues (n = 3). Scale bar = 1 μm. B, Immunohistochemical analyses of endothelial markers CD31, CD34 and vWF in liver tissues (n = 3). Scale bar = 100 μm. C, Western blot analysis of endothelial markers and angiogenic factors (n = 3). D, Immunofluorescence analysis of endothelial markers CD31 accompanied by HIF‐1α, VEGF‐A, Smo and PROX1 in liver tissues (n = 3). Scale bar = 20 μm. * P

    Techniques Used: Mouse Assay, Immunohistochemistry, Western Blot, Immunofluorescence

    Curcumol inhibits LSEC angiogenesis in vitro. Liver sinusoidal endothelial cells (LSECs) were treated with DMSO or VEGF‐A (40 ng/mL) and different concentrations of curcumol for 24 h. A, Cell viability was determined using MTT. Data were expressed as percentage of control value (n = 6). B, Primary LSECs were cultured on collagen‐covered plates, and the fenestrae were observed with scanning electron microscope scale. Day 1 LSECs show numerous fenestrae (arrowhead). Day 2 and 3 LSECs have few fenestrae. Primary LSECs treated with curcumol (20 μmol/L) for 24 h and cultured for 2 d presented sporadic fenestrae (n = 3). Bar = 5 μm. C, Real‐time PCR analysis of angiogenic cytokines. Data were expressed as fold of control value (n = 4). D, Western blot analyses of angiogenic cytokines (n = 3). E, Immunofluorescence analysis of angiogenic cytokine expression (n = 3). Scale bar = 20 μm. F, Tubulogenesis assay was visualized and quantified with ImageJ (n = 3). Scale bar = 500 μm. * P
    Figure Legend Snippet: Curcumol inhibits LSEC angiogenesis in vitro. Liver sinusoidal endothelial cells (LSECs) were treated with DMSO or VEGF‐A (40 ng/mL) and different concentrations of curcumol for 24 h. A, Cell viability was determined using MTT. Data were expressed as percentage of control value (n = 6). B, Primary LSECs were cultured on collagen‐covered plates, and the fenestrae were observed with scanning electron microscope scale. Day 1 LSECs show numerous fenestrae (arrowhead). Day 2 and 3 LSECs have few fenestrae. Primary LSECs treated with curcumol (20 μmol/L) for 24 h and cultured for 2 d presented sporadic fenestrae (n = 3). Bar = 5 μm. C, Real‐time PCR analysis of angiogenic cytokines. Data were expressed as fold of control value (n = 4). D, Western blot analyses of angiogenic cytokines (n = 3). E, Immunofluorescence analysis of angiogenic cytokine expression (n = 3). Scale bar = 20 μm. F, Tubulogenesis assay was visualized and quantified with ImageJ (n = 3). Scale bar = 500 μm. * P

    Techniques Used: In Vitro, MTT Assay, Cell Culture, Microscopy, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Expressing

    Curcumol inhibits liver sinusoidal endothelial cells (LSECs) angiogenesis via regulating PROX1. A, Real‐time PCR analysis of PROX1 mRNA in DMSO‐ and curcumol‐treated LSECs after the induction of VEGF‐A (n = 3). B, Western blot analysis of PROX1 protein expression in LSECs (n = 3). C and E, Immunofluorescence analysis of PROX1 and HIF‐1α protein expression in LSECs (n = 3). Scale bar = 20 μm. D, Western blot analysis of HIF‐1α and angiogenic properties of LSECs treated with the above models plus PROX1 plasmid (n = 3). F, LSECs were treated with protease inhibitor MG132 (10 mg/mL) for 6 h after drug treatment. Co‐IP assays were conducted with anti‐HIF‐1α, followed by detection of ubiquitin, HIF‐1α and PROX1. G, Tubulogenesis assay was visualized and quantified with ImageJ (n = 3). Scale bar = 500 μm. * P
    Figure Legend Snippet: Curcumol inhibits liver sinusoidal endothelial cells (LSECs) angiogenesis via regulating PROX1. A, Real‐time PCR analysis of PROX1 mRNA in DMSO‐ and curcumol‐treated LSECs after the induction of VEGF‐A (n = 3). B, Western blot analysis of PROX1 protein expression in LSECs (n = 3). C and E, Immunofluorescence analysis of PROX1 and HIF‐1α protein expression in LSECs (n = 3). Scale bar = 20 μm. D, Western blot analysis of HIF‐1α and angiogenic properties of LSECs treated with the above models plus PROX1 plasmid (n = 3). F, LSECs were treated with protease inhibitor MG132 (10 mg/mL) for 6 h after drug treatment. Co‐IP assays were conducted with anti‐HIF‐1α, followed by detection of ubiquitin, HIF‐1α and PROX1. G, Tubulogenesis assay was visualized and quantified with ImageJ (n = 3). Scale bar = 500 μm. * P

    Techniques Used: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Immunofluorescence, Plasmid Preparation, Protease Inhibitor, Co-Immunoprecipitation Assay

    16) Product Images from "Network Pharmacology Reveals the Mechanism of Activity of Tongqiao Huoxue Decoction Extract Against Middle Cerebral Artery Occlusion-Induced Cerebral Ischemia-Reperfusion Injury"

    Article Title: Network Pharmacology Reveals the Mechanism of Activity of Tongqiao Huoxue Decoction Extract Against Middle Cerebral Artery Occlusion-Induced Cerebral Ischemia-Reperfusion Injury

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2020.572624

    TQHXD strengthened angiogenesis in MCAO rats. (A) Representative band image (top) and quantitative analysis (bottom) of VEGF-A ( n = 3). (B) The mRNA expression of VEGF in cortex detected by qPCR. (C) Quantitative analysis for ELISA results of VEGF at 7 days. (D) The changes of rCBF for all groups at 1 and 7 d after reperfusion, n = 3. (E) Immunofluorescence of brain section. Angiogenesis was visualized by CD34 staining (red), and DNA was stained by 4′6,-diamidino-2-phenylindole (DAPI; blue). Red box implies zoom-in fluorescence. Right, the histograms indicate the percentages of CD34-expressing cells that were positive for indicated markers. Scale bar = 50 μm (×200 magnification). Data are presented as means ± SD. ## p
    Figure Legend Snippet: TQHXD strengthened angiogenesis in MCAO rats. (A) Representative band image (top) and quantitative analysis (bottom) of VEGF-A ( n = 3). (B) The mRNA expression of VEGF in cortex detected by qPCR. (C) Quantitative analysis for ELISA results of VEGF at 7 days. (D) The changes of rCBF for all groups at 1 and 7 d after reperfusion, n = 3. (E) Immunofluorescence of brain section. Angiogenesis was visualized by CD34 staining (red), and DNA was stained by 4′6,-diamidino-2-phenylindole (DAPI; blue). Red box implies zoom-in fluorescence. Right, the histograms indicate the percentages of CD34-expressing cells that were positive for indicated markers. Scale bar = 50 μm (×200 magnification). Data are presented as means ± SD. ## p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Fluorescence

    Experimental design and schedules. MCAO: middle cerebral artery occlusion; TQHXD: Tongqiao Huoxue Decoction; EB: Evans blue; ELISA: enzyme-linked immunosorbent assay; HE: hematoxylin-eosin staining; VEGF: vascular endothelial growth factor; FAK: focal adhesion kinase.
    Figure Legend Snippet: Experimental design and schedules. MCAO: middle cerebral artery occlusion; TQHXD: Tongqiao Huoxue Decoction; EB: Evans blue; ELISA: enzyme-linked immunosorbent assay; HE: hematoxylin-eosin staining; VEGF: vascular endothelial growth factor; FAK: focal adhesion kinase.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Staining

    TQHXD enhanced I/R-induced angiogenesis via activation of VEGF-A/VEGFR2-FAK-Paxillin signaling. (A) Neurological scores measured by Longa’s method ( n = 5). (B) The changes of rCBF for all groups at 7 days after reperfusion, n = 6. (C) Representative Western blot band (top) and quantitative analysis (bottom) of the expressions of FAK, p-FAK, Paxillin, and p-Paxillin ( n = 3). β-Actin was used as an internal reference. (D) Left, serial sections of the brain were subjected to hematoxylin and eosin (HE) staining, immunofluorescence for angiogenesis markers (CD34, red), and DNA was stained by 4′6-diamidino-2-phenylindole (DAPI; blue). White box implies zoom-in fluorescence. Right, the histograms indicate the percentages of CD34-positive cells. Scale bar = 50 μm (×200 magnification). White box implies zoom-in fluorescence. (E) The model illustrates the molecular mechanism of TQHXD on angiogenesis in MCAO injury rats. Data were expressed as mean ± SD. ## p
    Figure Legend Snippet: TQHXD enhanced I/R-induced angiogenesis via activation of VEGF-A/VEGFR2-FAK-Paxillin signaling. (A) Neurological scores measured by Longa’s method ( n = 5). (B) The changes of rCBF for all groups at 7 days after reperfusion, n = 6. (C) Representative Western blot band (top) and quantitative analysis (bottom) of the expressions of FAK, p-FAK, Paxillin, and p-Paxillin ( n = 3). β-Actin was used as an internal reference. (D) Left, serial sections of the brain were subjected to hematoxylin and eosin (HE) staining, immunofluorescence for angiogenesis markers (CD34, red), and DNA was stained by 4′6-diamidino-2-phenylindole (DAPI; blue). White box implies zoom-in fluorescence. Right, the histograms indicate the percentages of CD34-positive cells. Scale bar = 50 μm (×200 magnification). White box implies zoom-in fluorescence. (E) The model illustrates the molecular mechanism of TQHXD on angiogenesis in MCAO injury rats. Data were expressed as mean ± SD. ## p

    Techniques Used: Activation Assay, Western Blot, Staining, Immunofluorescence, Fluorescence

    17) Product Images from "MiR-145 inhibits tumor angiogenesis and growth by N-RAS and VEGF"

    Article Title: MiR-145 inhibits tumor angiogenesis and growth by N-RAS and VEGF

    Journal: Cell Cycle

    doi: 10.4161/cc.20598

    Figure 3. miR-145 targets N-RAS and VEGF-A. (A) Fluorescent images showed high transduction efficiency of lentivirus carrying miR-145 or miR-scr (upper panel). The miR-145 expression levels were detected by semi-quantitative RT-PCR (lower panel). (B) Schematic diagrams of putative miR-145 binding sites in the 3′UTRs of N-RAS and VEGF-A were shown in upper panel. The possible miR-145 binding sites in the 3′UTRs of the target genes were searched using algorithm KeyTar miRNA target prediction algorithm. The mutated nucleotides are shown in red. Luciferase activities of N-RAS and VEGFA reporter constructs in MDA-MB-231 cells transfected with miR-145 or miR-scr were normalized to cotransfected renilla luciferase plasmid activity (lower panel). *p
    Figure Legend Snippet: Figure 3. miR-145 targets N-RAS and VEGF-A. (A) Fluorescent images showed high transduction efficiency of lentivirus carrying miR-145 or miR-scr (upper panel). The miR-145 expression levels were detected by semi-quantitative RT-PCR (lower panel). (B) Schematic diagrams of putative miR-145 binding sites in the 3′UTRs of N-RAS and VEGF-A were shown in upper panel. The possible miR-145 binding sites in the 3′UTRs of the target genes were searched using algorithm KeyTar miRNA target prediction algorithm. The mutated nucleotides are shown in red. Luciferase activities of N-RAS and VEGFA reporter constructs in MDA-MB-231 cells transfected with miR-145 or miR-scr were normalized to cotransfected renilla luciferase plasmid activity (lower panel). *p

    Techniques Used: Transduction, Expressing, Quantitative RT-PCR, Binding Assay, Luciferase, Construct, Multiple Displacement Amplification, Transfection, Plasmid Preparation, Activity Assay

    18) Product Images from "mTORC1 drives HIF-1α and VEGF-A signalling via multiple mechanisms involving 4E-BP1, S6K1 and STAT3"

    Article Title: mTORC1 drives HIF-1α and VEGF-A signalling via multiple mechanisms involving 4E-BP1, S6K1 and STAT3

    Journal: Oncogene

    doi: 10.1038/onc.2014.164

    STAT3 is a direct substrate for mTORC1 A: mTORC1 kinase assay: An active mTORC1 complex was purified from insulin stimulated HEK293 cells grown under serum-starved conditions as described. 35 GST-STAT3 and GST-4E-BP1 were purified from serum-starved HEK293 cells, as was GST-Rheb, which was then loaded with GTPγS. Purified mTORC1 complex was incubated alongside potential substrates with and without GTPγS-Rheb (as indicated) for 1 h at 30°C with gentle agitation. SDS-PAGE and western blotting with phospho-specific antibodies was utilised to determine specific phosphorylation events as well as mTOR/Raptor purification. B: HEK293 cells were transfected with either Non-target control or STAT3 shRNA and treated with either 10 μg/ml insulin, 50 nM rapamycin (rap) or 1 μM KU-0063794 (KU). Western blotting was utilised to determine the efficiency of STAT3 knockdown and downstream effects. Q-PCR was utilised to determine HIF-1α mRNA levels (standardised to β-actin). No significant differences were observed between insulin stimulation or mTORC1 inhibition with rap/KU (indicated by NS). C: Schematic showing multiple mTORC1 inputs into angiogenesis. As demonstrated in this study, mTORC1 regulates the translation of HIF-1α via both 4E-BP1/eIF4E and S6K1. Although S6K1 can also promote HIF-1α translation, VEGF-A does not appear to be directly affected by active S6K1. mTORC1 also controls the transcription of HIF-1α mRNA in a rapamycin sensitive fashion via STAT3 phosphorylation, indicating that mTORC1 mediates angiogenesis via three distinct mechanisms.
    Figure Legend Snippet: STAT3 is a direct substrate for mTORC1 A: mTORC1 kinase assay: An active mTORC1 complex was purified from insulin stimulated HEK293 cells grown under serum-starved conditions as described. 35 GST-STAT3 and GST-4E-BP1 were purified from serum-starved HEK293 cells, as was GST-Rheb, which was then loaded with GTPγS. Purified mTORC1 complex was incubated alongside potential substrates with and without GTPγS-Rheb (as indicated) for 1 h at 30°C with gentle agitation. SDS-PAGE and western blotting with phospho-specific antibodies was utilised to determine specific phosphorylation events as well as mTOR/Raptor purification. B: HEK293 cells were transfected with either Non-target control or STAT3 shRNA and treated with either 10 μg/ml insulin, 50 nM rapamycin (rap) or 1 μM KU-0063794 (KU). Western blotting was utilised to determine the efficiency of STAT3 knockdown and downstream effects. Q-PCR was utilised to determine HIF-1α mRNA levels (standardised to β-actin). No significant differences were observed between insulin stimulation or mTORC1 inhibition with rap/KU (indicated by NS). C: Schematic showing multiple mTORC1 inputs into angiogenesis. As demonstrated in this study, mTORC1 regulates the translation of HIF-1α via both 4E-BP1/eIF4E and S6K1. Although S6K1 can also promote HIF-1α translation, VEGF-A does not appear to be directly affected by active S6K1. mTORC1 also controls the transcription of HIF-1α mRNA in a rapamycin sensitive fashion via STAT3 phosphorylation, indicating that mTORC1 mediates angiogenesis via three distinct mechanisms.

    Techniques Used: Kinase Assay, Purification, Incubation, SDS Page, Western Blot, Transfection, shRNA, Polymerase Chain Reaction, Inhibition

    eIF4E availability is a rate limiting factor in the synthesis of both HIF-1α and VEGF-A A B: Tsc2−/− MEFs were transfected with either pACATG-2 or mutant constructs of 4E-BP1 alongside the HIF-1α luciferase reporter. Lysates were analysed for HIF-1α induced luciferase activity (standardised to total protein levels). Graph indicates results from three independent experiments. eIF4E was immunoprecipitated from the remainder of the lysates cells using m 7 GTP beads and 4E-BP1 binding was assessed using western blotting. C: The TOS-mutant of 4E-BP1 was over-expressed in HEK293 cells cultured under hypoxia for the given time points. Lysates were analysed for total HIF-1α and VEGF-A levels using western blotting. D: HEK293 cells expressing empty vector/4E-BP1 mutant were cultured under hypoxia for 4 h, mRNA was extracted from the total lysates and Q-PCR was performed to determine HIF-1α mRNA levels (standardised to β-actin). * = p-value
    Figure Legend Snippet: eIF4E availability is a rate limiting factor in the synthesis of both HIF-1α and VEGF-A A B: Tsc2−/− MEFs were transfected with either pACATG-2 or mutant constructs of 4E-BP1 alongside the HIF-1α luciferase reporter. Lysates were analysed for HIF-1α induced luciferase activity (standardised to total protein levels). Graph indicates results from three independent experiments. eIF4E was immunoprecipitated from the remainder of the lysates cells using m 7 GTP beads and 4E-BP1 binding was assessed using western blotting. C: The TOS-mutant of 4E-BP1 was over-expressed in HEK293 cells cultured under hypoxia for the given time points. Lysates were analysed for total HIF-1α and VEGF-A levels using western blotting. D: HEK293 cells expressing empty vector/4E-BP1 mutant were cultured under hypoxia for 4 h, mRNA was extracted from the total lysates and Q-PCR was performed to determine HIF-1α mRNA levels (standardised to β-actin). * = p-value

    Techniques Used: Transfection, Mutagenesis, Construct, Luciferase, Activity Assay, Immunoprecipitation, Binding Assay, Western Blot, Cell Culture, Expressing, Plasmid Preparation, Polymerase Chain Reaction

    Rapamycin reduces HIF-1α and VEGF-A expression in renal cystadenoma cells from Tsc2+/− mice A: Paraffin-embedded kidney sections from five 12 month old Tsc2+/− mice were analysed by IHC. Thirty five cystadenomas were analysed for Tsc2 expression. Tsc2 expression was either undetectable or markedly reduced within these lesions. B: Tsc2+/− mice of 12 months old were treated with 10 mg/kg rapamycin (n=4) or vehicle (n=4) for two months. Paraffin-embedded kidney sections were stained using IHC with an antibody against phosphor-rpS6 (Ser235/236), HIF-1α or VEGF-A. Thirty renal lesions were scored for each treatment group. All lesions from mice treated with rapamycin showed a significant reduction in phospho-rpS6 and HIF-1α. VEGF-A was also reduced by rapamycin treatment although to a lesser extent.
    Figure Legend Snippet: Rapamycin reduces HIF-1α and VEGF-A expression in renal cystadenoma cells from Tsc2+/− mice A: Paraffin-embedded kidney sections from five 12 month old Tsc2+/− mice were analysed by IHC. Thirty five cystadenomas were analysed for Tsc2 expression. Tsc2 expression was either undetectable or markedly reduced within these lesions. B: Tsc2+/− mice of 12 months old were treated with 10 mg/kg rapamycin (n=4) or vehicle (n=4) for two months. Paraffin-embedded kidney sections were stained using IHC with an antibody against phosphor-rpS6 (Ser235/236), HIF-1α or VEGF-A. Thirty renal lesions were scored for each treatment group. All lesions from mice treated with rapamycin showed a significant reduction in phospho-rpS6 and HIF-1α. VEGF-A was also reduced by rapamycin treatment although to a lesser extent.

    Techniques Used: Expressing, Mouse Assay, Immunohistochemistry, Staining

    19) Product Images from "Acute Methylmercury Exposure and the Hypoxia-Inducible Factor-1α Signaling Pathway under Normoxic Conditions in the Rat Brain and Astrocytes in Vitro"

    Article Title: Acute Methylmercury Exposure and the Hypoxia-Inducible Factor-1α Signaling Pathway under Normoxic Conditions in the Rat Brain and Astrocytes in Vitro

    Journal: Environmental Health Perspectives

    doi: 10.1289/EHP5139

    Effects of pharmacologic and genetic manipulation of HIF- 1 α on cell proliferation. (A) Astrocytes were pretreated with CoCl 2 ( 200 μ M , 0.5 h ) and then treated with 10 μ M MeHg for 0.5 h . Western blotting was used to evaluate protein levels of HIF- 1 α , GLUT-1, EPO, and VEGF-A. (B) Effect of CoCl 2 pretreatment ( 200 μ M , 0.5 h ) on the cell proliferation was evaluated using an MTT assay. (C) Effect of 2-MeOE2 pretreatment ( 10 μ M , 0.5 h ) on the expression of HIF- 1 α , GLUT-1, EPO, and VEGF-A was detected by Western blotting. (D) Effect of 2-MeOE2 pretreatment ( 10 μ M , 0.5 h ) on the cell proliferation was evaluated using an MTT assay. For Western blotting analyses, representative blots are shown, and the intensities are presented as fold changes relative to the control group ( β -actin as the internal control). * p
    Figure Legend Snippet: Effects of pharmacologic and genetic manipulation of HIF- 1 α on cell proliferation. (A) Astrocytes were pretreated with CoCl 2 ( 200 μ M , 0.5 h ) and then treated with 10 μ M MeHg for 0.5 h . Western blotting was used to evaluate protein levels of HIF- 1 α , GLUT-1, EPO, and VEGF-A. (B) Effect of CoCl 2 pretreatment ( 200 μ M , 0.5 h ) on the cell proliferation was evaluated using an MTT assay. (C) Effect of 2-MeOE2 pretreatment ( 10 μ M , 0.5 h ) on the expression of HIF- 1 α , GLUT-1, EPO, and VEGF-A was detected by Western blotting. (D) Effect of 2-MeOE2 pretreatment ( 10 μ M , 0.5 h ) on the cell proliferation was evaluated using an MTT assay. For Western blotting analyses, representative blots are shown, and the intensities are presented as fold changes relative to the control group ( β -actin as the internal control). * p

    Techniques Used: Western Blot, MTT Assay, Expressing

    Effects of MeHg on the expression of HIF- 1 α – related proteins in astrocytes. (A) Western blotting for HIF- 1 α in astrocytes following MeHg (0, 1, 2.5, 5 or 10 μ M , 0.5 h ) exposure. (B) Western blotting for HIF- 1 β in astrocytes following MeHg (0, 1, 2.5, 5, or 10 μ M , 0.5 h ) exposure. (C) Effect of MeHg (0, 1, 2.5, 5, or 10 μ M , 0.5 h ) exposure on the expression of the downstream proteins of HIF- 1 α , including GLUT-1, EPO, and VEGF-A. β -actin was used as the internal control. Note: Data are presented as mean ± SD from three independent experiments ( n = 3 ). con, control (culture medium treatment without MeHg); EPO, erythropoietin; GLUT-1, glucose transporter 1; HIF- 1 α , Hypoxia-inducible factor- 1 α ; HIF- 1 β , Hypoxia-inducible factor- 1 β ; MeHg, methylmercury; VEGF-A, vascular endothelial growth factor A. * p
    Figure Legend Snippet: Effects of MeHg on the expression of HIF- 1 α – related proteins in astrocytes. (A) Western blotting for HIF- 1 α in astrocytes following MeHg (0, 1, 2.5, 5 or 10 μ M , 0.5 h ) exposure. (B) Western blotting for HIF- 1 β in astrocytes following MeHg (0, 1, 2.5, 5, or 10 μ M , 0.5 h ) exposure. (C) Effect of MeHg (0, 1, 2.5, 5, or 10 μ M , 0.5 h ) exposure on the expression of the downstream proteins of HIF- 1 α , including GLUT-1, EPO, and VEGF-A. β -actin was used as the internal control. Note: Data are presented as mean ± SD from three independent experiments ( n = 3 ). con, control (culture medium treatment without MeHg); EPO, erythropoietin; GLUT-1, glucose transporter 1; HIF- 1 α , Hypoxia-inducible factor- 1 α ; HIF- 1 β , Hypoxia-inducible factor- 1 β ; MeHg, methylmercury; VEGF-A, vascular endothelial growth factor A. * p

    Techniques Used: Expressing, Western Blot

    Protein expression of HIF- 1 α and downstream effectors following MeHg administration in in vivo rat models. Adult Sprague-Dawley rats were administered with MeHg (0, 2, 4, 6, 8, 10 mg / kg ) by intraperitoneal injection for 0.5 h . Whole brain lysates were analyzed by Western blotting for the indicated proteins. Note: Data are presented as mean ± SD ( n = 6 rats / group ). con, control (intraperitoneal injection of saline); EPO, erythropoietin; GLUT-1, glucose transporter 1; HIF- 1 α , Hypoxia-inducible factor- 1 α ; MeHg, methylmercury; VEGF-A, vascular endothelial growth factor A. * p
    Figure Legend Snippet: Protein expression of HIF- 1 α and downstream effectors following MeHg administration in in vivo rat models. Adult Sprague-Dawley rats were administered with MeHg (0, 2, 4, 6, 8, 10 mg / kg ) by intraperitoneal injection for 0.5 h . Whole brain lysates were analyzed by Western blotting for the indicated proteins. Note: Data are presented as mean ± SD ( n = 6 rats / group ). con, control (intraperitoneal injection of saline); EPO, erythropoietin; GLUT-1, glucose transporter 1; HIF- 1 α , Hypoxia-inducible factor- 1 α ; MeHg, methylmercury; VEGF-A, vascular endothelial growth factor A. * p

    Techniques Used: Expressing, In Vivo, Injection, Western Blot

    20) Product Images from "Paxillin regulates vascular endothelial growth factor A-induced in vitro angiogenesis of human umbilical vein endothelial cells"

    Article Title: Paxillin regulates vascular endothelial growth factor A-induced in vitro angiogenesis of human umbilical vein endothelial cells

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2014.2961

    Knockdown of paxillin inhibits the VEGF-A-induced migration of HUVECs. Cells were cultured on plates following either mock-transfection or transfection with scramble siRNA or siPXN-1, respectively. HUVECs (2×10 5 cells) were harvested and seeded into Transwell inserts. VEGF-A (20 ng/ml) was added to the lower well of a Boyden chamber. (A) Representative photomicrographs of the HUVECs in the lower well of the Boyden chamber stained with crystal violet. Magnification ×100. (B) Cell migration of the HUVECs. Cell migration was quantified by counting the number of migrated cells and expressed as a percentage of the cell migration in the control. Three independent experiments were performed. * P
    Figure Legend Snippet: Knockdown of paxillin inhibits the VEGF-A-induced migration of HUVECs. Cells were cultured on plates following either mock-transfection or transfection with scramble siRNA or siPXN-1, respectively. HUVECs (2×10 5 cells) were harvested and seeded into Transwell inserts. VEGF-A (20 ng/ml) was added to the lower well of a Boyden chamber. (A) Representative photomicrographs of the HUVECs in the lower well of the Boyden chamber stained with crystal violet. Magnification ×100. (B) Cell migration of the HUVECs. Cell migration was quantified by counting the number of migrated cells and expressed as a percentage of the cell migration in the control. Three independent experiments were performed. * P

    Techniques Used: Migration, Cell Culture, Transfection, Staining

    Knock down of paxillin inhibits the VEGF-A-induced proliferation of HUVECs. The HUVECs were cultured on plates following either mock-transfected or transfected with scramble siRNA or siPXN-1, respectively. VEGF-A (20 ng/ml) was added to the medium. (A) Representative photomicrographs of the HUVECs stained with EdU (red) and Hoechst 33342 (blue). Magnification ×200. (B) Percentage of EdU-positive cells. * P
    Figure Legend Snippet: Knock down of paxillin inhibits the VEGF-A-induced proliferation of HUVECs. The HUVECs were cultured on plates following either mock-transfected or transfected with scramble siRNA or siPXN-1, respectively. VEGF-A (20 ng/ml) was added to the medium. (A) Representative photomicrographs of the HUVECs stained with EdU (red) and Hoechst 33342 (blue). Magnification ×200. (B) Percentage of EdU-positive cells. * P

    Techniques Used: Cell Culture, Transfection, Staining

    Knockdown of paxillin inhibits the VEGF-A-induced adhesion of HUVECs. The HUVECs were seeded into 96-well plates following either mock-transfection or transfection with scramble siRNA or siPXN-1, respectively. VEGF-A (20 ng/ml) was added to the medium. (A) Representative photomicrographs of the HUVECs following siRNA and VEGF-A transfection. Magnification ×200. (B) OD at a wavelength of 490 nm of the HUVECs stained with MTT. Three independent experiments were performed. * P
    Figure Legend Snippet: Knockdown of paxillin inhibits the VEGF-A-induced adhesion of HUVECs. The HUVECs were seeded into 96-well plates following either mock-transfection or transfection with scramble siRNA or siPXN-1, respectively. VEGF-A (20 ng/ml) was added to the medium. (A) Representative photomicrographs of the HUVECs following siRNA and VEGF-A transfection. Magnification ×200. (B) OD at a wavelength of 490 nm of the HUVECs stained with MTT. Three independent experiments were performed. * P

    Techniques Used: Transfection, Staining, MTT Assay

    Tube formation of HUVECs is inhibited by siRNA-mediated paxillin knockdown. The HUVECs were seeded onto Matrigel 48 h after being either mock-transfected or transfected with scramble siRNA or siPXN-1, respectively. VEGF-A (20 ng/ml) was added to the medium. (A) Photomicrographs of the tubes; (B) Tube length was calculated using the Image-Pro Plus image processing system. Three independent experiments were performed. * P
    Figure Legend Snippet: Tube formation of HUVECs is inhibited by siRNA-mediated paxillin knockdown. The HUVECs were seeded onto Matrigel 48 h after being either mock-transfected or transfected with scramble siRNA or siPXN-1, respectively. VEGF-A (20 ng/ml) was added to the medium. (A) Photomicrographs of the tubes; (B) Tube length was calculated using the Image-Pro Plus image processing system. Three independent experiments were performed. * P

    Techniques Used: Transfection

    VEGF-A induces the phosphorylation of paxillin in HUVECs. The HUVECs were treated with 20 ng/ml VEGF-A for 0, 20, 40 and 60 min. Western blot analysis was performed to assess the phosphorylation of paxillin phosphorylated at PY118 and PY31 as well as total paxillin protein. Actin was used as a loading control. VEGF-A, vascular endothelial growth factor A; HUVECs, human umbilical vein endothelial cells. PY118, tyrosine 118; PY31, tyrosine31.
    Figure Legend Snippet: VEGF-A induces the phosphorylation of paxillin in HUVECs. The HUVECs were treated with 20 ng/ml VEGF-A for 0, 20, 40 and 60 min. Western blot analysis was performed to assess the phosphorylation of paxillin phosphorylated at PY118 and PY31 as well as total paxillin protein. Actin was used as a loading control. VEGF-A, vascular endothelial growth factor A; HUVECs, human umbilical vein endothelial cells. PY118, tyrosine 118; PY31, tyrosine31.

    Techniques Used: Western Blot

    21) Product Images from "Scutellaria barbata D. Don Inhibits Tumor Angiogenesis via Suppression of Hedgehog Pathway in a Mouse Model of Colorectal Cancer"

    Article Title: Scutellaria barbata D. Don Inhibits Tumor Angiogenesis via Suppression of Hedgehog Pathway in a Mouse Model of Colorectal Cancer

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms13089419

    Effect of EESB on the expression of VEGF-A and VEGFR2 in CRC xenograft mice. ( A ) The mRNA expression levels of Vascular endothelial growth factor-A (VEGF-A) and VEGF Receptor 2 (VEGFR2) were determined by RT-PCR. GAPDH was used as the internal control. The data of densitometric analysis were normalized to the mean mRNA expression of untreated control (100%); ( B ) Tumor tissues were processed for IHC staining for VEGF-A and VEGFR2. The photographs were representative images taken at a magnification of ×400. Quantification of IHC assay was represented as percentage of positively-stained cells. Data shown were averages with S.D. (error bars) from 10 individual mice in each group. * p
    Figure Legend Snippet: Effect of EESB on the expression of VEGF-A and VEGFR2 in CRC xenograft mice. ( A ) The mRNA expression levels of Vascular endothelial growth factor-A (VEGF-A) and VEGF Receptor 2 (VEGFR2) were determined by RT-PCR. GAPDH was used as the internal control. The data of densitometric analysis were normalized to the mean mRNA expression of untreated control (100%); ( B ) Tumor tissues were processed for IHC staining for VEGF-A and VEGFR2. The photographs were representative images taken at a magnification of ×400. Quantification of IHC assay was represented as percentage of positively-stained cells. Data shown were averages with S.D. (error bars) from 10 individual mice in each group. * p

    Techniques Used: Expressing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry, Staining

    22) Product Images from "Anti-cancer activity of Annexin V in murine melanoma model by suppressing tumor angiogenesis"

    Article Title: Anti-cancer activity of Annexin V in murine melanoma model by suppressing tumor angiogenesis

    Journal: Oncotarget

    doi: 10.18632/oncotarget.16645

    Annexin V downregulates VEGF expression at protein level, not at transcriptional level (A) Serum levels of VEGF in normal mice injected with 10.0 mg/kg Annexin V detected by ELISA (Mean ± SEM, n=8, **p
    Figure Legend Snippet: Annexin V downregulates VEGF expression at protein level, not at transcriptional level (A) Serum levels of VEGF in normal mice injected with 10.0 mg/kg Annexin V detected by ELISA (Mean ± SEM, n=8, **p

    Techniques Used: Expressing, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

    The negative correlation between Annexin V and VEGF expression in melanoma (A-B) Analysis of Annexin V and VEGF expression levels in normal melanocytes and most melanomas using Harlin melanoma dataset. The results showed a significant decrease of Annexin V levels and a remarkable rise of VEGF levels in most melanomas, compared to the normal group. (Mean ± SEM, n=8, *p
    Figure Legend Snippet: The negative correlation between Annexin V and VEGF expression in melanoma (A-B) Analysis of Annexin V and VEGF expression levels in normal melanocytes and most melanomas using Harlin melanoma dataset. The results showed a significant decrease of Annexin V levels and a remarkable rise of VEGF levels in most melanomas, compared to the normal group. (Mean ± SEM, n=8, *p

    Techniques Used: Expressing

    23) Product Images from "Essential Contribution of Macrophage Tie2 Signalling in a Murine Model of Laser-Induced Choroidal Neovascularization"

    Article Title: Essential Contribution of Macrophage Tie2 Signalling in a Murine Model of Laser-Induced Choroidal Neovascularization

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-66580-y

    ( a ) Quantitative reverse transcription polymerase chain reaction (RT-PCR) to assess mRNA expression of VEGF-A, bFGF and IL-10 with the total RNAs obtained from the control/TEM-KO mice choroids at different time points (0, 1, 3, 5, 7, and 14d). ( b ) AKT, p-AKT, ERK, p-ERK, VEGF-A and cleaved caspase-3 protein expression was detected using Western blot in the injured choroid plexus from the control/TEM-KO mice. ( c ) The mean grey value of the Western blot was determined and described as mean ± SEM. Cropped blots are displayed in the figure, and full-length gels and blots were included in the Supplementary information file. ( d ) In the flow cytometry detection, the apoptotic rate of the CD31 + cells of the mice choroid were tested at 0, 5, 7, and 14d after laser injury. N = 30 (control group); n = 30 (TEM-KO group). The left superior two images represent the viable cells and the CD31 + cells among the viable cells. * p
    Figure Legend Snippet: ( a ) Quantitative reverse transcription polymerase chain reaction (RT-PCR) to assess mRNA expression of VEGF-A, bFGF and IL-10 with the total RNAs obtained from the control/TEM-KO mice choroids at different time points (0, 1, 3, 5, 7, and 14d). ( b ) AKT, p-AKT, ERK, p-ERK, VEGF-A and cleaved caspase-3 protein expression was detected using Western blot in the injured choroid plexus from the control/TEM-KO mice. ( c ) The mean grey value of the Western blot was determined and described as mean ± SEM. Cropped blots are displayed in the figure, and full-length gels and blots were included in the Supplementary information file. ( d ) In the flow cytometry detection, the apoptotic rate of the CD31 + cells of the mice choroid were tested at 0, 5, 7, and 14d after laser injury. N = 30 (control group); n = 30 (TEM-KO group). The left superior two images represent the viable cells and the CD31 + cells among the viable cells. * p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Transmission Electron Microscopy, Mouse Assay, Western Blot, Flow Cytometry

    The effects of the TEM supernatant on the b.End3 cells. The b.End3 cells were incubated with DMEM, macrophage supernatant from the control mice (M Tie2+ ) and macrophage supernatant from the TEM-KO mice (M Tie2− ), respectively. ( a ) In the scratch wound assay, the b.End3 cells were treated with the macrophage supernatant for 12 h and 24 h. Statistical analysis was performed using one-way ANOVA. Scale bar: 100 μm. ( b ) In the transwell migration assay, the b.End3 cells in the different study groups were incubated for 24 h. Scale bar: 100 μm. ( c ) In the tube formation assay, the b.End3 cells in the different study groups were incubated for 6 h. Scale bar: 200 μm. ( d ) Representative results of p-AKT, AKT, p-ERK, ERK and VEGF-A are presented, as determined by Western blot, from three independent experiments. ( e ) In the annexin V/PI dual-staining flow cytometry detection, the apoptosis rate of the b.End3 cells was tested after 6, 12, and 24 h incubation with the macrophage supernatant. ( f ) Representative results, as determined by Western blot, from three independent experiments are presented. The relative cleaved caspase-3 protein expression of the b.End3 cells under different macrophage supernatants is presented. Cropped blots are displayed in the figure, and full-length gels and blots were included in Supplementary Information file. All values represent mean ± SEM. * p
    Figure Legend Snippet: The effects of the TEM supernatant on the b.End3 cells. The b.End3 cells were incubated with DMEM, macrophage supernatant from the control mice (M Tie2+ ) and macrophage supernatant from the TEM-KO mice (M Tie2− ), respectively. ( a ) In the scratch wound assay, the b.End3 cells were treated with the macrophage supernatant for 12 h and 24 h. Statistical analysis was performed using one-way ANOVA. Scale bar: 100 μm. ( b ) In the transwell migration assay, the b.End3 cells in the different study groups were incubated for 24 h. Scale bar: 100 μm. ( c ) In the tube formation assay, the b.End3 cells in the different study groups were incubated for 6 h. Scale bar: 200 μm. ( d ) Representative results of p-AKT, AKT, p-ERK, ERK and VEGF-A are presented, as determined by Western blot, from three independent experiments. ( e ) In the annexin V/PI dual-staining flow cytometry detection, the apoptosis rate of the b.End3 cells was tested after 6, 12, and 24 h incubation with the macrophage supernatant. ( f ) Representative results, as determined by Western blot, from three independent experiments are presented. The relative cleaved caspase-3 protein expression of the b.End3 cells under different macrophage supernatants is presented. Cropped blots are displayed in the figure, and full-length gels and blots were included in Supplementary Information file. All values represent mean ± SEM. * p

    Techniques Used: Transmission Electron Microscopy, Incubation, Mouse Assay, Scratch Wound Assay Assay, Transwell Migration Assay, Tube Formation Assay, Western Blot, Staining, Flow Cytometry, Expressing

    24) Product Images from "Annexin A12–26 Treatment Improves Skin Heterologous Transplantation by Modulating Inflammation and Angiogenesis Processes"

    Article Title: Annexin A12–26 Treatment Improves Skin Heterologous Transplantation by Modulating Inflammation and Angiogenesis Processes

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2018.01015

    AnxA1 2–26 increases angiogenesis in a dorsal chamber model in BALB/c mice. Mice were topically treated with Saline (10 μL), AnxA1 2–26 (1 mg/kg), and/or VEGF-A (10 ng/10 μL) in the dorsal skin. The treatments were administrated once per day, every 2 days, resulting three applications in each mouse. Representative images of the microcirculatory network of dorsal skin were obtained before (day 4) and after (day 9) treatments (A) . The images in the upper panel represent the stained normal tissue and in the lower panel, the same computational images obtained after inverting the colors are displayed (A) . The quantification of vessels is represented in B . The values express the mean ± SEM of five animals per group (ANOVA followed by Bonferroni’s Multiple Comparison Test). ∗ p
    Figure Legend Snippet: AnxA1 2–26 increases angiogenesis in a dorsal chamber model in BALB/c mice. Mice were topically treated with Saline (10 μL), AnxA1 2–26 (1 mg/kg), and/or VEGF-A (10 ng/10 μL) in the dorsal skin. The treatments were administrated once per day, every 2 days, resulting three applications in each mouse. Representative images of the microcirculatory network of dorsal skin were obtained before (day 4) and after (day 9) treatments (A) . The images in the upper panel represent the stained normal tissue and in the lower panel, the same computational images obtained after inverting the colors are displayed (A) . The quantification of vessels is represented in B . The values express the mean ± SEM of five animals per group (ANOVA followed by Bonferroni’s Multiple Comparison Test). ∗ p

    Techniques Used: Mouse Assay, Staining

    AnxA1 2–26 does not induce tube formation. HUVECs (2 × 10 4 cells/well) were incubated with PBS (control), AnxA1 2–26 (30 μM), and/or VEGF (50 ng/mL) for 6 h on Matrigel ® and the number of tube structures were quantified using an optical microscope (A,B) . PECAM-1 expression was evaluated by flow cytometry (C) . Scale bar = 10 μm. Results are expressed as the mean ± SEM of two independent experiments in triplicate (ANOVA followed by the Tukey’s multiple comparisons test). ∗ p
    Figure Legend Snippet: AnxA1 2–26 does not induce tube formation. HUVECs (2 × 10 4 cells/well) were incubated with PBS (control), AnxA1 2–26 (30 μM), and/or VEGF (50 ng/mL) for 6 h on Matrigel ® and the number of tube structures were quantified using an optical microscope (A,B) . PECAM-1 expression was evaluated by flow cytometry (C) . Scale bar = 10 μm. Results are expressed as the mean ± SEM of two independent experiments in triplicate (ANOVA followed by the Tukey’s multiple comparisons test). ∗ p

    Techniques Used: Incubation, Microscopy, Expressing, Flow Cytometry, Cytometry

    AnxA1 2–26 increases endothelial cell migration and actin polymerization. HUVECs (1 × 10 4 cells/well) were incubated with PBS (control), AnxA1 2–26 (30 μM), and/or VEGF-A (10 or 50 ng/mL) and cell proliferation was evaluated at 24, 48, and 72 h. Results are expressed as the mean ± SEM of cells of two independent experiments in triplicate (A) . HUVECs were incubated with different treatments for 48 h, later labeled with PI (50 μg/mL) and the cell cycle phases were evaluated (ANOVA followed by the Tukey’s multiple comparisons test) (B) . HUVEC migration was evaluated after 12 h of incubation with PBS (control), AnxA1 2–26 (1, 10, or 30 μM) and/or VEGF-A (50 ng/mL). Cell migration was monitored with images obtained before (0 h) and after (12 h) treatments (C,D) . HUVECs (1 × 10 4 cells/well) were incubated with different treatments for 2 h and later incubated with rhodamine-phalloidin to evaluate actin polymerization. The intensity of fluorescence was monitored using a fluorescent plate reader (E) and by confocal microscopy (F) . Scale bar = 10 μm. Results are expressed as the mean ± SEM of cells of two independent experiments in triplicate (ANOVA followed by the Bonferroni’s test). ∗ p
    Figure Legend Snippet: AnxA1 2–26 increases endothelial cell migration and actin polymerization. HUVECs (1 × 10 4 cells/well) were incubated with PBS (control), AnxA1 2–26 (30 μM), and/or VEGF-A (10 or 50 ng/mL) and cell proliferation was evaluated at 24, 48, and 72 h. Results are expressed as the mean ± SEM of cells of two independent experiments in triplicate (A) . HUVECs were incubated with different treatments for 48 h, later labeled with PI (50 μg/mL) and the cell cycle phases were evaluated (ANOVA followed by the Tukey’s multiple comparisons test) (B) . HUVEC migration was evaluated after 12 h of incubation with PBS (control), AnxA1 2–26 (1, 10, or 30 μM) and/or VEGF-A (50 ng/mL). Cell migration was monitored with images obtained before (0 h) and after (12 h) treatments (C,D) . HUVECs (1 × 10 4 cells/well) were incubated with different treatments for 2 h and later incubated with rhodamine-phalloidin to evaluate actin polymerization. The intensity of fluorescence was monitored using a fluorescent plate reader (E) and by confocal microscopy (F) . Scale bar = 10 μm. Results are expressed as the mean ± SEM of cells of two independent experiments in triplicate (ANOVA followed by the Bonferroni’s test). ∗ p

    Techniques Used: Migration, Incubation, Labeling, Fluorescence, Confocal Microscopy

    VEGF-A and AnxA1 2–26 decreases FPR1 expression on HUVECs. FPR1 was detected in the plasma membrane (arrows) and cytoplasm of cells under all experimental conditions (A) . Negative control (B) . Density of FPR1 immunogold particles in HUVECs (C) . Scale bar = 0.5 μm. Data are mean ± SEM of distinct cells analyzed ( n = 12–20/group) for each condition (ANOVA followed by the Bonferroni’s test). ∗ p
    Figure Legend Snippet: VEGF-A and AnxA1 2–26 decreases FPR1 expression on HUVECs. FPR1 was detected in the plasma membrane (arrows) and cytoplasm of cells under all experimental conditions (A) . Negative control (B) . Density of FPR1 immunogold particles in HUVECs (C) . Scale bar = 0.5 μm. Data are mean ± SEM of distinct cells analyzed ( n = 12–20/group) for each condition (ANOVA followed by the Bonferroni’s test). ∗ p

    Techniques Used: Expressing, Negative Control

    AnxA1 2–26 treatment improves the heterologous transplantation and induces angiogenesis. Histopathological analyses of skin transplanted fragments without (PBS) and with AnxA1 2–26 peptide treatment after 3 (A) , 10 (B) , 15 (C) , and 60 (D) days post-surgery. Cell infiltration (E) , TGF-β (F) , α-SMA (G) , FGF-b (H) , and VEGF-A (I) gene expression and VEGF-A protein (J) in the transplanted tissue. Host tissue (TH), transplanted scaffold (Sc), vessels ( ∗ ), fibroblasts (arrows). The inserts show high magnifications of the fibroblasts. The values express the mean ± SEM of five animals per group (ANOVA followed by the Bonferroni’s test). ∗ p
    Figure Legend Snippet: AnxA1 2–26 treatment improves the heterologous transplantation and induces angiogenesis. Histopathological analyses of skin transplanted fragments without (PBS) and with AnxA1 2–26 peptide treatment after 3 (A) , 10 (B) , 15 (C) , and 60 (D) days post-surgery. Cell infiltration (E) , TGF-β (F) , α-SMA (G) , FGF-b (H) , and VEGF-A (I) gene expression and VEGF-A protein (J) in the transplanted tissue. Host tissue (TH), transplanted scaffold (Sc), vessels ( ∗ ), fibroblasts (arrows). The inserts show high magnifications of the fibroblasts. The values express the mean ± SEM of five animals per group (ANOVA followed by the Bonferroni’s test). ∗ p

    Techniques Used: Transplantation Assay, Expressing

    25) Product Images from "Ellagic Acid Inhibits Bladder Cancer Invasiveness and In Vivo Tumor Growth"

    Article Title: Ellagic Acid Inhibits Bladder Cancer Invasiveness and In Vivo Tumor Growth

    Journal: Nutrients

    doi: 10.3390/nu8110744

    Treatment with EA inhibits T24 bladder cancer cell invasion in response to VEGF-A, but not to EGF. Invasion of T24 cells (2 × 10 5 cells/chamber, 2 h incubation), non-stimulated (CTR) or exposed to EA IC 25 (10 µM) in response to VEGF-A (50 ng/mL) or to EGF (50 ng/mL), was tested in Boyden chambers containing matrigel coated filters. Invading cells were counted in six random microscopic fields for each experimental condition. The histogram represents the arithmetic mean values of migrated cells/microscopic field ± SD of three independent determinations. Results of the statistical analysis performed by one-way ANOVA, followed by Bonferroni’s post-test for multiple comparison, were as follows: VEGF-A vs. CTR or EA, p
    Figure Legend Snippet: Treatment with EA inhibits T24 bladder cancer cell invasion in response to VEGF-A, but not to EGF. Invasion of T24 cells (2 × 10 5 cells/chamber, 2 h incubation), non-stimulated (CTR) or exposed to EA IC 25 (10 µM) in response to VEGF-A (50 ng/mL) or to EGF (50 ng/mL), was tested in Boyden chambers containing matrigel coated filters. Invading cells were counted in six random microscopic fields for each experimental condition. The histogram represents the arithmetic mean values of migrated cells/microscopic field ± SD of three independent determinations. Results of the statistical analysis performed by one-way ANOVA, followed by Bonferroni’s post-test for multiple comparison, were as follows: VEGF-A vs. CTR or EA, p

    Techniques Used: Incubation

    Treatment with EA inhibits migration of UM-UC-3 cells in response to VEGF-A but not to EGF. Migration of UM-UC-3 cells (2 × 10 5 cells/chamber, 18 h incubation), non-stimulated (CTR) or exposed to EA IC 25 (20 µM) in response to VEGF-A or EGF (50 ng/mL) was tested in Boyden chambers containing gelatin coated filters. Migrating cells were counted in six random microscopic fields for each experimental condition. The histogram represents the arithmetic mean values of migrated cells/microscopic field ± SD of three independent determinations. Results of the statistical analysis using one-way ANOVA, followed by Bonferroni’s post-test, were as follows: VEGF-A vs. CTR or EA, p
    Figure Legend Snippet: Treatment with EA inhibits migration of UM-UC-3 cells in response to VEGF-A but not to EGF. Migration of UM-UC-3 cells (2 × 10 5 cells/chamber, 18 h incubation), non-stimulated (CTR) or exposed to EA IC 25 (20 µM) in response to VEGF-A or EGF (50 ng/mL) was tested in Boyden chambers containing gelatin coated filters. Migrating cells were counted in six random microscopic fields for each experimental condition. The histogram represents the arithmetic mean values of migrated cells/microscopic field ± SD of three independent determinations. Results of the statistical analysis using one-way ANOVA, followed by Bonferroni’s post-test, were as follows: VEGF-A vs. CTR or EA, p

    Techniques Used: Migration, Incubation

    Inhibitory effect of EA on UM-UC-3 cell invasion in response to VEGF-A. Matrigel invasion assay. Invasion of UM-UC-3 cells (2 × 10 5 cells/chamber, 4 h incubation), non-stimulated (CTR) or exposed to EA IC 25 (20 µM) in response to VEGF-A (50 ng/mL) was tested in Boyden chambers containing matrigel coated filters ( A , B ) or by spheroid invasion assay ( C , D ). For matrigel invasion test, invading cells were counted in six random microscopic fields for each experimental condition. Histogram represents the arithmetic mean values of migrated cells/microscopic field ± SD of three independent determinations. Results of the statistical analysis using one-way ANOVA, followed by Bonferroni’s post-test, were as follows: VEGF-A vs. CTR or EA, p
    Figure Legend Snippet: Inhibitory effect of EA on UM-UC-3 cell invasion in response to VEGF-A. Matrigel invasion assay. Invasion of UM-UC-3 cells (2 × 10 5 cells/chamber, 4 h incubation), non-stimulated (CTR) or exposed to EA IC 25 (20 µM) in response to VEGF-A (50 ng/mL) was tested in Boyden chambers containing matrigel coated filters ( A , B ) or by spheroid invasion assay ( C , D ). For matrigel invasion test, invading cells were counted in six random microscopic fields for each experimental condition. Histogram represents the arithmetic mean values of migrated cells/microscopic field ± SD of three independent determinations. Results of the statistical analysis using one-way ANOVA, followed by Bonferroni’s post-test, were as follows: VEGF-A vs. CTR or EA, p

    Techniques Used: Invasion Assay, Incubation

    Treatment with EA reduces VEGFR-2 expression. ( A ) VEGF-A levels released in tumor cell culture supernatants. Quantification of the amount of VEGF-A in the concentrated supernatants of bladder cancer cell lines was performed using Maxisorp Nunc immunoplates coated with goat anti-VEGF-A IgGs. Results are the mean (± SD) of three independent determinations; ( B ) Immunoblot analysis of VEGFR-2. Western blot analysis of the levels of VEGFR-2 expressed in control (CTR) and in bladder cancer cell lines, treated with a vehicle (CTR) or exposed to EA for 24 h, at concentrations in the range of IC 50 values for each cell line (i.e., 20 µM, T24; 40 µM, UM-UC-3; 27 µM 5637; 60 µM HT-1376). HUVEC were loaded as a positive control and β-actin as a loading control; ( C ) Densitometric analysis. The relative levels of VEGFR-2 were calculated by densitometric analysis and normalized using β-actin expression in each sample. The histogram represents the ratios between the optical densities (O.D.) of VEGFR-2 in CTR or EA treated groups and β-actin. Results are the mean (±SD) of three independent experiments. Student’s t -test analysis: EA vs. CTR, p
    Figure Legend Snippet: Treatment with EA reduces VEGFR-2 expression. ( A ) VEGF-A levels released in tumor cell culture supernatants. Quantification of the amount of VEGF-A in the concentrated supernatants of bladder cancer cell lines was performed using Maxisorp Nunc immunoplates coated with goat anti-VEGF-A IgGs. Results are the mean (± SD) of three independent determinations; ( B ) Immunoblot analysis of VEGFR-2. Western blot analysis of the levels of VEGFR-2 expressed in control (CTR) and in bladder cancer cell lines, treated with a vehicle (CTR) or exposed to EA for 24 h, at concentrations in the range of IC 50 values for each cell line (i.e., 20 µM, T24; 40 µM, UM-UC-3; 27 µM 5637; 60 µM HT-1376). HUVEC were loaded as a positive control and β-actin as a loading control; ( C ) Densitometric analysis. The relative levels of VEGFR-2 were calculated by densitometric analysis and normalized using β-actin expression in each sample. The histogram represents the ratios between the optical densities (O.D.) of VEGFR-2 in CTR or EA treated groups and β-actin. Results are the mean (±SD) of three independent experiments. Student’s t -test analysis: EA vs. CTR, p

    Techniques Used: Expressing, Cell Culture, Western Blot, Positive Control

    Inhibitory effect of EA on 5637 and HT-1376 cell invasion in response to VEGF-A. Invasion of 5637 ( A , C ) and HT-1376 ( B , D ) cells (2 × 10 5 cells/chamber, 18 h incubation), non-stimulated (CTR) or exposed to EA IC 25 of each cell line (13.5 µM for 5637 cells and 30 µM for HT-1376) in response to VEGF-A (50 ng/mL) was tested in Boyden chambers containing matrigel coated filters. Invading cells were counted in six random microscopic fields for each experimental condition. Histograms represent the arithmetic mean values of migrated cells/microscopic field ± SD of three independent determinations. Results of the statistical analysis using one-way ANOVA, followed by Bonferroni’s post-test, were as follows for both cell lines: VEGF-A vs. CTR or EA, p
    Figure Legend Snippet: Inhibitory effect of EA on 5637 and HT-1376 cell invasion in response to VEGF-A. Invasion of 5637 ( A , C ) and HT-1376 ( B , D ) cells (2 × 10 5 cells/chamber, 18 h incubation), non-stimulated (CTR) or exposed to EA IC 25 of each cell line (13.5 µM for 5637 cells and 30 µM for HT-1376) in response to VEGF-A (50 ng/mL) was tested in Boyden chambers containing matrigel coated filters. Invading cells were counted in six random microscopic fields for each experimental condition. Histograms represent the arithmetic mean values of migrated cells/microscopic field ± SD of three independent determinations. Results of the statistical analysis using one-way ANOVA, followed by Bonferroni’s post-test, were as follows for both cell lines: VEGF-A vs. CTR or EA, p

    Techniques Used: Incubation

    26) Product Images from "Ulinastatin alleviates traumatic brain injury by reducing endothelin-1"

    Article Title: Ulinastatin alleviates traumatic brain injury by reducing endothelin-1

    Journal: Translational Neuroscience

    doi: 10.1515/tnsci-2021-0001

    UTI decreases expression of inflammatory mediators in TBI rats. (a–d) Representative images of IHC staining of VEGF and MMP-9 on resected brain tissues of the different experimental groups ( n = 3, each group). Insets in (a–d) show images obtained under higher magnification. Scale bar = 30 µm; (d) representative images of immunoblots showing expression of VEGF and MMP-9 in the indicated experimental groups. β-Actin was used as a loading control; (e) quantification of relative expression of VEGF and MMP-9 in the indicated experimental groups. Densitometry analysis of blots shown in (d) was done using Image J and data were normalized to expression of β-actin. Error bars are standard deviation; * p
    Figure Legend Snippet: UTI decreases expression of inflammatory mediators in TBI rats. (a–d) Representative images of IHC staining of VEGF and MMP-9 on resected brain tissues of the different experimental groups ( n = 3, each group). Insets in (a–d) show images obtained under higher magnification. Scale bar = 30 µm; (d) representative images of immunoblots showing expression of VEGF and MMP-9 in the indicated experimental groups. β-Actin was used as a loading control; (e) quantification of relative expression of VEGF and MMP-9 in the indicated experimental groups. Densitometry analysis of blots shown in (d) was done using Image J and data were normalized to expression of β-actin. Error bars are standard deviation; * p

    Techniques Used: Expressing, Immunohistochemistry, Staining, Western Blot, Standard Deviation

    27) Product Images from "VEGF/VEGFR-2 upregulates EZH2 expression in lung adenocarcinoma cells and EZH2 depletion enhances the response to platinum-based and VEGFR-2–targeted therapy"

    Article Title: VEGF/VEGFR-2 upregulates EZH2 expression in lung adenocarcinoma cells and EZH2 depletion enhances the response to platinum-based and VEGFR-2–targeted therapy

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    doi: 10.1158/1078-0432.CCR-13-1916

    Knockdown of VEGFR-2 expression by treatment with siRNA decreased the expression of EZH2 and H3K27me3 in lung adenocarcinoma cell lines stimulated by VEGF. A, and B, Western blots of VEGFR-2 ( siKDR ), EZH2, H3K27me3, and H3 expression in the lung adenocarcinoma
    Figure Legend Snippet: Knockdown of VEGFR-2 expression by treatment with siRNA decreased the expression of EZH2 and H3K27me3 in lung adenocarcinoma cell lines stimulated by VEGF. A, and B, Western blots of VEGFR-2 ( siKDR ), EZH2, H3K27me3, and H3 expression in the lung adenocarcinoma

    Techniques Used: Expressing, Western Blot

    VEGF stimulation leads to significantly increased expression of EZH2 and increased methylation of H3K27 in lung adenocarcinoma cell lines overexpressing VEGFR-2. A, Western blot of EZH2, H3K27me3 and VEGFR-2 expression in lung adenocarcinoma cell lines
    Figure Legend Snippet: VEGF stimulation leads to significantly increased expression of EZH2 and increased methylation of H3K27 in lung adenocarcinoma cell lines overexpressing VEGFR-2. A, Western blot of EZH2, H3K27me3 and VEGFR-2 expression in lung adenocarcinoma cell lines

    Techniques Used: Expressing, Methylation, Western Blot

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    Incubation:

    Article Title: Hepatoma-derived growth factor and its role in keloid pathogenesis
    Article Snippet: In total, 50 μg of whole-cell extract was separated by 14% or 8% SDS-PAGE under reducing conditions and electroblotted onto a nitrocellulose membrane. .. The blots were incubated with test antibodies, including mouse and rabbit anti-HDGF, mouse anti-PCNA (Santa Cruz, Biotechnology, Inc., CA, USA), mouse anti-vascular endothelial growth factor (VEGF), rabbit anti-p44/p42 mitogen-activated protein kinase (MAPK) and mouse anti-phospho p44/p42 MAPK (Cell Signaling Technology, Inc., MA, USA). .. The blots were visualized with a chemiluminescence-based photoblot system (Amersham Biosciences, Buckinghamshire, UK).

    Article Title: Anti-cancer activity of Annexin V in murine melanoma model by suppressing tumor angiogenesis
    Article Snippet: Supernatant was separated using 12% SDS-PAGE andtransferred to PVDF membranes (Millipore, Bedford, US). .. After that, the membranes were blocked in non-fat 5% milk and then incubated with primary antibodies aganst VEGF-A (Cell signaling) and α-Tublin (Abgent), followed by suitablesecondary antibody conjugated with horseradish peroxidase. .. Reactive bandswere detected with an ECL Western Blot Detection Kit (CST) and visualized with a Tanon Imager program (Tanon, China).

    Expressing:

    Article Title: Transcriptional factor OCT4 promotes esophageal cancer metastasis by inducing epithelial-mesenchymal transition through VEGF-C/VEGFR-3 signaling pathway
    Article Snippet: The harvested cells were used to extract total cellular protein according to the manufacturer's instructions of the protein extraction reagent kit (Pierce Biotechnology, Inc., Rockford, IL, USA). .. The expression levels of proteins were detected by western blotting using the primary antibodies, including the mouse anti-OCT4 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); the mouse anti-E-cadherin, mouse anti-N-cadherin, mouse anti-Vimentin, rabbit anti-VEGF-C (Cell Signaling Technology, Danvers, MA, USA); the mouse anti-VEGFR-1, mouse anti-VEGFR-2, mouse anti-VEGFR-3, rabbit anti-phospho-VEGFR-1, rabbit anti-phospho-VEGFR-2, rabbit anti-phospho-VEGFR-3 (Cell Applications Inc., CA, USA). .. Chromatin immunoprecipitation (ChIP) assay The shRNA vector-transfected Eca109 cells were seeded in 6-well plates at 1 × 106 cells/well and subjected to prepare the chromatin samples after incubated with 1% formaldehyde for 10 min at room temperature.

    Western Blot:

    Article Title: Transcriptional factor OCT4 promotes esophageal cancer metastasis by inducing epithelial-mesenchymal transition through VEGF-C/VEGFR-3 signaling pathway
    Article Snippet: The harvested cells were used to extract total cellular protein according to the manufacturer's instructions of the protein extraction reagent kit (Pierce Biotechnology, Inc., Rockford, IL, USA). .. The expression levels of proteins were detected by western blotting using the primary antibodies, including the mouse anti-OCT4 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); the mouse anti-E-cadherin, mouse anti-N-cadherin, mouse anti-Vimentin, rabbit anti-VEGF-C (Cell Signaling Technology, Danvers, MA, USA); the mouse anti-VEGFR-1, mouse anti-VEGFR-2, mouse anti-VEGFR-3, rabbit anti-phospho-VEGFR-1, rabbit anti-phospho-VEGFR-2, rabbit anti-phospho-VEGFR-3 (Cell Applications Inc., CA, USA). .. Chromatin immunoprecipitation (ChIP) assay The shRNA vector-transfected Eca109 cells were seeded in 6-well plates at 1 × 106 cells/well and subjected to prepare the chromatin samples after incubated with 1% formaldehyde for 10 min at room temperature.

    Immunohistochemistry:

    Article Title: Protective effects of sodium tanshinone IIA sulfonate on cardiac function after myocardial infarction in mice
    Article Snippet: We used average optical density (AOD) to quantify the expression level. .. Primary antibodies for immunohistochemistry were as follows: TGF-β, TNF-α, IL-1β (Abcam, USA), Bcl-2, Bax, α-SMA, CD31, VEGF (Cell Signaling Technology, USA). .. Total protein in vivo was obtained from left ventricular myocardial tissues.

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    Cell Signaling Technology Inc anti vegfr1
    miR-374 directly targets <t>VEGFR1</t> and PKG-1 (A, C). Sequence alignments of 3′-UTRs of mammalian VEGFR1 and PKG-1. miR-374 target region of VEGFR1 and PKG-1 3′-UTRs were well conserved. The dual luciferase reporter vector (pmiR-GLO) harboring the VEGFR1 or PKG-1 3′-UTR was cotransfected with NC or miR-374 (20 nM each), into HEK293 cells. The relative firefly luciferase activity was measured and normalized to that of Renilla luciferase. miR-139 did not have a specific seed region for the 3′-UTRs of VEGFR1 and PKG-1, and was therefore used as another negative control. (B, D) qRT-PCR analysis measuring the expression of VEGFR1 and PKG-1 mRNA in NRVMs transfected with either NC or miR-374 . (E, F) Western blot analysis measuring VEGFR1 and PKG-1 protein expression in NC or miR-374 -transfected NRVMs. The expression levels of the target proteins were estimated by measuring band densities using NIH ImageJ software. GAPDH was used as a loading control. Data are shown as fold changes ± SDs versus the control group (n = 3). Significance was measured via two-way ANOVA. *P
    Anti Vegfr1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc vegfr 2
    NCTD inhibits the expression of VEGF-A, VEGF-C, VEGF-D, <t>VEGFR-2</t> and VEGFR-3 proteins/mRNAs of the in-situ colonic xenografts in vivo. a Western-blotting: the expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 proteins in NCTD, Sorafenib, or NCTD + Sorafenib group was significantly downregulated as compared to control group (* P
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    Reversal effect of cholesterol on the inhibition of <t>VEGFR2</t> and mTOR activities by SERM. (A) and (B) HUVEC were treated with tamoxifen (TMX) or toremifene (TRM) with or without cholesterol (5 μg/ml)/cyclodextrin (0.1%) complex (Chol/CD) for 24
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    miR-374 directly targets VEGFR1 and PKG-1 (A, C). Sequence alignments of 3′-UTRs of mammalian VEGFR1 and PKG-1. miR-374 target region of VEGFR1 and PKG-1 3′-UTRs were well conserved. The dual luciferase reporter vector (pmiR-GLO) harboring the VEGFR1 or PKG-1 3′-UTR was cotransfected with NC or miR-374 (20 nM each), into HEK293 cells. The relative firefly luciferase activity was measured and normalized to that of Renilla luciferase. miR-139 did not have a specific seed region for the 3′-UTRs of VEGFR1 and PKG-1, and was therefore used as another negative control. (B, D) qRT-PCR analysis measuring the expression of VEGFR1 and PKG-1 mRNA in NRVMs transfected with either NC or miR-374 . (E, F) Western blot analysis measuring VEGFR1 and PKG-1 protein expression in NC or miR-374 -transfected NRVMs. The expression levels of the target proteins were estimated by measuring band densities using NIH ImageJ software. GAPDH was used as a loading control. Data are shown as fold changes ± SDs versus the control group (n = 3). Significance was measured via two-way ANOVA. *P

    Journal: BMB Reports

    Article Title: miR-374 promotes myocardial hypertrophy by negatively regulating vascular endothelial growth factor receptor-1 signaling

    doi: 10.5483/BMBRep.2017.50.4.165

    Figure Lengend Snippet: miR-374 directly targets VEGFR1 and PKG-1 (A, C). Sequence alignments of 3′-UTRs of mammalian VEGFR1 and PKG-1. miR-374 target region of VEGFR1 and PKG-1 3′-UTRs were well conserved. The dual luciferase reporter vector (pmiR-GLO) harboring the VEGFR1 or PKG-1 3′-UTR was cotransfected with NC or miR-374 (20 nM each), into HEK293 cells. The relative firefly luciferase activity was measured and normalized to that of Renilla luciferase. miR-139 did not have a specific seed region for the 3′-UTRs of VEGFR1 and PKG-1, and was therefore used as another negative control. (B, D) qRT-PCR analysis measuring the expression of VEGFR1 and PKG-1 mRNA in NRVMs transfected with either NC or miR-374 . (E, F) Western blot analysis measuring VEGFR1 and PKG-1 protein expression in NC or miR-374 -transfected NRVMs. The expression levels of the target proteins were estimated by measuring band densities using NIH ImageJ software. GAPDH was used as a loading control. Data are shown as fold changes ± SDs versus the control group (n = 3). Significance was measured via two-way ANOVA. *P

    Article Snippet: Membranes were then incubated overnight at 4°C with the following primary antibodies: anti-VEGFR1, anti-PKG-1, anti-phospho (p)-CaMKII (Thr286) (Cell Signaling Technology, MA, USA), anti-p-NFATc3, anti-p-NFATc4, anti-α-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

    Techniques: Sequencing, Luciferase, Plasmid Preparation, Activity Assay, Negative Control, Quantitative RT-PCR, Expressing, Transfection, Western Blot, Software

    Inhibition of VEGFR1 by siRNA induced cardiomyocyte hypertrophy. (A) Representative photographs of NRVMs transfected with either 50 nM si-NC or 50 nM si-VEGFR1 in the presence or absence of ET-1. Sarcomeric organization of the cardiomyocytes was visualized by staining with an anti-α-actinin antibody. (B) Cell surface areas were measured by using NIH ImageJ software (n = 100 cells). Scale bar, 100 μm. Significance was measured by two-way ANOVA. *P

    Journal: BMB Reports

    Article Title: miR-374 promotes myocardial hypertrophy by negatively regulating vascular endothelial growth factor receptor-1 signaling

    doi: 10.5483/BMBRep.2017.50.4.165

    Figure Lengend Snippet: Inhibition of VEGFR1 by siRNA induced cardiomyocyte hypertrophy. (A) Representative photographs of NRVMs transfected with either 50 nM si-NC or 50 nM si-VEGFR1 in the presence or absence of ET-1. Sarcomeric organization of the cardiomyocytes was visualized by staining with an anti-α-actinin antibody. (B) Cell surface areas were measured by using NIH ImageJ software (n = 100 cells). Scale bar, 100 μm. Significance was measured by two-way ANOVA. *P

    Article Snippet: Membranes were then incubated overnight at 4°C with the following primary antibodies: anti-VEGFR1, anti-PKG-1, anti-phospho (p)-CaMKII (Thr286) (Cell Signaling Technology, MA, USA), anti-p-NFATc3, anti-p-NFATc4, anti-α-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

    Techniques: Inhibition, Transfection, Staining, Software

    NCTD inhibits the expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 proteins/mRNAs of the in-situ colonic xenografts in vivo. a Western-blotting: the expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 proteins in NCTD, Sorafenib, or NCTD + Sorafenib group was significantly downregulated as compared to control group (* P

    Journal: BMC Cancer

    Article Title: A potential small-molecule synthetic antilymphangiogenic agent norcantharidin inhibits tumor growth and lymphangiogenesis of human colonic adenocarcinomas through blocking VEGF-A,-C,-D/VEGFR-2,-3 “multi-points priming” mechanisms in vitro and in vivo

    doi: 10.1186/s12885-015-1521-5

    Figure Lengend Snippet: NCTD inhibits the expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 proteins/mRNAs of the in-situ colonic xenografts in vivo. a Western-blotting: the expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 proteins in NCTD, Sorafenib, or NCTD + Sorafenib group was significantly downregulated as compared to control group (* P

    Article Snippet: It was shown that HT-29 cells may promote VEGF-C, VEGF-D and VEGFR-3 secreted from tumor and/or stromal cells or indirectly promote VEGF-A and VEGFR-2 secreted from tumor and/or stromal cells, so accelerate the lymphatic tube formation of HDLECs, and the lymphangiogenesis and tumor growth of the in-situ colonic xenografts; NCTD or in combination with mF4-31C1 or Sorafenib markedly downregulated the expression of VEGF-C, VEGF-D and VEGFR-3 other than VEGF-A and VEGFR-2 proteins/mRNAs of the co-culture system in vitro and the in-situ colonic xenografts in vivo .

    Techniques: Expressing, In Situ, In Vivo, Western Blot

    The expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 proteins/mRNAs of HCACCs and the co-culture system and the effect of NCTD on expression of these proteins/ mRNAs by western blotting ( a and b ) and fluorescent quantitative RT-PCR ( c and d ) in vitro . a and b Protein expression by western blotting: the expression of VEGF-C, VEGF-D and VEGFR-3 proteins of the co-culture system was higher than those of alone HCACC culture (* P = 0.001), but there was no difference on VEGF-A and VEGFR-2 expression between alone HCACC culture and the co-culture system. After treatment with NCTD, mF4-31C1 or NCTD + mF4-31C1, the expression of VEGF-C, VEGF-D and VEGFR-3 proteins of the co-culture system was downregulated significantly as compared to control group (all * P

    Journal: BMC Cancer

    Article Title: A potential small-molecule synthetic antilymphangiogenic agent norcantharidin inhibits tumor growth and lymphangiogenesis of human colonic adenocarcinomas through blocking VEGF-A,-C,-D/VEGFR-2,-3 “multi-points priming” mechanisms in vitro and in vivo

    doi: 10.1186/s12885-015-1521-5

    Figure Lengend Snippet: The expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 proteins/mRNAs of HCACCs and the co-culture system and the effect of NCTD on expression of these proteins/ mRNAs by western blotting ( a and b ) and fluorescent quantitative RT-PCR ( c and d ) in vitro . a and b Protein expression by western blotting: the expression of VEGF-C, VEGF-D and VEGFR-3 proteins of the co-culture system was higher than those of alone HCACC culture (* P = 0.001), but there was no difference on VEGF-A and VEGFR-2 expression between alone HCACC culture and the co-culture system. After treatment with NCTD, mF4-31C1 or NCTD + mF4-31C1, the expression of VEGF-C, VEGF-D and VEGFR-3 proteins of the co-culture system was downregulated significantly as compared to control group (all * P

    Article Snippet: It was shown that HT-29 cells may promote VEGF-C, VEGF-D and VEGFR-3 secreted from tumor and/or stromal cells or indirectly promote VEGF-A and VEGFR-2 secreted from tumor and/or stromal cells, so accelerate the lymphatic tube formation of HDLECs, and the lymphangiogenesis and tumor growth of the in-situ colonic xenografts; NCTD or in combination with mF4-31C1 or Sorafenib markedly downregulated the expression of VEGF-C, VEGF-D and VEGFR-3 other than VEGF-A and VEGFR-2 proteins/mRNAs of the co-culture system in vitro and the in-situ colonic xenografts in vivo .

    Techniques: Expressing, Co-Culture Assay, Western Blot, Quantitative RT-PCR, In Vitro

    The expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 protein products of HCACCs and the co-culture system of each group and the effect of NCTD on expression of these protein products in vitro (S-P staining, magnification × 200). a The expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 protein products of HCACCs and the co-culture system of each group. The expression of VEGF-C, VEGF-D and VEGFR-3 protein products (brown staining in cytoplasm) of the co-culture system was higher than those of alone HCACC culture (* P = 0.001); but there is no difference on the expression of VEGF-A and VEGFR-2 protein products between alone HCACC culture and the co-culture system. b The inhibitory effect of NCTD on expression of these protein products of the co-culture system. The expression of VEGF-C, VEGF-D and VEGFR-3 protein products in NCTD, mF4-31C1 or NCTD + mF4-31C1 group was downregulated significantly as compared to control group (* P

    Journal: BMC Cancer

    Article Title: A potential small-molecule synthetic antilymphangiogenic agent norcantharidin inhibits tumor growth and lymphangiogenesis of human colonic adenocarcinomas through blocking VEGF-A,-C,-D/VEGFR-2,-3 “multi-points priming” mechanisms in vitro and in vivo

    doi: 10.1186/s12885-015-1521-5

    Figure Lengend Snippet: The expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 protein products of HCACCs and the co-culture system of each group and the effect of NCTD on expression of these protein products in vitro (S-P staining, magnification × 200). a The expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 protein products of HCACCs and the co-culture system of each group. The expression of VEGF-C, VEGF-D and VEGFR-3 protein products (brown staining in cytoplasm) of the co-culture system was higher than those of alone HCACC culture (* P = 0.001); but there is no difference on the expression of VEGF-A and VEGFR-2 protein products between alone HCACC culture and the co-culture system. b The inhibitory effect of NCTD on expression of these protein products of the co-culture system. The expression of VEGF-C, VEGF-D and VEGFR-3 protein products in NCTD, mF4-31C1 or NCTD + mF4-31C1 group was downregulated significantly as compared to control group (* P

    Article Snippet: It was shown that HT-29 cells may promote VEGF-C, VEGF-D and VEGFR-3 secreted from tumor and/or stromal cells or indirectly promote VEGF-A and VEGFR-2 secreted from tumor and/or stromal cells, so accelerate the lymphatic tube formation of HDLECs, and the lymphangiogenesis and tumor growth of the in-situ colonic xenografts; NCTD or in combination with mF4-31C1 or Sorafenib markedly downregulated the expression of VEGF-C, VEGF-D and VEGFR-3 other than VEGF-A and VEGFR-2 proteins/mRNAs of the co-culture system in vitro and the in-situ colonic xenografts in vivo .

    Techniques: Expressing, Co-Culture Assay, In Vitro, Staining

    Reversal effect of cholesterol on the inhibition of VEGFR2 and mTOR activities by SERM. (A) and (B) HUVEC were treated with tamoxifen (TMX) or toremifene (TRM) with or without cholesterol (5 μg/ml)/cyclodextrin (0.1%) complex (Chol/CD) for 24

    Journal: Cancer letters

    Article Title: Inhibition of angiogenesis by selective estrogen receptor modulators through blockade of cholesterol trafficking rather than estrogen receptor antagonism

    doi: 10.1016/j.canlet.2015.03.022

    Figure Lengend Snippet: Reversal effect of cholesterol on the inhibition of VEGFR2 and mTOR activities by SERM. (A) and (B) HUVEC were treated with tamoxifen (TMX) or toremifene (TRM) with or without cholesterol (5 μg/ml)/cyclodextrin (0.1%) complex (Chol/CD) for 24

    Article Snippet: The blots were blocked with 5% non-fat dried milk at room temperature for 1 h, incubated with the primary antibodies including anti-VEGFR2 (Cell Signaling Technology), anti-phospho-VEGFR2 (Tyr1175, Cell Signaling Technology), anti-FGFR1 (Cell Signaling Technology), anti-mTOR (Cell Signaling Technology), anti-phospho-mTOR (Ser2448, Cell Signaling Technology) anti-S6K (Cell Signaling Technology), anti-phospho-S6K (Thr389, Cell Signaling Technology), anti-PDGFRβ (Santa Cruz Biotechnologies), anti-actin (Santa Cruz Biotechnologies) or anti-α-tubulin (Santa Cruz Biotechnologies) antibodies overnight at 4°C and then incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. The immune-complexes were detected using enhanced chemiluminescence (ECL) detection reagent (GE Healthcare, Pittsburgh, PA).

    Techniques: Inhibition

    Effects of SERM and cholesterol on the subcellular localization of VEGFR2 and mTOR in HUVEC. (A) HUVEC were treated with or without 1 μM tamoxifen (TMX) for 24 h and subcellular localization of VEGFR2 was assessed under a confocal microscope.

    Journal: Cancer letters

    Article Title: Inhibition of angiogenesis by selective estrogen receptor modulators through blockade of cholesterol trafficking rather than estrogen receptor antagonism

    doi: 10.1016/j.canlet.2015.03.022

    Figure Lengend Snippet: Effects of SERM and cholesterol on the subcellular localization of VEGFR2 and mTOR in HUVEC. (A) HUVEC were treated with or without 1 μM tamoxifen (TMX) for 24 h and subcellular localization of VEGFR2 was assessed under a confocal microscope.

    Article Snippet: The blots were blocked with 5% non-fat dried milk at room temperature for 1 h, incubated with the primary antibodies including anti-VEGFR2 (Cell Signaling Technology), anti-phospho-VEGFR2 (Tyr1175, Cell Signaling Technology), anti-FGFR1 (Cell Signaling Technology), anti-mTOR (Cell Signaling Technology), anti-phospho-mTOR (Ser2448, Cell Signaling Technology) anti-S6K (Cell Signaling Technology), anti-phospho-S6K (Thr389, Cell Signaling Technology), anti-PDGFRβ (Santa Cruz Biotechnologies), anti-actin (Santa Cruz Biotechnologies) or anti-α-tubulin (Santa Cruz Biotechnologies) antibodies overnight at 4°C and then incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. The immune-complexes were detected using enhanced chemiluminescence (ECL) detection reagent (GE Healthcare, Pittsburgh, PA).

    Techniques: Microscopy

    Effect of SERM on VEGFR2 glycosylation and mTORC1 pathway in HUVEC. (A) HUVEC were treated with SERM including tamoxifen (TMX, 5 μM), toremifene (TRM, 5 μM) and clomifene (CLM, 5 μM) for 24 h and VEGFR2 glycosylation was assessed

    Journal: Cancer letters

    Article Title: Inhibition of angiogenesis by selective estrogen receptor modulators through blockade of cholesterol trafficking rather than estrogen receptor antagonism

    doi: 10.1016/j.canlet.2015.03.022

    Figure Lengend Snippet: Effect of SERM on VEGFR2 glycosylation and mTORC1 pathway in HUVEC. (A) HUVEC were treated with SERM including tamoxifen (TMX, 5 μM), toremifene (TRM, 5 μM) and clomifene (CLM, 5 μM) for 24 h and VEGFR2 glycosylation was assessed

    Article Snippet: The blots were blocked with 5% non-fat dried milk at room temperature for 1 h, incubated with the primary antibodies including anti-VEGFR2 (Cell Signaling Technology), anti-phospho-VEGFR2 (Tyr1175, Cell Signaling Technology), anti-FGFR1 (Cell Signaling Technology), anti-mTOR (Cell Signaling Technology), anti-phospho-mTOR (Ser2448, Cell Signaling Technology) anti-S6K (Cell Signaling Technology), anti-phospho-S6K (Thr389, Cell Signaling Technology), anti-PDGFRβ (Santa Cruz Biotechnologies), anti-actin (Santa Cruz Biotechnologies) or anti-α-tubulin (Santa Cruz Biotechnologies) antibodies overnight at 4°C and then incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. The immune-complexes were detected using enhanced chemiluminescence (ECL) detection reagent (GE Healthcare, Pittsburgh, PA).

    Techniques:

    Asymmetric complex formation induces reciprocal allosteric changes in enzyme and substrate kinases. a , c , Overlays of 1 H/ 13 C HMQC (leucine/valine region) spectra of 0.4 mM isotopically labeled FGFR2K WT ( a ; blue) or FGFR2K K659E ( c ; red) either alone or together with 0.8 mM unlabeled substrate kinase (that is, FGFR2K R577E/R678E ). Peaks sustaining > 20% loss of intensity are boxed. Experiments were performed independently twice with similar results. b,d , R ex values (with range depicted by a boxed colored bar) derived from CPMG relaxation dispersion experiments for FGFR2K WT ( b ) or FGFR2K K659E ( d ) mixed with unlabeled FGFR2K R577E/R678E mapped onto the enzyme-acting kinase in the asymmetric complex crystal structure. e , Changes in R ex values of selected residues in FGFR2K WT or FGFR2K K659E enzyme kinase induced upon addition of substrate (that is, FGFR2K R577E/R678E ). f , Reductions in CPMG-derived R ex values in FGFR2K K659E enzyme kinase when A-loop tyrosines (annotated YY) of FGFR2K R577E/R678E substrate kinase are substituted to YF, FY and FF. e , f , n . a , c , e , f , Isotopically enriched kinases contained in mixtures are indicated by asterisks. g – j , Induced-fit model for A-loop-tyrosine transphosphorylation. g , Asymmetric complex formation of FGFR kinases (enzyme and substrate in green and blue, respectively) is thermodynamically inhibited by a charge repulsion between K659 in the enzyme-acting kinase and R669 in the incoming substrate-acting kinase (both residues highlighted in pink). h , Energetic gains in extracellular FGF-induced FGFR dimerization offset these repulsive forces, facilitating formation of a C lobe–C lobe-mediated asymmetric kinase dimer. HS, heparan sulfate. i , Asymmetric complex formation imparts upon the substrate A-loop a more phosphorylatable conformation (indicated as a change in color to yellow). j , This encourages the A-loop of the enzyme to adopt the active state (depicted by a change in color to red), resulting in the formation of an A-loop-tyrosine transphosphorylation complex as revealed by the crystal structure. k , l , Immunoblot analyses of L6 myoblast cell lines overexpressing either full-length mouse wild-type VEGFR2 ( k ) or human wild-type insulin receptor (IR) ( l ), together with variants harboring either a R1080G substitution ( k ) or a G1211R substitution ( l ) (in each case corresponding to FGFR2 R678) plus dimer-disrupting substitutions R929E/R932E and D1079R ( k ) or R1116E/R1119E and D1210R substitutions ( l ) (in each case corresponding to FGFR2 R577E/R580E and D677R). Cells were stimulated with either VEGF ( k ) or insulin ( l ) at the concentrations shown. Whole-cell lysates were analyzed by immunoblotting using antibodies specific for p-VEGFR2, VEGFR2 ( k ) or antibodies specific for phosphorylated human insulin receptor (p-hIR) or human insulin receptor (hIR) ( l ). k , l .

    Journal: Nature chemical biology

    Article Title: Molecular basis for receptor tyrosine kinase A-loop tyrosine transphosphorylation

    doi: 10.1038/s41589-019-0455-7

    Figure Lengend Snippet: Asymmetric complex formation induces reciprocal allosteric changes in enzyme and substrate kinases. a , c , Overlays of 1 H/ 13 C HMQC (leucine/valine region) spectra of 0.4 mM isotopically labeled FGFR2K WT ( a ; blue) or FGFR2K K659E ( c ; red) either alone or together with 0.8 mM unlabeled substrate kinase (that is, FGFR2K R577E/R678E ). Peaks sustaining > 20% loss of intensity are boxed. Experiments were performed independently twice with similar results. b,d , R ex values (with range depicted by a boxed colored bar) derived from CPMG relaxation dispersion experiments for FGFR2K WT ( b ) or FGFR2K K659E ( d ) mixed with unlabeled FGFR2K R577E/R678E mapped onto the enzyme-acting kinase in the asymmetric complex crystal structure. e , Changes in R ex values of selected residues in FGFR2K WT or FGFR2K K659E enzyme kinase induced upon addition of substrate (that is, FGFR2K R577E/R678E ). f , Reductions in CPMG-derived R ex values in FGFR2K K659E enzyme kinase when A-loop tyrosines (annotated YY) of FGFR2K R577E/R678E substrate kinase are substituted to YF, FY and FF. e , f , n . a , c , e , f , Isotopically enriched kinases contained in mixtures are indicated by asterisks. g – j , Induced-fit model for A-loop-tyrosine transphosphorylation. g , Asymmetric complex formation of FGFR kinases (enzyme and substrate in green and blue, respectively) is thermodynamically inhibited by a charge repulsion between K659 in the enzyme-acting kinase and R669 in the incoming substrate-acting kinase (both residues highlighted in pink). h , Energetic gains in extracellular FGF-induced FGFR dimerization offset these repulsive forces, facilitating formation of a C lobe–C lobe-mediated asymmetric kinase dimer. HS, heparan sulfate. i , Asymmetric complex formation imparts upon the substrate A-loop a more phosphorylatable conformation (indicated as a change in color to yellow). j , This encourages the A-loop of the enzyme to adopt the active state (depicted by a change in color to red), resulting in the formation of an A-loop-tyrosine transphosphorylation complex as revealed by the crystal structure. k , l , Immunoblot analyses of L6 myoblast cell lines overexpressing either full-length mouse wild-type VEGFR2 ( k ) or human wild-type insulin receptor (IR) ( l ), together with variants harboring either a R1080G substitution ( k ) or a G1211R substitution ( l ) (in each case corresponding to FGFR2 R678) plus dimer-disrupting substitutions R929E/R932E and D1079R ( k ) or R1116E/R1119E and D1210R substitutions ( l ) (in each case corresponding to FGFR2 R577E/R580E and D677R). Cells were stimulated with either VEGF ( k ) or insulin ( l ) at the concentrations shown. Whole-cell lysates were analyzed by immunoblotting using antibodies specific for p-VEGFR2, VEGFR2 ( k ) or antibodies specific for phosphorylated human insulin receptor (p-hIR) or human insulin receptor (hIR) ( l ). k , l .

    Article Snippet: Anti-FGFR2 (11835), anti-FGFR3 (4574), anti-FGFR4 (8562), anti-p-PLCγ (Y783; 2821), anti-PLCγ (2822), anti-p-FRS2 (Y436; 3861), anti-p-ERK1/2 (T202/Y204; 4370) and anti-p-VEGFR2 (Y1059; 3817), anti-phosphorylated insulin receptor β (Y1146; 3021) and anti-insulin receptor β (3025) antibodies were purchased from Cell Signaling Technologies; anti-ERK2 (sc-153) was obtained from Santa Cruz and an anti-β-tubulin antibody (PA1–41331) was purchased from Thermo Fisher Scientific.

    Techniques: Labeling, Derivative Assay