rabbit anti egfl7  (Danaher Inc)

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: 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

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: Frontiers in Oncology

Article Title: Novel Expression of EGFL7 in Osteosarcoma and Sensitivity to Cisplatin

doi: 10.3389/fonc.2020.00074

Figure Lengend Snippet: Compared with CS, EGFL7 is highly expressed in OS. (A) Typical pathological features of OS and CS revealed by HE staining, tumor-like osteogenesis can be seen in OS and cartilage-like matrix in CS. (B,C) Immunohistochemical of EGFL7 protein expression in OS tissue ( n = 2) and CS controls ( n = 2), comparison of IHC results from different magnification (100 and 200 ×) and its significance. High expression of EGFL7 (++) was found in OS tissues but negative in CS, the arrow refers to the positive area. The semi-quantitative statistical analysis based on IHC results shows that there is a significant difference in the expression of EGFL7 protein between OS and CS. (D,E) Western blot results showed that EGFL7 protein was overexpressed in OS tissue compared with CS, Student's t -test shows that the difference between OS and CS has obvious statistical significance. The abundance of EGFL7 protein in OS tissue was similar to that of GAPDH protein. (F) RT-PCR results showed that the transcription level of EGFL7 mRNA in OS tissues was significantly higher than that in CS tissues, and the difference was statistically significant. (G) Immunohistochemical results of chondrosarcoma and osteosarcoma showed that EGFL7 was highly expressed in osteosarcoma. (H) The immunohistochemical results of chondrosarcoma and osteosarcoma showed that CD34 was highly expressed in osteosarcoma, indicating that there were more abundant blood vessels in osteosarcoma. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rabbit anti-human-EGFL7 primary antibodies (19291-1-AP, Proteintech, USA) and rabbit anti-human CD34 antibody (ab81289, Abcam, UK) were used at a dilution of 1:100.

Techniques: Staining, Immunohistochemical staining, Expressing, Comparison, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Oncology

Article Title: Novel Expression of EGFL7 in Osteosarcoma and Sensitivity to Cisplatin

doi: 10.3389/fonc.2020.00074

Figure Lengend Snippet: The primers used for Q-PCR

Article Snippet: Rabbit anti-human-EGFL7 primary antibodies (19291-1-AP, Proteintech, USA) and rabbit anti-human CD34 antibody (ab81289, Abcam, UK) were used at a dilution of 1:100.

Techniques:

Journal: Frontiers in Oncology

Article Title: Novel Expression of EGFL7 in Osteosarcoma and Sensitivity to Cisplatin

doi: 10.3389/fonc.2020.00074

Figure Lengend Snippet: Upregulation of EGFL7 expression in OS cell line compared with ECs and CS cell line. (A,B) Western blot results showed that EGFL7 protein was overexpressed in all OS cell lines and HUVECs compared to SW1353. Statistical analysis showed that the expression of EGFL7 in OS cell lines was significantly different from that in SW1353. Similarly, the expression of EGFL7 protein in HUVEC was significantly different from that in SW1353. (C) RT-PCR results showed that the transcription level of EGFL7 mRNA in OS cell lines was significantly higher than HUVEC, there are also differences between HUVEC and SW1353, and the difference was all statistically significant. PCR and western blot experiments were all performed in triplicate. (D) The ELISA results of EGFL7 in cell culture medium also confirmed that there was indeed a high expression of EGFL7 protein in osteosarcoma cells. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rabbit anti-human-EGFL7 primary antibodies (19291-1-AP, Proteintech, USA) and rabbit anti-human CD34 antibody (ab81289, Abcam, UK) were used at a dilution of 1:100.

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Frontiers in Oncology

Article Title: Novel Expression of EGFL7 in Osteosarcoma and Sensitivity to Cisplatin

doi: 10.3389/fonc.2020.00074

Figure Lengend Snippet: Immunofluorescence technique was used to verify the expression of EGFL7 from histological and cytological aspects, respectively. (A) As seen in these representative images, the EGFL7 was labeled by special anti-EGFL7 antibody, and the cell nuclei were stained by DAPI. The merged image shows that green fluorescent signals for EGFL7 protein were expressed in both tumor tissues and vascular endothelial cells, and we have observed this expression from multiple samples and different optical multiples, respectively. White arrows indicate positive areas. (B) From the immunofluorescence results of OS cells, the EGFL7 protein was labeled by red fluorescent, the cell nuclei were stained by DAPI. The expression of EGFL7 protein in OS cells is located in the cytoplasm.

Article Snippet: Rabbit anti-human-EGFL7 primary antibodies (19291-1-AP, Proteintech, USA) and rabbit anti-human CD34 antibody (ab81289, Abcam, UK) were used at a dilution of 1:100.

Techniques: Immunofluorescence, Expressing, Labeling, Staining

Journal: Frontiers in Oncology

Article Title: Novel Expression of EGFL7 in Osteosarcoma and Sensitivity to Cisplatin

doi: 10.3389/fonc.2020.00074

Figure Lengend Snippet: The expression of EGFL7 deregulated in OS cells after cisplatin intervention. (A) Intervention of four osteosarcoma cell lines with different gradient concentration of cisplatin. Under light microscope, the number of cells changed significantly with different concentrations of cisplatin. The toxicity of cisplatin was detected by CCK-8 kit and the corresponding IC50 of various cells was calculated. (B) The results showed that cisplatin could significantly affect the proliferation rate of tumor cell lines. (C,D) Western blot was assessed to compare EGFL7 protein in OS cells before and after Cisplatin intervention, the results showed that the expression of EGFL7 protein deregulated after Cisplatin intervention. Paired T test showed that EGFL7 protein expression in OS cells was significantly deregulated after cisplatin intervention and the difference was statistically significant. (E) RT-PCR results showed that the transcription level of EGFL7 mRNA in OS cell lines was significantly deregulated after cisplatin intervention and the difference was statistically significant. PCR and western blot experiments were all performed in triplicate. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rabbit anti-human-EGFL7 primary antibodies (19291-1-AP, Proteintech, USA) and rabbit anti-human CD34 antibody (ab81289, Abcam, UK) were used at a dilution of 1:100.

Techniques: Expressing, Concentration Assay, Light Microscopy, CCK-8 Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Oncology

Article Title: Novel Expression of EGFL7 in Osteosarcoma and Sensitivity to Cisplatin

doi: 10.3389/fonc.2020.00074

Figure Lengend Snippet: Chemotherapy deregulated expression of EGFL7 in osteosarcoma. (A) Samples obtained by biopsy before chemotherapy and by tumor resection after chemotherapy in the same patient. Pre-chemotherapy tissue specimens showed fish-like appearance, while post-chemotherapy tumor tissue showed obvious bone repair changes. (B,C) Immunohistochemical staining of the same patient's tumors before and after chemotherapy showed that there was almost no expression of EGFL7 in the tumors after chemotherapy (black arrows represent positive areas). The semi-quantitative statistical analysis based on IHC results shows that there is a significant difference in the expression of EGFL7 between pre-chemotherapy and post-chemotherapy. (D,E) Western blot results showed that EGFL7 protein was almost no expression in post-chemotherapy tissue compared with pre-chemotherapy tissue, Student's t -test shows that the difference has obvious statistical significance. (F) RT-PCR results showed that the transcription level of EGFL7 mRNA in post-chemotherapy tissue was significantly deregulated than that in pre-chemotherapy tissue, and the difference was statistically significant. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rabbit anti-human-EGFL7 primary antibodies (19291-1-AP, Proteintech, USA) and rabbit anti-human CD34 antibody (ab81289, Abcam, UK) were used at a dilution of 1:100.

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction