gene exp vegfa hs00900055 m1  (Thermo Fisher)


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    Thermo Fisher gene exp vegfa hs00900055 m1
    OPG-Fas interaction mediates the OPG-induced phenotypic response of PASMC. TaqMan expression of ( a ) <t>VEGFA,</t> ( b ) PDGFRA, ( c ) TNC, ( d ) Cav1 and ( e ) TRAIL in response to OPG in the presence (hash bars) or absence (Grey bars) of anti-Fas neutralising antibody (1500 ng ml −1 ). Panel ( f ) demonstrates OPG inhibition of FasL and TRAIL-induced apoptosis in HT1080 cells. g PASMC migration following 6 h stimulation with PDGF (20 ng ml −1 ), OPG (30 ng ml −1 ) or 0.2% FCS (serum-free media, SFM), in the presence or absence of Fas neutralising antibody. h Proliferation of PASMCs following stimulation with OPG for 72 h in the presence or absence of Fas neutralising antibody and/or TRAIL neutralising antibody (0.5 nM). Proliferation expressed as a percentage of proliferation to PDGF. Bars represent the mean with error bars showing the standard error of the mean. Dots represent experimental repeats, Panels ( a – e ) ( n = 4), panel ( f ) ( n = 3), panel ( g ) ( n = 4), panel (h) ( n = 4 for SFM, 10 for PDGF OPG stimulations) * p
    Gene Exp Vegfa Hs00900055 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension"

    Article Title: A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13139-9

    OPG-Fas interaction mediates the OPG-induced phenotypic response of PASMC. TaqMan expression of ( a ) VEGFA, ( b ) PDGFRA, ( c ) TNC, ( d ) Cav1 and ( e ) TRAIL in response to OPG in the presence (hash bars) or absence (Grey bars) of anti-Fas neutralising antibody (1500 ng ml −1 ). Panel ( f ) demonstrates OPG inhibition of FasL and TRAIL-induced apoptosis in HT1080 cells. g PASMC migration following 6 h stimulation with PDGF (20 ng ml −1 ), OPG (30 ng ml −1 ) or 0.2% FCS (serum-free media, SFM), in the presence or absence of Fas neutralising antibody. h Proliferation of PASMCs following stimulation with OPG for 72 h in the presence or absence of Fas neutralising antibody and/or TRAIL neutralising antibody (0.5 nM). Proliferation expressed as a percentage of proliferation to PDGF. Bars represent the mean with error bars showing the standard error of the mean. Dots represent experimental repeats, Panels ( a – e ) ( n = 4), panel ( f ) ( n = 3), panel ( g ) ( n = 4), panel (h) ( n = 4 for SFM, 10 for PDGF OPG stimulations) * p
    Figure Legend Snippet: OPG-Fas interaction mediates the OPG-induced phenotypic response of PASMC. TaqMan expression of ( a ) VEGFA, ( b ) PDGFRA, ( c ) TNC, ( d ) Cav1 and ( e ) TRAIL in response to OPG in the presence (hash bars) or absence (Grey bars) of anti-Fas neutralising antibody (1500 ng ml −1 ). Panel ( f ) demonstrates OPG inhibition of FasL and TRAIL-induced apoptosis in HT1080 cells. g PASMC migration following 6 h stimulation with PDGF (20 ng ml −1 ), OPG (30 ng ml −1 ) or 0.2% FCS (serum-free media, SFM), in the presence or absence of Fas neutralising antibody. h Proliferation of PASMCs following stimulation with OPG for 72 h in the presence or absence of Fas neutralising antibody and/or TRAIL neutralising antibody (0.5 nM). Proliferation expressed as a percentage of proliferation to PDGF. Bars represent the mean with error bars showing the standard error of the mean. Dots represent experimental repeats, Panels ( a – e ) ( n = 4), panel ( f ) ( n = 3), panel ( g ) ( n = 4), panel (h) ( n = 4 for SFM, 10 for PDGF OPG stimulations) * p

    Techniques Used: Expressing, Inhibition, Migration

    2) Product Images from "Interplay between VEGF-A and cMET signaling in human vestibular schwannomas and schwann cells "

    Article Title: Interplay between VEGF-A and cMET signaling in human vestibular schwannomas and schwann cells

    Journal: Cancer Biology & Therapy

    doi: 10.4161/15384047.2014.972765

    VEGF-A and cMET pathways interact at the molecular level. ( A ) Representative image of western blot showing expression of VEGFR2, cMET and VEGF-A for vehicle only and for siRNAs targeting VEGFA and MET genes in primary human SCs. ( B ) Protein expression of VEGF-A, cMET and VEGFR2 after VEGFA and MET siRNA treatment of cultured human SCs (n = 2–4 different cultures) and VS cells (n = 5 different cultures) quantified through protein gel blot analysis. All levels are normalized to vehicle only protein expression, being 100% (dashed line). * P
    Figure Legend Snippet: VEGF-A and cMET pathways interact at the molecular level. ( A ) Representative image of western blot showing expression of VEGFR2, cMET and VEGF-A for vehicle only and for siRNAs targeting VEGFA and MET genes in primary human SCs. ( B ) Protein expression of VEGF-A, cMET and VEGFR2 after VEGFA and MET siRNA treatment of cultured human SCs (n = 2–4 different cultures) and VS cells (n = 5 different cultures) quantified through protein gel blot analysis. All levels are normalized to vehicle only protein expression, being 100% (dashed line). * P

    Techniques Used: Western Blot, Expressing, Cell Culture

    HGF and VEGF-A pathways are aberrantly expressed and activated in VS. ( A ) Gene expression of VEGFA and its receptor KDR, and HGF and its receptor MET, in human VS (n = 8 different tumors) normalized to great auricular nerves (GAN, n = 7 different nerves) as measured through qPCR. * P
    Figure Legend Snippet: HGF and VEGF-A pathways are aberrantly expressed and activated in VS. ( A ) Gene expression of VEGFA and its receptor KDR, and HGF and its receptor MET, in human VS (n = 8 different tumors) normalized to great auricular nerves (GAN, n = 7 different nerves) as measured through qPCR. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    3) Product Images from "Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis"

    Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

    Journal: Cell Death and Differentiation

    doi: 10.1038/s41418-020-0505-4

    Relationship between microvessel density and NOTCH1, HES1, DLL4, MMP13, and VEGFA levels. Levels of tumor microvessel density (MVD) correlate with mRNA expression of NOTCH1 ( a ), HES1 ( b ), DLL4 ( c ), and MMP13 ( e ), but not with those of VEGFA ( d ), in a collection of human intrahepatic cholangiocarcinoma (iCCA) samples ( n = 42). Linear regression analysis was used. f Representative examples of human iCCA specimens with high and low MVD.
    Figure Legend Snippet: Relationship between microvessel density and NOTCH1, HES1, DLL4, MMP13, and VEGFA levels. Levels of tumor microvessel density (MVD) correlate with mRNA expression of NOTCH1 ( a ), HES1 ( b ), DLL4 ( c ), and MMP13 ( e ), but not with those of VEGFA ( d ), in a collection of human intrahepatic cholangiocarcinoma (iCCA) samples ( n = 42). Linear regression analysis was used. f Representative examples of human iCCA specimens with high and low MVD.

    Techniques Used: Expressing

    LY3039478 inhibits VEGFA, CD31, and DLL4 expression in the iCCA PDX model. a Western blot analysis and semiquantitative evaluation of DLL4, VEGFA, and CD31 expression in PDX mice tissues by densitometry analysis of protein bands reveals a downregulation of DLL4, VEGFA, and CD31 protein expression in PDX mice treated with GSI. The bands were measured compared with the housekeeping GAPDH protein band, for each tissue. Average value of DLL4, VEGFA, and CD31 expression levels among all mouse treated with LY3039478 or vehicle is reported in the graph. P value showed versus vehicle treatment. Tissues PDX mice n = 10 for vehicle treatment in gray, n = 10 for LY3039478 treatment in black. b Representative images with immunofluorescence staining show DLL4 and CD31 downregulation in representative images of PDX tissues treated with LY30349478. DLL4 (green) and CD31 (red) and overlapping staining (yellow) were immunolocalized in PDX tissues. The yellow arrows highlight the detail of the co-localization of DLL4 and CD31 in PDX tissues (#4, #14, #24) not treated with LY339478. DAPI, 4′,6‐diamidino‐2‐phenylindole. c Immunofluorescence staining with MMP13 in red and nucleus in DAPI shown a significantly reduction of MMP13 in iCCA PDX tissues treated with LY3039478. Magnifications: ×20; inset ×60. d Representative images demonstrate a significant ( P
    Figure Legend Snippet: LY3039478 inhibits VEGFA, CD31, and DLL4 expression in the iCCA PDX model. a Western blot analysis and semiquantitative evaluation of DLL4, VEGFA, and CD31 expression in PDX mice tissues by densitometry analysis of protein bands reveals a downregulation of DLL4, VEGFA, and CD31 protein expression in PDX mice treated with GSI. The bands were measured compared with the housekeeping GAPDH protein band, for each tissue. Average value of DLL4, VEGFA, and CD31 expression levels among all mouse treated with LY3039478 or vehicle is reported in the graph. P value showed versus vehicle treatment. Tissues PDX mice n = 10 for vehicle treatment in gray, n = 10 for LY3039478 treatment in black. b Representative images with immunofluorescence staining show DLL4 and CD31 downregulation in representative images of PDX tissues treated with LY30349478. DLL4 (green) and CD31 (red) and overlapping staining (yellow) were immunolocalized in PDX tissues. The yellow arrows highlight the detail of the co-localization of DLL4 and CD31 in PDX tissues (#4, #14, #24) not treated with LY339478. DAPI, 4′,6‐diamidino‐2‐phenylindole. c Immunofluorescence staining with MMP13 in red and nucleus in DAPI shown a significantly reduction of MMP13 in iCCA PDX tissues treated with LY3039478. Magnifications: ×20; inset ×60. d Representative images demonstrate a significant ( P

    Techniques Used: Expressing, Western Blot, Mouse Assay, Immunofluorescence, Staining

    NOTCH1 , HES1 , DLL4 , VEGFA , and MMP13 mRNA expression in iCCA patients. a Analysis of 31 primary tumors from iCCA patients and matched surrounding normal liver tissues downloaded from the GEO database (GSE107943). Mean expression data were expressed in RPKM (Reads Per Kilobase Million). *** P
    Figure Legend Snippet: NOTCH1 , HES1 , DLL4 , VEGFA , and MMP13 mRNA expression in iCCA patients. a Analysis of 31 primary tumors from iCCA patients and matched surrounding normal liver tissues downloaded from the GEO database (GSE107943). Mean expression data were expressed in RPKM (Reads Per Kilobase Million). *** P

    Techniques Used: Expressing

    4) Product Images from "Nuclear Heparanase Regulates Chromatin Remodeling, Gene Expression and PTEN Tumor Suppressor Function"

    Article Title: Nuclear Heparanase Regulates Chromatin Remodeling, Gene Expression and PTEN Tumor Suppressor Function

    Journal: Cells

    doi: 10.3390/cells9092038

    Heparanase enhances chromatin accessibility. ( A ) To determine localization of heparanase, sequential cell fractionation of myeloma cells was used to isolate cytoplasmic (cyto), nucleus-soluble (sol), and nucleus-chromatin (chrom) fractions from CAG wild-type (HPSE Lo) and transfected CAG HPSE Hi cells prior to Western blot analysis. GAPDH and histone H3 (H3) were probed to assess the quality of the fractions. ( B ) Chromatin accessibility in CAG HPSE knockdown (KD) and HPSE Hi cells were assessed by sensitivity to micrococcal nuclease (MNase) digestion. Nuclei from KD and HPSE Hi cells were prepared and digested with 0.1 U/μL MNase and subjected to electrophoresis. Black arrows denote Tri (T), Di (D) and Mono (M)—nucleosomes. ( C ) Nuclei from CAG HPSE KD and RPMI-8226 myeloma cells were treated for 2 h with or without rHPSE before being subjected to Mnase digestion for chromatin accessibility. ( D ) CAG HPSE Hi cells were incubated with 20 µM of the HPSE inhibitor OGT2115 and chromatin accessibility was assessed following exposure of nuclei to MNase. ( E ) mRNA expression analysis by RT-PCR of syndecan-1 ( SDC1 ), vascular endothelial growth factor A ( VEGFA ), matrix metalloproteinase-9 ( MMP9 ) and cyclin D1 ( CCDN1 ) in CAG HPSE Hi cells in the presence or absence of OGT2115, compared to respective control. * p
    Figure Legend Snippet: Heparanase enhances chromatin accessibility. ( A ) To determine localization of heparanase, sequential cell fractionation of myeloma cells was used to isolate cytoplasmic (cyto), nucleus-soluble (sol), and nucleus-chromatin (chrom) fractions from CAG wild-type (HPSE Lo) and transfected CAG HPSE Hi cells prior to Western blot analysis. GAPDH and histone H3 (H3) were probed to assess the quality of the fractions. ( B ) Chromatin accessibility in CAG HPSE knockdown (KD) and HPSE Hi cells were assessed by sensitivity to micrococcal nuclease (MNase) digestion. Nuclei from KD and HPSE Hi cells were prepared and digested with 0.1 U/μL MNase and subjected to electrophoresis. Black arrows denote Tri (T), Di (D) and Mono (M)—nucleosomes. ( C ) Nuclei from CAG HPSE KD and RPMI-8226 myeloma cells were treated for 2 h with or without rHPSE before being subjected to Mnase digestion for chromatin accessibility. ( D ) CAG HPSE Hi cells were incubated with 20 µM of the HPSE inhibitor OGT2115 and chromatin accessibility was assessed following exposure of nuclei to MNase. ( E ) mRNA expression analysis by RT-PCR of syndecan-1 ( SDC1 ), vascular endothelial growth factor A ( VEGFA ), matrix metalloproteinase-9 ( MMP9 ) and cyclin D1 ( CCDN1 ) in CAG HPSE Hi cells in the presence or absence of OGT2115, compared to respective control. * p

    Techniques Used: Cell Fractionation, Transfection, Western Blot, Electrophoresis, Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction

    5) Product Images from "Preclinical validation of the small molecule drug quininib as a novel therapeutic for colorectal cancer"

    Article Title: Preclinical validation of the small molecule drug quininib as a novel therapeutic for colorectal cancer

    Journal: Scientific Reports

    doi: 10.1038/srep34523

    Quininib reduces angiogenic gene expression in HT-29luc2 xenografts. Changes in gene expression of IL-6 ( A ), IL-8 ( B ), VEGFA ( C ), NRP2 ( D ), Epiregulin ( E ) and FGF1 ( F ) were measured by RT-PCR. Error bars graphed are mean + SEM. Statistical analysis was performed by ANOVA and Dunnett’s multiple comparison test. *p
    Figure Legend Snippet: Quininib reduces angiogenic gene expression in HT-29luc2 xenografts. Changes in gene expression of IL-6 ( A ), IL-8 ( B ), VEGFA ( C ), NRP2 ( D ), Epiregulin ( E ) and FGF1 ( F ) were measured by RT-PCR. Error bars graphed are mean + SEM. Statistical analysis was performed by ANOVA and Dunnett’s multiple comparison test. *p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    6) Product Images from "Efficacy of tumor-targeting Salmonella typhimurium A1-R in combination with anti-angiogenesis therapy on a pancreatic cancer patient-derived orthotopic xenograft (PDOX) and cell line mouse models"

    Article Title: Efficacy of tumor-targeting Salmonella typhimurium A1-R in combination with anti-angiogenesis therapy on a pancreatic cancer patient-derived orthotopic xenograft (PDOX) and cell line mouse models

    Journal: Oncotarget

    doi:

    Efficacy of BEV treatment on pancreatic cancer cell lines which have different levels of VEGFA and VEGFR2 expression (A) Schema of treatments on subcutaneous tumors. Based on RT-PCR results, MiaPaCa-2 was determined to be VEGFA-positive and VEGFR2-positive. Panc-1 was determined as VEGFA-negative and VEGFR2-negative. In order to determine the efficacy of BEV on pancreatic cancer cell lines with different levels of VEGFA and VEGFR2 expression, subcutaneous tumors from MiaPaCa-2 and Panc-1 cells were grown in nude mice and randomized to 3 groups as described in the Materials and Methods. (B) BEV significantly reduced the growth of the MiaPaCa-2 tumor compared to the control on Day 22 (p
    Figure Legend Snippet: Efficacy of BEV treatment on pancreatic cancer cell lines which have different levels of VEGFA and VEGFR2 expression (A) Schema of treatments on subcutaneous tumors. Based on RT-PCR results, MiaPaCa-2 was determined to be VEGFA-positive and VEGFR2-positive. Panc-1 was determined as VEGFA-negative and VEGFR2-negative. In order to determine the efficacy of BEV on pancreatic cancer cell lines with different levels of VEGFA and VEGFR2 expression, subcutaneous tumors from MiaPaCa-2 and Panc-1 cells were grown in nude mice and randomized to 3 groups as described in the Materials and Methods. (B) BEV significantly reduced the growth of the MiaPaCa-2 tumor compared to the control on Day 22 (p

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

    mRNA expression of VEGFA (A), VEGFR1 (B) and VEGFR2 (C) in pancreatic cancer cell lines mRNA expression was determined with real-time RT-PCR. MiaPaCa-2 significantly expressed VEGFA more than other cell lines (p
    Figure Legend Snippet: mRNA expression of VEGFA (A), VEGFR1 (B) and VEGFR2 (C) in pancreatic cancer cell lines mRNA expression was determined with real-time RT-PCR. MiaPaCa-2 significantly expressed VEGFA more than other cell lines (p

    Techniques Used: Expressing, Quantitative RT-PCR

    7) Product Images from "Hypoxic gene expression in chronic hepatitis B virus infected patients is not observed in state-of-the-art in vitro and mouse infection models"

    Article Title: Hypoxic gene expression in chronic hepatitis B virus infected patients is not observed in state-of-the-art in vitro and mouse infection models

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-70865-7

    Effect of silencing viral transcription in HBV transgenic mice on hypoxia target gene transcripts. HBV transgenic mice (n = 6 per group) were treated with liver directed siRNAs targeting the HBx region (siHBV) which is commonly shared by all viral RNAs or with a control siRNA (siCtrl). Seven days later we assessed the efficacy of siHBV silencing by quantifying: serum HBeAg levels ( a ), HBV RNAs in the liver and hypoxia target gene ( CAIX, VEGFA, GLUT1 and PHD2 ) RNAs ( b ). Hypoxia target genes values are expressed as ΔCt values by subtracting the Ct value of the housekeeping gene β-actin from Ct value of the gene of interest. Mann Whitney ( a ) or 2-way-ANOVA with Bonferroni correction ( b ) analyses were applied with p
    Figure Legend Snippet: Effect of silencing viral transcription in HBV transgenic mice on hypoxia target gene transcripts. HBV transgenic mice (n = 6 per group) were treated with liver directed siRNAs targeting the HBx region (siHBV) which is commonly shared by all viral RNAs or with a control siRNA (siCtrl). Seven days later we assessed the efficacy of siHBV silencing by quantifying: serum HBeAg levels ( a ), HBV RNAs in the liver and hypoxia target gene ( CAIX, VEGFA, GLUT1 and PHD2 ) RNAs ( b ). Hypoxia target genes values are expressed as ΔCt values by subtracting the Ct value of the housekeeping gene β-actin from Ct value of the gene of interest. Mann Whitney ( a ) or 2-way-ANOVA with Bonferroni correction ( b ) analyses were applied with p

    Techniques Used: Transgenic Assay, Mouse Assay, MANN-WHITNEY

    8) Product Images from "Delivery of VEGFA in bone marrow stromal cells seeded in copolymer scaffold enhances angiogenesis, but is inadequate for osteogenesis as compared with the dual delivery of VEGFA and BMP2 in a subcutaneous mouse model"

    Article Title: Delivery of VEGFA in bone marrow stromal cells seeded in copolymer scaffold enhances angiogenesis, but is inadequate for osteogenesis as compared with the dual delivery of VEGFA and BMP2 in a subcutaneous mouse model

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-018-0778-4

    Adenoviral (ad) vector-mediated combined delivery of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor A (VEGFA) led to upregulation of osteogenic and angiogenic molecules in vitro . ad-GFP, ad-VEGFA, or ad-BMP2 + VEGFA BMSC (5 × 10 4 ) were seeded in each scaffold and harvested at 3 and 14 days for custom polymerase chain reaction (PCR) array and TaqMan-based qRT-PCR. a , b Unsupervised hierarchical clustering demonstrated two separate clusters consisting of replicates of ad-BMP2 + VEGFA, and replicates of ad-GFP and ad-VEGFA BMSC at both time points. c ANOVA disclosed that ALPL , BMP7 , BMP6 , FLT1 , and PGF mRNA levels were found to be significantly upregulated at 3 days in ad-BMP2 + VEGFA BMSC. d More osteogenic markers were significantly induced at 14 days in ad-BMP2 + VEGFA BMSC compared with the ad-GFP and ad-VEGFA BMSC groups. Error bars represent SEM of three biological replicates ( n = 3) performed in three technical replicates. ANOVA test with Bonferroni post hoc analysis was applied in c and d. e Representative images demonstrating higher alkaline phosphatase activity in ad-BMP2 + VEGFA BMSC in monolayer culture as compared with the ad-GFP and ad-VEGFA BMSC at 3 and 14 days. Experiments were repeated at least three times. *** p
    Figure Legend Snippet: Adenoviral (ad) vector-mediated combined delivery of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor A (VEGFA) led to upregulation of osteogenic and angiogenic molecules in vitro . ad-GFP, ad-VEGFA, or ad-BMP2 + VEGFA BMSC (5 × 10 4 ) were seeded in each scaffold and harvested at 3 and 14 days for custom polymerase chain reaction (PCR) array and TaqMan-based qRT-PCR. a , b Unsupervised hierarchical clustering demonstrated two separate clusters consisting of replicates of ad-BMP2 + VEGFA, and replicates of ad-GFP and ad-VEGFA BMSC at both time points. c ANOVA disclosed that ALPL , BMP7 , BMP6 , FLT1 , and PGF mRNA levels were found to be significantly upregulated at 3 days in ad-BMP2 + VEGFA BMSC. d More osteogenic markers were significantly induced at 14 days in ad-BMP2 + VEGFA BMSC compared with the ad-GFP and ad-VEGFA BMSC groups. Error bars represent SEM of three biological replicates ( n = 3) performed in three technical replicates. ANOVA test with Bonferroni post hoc analysis was applied in c and d. e Representative images demonstrating higher alkaline phosphatase activity in ad-BMP2 + VEGFA BMSC in monolayer culture as compared with the ad-GFP and ad-VEGFA BMSC at 3 and 14 days. Experiments were repeated at least three times. *** p

    Techniques Used: Plasmid Preparation, In Vitro, Polymerase Chain Reaction, Quantitative RT-PCR, Activity Assay

    Combined delivery of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor A (VEGFA) induced ectopic bone formation in scaffold explants in a subcutaneous NOD/SCID mouse model. The ability of VEGFA alone or in combination with BMP2 to induce ectopic bone formation was investigated in NOD/SCID mice by subcutaneously implanting scaffolds seeded with 5 × 10 5 BMSC from different experimental groups. Scaffold explants at 8 weeks were examined by μCT to evaluate ectopic bone formation. Negligible amounts of radiopaque bone-like structures were detected in a untransduced, b ad-GFP, and c ad-VEGFA BMSC. In contrast, numerous bony-like radiopaque structures were found at the periphery as well as within the scaffolds of ad-BMP2 + VEGFA explants ( d , cross sectional view in e ). f Quantification of the radiopaque bone-like structures in the scaffold explants revealed significant amounts of bone formation in ad-BMP2 + VEGFA scaffolds. g – n Representative images of H E stained FFPE sections of scaffold explants from different experimental groups at 2 and 8 weeks. g – l Formation of bony structures was not detected in untransduced, ad-GFP, or ad-VEGFA groups at either 2 or 8 weeks. m In the ad-BMP2 + VEGFA BMSC group, formation of bony structures (black arrows) could be seen as early as 2 weeks, mostly at the periphery of the scaffold explants. n Extensive bone formation (green arrows) with bony trabeculae extending inside the scaffolds was found at 8 weeks in ad-BMP2 + VEGFA BMSC. The structure consisted of numerous osteocyte-like cells at both 2 (black arrowheads, inset in m ) and 8 weeks (green arrowheads, inset in n ). ANOVA with Bonferroni post hoc analysis was carried out for statistical analysis in ( f ). Error bars represent SEM. ** p
    Figure Legend Snippet: Combined delivery of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor A (VEGFA) induced ectopic bone formation in scaffold explants in a subcutaneous NOD/SCID mouse model. The ability of VEGFA alone or in combination with BMP2 to induce ectopic bone formation was investigated in NOD/SCID mice by subcutaneously implanting scaffolds seeded with 5 × 10 5 BMSC from different experimental groups. Scaffold explants at 8 weeks were examined by μCT to evaluate ectopic bone formation. Negligible amounts of radiopaque bone-like structures were detected in a untransduced, b ad-GFP, and c ad-VEGFA BMSC. In contrast, numerous bony-like radiopaque structures were found at the periphery as well as within the scaffolds of ad-BMP2 + VEGFA explants ( d , cross sectional view in e ). f Quantification of the radiopaque bone-like structures in the scaffold explants revealed significant amounts of bone formation in ad-BMP2 + VEGFA scaffolds. g – n Representative images of H E stained FFPE sections of scaffold explants from different experimental groups at 2 and 8 weeks. g – l Formation of bony structures was not detected in untransduced, ad-GFP, or ad-VEGFA groups at either 2 or 8 weeks. m In the ad-BMP2 + VEGFA BMSC group, formation of bony structures (black arrows) could be seen as early as 2 weeks, mostly at the periphery of the scaffold explants. n Extensive bone formation (green arrows) with bony trabeculae extending inside the scaffolds was found at 8 weeks in ad-BMP2 + VEGFA BMSC. The structure consisted of numerous osteocyte-like cells at both 2 (black arrowheads, inset in m ) and 8 weeks (green arrowheads, inset in n ). ANOVA with Bonferroni post hoc analysis was carried out for statistical analysis in ( f ). Error bars represent SEM. ** p

    Techniques Used: Mouse Assay, Staining, Formalin-fixed Paraffin-Embedded

    Adenoviral (ad) vector-mediated expression of vascular endothelial growth factor A (VEGFA) alone and in combination with bone morphogenetic protein 2 (BMP2) was associated with enhanced angiogenesis in scaffold explants. a Representative images at 2 weeks revealed more reddish scaffold explants with multiple capillary/vessel like structures (black arrows) radiating from the periphery in ad-VEGFA and ad-BMP2 + VEGFA groups compared with the other control explants. b – e Frozen sections of scaffold explants at 2 weeks were stained with anti-CD31 targeting mouse CD31 protein and examined for capillary-like structures. Few CD31-positive structures were found in untransduced and ad-GFP scaffold explants ( b and c , white arrows). However, in ad-VEGFA and ad-BMP2 + VEGFA explants, many CD31-positive capillary-like structures (white arrows) were seen both at the periphery (not shown in the figure) as well as within the scaffolds ( d , e ). Red arrowheads indicate remnants of the scaffold material. f Quantification of CD31-positive area in the scaffold explants at 2 weeks demonstrated significantly more CD31-positive structures in ad-VEGFA and ad-BMP2 + VEGFA (ad-B + V) scaffolds than in the untransduced and ad-GFP explants. ANOVA test with Bonferroni post hoc analysis was used for statistical analysis in ( e ). Error bars represent SEM. *** p
    Figure Legend Snippet: Adenoviral (ad) vector-mediated expression of vascular endothelial growth factor A (VEGFA) alone and in combination with bone morphogenetic protein 2 (BMP2) was associated with enhanced angiogenesis in scaffold explants. a Representative images at 2 weeks revealed more reddish scaffold explants with multiple capillary/vessel like structures (black arrows) radiating from the periphery in ad-VEGFA and ad-BMP2 + VEGFA groups compared with the other control explants. b – e Frozen sections of scaffold explants at 2 weeks were stained with anti-CD31 targeting mouse CD31 protein and examined for capillary-like structures. Few CD31-positive structures were found in untransduced and ad-GFP scaffold explants ( b and c , white arrows). However, in ad-VEGFA and ad-BMP2 + VEGFA explants, many CD31-positive capillary-like structures (white arrows) were seen both at the periphery (not shown in the figure) as well as within the scaffolds ( d , e ). Red arrowheads indicate remnants of the scaffold material. f Quantification of CD31-positive area in the scaffold explants at 2 weeks demonstrated significantly more CD31-positive structures in ad-VEGFA and ad-BMP2 + VEGFA (ad-B + V) scaffolds than in the untransduced and ad-GFP explants. ANOVA test with Bonferroni post hoc analysis was used for statistical analysis in ( e ). Error bars represent SEM. *** p

    Techniques Used: Plasmid Preparation, Expressing, Staining

    VEGFA and BMP2 adenoviral vectors (ad) respectively upregulated the expression of vascular endothelial growth factor A (VEGFA) and bone morphogenetic protein 2 (BMP2) in BMSC. Representative immunofluorescence images of BMSC in monolayer transduced with ad-GFP ( a ), ad-VEGFA ( b ), and ad-VEGFA + BMP2 ( c – e ) adenoviral particles. BMSC (5× 10 4 ) were seeded in each scaffold and harvested after 3 and 14 days for mRNA and protein analyses. Significant upregulation of VEGFA mRNA ( f ) and VEGFA protein ( h ) levels were found at both time points in both ad-VEGFA and ad-BMP2 + ad-VEGFA BMSC compared with the ad-GFP BMSC. Similarly, significant upregulation of BMP2 mRNA ( g ) was found in ad-BMP2 + VEGFA BMSC compared with the ad-GFP and ad-VEGFA BMSC at both time points. VEGFA and BMP2 mRNA levels were normalized to GAPDH mRNA level. i ELISA disclosed higher levels of secreted BMP2 in the culture supernatant of ad-BMP2 BMSC compared with the ad-GFP BMSC at both 3 and 14 days. Error bars in ( f ) and ( g ) represent SEM of three repeated experiments ( n = 3) performed in three technical replicates. ANOVA test with Bonferroni post hoc analysis was used for statistical analysis in ( f ) and ( g ) and Student’s t test was performed in ( i ). Error bars in ( i ) represent SEM of three repeated experiments ( n = 3). *** p
    Figure Legend Snippet: VEGFA and BMP2 adenoviral vectors (ad) respectively upregulated the expression of vascular endothelial growth factor A (VEGFA) and bone morphogenetic protein 2 (BMP2) in BMSC. Representative immunofluorescence images of BMSC in monolayer transduced with ad-GFP ( a ), ad-VEGFA ( b ), and ad-VEGFA + BMP2 ( c – e ) adenoviral particles. BMSC (5× 10 4 ) were seeded in each scaffold and harvested after 3 and 14 days for mRNA and protein analyses. Significant upregulation of VEGFA mRNA ( f ) and VEGFA protein ( h ) levels were found at both time points in both ad-VEGFA and ad-BMP2 + ad-VEGFA BMSC compared with the ad-GFP BMSC. Similarly, significant upregulation of BMP2 mRNA ( g ) was found in ad-BMP2 + VEGFA BMSC compared with the ad-GFP and ad-VEGFA BMSC at both time points. VEGFA and BMP2 mRNA levels were normalized to GAPDH mRNA level. i ELISA disclosed higher levels of secreted BMP2 in the culture supernatant of ad-BMP2 BMSC compared with the ad-GFP BMSC at both 3 and 14 days. Error bars in ( f ) and ( g ) represent SEM of three repeated experiments ( n = 3) performed in three technical replicates. ANOVA test with Bonferroni post hoc analysis was used for statistical analysis in ( f ) and ( g ) and Student’s t test was performed in ( i ). Error bars in ( i ) represent SEM of three repeated experiments ( n = 3). *** p

    Techniques Used: Expressing, Immunofluorescence, Transduction, Enzyme-linked Immunosorbent Assay

    Bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor A (VEGFA) co-expression-mediated bone formation is associated with upregulation of osteogenic and angiogenic markers in the in vivo scaffold explants. Custom PCR array was used to examine differentially expressed osteogenesis- and angiogenesis-related genes in scaffold explants at 2 weeks. ANOVA revealed upregulation of a number of osteogenesis- and angiogenesis-related genes such as a ALPL , b RUNX2 , c SPP1 ( osteopontin ), d ANGPT1 , and e PGF in ad-BMP2 + VEGFA explants compared with the ad-GFP and ad-VEGFA explants. ANOVA test with Bonferroni post hoc analysis was undertaken for statistical analysis. ** p
    Figure Legend Snippet: Bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor A (VEGFA) co-expression-mediated bone formation is associated with upregulation of osteogenic and angiogenic markers in the in vivo scaffold explants. Custom PCR array was used to examine differentially expressed osteogenesis- and angiogenesis-related genes in scaffold explants at 2 weeks. ANOVA revealed upregulation of a number of osteogenesis- and angiogenesis-related genes such as a ALPL , b RUNX2 , c SPP1 ( osteopontin ), d ANGPT1 , and e PGF in ad-BMP2 + VEGFA explants compared with the ad-GFP and ad-VEGFA explants. ANOVA test with Bonferroni post hoc analysis was undertaken for statistical analysis. ** p

    Techniques Used: Expressing, In Vivo, Polymerase Chain Reaction

    9) Product Images from "YAP1 and TAZ negatively control bone angiogenesis by limiting hypoxia-inducible factor signaling in endothelial cells"

    Article Title: YAP1 and TAZ negatively control bone angiogenesis by limiting hypoxia-inducible factor signaling in endothelial cells

    Journal: eLife

    doi: 10.7554/eLife.50770

    Yap1 and Taz inhibit HIF1α-controlled gene expression. ( A ) Decreased Yap1 and Taz protein levels in siYAP1 and siTAZ -transfected HUVECs. ( B ) Reduced expression of YAP1 , TAZ and HIF1A transcripts in HUVECs transfected with the indicated siRNAs. ( C ) XBP1 expression is increased in siYAP1/TAZ -treated HUVECs and normalized by siHIF1A, whereas baseline XBP1 is not altered by HIF1A knockdown alone (n = 3–4; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( D ) Expression of the Yap1/Taz targets Ctgf, Cyr61 and the HIF1α targets Vegfa, Angptl4 in freshly isolated type H and type L EC subpopulations from P21 Hif1a iΔEC littermate control long bone. Source data for Figure 5—figure supplement 1B,C,D .
    Figure Legend Snippet: Yap1 and Taz inhibit HIF1α-controlled gene expression. ( A ) Decreased Yap1 and Taz protein levels in siYAP1 and siTAZ -transfected HUVECs. ( B ) Reduced expression of YAP1 , TAZ and HIF1A transcripts in HUVECs transfected with the indicated siRNAs. ( C ) XBP1 expression is increased in siYAP1/TAZ -treated HUVECs and normalized by siHIF1A, whereas baseline XBP1 is not altered by HIF1A knockdown alone (n = 3–4; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( D ) Expression of the Yap1/Taz targets Ctgf, Cyr61 and the HIF1α targets Vegfa, Angptl4 in freshly isolated type H and type L EC subpopulations from P21 Hif1a iΔEC littermate control long bone. Source data for Figure 5—figure supplement 1B,C,D .

    Techniques Used: Expressing, Transfection, Two Tailed Test, Isolation

    10) Product Images from "Hypoxic gene expression in chronic hepatitis B infected patients is not observed in state-of-art in vitro and mouse infection models"

    Article Title: Hypoxic gene expression in chronic hepatitis B infected patients is not observed in state-of-art in vitro and mouse infection models

    Journal: bioRxiv

    doi: 10.1101/2020.03.27.011841

    Effect of HBx on HIF expression and transcriptional activity in HepaRG cells HepaRG cells encoding HBx were incubated with Tet (50 μM) for 24 h and HBx protein and Smc6 expression detected by western blot ( a ). HepaRG cells encoding WT or mutated HBx (STOP) were incubated with or without Tet (50 μM, 24 h) and cultured under 20% or 1% oxygen conditions for 24h. Cells were lysed and expression of HIF-1, HIF-2, Carbonic anhydrase IX ( CAIX ) and housekeeping gene B-actin assessed by western blotting ( b ) and mRNA levels of HIF-1, HIF-2 and several HIF target genes ( CAIX, BNIP3, VEGFA and GLUT1 ) quantified by qPCR ( c ). HepaRG cells encoding wild type HBx were incubated with Tet (50 μM, 24 h) and cultured at 20% or 1% oxygen for 24h. The hypoxic cultures were returned to 20% oxygen. After 10 or 20 mins, cells were lysed and screened for HIF-1α or HIF-1α and housekeeping gene B-actin expression ( d ). The data is shown from a single experiment and is representative of three independent experiments and represents mean ! standard deviation. Normality distribution was assessed by D’Agostino-Pearson test; 2-way-ANOVA with Bonferroni correction was applied with p
    Figure Legend Snippet: Effect of HBx on HIF expression and transcriptional activity in HepaRG cells HepaRG cells encoding HBx were incubated with Tet (50 μM) for 24 h and HBx protein and Smc6 expression detected by western blot ( a ). HepaRG cells encoding WT or mutated HBx (STOP) were incubated with or without Tet (50 μM, 24 h) and cultured under 20% or 1% oxygen conditions for 24h. Cells were lysed and expression of HIF-1, HIF-2, Carbonic anhydrase IX ( CAIX ) and housekeeping gene B-actin assessed by western blotting ( b ) and mRNA levels of HIF-1, HIF-2 and several HIF target genes ( CAIX, BNIP3, VEGFA and GLUT1 ) quantified by qPCR ( c ). HepaRG cells encoding wild type HBx were incubated with Tet (50 μM, 24 h) and cultured at 20% or 1% oxygen for 24h. The hypoxic cultures were returned to 20% oxygen. After 10 or 20 mins, cells were lysed and screened for HIF-1α or HIF-1α and housekeeping gene B-actin expression ( d ). The data is shown from a single experiment and is representative of three independent experiments and represents mean ! standard deviation. Normality distribution was assessed by D’Agostino-Pearson test; 2-way-ANOVA with Bonferroni correction was applied with p

    Techniques Used: Expressing, Activity Assay, Incubation, Western Blot, Cell Culture, Real-time Polymerase Chain Reaction, Standard Deviation

    Effect of silencing viral transcription in HBV transgenic mice on hypoxia target gene transcripts. HBV transgenic mice (n=6 per group) were treated with liver directed siRNAs targeting the HBx region (siHBV) which is commonly shared by all viral RNAs or with a control siRNA (siCtrl). Seven days later we assessed the efficacy of siHBV silencing by quantifying: serum HBeAg levels ( a ), HBV RNAs in the liver ( b ) and hypoxia target gene (CAIX, VEGFA, GLUT1 and PHD2) RNAs ( d ). Hypoxia target genes values are expressed as ΔCt values by subtracting the Ct value of the housekeeping gene ß-actin from Ct value of the gene of interest. Mann Whitney test (a) or 2-way-ANOVA with Bonferroni correction (b) test were applied with p
    Figure Legend Snippet: Effect of silencing viral transcription in HBV transgenic mice on hypoxia target gene transcripts. HBV transgenic mice (n=6 per group) were treated with liver directed siRNAs targeting the HBx region (siHBV) which is commonly shared by all viral RNAs or with a control siRNA (siCtrl). Seven days later we assessed the efficacy of siHBV silencing by quantifying: serum HBeAg levels ( a ), HBV RNAs in the liver ( b ) and hypoxia target gene (CAIX, VEGFA, GLUT1 and PHD2) RNAs ( d ). Hypoxia target genes values are expressed as ΔCt values by subtracting the Ct value of the housekeeping gene ß-actin from Ct value of the gene of interest. Mann Whitney test (a) or 2-way-ANOVA with Bonferroni correction (b) test were applied with p

    Techniques Used: Transgenic Assay, Mouse Assay, MANN-WHITNEY

    11) Product Images from "VEGFD Protects Retinal Ganglion Cells and, consequently, Capillaries against Excitotoxic Injury"

    Article Title: VEGFD Protects Retinal Ganglion Cells and, consequently, Capillaries against Excitotoxic Injury

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2019.12.009

    NMDA-Triggered Excitotoxicity Reduces VEGFD Expression in RGCs (A) qRT-PCR analysis of vegfd , vegfc , vegfa , flt4 , flt1 , and kdr expression in retinal homogenates after intravitreal injection of NMDA. Unpaired t test. n = 6. vegfd , p = 0.0085; vegfc , p = 0.2052; vegfa , p = 0.8524; flt4 , p = 0.9414; flt1 , p = 0.6074; kdr , p = 0.2016. (B) Representative images of human retinal whole mounts. Retinas were immunolabeled with antibodies against Brn3a (red) and VEGFD (green). Scale bars, 10 μm. Negative controls of human retinas were immunolabeled only with secondary antibodies. (C) Representative bands of VEGFD, VEGFA, and β-actin cDNA products following qRT-PCR analysis of human retinal extracts obtained from two donors (subject 1, subject 2). (D) Quantification of VEGFD protein levels in cells in the ganglion cell layer of retinas of mice injected as indicated. Unpaired t test. n = 3. PBS versus NMDA, p = 0.0192. (E) Representative images of mouse sagittal retina sections at d7 after intravitreal injections of NMDA or PBS. Retinas were immunolabeled for VEGFD (green). Nuclei were labeled with Hoechst (blue). GCL, ganglion cell layer. Scale bar, 20 μm. (F) qRT-PCR analysis of vegfd , vegfc , vegfa , and flt4 expression in brain ECs (b.END3) 24 h after 20 μM NMDA treatment. Unpaired t test. n = 3. vegfd , p = 0.1744; vegfc , p = 0.7598; vegfa , p = 0.8998; flt4 , p = 0.4457. (G) qRT-PCR analysis of vegfd , vegfc , vegfa , and flt4 expression in primary astrocytes 24 h after 20 μM NMDA treatment. Unpaired t test. n = 3. vegfd , p = 0.8716; vegfc , p = 0.4918; vegfa , p = 0.9902; flt4 , p = 0.9331. Bars represent mean ± SEM. Dots represent single values. **p
    Figure Legend Snippet: NMDA-Triggered Excitotoxicity Reduces VEGFD Expression in RGCs (A) qRT-PCR analysis of vegfd , vegfc , vegfa , flt4 , flt1 , and kdr expression in retinal homogenates after intravitreal injection of NMDA. Unpaired t test. n = 6. vegfd , p = 0.0085; vegfc , p = 0.2052; vegfa , p = 0.8524; flt4 , p = 0.9414; flt1 , p = 0.6074; kdr , p = 0.2016. (B) Representative images of human retinal whole mounts. Retinas were immunolabeled with antibodies against Brn3a (red) and VEGFD (green). Scale bars, 10 μm. Negative controls of human retinas were immunolabeled only with secondary antibodies. (C) Representative bands of VEGFD, VEGFA, and β-actin cDNA products following qRT-PCR analysis of human retinal extracts obtained from two donors (subject 1, subject 2). (D) Quantification of VEGFD protein levels in cells in the ganglion cell layer of retinas of mice injected as indicated. Unpaired t test. n = 3. PBS versus NMDA, p = 0.0192. (E) Representative images of mouse sagittal retina sections at d7 after intravitreal injections of NMDA or PBS. Retinas were immunolabeled for VEGFD (green). Nuclei were labeled with Hoechst (blue). GCL, ganglion cell layer. Scale bar, 20 μm. (F) qRT-PCR analysis of vegfd , vegfc , vegfa , and flt4 expression in brain ECs (b.END3) 24 h after 20 μM NMDA treatment. Unpaired t test. n = 3. vegfd , p = 0.1744; vegfc , p = 0.7598; vegfa , p = 0.8998; flt4 , p = 0.4457. (G) qRT-PCR analysis of vegfd , vegfc , vegfa , and flt4 expression in primary astrocytes 24 h after 20 μM NMDA treatment. Unpaired t test. n = 3. vegfd , p = 0.8716; vegfc , p = 0.4918; vegfa , p = 0.9902; flt4 , p = 0.9331. Bars represent mean ± SEM. Dots represent single values. **p

    Techniques Used: Expressing, Quantitative RT-PCR, Injection, Immunolabeling, Mouse Assay, Labeling

    12) Product Images from "Fabrication of New Hybrid Scaffolds for in vivo Perivascular Application to Treat Limb Ischemia"

    Article Title: Fabrication of New Hybrid Scaffolds for in vivo Perivascular Application to Treat Limb Ischemia

    Journal: Frontiers in Cardiovascular Medicine

    doi: 10.3389/fcvm.2020.598890

    Effect of 3D culture on poly(ε-caprolactone) with gelatin nanofibers (PCL-GL) and polylactic-co-glycolic acid with gelatin nanofibers (PLGA-GL) on APCs. Bar graphs show the average of 3 biological replicates comparing the 3D conditions to the 2D monolayer culture on petri dish. (A) Representative fluorescence images of specific phenotype markers (NG2, PDGFR-β and Vimentin) in APCs seeded, respectively, on PCL- and PLGA-based scaffolds; images showed that all the cells were positive for the indicated markers. (B,C) Expression of differentiation (PDGFRB, ACTA2/SMA, TGLN/SM22A, COL1A1, and MYH11) molecules. (D) Expression of angiogenic miRs: miR132, miR210-3p, and miR532. (E) BACH1, FGF, VEGFA, and ANG-1 molecules. (F) Bar graph shows the secreted of VEGFA and ANG-1. * p
    Figure Legend Snippet: Effect of 3D culture on poly(ε-caprolactone) with gelatin nanofibers (PCL-GL) and polylactic-co-glycolic acid with gelatin nanofibers (PLGA-GL) on APCs. Bar graphs show the average of 3 biological replicates comparing the 3D conditions to the 2D monolayer culture on petri dish. (A) Representative fluorescence images of specific phenotype markers (NG2, PDGFR-β and Vimentin) in APCs seeded, respectively, on PCL- and PLGA-based scaffolds; images showed that all the cells were positive for the indicated markers. (B,C) Expression of differentiation (PDGFRB, ACTA2/SMA, TGLN/SM22A, COL1A1, and MYH11) molecules. (D) Expression of angiogenic miRs: miR132, miR210-3p, and miR532. (E) BACH1, FGF, VEGFA, and ANG-1 molecules. (F) Bar graph shows the secreted of VEGFA and ANG-1. * p

    Techniques Used: Fluorescence, Expressing

    13) Product Images from "Attenuation of periostin in retinal Müller glia by TNF-α and IFN-γ"

    Article Title: Attenuation of periostin in retinal Müller glia by TNF-α and IFN-γ

    Journal: International Journal of Ophthalmology

    doi: 10.18240/ijo.2019.02.05

    Periostin-knockdown attenuated VEGFA mRNA expression levels in MIO-M1 cells qRT-PCR results showed in bar graph revealed downregulation in MIO-M1 cells of periostin (A) and VEGFA (B) at mRNA expression levels after periostin knockdown. Results were normalized to human GAPDH mRNA expression levels correspondingly, and data were represented the mean±SEM ( n =4). b P
    Figure Legend Snippet: Periostin-knockdown attenuated VEGFA mRNA expression levels in MIO-M1 cells qRT-PCR results showed in bar graph revealed downregulation in MIO-M1 cells of periostin (A) and VEGFA (B) at mRNA expression levels after periostin knockdown. Results were normalized to human GAPDH mRNA expression levels correspondingly, and data were represented the mean±SEM ( n =4). b P

    Techniques Used: Expressing, Quantitative RT-PCR

    The effect of IFN-γ and TNF-α stimulation to the mRNA levels of VEGFA in MIO-M1 cells Fold change showed in bar plots in the expression for VEGFA in Müller cells after stimulation by IFN-γ and TNF-α by different concentration, 2, 20, 200 ng/mL for IFN-γ (A) and 0.1, 1, 10 ng/mL for TNF-α (B) respectively after 8h and 24h stimulation. GAPDH was used as normalization in all qRT-PCR results. Data were represented the mean±SEM ( n =4). b P
    Figure Legend Snippet: The effect of IFN-γ and TNF-α stimulation to the mRNA levels of VEGFA in MIO-M1 cells Fold change showed in bar plots in the expression for VEGFA in Müller cells after stimulation by IFN-γ and TNF-α by different concentration, 2, 20, 200 ng/mL for IFN-γ (A) and 0.1, 1, 10 ng/mL for TNF-α (B) respectively after 8h and 24h stimulation. GAPDH was used as normalization in all qRT-PCR results. Data were represented the mean±SEM ( n =4). b P

    Techniques Used: Expressing, Concentration Assay, Quantitative RT-PCR

    mRNA expression of VEGFA had no relevance with deficiency of periostin (periostin KO mice) in OIR retinas qRT-PCR results showed in bar plots for mRNA expression levels of periostin was remarkably downregulated in KO mice compared to WT control mice (A). No change was detected for VEGFA expression both in periostin KO mice and WT control mice (B). Results were normalized to mouse GAPDH mRNA expression levels correspondingly, and data were represented the mean±SEM ( n =4). b P
    Figure Legend Snippet: mRNA expression of VEGFA had no relevance with deficiency of periostin (periostin KO mice) in OIR retinas qRT-PCR results showed in bar plots for mRNA expression levels of periostin was remarkably downregulated in KO mice compared to WT control mice (A). No change was detected for VEGFA expression both in periostin KO mice and WT control mice (B). Results were normalized to mouse GAPDH mRNA expression levels correspondingly, and data were represented the mean±SEM ( n =4). b P

    Techniques Used: Expressing, Mouse Assay, Quantitative RT-PCR

    14) Product Images from "Plasma microvesicle analysis identifies microRNA 129-5p as a biomarker of heart failure in univentricular heart disease"

    Article Title: Plasma microvesicle analysis identifies microRNA 129-5p as a biomarker of heart failure in univentricular heart disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0183624

    miR129-5p expression in hESC-derived cardiomyocytes with hypoxia. A) hESC-derived cardiomyocytes were cultured in 1% O 2 and relative expression of vascular endothelial growth factor A (VEGFA) and miR129-5p were assessed by qPCR. Upregulation of hypoxia-mediated VEGFA and downregulation of miR129-5p was seen. Data shown are mean±SEM (n = 3); *p
    Figure Legend Snippet: miR129-5p expression in hESC-derived cardiomyocytes with hypoxia. A) hESC-derived cardiomyocytes were cultured in 1% O 2 and relative expression of vascular endothelial growth factor A (VEGFA) and miR129-5p were assessed by qPCR. Upregulation of hypoxia-mediated VEGFA and downregulation of miR129-5p was seen. Data shown are mean±SEM (n = 3); *p

    Techniques Used: Expressing, Derivative Assay, Cell Culture, Real-time Polymerase Chain Reaction

    15) Product Images from "Inspiration from heart development: Biomimetic development of functional human cardiac organoids"

    Article Title: Inspiration from heart development: Biomimetic development of functional human cardiac organoids

    Journal: Biomaterials

    doi: 10.1016/j.biomaterials.2017.07.021

    Human cardiac organoids display phenotypic channel functionality and organotypic responses to physiological/pathological stimuli. Cardiac organoids showed functional calcium handling. (A) Verapamil, which blocks the L-type calcium channel, displayed significant phenotypic reduction of calcium transient amplitude without inducing arrhythmia in cardiac organoids; n=4 organoids. (B) The use of ryanodine, known to block the ryanodine receptor of the sarcoplasmic reticulum and reduce endogenous calcium release, showed a significant decrease in calcium transient amplitude and a significant increase time to peak calcium; n=3 organoids. (C) Blockade of the hERG potassium channel using E-4031 showed a significant drop in calcium transient peak and a significant increase in time to 50% calcium decay; n=3 organoids. (D) A significant increase in beat rate after application of isoproterenol indicated intact β-adrenergic signaling in cardiac organoids; n=5 organoids. (E) Cardiac organoids showed organotypic transcription responses to ischemic stimuli; n=3 experiments; 10–15 microtissues/experiment. Cardiac organoids and hiPSC-CM spheroids showed increased VEGFA expression in response to ischemic stimulus. IL6 expression, a prominent interleukin in ischemic heart failure, increased in cardiac organoids in ischemia, while it was not detected in hiPSC-CM spheroids. hiPSC-CM-human induced pluripotent stem cell-derived cardiomyocytes. Cardiac organoids were fabricated using a developing stage cell ratio and were used at D10. Asterisk represents significant difference, p
    Figure Legend Snippet: Human cardiac organoids display phenotypic channel functionality and organotypic responses to physiological/pathological stimuli. Cardiac organoids showed functional calcium handling. (A) Verapamil, which blocks the L-type calcium channel, displayed significant phenotypic reduction of calcium transient amplitude without inducing arrhythmia in cardiac organoids; n=4 organoids. (B) The use of ryanodine, known to block the ryanodine receptor of the sarcoplasmic reticulum and reduce endogenous calcium release, showed a significant decrease in calcium transient amplitude and a significant increase time to peak calcium; n=3 organoids. (C) Blockade of the hERG potassium channel using E-4031 showed a significant drop in calcium transient peak and a significant increase in time to 50% calcium decay; n=3 organoids. (D) A significant increase in beat rate after application of isoproterenol indicated intact β-adrenergic signaling in cardiac organoids; n=5 organoids. (E) Cardiac organoids showed organotypic transcription responses to ischemic stimuli; n=3 experiments; 10–15 microtissues/experiment. Cardiac organoids and hiPSC-CM spheroids showed increased VEGFA expression in response to ischemic stimulus. IL6 expression, a prominent interleukin in ischemic heart failure, increased in cardiac organoids in ischemia, while it was not detected in hiPSC-CM spheroids. hiPSC-CM-human induced pluripotent stem cell-derived cardiomyocytes. Cardiac organoids were fabricated using a developing stage cell ratio and were used at D10. Asterisk represents significant difference, p

    Techniques Used: Functional Assay, Blocking Assay, Expressing, Derivative Assay

    16) Product Images from "Counterbalancing anti-adhesive effects of Tenascin-C through fibronectin expression in endothelial cells"

    Article Title: Counterbalancing anti-adhesive effects of Tenascin-C through fibronectin expression in endothelial cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13008-9

    Schematic of TNC-regulated pro-angiogenic signalling in endothelial cells. TNC inhibits endothelial cell adhesion, spreading and stress fibre formation (F-actin polymerization). This effect can lead to anoikis, or be counterbalanced by upregulation of Wnt target genes including FN and VEGFA. Autocrine FN (EDA) drives α5β1-dependent FN fibrillogenesis, promotes junction stabilization and impacts capillary morphogenesis.
    Figure Legend Snippet: Schematic of TNC-regulated pro-angiogenic signalling in endothelial cells. TNC inhibits endothelial cell adhesion, spreading and stress fibre formation (F-actin polymerization). This effect can lead to anoikis, or be counterbalanced by upregulation of Wnt target genes including FN and VEGFA. Autocrine FN (EDA) drives α5β1-dependent FN fibrillogenesis, promotes junction stabilization and impacts capillary morphogenesis.

    Techniques Used:

    The effect of TNC pre-conditioned HUVEC-derived matrices on naive HUVECs. ( a ) A local contrast map of representative phase contrast images of naïve HUVECs seeded onto matrices prepared from HUVECs plated on non-coated (NC) or TNC-coated substrates (top) for 24 h. Color-coded correlation length (µm) is shown (below). ( b ) Distribution of correlation length is plotted. At least 15 images were quantified for each condition. ( c ) Time course of VEGFA mRNA expression in HUVECs plated for the indicated time on non-coated or TNC-pretreated dishes. Fold change over NC was calculated using the ΔΔCt method (±S.D., n = 3). ( d ) Immunofluorescence staining of VEGFA, FN and nuclei in HUVECs plated for 5 days on non-coated or TNC-coated coverslips. Dotted squares in upper panels depict zoomed areas. VEGFA could be detected in focal adhesion-like structures in areas of low cell density. Bar = 50 µm (upper panels), 10 µm (zoomed area and lower panels).
    Figure Legend Snippet: The effect of TNC pre-conditioned HUVEC-derived matrices on naive HUVECs. ( a ) A local contrast map of representative phase contrast images of naïve HUVECs seeded onto matrices prepared from HUVECs plated on non-coated (NC) or TNC-coated substrates (top) for 24 h. Color-coded correlation length (µm) is shown (below). ( b ) Distribution of correlation length is plotted. At least 15 images were quantified for each condition. ( c ) Time course of VEGFA mRNA expression in HUVECs plated for the indicated time on non-coated or TNC-pretreated dishes. Fold change over NC was calculated using the ΔΔCt method (±S.D., n = 3). ( d ) Immunofluorescence staining of VEGFA, FN and nuclei in HUVECs plated for 5 days on non-coated or TNC-coated coverslips. Dotted squares in upper panels depict zoomed areas. VEGFA could be detected in focal adhesion-like structures in areas of low cell density. Bar = 50 µm (upper panels), 10 µm (zoomed area and lower panels).

    Techniques Used: Derivative Assay, Expressing, Immunofluorescence, Staining

    17) Product Images from "Functional inhibition of acid sphingomyelinase by Fluphenazine triggers hypoxia-specific tumor cell death"

    Article Title: Functional inhibition of acid sphingomyelinase by Fluphenazine triggers hypoxia-specific tumor cell death

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2017.130

    Fluphenazine induces HIF overactivation in conditions of high HIF background levels. ( a ) HIF activity reporter cells HCT116-HRE-Luc were treated either with DMSO control or 10 μ M Fluphenazine and incubated for 16 h in normoxia, hypoxia or in normoxia with the HIF-PH inhibitor DFO. After incubation, cells were lysed and luciferase activity was measured. Bars show mean with S.D. ( n =3). ( b ) HCT116 cells were treated for 24 h with or without DFO (1 mM) and additionally with either DMSO control or Fluphenazine (5 μ M). Gene expression analysis was performed for three HIF1 target genes (SLC2A3, VEGFA and BNIP3) by real-time quantitative PCR (RT-qPCR). hRP-L32 was used as reference gene and relative expression level were normalized to the untreated control (no DFO, DMSO). Bars show mean with S.D. ( n =3). ( c ) HCT116-HRE-Luc cells were treated with either Ethanol control or 100 μ M SM (18:1/16) and incubated for 24 h either in normoxia or hypoxia. After incubation, cells were lysed and luciferase activity was measured ( n =3). ( d ) RT-qPCR analysis of HIF-1-a mRNA level of HCT116 Spheroids treated for 24 h in normoxia or hypoxia with either DMSO control or 5 μ M Fluphenazine. hRP-L32 was used as reference gene and the relative expression level were normalized with the untreated control (normoxia DMSO control). Bars show mean with S.D. ( n =3). ( e ) Western blotting analysis of HIF-1-a protein expression in HCT116 spheroids or HCT116 cells grown in 2D that were treated for 24 h with either DMSO control or 5 μ M Fluphenazine under normoxia, hypoxia or normoxia+1 mM DFO. Representative data of multiple experiments are shown ( n =3). Intensity values were normalized to loading control and DMSO-treated controls under hypoxia (upper row) or DMSO controls co-incubated with DFO (lower row). Beta-actin was used as an internal control (not shown). * = P -value between 0.01 and 0.05, ** = P -value between 0.001 and 0.01, *** = P -value between 0.0001 and 0.001, **** = P -value smaller 0.0001
    Figure Legend Snippet: Fluphenazine induces HIF overactivation in conditions of high HIF background levels. ( a ) HIF activity reporter cells HCT116-HRE-Luc were treated either with DMSO control or 10 μ M Fluphenazine and incubated for 16 h in normoxia, hypoxia or in normoxia with the HIF-PH inhibitor DFO. After incubation, cells were lysed and luciferase activity was measured. Bars show mean with S.D. ( n =3). ( b ) HCT116 cells were treated for 24 h with or without DFO (1 mM) and additionally with either DMSO control or Fluphenazine (5 μ M). Gene expression analysis was performed for three HIF1 target genes (SLC2A3, VEGFA and BNIP3) by real-time quantitative PCR (RT-qPCR). hRP-L32 was used as reference gene and relative expression level were normalized to the untreated control (no DFO, DMSO). Bars show mean with S.D. ( n =3). ( c ) HCT116-HRE-Luc cells were treated with either Ethanol control or 100 μ M SM (18:1/16) and incubated for 24 h either in normoxia or hypoxia. After incubation, cells were lysed and luciferase activity was measured ( n =3). ( d ) RT-qPCR analysis of HIF-1-a mRNA level of HCT116 Spheroids treated for 24 h in normoxia or hypoxia with either DMSO control or 5 μ M Fluphenazine. hRP-L32 was used as reference gene and the relative expression level were normalized with the untreated control (normoxia DMSO control). Bars show mean with S.D. ( n =3). ( e ) Western blotting analysis of HIF-1-a protein expression in HCT116 spheroids or HCT116 cells grown in 2D that were treated for 24 h with either DMSO control or 5 μ M Fluphenazine under normoxia, hypoxia or normoxia+1 mM DFO. Representative data of multiple experiments are shown ( n =3). Intensity values were normalized to loading control and DMSO-treated controls under hypoxia (upper row) or DMSO controls co-incubated with DFO (lower row). Beta-actin was used as an internal control (not shown). * = P -value between 0.01 and 0.05, ** = P -value between 0.001 and 0.01, *** = P -value between 0.0001 and 0.001, **** = P -value smaller 0.0001

    Techniques Used: Activity Assay, Incubation, Luciferase, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

    18) Product Images from "Delivery of VEGFA in bone marrow stromal cells seeded in copolymer scaffold enhances angiogenesis, but is inadequate for osteogenesis as compared with the dual delivery of VEGFA and BMP2 in a subcutaneous mouse model"

    Article Title: Delivery of VEGFA in bone marrow stromal cells seeded in copolymer scaffold enhances angiogenesis, but is inadequate for osteogenesis as compared with the dual delivery of VEGFA and BMP2 in a subcutaneous mouse model

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-018-0778-4

    Adenoviral (ad) vector-mediated combined delivery of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor A (VEGFA) led to upregulation of osteogenic and angiogenic molecules in vitro . ad-GFP, ad-VEGFA, or ad-BMP2 + VEGFA BMSC (5 × 10 4 ) were seeded in each scaffold and harvested at 3 and 14 days for custom polymerase chain reaction (PCR) array and TaqMan-based qRT-PCR. a , b Unsupervised hierarchical clustering demonstrated two separate clusters consisting of replicates of ad-BMP2 + VEGFA, and replicates of ad-GFP and ad-VEGFA BMSC at both time points. c ANOVA disclosed that ALPL , BMP7 , BMP6 , FLT1 , and PGF mRNA levels were found to be significantly upregulated at 3 days in ad-BMP2 + VEGFA BMSC. d More osteogenic markers were significantly induced at 14 days in ad-BMP2 + VEGFA BMSC compared with the ad-GFP and ad-VEGFA BMSC groups. Error bars represent SEM of three biological replicates ( n = 3) performed in three technical replicates. ANOVA test with Bonferroni post hoc analysis was applied in c and d. e Representative images demonstrating higher alkaline phosphatase activity in ad-BMP2 + VEGFA BMSC in monolayer culture as compared with the ad-GFP and ad-VEGFA BMSC at 3 and 14 days. Experiments were repeated at least three times. *** p
    Figure Legend Snippet: Adenoviral (ad) vector-mediated combined delivery of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor A (VEGFA) led to upregulation of osteogenic and angiogenic molecules in vitro . ad-GFP, ad-VEGFA, or ad-BMP2 + VEGFA BMSC (5 × 10 4 ) were seeded in each scaffold and harvested at 3 and 14 days for custom polymerase chain reaction (PCR) array and TaqMan-based qRT-PCR. a , b Unsupervised hierarchical clustering demonstrated two separate clusters consisting of replicates of ad-BMP2 + VEGFA, and replicates of ad-GFP and ad-VEGFA BMSC at both time points. c ANOVA disclosed that ALPL , BMP7 , BMP6 , FLT1 , and PGF mRNA levels were found to be significantly upregulated at 3 days in ad-BMP2 + VEGFA BMSC. d More osteogenic markers were significantly induced at 14 days in ad-BMP2 + VEGFA BMSC compared with the ad-GFP and ad-VEGFA BMSC groups. Error bars represent SEM of three biological replicates ( n = 3) performed in three technical replicates. ANOVA test with Bonferroni post hoc analysis was applied in c and d. e Representative images demonstrating higher alkaline phosphatase activity in ad-BMP2 + VEGFA BMSC in monolayer culture as compared with the ad-GFP and ad-VEGFA BMSC at 3 and 14 days. Experiments were repeated at least three times. *** p

    Techniques Used: Plasmid Preparation, In Vitro, Polymerase Chain Reaction, Quantitative RT-PCR, Activity Assay

    Combined delivery of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor A (VEGFA) induced ectopic bone formation in scaffold explants in a subcutaneous NOD/SCID mouse model. The ability of VEGFA alone or in combination with BMP2 to induce ectopic bone formation was investigated in NOD/SCID mice by subcutaneously implanting scaffolds seeded with 5 × 10 5 BMSC from different experimental groups. Scaffold explants at 8 weeks were examined by μCT to evaluate ectopic bone formation. Negligible amounts of radiopaque bone-like structures were detected in a untransduced, b ad-GFP, and c ad-VEGFA BMSC. In contrast, numerous bony-like radiopaque structures were found at the periphery as well as within the scaffolds of ad-BMP2 + VEGFA explants ( d , cross sectional view in e ). f Quantification of the radiopaque bone-like structures in the scaffold explants revealed significant amounts of bone formation in ad-BMP2 + VEGFA scaffolds. g – n Representative images of H E stained FFPE sections of scaffold explants from different experimental groups at 2 and 8 weeks. g – l Formation of bony structures was not detected in untransduced, ad-GFP, or ad-VEGFA groups at either 2 or 8 weeks. m In the ad-BMP2 + VEGFA BMSC group, formation of bony structures (black arrows) could be seen as early as 2 weeks, mostly at the periphery of the scaffold explants. n Extensive bone formation (green arrows) with bony trabeculae extending inside the scaffolds was found at 8 weeks in ad-BMP2 + VEGFA BMSC. The structure consisted of numerous osteocyte-like cells at both 2 (black arrowheads, inset in m ) and 8 weeks (green arrowheads, inset in n ). ANOVA with Bonferroni post hoc analysis was carried out for statistical analysis in ( f ). Error bars represent SEM. ** p
    Figure Legend Snippet: Combined delivery of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor A (VEGFA) induced ectopic bone formation in scaffold explants in a subcutaneous NOD/SCID mouse model. The ability of VEGFA alone or in combination with BMP2 to induce ectopic bone formation was investigated in NOD/SCID mice by subcutaneously implanting scaffolds seeded with 5 × 10 5 BMSC from different experimental groups. Scaffold explants at 8 weeks were examined by μCT to evaluate ectopic bone formation. Negligible amounts of radiopaque bone-like structures were detected in a untransduced, b ad-GFP, and c ad-VEGFA BMSC. In contrast, numerous bony-like radiopaque structures were found at the periphery as well as within the scaffolds of ad-BMP2 + VEGFA explants ( d , cross sectional view in e ). f Quantification of the radiopaque bone-like structures in the scaffold explants revealed significant amounts of bone formation in ad-BMP2 + VEGFA scaffolds. g – n Representative images of H E stained FFPE sections of scaffold explants from different experimental groups at 2 and 8 weeks. g – l Formation of bony structures was not detected in untransduced, ad-GFP, or ad-VEGFA groups at either 2 or 8 weeks. m In the ad-BMP2 + VEGFA BMSC group, formation of bony structures (black arrows) could be seen as early as 2 weeks, mostly at the periphery of the scaffold explants. n Extensive bone formation (green arrows) with bony trabeculae extending inside the scaffolds was found at 8 weeks in ad-BMP2 + VEGFA BMSC. The structure consisted of numerous osteocyte-like cells at both 2 (black arrowheads, inset in m ) and 8 weeks (green arrowheads, inset in n ). ANOVA with Bonferroni post hoc analysis was carried out for statistical analysis in ( f ). Error bars represent SEM. ** p

    Techniques Used: Mouse Assay, Staining, Formalin-fixed Paraffin-Embedded

    Adenoviral (ad) vector-mediated expression of vascular endothelial growth factor A (VEGFA) alone and in combination with bone morphogenetic protein 2 (BMP2) was associated with enhanced angiogenesis in scaffold explants. a Representative images at 2 weeks revealed more reddish scaffold explants with multiple capillary/vessel like structures (black arrows) radiating from the periphery in ad-VEGFA and ad-BMP2 + VEGFA groups compared with the other control explants. b – e Frozen sections of scaffold explants at 2 weeks were stained with anti-CD31 targeting mouse CD31 protein and examined for capillary-like structures. Few CD31-positive structures were found in untransduced and ad-GFP scaffold explants ( b and c , white arrows). However, in ad-VEGFA and ad-BMP2 + VEGFA explants, many CD31-positive capillary-like structures (white arrows) were seen both at the periphery (not shown in the figure) as well as within the scaffolds ( d , e ). Red arrowheads indicate remnants of the scaffold material. f Quantification of CD31-positive area in the scaffold explants at 2 weeks demonstrated significantly more CD31-positive structures in ad-VEGFA and ad-BMP2 + VEGFA (ad-B + V) scaffolds than in the untransduced and ad-GFP explants. ANOVA test with Bonferroni post hoc analysis was used for statistical analysis in ( e ). Error bars represent SEM. *** p
    Figure Legend Snippet: Adenoviral (ad) vector-mediated expression of vascular endothelial growth factor A (VEGFA) alone and in combination with bone morphogenetic protein 2 (BMP2) was associated with enhanced angiogenesis in scaffold explants. a Representative images at 2 weeks revealed more reddish scaffold explants with multiple capillary/vessel like structures (black arrows) radiating from the periphery in ad-VEGFA and ad-BMP2 + VEGFA groups compared with the other control explants. b – e Frozen sections of scaffold explants at 2 weeks were stained with anti-CD31 targeting mouse CD31 protein and examined for capillary-like structures. Few CD31-positive structures were found in untransduced and ad-GFP scaffold explants ( b and c , white arrows). However, in ad-VEGFA and ad-BMP2 + VEGFA explants, many CD31-positive capillary-like structures (white arrows) were seen both at the periphery (not shown in the figure) as well as within the scaffolds ( d , e ). Red arrowheads indicate remnants of the scaffold material. f Quantification of CD31-positive area in the scaffold explants at 2 weeks demonstrated significantly more CD31-positive structures in ad-VEGFA and ad-BMP2 + VEGFA (ad-B + V) scaffolds than in the untransduced and ad-GFP explants. ANOVA test with Bonferroni post hoc analysis was used for statistical analysis in ( e ). Error bars represent SEM. *** p

    Techniques Used: Plasmid Preparation, Expressing, Staining

    VEGFA and BMP2 adenoviral vectors (ad) respectively upregulated the expression of vascular endothelial growth factor A (VEGFA) and bone morphogenetic protein 2 (BMP2) in BMSC. Representative immunofluorescence images of BMSC in monolayer transduced with ad-GFP ( a ), ad-VEGFA ( b ), and ad-VEGFA + BMP2 ( c – e ) adenoviral particles. BMSC (5× 10 4 ) were seeded in each scaffold and harvested after 3 and 14 days for mRNA and protein analyses. Significant upregulation of VEGFA mRNA ( f ) and VEGFA protein ( h ) levels were found at both time points in both ad-VEGFA and ad-BMP2 + ad-VEGFA BMSC compared with the ad-GFP BMSC. Similarly, significant upregulation of BMP2 mRNA ( g ) was found in ad-BMP2 + VEGFA BMSC compared with the ad-GFP and ad-VEGFA BMSC at both time points. VEGFA and BMP2 mRNA levels were normalized to GAPDH mRNA level. i ELISA disclosed higher levels of secreted BMP2 in the culture supernatant of ad-BMP2 BMSC compared with the ad-GFP BMSC at both 3 and 14 days. Error bars in ( f ) and ( g ) represent SEM of three repeated experiments ( n = 3) performed in three technical replicates. ANOVA test with Bonferroni post hoc analysis was used for statistical analysis in ( f ) and ( g ) and Student’s t test was performed in ( i ). Error bars in ( i ) represent SEM of three repeated experiments ( n = 3). *** p
    Figure Legend Snippet: VEGFA and BMP2 adenoviral vectors (ad) respectively upregulated the expression of vascular endothelial growth factor A (VEGFA) and bone morphogenetic protein 2 (BMP2) in BMSC. Representative immunofluorescence images of BMSC in monolayer transduced with ad-GFP ( a ), ad-VEGFA ( b ), and ad-VEGFA + BMP2 ( c – e ) adenoviral particles. BMSC (5× 10 4 ) were seeded in each scaffold and harvested after 3 and 14 days for mRNA and protein analyses. Significant upregulation of VEGFA mRNA ( f ) and VEGFA protein ( h ) levels were found at both time points in both ad-VEGFA and ad-BMP2 + ad-VEGFA BMSC compared with the ad-GFP BMSC. Similarly, significant upregulation of BMP2 mRNA ( g ) was found in ad-BMP2 + VEGFA BMSC compared with the ad-GFP and ad-VEGFA BMSC at both time points. VEGFA and BMP2 mRNA levels were normalized to GAPDH mRNA level. i ELISA disclosed higher levels of secreted BMP2 in the culture supernatant of ad-BMP2 BMSC compared with the ad-GFP BMSC at both 3 and 14 days. Error bars in ( f ) and ( g ) represent SEM of three repeated experiments ( n = 3) performed in three technical replicates. ANOVA test with Bonferroni post hoc analysis was used for statistical analysis in ( f ) and ( g ) and Student’s t test was performed in ( i ). Error bars in ( i ) represent SEM of three repeated experiments ( n = 3). *** p

    Techniques Used: Expressing, Immunofluorescence, Transduction, Enzyme-linked Immunosorbent Assay

    Bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor A (VEGFA) co-expression-mediated bone formation is associated with upregulation of osteogenic and angiogenic markers in the in vivo scaffold explants. Custom PCR array was used to examine differentially expressed osteogenesis- and angiogenesis-related genes in scaffold explants at 2 weeks. ANOVA revealed upregulation of a number of osteogenesis- and angiogenesis-related genes such as a ALPL , b RUNX2 , c SPP1 ( osteopontin ), d ANGPT1 , and e PGF in ad-BMP2 + VEGFA explants compared with the ad-GFP and ad-VEGFA explants. ANOVA test with Bonferroni post hoc analysis was undertaken for statistical analysis. ** p
    Figure Legend Snippet: Bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor A (VEGFA) co-expression-mediated bone formation is associated with upregulation of osteogenic and angiogenic markers in the in vivo scaffold explants. Custom PCR array was used to examine differentially expressed osteogenesis- and angiogenesis-related genes in scaffold explants at 2 weeks. ANOVA revealed upregulation of a number of osteogenesis- and angiogenesis-related genes such as a ALPL , b RUNX2 , c SPP1 ( osteopontin ), d ANGPT1 , and e PGF in ad-BMP2 + VEGFA explants compared with the ad-GFP and ad-VEGFA explants. ANOVA test with Bonferroni post hoc analysis was undertaken for statistical analysis. ** p

    Techniques Used: Expressing, In Vivo, Polymerase Chain Reaction

    19) Product Images from "VEGFA and NFE2L2 Gene Expression and Regulation by MicroRNAs in Thyroid Papillary Cancer and Colloid Goiter"

    Article Title: VEGFA and NFE2L2 Gene Expression and Regulation by MicroRNAs in Thyroid Papillary Cancer and Colloid Goiter

    Journal: Genes

    doi: 10.3390/genes11090954

    Protein expression of VEGFA in tumor, goiter, and normal tissue samples. VEGFA concentration was Log10 transformed ( y -axis). Mann–Whitney test (* p value).
    Figure Legend Snippet: Protein expression of VEGFA in tumor, goiter, and normal tissue samples. VEGFA concentration was Log10 transformed ( y -axis). Mann–Whitney test (* p value).

    Techniques Used: Expressing, Concentration Assay, Transformation Assay, MANN-WHITNEY

    Expression levels of ( A ) VEGFA ( Vascular Endothelial Growth Factor A ), ( B ) NFE2L2 (Nuclear Factor (Erythroid-derived 2)-Like 2 ), ( C ) miR-17-5p, and ( D ) miR-612 in tumor and goiter tissues and their respective adjacent tissues. Data are presented as median with interquartile range (25% percentile and 75% percentile). The relative expression value was Log2 transformed ( y -axis). Calibrator (normal tissue) log RQ = 1. *, Statistically significant (panel A, Mann–Whitney, p
    Figure Legend Snippet: Expression levels of ( A ) VEGFA ( Vascular Endothelial Growth Factor A ), ( B ) NFE2L2 (Nuclear Factor (Erythroid-derived 2)-Like 2 ), ( C ) miR-17-5p, and ( D ) miR-612 in tumor and goiter tissues and their respective adjacent tissues. Data are presented as median with interquartile range (25% percentile and 75% percentile). The relative expression value was Log2 transformed ( y -axis). Calibrator (normal tissue) log RQ = 1. *, Statistically significant (panel A, Mann–Whitney, p

    Techniques Used: Expressing, Derivative Assay, Transformation Assay, MANN-WHITNEY

    Results of the transfection assay on the TPC-1 cell line using mirVana™ miRNA Inhibitor for miR-17-5p, and mirVana™ miRNA Mimic for miR-612. VEGFA expression did not differ between cells transfected and non-transfected with miR-612 ( A ) and the inhibitor for miR-17-5p ( C ). NFE2L2 expression did not differ between cells transfected and non-transfected with miR-612 ( B ); the inhibition of miR-17-5p resulted in approximately 73% inhibition of NFE2L2 expression ( D ). *, approximately 73% inhibition of gene expression in relation to the negative control. Calibrator (negative control) log RQ = 1.
    Figure Legend Snippet: Results of the transfection assay on the TPC-1 cell line using mirVana™ miRNA Inhibitor for miR-17-5p, and mirVana™ miRNA Mimic for miR-612. VEGFA expression did not differ between cells transfected and non-transfected with miR-612 ( A ) and the inhibitor for miR-17-5p ( C ). NFE2L2 expression did not differ between cells transfected and non-transfected with miR-612 ( B ); the inhibition of miR-17-5p resulted in approximately 73% inhibition of NFE2L2 expression ( D ). *, approximately 73% inhibition of gene expression in relation to the negative control. Calibrator (negative control) log RQ = 1.

    Techniques Used: Transfection, Expressing, Inhibition, Negative Control

    20) Product Images from "Metabolic flux-driven sialylation alters internalization, recycling, and drug sensitivity of the epidermal growth factor receptor (EGFR) in SW1990 pancreatic cancer cells"

    Article Title: Metabolic flux-driven sialylation alters internalization, recycling, and drug sensitivity of the epidermal growth factor receptor (EGFR) in SW1990 pancreatic cancer cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.11582

    RT-PCR analysis of SW1990 cells treated with and without 100 μM of 1,3,4- O -Bu 3 ManNAc showed ( A ) a significant decrease in expression of STAT3 associated genes BCL3, MMP2 and MMP7 whereas ( B ) VEGFA and MYC, which can also be regulated by STAT3, showed increased expression (VEGFA) or was not affected (MYC). At least 3 biological replicates were carried out for each experiment with data expressed as mean ± standard error mean (SEM). * indicates a p value of
    Figure Legend Snippet: RT-PCR analysis of SW1990 cells treated with and without 100 μM of 1,3,4- O -Bu 3 ManNAc showed ( A ) a significant decrease in expression of STAT3 associated genes BCL3, MMP2 and MMP7 whereas ( B ) VEGFA and MYC, which can also be regulated by STAT3, showed increased expression (VEGFA) or was not affected (MYC). At least 3 biological replicates were carried out for each experiment with data expressed as mean ± standard error mean (SEM). * indicates a p value of

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

    21) Product Images from "Regulation of proinflammatory genes by the circulating microRNA hsa-miR-939"

    Article Title: Regulation of proinflammatory genes by the circulating microRNA hsa-miR-939

    Journal: Scientific Reports

    doi: 10.1038/srep30976

    Expression levels of miR-939 target genes IL-6, TNFα and VEGFA in THP-1 cells transfected with miR-939 followed by 6 hours of stimulation with LPS. ( A–C ) Taqman analysis of endogenous levels of IL-6, TNFα and VEGFA, after LPS induction, showed that transfection with miR-939 reduced IL-6 transcripts compared to miR- control. ( D–F ) Overexpression of miR-939 decreased secreted IL-6, and VEGFA. ELISA using cell culture supernatants of THP-1 cells stimulated with LPS showed lower levels of these two proinflammatory mediators secreted by miR-939 transfected cells compared to control miRNA transfection. Significance was determined by one-way ANOVA with Dunnet’s post hoc test *p
    Figure Legend Snippet: Expression levels of miR-939 target genes IL-6, TNFα and VEGFA in THP-1 cells transfected with miR-939 followed by 6 hours of stimulation with LPS. ( A–C ) Taqman analysis of endogenous levels of IL-6, TNFα and VEGFA, after LPS induction, showed that transfection with miR-939 reduced IL-6 transcripts compared to miR- control. ( D–F ) Overexpression of miR-939 decreased secreted IL-6, and VEGFA. ELISA using cell culture supernatants of THP-1 cells stimulated with LPS showed lower levels of these two proinflammatory mediators secreted by miR-939 transfected cells compared to control miRNA transfection. Significance was determined by one-way ANOVA with Dunnet’s post hoc test *p

    Techniques Used: Expressing, Transfection, Over Expression, Enzyme-linked Immunosorbent Assay, Cell Culture

    Relative expressions of VEGFA and TNFα mRNA in whole blood from CRPS patients and control individuals. Taqman analysis of target mRNAs of miR-939 that were consistently detectable in whole blood, showed a significant increase of VEGFA mRNA in patients with CRPS compared to control samples. TNFα mRNA levels were not significant between CRPS and control samples. GAPDH was used as the normalizer. Data represent mean ± SEM CRPS (n = 37) and control (n = 18). Statistical significance was calculated using Student t-test **p
    Figure Legend Snippet: Relative expressions of VEGFA and TNFα mRNA in whole blood from CRPS patients and control individuals. Taqman analysis of target mRNAs of miR-939 that were consistently detectable in whole blood, showed a significant increase of VEGFA mRNA in patients with CRPS compared to control samples. TNFα mRNA levels were not significant between CRPS and control samples. GAPDH was used as the normalizer. Data represent mean ± SEM CRPS (n = 37) and control (n = 18). Statistical significance was calculated using Student t-test **p

    Techniques Used:

    22) Product Images from "Cardiovascular Risk Factors and Differential Transcriptomic Profile of the Subcutaneous and Visceral Adipose Tissue and Their Resident Stem Cells"

    Article Title: Cardiovascular Risk Factors and Differential Transcriptomic Profile of the Subcutaneous and Visceral Adipose Tissue and Their Resident Stem Cells

    Journal: Cells

    doi: 10.3390/cells9102235

    Box-plot diagram showing adipose-derived stem cells (ASCs)-transcriptomic profile. ( A ) Differential mRNA expression of TF in SAT-ASC and VAT-ASC from diabetic with obesity and non-diabetic patients; ( B ) Differential mRNA expression of VEGFA in SAT-ASC and VAT-ASC from obese diabetic and non-diabetic patients; ( C ) Differential miR126 expression in SAT-ASC and VAT-ASC from obese diabetic and non-diabetic patients; ( D ) Differential miR145 expression in SAT-ASC and VAT-ASC from diabetic with obesity and non-diabetic patients. (* p
    Figure Legend Snippet: Box-plot diagram showing adipose-derived stem cells (ASCs)-transcriptomic profile. ( A ) Differential mRNA expression of TF in SAT-ASC and VAT-ASC from diabetic with obesity and non-diabetic patients; ( B ) Differential mRNA expression of VEGFA in SAT-ASC and VAT-ASC from obese diabetic and non-diabetic patients; ( C ) Differential miR126 expression in SAT-ASC and VAT-ASC from obese diabetic and non-diabetic patients; ( D ) Differential miR145 expression in SAT-ASC and VAT-ASC from diabetic with obesity and non-diabetic patients. (* p

    Techniques Used: Derivative Assay, Expressing

    TF and VEGF expression in in Subcutaneous or Visceral Derived ASCs from Diabetic/No Diabetic Patients with Obesity. ( A ) TF and VEGFA expression in ASCs obtained from SAT (S) or VAT (V) in obese patients (OB) in presence (DM) or absence (nDM) of diabetes. Western blot analysis of TF and VEGFA proteins in ASCs. To test for equal loading Western blots were reproved by β-actin; ( B ) Quantitative analysis of TF and VEGFA to β-actin relative levels.
    Figure Legend Snippet: TF and VEGF expression in in Subcutaneous or Visceral Derived ASCs from Diabetic/No Diabetic Patients with Obesity. ( A ) TF and VEGFA expression in ASCs obtained from SAT (S) or VAT (V) in obese patients (OB) in presence (DM) or absence (nDM) of diabetes. Western blot analysis of TF and VEGFA proteins in ASCs. To test for equal loading Western blots were reproved by β-actin; ( B ) Quantitative analysis of TF and VEGFA to β-actin relative levels.

    Techniques Used: Expressing, Derivative Assay, Western Blot

    Line regression between expression of validated genes and body mass index (BMI). Correlations were determined by linear correlations. ( A ) Line regression between AGER and body mass index; ( B ) Line regression between ANGPT2 and body mass index; ( C ) Line regression between CD68 and body mass index; ( D ) Line regression between IFI30 and body mass index; ( E ) Line regression between NOCH3 and body mass index; ( F ) Line regression between SPP1 and body mass index; ( G ) Line regression between DLL4 and body mass index; ( H ) Line regression between PLIN2 and body mass index; ( I ) Line regression between VEGFA and body mass index; ( J ) Line regression between PINPLA2 and body mass index.
    Figure Legend Snippet: Line regression between expression of validated genes and body mass index (BMI). Correlations were determined by linear correlations. ( A ) Line regression between AGER and body mass index; ( B ) Line regression between ANGPT2 and body mass index; ( C ) Line regression between CD68 and body mass index; ( D ) Line regression between IFI30 and body mass index; ( E ) Line regression between NOCH3 and body mass index; ( F ) Line regression between SPP1 and body mass index; ( G ) Line regression between DLL4 and body mass index; ( H ) Line regression between PLIN2 and body mass index; ( I ) Line regression between VEGFA and body mass index; ( J ) Line regression between PINPLA2 and body mass index.

    Techniques Used: Expressing

    23) Product Images from "Enhancing Osteoconductivity of Fibrin Gels with Apatite-Coated Polymer Microspheres"

    Article Title: Enhancing Osteoconductivity of Fibrin Gels with Apatite-Coated Polymer Microspheres

    Journal: Tissue Engineering. Part A

    doi: 10.1089/ten.tea.2012.0288

    Gene expression for hMSCs entrapped in fibrin gels after 7-, 14- and 21-day culture. (A) RUNX2 ; (B) SP7 ; and (C) VEGFA . Chart values represent mean±SD; n =4; * p
    Figure Legend Snippet: Gene expression for hMSCs entrapped in fibrin gels after 7-, 14- and 21-day culture. (A) RUNX2 ; (B) SP7 ; and (C) VEGFA . Chart values represent mean±SD; n =4; * p

    Techniques Used: Expressing

    24) Product Images from "MiR-578 and miR-573 as potential players in BRCA-related breast cancer angiogenesis"

    Article Title: MiR-578 and miR-573 as potential players in BRCA-related breast cancer angiogenesis

    Journal: Oncotarget

    doi:

    (A) HEK293 cells were co-transfected with the indicated reporter constructs. The Luciferase Activity was normalized to the level of Renilla luciferase (pRNL) and the Relative Luciferase Activity (RLA) was calibrated to 1 that refers the RLA of cells transfected with the hsa-miR-negative control as reported in Materials and Methods; cells were transfected with hsa-miR-578, hsa-miR-573 and negative control miRNA mimic (B) HEK293 (C) MCF-7 and (D) SUM149PT. Cells transfected with the negative control were calibrated to 1 and relative expression of VEGFA, FAK, ANGPT2 and HIF1A genes was explored.
    Figure Legend Snippet: (A) HEK293 cells were co-transfected with the indicated reporter constructs. The Luciferase Activity was normalized to the level of Renilla luciferase (pRNL) and the Relative Luciferase Activity (RLA) was calibrated to 1 that refers the RLA of cells transfected with the hsa-miR-negative control as reported in Materials and Methods; cells were transfected with hsa-miR-578, hsa-miR-573 and negative control miRNA mimic (B) HEK293 (C) MCF-7 and (D) SUM149PT. Cells transfected with the negative control were calibrated to 1 and relative expression of VEGFA, FAK, ANGPT2 and HIF1A genes was explored.

    Techniques Used: Transfection, Construct, Luciferase, Activity Assay, Negative Control, Expressing

    Mean expression levels with S.E.M. of both miR-578, miR-573 and their target genes VEGFA, HIF1A, FAK and ANGPT2 in BRCA1/2 carriers and BRCAX associated tumors.
    Figure Legend Snippet: Mean expression levels with S.E.M. of both miR-578, miR-573 and their target genes VEGFA, HIF1A, FAK and ANGPT2 in BRCA1/2 carriers and BRCAX associated tumors.

    Techniques Used: Expressing

    25) Product Images from "Angiogenesis in adenosquamous cancer of pancreas"

    Article Title: Angiogenesis in adenosquamous cancer of pancreas

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21319

    Bar graphs show log2 fold changes of differentially expressed miRNAs and genes between ASCP and PDAC cases Higher VEGFA, HIF1A, Ang-2 expression and lower miR-122-5p levels were detected in ASCP with respect to PDAC (A) . Lower Tie-2, Ang-1 transcript levels and higher miR-21-5p, miR-27a-3p and miR-181a-5p were observed in ASCP with respect to PDAC (B) .
    Figure Legend Snippet: Bar graphs show log2 fold changes of differentially expressed miRNAs and genes between ASCP and PDAC cases Higher VEGFA, HIF1A, Ang-2 expression and lower miR-122-5p levels were detected in ASCP with respect to PDAC (A) . Lower Tie-2, Ang-1 transcript levels and higher miR-21-5p, miR-27a-3p and miR-181a-5p were observed in ASCP with respect to PDAC (B) .

    Techniques Used: Expressing

    26) Product Images from "Bones in human CYP26B1 deficiency and rats with hypervitaminosis A phenocopy Vegfa overexpression"

    Article Title: Bones in human CYP26B1 deficiency and rats with hypervitaminosis A phenocopy Vegfa overexpression

    Journal: Bone Reports

    doi: 10.1016/j.bonr.2018.06.006

    Early pathological features of hypervitaminosis A in young rats. a) Plasma levels of Vegfa, Tnfa and soluble Icam 1 (sIcam1) protein at day 1, 2 and 7 into hypervitaminosis A, determined by ELISA. n = 4–5/group (day 1 and day 2) and n = 9–10/group (day 7). Student's t -test; p
    Figure Legend Snippet: Early pathological features of hypervitaminosis A in young rats. a) Plasma levels of Vegfa, Tnfa and soluble Icam 1 (sIcam1) protein at day 1, 2 and 7 into hypervitaminosis A, determined by ELISA. n = 4–5/group (day 1 and day 2) and n = 9–10/group (day 7). Student's t -test; p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Normal human endothelial cell and osteoblast response to retinoic acid (RA). a) CYP26B1 mRNA expression in human dermal microvascular endothelial cells (HDMEC) and human primary osteoblasts (HOB) treated with or without 400 nM RA for 24 h (h). b) VEGFA mRNA expression in HDMEC and HOB cells treated with or without 400 nM RA for 24 h or 48 h. c) Representative pictures of HDMEC cells treated with or without 400 nM RA for 24 and 48 h. Yellow arrowheads indicate RA induced cell contraction/slimming noticed at both time points. RA did not induce apoptosis as determined by caspase-3 activity. d) Immunofluorescent VE-Cadherin staining of HDMEC cells treated with or without 400 nM RA for 48 h. White arrowheads indicate RA induced gaps in VE-cadherin staining at cell-cell junctions. RA reduced VE-Cadherin staining between endothelial cells. n = 4/treatment. Bar 25 μm. Results are presented as mean ± SD. Student's t -test; p
    Figure Legend Snippet: Normal human endothelial cell and osteoblast response to retinoic acid (RA). a) CYP26B1 mRNA expression in human dermal microvascular endothelial cells (HDMEC) and human primary osteoblasts (HOB) treated with or without 400 nM RA for 24 h (h). b) VEGFA mRNA expression in HDMEC and HOB cells treated with or without 400 nM RA for 24 h or 48 h. c) Representative pictures of HDMEC cells treated with or without 400 nM RA for 24 and 48 h. Yellow arrowheads indicate RA induced cell contraction/slimming noticed at both time points. RA did not induce apoptosis as determined by caspase-3 activity. d) Immunofluorescent VE-Cadherin staining of HDMEC cells treated with or without 400 nM RA for 48 h. White arrowheads indicate RA induced gaps in VE-cadherin staining at cell-cell junctions. RA reduced VE-Cadherin staining between endothelial cells. n = 4/treatment. Bar 25 μm. Results are presented as mean ± SD. Student's t -test; p

    Techniques Used: Expressing, Activity Assay, Staining

    Femur phenotype from an individual with CYP26B1 insufficiency. a) Cathepsin K immunohistochemical staining (brown) of pathological periosteal osteoclast accumulation in sections of fetal bones from an individual with CYP26B1 insufficiency, compared with periosteal osteoclast accumulation from a typical hypervitaminosis A rat bone. Bar = 100 μm. b) Hematoxylin stained section of femur bone tissue from this individual with CYP26B1 insufficiency show angulation at midpoint. Asterisk highlight “striped” bone formation at the angulation point. Bar = 200 μm. c) Osteopontin, PECAM1 and VEGFA immunohistochemical staining (brown) of this human femur bone tissue at the angulation point. Lower panel show pictures at higher magnification from boxed area. d) Dentin matrix protein 1 immunohistochemical staining (brown) of osteocytes and canaliculi in bone tissue at the angulation point of this human fetal femur.
    Figure Legend Snippet: Femur phenotype from an individual with CYP26B1 insufficiency. a) Cathepsin K immunohistochemical staining (brown) of pathological periosteal osteoclast accumulation in sections of fetal bones from an individual with CYP26B1 insufficiency, compared with periosteal osteoclast accumulation from a typical hypervitaminosis A rat bone. Bar = 100 μm. b) Hematoxylin stained section of femur bone tissue from this individual with CYP26B1 insufficiency show angulation at midpoint. Asterisk highlight “striped” bone formation at the angulation point. Bar = 200 μm. c) Osteopontin, PECAM1 and VEGFA immunohistochemical staining (brown) of this human femur bone tissue at the angulation point. Lower panel show pictures at higher magnification from boxed area. d) Dentin matrix protein 1 immunohistochemical staining (brown) of osteocytes and canaliculi in bone tissue at the angulation point of this human fetal femur.

    Techniques Used: Immunohistochemistry, Staining

    27) Product Images from "Interplay between miR-574-3p and hnRNP L regulates VEGFA mRNA translation and tumorigenesis"

    Article Title: Interplay between miR-574-3p and hnRNP L regulates VEGFA mRNA translation and tumorigenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx440

    miR-574-3p represses hnRNP L binding to VEGFA mRNA and translational activation in normoxia. ( A ) Bioinformatic identification of CA-rich miRNAs and their expression in U937 cells. qRT-PCR was performed and expression normalized to RNU6B. ( B ) Sequence of precursor miR-574 stem–loop. Guide strand miR-574-5p (blue) and passenger strand miR-574-3p (red) are highlighted; seed and CARE regions are underlined. ( C ) Interaction of miR-574-3p with hnRNP L and RISC shown by percentage of binding relative to total input. U937 cells were cultured under normoxic condition for 24 h. Lysates with the same amount of total protein were subjected to IP with anti-hnRNP L, -Ago2 or pre-immune IgG antibodies, and then qRT-PCR using miR-574-3p-specific probe. Input RNA from the same amount of lysates was used as normalizer. ( D ) Endogenous miR-574-3p inhibits VEGF-A expression. U937 cells were transfected with negative control anti-miR, anti-miR-574-3p, -297 (200 nM), or both (100 nM/each) for 48 h, and lysates immunoblotted with anti-VEGF-A and -β-actin antibodies. VEGFA and β-actin mRNA was determined by semi-quantitative RT-PCR (equal total RNA for RT; 25 and 15 cycles for PCR of VEGFA and actin β-mRNA, respectively). ( E ) Inactivation of miR-574-3p increases VEGFA mRNA translation. U937 cells were transfected with anti-miR-574-3p or control anti-miR (200 nM) for 48 h. Ribosome-free and -bound mRNA were fractionated on a polysome cushion (rRNA was shown), and total RNA extracts were subjected to qRT-PCR using VEGFA- and β-actin -specific probes. ( F ) Endogenous miR-574-3p reduces the interaction between hnRNP L and VEGFA mRNA. U937 cells were transfected with anti-miR-574-3p or control (200 nM) for 24 h. Lysates were subjected to RIP with anti-hnRNP L antibody coupled with qRT-PCR using VEGFA -specific probe. ( G ) Endogenous miR-574-3p inhibits expression of HSR-bearing reporter. FLuc reporter bearing the VEGFA HSR was co-transfected into U937 cells with anti-miR-574-3p or anti-miR-297, or both, for 48 h in normoxic condition. FLuc activity was normalized by RLuc and expressed as percentage of control. FLuc mRNA level was shown by normalization with RLuc mRNA. ( H ) Schematic showing effects of CA-rich miR-574-3p and hnRNP L on normoxic VEGF-A expression. VEGFA mRNA translation is inhibited by miR-297-RISC binding to VEGFA 3΄UTR CARE, and by miR-574-3p binding to hnRNP L as RNA decoy in normoxia. In (A, C–G), data are presented as mean ± SD ( n = 3; * P ≤ 0.05, ** P ≤ 0.01, Student's t -test).
    Figure Legend Snippet: miR-574-3p represses hnRNP L binding to VEGFA mRNA and translational activation in normoxia. ( A ) Bioinformatic identification of CA-rich miRNAs and their expression in U937 cells. qRT-PCR was performed and expression normalized to RNU6B. ( B ) Sequence of precursor miR-574 stem–loop. Guide strand miR-574-5p (blue) and passenger strand miR-574-3p (red) are highlighted; seed and CARE regions are underlined. ( C ) Interaction of miR-574-3p with hnRNP L and RISC shown by percentage of binding relative to total input. U937 cells were cultured under normoxic condition for 24 h. Lysates with the same amount of total protein were subjected to IP with anti-hnRNP L, -Ago2 or pre-immune IgG antibodies, and then qRT-PCR using miR-574-3p-specific probe. Input RNA from the same amount of lysates was used as normalizer. ( D ) Endogenous miR-574-3p inhibits VEGF-A expression. U937 cells were transfected with negative control anti-miR, anti-miR-574-3p, -297 (200 nM), or both (100 nM/each) for 48 h, and lysates immunoblotted with anti-VEGF-A and -β-actin antibodies. VEGFA and β-actin mRNA was determined by semi-quantitative RT-PCR (equal total RNA for RT; 25 and 15 cycles for PCR of VEGFA and actin β-mRNA, respectively). ( E ) Inactivation of miR-574-3p increases VEGFA mRNA translation. U937 cells were transfected with anti-miR-574-3p or control anti-miR (200 nM) for 48 h. Ribosome-free and -bound mRNA were fractionated on a polysome cushion (rRNA was shown), and total RNA extracts were subjected to qRT-PCR using VEGFA- and β-actin -specific probes. ( F ) Endogenous miR-574-3p reduces the interaction between hnRNP L and VEGFA mRNA. U937 cells were transfected with anti-miR-574-3p or control (200 nM) for 24 h. Lysates were subjected to RIP with anti-hnRNP L antibody coupled with qRT-PCR using VEGFA -specific probe. ( G ) Endogenous miR-574-3p inhibits expression of HSR-bearing reporter. FLuc reporter bearing the VEGFA HSR was co-transfected into U937 cells with anti-miR-574-3p or anti-miR-297, or both, for 48 h in normoxic condition. FLuc activity was normalized by RLuc and expressed as percentage of control. FLuc mRNA level was shown by normalization with RLuc mRNA. ( H ) Schematic showing effects of CA-rich miR-574-3p and hnRNP L on normoxic VEGF-A expression. VEGFA mRNA translation is inhibited by miR-297-RISC binding to VEGFA 3΄UTR CARE, and by miR-574-3p binding to hnRNP L as RNA decoy in normoxia. In (A, C–G), data are presented as mean ± SD ( n = 3; * P ≤ 0.05, ** P ≤ 0.01, Student's t -test).

    Techniques Used: Binding Assay, Activation Assay, Expressing, Quantitative RT-PCR, Sequencing, Cell Culture, Transfection, Negative Control, Polymerase Chain Reaction, Activity Assay

    miR-574-3p binds multiple hnRNP L domains and inhibits binding to mRNA targets and HILDA complex formation. ( A ) hnRNP L and miR-574-3p form stable complexes. 32 P-labeled miR-574-3p, miR-574-5p, or CARE mutant miR-574-3p were incubated with increasing amounts of recombinant hnRNP L or variants (0, 20, 50, 100, 200 and 500 nM), and RNA-protein complexes resolved by electrophoresis on a nondenaturing 5% polyacrylamide gel. ( B ) miR-574-3p-associated hnRNP L does not bind DRBP76 in hypoxia. U937 cells were cultured in hypoxia for 24 h. Lysates were IPed using anti-hnRNP L and -DRBP76 antibodies, and qRT-PCR was done with probes against miR-574-3p, miR-574-5p and miR-17. Data are presented as mean ± SD ( n = 3, Student's t -test). ( C ) miR-574-3p blocks HILDA complex assembly. U937 cells were transfected with miR-574-3p or control miR, and lysates IPed with anti-hnRNP L antibody and immunoblotted using anti-DRBP76, -hnRNP A2/B1 and -hnRNP L antibodies. ( D ) Schematic depicting interaction of hnRNP L and miR-574-3p and its downstream consequences. miR-574-3p CARE interacts with RRM1,2 and RRM3,4, and prevents binding to VEGFA mRNA CARE and DRBP76, respectively.
    Figure Legend Snippet: miR-574-3p binds multiple hnRNP L domains and inhibits binding to mRNA targets and HILDA complex formation. ( A ) hnRNP L and miR-574-3p form stable complexes. 32 P-labeled miR-574-3p, miR-574-5p, or CARE mutant miR-574-3p were incubated with increasing amounts of recombinant hnRNP L or variants (0, 20, 50, 100, 200 and 500 nM), and RNA-protein complexes resolved by electrophoresis on a nondenaturing 5% polyacrylamide gel. ( B ) miR-574-3p-associated hnRNP L does not bind DRBP76 in hypoxia. U937 cells were cultured in hypoxia for 24 h. Lysates were IPed using anti-hnRNP L and -DRBP76 antibodies, and qRT-PCR was done with probes against miR-574-3p, miR-574-5p and miR-17. Data are presented as mean ± SD ( n = 3, Student's t -test). ( C ) miR-574-3p blocks HILDA complex assembly. U937 cells were transfected with miR-574-3p or control miR, and lysates IPed with anti-hnRNP L antibody and immunoblotted using anti-DRBP76, -hnRNP A2/B1 and -hnRNP L antibodies. ( D ) Schematic depicting interaction of hnRNP L and miR-574-3p and its downstream consequences. miR-574-3p CARE interacts with RRM1,2 and RRM3,4, and prevents binding to VEGFA mRNA CARE and DRBP76, respectively.

    Techniques Used: Binding Assay, Labeling, Mutagenesis, Incubation, Recombinant, Electrophoresis, Cell Culture, Quantitative RT-PCR, Transfection

    CARE-dependent inhibition of hnRNP L by miR-574-3p reduces VEGF-A expression in hypoxia. ( A ) Overexpression of miR-574-3p represses polysome loading of VEGFA mRNA in hypoxia. U937 cells were transfected with miR-574-3p or control miR (200 nM) for 48 h in hypoxia, and lysates subjected to sucrose density gradient fractionation. RNA isolated from each fraction was subjected to qRT-PCR to determine VEGFA mRNA distribution. Inset: VEGF-A expression was measured by immunoblot. ( B ) CARE is required for miR-574-3p-mediated inhibition of VEGF-A in hypoxia. U937 cells were transfected with wild-type and mutant miR-574-3p, miR-297, or hnRNP L siRNA (200 nM) as indicated. After 48 h, cell lysates were subjected to immunoblot analysis with anti-VEGF-A or -β-actin antibody, and quantitated by densitometry. ( C ) Overexpression of miR-574-3p inhibits translation of HSR-bearing reporter in hypoxia. FLuc reporter bearing VEGFA HSR was co-transfected into U937 cells with RLuc reporter and wild-type and mutant miR-574-3p, miR-297 or both for 48 h. FLuc activity was normalized by RLuc expression. ( D ) Overexpression of hnRNP L overcomes miR-574-3p-mediated repression of VEGF-A in hypoxia. U937 cells were co-transfected with pcDNA3-Myc-hnRNPL or the H 105 A mutant and with miR-574-3p or miR-297, or both, for 48 h in Hpx. Lysates were subjected to immunoblot analysis with anti-VEGF-A or -β-actin antibodies. ( E ) Overexpressed hnRNP L reverses miR-574-3p-mediated repression of HSR-bearing reporter in hypoxia. FLuc reporter bearing V EGFA HSR was co-transfected into U937 cells with miR-574-3p, miR-297, or both (or control miR) in the presence of pcDNA3-Myc-hnRNPL or its H 105 A mutant for 48 h in Hpx. FLuc was normalized by RLuc expression. In (A–E), data are presented as mean ± SD ( n = 3; * P ≤ 0.05, Student's t -test).
    Figure Legend Snippet: CARE-dependent inhibition of hnRNP L by miR-574-3p reduces VEGF-A expression in hypoxia. ( A ) Overexpression of miR-574-3p represses polysome loading of VEGFA mRNA in hypoxia. U937 cells were transfected with miR-574-3p or control miR (200 nM) for 48 h in hypoxia, and lysates subjected to sucrose density gradient fractionation. RNA isolated from each fraction was subjected to qRT-PCR to determine VEGFA mRNA distribution. Inset: VEGF-A expression was measured by immunoblot. ( B ) CARE is required for miR-574-3p-mediated inhibition of VEGF-A in hypoxia. U937 cells were transfected with wild-type and mutant miR-574-3p, miR-297, or hnRNP L siRNA (200 nM) as indicated. After 48 h, cell lysates were subjected to immunoblot analysis with anti-VEGF-A or -β-actin antibody, and quantitated by densitometry. ( C ) Overexpression of miR-574-3p inhibits translation of HSR-bearing reporter in hypoxia. FLuc reporter bearing VEGFA HSR was co-transfected into U937 cells with RLuc reporter and wild-type and mutant miR-574-3p, miR-297 or both for 48 h. FLuc activity was normalized by RLuc expression. ( D ) Overexpression of hnRNP L overcomes miR-574-3p-mediated repression of VEGF-A in hypoxia. U937 cells were co-transfected with pcDNA3-Myc-hnRNPL or the H 105 A mutant and with miR-574-3p or miR-297, or both, for 48 h in Hpx. Lysates were subjected to immunoblot analysis with anti-VEGF-A or -β-actin antibodies. ( E ) Overexpressed hnRNP L reverses miR-574-3p-mediated repression of HSR-bearing reporter in hypoxia. FLuc reporter bearing V EGFA HSR was co-transfected into U937 cells with miR-574-3p, miR-297, or both (or control miR) in the presence of pcDNA3-Myc-hnRNPL or its H 105 A mutant for 48 h in Hpx. FLuc was normalized by RLuc expression. In (A–E), data are presented as mean ± SD ( n = 3; * P ≤ 0.05, Student's t -test).

    Techniques Used: Inhibition, Expressing, Over Expression, Transfection, Fractionation, Isolation, Quantitative RT-PCR, Mutagenesis, Activity Assay

    28) Product Images from "Differential expression of angiogenesis-related miRNAs and VEGFA in cirrhosis and hepatocellular carcinoma"

    Article Title: Differential expression of angiogenesis-related miRNAs and VEGFA in cirrhosis and hepatocellular carcinoma

    Journal: Archives of Medical Science : AMS

    doi: 10.5114/aoms.2020.97967

    Representative RQ median of VEGFA gene expression in liver cirrhosis and hepatocellular carcinoma tissues
    Figure Legend Snippet: Representative RQ median of VEGFA gene expression in liver cirrhosis and hepatocellular carcinoma tissues

    Techniques Used: Expressing

    29) Product Images from "Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation"

    Article Title: Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0147507

    BMP2 over-expression led to up-regulation of osteogenic and angiogenic molecules in vitro. 5 × 10 4 ad-BMP2 or ad-GFP BMSC were seeded in each Poly(LLA-co-CL) scaffolds and harvested after 3 and 14 days for custom PCR array and TaqMan based qRT-PCR. (A) With SAM analysis, ALPL and FLT1 mRNA levels were found to be significantly (FDR = 0) up-regulated at 3-days in ad-BMP2 BMSC. (B) More osteogenic and angiogenic markers were significantly induced at 14-days in ad-BMP2 BMSC as compared to the ad-GFP BMSC group. (C and D) Array results for ALPL , RUNX2 , BGLAP and VEGFA genes were independently validated in ad-BMP2 BMSC by using TaqMan qRT- PCR. Error bars represent SEM of 3 repeated experiments ( n = 3) done in 3 technical replicates. ANOVA test with Bonferroni post hoc analysis was performed for statistical analysis in C and D. ***, p
    Figure Legend Snippet: BMP2 over-expression led to up-regulation of osteogenic and angiogenic molecules in vitro. 5 × 10 4 ad-BMP2 or ad-GFP BMSC were seeded in each Poly(LLA-co-CL) scaffolds and harvested after 3 and 14 days for custom PCR array and TaqMan based qRT-PCR. (A) With SAM analysis, ALPL and FLT1 mRNA levels were found to be significantly (FDR = 0) up-regulated at 3-days in ad-BMP2 BMSC. (B) More osteogenic and angiogenic markers were significantly induced at 14-days in ad-BMP2 BMSC as compared to the ad-GFP BMSC group. (C and D) Array results for ALPL , RUNX2 , BGLAP and VEGFA genes were independently validated in ad-BMP2 BMSC by using TaqMan qRT- PCR. Error bars represent SEM of 3 repeated experiments ( n = 3) done in 3 technical replicates. ANOVA test with Bonferroni post hoc analysis was performed for statistical analysis in C and D. ***, p

    Techniques Used: Over Expression, In Vitro, Polymerase Chain Reaction, Quantitative RT-PCR

    30) Product Images from "Novel 1,3,4-thiadiazole–chalcone hybrids containing catechol moiety: synthesis, antioxidant activity, cytotoxicity and DNA interaction studies"

    Article Title: Novel 1,3,4-thiadiazole–chalcone hybrids containing catechol moiety: synthesis, antioxidant activity, cytotoxicity and DNA interaction studies

    Journal: MedChemComm

    doi: 10.1039/c8md00316e

    Changes in expression levels of MMP2 gene (A), MMP9 gene (B), TIMP3 gene (C), VEGFA gene (D), miR-21 (E), miR-133b (F), miR-155 (G), and miR-206 (H) in HeLa cells exposed to subtoxic IC 20 concentrations of the compounds 5a , 5c , 5f , and 5m (10 μM for each compound) for 24 h. Representative graphs are shown.
    Figure Legend Snippet: Changes in expression levels of MMP2 gene (A), MMP9 gene (B), TIMP3 gene (C), VEGFA gene (D), miR-21 (E), miR-133b (F), miR-155 (G), and miR-206 (H) in HeLa cells exposed to subtoxic IC 20 concentrations of the compounds 5a , 5c , 5f , and 5m (10 μM for each compound) for 24 h. Representative graphs are shown.

    Techniques Used: Expressing

    31) Product Images from "Hepatitis B Virus X Protein Upregulates mTOR Signaling through IKK? to Increase Cell Proliferation and VEGF Production in Hepatocellular Carcinoma"

    Article Title: Hepatitis B Virus X Protein Upregulates mTOR Signaling through IKK? to Increase Cell Proliferation and VEGF Production in Hepatocellular Carcinoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0041931

    Expression of VEGF is increased in Hep3Bx and HepG2x cells and is further enhanced by TNF-α and blocked by IKKβ inhibitor Bay 11-7082 or the mTOR inhibitor rapamycin. (A). The expression levels of secreted VEGF in the culture medium of Hep3B, Hep3Bx, HepG2, and HepG2x cells were measured by ELISA assay as described in the Methods . (B). The expression levels of VEGFA mRNA were assessed in Hep3B, Hep3Bx, HepG2, and HepG2x cells using semi-quantitative RT-PCR (left) or real-time RT-PCR (right) as described in the Methods . (C). The amounts of secreted VEGF in the culture medium of Hep3B, Hep3Bx, HepG2, or HepG2x cells treated with or without TNF-α in the presence or absence of Bay11-7082 or rapamycin were measured by ELISA assay. Data are shown as means ± S.D. of three experiments. Comparisons were made between different groups as indicated. * P
    Figure Legend Snippet: Expression of VEGF is increased in Hep3Bx and HepG2x cells and is further enhanced by TNF-α and blocked by IKKβ inhibitor Bay 11-7082 or the mTOR inhibitor rapamycin. (A). The expression levels of secreted VEGF in the culture medium of Hep3B, Hep3Bx, HepG2, and HepG2x cells were measured by ELISA assay as described in the Methods . (B). The expression levels of VEGFA mRNA were assessed in Hep3B, Hep3Bx, HepG2, and HepG2x cells using semi-quantitative RT-PCR (left) or real-time RT-PCR (right) as described in the Methods . (C). The amounts of secreted VEGF in the culture medium of Hep3B, Hep3Bx, HepG2, or HepG2x cells treated with or without TNF-α in the presence or absence of Bay11-7082 or rapamycin were measured by ELISA assay. Data are shown as means ± S.D. of three experiments. Comparisons were made between different groups as indicated. * P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

    32) Product Images from "Proangiogenic Hypoxia-Mimicking Agents Attenuate Osteogenic Potential of Adipose Stem/Stromal Cells"

    Article Title: Proangiogenic Hypoxia-Mimicking Agents Attenuate Osteogenic Potential of Adipose Stem/Stromal Cells

    Journal: Tissue Engineering and Regenerative Medicine

    doi: 10.1007/s13770-020-00259-3

    Expression of VEGFA and osteogenic genes in response to PHIs in both maintenance and osteogenic induction conditions; after 1 week of treatment, VEGFA ( A ) was upregulated with DMOG and to a lower extent with baicalein in both MM and OM. Relative gene expression levels for RUNX2 ( B ), ALPL ( C ), and COL1A1 ( D ) declined with both DMOG and baicalein. Baicalein revealed a trend for upregulating BMP2 and SPP1 ( E , F ). Column graphs show observed mean and SD for three independent biological replicates (dots), * p
    Figure Legend Snippet: Expression of VEGFA and osteogenic genes in response to PHIs in both maintenance and osteogenic induction conditions; after 1 week of treatment, VEGFA ( A ) was upregulated with DMOG and to a lower extent with baicalein in both MM and OM. Relative gene expression levels for RUNX2 ( B ), ALPL ( C ), and COL1A1 ( D ) declined with both DMOG and baicalein. Baicalein revealed a trend for upregulating BMP2 and SPP1 ( E , F ). Column graphs show observed mean and SD for three independent biological replicates (dots), * p

    Techniques Used: Expressing

    33) Product Images from "MicroRNA response to hypoxic stress in soft tissue sarcoma cells: microRNA mediated regulation of HIF3?"

    Article Title: MicroRNA response to hypoxic stress in soft tissue sarcoma cells: microRNA mediated regulation of HIF3?

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-14-429

    Hypoxia induces HIF1α protein levels and CA9 and VEGFA transcription in soft tissue sarcoma cell lines. (A) HIF1α protein levels are stabilized and increase during hypoxia. Cell lines were cultured under hypoxia (H) or normoxia (N), after the indicated times cell lysates were prepared and analysed by Western blotting for HIF1α expression. HIF1α levels peak at 6 hours of hypoxia. Upon prolonged hypoxia (24 and 48 hours) HIF1α protein levels decrease, but remain increased compared to the HIF1α levels detected in cells cultured under normoxic conditions. β-Actin was used as a loading control. (B,C) mRNA levels of HIF1 target genes CA9 (B) and VEGFA (C) are upregulated during hypoxia (grey bars) compared to normoxia (black bars). CA9 and VEGFA expression was determined by RT-PCR and normalized to the expression of HPRT. Bars indicate average fold change of mRNA expression ± S.D.(n = 2) compared to the levels detected at 0 h which are arbitrarily set at 1. Statistical significance between CA9 and VEGF expression in normoxic and hypoxic samples at the various time points was determined by two-sample t-tests: * = p
    Figure Legend Snippet: Hypoxia induces HIF1α protein levels and CA9 and VEGFA transcription in soft tissue sarcoma cell lines. (A) HIF1α protein levels are stabilized and increase during hypoxia. Cell lines were cultured under hypoxia (H) or normoxia (N), after the indicated times cell lysates were prepared and analysed by Western blotting for HIF1α expression. HIF1α levels peak at 6 hours of hypoxia. Upon prolonged hypoxia (24 and 48 hours) HIF1α protein levels decrease, but remain increased compared to the HIF1α levels detected in cells cultured under normoxic conditions. β-Actin was used as a loading control. (B,C) mRNA levels of HIF1 target genes CA9 (B) and VEGFA (C) are upregulated during hypoxia (grey bars) compared to normoxia (black bars). CA9 and VEGFA expression was determined by RT-PCR and normalized to the expression of HPRT. Bars indicate average fold change of mRNA expression ± S.D.(n = 2) compared to the levels detected at 0 h which are arbitrarily set at 1. Statistical significance between CA9 and VEGF expression in normoxic and hypoxic samples at the various time points was determined by two-sample t-tests: * = p

    Techniques Used: Cell Culture, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

    34) Product Images from "The hypoxia-inducible miR-429 regulates hypoxia-inducible factor-1α expression in human endothelial cells through a negative feedback loop"

    Article Title: The hypoxia-inducible miR-429 regulates hypoxia-inducible factor-1α expression in human endothelial cells through a negative feedback loop

    Journal: The FASEB Journal

    doi: 10.1096/fj.14-267054

    Hypoxia induces dynamic changes of expression profiles of HIF1A ( A ), FIH1 ( B ), PHD2 ( C ), and VEGFA ( D ) in HUVECs. The mRNA levels were monitored in quantitative real-time PCR experiments. The results from 3 independent experiments ( n = 32) are plotted
    Figure Legend Snippet: Hypoxia induces dynamic changes of expression profiles of HIF1A ( A ), FIH1 ( B ), PHD2 ( C ), and VEGFA ( D ) in HUVECs. The mRNA levels were monitored in quantitative real-time PCR experiments. The results from 3 independent experiments ( n = 32) are plotted

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Hypoxia induces dynamic changes of protein levels of HIF-1 α ( A ), FIH-1 ( B ), PHD2 ( C ), and VEGFA ( D ) in HUVECs. The bar graphs below show the relative protein amounts at each time point. The protein levels were detected with SDS-PAGE and Western
    Figure Legend Snippet: Hypoxia induces dynamic changes of protein levels of HIF-1 α ( A ), FIH-1 ( B ), PHD2 ( C ), and VEGFA ( D ) in HUVECs. The bar graphs below show the relative protein amounts at each time point. The protein levels were detected with SDS-PAGE and Western

    Techniques Used: SDS Page, Western Blot

    A ) miR-429 modulates VEGFA expression indirectly through changing HIF1A levels. HUVECs were transfected with miR-429 analog or inhibitor, and the mRNA levels were monitored in quantitative real-time PCR experiments in normoxic conditions and after 4 and
    Figure Legend Snippet: A ) miR-429 modulates VEGFA expression indirectly through changing HIF1A levels. HUVECs were transfected with miR-429 analog or inhibitor, and the mRNA levels were monitored in quantitative real-time PCR experiments in normoxic conditions and after 4 and

    Techniques Used: Expressing, Transfection, Real-time Polymerase Chain Reaction

    35) Product Images from "Mesenchymal Stem Cell Spheroids Retain Osteogenic Phenotype Through α2β1 Signaling"

    Article Title: Mesenchymal Stem Cell Spheroids Retain Osteogenic Phenotype Through α2β1 Signaling

    Journal: Stem Cells Translational Medicine

    doi: 10.5966/sctm.2015-0412

    The proangiogenic potential of mesenchymal stem cell spheroids is dependent on osteogenic induction prior to encapsulation in collagen gels. Cells were cultured in growth media or osteogenic media for 12 days prior to being formed into spheroids or replated in adherent monolayers and analyzed for normalized VEGF secretion (A) , total vascular endothelial growth factor secretion (B) , and VEGFA gene expression (C) . Chart values represent mean ± SD for n = 4. Significance is denoted by alphabetical letterings; groups with no significance are linked by the same letters, and groups with significance do not share the same letters. Abbreviations: GM, growth media; OM, osteogenic media; VEGF, vascular endothelial growth factor.
    Figure Legend Snippet: The proangiogenic potential of mesenchymal stem cell spheroids is dependent on osteogenic induction prior to encapsulation in collagen gels. Cells were cultured in growth media or osteogenic media for 12 days prior to being formed into spheroids or replated in adherent monolayers and analyzed for normalized VEGF secretion (A) , total vascular endothelial growth factor secretion (B) , and VEGFA gene expression (C) . Chart values represent mean ± SD for n = 4. Significance is denoted by alphabetical letterings; groups with no significance are linked by the same letters, and groups with significance do not share the same letters. Abbreviations: GM, growth media; OM, osteogenic media; VEGF, vascular endothelial growth factor.

    Techniques Used: Cell Culture, Expressing

    36) Product Images from "Bones in human CYP26B1 deficiency and rats with hypervitaminosis A phenocopy Vegfa overexpression"

    Article Title: Bones in human CYP26B1 deficiency and rats with hypervitaminosis A phenocopy Vegfa overexpression

    Journal: Bone Reports

    doi: 10.1016/j.bonr.2018.06.006

    Early pathological features of hypervitaminosis A in young rats. a) Plasma levels of Vegfa, Tnfa and soluble Icam 1 (sIcam1) protein at day 1, 2 and 7 into hypervitaminosis A, determined by ELISA. n = 4–5/group (day 1 and day 2) and n = 9–10/group (day 7). Student's t -test; p
    Figure Legend Snippet: Early pathological features of hypervitaminosis A in young rats. a) Plasma levels of Vegfa, Tnfa and soluble Icam 1 (sIcam1) protein at day 1, 2 and 7 into hypervitaminosis A, determined by ELISA. n = 4–5/group (day 1 and day 2) and n = 9–10/group (day 7). Student's t -test; p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Normal human endothelial cell and osteoblast response to retinoic acid (RA). a) CYP26B1 mRNA expression in human dermal microvascular endothelial cells (HDMEC) and human primary osteoblasts (HOB) treated with or without 400 nM RA for 24 h (h). b) VEGFA mRNA expression in HDMEC and HOB cells treated with or without 400 nM RA for 24 h or 48 h. c) Representative pictures of HDMEC cells treated with or without 400 nM RA for 24 and 48 h. Yellow arrowheads indicate RA induced cell contraction/slimming noticed at both time points. RA did not induce apoptosis as determined by caspase-3 activity. d) Immunofluorescent VE-Cadherin staining of HDMEC cells treated with or without 400 nM RA for 48 h. White arrowheads indicate RA induced gaps in VE-cadherin staining at cell-cell junctions. RA reduced VE-Cadherin staining between endothelial cells. n = 4/treatment. Bar 25 μm. Results are presented as mean ± SD. Student's t -test; p
    Figure Legend Snippet: Normal human endothelial cell and osteoblast response to retinoic acid (RA). a) CYP26B1 mRNA expression in human dermal microvascular endothelial cells (HDMEC) and human primary osteoblasts (HOB) treated with or without 400 nM RA for 24 h (h). b) VEGFA mRNA expression in HDMEC and HOB cells treated with or without 400 nM RA for 24 h or 48 h. c) Representative pictures of HDMEC cells treated with or without 400 nM RA for 24 and 48 h. Yellow arrowheads indicate RA induced cell contraction/slimming noticed at both time points. RA did not induce apoptosis as determined by caspase-3 activity. d) Immunofluorescent VE-Cadherin staining of HDMEC cells treated with or without 400 nM RA for 48 h. White arrowheads indicate RA induced gaps in VE-cadherin staining at cell-cell junctions. RA reduced VE-Cadherin staining between endothelial cells. n = 4/treatment. Bar 25 μm. Results are presented as mean ± SD. Student's t -test; p

    Techniques Used: Expressing, Activity Assay, Staining

    Femur phenotype from an individual with CYP26B1 insufficiency. a) Cathepsin K immunohistochemical staining (brown) of pathological periosteal osteoclast accumulation in sections of fetal bones from an individual with CYP26B1 insufficiency, compared with periosteal osteoclast accumulation from a typical hypervitaminosis A rat bone. Bar = 100 μm. b) Hematoxylin stained section of femur bone tissue from this individual with CYP26B1 insufficiency show angulation at midpoint. Asterisk highlight “striped” bone formation at the angulation point. Bar = 200 μm. c) Osteopontin, PECAM1 and VEGFA immunohistochemical staining (brown) of this human femur bone tissue at the angulation point. Lower panel show pictures at higher magnification from boxed area. d) Dentin matrix protein 1 immunohistochemical staining (brown) of osteocytes and canaliculi in bone tissue at the angulation point of this human fetal femur.
    Figure Legend Snippet: Femur phenotype from an individual with CYP26B1 insufficiency. a) Cathepsin K immunohistochemical staining (brown) of pathological periosteal osteoclast accumulation in sections of fetal bones from an individual with CYP26B1 insufficiency, compared with periosteal osteoclast accumulation from a typical hypervitaminosis A rat bone. Bar = 100 μm. b) Hematoxylin stained section of femur bone tissue from this individual with CYP26B1 insufficiency show angulation at midpoint. Asterisk highlight “striped” bone formation at the angulation point. Bar = 200 μm. c) Osteopontin, PECAM1 and VEGFA immunohistochemical staining (brown) of this human femur bone tissue at the angulation point. Lower panel show pictures at higher magnification from boxed area. d) Dentin matrix protein 1 immunohistochemical staining (brown) of osteocytes and canaliculi in bone tissue at the angulation point of this human fetal femur.

    Techniques Used: Immunohistochemistry, Staining

    37) Product Images from "PRSS21/Testisin inhibits ovarian tumor metastasis and antagonizes proangiogenic angiopoietins ANG2 and ANGPTL4"

    Article Title: PRSS21/Testisin inhibits ovarian tumor metastasis and antagonizes proangiogenic angiopoietins ANG2 and ANGPTL4

    Journal: Journal of molecular medicine (Berlin, Germany)

    doi: 10.1007/s00109-019-01763-3

    Testisin proteolytic activity reduces ANG2 and ANGPTL4 gene expression in ES-2-Luc cells. qPCR validation study of genes identified to be up- or down-regulated at least 2-fold by the TaqMan Angiogenesis Array: a . ANG1, ANG2, ANGPTL4, PECAM1/CD31, ANGPTL2, PDGFB, VEGFA, and TIE2. mRNA expression was normalized to GAPDH and expressed relative to ES-2-Luc-Ctl cells. All graphs show mean +/− SEM and are the average of at least 2 independent experiments from triplicate wells, *p
    Figure Legend Snippet: Testisin proteolytic activity reduces ANG2 and ANGPTL4 gene expression in ES-2-Luc cells. qPCR validation study of genes identified to be up- or down-regulated at least 2-fold by the TaqMan Angiogenesis Array: a . ANG1, ANG2, ANGPTL4, PECAM1/CD31, ANGPTL2, PDGFB, VEGFA, and TIE2. mRNA expression was normalized to GAPDH and expressed relative to ES-2-Luc-Ctl cells. All graphs show mean +/− SEM and are the average of at least 2 independent experiments from triplicate wells, *p

    Techniques Used: Activity Assay, Expressing, Real-time Polymerase Chain Reaction

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: Interplay between VEGF-A and cMET signaling in human vestibular schwannomas and schwann cells
    Article Snippet: The RNA was reverse-transcribed to cDNA with TaqMan Reverse Transcription Reagent Kit (Applied Biosystems, #4304134) following the manufacturer's protocol. .. The cDNA was stored at either 4°C for short term or −20°C for long term. qPCR was performed with TaqMan primers and 6-Carboxyfluorescein (6-FAM) linked fluorescent probes (Applied Biosystems) for VEGFA (#Hs00900055_m1), HGF (#Hs00300159_m1), KDR (#Hs00911700_m1) and MET (#Hs01565582_g1). ..

    Article Title: A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension
    Article Snippet: Purified RNA was reverse transcribed with the High Capacity RNA-to-cDNA Kit (Life Technologies). .. Gene expression was measured by performing TaqMan PCR using Gene Expression MasterMix (Applied Biosystems) for, Cav-1 (Hs00971716_m1), PDGFRa (Hs00998018_m1), TNC (Hs01115665_m1), TRAIL (Hs00921974_m1, Rn0059556_m1, Mn01182929_m1), VEGFA (Hs00900055_m1), VIPR1 (Hs00270351_m1), Fas (Hs00236330_m1, Rn00685720_m1) and OPG (Mn01205928_m1, Rn00563499_m1) on the 7900HT fast real time PCR system (Applied Biosystems). .. Gene expression was calculated using the ∆∆CT comparative quantification method with 18 S rRNA (Hs03003631_g1) as an endogenous control.

    Article Title: Hypoxic gene expression in chronic hepatitis B virus infected patients is not observed in state-of-the-art in vitro and mouse infection models
    Article Snippet: The amplification conditions were: 95 °C for 2 min (enzyme activation), followed by 45 cycles of amplification (95 °C for 5 s; 60 °C for 30 s). .. HIF target genes were amplified using TaqMan® Gene Expression assays (CAIX [Hs00154208_m1]; VEGFA [Hs00900055_m1]; BNIP3 [Hs00969291_m1] and GLUT1 [Hs00892681_m1]) (Thermo Fisher) and amplified using a Taqman real-time PCR protocol (qPCRBIO probe, PCR Biosystems) using the same conditions as listed above. .. HBV transgenic mice and siRNA delivery Animal experiments were conducted in accordance with the German regulations of the Society for Laboratory Animal Science (GV-SOLAS) and the European Health Law of the Federation of Laboratory Animal Science Associations (FELASA).

    Expressing:

    Article Title: A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension
    Article Snippet: Purified RNA was reverse transcribed with the High Capacity RNA-to-cDNA Kit (Life Technologies). .. Gene expression was measured by performing TaqMan PCR using Gene Expression MasterMix (Applied Biosystems) for, Cav-1 (Hs00971716_m1), PDGFRa (Hs00998018_m1), TNC (Hs01115665_m1), TRAIL (Hs00921974_m1, Rn0059556_m1, Mn01182929_m1), VEGFA (Hs00900055_m1), VIPR1 (Hs00270351_m1), Fas (Hs00236330_m1, Rn00685720_m1) and OPG (Mn01205928_m1, Rn00563499_m1) on the 7900HT fast real time PCR system (Applied Biosystems). .. Gene expression was calculated using the ∆∆CT comparative quantification method with 18 S rRNA (Hs03003631_g1) as an endogenous control.

    Article Title: Hypoxic gene expression in chronic hepatitis B virus infected patients is not observed in state-of-the-art in vitro and mouse infection models
    Article Snippet: The amplification conditions were: 95 °C for 2 min (enzyme activation), followed by 45 cycles of amplification (95 °C for 5 s; 60 °C for 30 s). .. HIF target genes were amplified using TaqMan® Gene Expression assays (CAIX [Hs00154208_m1]; VEGFA [Hs00900055_m1]; BNIP3 [Hs00969291_m1] and GLUT1 [Hs00892681_m1]) (Thermo Fisher) and amplified using a Taqman real-time PCR protocol (qPCRBIO probe, PCR Biosystems) using the same conditions as listed above. .. HBV transgenic mice and siRNA delivery Animal experiments were conducted in accordance with the German regulations of the Society for Laboratory Animal Science (GV-SOLAS) and the European Health Law of the Federation of Laboratory Animal Science Associations (FELASA).

    Article Title: Efficacy of tumor-targeting Salmonella typhimurium A1-R in combination with anti-angiogenesis therapy on a pancreatic cancer patient-derived orthotopic xenograft (PDOX) and cell line mouse models
    Article Snippet: Real-time RT-PCR Total RNA was extracted from human pancreatic cancer cell lines (BxPC-3, Capan-1, Hs766T, MiaPaCa-2 and Panc-1) using TRIzol (Invitrogen, Carlsbad, CA, USA), followed by on-column clean up with the RNA spin mini kit (GE Healthcare BioSciences, Little Chalfont, UK). .. Total RNA (2 μg) was reverse transcribed using the High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, USA) for complementary DNA (cDNA) synthesis. cDNA (2 μl) in a final volume of 20 μl,was amplified using the following Taqman Gene Expression assays (Applied Biosystems): VEGFA (Hs00900055_m1); VEGFR1 (Hs01052961_m1); VEGFR2 (Hs00911700_m1); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH ) endogenous control (Hs99999905_m1). .. All reactions were performed in triplicate using ABI 7900 HT Fast (Applied Biosystems).

    Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis
    Article Snippet: The primers used were as follows: MMP13 Human PrimePCR™ SYBR® Green Assay ID: qHsaCIP0026824 (Biorad); Hs_NOTCH1_2_SG QuantiTect Primer Assay ID: QT01005109; Hs_HES1_1_SG QuantiTect Primer Assay ID: QT00039648; Hs_DLL4_1_SG QuantiTect Primer Assay ID: QT00081004; Hs_GAPDH_1_SG QuantiTect Primer Assay ID: QT00079247 (Qiagen) and primer sequences for VEGFA: forward, 5′-CAGATGTCCCGGCGAAGA-3′; reverse, 5′-GAGGGCGAGTCCCAGGAA-3′. .. For human iCCA samples, mRNA expression of the genes of interest was detected by qRT-PCR using validated TaqMan Gene Expression Assays for human NOTCH1 ( Hs01062014_m1 ) , NOTCH2 ( Hs01050702_m1), NOTCH3 (Hs01128537_m1), NOTCH4 (Hs00965889_m1), DLL4 ( Hs00184092_m1), HES1 (Hs00172878_m1), VEGFA (Hs00900055_m1), MMP13 (Hs00942584_m1), RBPJ (Hs00794653_m1), and β-actin (Hs01060665_g1) genes (ThermoFisher Scientific). .. PCR reactions were performed with 100 ng of cDNA of the collected samples, using an ABI Prism 7000 Sequence Detection System with TaqMan Universal PCR Master Mix (Applied Biosystems).

    Article Title: Delivery of VEGFA in bone marrow stromal cells seeded in copolymer scaffold enhances angiogenesis, but is inadequate for osteogenesis as compared with the dual delivery of VEGFA and BMP2 in a subcutaneous mouse model
    Article Snippet: Three hundred nanograms of total RNA were converted to cDNA by reverse-transcription reaction using a high capacity cDNA Archive Kit (Applied Biosystems, Carlsbad, CA, USA). .. VEGFA (Hs00900055_m1) and BMP2 (Hs00154192_m1) TaqMan assays were used to verify the expression of VEGFA and BMP2 mRNA levels in adenovirus-transduced BMSC. .. The in vitro PCR array results were independently validated by qRT-PCR, using TaqMan assays for selected key genes: ALPL (Hs01029144_m1), RUNX2 (Hs00231692_m1), and SPP1 (Hs00959010_m1).

    Polymerase Chain Reaction:

    Article Title: A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension
    Article Snippet: Purified RNA was reverse transcribed with the High Capacity RNA-to-cDNA Kit (Life Technologies). .. Gene expression was measured by performing TaqMan PCR using Gene Expression MasterMix (Applied Biosystems) for, Cav-1 (Hs00971716_m1), PDGFRa (Hs00998018_m1), TNC (Hs01115665_m1), TRAIL (Hs00921974_m1, Rn0059556_m1, Mn01182929_m1), VEGFA (Hs00900055_m1), VIPR1 (Hs00270351_m1), Fas (Hs00236330_m1, Rn00685720_m1) and OPG (Mn01205928_m1, Rn00563499_m1) on the 7900HT fast real time PCR system (Applied Biosystems). .. Gene expression was calculated using the ∆∆CT comparative quantification method with 18 S rRNA (Hs03003631_g1) as an endogenous control.

    other:

    Article Title: Nuclear Heparanase Regulates Chromatin Remodeling, Gene Expression and PTEN Tumor Suppressor Function
    Article Snippet: The primers utilized were HPSE (Hs00935036_m1), SDC1 (Hs01045460_g1), MMP9 (Hs00957562_m1), VEGFA (Hs00900055_m1), CCND1 (Hs00765553_m1) and GAPDH (Hs02786624_g1).

    Article Title: Preclinical validation of the small molecule drug quininib as a novel therapeutic for colorectal cancer
    Article Snippet: Specific primers were used for VEGFA (Hs00900055_m1), EREG (Hs00914313_m1), NRP2 (Hs00187290_m1), FGF1 (Hs00265254_m1), IL6 (Hs00985639_m1) and IL8 (Hs01567912_m1) (ABI Biosystems, CA, USA).

    Amplification:

    Article Title: Hypoxic gene expression in chronic hepatitis B virus infected patients is not observed in state-of-the-art in vitro and mouse infection models
    Article Snippet: The amplification conditions were: 95 °C for 2 min (enzyme activation), followed by 45 cycles of amplification (95 °C for 5 s; 60 °C for 30 s). .. HIF target genes were amplified using TaqMan® Gene Expression assays (CAIX [Hs00154208_m1]; VEGFA [Hs00900055_m1]; BNIP3 [Hs00969291_m1] and GLUT1 [Hs00892681_m1]) (Thermo Fisher) and amplified using a Taqman real-time PCR protocol (qPCRBIO probe, PCR Biosystems) using the same conditions as listed above. .. HBV transgenic mice and siRNA delivery Animal experiments were conducted in accordance with the German regulations of the Society for Laboratory Animal Science (GV-SOLAS) and the European Health Law of the Federation of Laboratory Animal Science Associations (FELASA).

    Article Title: Efficacy of tumor-targeting Salmonella typhimurium A1-R in combination with anti-angiogenesis therapy on a pancreatic cancer patient-derived orthotopic xenograft (PDOX) and cell line mouse models
    Article Snippet: Real-time RT-PCR Total RNA was extracted from human pancreatic cancer cell lines (BxPC-3, Capan-1, Hs766T, MiaPaCa-2 and Panc-1) using TRIzol (Invitrogen, Carlsbad, CA, USA), followed by on-column clean up with the RNA spin mini kit (GE Healthcare BioSciences, Little Chalfont, UK). .. Total RNA (2 μg) was reverse transcribed using the High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, USA) for complementary DNA (cDNA) synthesis. cDNA (2 μl) in a final volume of 20 μl,was amplified using the following Taqman Gene Expression assays (Applied Biosystems): VEGFA (Hs00900055_m1); VEGFR1 (Hs01052961_m1); VEGFR2 (Hs00911700_m1); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH ) endogenous control (Hs99999905_m1). .. All reactions were performed in triplicate using ABI 7900 HT Fast (Applied Biosystems).

    Quantitative RT-PCR:

    Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis
    Article Snippet: The primers used were as follows: MMP13 Human PrimePCR™ SYBR® Green Assay ID: qHsaCIP0026824 (Biorad); Hs_NOTCH1_2_SG QuantiTect Primer Assay ID: QT01005109; Hs_HES1_1_SG QuantiTect Primer Assay ID: QT00039648; Hs_DLL4_1_SG QuantiTect Primer Assay ID: QT00081004; Hs_GAPDH_1_SG QuantiTect Primer Assay ID: QT00079247 (Qiagen) and primer sequences for VEGFA: forward, 5′-CAGATGTCCCGGCGAAGA-3′; reverse, 5′-GAGGGCGAGTCCCAGGAA-3′. .. For human iCCA samples, mRNA expression of the genes of interest was detected by qRT-PCR using validated TaqMan Gene Expression Assays for human NOTCH1 ( Hs01062014_m1 ) , NOTCH2 ( Hs01050702_m1), NOTCH3 (Hs01128537_m1), NOTCH4 (Hs00965889_m1), DLL4 ( Hs00184092_m1), HES1 (Hs00172878_m1), VEGFA (Hs00900055_m1), MMP13 (Hs00942584_m1), RBPJ (Hs00794653_m1), and β-actin (Hs01060665_g1) genes (ThermoFisher Scientific). .. PCR reactions were performed with 100 ng of cDNA of the collected samples, using an ABI Prism 7000 Sequence Detection System with TaqMan Universal PCR Master Mix (Applied Biosystems).

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    Thermo Fisher gene exp vegfa hs00900055 m1
    OPG-Fas interaction mediates the OPG-induced phenotypic response of PASMC. TaqMan expression of ( a ) <t>VEGFA,</t> ( b ) PDGFRA, ( c ) TNC, ( d ) Cav1 and ( e ) TRAIL in response to OPG in the presence (hash bars) or absence (Grey bars) of anti-Fas neutralising antibody (1500 ng ml −1 ). Panel ( f ) demonstrates OPG inhibition of FasL and TRAIL-induced apoptosis in HT1080 cells. g PASMC migration following 6 h stimulation with PDGF (20 ng ml −1 ), OPG (30 ng ml −1 ) or 0.2% FCS (serum-free media, SFM), in the presence or absence of Fas neutralising antibody. h Proliferation of PASMCs following stimulation with OPG for 72 h in the presence or absence of Fas neutralising antibody and/or TRAIL neutralising antibody (0.5 nM). Proliferation expressed as a percentage of proliferation to PDGF. Bars represent the mean with error bars showing the standard error of the mean. Dots represent experimental repeats, Panels ( a – e ) ( n = 4), panel ( f ) ( n = 3), panel ( g ) ( n = 4), panel (h) ( n = 4 for SFM, 10 for PDGF OPG stimulations) * p
    Gene Exp Vegfa Hs00900055 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    OPG-Fas interaction mediates the OPG-induced phenotypic response of PASMC. TaqMan expression of ( a ) VEGFA, ( b ) PDGFRA, ( c ) TNC, ( d ) Cav1 and ( e ) TRAIL in response to OPG in the presence (hash bars) or absence (Grey bars) of anti-Fas neutralising antibody (1500 ng ml −1 ). Panel ( f ) demonstrates OPG inhibition of FasL and TRAIL-induced apoptosis in HT1080 cells. g PASMC migration following 6 h stimulation with PDGF (20 ng ml −1 ), OPG (30 ng ml −1 ) or 0.2% FCS (serum-free media, SFM), in the presence or absence of Fas neutralising antibody. h Proliferation of PASMCs following stimulation with OPG for 72 h in the presence or absence of Fas neutralising antibody and/or TRAIL neutralising antibody (0.5 nM). Proliferation expressed as a percentage of proliferation to PDGF. Bars represent the mean with error bars showing the standard error of the mean. Dots represent experimental repeats, Panels ( a – e ) ( n = 4), panel ( f ) ( n = 3), panel ( g ) ( n = 4), panel (h) ( n = 4 for SFM, 10 for PDGF OPG stimulations) * p

    Journal: Nature Communications

    Article Title: A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension

    doi: 10.1038/s41467-019-13139-9

    Figure Lengend Snippet: OPG-Fas interaction mediates the OPG-induced phenotypic response of PASMC. TaqMan expression of ( a ) VEGFA, ( b ) PDGFRA, ( c ) TNC, ( d ) Cav1 and ( e ) TRAIL in response to OPG in the presence (hash bars) or absence (Grey bars) of anti-Fas neutralising antibody (1500 ng ml −1 ). Panel ( f ) demonstrates OPG inhibition of FasL and TRAIL-induced apoptosis in HT1080 cells. g PASMC migration following 6 h stimulation with PDGF (20 ng ml −1 ), OPG (30 ng ml −1 ) or 0.2% FCS (serum-free media, SFM), in the presence or absence of Fas neutralising antibody. h Proliferation of PASMCs following stimulation with OPG for 72 h in the presence or absence of Fas neutralising antibody and/or TRAIL neutralising antibody (0.5 nM). Proliferation expressed as a percentage of proliferation to PDGF. Bars represent the mean with error bars showing the standard error of the mean. Dots represent experimental repeats, Panels ( a – e ) ( n = 4), panel ( f ) ( n = 3), panel ( g ) ( n = 4), panel (h) ( n = 4 for SFM, 10 for PDGF OPG stimulations) * p

    Article Snippet: Gene expression was measured by performing TaqMan PCR using Gene Expression MasterMix (Applied Biosystems) for, Cav-1 (Hs00971716_m1), PDGFRa (Hs00998018_m1), TNC (Hs01115665_m1), TRAIL (Hs00921974_m1, Rn0059556_m1, Mn01182929_m1), VEGFA (Hs00900055_m1), VIPR1 (Hs00270351_m1), Fas (Hs00236330_m1, Rn00685720_m1) and OPG (Mn01205928_m1, Rn00563499_m1) on the 7900HT fast real time PCR system (Applied Biosystems).

    Techniques: Expressing, Inhibition, Migration

    VEGF-A and cMET pathways interact at the molecular level. ( A ) Representative image of western blot showing expression of VEGFR2, cMET and VEGF-A for vehicle only and for siRNAs targeting VEGFA and MET genes in primary human SCs. ( B ) Protein expression of VEGF-A, cMET and VEGFR2 after VEGFA and MET siRNA treatment of cultured human SCs (n = 2–4 different cultures) and VS cells (n = 5 different cultures) quantified through protein gel blot analysis. All levels are normalized to vehicle only protein expression, being 100% (dashed line). * P

    Journal: Cancer Biology & Therapy

    Article Title: Interplay between VEGF-A and cMET signaling in human vestibular schwannomas and schwann cells

    doi: 10.4161/15384047.2014.972765

    Figure Lengend Snippet: VEGF-A and cMET pathways interact at the molecular level. ( A ) Representative image of western blot showing expression of VEGFR2, cMET and VEGF-A for vehicle only and for siRNAs targeting VEGFA and MET genes in primary human SCs. ( B ) Protein expression of VEGF-A, cMET and VEGFR2 after VEGFA and MET siRNA treatment of cultured human SCs (n = 2–4 different cultures) and VS cells (n = 5 different cultures) quantified through protein gel blot analysis. All levels are normalized to vehicle only protein expression, being 100% (dashed line). * P

    Article Snippet: The cDNA was stored at either 4°C for short term or −20°C for long term. qPCR was performed with TaqMan primers and 6-Carboxyfluorescein (6-FAM) linked fluorescent probes (Applied Biosystems) for VEGFA (#Hs00900055_m1), HGF (#Hs00300159_m1), KDR (#Hs00911700_m1) and MET (#Hs01565582_g1).

    Techniques: Western Blot, Expressing, Cell Culture

    HGF and VEGF-A pathways are aberrantly expressed and activated in VS. ( A ) Gene expression of VEGFA and its receptor KDR, and HGF and its receptor MET, in human VS (n = 8 different tumors) normalized to great auricular nerves (GAN, n = 7 different nerves) as measured through qPCR. * P

    Journal: Cancer Biology & Therapy

    Article Title: Interplay between VEGF-A and cMET signaling in human vestibular schwannomas and schwann cells

    doi: 10.4161/15384047.2014.972765

    Figure Lengend Snippet: HGF and VEGF-A pathways are aberrantly expressed and activated in VS. ( A ) Gene expression of VEGFA and its receptor KDR, and HGF and its receptor MET, in human VS (n = 8 different tumors) normalized to great auricular nerves (GAN, n = 7 different nerves) as measured through qPCR. * P

    Article Snippet: The cDNA was stored at either 4°C for short term or −20°C for long term. qPCR was performed with TaqMan primers and 6-Carboxyfluorescein (6-FAM) linked fluorescent probes (Applied Biosystems) for VEGFA (#Hs00900055_m1), HGF (#Hs00300159_m1), KDR (#Hs00911700_m1) and MET (#Hs01565582_g1).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Relationship between microvessel density and NOTCH1, HES1, DLL4, MMP13, and VEGFA levels. Levels of tumor microvessel density (MVD) correlate with mRNA expression of NOTCH1 ( a ), HES1 ( b ), DLL4 ( c ), and MMP13 ( e ), but not with those of VEGFA ( d ), in a collection of human intrahepatic cholangiocarcinoma (iCCA) samples ( n = 42). Linear regression analysis was used. f Representative examples of human iCCA specimens with high and low MVD.

    Journal: Cell Death and Differentiation

    Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

    doi: 10.1038/s41418-020-0505-4

    Figure Lengend Snippet: Relationship between microvessel density and NOTCH1, HES1, DLL4, MMP13, and VEGFA levels. Levels of tumor microvessel density (MVD) correlate with mRNA expression of NOTCH1 ( a ), HES1 ( b ), DLL4 ( c ), and MMP13 ( e ), but not with those of VEGFA ( d ), in a collection of human intrahepatic cholangiocarcinoma (iCCA) samples ( n = 42). Linear regression analysis was used. f Representative examples of human iCCA specimens with high and low MVD.

    Article Snippet: For human iCCA samples, mRNA expression of the genes of interest was detected by qRT-PCR using validated TaqMan Gene Expression Assays for human NOTCH1 ( Hs01062014_m1 ) , NOTCH2 ( Hs01050702_m1), NOTCH3 (Hs01128537_m1), NOTCH4 (Hs00965889_m1), DLL4 ( Hs00184092_m1), HES1 (Hs00172878_m1), VEGFA (Hs00900055_m1), MMP13 (Hs00942584_m1), RBPJ (Hs00794653_m1), and β-actin (Hs01060665_g1) genes (ThermoFisher Scientific).

    Techniques: Expressing

    LY3039478 inhibits VEGFA, CD31, and DLL4 expression in the iCCA PDX model. a Western blot analysis and semiquantitative evaluation of DLL4, VEGFA, and CD31 expression in PDX mice tissues by densitometry analysis of protein bands reveals a downregulation of DLL4, VEGFA, and CD31 protein expression in PDX mice treated with GSI. The bands were measured compared with the housekeeping GAPDH protein band, for each tissue. Average value of DLL4, VEGFA, and CD31 expression levels among all mouse treated with LY3039478 or vehicle is reported in the graph. P value showed versus vehicle treatment. Tissues PDX mice n = 10 for vehicle treatment in gray, n = 10 for LY3039478 treatment in black. b Representative images with immunofluorescence staining show DLL4 and CD31 downregulation in representative images of PDX tissues treated with LY30349478. DLL4 (green) and CD31 (red) and overlapping staining (yellow) were immunolocalized in PDX tissues. The yellow arrows highlight the detail of the co-localization of DLL4 and CD31 in PDX tissues (#4, #14, #24) not treated with LY339478. DAPI, 4′,6‐diamidino‐2‐phenylindole. c Immunofluorescence staining with MMP13 in red and nucleus in DAPI shown a significantly reduction of MMP13 in iCCA PDX tissues treated with LY3039478. Magnifications: ×20; inset ×60. d Representative images demonstrate a significant ( P

    Journal: Cell Death and Differentiation

    Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

    doi: 10.1038/s41418-020-0505-4

    Figure Lengend Snippet: LY3039478 inhibits VEGFA, CD31, and DLL4 expression in the iCCA PDX model. a Western blot analysis and semiquantitative evaluation of DLL4, VEGFA, and CD31 expression in PDX mice tissues by densitometry analysis of protein bands reveals a downregulation of DLL4, VEGFA, and CD31 protein expression in PDX mice treated with GSI. The bands were measured compared with the housekeeping GAPDH protein band, for each tissue. Average value of DLL4, VEGFA, and CD31 expression levels among all mouse treated with LY3039478 or vehicle is reported in the graph. P value showed versus vehicle treatment. Tissues PDX mice n = 10 for vehicle treatment in gray, n = 10 for LY3039478 treatment in black. b Representative images with immunofluorescence staining show DLL4 and CD31 downregulation in representative images of PDX tissues treated with LY30349478. DLL4 (green) and CD31 (red) and overlapping staining (yellow) were immunolocalized in PDX tissues. The yellow arrows highlight the detail of the co-localization of DLL4 and CD31 in PDX tissues (#4, #14, #24) not treated with LY339478. DAPI, 4′,6‐diamidino‐2‐phenylindole. c Immunofluorescence staining with MMP13 in red and nucleus in DAPI shown a significantly reduction of MMP13 in iCCA PDX tissues treated with LY3039478. Magnifications: ×20; inset ×60. d Representative images demonstrate a significant ( P

    Article Snippet: For human iCCA samples, mRNA expression of the genes of interest was detected by qRT-PCR using validated TaqMan Gene Expression Assays for human NOTCH1 ( Hs01062014_m1 ) , NOTCH2 ( Hs01050702_m1), NOTCH3 (Hs01128537_m1), NOTCH4 (Hs00965889_m1), DLL4 ( Hs00184092_m1), HES1 (Hs00172878_m1), VEGFA (Hs00900055_m1), MMP13 (Hs00942584_m1), RBPJ (Hs00794653_m1), and β-actin (Hs01060665_g1) genes (ThermoFisher Scientific).

    Techniques: Expressing, Western Blot, Mouse Assay, Immunofluorescence, Staining

    NOTCH1 , HES1 , DLL4 , VEGFA , and MMP13 mRNA expression in iCCA patients. a Analysis of 31 primary tumors from iCCA patients and matched surrounding normal liver tissues downloaded from the GEO database (GSE107943). Mean expression data were expressed in RPKM (Reads Per Kilobase Million). *** P

    Journal: Cell Death and Differentiation

    Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

    doi: 10.1038/s41418-020-0505-4

    Figure Lengend Snippet: NOTCH1 , HES1 , DLL4 , VEGFA , and MMP13 mRNA expression in iCCA patients. a Analysis of 31 primary tumors from iCCA patients and matched surrounding normal liver tissues downloaded from the GEO database (GSE107943). Mean expression data were expressed in RPKM (Reads Per Kilobase Million). *** P

    Article Snippet: For human iCCA samples, mRNA expression of the genes of interest was detected by qRT-PCR using validated TaqMan Gene Expression Assays for human NOTCH1 ( Hs01062014_m1 ) , NOTCH2 ( Hs01050702_m1), NOTCH3 (Hs01128537_m1), NOTCH4 (Hs00965889_m1), DLL4 ( Hs00184092_m1), HES1 (Hs00172878_m1), VEGFA (Hs00900055_m1), MMP13 (Hs00942584_m1), RBPJ (Hs00794653_m1), and β-actin (Hs01060665_g1) genes (ThermoFisher Scientific).

    Techniques: Expressing

    Heparanase enhances chromatin accessibility. ( A ) To determine localization of heparanase, sequential cell fractionation of myeloma cells was used to isolate cytoplasmic (cyto), nucleus-soluble (sol), and nucleus-chromatin (chrom) fractions from CAG wild-type (HPSE Lo) and transfected CAG HPSE Hi cells prior to Western blot analysis. GAPDH and histone H3 (H3) were probed to assess the quality of the fractions. ( B ) Chromatin accessibility in CAG HPSE knockdown (KD) and HPSE Hi cells were assessed by sensitivity to micrococcal nuclease (MNase) digestion. Nuclei from KD and HPSE Hi cells were prepared and digested with 0.1 U/μL MNase and subjected to electrophoresis. Black arrows denote Tri (T), Di (D) and Mono (M)—nucleosomes. ( C ) Nuclei from CAG HPSE KD and RPMI-8226 myeloma cells were treated for 2 h with or without rHPSE before being subjected to Mnase digestion for chromatin accessibility. ( D ) CAG HPSE Hi cells were incubated with 20 µM of the HPSE inhibitor OGT2115 and chromatin accessibility was assessed following exposure of nuclei to MNase. ( E ) mRNA expression analysis by RT-PCR of syndecan-1 ( SDC1 ), vascular endothelial growth factor A ( VEGFA ), matrix metalloproteinase-9 ( MMP9 ) and cyclin D1 ( CCDN1 ) in CAG HPSE Hi cells in the presence or absence of OGT2115, compared to respective control. * p

    Journal: Cells

    Article Title: Nuclear Heparanase Regulates Chromatin Remodeling, Gene Expression and PTEN Tumor Suppressor Function

    doi: 10.3390/cells9092038

    Figure Lengend Snippet: Heparanase enhances chromatin accessibility. ( A ) To determine localization of heparanase, sequential cell fractionation of myeloma cells was used to isolate cytoplasmic (cyto), nucleus-soluble (sol), and nucleus-chromatin (chrom) fractions from CAG wild-type (HPSE Lo) and transfected CAG HPSE Hi cells prior to Western blot analysis. GAPDH and histone H3 (H3) were probed to assess the quality of the fractions. ( B ) Chromatin accessibility in CAG HPSE knockdown (KD) and HPSE Hi cells were assessed by sensitivity to micrococcal nuclease (MNase) digestion. Nuclei from KD and HPSE Hi cells were prepared and digested with 0.1 U/μL MNase and subjected to electrophoresis. Black arrows denote Tri (T), Di (D) and Mono (M)—nucleosomes. ( C ) Nuclei from CAG HPSE KD and RPMI-8226 myeloma cells were treated for 2 h with or without rHPSE before being subjected to Mnase digestion for chromatin accessibility. ( D ) CAG HPSE Hi cells were incubated with 20 µM of the HPSE inhibitor OGT2115 and chromatin accessibility was assessed following exposure of nuclei to MNase. ( E ) mRNA expression analysis by RT-PCR of syndecan-1 ( SDC1 ), vascular endothelial growth factor A ( VEGFA ), matrix metalloproteinase-9 ( MMP9 ) and cyclin D1 ( CCDN1 ) in CAG HPSE Hi cells in the presence or absence of OGT2115, compared to respective control. * p

    Article Snippet: The primers utilized were HPSE (Hs00935036_m1), SDC1 (Hs01045460_g1), MMP9 (Hs00957562_m1), VEGFA (Hs00900055_m1), CCND1 (Hs00765553_m1) and GAPDH (Hs02786624_g1).

    Techniques: Cell Fractionation, Transfection, Western Blot, Electrophoresis, Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction