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

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: Comparison of clinical and molecular characteristics by EGFL7 -expresser status of younger adult patients (age <60 y) with de novo CN-AML

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Comparison

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

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: Prognostic significance of EGFL7 in younger and older CN-AML patients. (A and B) Impact of EGFL7 expression levels on DFS of younger (age <60 y) (A) and older (age ≥60 y) (B) adult patients. (C) DFS according to EGFL7 risk group in older CN-AML patients. The favorable risk group comprised patients with EGFL7 low expression/high methylation; the unfavorable risk group comprised the remaining patients (high expression/low methylation, high expression/high methylation, low expression/low methylation). The median values of EGFL7 expression and EGFL7 promoter methylation were used as cutoffs.

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Expressing, Methylation

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

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: Prognostic significance of EGFL7 in younger and older CN-AML patients. (A and B) The association of EGFL7 expression levels with OS and EFS of younger adult patients (age <60 y) (A) and older patients (age ≥60 y) (B). (C) OS and EFS according to EGFL7 risk group in older CN-AML patients. The favorable risk group was comprised of patients with EGFL7 low expression/high promoter methylation; the unfavorable risk group included the remaining patients (high expression/low promoter methylation, high expression/high promoter methylation, low expression/low promoter methylation). The median values of EGFL7 expression and EGFL7 promoter methylation were used as the high/low cut points.

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Expressing, Methylation

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

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: Treatment outcomes according to EGFL7 expression in 374 younger adult patients (age <60 y) with de novo CN-AML

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Expressing

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

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: Comparison of clinical and molecular characteristics by EGFL7 -expresser status of older patients (age ≥60 y) with de novo CN-AML

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Comparison

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

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: Treatment outcomes according to EGFL7 expression in 198 older patients (age ≥60 y) with de novo CN-AML

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Expressing

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

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: Univariable models for expression of EGFL7 and miR-126 and associations with outcome in 300 younger adults and 171 older patients with CN-AML

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Expressing

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

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: Treatment outcomes according to EGFL7 risk group in 126 older patients (age ≥60 y) with de novo CN-AML

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques:

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

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: Multivariable analyses of outcome according to the EGFL7 risk group in 126 older patients (age ≥60 y) with de novo CN-AML

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques:

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

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: Multivariable analyses of event-free survival according to the EGFL7 risk group in 126 older patients (age ≥60 y) with de novo CN-AML

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques:

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

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: Cytogenetic and molecular characteristics of the leukapheresis samples from AML patients that were profiled for EGFL7 expression and were used in functional studies

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Expressing, Functional Assay

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

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: EGFL7 is up-regulated in human and mouse AML cells. (A) NBM samples from healthy donors (n = 3) were compared with leukapheresis samples of AML patients (ptAML, n = 11). EGFL7 levels were measured in AML samples by real-time RT-PCR, and the results were normalized to β-ACTIN RNA levels. (B) Mean ± SD of EGFL7 mRNA expression levels between NBM and AML in aggregate. *P < 0.05. (C) EGFL7 protein levels in human NBM and leukapheresis samples of AML patients were determined by immunoblotting with GAPDH as loading control. (D) Normal BM from WT mice (n = 4) was compared with murine AML blasts of the MllPTD/WTFlt3ITD/WT mouse model (mAML, n = 4) for the detection of mouse Egfl7 mRNA by RT-PCR with β-Actin as internal control. (E) Mean ± SD of Egfl7 mRNA between murine NBM and murine AML blasts, in aggregate. **P < 0.01. (F) Mouse Egfl7 protein levels in WT murine controls (n = 4) and murine AML blasts from the MllPTD/WTFlt3ITD/WT mouse model (mAML) (n = 4) were assessed by immunoblotting using Gapdh as loading control. (G) Immunohistochemistry of Egfl7 in NBM of WT (n = 3) control mice vs. BM from MllPTD/WTFlt3ITD/WT leukemic mice (n = 3) using an Egfl7-specific antibody or no antibody controls. (Original magnification: 100×; Insets with same areas across the samples are magnified at same strength.) (H) Percent of Egfl7+ cells in NBM of WT mice vs. BM from MllPTD/WTFlt3ITD/WT leukemic mice. ****P < 0.0001. (I and J) EGFL7 mRNA (I) and EGFL7 protein (J) expression levels in four human AML cell lines (EOL1, OCI-AML3, MV4-11, and Kasumi-1). The relative expression of EGFL7 mRNA was measured with real-time RT-PCR normalized to β-ACTIN. For immunoblotting, β-Tubulin was used as loading control.

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry

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

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: EGFL7 is a secreted protein and is increased in the serum of some AML patients. (A) Blasts of AML patients (ptAML, n = 3) were cultured in SFEM medium + 10% FBS supplemented with cytokines for 24 h. The EGFL7 protein level in the cell-culture supernatant was detected by ELISA and was compared with that in medium from wells without cultured cells. **P < 0.01, *P < 0.05. (B) Blasts of AML patients (n = 3) were cultured in SFEM medium + 10% FBS supplemented with cytokines for 24 h. The EGFL7 protein in the cell-culture supernatant was assessed by immunoblotting with rEGFL7 as a positive control and medium from wells without cultured cells as a negative control. Ponceau S staining shows the loading control for protein. (C) EGFL7 protein levels in sera from normal healthy controls (sN, n = 6) and AML patients (sAML, n = 6) were determined by the EGFL7 ELISA kit. SFEM medium alone and 10% FBS serve as blank controls. *P < 0.05, **P < 0.01. (D) An equal volume of serum from AML patients (sAML, n = 12) or normal healthy donors (sN, n = 6) was subjected to the separation of exosomal vs. nonexosomal eluant using the ExoQuick kit (System Biosciences). EGFL7 protein levels in both isolated exosomes and the supernatant were determined by immunoblotting. SFEM containing 10% FBS served as a negative control, and rEGFL7 was used as a positive control. Ponceau S staining shows the loading of proteins.

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Positive Control, Negative Control, Staining, Control, Isolation

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

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: EGFL7 stimulates the proliferation of human and mouse AML cells. (A and B) BM cells from WT (A) and MllPTD/WTFlt3ITD/WT (B) mice were treated without (Unstim) or with 0.1, 0.25, or 0.5 μM rEgfl7 in Iscove’s Modified Dulbecco’s Medium (IMDM) + 2% BSA for 24, 48, 72, or 96 h. At the indicated time points, the number of viable cells was determined by Trypan blue dye exclusion assay. Each condition was repeated in triplicate. *P < 0.05, **P < 0.01; NS, not significant. (C) Blasts of the indicated AML patients (20,000 cells) were mixed with methylcellulose medium in the absence or presence of 0.25 μM rEGFL7 and were plated onto 2-cm dishes for 10 d. Colonies with more than 50 cells were enumerated using a light microscope. Each condition for each patient (n = 4) was plated in triplicate; **P < 0.01, ***P < 0.001. (D) Kasumi-1 cells were stimulated with 100 nM rEGFL7 for 4 h in RPMI1640 with 10% FBS. Cell proliferation was assessed using APC-BrdU/7AAD staining coupled with flow cytometry; *P < 0.05. (E) Kasumi-1 cells (2,500 cells) were mixed with methylcellulose medium in the absence or presence of 100 nM recombinant human EGFL7 and were scored after 10 d. Each condition was plated in triplicate in three independent experiments. *P < 0.05. (F) Blasts from AML patients (n = 4) were cultured in SFEM + 2% BSA in the absence or presence of 0.25 μM rEGFL7 for 20 min. Total proteins were extracted for immunoblotting of pAKT-S473 and total AKT. GAPDH was used as loading control. (G) AML blasts from MllPTD/WTFlt3ITD/WT mice (n = 3) were cultured in IMDM medium + 2% BSA in the absence or presence of 0.25 μM rEgfl7 for 20 min. Total proteins were extracted for immunoblotting of pAkt-S473 and total Akt. Gapdh was used as loading control. (H) Exponentially growing Kasumi-1 cells were starved in serum-free RPMI1640 medium for 1 h, followed by the addition of 100 nM recombinant EGFL7 for 5 min. Total proteins were extracted for immunoblotting of pAKT-S473, total AKT, and GAPDH.

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Modification, Exclusion Assay, Light Microscopy, Staining, Flow Cytometry, Recombinant, Cell Culture, Western Blot, Control

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

Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia

doi: 10.1073/pnas.1703142114

Figure Lengend Snippet: EGFL7 inhibition results in decreased human AML cell growth without affecting normal hematopoietic cells. (A) Blasts from AML patients were cultured in SFEM with 10% FBS in the presence of 50 μg/mL of normal human IgG or anti-EGFL7 (@E7) antibody for 1 h. Total proteins were extracted for immunoblotting of pAKT-S473 and total AKT. GAPDH was used as loading control. (B) Human primary blasts (400,000) from AML patients (n = 4) were treated with 2, 10, 50, or 250 μg/mL of IgG control or anti-EGFL7 antibody in SFEM containing 10% FBS and cytokines for 2 h. Twenty thousand cells were plated in triplicate in methylcellulose medium and scored after 14 d of culture for mean ± SD colony numbers. *P < 0.05, **P < 0.01. (C) Forty-eight hours after IgG control vs. anti-EGFL7 antibody treatment (50 μg/mL) of AML cell lines, apoptosis was evaluated by Annexin V/7AAD staining, (D and E) Cell proliferation was measured using BrdU incorporation (D), and differentiation analysis was evaluated by CD11b expression (E). CD11b is depicted as the ratio of the CD11b expression value of the examined sample to the CD11b expression value of the corresponding IgG-treated control. *P < 0.05, **P < 0.01. (F) CD34+ CB cells from four different donors were plated in methylcellulose medium in the presence of increasing concentrations (2, 10, 50, or 250 μg/mL) of human IgG or anti-EGFL7 and were scored after 14 d of culture. Along with total number of colonies, colony types [erythroid burst-forming units (BFU), granulocyte/monocyte (GM), or granulocyte/erythrocyte/monocyte/megakaryocyte colonies (GEMM)] were enumerated also.

Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.

Techniques: Inhibition, Cell Culture, Western Blot, Control, Staining, BrdU Incorporation Assay, Expressing

Journal: Arthritis Research & Therapy

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

doi: 10.1186/ar4349

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

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

Techniques: Sandwich ELISA, MANN-WHITNEY

Journal: Arthritis Research & Therapy

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

doi: 10.1186/ar4349

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

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

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

Journal: Arthritis Research & Therapy

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

doi: 10.1186/ar4349

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

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

Techniques: Expressing, Derivative Assay, Western Blot, Control