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: Scientific Reports

Article Title: Epidermal Growth Factor Like-domain 7 and miR-126 are abnormally expressed in diffuse Systemic Sclerosis fibroblasts

doi: 10.1038/s41598-019-39485-8

Figure Lengend Snippet: EGFL7 immunostaining in HC and dcSSc skin. ( A , D ) In HC skin, strong EGFL7 expression was detected in ECs and pericytes of vessels (black arrows), in fibroblasts of papillary and reticular dermis (black open arrows), and cells of the basal layer of the epidermis and keratinocytes. ( B , E ) In EOS dcSSc skin, strong EGFL7 expression was detected in ECs and pericytes of vessels (black arrows), in fibroblasts of papillary and reticular dermis and epidermal (black open arrows), cells of the basal layer of the epidermis and keratinocytes as in the HC skin. ECs of the most vessels were strong immunostained for EGFL7. However, ECs of a few vessels displayed a weaker immunostaining intensity for EGFL7 (blu arrows). Inset shows EGFL7 positive fibroblast ( C and F ) In LSS dcSSc skin, the remaining microvessels displayed a weak (or absent) immunostaining for EGFL7. Furthermore, fibroblasts of papillary and reticular dermis and cells of the basal layer of the epidermis and keratinocytes displayed a weak (or absent) immunostaining for EGFL7. Inset shows EGFL7 weak positive fibroblast. Semiquantitative scoring was performed independently by 2 blinded observers, based on the observation of immunostained skin sections. Scores are defined as follows: +++ intense staining; +++/− intense staining but not homogeneous positive staining; ++ moderate staining; + weak staining; +/− weak staining but not homogeneous positive staining.

Article Snippet: Non specific antibody binding was blocked with 10% casein in PBS for 20 min. All cells were incubated for 1 hr with a rabbit polyclonal anti-EGFL7 (Bioss) and mouse monoclonal α-SMA antibody (abcam).

Techniques: Immunostaining, Expressing, Staining

Journal: Scientific Reports

Article Title: Epidermal Growth Factor Like-domain 7 and miR-126 are abnormally expressed in diffuse Systemic Sclerosis fibroblasts

doi: 10.1038/s41598-019-39485-8

Figure Lengend Snippet: Abnormal expression of EGFL7 mRNA and protein levels in FBs of SSc patients ( A–C ). FBs isolated from the skin of EOS SSc patients, express a statistically significant higher levels of EGFL7 mRNA, when compared to HC and LSS SSc-FBs ( *** p = 0.0001). The experiments were performed in triplicate for each patient and HC. Bars represent mean values ± SEM (N = 10 for each group) ( A ). 18 s gene served as the control. WB analysis of EGFL7 ( B ). These results confirm at protein level the results of qRT-PCR analysis. Blots were stripped using Re-Blot Plus Western Blot recycling kit (Chemicon International, USA) and re-probed with anti-mouse IgG β-actin antibody (Sigma-Aldrich, USA) to confirm similar loading of the gels and efficiency in electrophoretic transfer. The experiments were performed in triplicate for each patient and HC. Full length blot is represented in Supplementary Fig. . Immunoreactive bands were acquired by chemidoc (ImageLab). Densitometric analysis of the bands was performed using ImageJ software (NIH; online at http://rsbweb.nih.gov/ij ) ( C ). IF on cultured FBs at 60% of confluence. Negative controls were obtained by omitting the primary antibody.

Article Snippet: Non specific antibody binding was blocked with 10% casein in PBS for 20 min. All cells were incubated for 1 hr with a rabbit polyclonal anti-EGFL7 (Bioss) and mouse monoclonal α-SMA antibody (abcam).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Western Blot, Software, Cell Culture

Journal: Scientific Reports

Article Title: Epidermal Growth Factor Like-domain 7 and miR-126 are abnormally expressed in diffuse Systemic Sclerosis fibroblasts

doi: 10.1038/s41598-019-39485-8

Figure Lengend Snippet: EGFL7 is inducible by TGF-β on HC-FBs and decreases the expression of COL1A1 ( A–G ). The experiments were performed in triplicate. Bars represent mean values ± SEM before and after stimulation with 10 ng/ml of TGF-β for 48 hrs. The absence of rh proteins from the medium of stimulation was considered as a negative control. HC-FBs, after stimulation by TGF-β, expressed significantly higher levels of EGFL7 RNA, when compared to untreated HC-FBs ( *** p < 0.0001). On the contrary, TGF-β treated EOS SSc-FBs did not show any difference in the EGFL7-RNA levels, when compared to the untreated EOS SSc-FBs. No effect was observed, on the EGFL7 RNA levels for the TGF-β treated and untreated LSS SSc-FBs ( A ). The levels of TGF-β in untreated HC-FBs were set to 100% and all the results were normalized to this value. After EGFL7 stimulation, we observed a dose-related fashion decrease of the expression of COL1A1 ( * p = 0.018; *** p = 0.0001 for RNA levels), both in RNA and protein levels, in SSc-FBs. On the contrary, when HC-FBs were treated with the same rhEGFL7 concentrations, the expression of COL1A1 protein did not change at any concentration of EGFL7 stimulation ( B,C ). Full length blot is represented in Supplementary Fig. . Densitometric analysis of the bands was performed using ImageJ software (NIH; online at http://rsbweb.nih.gov/ij ) ( D ).

Article Snippet: Non specific antibody binding was blocked with 10% casein in PBS for 20 min. All cells were incubated for 1 hr with a rabbit polyclonal anti-EGFL7 (Bioss) and mouse monoclonal α-SMA antibody (abcam).

Techniques: Expressing, Negative Control, Concentration Assay, Software

Journal: Scientific Reports

Article Title: Epidermal Growth Factor Like-domain 7 and miR-126 are abnormally expressed in diffuse Systemic Sclerosis fibroblasts

doi: 10.1038/s41598-019-39485-8

Figure Lengend Snippet: EGFL7 knockdown in HC- and SSc-FBs up-regulates Col1A1 expression after TGF-β stimulation. SSc-FBs were transfected with specific EGFL7-siRNA (siRNA) or non-targeting siRNA (scr), and EGFL7 mRNA expression was evaluated by qRT-PCR analysis. The cells transfected with EGFL7-siRNA showed a decreased expression of EGFL7 mRNA levels when compared with cells transfected with scr siRNA ( A ). After TGF-β stimulation, the silenced cells show an overexpression of COL1A1, suggesting the anti-fibrotic role of EGFL7, confirming the effects observed in Fig. . Each experimental condition was performed in triplicate. Bars represent mean values ± SEM.

Article Snippet: Non specific antibody binding was blocked with 10% casein in PBS for 20 min. All cells were incubated for 1 hr with a rabbit polyclonal anti-EGFL7 (Bioss) and mouse monoclonal α-SMA antibody (abcam).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Over Expression

Journal: Scientific Reports

Article Title: Epidermal Growth Factor Like-domain 7 and miR-126 are abnormally expressed in diffuse Systemic Sclerosis fibroblasts

doi: 10.1038/s41598-019-39485-8

Figure Lengend Snippet: EGFL7 promotes migration and invasion but not proliferation of EOS dcSSc-FBs. At basal state EOS SSc-FBs, expressed a significantly lower ki67 transcript, when compared to that of HC. EGFL7 did not modulates the proliferation of treated HC- and EOS-FBs ( A ). Furthermore, we assessed the migratory ability and invasion of our cells and we found that HC- and EOS dcSSc-FBs migration and invasion were significantly induced by EGFL7 in a concentration dependent-manner (1–100 ng/ml) (p = 0.013 and p = 0.0044 and p = 0.0020) ( B ). All assays were independently performed in triplicate and repeated at least thrice. Bars represent mean values ± SEM. Representative photomicrographs show SSc-FBs migration at basal state ( C ), following stimulation with EGFL7 1 ng/ml ( D ) and EGFL7 100 ng/ml ( E ). Original magnification X20.

Article Snippet: Non specific antibody binding was blocked with 10% casein in PBS for 20 min. All cells were incubated for 1 hr with a rabbit polyclonal anti-EGFL7 (Bioss) and mouse monoclonal α-SMA antibody (abcam).

Techniques: Migration, Concentration Assay

Journal: Scientific Reports

Article Title: Epidermal Growth Factor Like-domain 7 and miR-126 are abnormally expressed in diffuse Systemic Sclerosis fibroblasts

doi: 10.1038/s41598-019-39485-8

Figure Lengend Snippet: SSc-FBs are responsible for impaired angiogenesis in an organotypic co-culture assay system in vitro but EGFL7 restores angiogenesis. Angiogenesis was not affected when primary HC-FBs were co-cultured with HUVECS ( A ). Interestingly, when primary SSc-FBs were co-cultured with HUVECs, angiogenesis was impaired when compared with the tubule formation obtained from co-cultured HC-FBs with HUVECs ( B–C ). EGFL7 increases the tubule formation when added to the cultured medium (100 ng/ml) thus adding EGFL7 to the list of the pro-angiogenic molecules that rescue angiogenesis defects in SSc patients ( E–G ). VEGF (25 ng/ml) was used as positive control ( F–K ). Representative microscopic fields at 4X magnification from triplicate wells. Vascular junction and tubule number and total tubule length were analysed by Angiosys System (TCS CellWorks, USA) ( D – H – L ). Bars represent mean values ± SEM (measurment of 12 microscopic fields from triplicate wells). Statistical analysis was performed by unpaired and paired t test, two tailed. * p < 0.05; ** p < 0.001; *** p < 0.0001.

Article Snippet: Non specific antibody binding was blocked with 10% casein in PBS for 20 min. All cells were incubated for 1 hr with a rabbit polyclonal anti-EGFL7 (Bioss) and mouse monoclonal α-SMA antibody (abcam).

Techniques: Co-culture Assay, In Vitro, Cell Culture, Positive Control, Two Tailed Test

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: EGFL7 participates in regulating biological behavior of growth hormone-secreting pituitary adenomas via Notch2/DLL3 signaling pathway.

doi: 10.1177/1010428317706203

Figure Lengend Snippet: Figure 3. EGFL7 protein is highly expressed in invasive GHPA tissue. (a) Representative western blots of EGFL7 and Notch2 in invasive and non-invasive GHPA. Blots were reprobed with anti-GAPGH antibody to ensure equal loading. (b) Quantitative analysis of western blots showed that EGFL7 and Notch2 expression was markedly higher in invasive GHPA than in non-invasive GHPA. (c) Mean H-scores°±°SD of EGFL7 staining of tissue microarrays. *p < 0.05 versus normal pituitary; **p < 0.001 versus normal pituitary; #p < 0.05 versus non-invasive pituitary adenoma. (d) Representative images of EGFL7 staining of a tissue microarray showing that cytoplasmic EGFL7 staining was significantly higher in invasive GHPA than in non-invasive GHPA and normal pituitary tissue. EGFL7- positive endothelial cells can be observed in immunostained slides (red arrows) (scale bar: 60 µm).

Article Snippet: Membranes were then blocked in Tris-buffered saline (TBS) buffer containing 5% degreased milk for 1 h at room temperature and incubated with primary antibody overnight at 4°C, rabbit polyclonal anti-EGFL7 antibody (1:500, H-90 sc-66874; Santa Cruz Biotechnology; 1:500, 19291-1-AP; Proteintech, Chicago, IL, USA), mouse monoclonal anti-EGFL7 antibody (1:2000, 2H2 sc-101349; Santa Cruz Biotechnology, California, USA), rabbit polyclonal anti-Notch1 (1:2000, ab27526; Abcam, Cambridge, USA), rabbit monoclonal anti-Notch1 (1:5000, ab194123; Abcam, Cambridge, USA), rabbit polyclonal anti-Notch2 (1:2000, ab8926; Abcam, Cambridge, USA), rabbit monoclonal anti-Notch4 (1:2000, ab184742; Abcam, Cambridge, USA), rabbit polyclonal anti-DLL3 (1:2000, ab10554; Abcam, Cambridge, USA), rabbit polyclonal antiDLL3 (1:2000, ab63707; Abcam, Cambridge, USA), rabbit polyclonal anti-DLL4 (1:2000, ab7280; Abcam, Cambridge, USA), and GAPDH (1:8000; Sigma, St Louis, MO, USA) followed by secondary antibodies tagged with horseradish peroxidase (Abcam, Cambridge, USA).

Techniques: Western Blot, Expressing, Staining, Microarray

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: EGFL7 participates in regulating biological behavior of growth hormone-secreting pituitary adenomas via Notch2/DLL3 signaling pathway.

doi: 10.1177/1010428317706203

Figure Lengend Snippet: Figure 4. Lentivirus-mediated knockdown of endogenous EGFL7 expression and differential expression of Notch pathway. (a) Representative western blots of downregulated EGFL7 expression by RNAi and differential expression of Notch pathway. (b) and (c) Quantitative analysis of western blots showed GH3 cells transfected with sh-C or sh-B downregulated EGFL7 expression more efficiently. Knockdown of EGFL7 induced low expression of Notch2/DLL3. However, there are no significant alteration in Notch1, 4 and DLL1, 4. *p < 0.05 versus control and non-silence shRNA. (d) qRT–PCR analysis demonstrated a significantly reduction in EGFL7 mRNA expression after transfected with sh-C and sh-B. *p < 0.05 versus control and non-silence shRNA.

Article Snippet: Membranes were then blocked in Tris-buffered saline (TBS) buffer containing 5% degreased milk for 1 h at room temperature and incubated with primary antibody overnight at 4°C, rabbit polyclonal anti-EGFL7 antibody (1:500, H-90 sc-66874; Santa Cruz Biotechnology; 1:500, 19291-1-AP; Proteintech, Chicago, IL, USA), mouse monoclonal anti-EGFL7 antibody (1:2000, 2H2 sc-101349; Santa Cruz Biotechnology, California, USA), rabbit polyclonal anti-Notch1 (1:2000, ab27526; Abcam, Cambridge, USA), rabbit monoclonal anti-Notch1 (1:5000, ab194123; Abcam, Cambridge, USA), rabbit polyclonal anti-Notch2 (1:2000, ab8926; Abcam, Cambridge, USA), rabbit monoclonal anti-Notch4 (1:2000, ab184742; Abcam, Cambridge, USA), rabbit polyclonal anti-DLL3 (1:2000, ab10554; Abcam, Cambridge, USA), rabbit polyclonal antiDLL3 (1:2000, ab63707; Abcam, Cambridge, USA), rabbit polyclonal anti-DLL4 (1:2000, ab7280; Abcam, Cambridge, USA), and GAPDH (1:8000; Sigma, St Louis, MO, USA) followed by secondary antibodies tagged with horseradish peroxidase (Abcam, Cambridge, USA).

Techniques: Knockdown, Expressing, Quantitative Proteomics, Western Blot, Transfection, Control, shRNA, Quantitative RT-PCR

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: EGFL7 participates in regulating biological behavior of growth hormone-secreting pituitary adenomas via Notch2/DLL3 signaling pathway.

doi: 10.1177/1010428317706203

Figure Lengend Snippet: Figure 5. EGFL7 downregulation suppressed invasion and proliferation of GH3 cells. (a) Representative transwell invasion assays of GH3 cells were performed after knockdown of EGFL7. (b) Quantitative analysis indicated the invasion of GH3 cells decreased by about threefold with sh-C transfection and decreased twofold with sh-B transfectioncompared to transfected with non-silence shRNA. (c) MTT assay revealed that knockdown of EGFL7 suppresses GH3 cell proliferation rate.

Article Snippet: Membranes were then blocked in Tris-buffered saline (TBS) buffer containing 5% degreased milk for 1 h at room temperature and incubated with primary antibody overnight at 4°C, rabbit polyclonal anti-EGFL7 antibody (1:500, H-90 sc-66874; Santa Cruz Biotechnology; 1:500, 19291-1-AP; Proteintech, Chicago, IL, USA), mouse monoclonal anti-EGFL7 antibody (1:2000, 2H2 sc-101349; Santa Cruz Biotechnology, California, USA), rabbit polyclonal anti-Notch1 (1:2000, ab27526; Abcam, Cambridge, USA), rabbit monoclonal anti-Notch1 (1:5000, ab194123; Abcam, Cambridge, USA), rabbit polyclonal anti-Notch2 (1:2000, ab8926; Abcam, Cambridge, USA), rabbit monoclonal anti-Notch4 (1:2000, ab184742; Abcam, Cambridge, USA), rabbit polyclonal anti-DLL3 (1:2000, ab10554; Abcam, Cambridge, USA), rabbit polyclonal antiDLL3 (1:2000, ab63707; Abcam, Cambridge, USA), rabbit polyclonal anti-DLL4 (1:2000, ab7280; Abcam, Cambridge, USA), and GAPDH (1:8000; Sigma, St Louis, MO, USA) followed by secondary antibodies tagged with horseradish peroxidase (Abcam, Cambridge, USA).

Techniques: Knockdown, Transfection, shRNA, MTT Assay

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: EGFL7 participates in regulating biological behavior of growth hormone-secreting pituitary adenomas via Notch2/DLL3 signaling pathway.

doi: 10.1177/1010428317706203

Figure Lengend Snippet: Figure 3. EGFL7 protein is highly expressed in invasive GHPA tissue. (a) Representative western blots of EGFL7 and Notch2 in invasive and non-invasive GHPA. Blots were reprobed with anti-GAPGH antibody to ensure equal loading. (b) Quantitative analysis of western blots showed that EGFL7 and Notch2 expression was markedly higher in invasive GHPA than in non-invasive GHPA. (c) Mean H-scores°±°SD of EGFL7 staining of tissue microarrays. *p < 0.05 versus normal pituitary; **p < 0.001 versus normal pituitary; #p < 0.05 versus non-invasive pituitary adenoma. (d) Representative images of EGFL7 staining of a tissue microarray showing that cytoplasmic EGFL7 staining was significantly higher in invasive GHPA than in non-invasive GHPA and normal pituitary tissue. EGFL7- positive endothelial cells can be observed in immunostained slides (red arrows) (scale bar: 60 µm).

Article Snippet: Membranes were then blocked in Tris-buffered saline (TBS) buffer containing 5% degreased milk for 1 h at room temperature and incubated with primary antibody overnight at 4°C, rabbit polyclonal anti-EGFL7 antibody (1:500, H-90 sc-66874; Santa Cruz Biotechnology; 1:500, 19291-1-AP; Proteintech, Chicago, IL, USA), mouse monoclonal anti-EGFL7 antibody (1:2000, 2H2 sc-101349; Santa Cruz Biotechnology, California, USA), rabbit polyclonal anti-Notch1 (1:2000, ab27526; Abcam, Cambridge, USA), rabbit monoclonal anti-Notch1 (1:5000, ab194123; Abcam, Cambridge, USA), rabbit polyclonal anti-Notch2 (1:2000, ab8926; Abcam, Cambridge, USA), rabbit monoclonal anti-Notch4 (1:2000, ab184742; Abcam, Cambridge, USA), rabbit polyclonal anti-DLL3 (1:2000, ab10554; Abcam, Cambridge, USA), rabbit polyclonal antiDLL3 (1:2000, ab63707; Abcam, Cambridge, USA), rabbit polyclonal anti-DLL4 (1:2000, ab7280; Abcam, Cambridge, USA), and GAPDH (1:8000; Sigma, St Louis, MO, USA) followed by secondary antibodies tagged with horseradish peroxidase (Abcam, Cambridge, USA).

Techniques: Western Blot, Expressing, Staining, Microarray

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: EGFL7 participates in regulating biological behavior of growth hormone-secreting pituitary adenomas via Notch2/DLL3 signaling pathway.

doi: 10.1177/1010428317706203

Figure Lengend Snippet: Figure 4. Lentivirus-mediated knockdown of endogenous EGFL7 expression and differential expression of Notch pathway. (a) Representative western blots of downregulated EGFL7 expression by RNAi and differential expression of Notch pathway. (b) and (c) Quantitative analysis of western blots showed GH3 cells transfected with sh-C or sh-B downregulated EGFL7 expression more efficiently. Knockdown of EGFL7 induced low expression of Notch2/DLL3. However, there are no significant alteration in Notch1, 4 and DLL1, 4. *p < 0.05 versus control and non-silence shRNA. (d) qRT–PCR analysis demonstrated a significantly reduction in EGFL7 mRNA expression after transfected with sh-C and sh-B. *p < 0.05 versus control and non-silence shRNA.

Article Snippet: Membranes were then blocked in Tris-buffered saline (TBS) buffer containing 5% degreased milk for 1 h at room temperature and incubated with primary antibody overnight at 4°C, rabbit polyclonal anti-EGFL7 antibody (1:500, H-90 sc-66874; Santa Cruz Biotechnology; 1:500, 19291-1-AP; Proteintech, Chicago, IL, USA), mouse monoclonal anti-EGFL7 antibody (1:2000, 2H2 sc-101349; Santa Cruz Biotechnology, California, USA), rabbit polyclonal anti-Notch1 (1:2000, ab27526; Abcam, Cambridge, USA), rabbit monoclonal anti-Notch1 (1:5000, ab194123; Abcam, Cambridge, USA), rabbit polyclonal anti-Notch2 (1:2000, ab8926; Abcam, Cambridge, USA), rabbit monoclonal anti-Notch4 (1:2000, ab184742; Abcam, Cambridge, USA), rabbit polyclonal anti-DLL3 (1:2000, ab10554; Abcam, Cambridge, USA), rabbit polyclonal antiDLL3 (1:2000, ab63707; Abcam, Cambridge, USA), rabbit polyclonal anti-DLL4 (1:2000, ab7280; Abcam, Cambridge, USA), and GAPDH (1:8000; Sigma, St Louis, MO, USA) followed by secondary antibodies tagged with horseradish peroxidase (Abcam, Cambridge, USA).

Techniques: Knockdown, Expressing, Quantitative Proteomics, Western Blot, Transfection, Control, shRNA, Quantitative RT-PCR

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: EGFL7 participates in regulating biological behavior of growth hormone-secreting pituitary adenomas via Notch2/DLL3 signaling pathway.

doi: 10.1177/1010428317706203

Figure Lengend Snippet: Figure 5. EGFL7 downregulation suppressed invasion and proliferation of GH3 cells. (a) Representative transwell invasion assays of GH3 cells were performed after knockdown of EGFL7. (b) Quantitative analysis indicated the invasion of GH3 cells decreased by about threefold with sh-C transfection and decreased twofold with sh-B transfectioncompared to transfected with non-silence shRNA. (c) MTT assay revealed that knockdown of EGFL7 suppresses GH3 cell proliferation rate.

Article Snippet: Membranes were then blocked in Tris-buffered saline (TBS) buffer containing 5% degreased milk for 1 h at room temperature and incubated with primary antibody overnight at 4°C, rabbit polyclonal anti-EGFL7 antibody (1:500, H-90 sc-66874; Santa Cruz Biotechnology; 1:500, 19291-1-AP; Proteintech, Chicago, IL, USA), mouse monoclonal anti-EGFL7 antibody (1:2000, 2H2 sc-101349; Santa Cruz Biotechnology, California, USA), rabbit polyclonal anti-Notch1 (1:2000, ab27526; Abcam, Cambridge, USA), rabbit monoclonal anti-Notch1 (1:5000, ab194123; Abcam, Cambridge, USA), rabbit polyclonal anti-Notch2 (1:2000, ab8926; Abcam, Cambridge, USA), rabbit monoclonal anti-Notch4 (1:2000, ab184742; Abcam, Cambridge, USA), rabbit polyclonal anti-DLL3 (1:2000, ab10554; Abcam, Cambridge, USA), rabbit polyclonal antiDLL3 (1:2000, ab63707; Abcam, Cambridge, USA), rabbit polyclonal anti-DLL4 (1:2000, ab7280; Abcam, Cambridge, USA), and GAPDH (1:8000; Sigma, St Louis, MO, USA) followed by secondary antibodies tagged with horseradish peroxidase (Abcam, Cambridge, USA).

Techniques: Knockdown, Transfection, shRNA, MTT Assay

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: Blood Advances

Article Title: The EGFL7-ITGB3-KLF2 axis enhances survival of multiple myeloma in preclinical models

doi: 10.1182/bloodadvances.2019001002

Figure Lengend Snippet: EGFL7 is a survival factor in myeloma cells (A) Fold change in EGFL7 gene expression of the human stromal cell line HS-5 and the MM cell lines U-266, RPMI8226, (abbreviated RPMI), MM.1S, H929, and KMS11, as determined by RT-PCR when compared with the EGFL7 expression in the human myeloid leukemia HL60 cells. (B) A representative western blot is shown for EGFL7, with b-ACTIN as a control, in tumor lysates from mice injected with indicated RPMI cells. (C) Representative images of RPMI8226 cells stained for EGFL7 (green fluorescence) and 4′,6-diamidino-2-phenylindole (DAPI; blue nuclear staining) by immunofluorescent staining. (D) Fold change in EGFL7 gene expression of MACS-isolated CD138+ cells from normal donors when compared with the EGFL7 expression in CD138− cells. (E) Fold change in EGFL7 gene expression as determined by RT-PCR in human cell samples of patients with MM (patients #1 and #2 MM patient sample at diagnosis and patient #3 MM patient sample at refractory stage of the disease; for more clinical details, see supplemental Figure 1) when compared with EGFL7 gene expression found in human BMMCs of healthy donors (n = 3/condition). (F) Western blot analysis of EGFL7 and b-ACTIN as a control in indicated cell population from healthy volunteers and patients with MM. (Upper) Band quantification using the ImageJ program. (Lower) Representative western blots of the same samples. (G-H) EGFL7 (EGFL OE), EGFL7 knockdown (KD), or Mock MM cells (RPMI8226, MM.1S, and U-266) were generated. (G) Fold change in EGFL7 gene expression when compared with Mock cells, as determined by RT-PCR (n = 3/condition). (H) Cells were counted 24 hours after cell seeding after trypan blue exclusion (n = 6/group). (I-J) RPMI8226 cells (OE, KD, Mock) were stained with Annexin V-FITC and PI and analyzed 48 hours after cell plating by FACS. (I) A representative FACS blot is shown for each condition (n = 6/group). (J) Percentage of Annexin V+ and PI− cells (as a measure of early and late apoptosis) by FACS after 48 hours (n = 3/group). (K) Representative western blot analysis of the expression of phosphorylated AKT, BAX, and b-ACTIN (control) in cell lysates of Mock, EGFL7 OE, and EGFL7 KD RPMI8226 cells. (L) Fold change in caspase 3/7 activity of EGFL7 OE or KD cells when compared with Mock RPMI8226 cells 48 hours after cell plating, as determined by Versa Max (n = 6/condition). (M-N) EGFL7 OE and wild-type (RPMI) cells were treated with various concentrations of the AKT1/2 inhibitor. (M) Proliferation was determined using the CCK-8 kit after 24 hours in culture. % Absorbance indicates the rate of viable cells (n = 6/condition). (N) Fold increase in EGFL7 expression after AKT1/2 inhibitor treatment when compared with EGFL7 expression in nontreated cells. (O-P) MM cells were cocultured with a confluent layer of GFPpos-EGFL7 Mock, EGFL7 OE, or EGFL7 KD ECs in direct contact, and the percentage of GFP-MM cells was determined after 24 hours of coculture (n = 3/condition). (O) Percentage of GFP− MM cells in the adherent fraction. (P) Absolute number of nonadherent suspension cells retrieved from the cocultures. As for RT-PCR data, transcripts were normalized to b-ACTIN. Graphs represent averages from 3 independently prepared templates per condition. Experiments were repeated twice with similar results. Data are represented as mean ± SEM. * P ≤ .05; ** P ≤ .01; *** P ≤ .001. P values were determined using a Student t test.

Article Snippet: Cells were incubated overnight with EGFL7 primary Ab (rabbit anti-Goat; Santa Cruz Biotech, sc-34416) at 4°C.

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Control, Injection, Staining, Fluorescence, Isolation, Biomarker Discovery, Knockdown, Generated, Activity Assay, CCK-8 Assay, Suspension

Journal: Blood Advances

Article Title: The EGFL7-ITGB3-KLF2 axis enhances survival of multiple myeloma in preclinical models

doi: 10.1182/bloodadvances.2019001002

Figure Lengend Snippet: EGFL7 augments MM growth in vivo. (A) Human RPMI8226 wild-type cells were injected s.c. into NOD/SCID mice (n = 6/group). (B) Human EGFL7 plasma levels were measured in murine plasma samples at indicated times by enzyme-linked immunosorbent assay in (n = 6). (C) Representative western blotting for EGFL7 and b-ACTIN in tumors extracted from tumors injected with Mock, EGFL7 OE, or KD RPMI8226 cells. (D-E) The size of tumors that formed after s.c. injection of Mock, EGFL7 OE, or KD cells after day 53. (D) Tumor weight was measured (left panel; n = 6/group). Representative macroscopic tumor images were taken on day 53 postinoculation (right panel). (E) Representative hematoxylin and eosin-stained tumor sections demonstrating increased necrotic areas (yellow lines) in tumors established with EGFL7 KD cells (scale bar, 200 µm). (F-G) RPMI8226 wild-type tumor-bearing NOD/SCID mice were injected with/without neutralizing Abs against EGFL7 (anti-EGFL7) starting after visible tumors had been established by day 25 (n = 6/group). (F) Tumor weight on day 53 after initial tumor inoculation (left panel). Representative macroscopic images of tumors (right panels). (G) Histological images of hematoxylin and eosin–stained tumor sections (scale bar of the insert, 100 µm). Graphs show mean ± SEM. Significance was calculated by Student t test, * P ≤ .05; ** P ≤ .01.

Article Snippet: Cells were incubated overnight with EGFL7 primary Ab (rabbit anti-Goat; Santa Cruz Biotech, sc-34416) at 4°C.

Techniques: In Vivo, Injection, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Western Blot, Staining

Journal: Blood Advances

Article Title: The EGFL7-ITGB3-KLF2 axis enhances survival of multiple myeloma in preclinical models

doi: 10.1182/bloodadvances.2019001002

Figure Lengend Snippet: Increased circulating and tissue-bound EGFL7 in the murine Vk*MYC model of MM. (A-F) MACS-isolated CD138+ Vk*MYC BM and spleen cells were injected into C57BL/6J mice via the tail vein. C57BL/6J mice transplanted with Vk*MYC tumor cells (Vk*MYC mice) and control mice (WT) that did not receive tumor cells were analyzed 25 days after cell inoculation. (A) Representative macroscopic images of whole spleens (left panel) and total spleen weight (right panel; n = 6/group) of WT and Vk*MYC mice. (B) Spleen and BM cells from transplanted Vk*MYC recipient or WT mice were stained for the plasma cell marker CD138 and the B-cell marker B220. Percentage of CD138+ cells per total BM or spleen cells and percentage of B220+ cells per total BM cells are given in the left panels. Representative FACS blots of CD138+ cells in BM and spleen are shown in the right panels. (C) Immunohistochemistry analysis of EGFL7 in BM and spleen sections of WT and VK*MYC mice (scale bar of the insert, 10 µm). Hematoxylin and eosin–stained sections (H&E; scale bar of the insert, 20 µm) show massive infiltration of MM cells in the tissue section of Vk*MYC mice. (D) Representative western blot for EGFL7 and b-ACTIN in BM and spleen cell lysates of WT and Vk*MYC mice. Two independent experiments were performed. (E) Fold change of EGFL7 expression in total BMNCs, MACS-isolated CD31+ BM cells, and splenocytes of Vk*MYC mice when compared with the EGFL7 expression in spleen and BM of nontumor-bearing mice (n = 2/group). Transcripts normalized to β-ACTIN. Graphs represent averages from 3 independently prepared templates per condition. Experiments were repeated twice, with similar results. (F) Plasma mouse EGFL7 as determined by enzyme-linked immunosorbent assay (n = 6/group). (G-J) Vk*MYC BM and spleen cells of donor mice with a tumor load of 30% in the BM were injected into C57BL/6J mice via the tail vein. VK*MYC transplanted mice were left untreated for 21 days to ensure sufficient tumor load. Then, 3 mice were treated with neutralizing Abs against EGFL7 (anti-EGFL7) or with isotype (nonbinding) control Ab at 1.5 mg/kg IV. On day 42, mice were euthanized. (G) Spleen weight was determined. (H) Frequency of B220−CD138+ MM cells in splenocytes as determined by FACS. (J) Hematoxylin and eosin–stained sections (scale bar of the insert, 50 µm) showing mild infiltration of MM cells in the BM. (I) Frequency of B220−CD138+ MM cells in BM of Vk*MYC mice treated with anti-EGFL7 Ab or isotype controls (n = 3/group). The data represent the data of 1 experiment. A similar experiment was performed independently, showing similar results. Graphs show mean ± SEM. Significance was calculated by Student t test, * P ≤ .05; ** P ≤ .01.

Article Snippet: Cells were incubated overnight with EGFL7 primary Ab (rabbit anti-Goat; Santa Cruz Biotech, sc-34416) at 4°C.

Techniques: Isolation, Injection, Control, Staining, Clinical Proteomics, Marker, Immunohistochemistry, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Blood Advances

Article Title: The EGFL7-ITGB3-KLF2 axis enhances survival of multiple myeloma in preclinical models

doi: 10.1182/bloodadvances.2019001002

Figure Lengend Snippet: ITGB3+ MM cells adhere to immobilized EGFL7 via its RGD region (A) Fold change in ITGB3 expression of HUVEC, HEL, RPMI8226, U-266, MM.1S, and HS-5 cells when compared with ITGB3 expression in HL60 cells using RT-PCR. Transcripts normalized to b-ACTIN. Graphs represent averages from 3 independently prepared templates per condition. Experiments were repeated twice with similar results. (B) Representative FACS plots showing gating for PI negative cells. Histogram showing ITGB3 levels on indicated MM cell lines. (C) Percentage of adhesion of RPMI8226 cells after 4 hours on precoated plates with deposited ECM from cells infected with AdNull (Mock), AdEGFL7 full-form (EGFL7), or Addeleted RGD (delRGD), or precoated with rec. murine EGFL7 (rec. EGFL7) or with rec. FN. The percentage of adherent cells was quantified after cellular staining with crystal violet and absorbance measurement at 550 nm. Cumulative data of 2 independent experiments are shown (n = 5/condition per experiment). (D) Representative FACS histogram of ITGB3 expression using RPMI8226 cells stably transduced with ITGB3 (ITGB3 OE) in the left panel. The right panel shows the percentage of ITGB3 expression on ITGB3 OE cells, as determined by FACS (n = 6/group). (E) Representative western blot image of Tyr747, FAK EGFL7, and b-ACTIN proteins in EGFL7 OE/KD, and ITGB3 OE/KD in RPMI8226 cells. Two independent experiments were performed. (F) Human ITGB3 OE or ITGB3 KD cells were cultured on EGFL7- and FN-coated plates for 4 hours with/without the ITGB3 inhibitor Cilengitide (Celin). Quantification of adherence using crystal violet absorbance of adherent cells (n = 5 wells/condition). (G) Cell proliferation rate was determined after 24 hours by counting Trypan blue-negative ITGB3 OE and Mock MM cells (n = 3/wells per condition). (H) EGFL7 OE or EGFL7 KD MM cells were coinfected with Mock vector (−ITGB3) or ITGB3 OE (+ITGB3) vectors. Cells were counted after 24 hours in culture (n = 6/condition). Experiments were repeated at least twice. Graphs show mean ± SEM. Significance was calculated by Student t test, * P ≤ .05; ** P ≤ .01.

Article Snippet: Cells were incubated overnight with EGFL7 primary Ab (rabbit anti-Goat; Santa Cruz Biotech, sc-34416) at 4°C.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Infection, Staining, Stable Transfection, Transduction, Western Blot, Cell Culture, Plasmid Preparation

Journal: Blood Advances

Article Title: The EGFL7-ITGB3-KLF2 axis enhances survival of multiple myeloma in preclinical models

doi: 10.1182/bloodadvances.2019001002

Figure Lengend Snippet: EGFL7 loss in MM cells overrides BTZ resistance (A-B) RPMI8226, U-266, and MM.1S cells were cultured for 24 hours in the presence of BTZ at indicated concentrations. (A) Fold change in EGFL7 gene expression of BTZ-treated MM cells when compared with non-BTZ-treated cultures, as determined by RT-PCR. Transcripts normalized to β-ACTIN. Graphs represent averages from independently prepared templates per condition. (B) Viable MM cells were counted 24 hours after BTZ treatment, using Trypan blue (n = 6/condition). (C) RPMI8226 cells were treated with rec. EGFL7 in the presence/absence of 5 nM BTZ. Cells were counted after 24 hours in culture (n = 6/group). (D) EGFL7 OE and Mock RPMI8226 cells were treated with/without BTZ. Cells were counted after 24 hours (n = 6/group). (E) RPMI Mock cells were cultured with/without neutralizing Abs against EGFL7 in the presence or absence of 10 nM BTZ. Cell proliferation was assessed using the CCK-8 kit after 24 hours in culture (n = 6/condition). (F-G) RPMI8226, U-266, and MM.1S cells were treated with/without BTZ for 24 hours. (F) Fold change in ITGB3 expression in BTZ-treated cells when compared with non-BTZ-treated cells after 24 hours determined by RT-PCR. Gene expression was normalized to β-ACTIN expression. Graphs represent averages from 3 independently prepared templates per condition. Experiments were repeated twice with similar results. (G) Representative FACS histograms showing ITGB3 expression in PI-negative gated cells after being cultured with or without BTZ for 24 hours. (H) Percentage of ITGB3-positive cells in BTZ or non-BTZ cultures, as determined by FACS (n = 6/condition). (I) Mock, EGFL7 KD, and EGFL7 OE RPMI cells were treated with/without BTZ for 24 hours in vitro. Washed RPMI8226 OE, Mock, or KD cells were allowed to adhere to plastic for 4 hours. Adherent cells were quantified using a fluorescence plate reader. Results are shown as percentage adhesion over the input. Experiment was run twice in triplicate (n = 6/condition). (J) Absolute number of GFP+ RPMI8226 cells per well after 4 hours of culture on EGFL7 OE or EGFL7 KD BMEC1 cells. (K) RPMI8226 cells were cultured in the presence or absence of BTZ with/without the integrin inhibitor Cilengitide (Celin; n = 6/group). The number of viable cells was counted after 24 hours. The experiment was run once in triplicate (n = 6/condition). (L) Mock, ITGB3 OE, and ITGB3 KD RPMI cells were treated with/without BTZ. Viable cells were counted using the Trypan exclusion assay (n = 6/group). The experiment was repeated twice. (M) Mock, EGFL7 OE, and EGFL7 KD RPMI cells concomitantly infected with ITGB3 OE or KD plasmids were treated for 24 hours with/without BTZ (n = 6/group). The number of viable cells was determined. The experiment was performed twice. (N) Study treatment scheme. Mock, EGFL7 OE, and EGFL7 KD RPMI MM cells concomitantly infected with/without ITGB3 OE or KD plasmids were injected s.c. into NOD/SCID mice, and tumors were established. Starting on the 25th day (treatment day 1), tumor-carrying mice were randomized according to tumor size. A total of 5 injections of PBS/dimethyl sulfoxide carrier, or BTZ (1 mg/kg) were given twice per week (n = 5 mice per group). Tumor weight was determined on day 36 (n = 5-7/group). Graphs show mean ± SEM. Significance was calculated by Student t test, * P ≤ .05; ** P ≤ .01.

Article Snippet: Cells were incubated overnight with EGFL7 primary Ab (rabbit anti-Goat; Santa Cruz Biotech, sc-34416) at 4°C.

Techniques: Cell Culture, Gene Expression, Reverse Transcription Polymerase Chain Reaction, CCK-8 Assay, Expressing, In Vitro, Fluorescence, Exclusion Assay, Infection, Injection

Journal: Blood Advances

Article Title: The EGFL7-ITGB3-KLF2 axis enhances survival of multiple myeloma in preclinical models

doi: 10.1182/bloodadvances.2019001002

Figure Lengend Snippet: EGFL7, a KLF2 downstream target, promotes primary MM survival. (A) Fold change in ITGB3, EGFL7, and KLF2 expression in ITGB3 OE RPMI cells when compared with Mock cells, as determined by RT-PCR (n = 3/group). (B) Fold change in KLF2 expression in Mock, si-KLF2, and KLF2 OE RPMI cells as determined by RT-PCR (n = 3/group). (C) Fold change in EGFL7 expression in KLF2 OE or si-KLF2 RPMI cells when compared with KLF2 Mock or si-KLF2 cells, respectively, as determined by RT-PCR. (D) Representative western blot for indicated proteins from lysates of si-Ctrl, si-KLF2, Mock, and KLF2 OE cells. (E) Viable cell quantification of KLF2 OE and Mock cells after 24 hours in culture (n = 6/group). (F) KLF2 OE, Mock, si-Ctrl, and si-KLF2 cells were cultured in the presence or absence of rec. EGFL7. Cells were counted after 24 hours (n = 6/group). (G) Fold change in KLF2 expression in BTZ-treated MM cells at the indicated concentration for 24 hours when compared with non-BTZ-treated controls, as evaluated by RT-PCR. (H) MM cells (EGFL7 OE/EGFL7 KD, KLF2 OE, KLF2 KD, or KLF2 OE + EGFL7 KD) were cultured with/without 10 µM BTZ. After 24 hours, viable cells were enumerated (n = 3/group). (I) Immunohistochemical staining for EGFL7 (background panel; scale bars, 50 µm) and hematoxylin and eosin (insert; scale bars, 10 µm) in BM sections of patients with MM. For patient details, see supplemental Figure 1. (J) KLF2 expression determined by RT-PCR in total BMMCs, MACS-isolated BM CD138+ and CD138− cells derived from MM patients #1, #2, and #3. KLF2 expression was given as a fold change to the expression found in MACS-isolated CD138− cells from a healthy donor as the comparator. (K) EGFL7 KD or OE was achieved in human primary MACS-isolated CD138+ MM cells using a lentiviral virus–containing GFP. Cell proliferation was monitored by GFP positivity using FACS after 24 hours (n = 3/group). (L) EGFL7 OE, KD, KLF2 OE or KD was achieved in primary PCR in CD138+ MM patient cell samples (#1-#3). Cell proliferation of transduced cells was determined after 24 hours (n = 6/group). (M) Fold change in ITGB3 expression, as determined by RT-PCR. For all RT-PCR results, transcripts normalized to b-ACTIN. Experiments were repeated twice with similar results. (N) Model of the EGFL7-ITGB3-KLF2-EGFL7 axis in MM cells. EGFL7 partially through binding to ITGB3 on MM cells upregulates the transcription factor KLF2. In turn, KLF2 augments EGFL7 expression. This leads to a positive forward amplification loop that promotes MM survival, a mechanism that seems especially active under the pressure of chemotherapeutic drugs such as BTZ. We propose that EGFL7 contributes to BTZ-induced drug resistance. Data are represented as mean ± SEM. * P ≤ .05; ** P ≤ .01; *** P ≤ .001. P values were determined using a Student t test.

Article Snippet: Cells were incubated overnight with EGFL7 primary Ab (rabbit anti-Goat; Santa Cruz Biotech, sc-34416) at 4°C.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Cell Culture, Concentration Assay, Immunohistochemical staining, Staining, Isolation, Derivative Assay, Virus, Binding Assay, Amplification

Journal: Biomedicines

Article Title: Effect of Pravastatin on Placental Expression of Epidermal Growth Factor-like Domain 7 in Early-Onset Pre-Eclampsia: A New Potential Mechanism of Action

doi: 10.3390/biomedicines12081929

Figure Lengend Snippet: Primer sequences, amplicon size, and gene accession number.

Article Snippet: Membranes were then incubated overnight at 4 °C with rabbit anti-EGFL7 (Abcam, Cambridge, UK, cat. ab256451, 1:1000) or mouse anti-GAPDH (clone 6C5, Santa Cruz, CA, USA, cat. sc-32233, 1:2000), all diluted in TBS/T with 5% ( w / v ) bovine serum albumin (BSA).

Techniques: Amplification, Sequencing

Journal: Biomedicines

Article Title: Effect of Pravastatin on Placental Expression of Epidermal Growth Factor-like Domain 7 in Early-Onset Pre-Eclampsia: A New Potential Mechanism of Action

doi: 10.3390/biomedicines12081929

Figure Lengend Snippet: EGFL7 gene expression levels in chorionic villous explants immediately after dissection. ( A ): Representative phase-contrast images of villous explant samples from control and PE placenta. ( B ): qRT-PCR analysis demonstrating reduced expression of EGFL7 in PE villous explant samples compared to the healthy controls. Scale bar in panel images = 250 μM. Statistical analysis was performed using Mann–Whitney test (** p = 0.0044).

Article Snippet: Membranes were then incubated overnight at 4 °C with rabbit anti-EGFL7 (Abcam, Cambridge, UK, cat. ab256451, 1:1000) or mouse anti-GAPDH (clone 6C5, Santa Cruz, CA, USA, cat. sc-32233, 1:2000), all diluted in TBS/T with 5% ( w / v ) bovine serum albumin (BSA).

Techniques: Expressing, Dissection, Control, Quantitative RT-PCR, MANN-WHITNEY

Journal: Biomedicines

Article Title: Effect of Pravastatin on Placental Expression of Epidermal Growth Factor-like Domain 7 in Early-Onset Pre-Eclampsia: A New Potential Mechanism of Action

doi: 10.3390/biomedicines12081929

Figure Lengend Snippet: Effect of pravastatin administration to ex vivo chorionic villous explant cultures on EGFL7 expression. ( A ): Representative phase-contrast images of villous explants from healthy control and PE placenta after 24 h of culture in the presence or absence of 10 μM pravastatin (PRA). ( B ): qRT-PCR analysis showing that PRA treatment did not affect EGFL7 expression in both healthy control and PE villous cultures. Scale bar in panel images = 250 μM. Statistical analysis was performed using Student’s t test. − and + PRA: without or with 10 μM pravastatin.

Article Snippet: Membranes were then incubated overnight at 4 °C with rabbit anti-EGFL7 (Abcam, Cambridge, UK, cat. ab256451, 1:1000) or mouse anti-GAPDH (clone 6C5, Santa Cruz, CA, USA, cat. sc-32233, 1:2000), all diluted in TBS/T with 5% ( w / v ) bovine serum albumin (BSA).

Techniques: Ex Vivo, Expressing, Control, Quantitative RT-PCR

Journal: Biomedicines

Article Title: Effect of Pravastatin on Placental Expression of Epidermal Growth Factor-like Domain 7 in Early-Onset Pre-Eclampsia: A New Potential Mechanism of Action

doi: 10.3390/biomedicines12081929

Figure Lengend Snippet: Identification of high-responder and low-responder PE pregnancies following treatment of chorionic villous explant cultures with 10 μM pravastatin (PRA) for 24 h. ( A , B ): qRT-PCR analysis demonstrating increased expression of EGFL7 in high-responder PE villous explant cultures following PRA treatment ( A ) and no changes in low-responder PE villous cultures ( B ) when compared to villi cultured without PRA. ( C ): Western blot analysis of villous explant cultures with or without PRA, indicating increased expression of EGFL7 in high-responder PE villous cultures. ( D ): Quantification of Western blot analysis ( n = 3). Statistical analysis was performed using Student’s t test (* p = 0.0467 for qRT-PCR; * p = 0.028 for western blot). − and + PRA: without or with 10 μM pravastatin.

Article Snippet: Membranes were then incubated overnight at 4 °C with rabbit anti-EGFL7 (Abcam, Cambridge, UK, cat. ab256451, 1:1000) or mouse anti-GAPDH (clone 6C5, Santa Cruz, CA, USA, cat. sc-32233, 1:2000), all diluted in TBS/T with 5% ( w / v ) bovine serum albumin (BSA).

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Western Blot

Journal: Biomedicines

Article Title: Effect of Pravastatin on Placental Expression of Epidermal Growth Factor-like Domain 7 in Early-Onset Pre-Eclampsia: A New Potential Mechanism of Action

doi: 10.3390/biomedicines12081929

Figure Lengend Snippet: Maternal socio-demographic characteristics and biochemical findings of women with pre-eclampsia according to EGFL7 expression in response to pravastatin treatment.

Article Snippet: Membranes were then incubated overnight at 4 °C with rabbit anti-EGFL7 (Abcam, Cambridge, UK, cat. ab256451, 1:1000) or mouse anti-GAPDH (clone 6C5, Santa Cruz, CA, USA, cat. sc-32233, 1:2000), all diluted in TBS/T with 5% ( w / v ) bovine serum albumin (BSA).

Techniques: Expressing

Journal: Biomedicines

Article Title: Effect of Pravastatin on Placental Expression of Epidermal Growth Factor-like Domain 7 in Early-Onset Pre-Eclampsia: A New Potential Mechanism of Action

doi: 10.3390/biomedicines12081929

Figure Lengend Snippet: Perinatal and neonatal outcome of women with pre-eclampsia according to EGFL7 expression in response to pravastatin treatment.

Article Snippet: Membranes were then incubated overnight at 4 °C with rabbit anti-EGFL7 (Abcam, Cambridge, UK, cat. ab256451, 1:1000) or mouse anti-GAPDH (clone 6C5, Santa Cruz, CA, USA, cat. sc-32233, 1:2000), all diluted in TBS/T with 5% ( w / v ) bovine serum albumin (BSA).

Techniques: Expressing