gene exp vegfa mm00437306 m1  (Thermo Fisher)


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    Thermo Fisher gene exp vegfa mm00437306 m1
    A. Gating strategy for analyzing Sema3A expression among leukocytes, epithelial and endothelial cells in lung and dLN. B. Quantification of Sema3A positive cells in different cell populations in uninfected (n=4), and infected mice, at 3 days DPI (n=4) and 10 DPI (n=4) in lung (left panel) or dLN (right panel). Nrp1 Flox/Flox mice used. Data combined from two independent experiments. ns = not significant by two-way ANOVA. C. Quantification of Pkd1, Bnip3, <t>Vegfa,</t> Pdl1 and Sema3A mRNA level following 24 hour culture in 1% O2 chamber. D. Gating strategy (left) and representative histograms (right) analyzing Sema3A positive cell populations in B16.F10 tumors 11 days post-injection. Olfactory lobe is used as a positive control for Sema3A expression. E. Quantification of Sema3A positive cells in same experiment as in (D) in tumor, dLN and ndLN. F. Genomic organization of murine Sema3a gene, indicating where CRISPR guide RNA targets with red arrow (upper figure). MiSEQ sequence results for chosen Sema3A KO clone showing 4 base deletion in two alleles (46% of all reads), a frameshift in one allele (45% of all reads) and WT reads in 2% of all reads. G. RT-qPCR analysis show down- and up-regulation of Sema3A in Sema3A KO and OE cell lines, respectively. Normalized to Hprt. Experiment performed once. H. Intracellular staining shows no detectable expression of Sema3A in Sema3A KO cells, and expression in Sema3A OE cells, as expected. Experiment performed once, at low seeding density. I. Growth of WT, Sema3A KO and Sema3A OE B16.F10.Ova cell lines in normal, IFNγ or TNFα-rich media. Experiment performed once. Data indicate mean ± SD. ns = not significant by two-way ANOVA. Abbreviations: BEC, blood endothelial cells. dLN, draining lymph node. DPI, days post-infection. FRC, fibroblastic reticular cells. KO, knockout. LEC, lymphatic endothelial cells. ndLN, non-draining lymph node. OE, overexpressing. TME, tumor microenvironment. WT, wild-type.
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    Images

    1) Product Images from "Semaphorin 3A induces cytoskeletal paralysis in tumor-specific CD8+ T cells"

    Article Title: Semaphorin 3A induces cytoskeletal paralysis in tumor-specific CD8+ T cells

    Journal: bioRxiv

    doi: 10.1101/849083

    A. Gating strategy for analyzing Sema3A expression among leukocytes, epithelial and endothelial cells in lung and dLN. B. Quantification of Sema3A positive cells in different cell populations in uninfected (n=4), and infected mice, at 3 days DPI (n=4) and 10 DPI (n=4) in lung (left panel) or dLN (right panel). Nrp1 Flox/Flox mice used. Data combined from two independent experiments. ns = not significant by two-way ANOVA. C. Quantification of Pkd1, Bnip3, Vegfa, Pdl1 and Sema3A mRNA level following 24 hour culture in 1% O2 chamber. D. Gating strategy (left) and representative histograms (right) analyzing Sema3A positive cell populations in B16.F10 tumors 11 days post-injection. Olfactory lobe is used as a positive control for Sema3A expression. E. Quantification of Sema3A positive cells in same experiment as in (D) in tumor, dLN and ndLN. F. Genomic organization of murine Sema3a gene, indicating where CRISPR guide RNA targets with red arrow (upper figure). MiSEQ sequence results for chosen Sema3A KO clone showing 4 base deletion in two alleles (46% of all reads), a frameshift in one allele (45% of all reads) and WT reads in 2% of all reads. G. RT-qPCR analysis show down- and up-regulation of Sema3A in Sema3A KO and OE cell lines, respectively. Normalized to Hprt. Experiment performed once. H. Intracellular staining shows no detectable expression of Sema3A in Sema3A KO cells, and expression in Sema3A OE cells, as expected. Experiment performed once, at low seeding density. I. Growth of WT, Sema3A KO and Sema3A OE B16.F10.Ova cell lines in normal, IFNγ or TNFα-rich media. Experiment performed once. Data indicate mean ± SD. ns = not significant by two-way ANOVA. Abbreviations: BEC, blood endothelial cells. dLN, draining lymph node. DPI, days post-infection. FRC, fibroblastic reticular cells. KO, knockout. LEC, lymphatic endothelial cells. ndLN, non-draining lymph node. OE, overexpressing. TME, tumor microenvironment. WT, wild-type.
    Figure Legend Snippet: A. Gating strategy for analyzing Sema3A expression among leukocytes, epithelial and endothelial cells in lung and dLN. B. Quantification of Sema3A positive cells in different cell populations in uninfected (n=4), and infected mice, at 3 days DPI (n=4) and 10 DPI (n=4) in lung (left panel) or dLN (right panel). Nrp1 Flox/Flox mice used. Data combined from two independent experiments. ns = not significant by two-way ANOVA. C. Quantification of Pkd1, Bnip3, Vegfa, Pdl1 and Sema3A mRNA level following 24 hour culture in 1% O2 chamber. D. Gating strategy (left) and representative histograms (right) analyzing Sema3A positive cell populations in B16.F10 tumors 11 days post-injection. Olfactory lobe is used as a positive control for Sema3A expression. E. Quantification of Sema3A positive cells in same experiment as in (D) in tumor, dLN and ndLN. F. Genomic organization of murine Sema3a gene, indicating where CRISPR guide RNA targets with red arrow (upper figure). MiSEQ sequence results for chosen Sema3A KO clone showing 4 base deletion in two alleles (46% of all reads), a frameshift in one allele (45% of all reads) and WT reads in 2% of all reads. G. RT-qPCR analysis show down- and up-regulation of Sema3A in Sema3A KO and OE cell lines, respectively. Normalized to Hprt. Experiment performed once. H. Intracellular staining shows no detectable expression of Sema3A in Sema3A KO cells, and expression in Sema3A OE cells, as expected. Experiment performed once, at low seeding density. I. Growth of WT, Sema3A KO and Sema3A OE B16.F10.Ova cell lines in normal, IFNγ or TNFα-rich media. Experiment performed once. Data indicate mean ± SD. ns = not significant by two-way ANOVA. Abbreviations: BEC, blood endothelial cells. dLN, draining lymph node. DPI, days post-infection. FRC, fibroblastic reticular cells. KO, knockout. LEC, lymphatic endothelial cells. ndLN, non-draining lymph node. OE, overexpressing. TME, tumor microenvironment. WT, wild-type.

    Techniques Used: Expressing, Infection, Mouse Assay, Injection, Positive Control, CRISPR, Sequencing, Quantitative RT-PCR, Staining, Knock-Out

    2) Product Images from "Ferrochelatase regulates retinal neovascularization"

    Article Title: Ferrochelatase regulates retinal neovascularization

    Journal: bioRxiv

    doi: 10.1101/2020.06.02.129650

    Ferrochelatase (FECH) expression and vascular pathology in the oxygen-induced retinopathy (OIR) mouse model. A ) Scheme: Juvenile C57BL/6J mice at postnatal day (P) 7 were exposed to hyperoxia chamber for 5 days and returned to room air (normoxia) at P12. Retinal analysis was performed at various time points as indicated. Retinas were isolated for RNA analysis, or fixed, cryosectioned, and immunostained, and retinal sections or whole flatmounts were analyzed. B ) Retinal flatmount from OIR and control retinas stained with GS-IB4 (green; labels vasculature) and pimonidazole (red; labels hypoxic regions) showing the temporal pattern of hypoxia and formation of pathological neovascular tufts in OIR retina. C ) Gene expression analysis of Fech and Vegfa by qPCR of RNA derived from whole retinal samples. Temporal analysis indicated Fech and Vegfa are upregulated in OIR. C t valves from gene expression data were normalized to the housekeeping genes ( Tbp and Hprt ) and respective age matched untouched retina control (ΔΔC t method). All qPCR reactions were run in technical triplicate, N=3 biological replicates. Data presented as mean ± SEM, *p
    Figure Legend Snippet: Ferrochelatase (FECH) expression and vascular pathology in the oxygen-induced retinopathy (OIR) mouse model. A ) Scheme: Juvenile C57BL/6J mice at postnatal day (P) 7 were exposed to hyperoxia chamber for 5 days and returned to room air (normoxia) at P12. Retinal analysis was performed at various time points as indicated. Retinas were isolated for RNA analysis, or fixed, cryosectioned, and immunostained, and retinal sections or whole flatmounts were analyzed. B ) Retinal flatmount from OIR and control retinas stained with GS-IB4 (green; labels vasculature) and pimonidazole (red; labels hypoxic regions) showing the temporal pattern of hypoxia and formation of pathological neovascular tufts in OIR retina. C ) Gene expression analysis of Fech and Vegfa by qPCR of RNA derived from whole retinal samples. Temporal analysis indicated Fech and Vegfa are upregulated in OIR. C t valves from gene expression data were normalized to the housekeeping genes ( Tbp and Hprt ) and respective age matched untouched retina control (ΔΔC t method). All qPCR reactions were run in technical triplicate, N=3 biological replicates. Data presented as mean ± SEM, *p

    Techniques Used: Expressing, Mouse Assay, Isolation, Staining, Real-time Polymerase Chain Reaction, Derivative Assay

    3) Product Images from "Genome-wide redistribution of MeCP2 in dorsal root ganglia after peripheral nerve injury"

    Article Title: Genome-wide redistribution of MeCP2 in dorsal root ganglia after peripheral nerve injury

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-016-0073-5

    miR-126 regulates expression of Dnmt1 and Vegfa in vitro. a Relative expression of endogenous Dnmt1 and Vegfa mRNA in Neuro 2a cells transfected with miR-126 Gapdh was used as a normalizer. b Representative Western blot of Dnmt1 and Vegfa using lysate of Neuro 2a cells transfected with miR-126 precursor plasmid for 72 h. Overexpression of miR-126 decreased mRNA and protein levels of Dnmt1 and Vegfa; beta tubulin was used as the control. c Immunohistochemistry indicating transfection with miR-126 plasmid co-expressing GFP in Neuro 2a cells decreased Dnmt1 levels compared to untransfected cells 72 h post-transfection. Significance determined using unpaired Student’s t test, p value *
    Figure Legend Snippet: miR-126 regulates expression of Dnmt1 and Vegfa in vitro. a Relative expression of endogenous Dnmt1 and Vegfa mRNA in Neuro 2a cells transfected with miR-126 Gapdh was used as a normalizer. b Representative Western blot of Dnmt1 and Vegfa using lysate of Neuro 2a cells transfected with miR-126 precursor plasmid for 72 h. Overexpression of miR-126 decreased mRNA and protein levels of Dnmt1 and Vegfa; beta tubulin was used as the control. c Immunohistochemistry indicating transfection with miR-126 plasmid co-expressing GFP in Neuro 2a cells decreased Dnmt1 levels compared to untransfected cells 72 h post-transfection. Significance determined using unpaired Student’s t test, p value *

    Techniques Used: Expressing, In Vitro, Transfection, Western Blot, Plasmid Preparation, Over Expression, Immunohistochemistry

    Administration of exogenous miR-126 decreased Dnmt1 and Vegfa expression in vivo but did not alter pain sensitivity. a Mechanical sensitivity measured by von Frey filaments showed that intrathecal delivery of miR-126 did not alter the paw withdrawal threshold in SNI model mice. Arrows indicate daily intrathecal injections with 2 nmol miR-126 or control miRNA via catheter ( n = 5 for miR-126 and miR-control injected mice, n = 3 for PBS injected mice). b Confirmation of miR-126 delivery to DRG. A qPCR performed using DRG collected from mice injected with miR-126 or control miRNA showed an increase in miR-126 in mice that received miR-126 compared to miR-control injected mice indicating successful delivery. c Relative expression of Dnmt1 and Vegfa mRNA in the DRG of miR-126 and control injected mice. Increased miR-126 decreased the expression of endogenous Dnmt1 and Vegfa compared to miR-control injected mice. Significance determined using unpaired Student’s t test, p value, **
    Figure Legend Snippet: Administration of exogenous miR-126 decreased Dnmt1 and Vegfa expression in vivo but did not alter pain sensitivity. a Mechanical sensitivity measured by von Frey filaments showed that intrathecal delivery of miR-126 did not alter the paw withdrawal threshold in SNI model mice. Arrows indicate daily intrathecal injections with 2 nmol miR-126 or control miRNA via catheter ( n = 5 for miR-126 and miR-control injected mice, n = 3 for PBS injected mice). b Confirmation of miR-126 delivery to DRG. A qPCR performed using DRG collected from mice injected with miR-126 or control miRNA showed an increase in miR-126 in mice that received miR-126 compared to miR-control injected mice indicating successful delivery. c Relative expression of Dnmt1 and Vegfa mRNA in the DRG of miR-126 and control injected mice. Increased miR-126 decreased the expression of endogenous Dnmt1 and Vegfa compared to miR-control injected mice. Significance determined using unpaired Student’s t test, p value, **

    Techniques Used: Expressing, In Vivo, Mouse Assay, Injection, Real-time Polymerase Chain Reaction

    Expression of miR-126 and its target genes Dnmt1 and Vegfa in the DRG after nerve injury. a Relative expression of miR-126 determined by qPCR shows a reduction in miR-126 in SNI model compared to DRG from sham control. U6 was used for normalization ( n = 8 sham, n = 7 SNI). b Relative expression of Dnmt1 mRNA and c Vegfa transcripts showed an increase in the DRG after nerve injury compared to control ( n = 3). Gapdh was used as a normalizer. d Representative Western blot and quantification showed an increase of Dnmt1 protein in the DRG after nerve injury. e Western blot and quantification showed Vegfa protein was not significantly different in DRG after nerve injury ( n = 3 from pooled samples, three DRG were pooled for each sample). Significance determined using unpaired Student’s t test, p value *
    Figure Legend Snippet: Expression of miR-126 and its target genes Dnmt1 and Vegfa in the DRG after nerve injury. a Relative expression of miR-126 determined by qPCR shows a reduction in miR-126 in SNI model compared to DRG from sham control. U6 was used for normalization ( n = 8 sham, n = 7 SNI). b Relative expression of Dnmt1 mRNA and c Vegfa transcripts showed an increase in the DRG after nerve injury compared to control ( n = 3). Gapdh was used as a normalizer. d Representative Western blot and quantification showed an increase of Dnmt1 protein in the DRG after nerve injury. e Western blot and quantification showed Vegfa protein was not significantly different in DRG after nerve injury ( n = 3 from pooled samples, three DRG were pooled for each sample). Significance determined using unpaired Student’s t test, p value *

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot

    Expression of miR-126 and its target genes Dnmt1 and Vegfa in the DRG from Mecp2 -null mouse. a Relative expression of miR-126 in the DRG showed comparable expression in Mecp2 -null and wild-type littermates. U6 was used for normalization. b Dnmt1 and c Vegfa expression was decreased in the DRG from Mecp2 -null mouse, indicating MeCP2 has a role in regulating expression of Dnmt1 and Vegfa. Gapdh was used as a normalizer. Significance determined using unpaired Student’s t test, p value *
    Figure Legend Snippet: Expression of miR-126 and its target genes Dnmt1 and Vegfa in the DRG from Mecp2 -null mouse. a Relative expression of miR-126 in the DRG showed comparable expression in Mecp2 -null and wild-type littermates. U6 was used for normalization. b Dnmt1 and c Vegfa expression was decreased in the DRG from Mecp2 -null mouse, indicating MeCP2 has a role in regulating expression of Dnmt1 and Vegfa. Gapdh was used as a normalizer. Significance determined using unpaired Student’s t test, p value *

    Techniques Used: Expressing

    4) Product Images from "eNOS-induced vascular barrier disruption in retinopathy by c-Src activation and tyrosine phosphorylation of VE-cadherin"

    Article Title: eNOS-induced vascular barrier disruption in retinopathy by c-Src activation and tyrosine phosphorylation of VE-cadherin

    Journal: bioRxiv

    doi: 10.1101/2020.11.24.396077

    VEGFA induced eNOS phosphorylation and activity in vitro A. Effect of VEGFA (V; 100 ng/mL; 1, 5, 10 min), histamine (H; 10 μM, 1, 5, 10 min) or medium (C, control) on eNOS phosphorylation at S1177 in cultured Human Retinal Microvascular Endothelial Cells (HRMEC). B. Quantification of eNOS pS1177/total eNOS normalized to tubulin. Mean ±S.E.M. n = 3 independent experiments. * = p
    Figure Legend Snippet: VEGFA induced eNOS phosphorylation and activity in vitro A. Effect of VEGFA (V; 100 ng/mL; 1, 5, 10 min), histamine (H; 10 μM, 1, 5, 10 min) or medium (C, control) on eNOS phosphorylation at S1177 in cultured Human Retinal Microvascular Endothelial Cells (HRMEC). B. Quantification of eNOS pS1177/total eNOS normalized to tubulin. Mean ±S.E.M. n = 3 independent experiments. * = p

    Techniques Used: Activity Assay, In Vitro, Cell Culture

    Expression of Nos2, Nos3 and Vegfa in Nos3 +/+ and Nos3S1176A/S1176A retinas A-C. qPCR of Nos3 (A) , Nos2 (B) and Vegfa (C) expression in P17 normoxic and OIR-challenged Nos3 +/+ and Nos3 S1176A/S1176A mouse retinas. D,E. Relative quantities of Nos2 and Nos3 compared against standard curves of TBP and UBC in Nos3 +/+ and Nos3 S1176A/S1176A retinas. Mean ±S.E.M. n = 5 ( Nos3 +/+ ) and 5 ( Nos3 S1176A/S1176A ) mice. *, **, *** = p
    Figure Legend Snippet: Expression of Nos2, Nos3 and Vegfa in Nos3 +/+ and Nos3S1176A/S1176A retinas A-C. qPCR of Nos3 (A) , Nos2 (B) and Vegfa (C) expression in P17 normoxic and OIR-challenged Nos3 +/+ and Nos3 S1176A/S1176A mouse retinas. D,E. Relative quantities of Nos2 and Nos3 compared against standard curves of TBP and UBC in Nos3 +/+ and Nos3 S1176A/S1176A retinas. Mean ±S.E.M. n = 5 ( Nos3 +/+ ) and 5 ( Nos3 S1176A/S1176A ) mice. *, **, *** = p

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

    eNOS/NO modulates VE-cadherin Y685 phosphorylation via c-Src in a VEGFA/VEGFR2 dependent manner. VEGFA through VEGFR2 and its phosphosite Y1212 induces a chain of consecutive reactions in endothelial cells: phosphorylation of AKT at S473 and eNOS at S1176. The VEGFR2 phosphosite Y949 mediates phosphorylation of c-Src at Y418 and of VE-cadherin at Y685. Combined, these activating phosphorylation reactions disrupt the vascular barrier by dissociating VE-cadherin’s homophilic interactions, resulting in macromolecular leakage. eNOS/NO exacerbates this damage via an interaction with c-Src to enhance VE-cadherin Y685 phosphorylation and internalization.
    Figure Legend Snippet: eNOS/NO modulates VE-cadherin Y685 phosphorylation via c-Src in a VEGFA/VEGFR2 dependent manner. VEGFA through VEGFR2 and its phosphosite Y1212 induces a chain of consecutive reactions in endothelial cells: phosphorylation of AKT at S473 and eNOS at S1176. The VEGFR2 phosphosite Y949 mediates phosphorylation of c-Src at Y418 and of VE-cadherin at Y685. Combined, these activating phosphorylation reactions disrupt the vascular barrier by dissociating VE-cadherin’s homophilic interactions, resulting in macromolecular leakage. eNOS/NO exacerbates this damage via an interaction with c-Src to enhance VE-cadherin Y685 phosphorylation and internalization.

    Techniques Used:

    5) Product Images from "EphrinB2/EphB4 signaling regulates non‐sprouting angiogenesis by VEGF"

    Article Title: EphrinB2/EphB4 signaling regulates non‐sprouting angiogenesis by VEGF

    Journal: EMBO Reports

    doi: 10.15252/embr.201745054

    Expression of endogenous Fgf2 and endogenous and total Vegfa A–D Gene expression of Fgf2 (A) and total (B) or endogenous Vegfa (C) was quantified in skeletal muscles 3 days after myoblast implantation and expressed as fold‐change vs. control muscles. The relative magnitude of gene expression changes can be better appreciated by plotting the data on the same scale (D). Mean ± SEM; n = 4–6 independent samples/group; ** P
    Figure Legend Snippet: Expression of endogenous Fgf2 and endogenous and total Vegfa A–D Gene expression of Fgf2 (A) and total (B) or endogenous Vegfa (C) was quantified in skeletal muscles 3 days after myoblast implantation and expressed as fold‐change vs. control muscles. The relative magnitude of gene expression changes can be better appreciated by plotting the data on the same scale (D). Mean ± SEM; n = 4–6 independent samples/group; ** P

    Techniques Used: Expressing

    6) Product Images from "Different pro-angiogenic potential of γ-irradiated PBMC-derived secretome and its subfractions"

    Article Title: Different pro-angiogenic potential of γ-irradiated PBMC-derived secretome and its subfractions

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36928-6

    MNC aposec and EVs induce Vegfa and Ena-78 mRNA expression in aortas from diabetic mice. Aortic rings were stimulated with medium, MNC aposec , EVs, proteins, and lipids and ( A ) Vegfa and ( B ) Ena-78 expression normalized to the housekeeping gene Gapdh . Data are means +/− SD. Mean represents statistical analysis of three replicates; *p
    Figure Legend Snippet: MNC aposec and EVs induce Vegfa and Ena-78 mRNA expression in aortas from diabetic mice. Aortic rings were stimulated with medium, MNC aposec , EVs, proteins, and lipids and ( A ) Vegfa and ( B ) Ena-78 expression normalized to the housekeeping gene Gapdh . Data are means +/− SD. Mean represents statistical analysis of three replicates; *p

    Techniques Used: Expressing, Mouse Assay

    7) Product Images from "Epidermal Smad4 Deletion Results in Aberrant Wound Healing"

    Article Title: Epidermal Smad4 Deletion Results in Aberrant Wound Healing

    Journal: The American Journal of Pathology

    doi: 10.2353/ajpath.2010.090081

    Smad4 KO spontaneous tumors exhibited molecular changes similar to Smad4 KO wounds. qRT- PCR compares at least three Smad4 KO skins with at least ten tumors for mRNA of VEGFA ( A ), CCL5 ( B ), CCL20 ( C ), MMP14 ( D ), and TGFβ ( E ) are all significantly
    Figure Legend Snippet: Smad4 KO spontaneous tumors exhibited molecular changes similar to Smad4 KO wounds. qRT- PCR compares at least three Smad4 KO skins with at least ten tumors for mRNA of VEGFA ( A ), CCL5 ( B ), CCL20 ( C ), MMP14 ( D ), and TGFβ ( E ) are all significantly

    Techniques Used: Quantitative RT-PCR

    8) 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

    9) Product Images from "EphrinB2/EphB4 signaling regulates non‐sprouting angiogenesis by VEGF"

    Article Title: EphrinB2/EphB4 signaling regulates non‐sprouting angiogenesis by VEGF

    Journal: EMBO Reports

    doi: 10.15252/embr.201745054

    Expression of endogenous Fgf2 and endogenous and total Vegfa Gene expression of Fgf2 (A) and total (B) or endogenous Vegfa (C) was quantified in skeletal muscles 3 days after myoblast implantation and expressed as fold‐change vs. control muscles. The relative magnitude of gene expression changes can be better appreciated by plotting the data on the same scale (D). Mean ± SEM; n = 4–6 independent samples/group; ** P
    Figure Legend Snippet: Expression of endogenous Fgf2 and endogenous and total Vegfa Gene expression of Fgf2 (A) and total (B) or endogenous Vegfa (C) was quantified in skeletal muscles 3 days after myoblast implantation and expressed as fold‐change vs. control muscles. The relative magnitude of gene expression changes can be better appreciated by plotting the data on the same scale (D). Mean ± SEM; n = 4–6 independent samples/group; ** P

    Techniques Used: Expressing

    10) Product Images from "Angiotensin II Evokes Angiogenic Signals within Skeletal Muscle through Co-ordinated Effects on Skeletal Myocytes and Endothelial Cells"

    Article Title: Angiotensin II Evokes Angiogenic Signals within Skeletal Muscle through Co-ordinated Effects on Skeletal Myocytes and Endothelial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0085537

    Contribution of endogenous Ang II to muscle VEGF production and endothelial cell-muscle cell crosstalk. C2C12 myotubes and confluent cultures of microvascular endothelial cells were lysed, and endogenous basal levels of angiotensinogen, AT1R and AT2R were assessed by Western blot analysis (A). In all blots, two independent cultures are shown for each cell type. C2C12 myotubes (n = 3 per condition) were treated overnight with the AT1R inhibitor Losartan (0.1 µM or 1 µM) and VEGF transcript levels were assessed by qPCR (B). Representative image and the quantification of RT-PCR analysis of VEGFA isoform expression (VEGF 120 ∼170 bp; VEGF 164 ∼300 bp and VEGF 180 = not detected) in C2C12 myotubes and skeletal muscle endothelial cells (C). Two independent samples are shown for both C2C12 and endothelial cells (n = 6 for C2C12 and n = 13 for endothelial cells). Lysates of matrix bound proteins were assessed for VEGF and tubulin protein expression by Western blotting (D). Cell extract (Ex) was used as a comparator with extracts of matrix (M) alone. Three independent samples of matrix-associated protein extracts are shown. Tubulin was detectable within cell extract, but not in matrix-derived extracts. In (E), endothelial cells alone or previously treated with an inhibitor of the VEGFR2, were incubated on matrix which previously contained C2C12 myoblasts with or without losartan treatment. Representative image and quantification of phosphorylated p38 levels (n = 6 for E and E+M, n = 5 for E+M L and n = 4 for E V +M). Values are presented as mean ± SEM. One way ANOVA followed by Tukey’s multiple comparison test and student’s t-test were used to assess statistical significance which was set as p
    Figure Legend Snippet: Contribution of endogenous Ang II to muscle VEGF production and endothelial cell-muscle cell crosstalk. C2C12 myotubes and confluent cultures of microvascular endothelial cells were lysed, and endogenous basal levels of angiotensinogen, AT1R and AT2R were assessed by Western blot analysis (A). In all blots, two independent cultures are shown for each cell type. C2C12 myotubes (n = 3 per condition) were treated overnight with the AT1R inhibitor Losartan (0.1 µM or 1 µM) and VEGF transcript levels were assessed by qPCR (B). Representative image and the quantification of RT-PCR analysis of VEGFA isoform expression (VEGF 120 ∼170 bp; VEGF 164 ∼300 bp and VEGF 180 = not detected) in C2C12 myotubes and skeletal muscle endothelial cells (C). Two independent samples are shown for both C2C12 and endothelial cells (n = 6 for C2C12 and n = 13 for endothelial cells). Lysates of matrix bound proteins were assessed for VEGF and tubulin protein expression by Western blotting (D). Cell extract (Ex) was used as a comparator with extracts of matrix (M) alone. Three independent samples of matrix-associated protein extracts are shown. Tubulin was detectable within cell extract, but not in matrix-derived extracts. In (E), endothelial cells alone or previously treated with an inhibitor of the VEGFR2, were incubated on matrix which previously contained C2C12 myoblasts with or without losartan treatment. Representative image and quantification of phosphorylated p38 levels (n = 6 for E and E+M, n = 5 for E+M L and n = 4 for E V +M). Values are presented as mean ± SEM. One way ANOVA followed by Tukey’s multiple comparison test and student’s t-test were used to assess statistical significance which was set as p

    Techniques Used: Western Blot, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Derivative Assay, Incubation

    11) Product Images from "Different pro-angiogenic potential of γ-irradiated PBMC-derived secretome and its subfractions"

    Article Title: Different pro-angiogenic potential of γ-irradiated PBMC-derived secretome and its subfractions

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36928-6

    MNC aposec and EVs induce Vegfa and Ena-78 mRNA expression in aortas from diabetic mice. Aortic rings were stimulated with medium, MNC aposec , EVs, proteins, and lipids and ( A ) Vegfa and ( B ) Ena-78 expression normalized to the housekeeping gene Gapdh . Data are means +/− SD. Mean represents statistical analysis of three replicates; *p
    Figure Legend Snippet: MNC aposec and EVs induce Vegfa and Ena-78 mRNA expression in aortas from diabetic mice. Aortic rings were stimulated with medium, MNC aposec , EVs, proteins, and lipids and ( A ) Vegfa and ( B ) Ena-78 expression normalized to the housekeeping gene Gapdh . Data are means +/− SD. Mean represents statistical analysis of three replicates; *p

    Techniques Used: Expressing, Mouse Assay

    12) Product Images from "Angiopoietin 2 induces astrocyte apoptosis via αvβ5-integrin signaling in diabetic retinopathy"

    Article Title: Angiopoietin 2 induces astrocyte apoptosis via αvβ5-integrin signaling in diabetic retinopathy

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2015.347

    Inhibition of Ang2 reduces the astrocyte loss and vascular leakage in early diabetic retina. ( a – c ) Retinal mRNA was determined in 1, 3, 5, and 7 weeks from streptozotocin-induced diabetic mice (DM) and control mice (Con) retinas by qPCR, and normalized to Rn18s mRNA. ( a ) Ang2 mRNA. ( b ) Ang1 mRNA. ( c ) Vegfa mRNA. ( d – f ) Anti-Ang2-neutralizing antibody (Anti-Ang2 Ab, 1 μ g) or PBS was intravitreally injected to 2-week-old DM. Retinal astrocyte and retinal vascular leakage were evaluated 1 week after the injection in 3-week-old DM. ( d ) Focal astrocyte loss shown in diabetic retina (PBS) is rescued in anti-Ang2 Ab-injected diabetic mice (original magnification × 400; scale bar, 100 μ m). White arrowheads indicate loss of astrocytes on diabetic retinal vessels. ( e ) Relative astrocyte coverage (% Anti-Ang2 Ab) is calculated by colocalized area per IB4 + vascular area and normalized to the value of control mice. ( f ) Relative retinal vascular leakage (% Anti-Ang2 Ab) with FITC-dextran is shown normalized to the value of control mice. ( g ) Body weight and blood glucose level of the STZ-induced diabetic and non-diabetic control mice at 7 weeks. The sample size for each group is indicated on the bar graph. The bar graphs represent mean±S.E.M.; * P
    Figure Legend Snippet: Inhibition of Ang2 reduces the astrocyte loss and vascular leakage in early diabetic retina. ( a – c ) Retinal mRNA was determined in 1, 3, 5, and 7 weeks from streptozotocin-induced diabetic mice (DM) and control mice (Con) retinas by qPCR, and normalized to Rn18s mRNA. ( a ) Ang2 mRNA. ( b ) Ang1 mRNA. ( c ) Vegfa mRNA. ( d – f ) Anti-Ang2-neutralizing antibody (Anti-Ang2 Ab, 1 μ g) or PBS was intravitreally injected to 2-week-old DM. Retinal astrocyte and retinal vascular leakage were evaluated 1 week after the injection in 3-week-old DM. ( d ) Focal astrocyte loss shown in diabetic retina (PBS) is rescued in anti-Ang2 Ab-injected diabetic mice (original magnification × 400; scale bar, 100 μ m). White arrowheads indicate loss of astrocytes on diabetic retinal vessels. ( e ) Relative astrocyte coverage (% Anti-Ang2 Ab) is calculated by colocalized area per IB4 + vascular area and normalized to the value of control mice. ( f ) Relative retinal vascular leakage (% Anti-Ang2 Ab) with FITC-dextran is shown normalized to the value of control mice. ( g ) Body weight and blood glucose level of the STZ-induced diabetic and non-diabetic control mice at 7 weeks. The sample size for each group is indicated on the bar graph. The bar graphs represent mean±S.E.M.; * P

    Techniques Used: Inhibition, Mouse Assay, Real-time Polymerase Chain Reaction, Injection

    13) Product Images from "Metabolic origins of spatial organization in the tumor microenvironment"

    Article Title: Metabolic origins of spatial organization in the tumor microenvironment

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

    doi: 10.1073/pnas.1700600114

    Macrophages relay their positional information to endothelial cells and orchestrate efficient tube morphogenesis. ( A ) Our data suggest that, in a process resembling embryological organization, gradients of extracellular metabolites convey positional information that modifies TAM phenotypes. We asked whether additional signaling molecules, secreted by patterned macrophages, could relay positional information to other tumor cells. ( B ) Screening of 111 secreted chemokines revealed that ischemic macrophages express VEGFA. ( C ) Quantification of secreted chemokines confirmed this finding and the synergy between lactate and hypoxia (bars indicate SD from six biological replicates; ** P
    Figure Legend Snippet: Macrophages relay their positional information to endothelial cells and orchestrate efficient tube morphogenesis. ( A ) Our data suggest that, in a process resembling embryological organization, gradients of extracellular metabolites convey positional information that modifies TAM phenotypes. We asked whether additional signaling molecules, secreted by patterned macrophages, could relay positional information to other tumor cells. ( B ) Screening of 111 secreted chemokines revealed that ischemic macrophages express VEGFA. ( C ) Quantification of secreted chemokines confirmed this finding and the synergy between lactate and hypoxia (bars indicate SD from six biological replicates; ** P

    Techniques Used:

    14) Product Images from "Inflammatory Skin-Derived Cytokines Accelerate Osteoporosis in Mice with Persistent Skin Inflammation"

    Article Title: Inflammatory Skin-Derived Cytokines Accelerate Osteoporosis in Mice with Persistent Skin Inflammation

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21103620

    Bone mineral density (BMD) and expression of mRNAs associated to bone formation or destruction in the femur of KCASP1Tg and wild-type mice. BMD was scanned at the distal end of the right femur in KCASP1Tg and wild-type littermates of 5 to 52 weeks of age ( n = 4 per group). In KCASP1Tg mice, BMD decreased significantly compared to wild-type littermates at 12 weeks of age ( A ). The cortical bone width was measured in HE (upper panel × 40, lower panel × 100). Four parts ( n = 3 per group) were randomly selected and measured by using Analysis Application Hybrid cell count. The cortical width of the femur was significantly thinner in KCASP1Tg mouse, as determined by histological HE staining ( B,C ). Bone remodeling markers including TRACP-5b and osteocalcin were measured in the serum by a specific ELISA kit. The serum level of TRACP-5b is a sensitive marker of bone resorption, and was significantly increased in KCASP1Tg mice ( D ), but the biomarker of bone formation, serum osteocalcin, was unchanged (( E ), n = 8 per group). The expression of relevant mRNAs in the right femur was quantified by real time PCR, and the values were standardized by using GAPDH ( n = 6 per group). The expressions of genes involved in bone formation, such as alkaline phosphatase-( Alpl , ( F )), collagen type I ( Col1a1 , ( G )) and bone gamma-carboxyglutamate protein ( Bglap , osteocalcin, ( H )) were significantly decreased in KCASP1Tg mice. The mRNA levels of genes for osteoclast differentiation and activation, i.e., members of the tumor necrosis factor superfamily ( Tnfsf11 , RANKL , ( I )) and vascular endothelial growth factor ( Vegfa , ( J )) were unchanged in the model mice. All data are expressed as mean ± SD. *; p
    Figure Legend Snippet: Bone mineral density (BMD) and expression of mRNAs associated to bone formation or destruction in the femur of KCASP1Tg and wild-type mice. BMD was scanned at the distal end of the right femur in KCASP1Tg and wild-type littermates of 5 to 52 weeks of age ( n = 4 per group). In KCASP1Tg mice, BMD decreased significantly compared to wild-type littermates at 12 weeks of age ( A ). The cortical bone width was measured in HE (upper panel × 40, lower panel × 100). Four parts ( n = 3 per group) were randomly selected and measured by using Analysis Application Hybrid cell count. The cortical width of the femur was significantly thinner in KCASP1Tg mouse, as determined by histological HE staining ( B,C ). Bone remodeling markers including TRACP-5b and osteocalcin were measured in the serum by a specific ELISA kit. The serum level of TRACP-5b is a sensitive marker of bone resorption, and was significantly increased in KCASP1Tg mice ( D ), but the biomarker of bone formation, serum osteocalcin, was unchanged (( E ), n = 8 per group). The expression of relevant mRNAs in the right femur was quantified by real time PCR, and the values were standardized by using GAPDH ( n = 6 per group). The expressions of genes involved in bone formation, such as alkaline phosphatase-( Alpl , ( F )), collagen type I ( Col1a1 , ( G )) and bone gamma-carboxyglutamate protein ( Bglap , osteocalcin, ( H )) were significantly decreased in KCASP1Tg mice. The mRNA levels of genes for osteoclast differentiation and activation, i.e., members of the tumor necrosis factor superfamily ( Tnfsf11 , RANKL , ( I )) and vascular endothelial growth factor ( Vegfa , ( J )) were unchanged in the model mice. All data are expressed as mean ± SD. *; p

    Techniques Used: Expressing, Mouse Assay, Cell Counting, Staining, Enzyme-linked Immunosorbent Assay, Marker, Biomarker Assay, Real-time Polymerase Chain Reaction, Activation Assay

    15) Product Images from "Stanniocalcin-1 is a modifier of oxygen induced retinopathy severity"

    Article Title: Stanniocalcin-1 is a modifier of oxygen induced retinopathy severity

    Journal: Current eye research

    doi: 10.1080/02713683.2019.1645184

    Stc-1 −/− mice show higher levels of VEGFA compared to wild-type in a mouse model of oxygen induced retinopathy Quantitative real-time PCR shows that Stc-1 −/− mice had a significant increase in expression of VEGF-A (P=0.03)* at P17 compared to wild-type controls. Error bars represent SEM.
    Figure Legend Snippet: Stc-1 −/− mice show higher levels of VEGFA compared to wild-type in a mouse model of oxygen induced retinopathy Quantitative real-time PCR shows that Stc-1 −/− mice had a significant increase in expression of VEGF-A (P=0.03)* at P17 compared to wild-type controls. Error bars represent SEM.

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

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: YAP1 and TAZ negatively control bone angiogenesis by limiting hypoxia-inducible factor signaling in endothelial cells
    Article Snippet: RNA was reverse-transcribed using the iScript cDNA synthesis kit (Bio-Rad, Cat#1708890). .. Quantitative PCR was carried out using gene TaqMan Gene Expression Master Mix (ThermoFisher Scientific, Cat#4369016) and specific Taqman probes human: eukaryotic 18S rRNA (4319413E), VEGFA (Hs00900055_m1, ANGPTL4 (Hs01101127_m1), IGFBP2 (Hs01040719_m1), XBP1 (Hs00231936_m1), CTGF (Hs01026927_g1), CYR61(Hs00998500_g1), YAP1(Hs00902712_g1), WWTR1(Hs00210007_m1), HIF1A(Hs00153153_m1) and mouse probes: Vegfa (Mm00437306_m1), Angptl4 (Mm00480431_m1), Ctgf (Mm01192932_g1), Cyr61 (Mm00487499_g1) from ThermoFisher Scientific (Cat# 4331182) using a C1000 Touch Thermal cycler (BIORAD). .. Primer sequences for qPCR analysis of Yap1 and Wwtr 1 expression in freshly isolated bone ECs ( ) are provided in the Key Resources Table ( ).

    Article Title: eNOS-induced vascular barrier disruption in retinopathy by c-Src activation and tyrosine phosphorylation of VE-cadherin
    Article Snippet: Quantitative real-time PCR RNA from retinas were purified using RNeasy Kit (Qiagen). .. One microgram of RNA was reverse transcribed using SuperScript III (Invitrogen) and quantitative PCR were assayed using Mus musculus primers against Vegfa (Mm00437306_m1, ThermoFisher), Nos3 (Mm00435217_m1) and Nos2 (Mm00440502_m1). .. The expression levels were normalized against TATA binding protein (TBP) Mus musculus (Mm01277042_m1,ThermoFisher) and Ubiqutin C (UBC) Mus musculus (Mm02525934_g1, ThermoFisher).

    Expressing:

    Article Title: YAP1 and TAZ negatively control bone angiogenesis by limiting hypoxia-inducible factor signaling in endothelial cells
    Article Snippet: RNA was reverse-transcribed using the iScript cDNA synthesis kit (Bio-Rad, Cat#1708890). .. Quantitative PCR was carried out using gene TaqMan Gene Expression Master Mix (ThermoFisher Scientific, Cat#4369016) and specific Taqman probes human: eukaryotic 18S rRNA (4319413E), VEGFA (Hs00900055_m1, ANGPTL4 (Hs01101127_m1), IGFBP2 (Hs01040719_m1), XBP1 (Hs00231936_m1), CTGF (Hs01026927_g1), CYR61(Hs00998500_g1), YAP1(Hs00902712_g1), WWTR1(Hs00210007_m1), HIF1A(Hs00153153_m1) and mouse probes: Vegfa (Mm00437306_m1), Angptl4 (Mm00480431_m1), Ctgf (Mm01192932_g1), Cyr61 (Mm00487499_g1) from ThermoFisher Scientific (Cat# 4331182) using a C1000 Touch Thermal cycler (BIORAD). .. Primer sequences for qPCR analysis of Yap1 and Wwtr 1 expression in freshly isolated bone ECs ( ) are provided in the Key Resources Table ( ).

    Article Title: EphrinB2/EphB4 signaling regulates non‐sprouting angiogenesis by VEGF
    Article Snippet: RNA was reverse transcribed into cDNA using the Omniscript Reverse Transcription kit (Qiagen) at 37°C for 60 min. Quantitative real‐time PCR (qRT–PCR) was performed on an ABI 7300 Real‐Time PCR system (Applied Biosystems, Basel, Switzerland). .. Expression of genes of interest was determined using the following TaqMan Gene Expression assays (Applied Biosystems) according to manufacturer's instructions: mouse Tnfa (Mm00443258_m1); mouse Gapdh (Mm03302249_g1); mouse Pdgfb (Mm00440678_m1); mouse Pdgfrb (Mm00435545_m1); mouse Fgf2 (Mm01285715_m1); mouse Vegfa (Mm00437306_m1); human Igfbp3 (Hs00365742_g1); human Esm1 (Hs00199831_m1); and human Gapdh (Hs02758991_g1). .. In order to quantify endogenous Vegfa transcripts separately from those from the transduced myoblasts, a previously designed sets of primers and probe were used to detect a sequence on the mRNA 5′‐UTR, which is absent from the vector‐encoded transcripts .

    Chloramphenicol Acetyltransferase Assay:

    Article Title: YAP1 and TAZ negatively control bone angiogenesis by limiting hypoxia-inducible factor signaling in endothelial cells
    Article Snippet: RNA was reverse-transcribed using the iScript cDNA synthesis kit (Bio-Rad, Cat#1708890). .. Quantitative PCR was carried out using gene TaqMan Gene Expression Master Mix (ThermoFisher Scientific, Cat#4369016) and specific Taqman probes human: eukaryotic 18S rRNA (4319413E), VEGFA (Hs00900055_m1, ANGPTL4 (Hs01101127_m1), IGFBP2 (Hs01040719_m1), XBP1 (Hs00231936_m1), CTGF (Hs01026927_g1), CYR61(Hs00998500_g1), YAP1(Hs00902712_g1), WWTR1(Hs00210007_m1), HIF1A(Hs00153153_m1) and mouse probes: Vegfa (Mm00437306_m1), Angptl4 (Mm00480431_m1), Ctgf (Mm01192932_g1), Cyr61 (Mm00487499_g1) from ThermoFisher Scientific (Cat# 4331182) using a C1000 Touch Thermal cycler (BIORAD). .. Primer sequences for qPCR analysis of Yap1 and Wwtr 1 expression in freshly isolated bone ECs ( ) are provided in the Key Resources Table ( ).

    other:

    Article Title: Genome-wide redistribution of MeCP2 in dorsal root ganglia after peripheral nerve injury
    Article Snippet: The Assay ID for TaqMan primer probes used were Mm00599780_g1 (Dnmt1) and Mm00437306_m1 (Vegfa).

    Article Title: Ferrochelatase regulates retinal neovascularization
    Article Snippet: Primer/probe sets used were as follows: Vegfa (Mm00437306_m1), Fech (Mm00500394_m1) and housekeeping controls Hprt (Hs03024075_m1) and Tbp (Mm01277042_m1).

    Article Title: Different pro-angiogenic potential of γ-irradiated PBMC-derived secretome and its subfractions
    Article Snippet: Assay IDs were: Gapdh Mm99999915_g1; Vegfa Mm00437306_m1; Ena-78/Cxcl5 Mm00436451_g1.

    Article Title: Semaphorin 3A induces cytoskeletal paralysis in tumor-specific CD8+ T cells
    Article Snippet: The following TagMan probes were used: BNIP3 (Mm01275600-g1), HPRT (Mm03024075-m1), PDK1 (Mm00554300-m1), PDL1 (Mm00452054-m1), SEMA3A (Mm00436469-m1) and VEGFA (Mm00437306-m1).

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    Thermo Fisher gene exp vegfa mm00437306 m1
    A. Gating strategy for analyzing Sema3A expression among leukocytes, epithelial and endothelial cells in lung and dLN. B. Quantification of Sema3A positive cells in different cell populations in uninfected (n=4), and infected mice, at 3 days DPI (n=4) and 10 DPI (n=4) in lung (left panel) or dLN (right panel). Nrp1 Flox/Flox mice used. Data combined from two independent experiments. ns = not significant by two-way ANOVA. C. Quantification of Pkd1, Bnip3, <t>Vegfa,</t> Pdl1 and Sema3A mRNA level following 24 hour culture in 1% O2 chamber. D. Gating strategy (left) and representative histograms (right) analyzing Sema3A positive cell populations in B16.F10 tumors 11 days post-injection. Olfactory lobe is used as a positive control for Sema3A expression. E. Quantification of Sema3A positive cells in same experiment as in (D) in tumor, dLN and ndLN. F. Genomic organization of murine Sema3a gene, indicating where CRISPR guide RNA targets with red arrow (upper figure). MiSEQ sequence results for chosen Sema3A KO clone showing 4 base deletion in two alleles (46% of all reads), a frameshift in one allele (45% of all reads) and WT reads in 2% of all reads. G. RT-qPCR analysis show down- and up-regulation of Sema3A in Sema3A KO and OE cell lines, respectively. Normalized to Hprt. Experiment performed once. H. Intracellular staining shows no detectable expression of Sema3A in Sema3A KO cells, and expression in Sema3A OE cells, as expected. Experiment performed once, at low seeding density. I. Growth of WT, Sema3A KO and Sema3A OE B16.F10.Ova cell lines in normal, IFNγ or TNFα-rich media. Experiment performed once. Data indicate mean ± SD. ns = not significant by two-way ANOVA. Abbreviations: BEC, blood endothelial cells. dLN, draining lymph node. DPI, days post-infection. FRC, fibroblastic reticular cells. KO, knockout. LEC, lymphatic endothelial cells. ndLN, non-draining lymph node. OE, overexpressing. TME, tumor microenvironment. WT, wild-type.
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    A. Gating strategy for analyzing Sema3A expression among leukocytes, epithelial and endothelial cells in lung and dLN. B. Quantification of Sema3A positive cells in different cell populations in uninfected (n=4), and infected mice, at 3 days DPI (n=4) and 10 DPI (n=4) in lung (left panel) or dLN (right panel). Nrp1 Flox/Flox mice used. Data combined from two independent experiments. ns = not significant by two-way ANOVA. C. Quantification of Pkd1, Bnip3, Vegfa, Pdl1 and Sema3A mRNA level following 24 hour culture in 1% O2 chamber. D. Gating strategy (left) and representative histograms (right) analyzing Sema3A positive cell populations in B16.F10 tumors 11 days post-injection. Olfactory lobe is used as a positive control for Sema3A expression. E. Quantification of Sema3A positive cells in same experiment as in (D) in tumor, dLN and ndLN. F. Genomic organization of murine Sema3a gene, indicating where CRISPR guide RNA targets with red arrow (upper figure). MiSEQ sequence results for chosen Sema3A KO clone showing 4 base deletion in two alleles (46% of all reads), a frameshift in one allele (45% of all reads) and WT reads in 2% of all reads. G. RT-qPCR analysis show down- and up-regulation of Sema3A in Sema3A KO and OE cell lines, respectively. Normalized to Hprt. Experiment performed once. H. Intracellular staining shows no detectable expression of Sema3A in Sema3A KO cells, and expression in Sema3A OE cells, as expected. Experiment performed once, at low seeding density. I. Growth of WT, Sema3A KO and Sema3A OE B16.F10.Ova cell lines in normal, IFNγ or TNFα-rich media. Experiment performed once. Data indicate mean ± SD. ns = not significant by two-way ANOVA. Abbreviations: BEC, blood endothelial cells. dLN, draining lymph node. DPI, days post-infection. FRC, fibroblastic reticular cells. KO, knockout. LEC, lymphatic endothelial cells. ndLN, non-draining lymph node. OE, overexpressing. TME, tumor microenvironment. WT, wild-type.

    Journal: bioRxiv

    Article Title: Semaphorin 3A induces cytoskeletal paralysis in tumor-specific CD8+ T cells

    doi: 10.1101/849083

    Figure Lengend Snippet: A. Gating strategy for analyzing Sema3A expression among leukocytes, epithelial and endothelial cells in lung and dLN. B. Quantification of Sema3A positive cells in different cell populations in uninfected (n=4), and infected mice, at 3 days DPI (n=4) and 10 DPI (n=4) in lung (left panel) or dLN (right panel). Nrp1 Flox/Flox mice used. Data combined from two independent experiments. ns = not significant by two-way ANOVA. C. Quantification of Pkd1, Bnip3, Vegfa, Pdl1 and Sema3A mRNA level following 24 hour culture in 1% O2 chamber. D. Gating strategy (left) and representative histograms (right) analyzing Sema3A positive cell populations in B16.F10 tumors 11 days post-injection. Olfactory lobe is used as a positive control for Sema3A expression. E. Quantification of Sema3A positive cells in same experiment as in (D) in tumor, dLN and ndLN. F. Genomic organization of murine Sema3a gene, indicating where CRISPR guide RNA targets with red arrow (upper figure). MiSEQ sequence results for chosen Sema3A KO clone showing 4 base deletion in two alleles (46% of all reads), a frameshift in one allele (45% of all reads) and WT reads in 2% of all reads. G. RT-qPCR analysis show down- and up-regulation of Sema3A in Sema3A KO and OE cell lines, respectively. Normalized to Hprt. Experiment performed once. H. Intracellular staining shows no detectable expression of Sema3A in Sema3A KO cells, and expression in Sema3A OE cells, as expected. Experiment performed once, at low seeding density. I. Growth of WT, Sema3A KO and Sema3A OE B16.F10.Ova cell lines in normal, IFNγ or TNFα-rich media. Experiment performed once. Data indicate mean ± SD. ns = not significant by two-way ANOVA. Abbreviations: BEC, blood endothelial cells. dLN, draining lymph node. DPI, days post-infection. FRC, fibroblastic reticular cells. KO, knockout. LEC, lymphatic endothelial cells. ndLN, non-draining lymph node. OE, overexpressing. TME, tumor microenvironment. WT, wild-type.

    Article Snippet: The following TagMan probes were used: BNIP3 (Mm01275600-g1), HPRT (Mm03024075-m1), PDK1 (Mm00554300-m1), PDL1 (Mm00452054-m1), SEMA3A (Mm00436469-m1) and VEGFA (Mm00437306-m1).

    Techniques: Expressing, Infection, Mouse Assay, Injection, Positive Control, CRISPR, Sequencing, Quantitative RT-PCR, Staining, Knock-Out

    Ferrochelatase (FECH) expression and vascular pathology in the oxygen-induced retinopathy (OIR) mouse model. A ) Scheme: Juvenile C57BL/6J mice at postnatal day (P) 7 were exposed to hyperoxia chamber for 5 days and returned to room air (normoxia) at P12. Retinal analysis was performed at various time points as indicated. Retinas were isolated for RNA analysis, or fixed, cryosectioned, and immunostained, and retinal sections or whole flatmounts were analyzed. B ) Retinal flatmount from OIR and control retinas stained with GS-IB4 (green; labels vasculature) and pimonidazole (red; labels hypoxic regions) showing the temporal pattern of hypoxia and formation of pathological neovascular tufts in OIR retina. C ) Gene expression analysis of Fech and Vegfa by qPCR of RNA derived from whole retinal samples. Temporal analysis indicated Fech and Vegfa are upregulated in OIR. C t valves from gene expression data were normalized to the housekeeping genes ( Tbp and Hprt ) and respective age matched untouched retina control (ΔΔC t method). All qPCR reactions were run in technical triplicate, N=3 biological replicates. Data presented as mean ± SEM, *p

    Journal: bioRxiv

    Article Title: Ferrochelatase regulates retinal neovascularization

    doi: 10.1101/2020.06.02.129650

    Figure Lengend Snippet: Ferrochelatase (FECH) expression and vascular pathology in the oxygen-induced retinopathy (OIR) mouse model. A ) Scheme: Juvenile C57BL/6J mice at postnatal day (P) 7 were exposed to hyperoxia chamber for 5 days and returned to room air (normoxia) at P12. Retinal analysis was performed at various time points as indicated. Retinas were isolated for RNA analysis, or fixed, cryosectioned, and immunostained, and retinal sections or whole flatmounts were analyzed. B ) Retinal flatmount from OIR and control retinas stained with GS-IB4 (green; labels vasculature) and pimonidazole (red; labels hypoxic regions) showing the temporal pattern of hypoxia and formation of pathological neovascular tufts in OIR retina. C ) Gene expression analysis of Fech and Vegfa by qPCR of RNA derived from whole retinal samples. Temporal analysis indicated Fech and Vegfa are upregulated in OIR. C t valves from gene expression data were normalized to the housekeeping genes ( Tbp and Hprt ) and respective age matched untouched retina control (ΔΔC t method). All qPCR reactions were run in technical triplicate, N=3 biological replicates. Data presented as mean ± SEM, *p

    Article Snippet: Primer/probe sets used were as follows: Vegfa (Mm00437306_m1), Fech (Mm00500394_m1) and housekeeping controls Hprt (Hs03024075_m1) and Tbp (Mm01277042_m1).

    Techniques: Expressing, Mouse Assay, Isolation, Staining, Real-time Polymerase Chain Reaction, Derivative Assay

    miR-126 regulates expression of Dnmt1 and Vegfa in vitro. a Relative expression of endogenous Dnmt1 and Vegfa mRNA in Neuro 2a cells transfected with miR-126 Gapdh was used as a normalizer. b Representative Western blot of Dnmt1 and Vegfa using lysate of Neuro 2a cells transfected with miR-126 precursor plasmid for 72 h. Overexpression of miR-126 decreased mRNA and protein levels of Dnmt1 and Vegfa; beta tubulin was used as the control. c Immunohistochemistry indicating transfection with miR-126 plasmid co-expressing GFP in Neuro 2a cells decreased Dnmt1 levels compared to untransfected cells 72 h post-transfection. Significance determined using unpaired Student’s t test, p value *

    Journal: Epigenetics & Chromatin

    Article Title: Genome-wide redistribution of MeCP2 in dorsal root ganglia after peripheral nerve injury

    doi: 10.1186/s13072-016-0073-5

    Figure Lengend Snippet: miR-126 regulates expression of Dnmt1 and Vegfa in vitro. a Relative expression of endogenous Dnmt1 and Vegfa mRNA in Neuro 2a cells transfected with miR-126 Gapdh was used as a normalizer. b Representative Western blot of Dnmt1 and Vegfa using lysate of Neuro 2a cells transfected with miR-126 precursor plasmid for 72 h. Overexpression of miR-126 decreased mRNA and protein levels of Dnmt1 and Vegfa; beta tubulin was used as the control. c Immunohistochemistry indicating transfection with miR-126 plasmid co-expressing GFP in Neuro 2a cells decreased Dnmt1 levels compared to untransfected cells 72 h post-transfection. Significance determined using unpaired Student’s t test, p value *

    Article Snippet: The Assay ID for TaqMan primer probes used were Mm00599780_g1 (Dnmt1) and Mm00437306_m1 (Vegfa).

    Techniques: Expressing, In Vitro, Transfection, Western Blot, Plasmid Preparation, Over Expression, Immunohistochemistry

    Administration of exogenous miR-126 decreased Dnmt1 and Vegfa expression in vivo but did not alter pain sensitivity. a Mechanical sensitivity measured by von Frey filaments showed that intrathecal delivery of miR-126 did not alter the paw withdrawal threshold in SNI model mice. Arrows indicate daily intrathecal injections with 2 nmol miR-126 or control miRNA via catheter ( n = 5 for miR-126 and miR-control injected mice, n = 3 for PBS injected mice). b Confirmation of miR-126 delivery to DRG. A qPCR performed using DRG collected from mice injected with miR-126 or control miRNA showed an increase in miR-126 in mice that received miR-126 compared to miR-control injected mice indicating successful delivery. c Relative expression of Dnmt1 and Vegfa mRNA in the DRG of miR-126 and control injected mice. Increased miR-126 decreased the expression of endogenous Dnmt1 and Vegfa compared to miR-control injected mice. Significance determined using unpaired Student’s t test, p value, **

    Journal: Epigenetics & Chromatin

    Article Title: Genome-wide redistribution of MeCP2 in dorsal root ganglia after peripheral nerve injury

    doi: 10.1186/s13072-016-0073-5

    Figure Lengend Snippet: Administration of exogenous miR-126 decreased Dnmt1 and Vegfa expression in vivo but did not alter pain sensitivity. a Mechanical sensitivity measured by von Frey filaments showed that intrathecal delivery of miR-126 did not alter the paw withdrawal threshold in SNI model mice. Arrows indicate daily intrathecal injections with 2 nmol miR-126 or control miRNA via catheter ( n = 5 for miR-126 and miR-control injected mice, n = 3 for PBS injected mice). b Confirmation of miR-126 delivery to DRG. A qPCR performed using DRG collected from mice injected with miR-126 or control miRNA showed an increase in miR-126 in mice that received miR-126 compared to miR-control injected mice indicating successful delivery. c Relative expression of Dnmt1 and Vegfa mRNA in the DRG of miR-126 and control injected mice. Increased miR-126 decreased the expression of endogenous Dnmt1 and Vegfa compared to miR-control injected mice. Significance determined using unpaired Student’s t test, p value, **

    Article Snippet: The Assay ID for TaqMan primer probes used were Mm00599780_g1 (Dnmt1) and Mm00437306_m1 (Vegfa).

    Techniques: Expressing, In Vivo, Mouse Assay, Injection, Real-time Polymerase Chain Reaction

    Expression of miR-126 and its target genes Dnmt1 and Vegfa in the DRG after nerve injury. a Relative expression of miR-126 determined by qPCR shows a reduction in miR-126 in SNI model compared to DRG from sham control. U6 was used for normalization ( n = 8 sham, n = 7 SNI). b Relative expression of Dnmt1 mRNA and c Vegfa transcripts showed an increase in the DRG after nerve injury compared to control ( n = 3). Gapdh was used as a normalizer. d Representative Western blot and quantification showed an increase of Dnmt1 protein in the DRG after nerve injury. e Western blot and quantification showed Vegfa protein was not significantly different in DRG after nerve injury ( n = 3 from pooled samples, three DRG were pooled for each sample). Significance determined using unpaired Student’s t test, p value *

    Journal: Epigenetics & Chromatin

    Article Title: Genome-wide redistribution of MeCP2 in dorsal root ganglia after peripheral nerve injury

    doi: 10.1186/s13072-016-0073-5

    Figure Lengend Snippet: Expression of miR-126 and its target genes Dnmt1 and Vegfa in the DRG after nerve injury. a Relative expression of miR-126 determined by qPCR shows a reduction in miR-126 in SNI model compared to DRG from sham control. U6 was used for normalization ( n = 8 sham, n = 7 SNI). b Relative expression of Dnmt1 mRNA and c Vegfa transcripts showed an increase in the DRG after nerve injury compared to control ( n = 3). Gapdh was used as a normalizer. d Representative Western blot and quantification showed an increase of Dnmt1 protein in the DRG after nerve injury. e Western blot and quantification showed Vegfa protein was not significantly different in DRG after nerve injury ( n = 3 from pooled samples, three DRG were pooled for each sample). Significance determined using unpaired Student’s t test, p value *

    Article Snippet: The Assay ID for TaqMan primer probes used were Mm00599780_g1 (Dnmt1) and Mm00437306_m1 (Vegfa).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

    Expression of miR-126 and its target genes Dnmt1 and Vegfa in the DRG from Mecp2 -null mouse. a Relative expression of miR-126 in the DRG showed comparable expression in Mecp2 -null and wild-type littermates. U6 was used for normalization. b Dnmt1 and c Vegfa expression was decreased in the DRG from Mecp2 -null mouse, indicating MeCP2 has a role in regulating expression of Dnmt1 and Vegfa. Gapdh was used as a normalizer. Significance determined using unpaired Student’s t test, p value *

    Journal: Epigenetics & Chromatin

    Article Title: Genome-wide redistribution of MeCP2 in dorsal root ganglia after peripheral nerve injury

    doi: 10.1186/s13072-016-0073-5

    Figure Lengend Snippet: Expression of miR-126 and its target genes Dnmt1 and Vegfa in the DRG from Mecp2 -null mouse. a Relative expression of miR-126 in the DRG showed comparable expression in Mecp2 -null and wild-type littermates. U6 was used for normalization. b Dnmt1 and c Vegfa expression was decreased in the DRG from Mecp2 -null mouse, indicating MeCP2 has a role in regulating expression of Dnmt1 and Vegfa. Gapdh was used as a normalizer. Significance determined using unpaired Student’s t test, p value *

    Article Snippet: The Assay ID for TaqMan primer probes used were Mm00599780_g1 (Dnmt1) and Mm00437306_m1 (Vegfa).

    Techniques: Expressing

    VEGFA induced eNOS phosphorylation and activity in vitro A. Effect of VEGFA (V; 100 ng/mL; 1, 5, 10 min), histamine (H; 10 μM, 1, 5, 10 min) or medium (C, control) on eNOS phosphorylation at S1177 in cultured Human Retinal Microvascular Endothelial Cells (HRMEC). B. Quantification of eNOS pS1177/total eNOS normalized to tubulin. Mean ±S.E.M. n = 3 independent experiments. * = p

    Journal: bioRxiv

    Article Title: eNOS-induced vascular barrier disruption in retinopathy by c-Src activation and tyrosine phosphorylation of VE-cadherin

    doi: 10.1101/2020.11.24.396077

    Figure Lengend Snippet: VEGFA induced eNOS phosphorylation and activity in vitro A. Effect of VEGFA (V; 100 ng/mL; 1, 5, 10 min), histamine (H; 10 μM, 1, 5, 10 min) or medium (C, control) on eNOS phosphorylation at S1177 in cultured Human Retinal Microvascular Endothelial Cells (HRMEC). B. Quantification of eNOS pS1177/total eNOS normalized to tubulin. Mean ±S.E.M. n = 3 independent experiments. * = p

    Article Snippet: One microgram of RNA was reverse transcribed using SuperScript III (Invitrogen) and quantitative PCR were assayed using Mus musculus primers against Vegfa (Mm00437306_m1, ThermoFisher), Nos3 (Mm00435217_m1) and Nos2 (Mm00440502_m1).

    Techniques: Activity Assay, In Vitro, Cell Culture

    Expression of Nos2, Nos3 and Vegfa in Nos3 +/+ and Nos3S1176A/S1176A retinas A-C. qPCR of Nos3 (A) , Nos2 (B) and Vegfa (C) expression in P17 normoxic and OIR-challenged Nos3 +/+ and Nos3 S1176A/S1176A mouse retinas. D,E. Relative quantities of Nos2 and Nos3 compared against standard curves of TBP and UBC in Nos3 +/+ and Nos3 S1176A/S1176A retinas. Mean ±S.E.M. n = 5 ( Nos3 +/+ ) and 5 ( Nos3 S1176A/S1176A ) mice. *, **, *** = p

    Journal: bioRxiv

    Article Title: eNOS-induced vascular barrier disruption in retinopathy by c-Src activation and tyrosine phosphorylation of VE-cadherin

    doi: 10.1101/2020.11.24.396077

    Figure Lengend Snippet: Expression of Nos2, Nos3 and Vegfa in Nos3 +/+ and Nos3S1176A/S1176A retinas A-C. qPCR of Nos3 (A) , Nos2 (B) and Vegfa (C) expression in P17 normoxic and OIR-challenged Nos3 +/+ and Nos3 S1176A/S1176A mouse retinas. D,E. Relative quantities of Nos2 and Nos3 compared against standard curves of TBP and UBC in Nos3 +/+ and Nos3 S1176A/S1176A retinas. Mean ±S.E.M. n = 5 ( Nos3 +/+ ) and 5 ( Nos3 S1176A/S1176A ) mice. *, **, *** = p

    Article Snippet: One microgram of RNA was reverse transcribed using SuperScript III (Invitrogen) and quantitative PCR were assayed using Mus musculus primers against Vegfa (Mm00437306_m1, ThermoFisher), Nos3 (Mm00435217_m1) and Nos2 (Mm00440502_m1).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Mouse Assay

    eNOS/NO modulates VE-cadherin Y685 phosphorylation via c-Src in a VEGFA/VEGFR2 dependent manner. VEGFA through VEGFR2 and its phosphosite Y1212 induces a chain of consecutive reactions in endothelial cells: phosphorylation of AKT at S473 and eNOS at S1176. The VEGFR2 phosphosite Y949 mediates phosphorylation of c-Src at Y418 and of VE-cadherin at Y685. Combined, these activating phosphorylation reactions disrupt the vascular barrier by dissociating VE-cadherin’s homophilic interactions, resulting in macromolecular leakage. eNOS/NO exacerbates this damage via an interaction with c-Src to enhance VE-cadherin Y685 phosphorylation and internalization.

    Journal: bioRxiv

    Article Title: eNOS-induced vascular barrier disruption in retinopathy by c-Src activation and tyrosine phosphorylation of VE-cadherin

    doi: 10.1101/2020.11.24.396077

    Figure Lengend Snippet: eNOS/NO modulates VE-cadherin Y685 phosphorylation via c-Src in a VEGFA/VEGFR2 dependent manner. VEGFA through VEGFR2 and its phosphosite Y1212 induces a chain of consecutive reactions in endothelial cells: phosphorylation of AKT at S473 and eNOS at S1176. The VEGFR2 phosphosite Y949 mediates phosphorylation of c-Src at Y418 and of VE-cadherin at Y685. Combined, these activating phosphorylation reactions disrupt the vascular barrier by dissociating VE-cadherin’s homophilic interactions, resulting in macromolecular leakage. eNOS/NO exacerbates this damage via an interaction with c-Src to enhance VE-cadherin Y685 phosphorylation and internalization.

    Article Snippet: One microgram of RNA was reverse transcribed using SuperScript III (Invitrogen) and quantitative PCR were assayed using Mus musculus primers against Vegfa (Mm00437306_m1, ThermoFisher), Nos3 (Mm00435217_m1) and Nos2 (Mm00440502_m1).

    Techniques: