Journal: Molecular Cancer

Article Title: A positive feed-forward loop between LncRNA-URRCC and EGFL7/P-AKT/FOXO3 signaling promotes proliferation and metastasis of clear cell renal cell carcinoma

doi: 10.1186/s12943-019-0998-y

Figure Lengend Snippet: URRCC enhances EGFL7 level by mediating histone H3 acetylation of EGFL7 promoter. a : URRCC subnetwork according to the result of Target mRNA PCR Array. Genes colored in blue are down-regulated genes after that cells were transfected with sh-URRCC. Genes colored in red are up-regulated genes after that cells were transfected with sh-URRCC. The size of the gene round represents the fold change. b : The mRNA and protein levels of EGFL7 were detected by qRT-PCR and WB assays in A498 and OSRC-2 cells after transfection with sh-control or sh-URRCC. c : The mRNA and protein levels of EGFL7 were detected by qRT-PCR and WB assays in A498 and OSRC-2 cells after transfection with mock or oe-URRCC. d : EGFL7 expression in ccRCC and normal samples from TCGA KIRC dataset. e : Representative EGFL7 IHC staining of ccRCC tissues compared to paired normal renal tissues (200×, 400×). Black scale bar represents 100 μm (200×) or 50 μm (400×). f and g : Representative EGFL7 IHC staining of xenograft tumors from sh-control, sh-URRCC, mock, and oe-URRCC groups (200×, 400×). Black scale bar represents 100 μm (200×) or 50 μm (400×). h : qRT-PCR and WB analysis of EGFL7 in A498 and OSRC-2 cells treated with DMSO or trichostatin A (TSA) (50 nM or 100 nM) for 72 h ( n = 3). i : ChIP analyses of A498 cells transfected with sh-control or sh-URRCC were conducted on the EGFL7 promoter regions using anti-acetyl-histone H3 and anti-acetyl-histone H4. Enrichment was determined relative to input controls. The data are the means ± standard deviations of three independent biological replicates. j : ChIP analyses of A498 cells transfected with sh-control or sh-URRCC were conducted on the GAPDH promoter regions using anti-acetyl-histone H3 and anti-acetyl-histone H4. Enrichment was determined relative to input controls. k : ChIP analyses of OSRC-2 cells transfected with mock or oe-URRCC were conducted on the EGFL7 promoter regions using anti-acetyl-histone H3 and anti-acetyl-histone H4. Enrichment was determined relative to input controls. The data are the means ± standard deviations of three independent biological replicates. l : EGFL7 promoter region was enriched with H3K27Ac histone mark presented with UCSC data. m : WB analysis of H3K27ac and total H3 in A498 and OSRC-2 cells treated with DMSO or TSA, β-actin was used as a loading control

Article Snippet: Immunohistochemistry for the target molecules was performed on paraffin sections using a primary antibody against EGFL7 (1:50, proteintech, 19,291–1-AP), FOXO3 (1:500, Abcam, ab12162), P-AKT (1:50, Abcam, ab131443), Ki67 (1:500, Abcam, ab92742) and the proteins in situ were visualized with 3, 3-diaminobenzidine.

Techniques: Transfection, Quantitative RT-PCR, Control, Expressing, Immunohistochemistry

Journal: Molecular Cancer

Article Title: A positive feed-forward loop between LncRNA-URRCC and EGFL7/P-AKT/FOXO3 signaling promotes proliferation and metastasis of clear cell renal cell carcinoma

doi: 10.1186/s12943-019-0998-y

Figure Lengend Snippet: URRCC is associated with EGFL7/P-AKT/FOXO3 signaling pathway. a and b : WB analysis of T-AKT, P-AKT, and FOXO3 in URRCC-overexpressing or URRCC-inhibited A498 and OSRC-2 cells compared with sh-control or mock group, β-actin was used as a loading control. c : CCK8 assays of A498 cells transfected with mock/oe-URRCC and si-NC/si-EGFL7. d : Representative images and the numbers of invasive cells per high-power field reduced after the transfection with mock/oe-URRCC and si-NC/si-EGFL7 in A498 cells. e : WB analysis for EGFL7, T-AKT, P-AKT, and FOXO3 protein levels of A498 cells transfected with mock/oe-URRCC and si-NC/si-EGFL7. β-actin was used as a loading control

Article Snippet: Immunohistochemistry for the target molecules was performed on paraffin sections using a primary antibody against EGFL7 (1:50, proteintech, 19,291–1-AP), FOXO3 (1:500, Abcam, ab12162), P-AKT (1:50, Abcam, ab131443), Ki67 (1:500, Abcam, ab92742) and the proteins in situ were visualized with 3, 3-diaminobenzidine.

Techniques: Control, Transfection

Journal: Molecular Cancer

Article Title: A positive feed-forward loop between LncRNA-URRCC and EGFL7/P-AKT/FOXO3 signaling promotes proliferation and metastasis of clear cell renal cell carcinoma

doi: 10.1186/s12943-019-0998-y

Figure Lengend Snippet: A Schematic Diagram of LncRNA-URRCC-Based Signaling Pathway in ccRCC cells proliferation and invasion. LncRNA-URRCC upregulates AKT signaling by directly targeting EGFL7 via mediating histone H3 acetylation of EGFL7 promoter, and then inducing cell growth and invasion. AKT signaling downstream FOXO3 in turn downregulates URRCC by directly binding to its promoter

Article Snippet: Immunohistochemistry for the target molecules was performed on paraffin sections using a primary antibody against EGFL7 (1:50, proteintech, 19,291–1-AP), FOXO3 (1:500, Abcam, ab12162), P-AKT (1:50, Abcam, ab131443), Ki67 (1:500, Abcam, ab92742) and the proteins in situ were visualized with 3, 3-diaminobenzidine.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation *

doi: 10.1074/jbc.M116.731331

Figure Lengend Snippet: Egfl7 is repressed in endothelial cells under inflammatory conditions in vivo. A, left panel, in situ hybridization detection of Egfl7 transcripts in endothelial cell nuclei of adult mouse lungs (blue staining, arrows). Right panel and inset, CD31 immunostaining (brown, arrows) and hematoxylin counterstaining of a parallel section of the same area. Bar, 25 μm. B, expression levels of CD31 and Egfl7 transcripts in CD31− cells (white bars) and CD31+ cells (black bars) isolated from mouse lungs using immunoaffinity and measured by duplex RT-qPCR using a mouse CD31-FAM or a mouse Egfl7-FAM TaqMan probe mixed with a mouse β-actin-VIC probe (see “Experimental Procedures”). The results are plotted as quantities relative to CD31− controls values set to 1. RQ, relative quantities. C, LPS (5 mg/kg, +) or LPS-free PBS (−) was instilled in mice nostrils, and animals were sacrificed at the onset of treatment (0 h) or after 10 or 24 h; the lungs were dissected and processed for total RNA isolation. Expression levels of ICAM-1, VCAM-1, E-selectin, and Egfl7 were measured by duplex RT-qPCR using the indicated FAM-labeled TaqMan probe for the mouse transcript of interest and a mouse β-actin-VIC-labeled TaqMan probe and expressed as 2−ΔΔCT quantities relative to t = 0 h values set to 1. *, p < 0.05; **, p < 0.01; ***, p < 0,001. The results are representative of three experiments performed in triplicate. RQ, relative quantities. D, left panel, HUVEC were treated with increasing doses of LPS for 4 h and Egfl7 transcript levels assessed by duplex RT-qPCR. Right panel, expression levels of Egfl7 transcripts in primary HUVEC cells treated with 0.1 μg/ml of LPS for the indicated length of time and assessed by duplex RT-qPCR. E, TNFα (0.25 mg/kg, +) or LPS-free PBS (−) was instilled in mice nostrils and lungs processed as in C for the analysis of expression levels of ICAM-1, VCAM-1, E-selectin, and Egfl7 expressed as relative to t = 0 values set to 1. RQ, relative quantities. **, p < 0.01; ***, p < 0,001.

Article Snippet: Immunoblots The anti-human Egfl7 antibody (AF3638, lot WNK0109011) was from R&D Systems.

Techniques: In Vivo, In Situ Hybridization, Staining, Immunostaining, Expressing, Isolation, Quantitative RT-PCR, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation *

doi: 10.1074/jbc.M116.731331

Figure Lengend Snippet: Expression of Egfl7 in endothelial cells is repressed by pro-inflammatory cytokines. A and B, expression levels of Egfl7 measured by duplex RT-qPCR in HUVEC treated with PBS (□) or with 10 ng/ml TNFα (●) (A) for the indicated length of time or with PBS (□), 10 ng/ml IL1β (●), or 10 ng/ml IL6 (▵) (B). The results are representative of three experiments performed in triplicate. C, Western blotting analysis of endogenous Egfl7 or of actin in HUVEC treated with TNFα as in A for the indicated length of time. The numbers below indicate the TNFα treated/non-treated ratio of Egfl7 protein levels normalized to actin levels taken at the same time points and assessed by densitometry. The results are representative of two experiments. D, immunostaining of Egfl7 in confluent HUVEC monolayers treated for 4 h with or without TNFα. Bar, 25 μm. E, expression levels of Egfl7 measured by duplex RT-qPCR in HUVEC treated for 24 h with complete medium (EGM-2) or in basal medium supplemented with the indicated amounts of FGF-2 or VEGF-A165. The results are representative of two experiments performed in triplicate.

Article Snippet: Immunoblots The anti-human Egfl7 antibody (AF3638, lot WNK0109011) was from R&D Systems.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunostaining

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation *

doi: 10.1074/jbc.M116.731331

Figure Lengend Snippet: Egfl7 expression in endothelial cells is regulated by TNFα at the transcriptional level. A, Egfl7 transcript levels measured by duplex RT-qPCR in confluent HUVEC treated with 10 ng/ml TNFα (●) or with PBS (□) for 1 h and then with 10 μg/ml actinomycin D (ActD, t = 0) and assessed during the next 6 h. B, Egfl7 transcripts levels measured by duplex RT-qPCR in confluent HUVEC treated with DMSO or 10 μg/ml actinomycin D for 1 h before stimulation with or without 10 ng/ml TNFα for 6 h. *, p < 0.05; **, ns, non-significant. C, luciferase activities measured in HUVEC transfected with pGL3basic (Ctrl) or with the indicated reporter constructs containing fragments of the human egfl7 gene promoter and with the pCMV-β-Gal normalizing vector. The cells were then treated with 10 ng/ml TNFα (black bars) or with PBS (white bars) and lysed 18 h later. The letters correspond to conserved promoter regions (16). The numbers indicate the base position relative to the exon 1b transcription initiation site (2). Activities were normalized with β-galactosidase values, folds of induction were calculated using pGL3basic values as reference; the results are representative of three experiments performed in triplicate. **, p < 0.01; ***, p < 0.001; ns, non-significant.

Article Snippet: Immunoblots The anti-human Egfl7 antibody (AF3638, lot WNK0109011) was from R&D Systems.

Techniques: Expressing, Quantitative RT-PCR, Luciferase, Transfection, Construct, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation *

doi: 10.1074/jbc.M116.731331

Figure Lengend Snippet: Egfl7 repression by TNFα is mediated via the NF-κB pathway. A, confluent HUVEC were cultured in the presence or not of 5 ng/ml TNFα and of 10 μm of the NF-κB inhibitor BAY117085 (BAY) for 6 h, after which RNA were isolated and Egfl7 transcripts were quantified by duplex RT-qPCR. *, p < 0.05. B, HUVEC were transfected with pGL3basic (Ctrl) or with the −7585/+50Luc construct in the absence (NT) or presence of pRC-CMV (Ctrl) or of pRC-CMV-IκBα S32/36A vector and with pCMV-β-Gal. 24 h later, the cells were treated with 10 ng/ml TNFα (+, black bars) or with PBS (−, white bars) and lysed after 18 h, and luciferase and β-galactosidase activities were measured in cell extracts. The results are representative of two experiments performed in triplicate. *, p < 0.05; **, p < 0.01; ns, non-significant.

Article Snippet: Immunoblots The anti-human Egfl7 antibody (AF3638, lot WNK0109011) was from R&D Systems.

Techniques: Cell Culture, Isolation, Quantitative RT-PCR, Transfection, Construct, Plasmid Preparation, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation *

doi: 10.1074/jbc.M116.731331

Figure Lengend Snippet: Specific targeting and rescue of egfl7 do not affect expression of the miR-126 locus. A, HUVEC were transfected with siCtrl (white) or with two different siRNA targeting Egfl7: siEgfl7 #9 (black) or siEgfl7 #10 (gray) and cultured for 4 days. Left panel, total RNA were prepared and analyzed by duplex RT-qPCR for Egfl7 expression. ***, p < 0.001. Middle panel, protein extracts were prepared and analyzed for the presence of Egfl7 or actin by 12% SDS-PAGE and Western blotting. Right panel, HUVEC were treated as in A, and analysis of miR126-3p and miR126-5p expression was performed by RT-qPCR, using the U6 snRNA as normalizer. B, HUVEC were transfected with siCtrl or siEgfl7#10 as in A and then with pcDNA3 (pCtrl) or a pcDNA3-human Egfl7 expression plasmid (pEgfl7). Left panel, RT-qPCR analysis of Egfl7 expression on triplicate experimental samples. Right panel, RT-qPCR analysis of expression of miR126-3p and of miR126-5p in pCtrl- or in pEgfl7-transfected HUVEC on triplicate experimental samples. ***, p < 0.001; ns, non-significant.

Article Snippet: Immunoblots The anti-human Egfl7 antibody (AF3638, lot WNK0109011) was from R&D Systems.

Techniques: Expressing, Transfection, Cell Culture, Quantitative RT-PCR, SDS Page, Western Blot, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation *

doi: 10.1074/jbc.M116.731331

Figure Lengend Snippet: Egfl7 represses the activation of endothelial cells by TNFα. A, expression levels of ICAM-1 (left panel), VCAM-1 (middle panel), and E-selectin (right panel) measured using duplex RT-qPCR in HUVEC transfected with siCtrl (□) or siEgfl7 (●) and stimulated 3 days later with TNFα. The results are representative of two experiments performed in triplicate. At time 0, the levels of Egfl7, ICAM-1, VCAM-1, and E-selectin in the siEgfl7 condition relative to siCtrl condition were 0.31 ± 0.01, 3.18 ± 0.23, 1.00 ± 0.10, and 2.14 ± 0.29, respectively. B, left panel, HUVEC were transfected with pCtrl or pEgfl7 and treated or not for 6 h with TNFα and expression levels of Egfl7 assessed by RT-qPCR on triplicate experimental samples. The numbers indicate the pEgfl7/pCtrl ratio of Egfl7 expression. ***, p < 0.001. Right three panels, HUVEC were transfected with siCtrl or siEgfl7, rescued with pCtrl or pEgfl7, and stimulated or not 2 days later with TNFα for 6 h. Expression levels of ICAM-1, VCAM-1, and E-selectin were assessed by duplex RT-qPCR on triplicate experimental samples. *, p < 0.05; ***, p < 0.001. C, left panel, adhesion of fluorescently labeled Jurkat T-lymphocytes plated onto monolayers of HUVEC transfected 2 days earlier with pCtrl or pEgfl7 and stimulated or not with TNFα. The results are representative of two experiments performed in triplicate. Right panel, mean numbers of adherent fluorescent cells counted over 10 microscopic fields in each condition. *, p < 0.05; ns, non-significant

Article Snippet: Immunoblots The anti-human Egfl7 antibody (AF3638, lot WNK0109011) was from R&D Systems.

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Transfection, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation *

doi: 10.1074/jbc.M116.731331

Figure Lengend Snippet: Egfl7 regulates the activation of endothelial cells through the NF-κB and MEK/Erk pathways. A, left panel, microscopic fields of fluorescent Jurkat T-lymphocytes adhering onto HUVEC transfected 3 days earlier with siCtrl or siEgfl7. DMSO or BAY117085 (BAY, 10 μm) was added 20 h before plating T-lymphocytes. Right panel, mean numbers of adherent Jurkat T-lymphocytes counted in 15 non-overlapping microscope fields. *, p < 0.05. B, expression levels of ICAM-1, VCAM-1, and E-selectin transcripts measured using duplex RT-qPCR in HUVEC transfected with siCtrl or siEgfl7 and treated with DMSO or BAY117085 (10 μm). The results are representative of three experiments performed in triplicate. C, left panel, microscopic fields of fluorescent T-lymphocytes adhering onto HUVEC transfected 3 days earlier with siCtrl or siEgfl7. DMSO or U0126 (10 μm) was added 20 h before plating T-lymphocytes. Right panel, mean numbers of adherent Jurkat T-lymphocytes counted in 15 non-overlapping microscope fields. The results are representative of two experiments performed in triplicate. *, p < 0.05. D, expression levels of ICAM-1, VCAM-1, and E-selectin measured using duplex RT-qPCR in HUVEC transfected with siCtrl or siEgfl7 and treated with DMSO or 10 μm of the MEK1/2 inhibitor U0126 for 16 h. The results are representative of two experiments performed in triplicate. *, p < 0.05.

Article Snippet: Immunoblots The anti-human Egfl7 antibody (AF3638, lot WNK0109011) was from R&D Systems.

Techniques: Activation Assay, Transfection, Microscopy, Expressing, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation *

doi: 10.1074/jbc.M116.731331

Figure Lengend Snippet: Egfl7 prevents the activation of the NF-κB pathway in endothelial cells. A, HUVEC were transfected with siCtrl or siEgfl7, treated 4 days later with 10 ng/ml TNFα, and lysed at the indicated time points. The cell extracts were prepared, and proteins (10–20 μg) were analyzed by Western blotting using antibodies against the indicated proteins (left). The results are representative of three experiments. B, membranes were exposed to a Las3000 system, and intensities were quantified using the Multigauge V3.0 software.

Article Snippet: Immunoblots The anti-human Egfl7 antibody (AF3638, lot WNK0109011) was from R&D Systems.

Techniques: Activation Assay, Transfection, Western Blot, Software

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation *

doi: 10.1074/jbc.M116.731331

Figure Lengend Snippet: Egfl7 does not affect the IKK complex. HUVEC were transfected with siCtrl or siEgfl7, treated 4 days later with 10 ng/ml TNFα, and lysed at the indicated time points. Cell extracts were prepared, and proteins (10–20 μg) were analyzed by Western blotting using antibodies against the indicated proteins (left). The results are representative of three experiments.

Article Snippet: Immunoblots The anti-human Egfl7 antibody (AF3638, lot WNK0109011) was from R&D Systems.

Techniques: Transfection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation *

doi: 10.1074/jbc.M116.731331

Figure Lengend Snippet: Egfl7 regulates the degradation of IκBα by the proteasome. A, HUVEC were transfected with siCtrl or siEgfl7, treated 4 days later with 10 ng/ml TNFα, and lysed at the indicated time points. The cell extracts were prepared, and proteins (10–20 μg) were analyzed by Western blotting using antibodies against the indicated proteins (left). The results are representative of three experiments. B, HUVEC were transfected with siCtrl or siEgfl7 and treated 4 days later with the proteasome inhibitor MG132 for 2 h prior to adding 10 ng/ml TNFα for the indicated time. The cells were lysed, and proteins (5 μg) were analyzed by Western blotting against total IκBα or GAPDH. The results are representative of two experiments.

Article Snippet: Immunoblots The anti-human Egfl7 antibody (AF3638, lot WNK0109011) was from R&D Systems.

Techniques: Transfection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation *

doi: 10.1074/jbc.M116.731331

Figure Lengend Snippet: Egfl7 participates in a loop of regulation of endothelial activation during inflammation. In normal, unstimulated conditions (left panel), Egfl7 constitutively expressed by endothelial cells represses IκBα phosphorylation and degradation, thus promoting the accumulation of inactive NF-κB dimers complexed to IκBα. Egfl7 contributes to low expression levels of leukocyte adhesion molecules and low cell activation. Upon TNFα stimulation (right panel), activation of the NF-κB pathway leads to the repression of Egfl7, which participates in the destabilization of IκBα and triggers endothelial cell activation.

Article Snippet: Immunoblots The anti-human Egfl7 antibody (AF3638, lot WNK0109011) was from R&D Systems.

Techniques: Activation Assay, Phospho-proteomics, Expressing

Journal: bioRxiv

Article Title: Less is more - loss of EGFL7 improves memory by upregulation of VEGF-D

doi: 10.1101/2022.04.07.487327

Figure Lengend Snippet: ( a ) Immunofluorescence staining revealed EGFL7 expression in granule cells and larger blood vessels of the human dentate gyrus as well as larger neurons of the hilus. Scale bars represent 50 μm or 10 µm (magnification). ( b ) Furthermore, EGFL7 was expressed in the dentate gyrus of mice as detected by fluorescence in situ hybridization (FISH). Scale bars represent 50 µm or 10 µm (magnification). ( c ) Cells of murine hippocampi were isolated by fluorescence-activated cell sorting using a combination of the following markers: GFAP + /CD133 + /EGFR - for quiescent neural stem cells (qNSCs), GFAP + /CD133 + /EGFR + for active NSCs (aNSC), GLAST - /CD133 - /EGFR + for neural progenitor cells (NPCs), CD24 + for neuroblasts (NBs), Thy-GFP1 + for neurons and CD31 + for endothelial cells (ECs). Expression of Egfl7 was measured by quantitative reverse transcriptase-polymerase chain reaction using two housekeeping genes and data was plotted as normalized to unsorted hippocampus tissue (HC). Data are presented as mean values with 95% confidence interval (CI). ( d ) EGFL7-specific FISH probes in combination with cell type-specific markers verified EGFL7-expression in aNSCs/NPCs (Stmn1; arrowheads) and neurons (NeuN; arrowheads). Scale bars represent 10 µm.

Article Snippet: As primary antibody a polyclonal rabbit anti-human EGFL7 antibody (1:50, ReliaTech GmbH) was applied.

Techniques: Immunofluorescence, Staining, Expressing, Fluorescence, In Situ Hybridization, Isolation, FACS, Reverse Transcription, Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Less is more - loss of EGFL7 improves memory by upregulation of VEGF-D

doi: 10.1101/2022.04.07.487327

Figure Lengend Snippet: ( a ) Representative images of neural stem and progenitor cells isolated from mouse hippocampus (HC) and cultured as spheres. Scale bars represent 25 µm. ( b ) Size of EGFL7 -/- neurospheres was increased (58.95 ± 11.24 µm (n = 5) versus 43.85 ± 2.25 µm in wild-type control (WT); n = 4; p = 0.0317). ( c ) Fluorescence-activated cell sorting revealed an increase in activated neural stem cells (aNSCs) in EGFL7 -/- mice (5.56 ± 1.65% versus 2.20 ± 0.53% in WT, n = 3; p = 0.0499) but about equal amounts of quiescent qNSCs. ( d ) Flow cytometry-based cell cycle analysis of EGFL7 -/- and WT neurospheres yielded an increased amount of cells in the G2/M phase in EGFL7 -/- mice (33.65 ± 6.10% versus 25.10 ± 2.03%; n = 3; p = 0.0286). ( e ) The paradigm used for cell cycle analysis in vivo . ( f ) Representative images of the dentate gyrus 1 h post administration of CldU. Quantification of cells yielded an increased amount of cells in S phase in EGFL7 -/- mice (11.00 ± 2.71 versus 5.25 ± 1.71 cells per section in WT; n = 4; p = 0.0571). ( g ) Representative images of the dentate gyrus 24 h post administration of IdU and double-stained for Ki67/IdU. Quantification revealed sustained proliferation in EGFL7 -/- mice (17.25 ± 2.06 versus 10.00 ± 2.45 cells per section in WT; n = 4; p = 0.0286). Scale bars represent 90 µm. Statistical analysis was performed by Mann–Whitney U -test. Data are represented as mean ± SEM; * p < 0.05.

Article Snippet: As primary antibody a polyclonal rabbit anti-human EGFL7 antibody (1:50, ReliaTech GmbH) was applied.

Techniques: Isolation, Cell Culture, Control, Fluorescence, FACS, Flow Cytometry, Cell Cycle Assay, In Vivo, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Less is more - loss of EGFL7 improves memory by upregulation of VEGF-D

doi: 10.1101/2022.04.07.487327

Figure Lengend Snippet: ( a ) Markers used in immunofluorescence analyses (IF) to label specific cell types of neural stem cell (NSC) differentiation in the dentate gyrus (DG): GFAP/Nestin for NSCs (type 1), Stmn1 for activated aNSCs and neural precursor cells (NPCs, type 1, type 2), Mash1 for aNSCs/type 2a cells, DCX for type 2b/type 3 and immature neurons, NeuN for mature neurons and granule cells. ( b ) Experimental paradigms of applied neurogenesis assays. ( c ) Quantitative analyses revealed that NSCs (GFAP + /Nestin + /BrdU + ) remained unchanged in EGFL7 -/- mice (10.50 ± 3.51 versus 11.00 ± 1.41 cells per section in wild-type controls (WT); n = 4; p > 0.9999). ( d ) The number of type 2a (BrdU + /Mash1 + ) cells was increased (11.25 ± 1.71 versus 3.00 ± 0.82 in WT; n = 4; p = 0.0286). ( e ) Type 2b/type 3 (BrdU + /DCX + ) cells were decreased (7.00 ± 2.16 versus 14.75 ± 5.56 cells per section in WT; n = 4; p = 0.0286). ( f ) The amount of aNSCs/NPCs (BrdU + /Stmn1 + ) was strongly increased in the DG of EGFL7 -/- animals (18.25 ± 4.99 versus 5.25 ± 3.59 cells per section in WT; n = 4; p = 0.0286). ( g ) Last, more newborn neurons (BrdU + /NeuN + ) were formed in the DG of EGFL7 -/- mice (21.50 ± 4.12 versus 5.75 ± 4.35 cells per section in WT; n = 4; p = 0.0286). Statistical analysis was performed by Mann-Whitney U -test. Data are represented as mean ± SEM; * p < 0.05. Scale bars represent 60 µm.

Article Snippet: As primary antibody a polyclonal rabbit anti-human EGFL7 antibody (1:50, ReliaTech GmbH) was applied.

Techniques: Immunofluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Less is more - loss of EGFL7 improves memory by upregulation of VEGF-D

doi: 10.1101/2022.04.07.487327

Figure Lengend Snippet: ( a ) Description of the EGFL7 fl/fl;Ascl1-CreERT2 mouse model allowing for a tamoxifen-inducible, type 2a cell-specific knock-out of EGFL7. ( b ) Experimental paradigms of applied neurogenesis assays. ( c ) The amount of aNSCs/NPCs (BrdU + /Mash1 + ) were found to be increased (5.25 ± 2.63 versus 1.00 ± 1.15 cells per section in EGFL7 fl/fl ; n = 4; p = 0.0286). ( d ) The number of type 2b/type 3 cells (BrdU + /DCX + ) was decreased in EGFL7 del;Ascl1-CreERT2 mice (8.50 ± 5.51 versus 18.75 ± 2.22 in EGFL7 fl/fl ; n = 4; p = 0.0286), ( e ) while quantification of BrdU + /Stmn1 + activated neural stem and precursor cells (aNSCs/NPCs) revealed a significant upregulation in EGFL7 del ;Ascl1-CreERT2 mice (20.25 ± 3.10 versus 8.00 ± 4.24 cells per section in EGFL7 fl/fl control; n = 4; p = 0.0286). ( f ) Futhermore, the amount of adult-born neurons (BrdU + /NeuN + ) was increased (18.25 ± 6.80 versus 5.50 ± 2.65 cells per section in EGFL7 fl/fl ; n = 4; p = 0.0286). Statistical analysis was performed by Mann-Whitney U -test. Data are represented as mean ± SEM; * p < 0.05; Scale bars represent 60 µm.

Article Snippet: As primary antibody a polyclonal rabbit anti-human EGFL7 antibody (1:50, ReliaTech GmbH) was applied.

Techniques: Knock-Out, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Less is more - loss of EGFL7 improves memory by upregulation of VEGF-D

doi: 10.1101/2022.04.07.487327

Figure Lengend Snippet: ( a ) Volcano and ( b ) MA plots of RNA-sequencing-based transcriptomics in neural stem and precursor cells confirmed EGFL7 knock-out and identified an upregulation of the cytokine VEGF-D (n = 4 for each genotype; two datasets). Volcano plots illustrate the Log2 difference (i.e., fold change) versus -logP of the t-test. MA plots display the LOG2 difference versus the mean expression level (LOG2 RPKM). The VENN diagrams show the agreement of experiment 1 and 2 and the combined analysis of regulated candidates, based on P value and fold change. ( c ) Upregulation of VEGF-D upon EGFL7 knock-out was confirmed by quantitative reverse transcriptase-polymerase chain reaction (6.56 ± 2.85 versus 1.47 ± 0.61 in wild-type control (WT); n = 3; p = 0.039). ( d ) Expression of VEGF-D in the dentate gyrus of the hippocampus was visualized by immunofluorescent staining using a VEGF-D-specific antibody. Statistical analysis was performed by Student’s t-test. Data are represented as mean ± SEM; * p < 0.05. Scale bars represent 90 µm or 45 µm (magnifications).

Article Snippet: As primary antibody a polyclonal rabbit anti-human EGFL7 antibody (1:50, ReliaTech GmbH) was applied.

Techniques: RNA Sequencing, Knock-Out, Expressing, Reverse Transcription, Polymerase Chain Reaction, Control, Staining

Journal: bioRxiv

Article Title: Less is more - loss of EGFL7 improves memory by upregulation of VEGF-D

doi: 10.1101/2022.04.07.487327

Figure Lengend Snippet: ( a ) Representative pictures of analyzed spines of 10-week-old EGFL7 -/- and WT mice. ( b ) The analysis of DG spines of 10-week-old mice showed increased number of spines in EGFL7 -/- mice and ( c ) significantly more thin spines. ( d ) Representative pictures of analyzed spines of 20-week-old EGFL7 -/- and WT mice. ( e ) After 20 weeks the number of spines is unaffected. ( e) The numbers of thin, stubby and mushroom spines were also not affected. ( g ) The number of dendrites and their total length was found increased in EGFL7 -/- primary neuron cultures as detected by Map2 IF staining ( h ). ( i ) Timeline for analysis of neurogenesis in aged mice and quantification of BrdU + /NeuN + newborn neurons across different ages. Statistical analysis was performed by Mann–Whitney U -test. Data are represented as mean ± SD; * p < 0.05; n = 3. Scale bar represents 5 µm.

Article Snippet: As primary antibody a polyclonal rabbit anti-human EGFL7 antibody (1:50, ReliaTech GmbH) was applied.

Techniques: Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Less is more - loss of EGFL7 improves memory by upregulation of VEGF-D

doi: 10.1101/2022.04.07.487327

Figure Lengend Snippet: ( a ) In the Morris water maze (MWM), the latency to reach the visible platform, the proportion of time spent in the platform quarter and the swimming velocity were similar in both genotypes. ( b ) Body weights were similar throughout experiments. Exemplary cohort is shown as mean ± SD. ( c ) EGFL7 -/- mice reached the hidden platform faster. Escape latency was reduced considering the full-time course (AUCs) and late trials. ( d ) The proportion of time spent in the platform quarter after its removal was increased. For MWM sample sizes were n = 19 for EGFL7 -/- mice and n = 33 for wild-type littermate controls (WT). Data were compared by paired (visible platform) and unpaired two-sided Student’s t-tests, or 2-way ANOVA for time courses. ( e ) In the IntelliCage, EGFL7 -/- mice maintained longer avoidance of a previously punished corner (air puff), as revealed by a reduced proportion of nosepoke errors (n = 16 per genotype). ( f ) During preference learning in active module times (11-13:00 and 16-18:00 h each day), EGFL7 -/- mice showed faster relearning to prefer a specified corner upon switching to the opposite side (reversal learning), indicated by a higher accuracy of nosepokes (n = 16 per genotype). Overall activity is shown in ( Supplementary Fig. 8_2 ). ( g,h ) During periods in which the learning modules were inactive (doors remain closed, “default-times”), EGFL7 -/- mice retained higher preference of rewarding corners and the proportion of “memorizers” was higher, the latter defined as 35% accuracy of corner visits (random = 25%). Time course data are represented as mean ± SEM, summarizing data as mean ± SD. The boxes show the interquartile range, the line is the median, and whiskers are plotted minimum to maximum. Scatters represent individual mice. Time courses were compared by 2-way ANOVA and subsequent posthoc t-tests to assess genotype differences at specific time points without multiplicity adjustment for between group comparisons, AUCs were compared with 2-sided unpaired t-tests; * p < 0.05.

Article Snippet: As primary antibody a polyclonal rabbit anti-human EGFL7 antibody (1:50, ReliaTech GmbH) was applied.

Techniques: Activity Assay

Journal: Arthritis Research & Therapy

Article Title: Decreased expression of the endothelial cell-derived factor EGFL7 in systemic sclerosis: potential contribution to impaired angiogenesis and vasculogenesis

doi: 10.1186/ar4349

Figure Lengend Snippet: Serum levels of epidermal growth factor-like domain 7 (EGFL7) determined by colorimetric sandwich ELISA. (A) Serum EGFL7 levels in healthy controls, all patients with systemic sclerosis (SSc), patients with limited cutaneous SSc (lcSSc) and patients with diffuse cutaneous SSc (dcSSc). (B) Serum levels of EGFL7 in patients with SSc according to nailfold videocapillaroscopy pattern (early, active and late). Data are shown as box plots. Each box represents the 25th to 75th percentiles. Lines inside the boxes represent the median. Lines outside the boxes represent the 10th and the 90th percentiles. Circles indicate outliers, and asterisks indicate the extreme values. # P = 0.006 versus controls. Mann–Whitney U -test was used for statistical analysis. NS, not significant.

Article Snippet: The microplates were washed three times with PBS with 0.1% Tween-20, followed by the addition of mouse monoclonal antihuman EGFL7 as a detection antibody (1 μg/ml; Abnova) and horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG).

Techniques: Sandwich ELISA, MANN-WHITNEY

Journal: Arthritis Research & Therapy

Article Title: Decreased expression of the endothelial cell-derived factor EGFL7 in systemic sclerosis: potential contribution to impaired angiogenesis and vasculogenesis

doi: 10.1186/ar4349

Figure Lengend Snippet: Decreased expression of epidermal growth factor-like domain 7 (EGFL7) in the affected skin and dermal microvascular endothelial cells (MVEC) of patients with systemic sclerosis (SSc). (A-F) Representative microphotographs of skin sections from healthy controls ( A - C ; n = 10) and SSc patients ( D - F ; n = 16) immunostained for EGFL7 (red) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue) for nuclei. Arrows indicate microvessels. (G and H) Representative microphotographs of skin sections from healthy controls (G) and SSc patients (H) double immunostained for EGFL7 (red) and the pan-endothelial cell marker CD31 (green) and counterstained with DAPI (blue). Original magnification and scale bars are indicated in each panel. (I) Densitometric analysis of EGFL7 immunofluorescent staining in dermal microvessels expressed as optical density in arbitrary units (a.u.). Data are shown as box plots. Each box represents the 25th to 75th percentiles. Lines inside the boxes represent the median. Lines outside the boxes represent the 10th and the 90th percentiles. Mann–Whitney U -test was used for statistical analysis. (J) Western blotting of protein lysates from control dermal MVEC (n = 3) and SSc MVEC (n = 3) analyzed using anti-EGFL7 antibodies. Representative immunoblots are shown. The densitometric analysis of the bands normalized to α-tubulin is reported in the histograms. Data are mean ± standard error of the mean of optical density in a.u. Student t -test was used for statistical analysis.

Article Snippet: The microplates were washed three times with PBS with 0.1% Tween-20, followed by the addition of mouse monoclonal antihuman EGFL7 as a detection antibody (1 μg/ml; Abnova) and horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG).

Techniques: Expressing, Marker, Staining, MANN-WHITNEY, Western Blot, Control

Journal: Arthritis Research & Therapy

Article Title: Decreased expression of the endothelial cell-derived factor EGFL7 in systemic sclerosis: potential contribution to impaired angiogenesis and vasculogenesis

doi: 10.1186/ar4349

Figure Lengend Snippet: Decreased expression of epidermal growth factor-like domain 7 (EGFL7) in late-outgrowth peripheral blood endothelial progenitor cell (EPC)-derived endothelial cells from patients with systemic sclerosis (SSc). Western blotting of protein lysates from control (n = 8) and SSc (n = 15) late-outgrowth peripheral blood EPC-derived endothelial cells analyzed using anti-EGFL7 antibodies. Representative immunoblots are shown. The densitometric analysis of the bands normalized to α-tubulin is reported in the histograms. Data are mean ± standard error of the mean of optical density in arbitrary units (a.u.). Student t -test was used for statistical analysis.

Article Snippet: The microplates were washed three times with PBS with 0.1% Tween-20, followed by the addition of mouse monoclonal antihuman EGFL7 as a detection antibody (1 μg/ml; Abnova) and horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG).

Techniques: Expressing, Derivative Assay, Western Blot, Control