recombinant egfl6 protein  (R&D Systems)

Journal: The Journal of Clinical Investigation

Article Title: Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells

doi: 10.1172/JCI175147

Figure Lengend Snippet: ( A and B ) Graphs represent the percentage of B, CD4 + , CD8 + , and CD11b + cells in BM ( A ) and spleen ( B ) of WT and Egfl6 mice. ( C ) Gating and quantification of Ly6G and Ly6C subsets of CD11b + BM and splenic cells from healthy C57BL/6J (WT) and Egfl6 mice. ( D ) Volcano plot showing differentially expressed genes (DEGs) between BM CD11b + cells of Egfl6 mice versus C57BL/6J (WT). P values determined via t test are plotted on the y axis. DEGs are colored in red. ( E ) Gating and quantification of BM-derived CD11b + Ly6G + Ly6C – cells stimulated with rGM-CSF ± rEgfl6. ( F ) qPCR analyses of indicated genes in sorted BM CD11b + cells stimulated with rGM-CSF + rEgfl6. Stimulation with rGM-CSF alone was used as control. ( G and H ) ELISA of Granzyme B (GZMB) in IL-2 + CD3/CD28 activated CD8 + T cells and cultured directly with rEgfl6-stimulated BM-derived MDSC cells or MDSC control at different ratio ( G ) or with the conditioned media (CM) of rEgfl6-stimulated BM-derived MDSC cells or MDSC control ( H ). Unstimulated CD8 + T cells were used as negative control. Results were analyzed using unpaired 2-tailed t test or 2-way ANOVA. Experiments were performed in triplicate. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were supplemented with 50 ng/mL recombinant mouse granulocyte macrophage colony stimulating factor (GM-CSF) (R&D System) alone or in combination with 200 ng/mL recombinant murine Egfl6 (rEgfl6) (Sino Biological) and cultured for 4 days in a humidified incubator at 37°C and 5% CO 2 .

Techniques: Derivative Assay, Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Negative Control

Journal: The Journal of Clinical Investigation

Article Title: Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells

doi: 10.1172/JCI175147

Figure Lengend Snippet: ( A ) Tumor volume changes (mm 3 ) and images of 2F8c and 2F8c-Egfl6 subcutaneous tumors resected and measured 3 weeks after tumor cell inoculation ( n = 6 mice per group). ( B ) Time-dependent body weight gain in mice i.p. injected with ID8-CV and ID8-Egfl6 tumors ( n = 8 mice per group). ( C ) Evaluation of peritoneal metastases of ID8-CV and ID8-Egfl6 that had a weight increase of over 35% of their original weight on the day of tumor cell injections ( n = 6 mice per group). ( D and E ) Kaplan-Meier overall survival analysis for 2F8c+/–Egfl6 ( D ) and ID8+/–Egfl6 ( E ). Survival statistics were calculated using log-rank analysis from Kaplan-Meier survival plots. ( F and G ) Flow cytometric evaluation and summary of PMN-MDSC (CD11b + Ly6G + Ly6C – ) ( F , top panel), M-MDSC (CD11b + Ly6G – Ly6C + ) ( F , bottom panel), and TAM (CD11b + F4/80 + CD206 + ) ( G ) in ID8+/–Egfl6 tumors. ( H ) Flow cytometric evaluation and quantification of CD8 T (CD45 + Thy1.2 + ) cells and their expression of IFN-γ in ID8+/–Egfl6 tumors. ( I and J ) ELISA of Granzyme B (GZMB) ( I ) and IFN ( J ) in IL-2 + CD3/CD28 activated CD8 T cells (Pos Control) and cultured directly with F4/80 + or Ly6G + cells isolated from ID8 and ID8-Egfl6 ascites at ratio of 1:1. ( K and L ) Time-dependent volume changes (mm 3 ) of 2F8c and 2F8c-Egfl6 tumor cells ( K ) or body-weight gain in mice i.p. injected with ID8 and ID8-Egfl6 tumor cells ( L ) and treated with anti-Ly6G/Ly6C Ab or IgG isotype control ( n = 6 mice per group). P values were calculated using unpaired 2-tailed t test, 1-way, or 2-way ANOVA with Tukey’s post test for multiple comparisons. Experiments were performed in triplicate. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were supplemented with 50 ng/mL recombinant mouse granulocyte macrophage colony stimulating factor (GM-CSF) (R&D System) alone or in combination with 200 ng/mL recombinant murine Egfl6 (rEgfl6) (Sino Biological) and cultured for 4 days in a humidified incubator at 37°C and 5% CO 2 .

Techniques: Injection, Expressing, Enzyme-linked Immunosorbent Assay, Control, Cell Culture, Isolation

Journal: The Journal of Clinical Investigation

Article Title: Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells

doi: 10.1172/JCI175147

Figure Lengend Snippet: ( A ) Volcano plot showing differentially expressed genes (DEGs) between CD11b + cells infiltrating 2F8c-Egfl6 versus 2F8c tumors. Negative Log 10 P values determined via t test are plotted on the y axis. ( B ) IPA protein analysis of Egfl6 treatment associated DEG pathways identified as significantly ( P < 0.05) upregulated (left panel) or downregulated (right panel). ( C and D ) Summary of PD-L1 expression determined by flow cytometry in infiltrating TAMs ( C ) and by qPCR in BM-derived macrophages polarized with different stimuli as indicated D . ( E ) Western blotting analysis of IL-10 and Cxcl2 in TAMs and PMN-MDSCs isolated from ID8+/–Egfl6 ascites. Actin was used as loading control. ( F ) ELISA of IFN-γ in CD8 + T cells cultured with the Ly6G + cells isolated from ID8+/–Egfl6 ascites in the absence/presence of IL-10 or Cxcl2 NAbs. ( G ) Western blotting showing the indicated protein expression in BM-isolated CD11b + cells treated with GM-CSF and rEgfl6 for 0, 7.5, and 15 minutes. β-Actin was used as loading control. Results are representative of at least 3 independent experiments. ( H and I ) ELISA showing IL-10 and Cxcl2 protein secretion in GM-CSF-treated BM CD11b + cells +/– rEgfl6 and/or Syk inhibitor (R406) ( H ), and GM-CSF-treated BM CD11b + cells +/– rEgfl6 and/or the integrin inhibitor Cyclo-RGD (c-RGD) ( I ). ( J ) Graph represents a ChIP assay performed with anti-Jun Ab followed by qPCR to measure IL-10 promoter in ID8+/–Egfl6 ascites. Data are presented as mean ± SEM. P values were calculated using unpaired 2-tailed t test or 1-way ANOVA with Tukey’s post test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. All results are representative of 3 independent experiments.

Article Snippet: Cells were supplemented with 50 ng/mL recombinant mouse granulocyte macrophage colony stimulating factor (GM-CSF) (R&D System) alone or in combination with 200 ng/mL recombinant murine Egfl6 (rEgfl6) (Sino Biological) and cultured for 4 days in a humidified incubator at 37°C and 5% CO 2 .

Techniques: Expressing, Flow Cytometry, Derivative Assay, Western Blot, Isolation, Control, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: The Journal of Clinical Investigation

Article Title: Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells

doi: 10.1172/JCI175147

Figure Lengend Snippet: ( A ) 2F8c and 2F8c-Egfl6 tumor growth in mice treated with anti-PD-L1 Ab or IgG isotype control Ab ( n = 8 mice per group). * P < 0.05, 2F8c + IgG versus 2F8c-Egfl6 + IgG; *** P < 0.001, 2F8c + anti-PD-L1 versus 2F8c + IgG. ( B ) Kaplan-Meier survival analysis for the indicated treatment groups. *** P < 0.001, 2F8c + anti-PD-L1 versus 2F8c + IgG. Survival statistics were calculated using log-rank (Mantel-Cox) analysis from Kaplan-Meier survival plots. ( C ) Flow cytometry quantification of intratumoral PMN-MDSCs (CD11b + Ly6G + Ly6C – ), M-MDSCs (CD11b + Ly6G – Ly6C + ), CD206 + TAMs, and CD8 + T cells in the indicated tumors. ( D – F ) qPCR analysis of mRNA expression of S100A9 , IL-10 , and Cxcl2 gene expression in ( D ) 2F8c-Egfl6 versus 2F8c, ( E ) anti-PD-L1–treated 2F8c versus IgG-treated 2F8c, ( F ) anti-PD-L1–treated 2F8c-Egfl6 versus IgG-treated 2F8c-Egfl6 tumor samples. ( G ) Representative images of IHC staining showing Cxcl2-expressing cells in control and a-PD-L1–treated tumor tissue sections. Graph represents the number of Cxcl2 + cells in the indicated tumors. Scale bars: 20 μm. Error bars show SEM. Experiments were performed in triplicate. Statistical significance was determined by unpaired 2-tailed t test, 1-way, or 2-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001.

Article Snippet: Cells were supplemented with 50 ng/mL recombinant mouse granulocyte macrophage colony stimulating factor (GM-CSF) (R&D System) alone or in combination with 200 ng/mL recombinant murine Egfl6 (rEgfl6) (Sino Biological) and cultured for 4 days in a humidified incubator at 37°C and 5% CO 2 .

Techniques: Control, Flow Cytometry, Expressing, Immunohistochemistry

Journal: The Journal of Clinical Investigation

Article Title: Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells

doi: 10.1172/JCI175147

Figure Lengend Snippet: ( A ) Volume changes (mm 3 ) and representative images of 2F8c-Egfl6 subcutaneous tumors treated with IgG isotype Ab (Control), a-PD-L1 Ab, and a-Egfl6 Ab, alone or in combination, were resected and measured 2 days after the last treatment ( n = 8 mice per group). ** P < 0.01, IgG Ab versus a-Egfl6 Ab; *** P < 0.001, anti-PD-L1 Ab versus a-Egfl6 Ab and IgG Ab versus anti-PD-L1+ a-Egfl6 Abs. ( B and C ) Kaplan-Meier overall survival analysis for 2F8c-Egfl6 ( B ) and ID8 p53–/– Brca2–/— -Egfl6 ( C ) mice receiving the indicated treatment. Survival statistics were calculated using the Log-rank (Mantel-Cox) test analysis. ( D – G ) Flow cytometric gating and quantification of CD206 + TAMs ( D ), PMN-MDSC (CD11b + Ly6G + Ly6C – ) ( E ), MHCII + TAMs ( F ), and CD8 + T (CD45 + Thy1.2 + ) ( G ) cells in 2F8c-Egfl6 and ID8 p53–/– Brca2–/— -Egfl6 tumors. Error bars show SEM. Experiments were performed in triplicate. Statistical significance was determined by unpaired 2-tailed t test or 2-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001.

Article Snippet: Cells were supplemented with 50 ng/mL recombinant mouse granulocyte macrophage colony stimulating factor (GM-CSF) (R&D System) alone or in combination with 200 ng/mL recombinant murine Egfl6 (rEgfl6) (Sino Biological) and cultured for 4 days in a humidified incubator at 37°C and 5% CO 2 .

Techniques: Control

Journal: The Journal of Clinical Investigation

Article Title: Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells

doi: 10.1172/JCI175147

Figure Lengend Snippet: ( A ) qPCR analyses of IL-10 and Cxcl2 in the indicated treated Egfl6 + 2F8c tumors. ( B ) IF images and quantification of IL-10 expression in the indicated treated Egfl6 + 2F8c tumors. P values were calculated using unpaired 2-tailed t test. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001 , **** P < 0.0001. All results are representative of 3 independent experiments. Scale bar: 30 μm.

Article Snippet: Cells were supplemented with 50 ng/mL recombinant mouse granulocyte macrophage colony stimulating factor (GM-CSF) (R&D System) alone or in combination with 200 ng/mL recombinant murine Egfl6 (rEgfl6) (Sino Biological) and cultured for 4 days in a humidified incubator at 37°C and 5% CO 2 .

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells

doi: 10.1172/JCI175147

Figure Lengend Snippet: ( A and B ) Gating and quantification of human CD11b + CD66b + ( A ) and CD11b + CD163 + CD64 + ( B ) cells in CD33 + cells isolated from ascites of patients with HGSOC and stimulated with rEGFL6 +/– c-RGD. ( C ) Cytokine array and densitometry of the CM of CD33 + ascites from patients with HGSOC stimulated with GM-CSF +/– rEGFL6. Spot intensities were calculated using ImageJ software. ( D ) Representative immunofluorescence images showing EGFL6 expression (red) and CD68 cell (green) localization in HGSOC tumor tissue sections ( n = 6 per group). DAPI stained nuclei. Graph represents the number of CD68-positive cells in tissues expressing high or low levels of EGFL6. Scale bar: 100 μm. ( E ) Spatial feature plots of EGFL6 and CD163 markers and spatial autocorrelation of selected genes. Moran’s I test, implemented in the Seurat FindSpatiallyVariableFeatures function, was applied to compute spatial autocorrelation of the expression of each gene. Data are from a previously published dataset . ( F – H ) Sorted correlation plots between mRNA expression of EGFL6 in CD45 – cells and mRNA expression of cytokines and surface proteins in the indicated immune cells. Correlation was computed using the Spearman’s correlation with the sample-wise averaged gene expression. Each dot represents the Spearman’s correlation coefficients of a gene, and the dots were sorted in ascending order. P values were calculated using unpaired 2-tailed t test, 1-way, or 2-way ANOVA with Tukey’s post test for multiple comparisons. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.

Article Snippet: Cells were supplemented with 50 ng/mL recombinant mouse granulocyte macrophage colony stimulating factor (GM-CSF) (R&D System) alone or in combination with 200 ng/mL recombinant murine Egfl6 (rEgfl6) (Sino Biological) and cultured for 4 days in a humidified incubator at 37°C and 5% CO 2 .

Techniques: Isolation, Software, Immunofluorescence, Expressing, Staining

Journal: Stem Cell Research & Therapy

Article Title: EGFL6 regulates angiogenesis and osteogenesis in distraction osteogenesis via Wnt/β-catenin signaling

doi: 10.1186/s13287-021-02487-3

Figure Lengend Snippet: Real-time PCR primer sequences used in the study

Article Snippet: Recombinant EGFL6 protein was purchased from R&D Systems (Cat no.8638-EG-050, R&D Systems Inc., Minneapolis, MN, USA).

Techniques: Real-time Polymerase Chain Reaction

Journal: Stem Cell Research & Therapy

Article Title: EGFL6 regulates angiogenesis and osteogenesis in distraction osteogenesis via Wnt/β-catenin signaling

doi: 10.1186/s13287-021-02487-3

Figure Lengend Snippet: Locally applied EGFL6 accelerates bone formation and consolidation in a rat model of tibia distraction osteogenesis (DO). a Overall schematic diagram illustrating the study design. DO was performed in three phases as indicated. Midway through the distraction phase on day 10, recombinant EGFL6 (200 ng/ml), or an equivalent volume of sterile PBS (control), was infused into the distracted area and then infused again every 2 days until the end of the distraction phase on day 15. Distraction was performed at a rate of 0.25 mm per 12 h. Asterisk (*) in a indicates that the tibia bone fragments were distracted for a total of 5 mm over a period of 10 days. b X-ray images (lateral view) of the distracted bones from representative cases after 2, 3, and 4 weeks of consolidation. Bright white angular areas in images are the densities of the metal monolateral external fixator. c, d Three-dimensional reconstructions ( c ) and internal longitudinal profiles ( d ) derived from micro-CT of distracted tibia bones from representative cases of EGFL6-treated and control rats after 2 and 4 weeks of consolidation. Light areas show the increased bone-tissue mineralization. e, f Quantitation analysis of bone-tissue mineralization showing the mean (±SD) percentage bone volume/total tissue volume (BV/TV) and mean (±SD) bone mineral density (BMD) in EGFL6-treated and control rats. Mineralization parameters were calculated from the micro-CT image data. Significant differences were evaluated by one-way ANOVA with post hoc Dunnett’s tests. * p < 0.05

Article Snippet: Recombinant EGFL6 protein was purchased from R&D Systems (Cat no.8638-EG-050, R&D Systems Inc., Minneapolis, MN, USA).

Techniques: Recombinant, Sterility, Control, Derivative Assay, Micro-CT, Quantitation Assay

Journal: Stem Cell Research & Therapy

Article Title: EGFL6 regulates angiogenesis and osteogenesis in distraction osteogenesis via Wnt/β-catenin signaling

doi: 10.1186/s13287-021-02487-3

Figure Lengend Snippet: Effects of different concentrations of EGFL6 protein on human umbilical cord vein endothelial cell (HUVEC) angiogenesis in vitro. a Phase-contrast images of HUVEC cultures treated with EGFL6 showing cell migration in the scratch-wound assay at the indicated times. Vertical dashed lines (white) demarcate the border between the wavefront of migrating cells and scratched area that was initially void of cells. b Quantitation (mean ± SD) of cell proliferation in response to EGFL6 (CCK-8 assay). c Mean percentage of cells migrating as a function of EGFL6 concentration in the scratch-wound assay. d, e Crystal violet-stained HUVECs that migrated in the transwell assay. Optical density (OD) of staining is relative to untreated control cells ( e ). f Relative quantification of capillary-like structures formed by HUVECs cultured with EGFL6 in the tube-formation assay. Values are relative to control values. g Phase-contrast images of HUVECs cultured with EGFL6 in the tube-formation assay. h Expression levels of Hif1a, VEGF-A, CD31, and EMCN genes in HUVECs treated with EGFL6 for 1 day, as evaluated by RT-PCR. The housekeeping gene GAPDH served as an internal control. i, j Quantitation of VEGF-A protein concentration in HUVECs treated with EGFL6 (200 ng/ml) for the indicated times. k, l Western blots of lysates from HUVECs treated with EGFL6. Blots were probed with antibodies against angiogenesis markers (Hif1a, VEGF-A, CD31, EMCN) and pathway markers (β-catenin, pβ-catenin, active β-catenin, and pGSK3β). GADPH is the loading control. Significant differences among groups were determined by one-way ANOVA and post hoc Dunnett’s test; * p < 0.05; ** p < 0.01; and *** p < 0.001. All immunoblots were cropped from the original here and in subsequent figures. Experimental HUVECs were treated with the indicated EGFL6 concentrations. Control and experimental conditions for all functional assays were the same, except controls lacked EGFL6. Histogram values are based on three replicated experiments, and error bars are SD here and in all subsequent figures. Scale bars for a, e, g , 250 μm

Article Snippet: Recombinant EGFL6 protein was purchased from R&D Systems (Cat no.8638-EG-050, R&D Systems Inc., Minneapolis, MN, USA).

Techniques: In Vitro, Migration, Scratch Wound Assay Assay, Quantitation Assay, CCK-8 Assay, Concentration Assay, Staining, Transwell Assay, Control, Quantitative Proteomics, Cell Culture, Tube Formation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Protein Concentration, Western Blot, Functional Assay

Journal: Stem Cell Research & Therapy

Article Title: EGFL6 regulates angiogenesis and osteogenesis in distraction osteogenesis via Wnt/β-catenin signaling

doi: 10.1186/s13287-021-02487-3

Figure Lengend Snippet: EGFL6 treatment enhances osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. a BMSC viability after treatment with different concentrations of EGFL6, as assessed by the CCK-8 assay. Values are means±SD. b Images of Alizarin Red S (AR-S)-stained BMSCs treated with EGFL6, showing increased mineralization (rust-colored deposits). Osteogenic differentiation of BMSCs was examined on day 14. c Images of alkaline phosphatase (ALP)-stained BMSCs treated with EGFL6. Osteogenic differentiation of BMSCs was examined on day 3. Insets in b and c show low-magnification images of entire culture well. Scale bars, 250 μm. BMSCs were treated with different concentrations of EGFL6 for 5 or 10 days. d Expression levels of angiogenesis- and osteogenesis-related markers in BMSCs following treatment with/without EGFL6 for 5 days, as evaluated by RT-PCR. The housekeeping gene GAPDH served as an internal control. e Western blots of lysates from cultured BMSCs treated with/without EGFL6 for 5 or 10 days. Blots were probed with antibodies against different markers for angiogenesis (VEGF-A), osteogenesis (BMP2, CXCR4, RUNX2), and the Wnt/b-catenin signaling pathway (b-catenin, pb-catenin, active β-catenin, and pGSK3β). f Quantitation of expression of angiogenesis-, osteogenesis-, and pathway-related marker proteins in panel e . Significant differences among groups were determined by one-way ANOVA and post hoc Dunnett’s test; * p < 0.05; ** p < 0.01; and *** p < 0.001

Article Snippet: Recombinant EGFL6 protein was purchased from R&D Systems (Cat no.8638-EG-050, R&D Systems Inc., Minneapolis, MN, USA).

Techniques: In Vitro, CCK-8 Assay, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Cell Culture, Quantitation Assay, Marker

Journal: Stem Cell Research & Therapy

Article Title: EGFL6 regulates angiogenesis and osteogenesis in distraction osteogenesis via Wnt/β-catenin signaling

doi: 10.1186/s13287-021-02487-3

Figure Lengend Snippet: EGFL6 treatment enhances osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro . a, c, e Immunofluorescent images of EGFL6-treated BMSCs stained for the osteogenic-specific protein RUNX2 ( a ), and pathway-specific protein β-catenin ( c ) and active β-catenin ( e ). Cells were counterstained with the nuclear stain DAPI (blue) and the cytoskeleton stain phalloidin (red). Scale bars, 100 μm. b, d, f Quantitation of mean relative levels of RUNX2 ( b ), β-catenin ( d ), and active β-catenin ( f ) in BMSCs treated with EGFL6 (200 ng/ml). Significant differences between experimental and control groups were evaluated by Student t tests; * p < 0.05; ** p < 0.01; and *** p < 0.001

Article Snippet: Recombinant EGFL6 protein was purchased from R&D Systems (Cat no.8638-EG-050, R&D Systems Inc., Minneapolis, MN, USA).

Techniques: In Vitro, Staining, Quantitation Assay, Control

Journal: Stem Cell Research & Therapy

Article Title: EGFL6 regulates angiogenesis and osteogenesis in distraction osteogenesis via Wnt/β-catenin signaling

doi: 10.1186/s13287-021-02487-3

Figure Lengend Snippet: Dickkopf-related protein 1 (DKK1) partially suppresses EGFL6-enhanced BMSC osteogenesis in vitro. a Light micrographs of ALP-stained BMSC cultures on day 3 of differentiation. BMSCs were treated with 200 ng/ml EGFL6 to enhance osteogenic differentiation, and then supplemented with/without 0.3 μg/ml DKK1. Insets show low-magnification images of entire culture well. Scale bars, 250 μm. b AR-S staining of differentiated BMSCs showing mineralization (red) on day 14 after DKK1 application. Insets show low-magnification images of entire culture well. Scale bars, 250 μm. c Western blots showing the expression of osteogenic-specific and Wnt/β-catenin signaling-related proteins in BMSCs treated with/without EGFL6 and with/without DKK1. GADPH is the loading control. d Quantitation of osteogenic-specific and Wnt/β-catenin signaling-related proteins normalized to control condition (NS; black-colored bars). e, f, g Immunofluorescent images of BMSCs stained for RUNX2 (green) or active β-catenin (green). BMSCs were cultured with 200 ng/ml EGFL6 to enhance BMSC osteogenesis, and then treated with/without 0.3 μg/ml DKK1, an antagonist of Wnt/β-catenin signaling. Scale bar, 100 μm. Quantitation of RUNX2 or active β-catenin immunofluorescent staining showing mean relative fluorescence of DKK1 + EGFL6 (blue-colored bars) and EGFL6 alone (pink-colored bars) conditions normalized to control fluorescence (no DKK1, no EGFL6; gray-colored bars). Significant differences were evaluated by one-way ANOVA and post hoc Dunnett’s tests for all panels; * p < 0.05; ** p < 0.01; and *** p < 0.001

Article Snippet: Recombinant EGFL6 protein was purchased from R&D Systems (Cat no.8638-EG-050, R&D Systems Inc., Minneapolis, MN, USA).

Techniques: In Vitro, Staining, Western Blot, Expressing, Control, Quantitation Assay, Cell Culture, Fluorescence

Journal: Stem Cell Research & Therapy

Article Title: EGFL6 regulates angiogenesis and osteogenesis in distraction osteogenesis via Wnt/β-catenin signaling

doi: 10.1186/s13287-021-02487-3

Figure Lengend Snippet: EGFL6 stimulated formation of bone after consolidation for 2 and 4 weeks in a rat tibia DO model. a–c Images of histological sections of regenerated bone obtained from the distraction zone (boxed areas in a ) of rats treated with EGFL6 or PBS (control). Sections were stained with hematoxylin and eosin (HE) ( a ), Masson trichrome stain ( b ), or Safranin O/Fast green stain ( c ) in order to indicate newly formed trabecular bone, fibrous tissue, and cartilaginous tissue, respectively, in the distracted area. Scale bars, 200 μm

Article Snippet: Recombinant EGFL6 protein was purchased from R&D Systems (Cat no.8638-EG-050, R&D Systems Inc., Minneapolis, MN, USA).

Techniques: Control, Staining

Journal: Stem Cell Research & Therapy

Article Title: EGFL6 regulates angiogenesis and osteogenesis in distraction osteogenesis via Wnt/β-catenin signaling

doi: 10.1186/s13287-021-02487-3

Figure Lengend Snippet: EGFL6 stimulated formation of bone and CD31 hi EMCN hi -positive type H vessels after consolidation for 2 and 4 weeks in a rat tibia DO model. a Sections were immunostained for osteocalcin (OCN), a hormone secreted by osteoblasts, and visualized with peroxidase-DAB. Quantitation of OCN-positive staining intensity in the distraction zone is summarized in the histograms on the right. b Sections were immunostained for VEGF-A, a key angiogenesis marker, and visualized with peroxidase-DAB. Quantitation of VEGF-A-positive staining intensity in the distraction zone after consolidation for 2 weeks is summarized in the histograms on the right. c Immunofluorescent images of regenerated bone sections obtained from the distraction zone immunostained for active β-catenin (green). The sections were counterstained with DAPI (blue), which stains nuclei of all cells. d Immunofluorescent images of regenerated bone sections obtained from the distraction zone immunostained for CD31 (red) or endomucin (EMCN, green). The sections were counterstained with DAPI (blue). Note that CD31 hi EMCN hi (yellow) vessels in EGFL6-treated rats are densely stained (arrows) compared to vessels in the controls. Scale bars for a–d , 200 μm. Significant differences were evaluated by Student t tests; * p < 0.05

Article Snippet: Recombinant EGFL6 protein was purchased from R&D Systems (Cat no.8638-EG-050, R&D Systems Inc., Minneapolis, MN, USA).

Techniques: Quantitation Assay, Staining, Marker

Journal: Stem Cell Research & Therapy

Article Title: EGFL6 regulates angiogenesis and osteogenesis in distraction osteogenesis via Wnt/β-catenin signaling

doi: 10.1186/s13287-021-02487-3

Figure Lengend Snippet: Working model of EGFL6-mediated signaling, illustrating the coupling of angiogenesis and osteogenesis in the rat DO model. During the consolidation phase of bone remodeling, type H vessels form alongside newly developing bone and extend toward the distraction gap. In the bone marrow microenvironment, multiple cell types secrete angiogenic factors to support type H vessel formation. Osteoblast-lineage cells and ECs secrete VEGF-A. EGFL6 secreted by osteoblasts enhances VEGF-A expression in ECs to promote cell migration, tube formation, and branching, which further stimulates the formation of type H vessels during early consolidation phase. As a key regulatory factor, EGFL6 also promotes osteogenic differentiation of BMSCs into osteoblast-lineage cells, activated by the Wnt/β-catenin signaling pathway. EGFL6 also increases expression of the osteogenic proteins RUNX2, BMP2, and OCN, leading to faster restoration of the bone defect in the DO model. Abbreviations: ECs, endothelial cells; BMSCs, bone marrow mesenchymal stem cells; EGFL6, epidermal growth factor-like domain-containing protein 6; VEGF-A, vascular endothelial growth factor; RUNX2, Runt-related transcription factor 2; BMP2, bone morphogenetic protein 2; OCN, osteocalcin

Article Snippet: Recombinant EGFL6 protein was purchased from R&D Systems (Cat no.8638-EG-050, R&D Systems Inc., Minneapolis, MN, USA).

Techniques: Expressing, Migration

Journal: Cell & Bioscience

Article Title: EGFL6 promotes colorectal cancer cell growth and mobility and the anti‐cancer property of anti-EGFL6 antibody

doi: 10.1186/s13578-021-00561-0

Figure Lengend Snippet: EGFL6 expresses in CRC patients and in early-developed colon carcinogenesis animal model. a EGFL6 (Epidermal growth factor-like protein 6) expression in human CRC (colorectal cancer) tissue with indicated stages under 100X magnification. EGFL6 staining score was “++” for stage I to IV. b EGFL6 staining scoring of non-tumor part and tumor part were showed. Normal group (n = 5) and human CRC group stage I (n = 6), stage II (n = 10), stage III (n = 9), stage IV (n = 9). c AOM (Azoxymethane) mouse model schedule. Total number of 35 eight-week-old A/J mice were randomly divided into control or AOM treatment group. Mice were sacrificed and colons were collected at 1, 2, 4, 8, and 18 weeks after the last AOM challenge to assess for methylene blue, H&E (hematoxylin and eosin stain) staining, and IHC (immunohistochemistry) staining of EGFL6. d Methylene blue staining of intestinal tissue (from anus to the cecum) to observe changes in the intestines. Stained with 0.05% methylene blue. e IHC staining of intestinal tissues in different time points after AOM stimulation. Scale bar represents 50 µm. **** p < 0.0001

Article Snippet: EGFL6 recombinant protein was purchased from Sino Biological (Beijing, China).

Techniques: Animal Model, Expressing, Staining, H&E Stain, Immunohistochemistry

Journal: Cell & Bioscience

Article Title: EGFL6 promotes colorectal cancer cell growth and mobility and the anti‐cancer property of anti-EGFL6 antibody

doi: 10.1186/s13578-021-00561-0

Figure Lengend Snippet: EGFL6 promotes CRC cell proliferation. a , b Relative EGFL6 mRNA level and protein expression of FHC (human normal colon epithelial cell), HCT116 (human colorectal carcinoma epithelial cell) and HT29 (human colorectal epithelial adenocarcinoma cell). c EGFL6 expression of HCT116 culture medium presenting in western blot. CM: condition medium. d The relative viability of HCT116 (2 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} × 10 3 cell/well) and HT29 (3 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} × 10 3 cell/well) treated from various concentrations of human recombinant EGFL6 (0.1, 1 or 10 ng/mL), incubated 5 days in 96 well for SRB. e Cell proliferation signals (p-ERK (extracellular signal–regulated kinase), p-AKT (protein kinase) protein expression after EGFL6 (10 ng/mL) treatment by different time point. f EGFL6 mRNA expression of HCT-116 and HT29 after knockdown EGFL6 . Incubated 48 h after siRNA treatment. g , h Tumor cell viability as well as ERK and AKT phosphorylation of HCT-116 and HT29 after EGFL6 knockdown. Incubated 48 h after siRNA treatment. * p < 0.05, ** p < 0.01, *** p < 0.005

Article Snippet: EGFL6 recombinant protein was purchased from Sino Biological (Beijing, China).

Techniques: Expressing, Western Blot, Recombinant, Incubation

Journal: Cell & Bioscience

Article Title: EGFL6 promotes colorectal cancer cell growth and mobility and the anti‐cancer property of anti-EGFL6 antibody

doi: 10.1186/s13578-021-00561-0

Figure Lengend Snippet: EGFL6 regulates CRC cell migration and invasion. a , b Colony formation assay to test the proliferation of HCT116 and HT29 after silencing EGFL6 . c , d The migration and invasion assay of HCT116 cell after silencing EGFL6 . Scale bar represents 100 µm. e The mRNA level of EGFL6 and ADAMTS1 (a disintegrin and metalloprotease with thrombospondin motif 1 ) under normoxia and hypoxic conditions to validate the signaling relationship between EGFL6 and HIF-1α . N = normoia, H = hypoxia. f HIF-1α and EGFL6 expression under hypoxia condition. N = normoia, H = hypoxia. g The mRNA expression of invasion and migration-associated MMP ( matrix metalloproteinase )-2, MMP-9, ADAMTS1 and Snail after silencing EGFL6. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.0001

Article Snippet: EGFL6 recombinant protein was purchased from Sino Biological (Beijing, China).

Techniques: Migration, Colony Assay, Invasion Assay, Expressing

Journal: Cell & Bioscience

Article Title: EGFL6 promotes colorectal cancer cell growth and mobility and the anti‐cancer property of anti-EGFL6 antibody

doi: 10.1186/s13578-021-00561-0

Figure Lengend Snippet: EGFL6 activates EGFR and integrin signaling in CRC cells. a EGFL6-induced cell proliferation ability under SB273005 (an αvβ3 integrin inhibitor), and Erlotinib (a receptor tyrosine kinase inhibitor on EGFR (epidermal growth factor receptor) treatment in HCT116 cell line. b , c The migration, invasion and proliferation-associated protein signaling of HCT116 and HT29 after EGFL6 treatment by time and after silencing of EGFL6 . d , e The mRNA expression of cell stemness maintenance associated gene, POU5F1 (POU class 5 homeobox 1) , NANOG and LIN28 after EGFL6 silencing in HCT116 and HT29. * p < 0.05, ** p < 0.01, *** p < 0.005

Article Snippet: EGFL6 recombinant protein was purchased from Sino Biological (Beijing, China).

Techniques: Migration, Expressing

Journal: Cell & Bioscience

Article Title: EGFL6 promotes colorectal cancer cell growth and mobility and the anti‐cancer property of anti-EGFL6 antibody

doi: 10.1186/s13578-021-00561-0

Figure Lengend Snippet: In vivo efficacy evaluation of EGFL6 antibody. a 16 six-week-old nude mice were injected subcutaneously with the same volume of Matrigel, and 1 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} × 10 7 of HCT-116 cells into the right flank of each animal. The tumor volume and body weight observation in HCT-116 xenograft model treated with two groups: control (IgG, i.p, twice/week, n = 8) and EGFL6-E5-IgG (10 mg/kg, iv, twice/week, n = 8). b Eight-week-old balb/c mice (NLAC, Taipei, Taiwan) were injected with CT-26 cells (2 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} × 10 5 cell/mouse) intravenously and then divided into three groups for treatments: control (IgG, 20 mg/kg, n = 8), EGFL6-E5-IgG (20 mg/kg, n = 6) and bevacizumab (20 mg/kg, n = 6). Antibodies were given every 5 days by tail vein injection. Body weight was monitored every 2 or 3 days. Scale bar represents 1 cm. c A total of 11 seven-week-old nude mice (NLAC, Taipei, Taiwan) were used in wound healing model, 3 groups of mice treated with Control IgG (20 mg/kg, n = 5), EGFL6-E5-IgG (20 mg/kg, n = 5), and bevacizumab (20 mg/kg, n = 5). Treatments were given intravenously for a consecutive five-on-two-off regimen. Observed for 1 week until Control healed. d A total of twelve nude mice (NLAC, Taipei, Taiwan) were divided into four groups. For basal group (n = 2), mice were injected with 500 µl matrigel subcutaneously. For control group (n = 5), mice were injected with 500 µl matrigel subcutaneously mixed with endothelial growth factor (EGF, 150 ng/mL) and heparin (10 µl). For indicated antibody group (n = 5), mice were injected with 500 µl matrigel subcutaneously mixed with endothelial growth factor (EGF, 150 ng/mL), heparin (10 µl), and treated anti-EGFL6 antibody (15 mg/kg, iv, q5D). * p < 0.05, ** p < 0.01

Article Snippet: EGFL6 recombinant protein was purchased from Sino Biological (Beijing, China).

Techniques: In Vivo, Injection

Journal: Cell & Bioscience

Article Title: EGFL6 promotes colorectal cancer cell growth and mobility and the anti‐cancer property of anti-EGFL6 antibody

doi: 10.1186/s13578-021-00561-0

Figure Lengend Snippet: k on and k off rate constants of E5 IgG targeting to EGFL6

Article Snippet: EGFL6 recombinant protein was purchased from Sino Biological (Beijing, China).

Techniques:

Journal: Cell & Bioscience

Article Title: EGFL6 promotes colorectal cancer cell growth and mobility and the anti‐cancer property of anti-EGFL6 antibody

doi: 10.1186/s13578-021-00561-0

Figure Lengend Snippet: Association of EGFL6 expression and clinical parameters in tumor tissues of CRC patients

Article Snippet: EGFL6 recombinant protein was purchased from Sino Biological (Beijing, China).

Techniques: Expressing

Journal: Cell reports

Article Title: Differential effects of EGFL6 on tumor versus wound angiogenesis

doi: 10.1016/j.celrep.2017.11.020

Figure Lengend Snippet: a, Human normal ovary, ovarian tumor, and healing wound tissues were dissociated, and isolated endothelial cells and samples were processed for microarray. b, Gene microarray of endothelial cells from normal ovary, healing-wound tissue, and ovarian tumor–associated endothelial cells. c, Expression of EGFL6 in human normal ovary, wound, and ovarian tumor samples. Representative images were taken from different samples. Scale bar =100 µm d, Validation of gene microarray data using q-PCR.(Normal ovary; n = 5, Ovarian tumor; n = 10, Wound; n = 7). Validation Error bars indicates SEM. *p<0.05 vs. Normal. See also Figure S1.

Article Snippet: The human EGFL6 recombinant protein was obtained from Sinobiological (Beijing, China).

Techniques: Isolation, Microarray, Expressing

Journal: Cell reports

Article Title: Differential effects of EGFL6 on tumor versus wound angiogenesis

doi: 10.1016/j.celrep.2017.11.020

Figure Lengend Snippet: a, Graph of wound area on mice treated with either control IgG antibody or DC101 (anti-VEGFR2) quantified on days 0 through 15 after a skin excision wound. (n=10 mice per group); error bars indicate SEM. *p<0.05 vs. Control IgG. b, One day after SKOV3ip1 tumor cell injection, a wound was created on the dorsal side of the mice. Animals were treated with either Control siRNA-CH or mEGFL6 siRNA-CH. Graphical depiction of wound areas quantified on days 0 through 15 after skin excision wound. c, Effect of mEGFL6 silencing on tumor growth; representative images of tumor burden. Tumor weights are shown in d and numbers of tumor nodules in e. (n=10 mice per group); error bars indicate SEM. *p<0.05 vs. Control siRNA. f, Hind limb ischemia. After arterial ligation, mice were separated into 3 groups (n = 5 mice per group): normal, ischemia-24 h, and 96 h. Blood flow was monitored before and after femoral artery ligation with use of serial color doppler. At each time point, tissue was harvested and frozen so that immunofluorescence staining could be performed. g, EGFL6 expression was increased in endothelial cells in the ischemic (hypoxic) condition compared with the normal conditions. Error bars indicate SEM. *p<0.05 vs. Normal. See also Figure S2.

Article Snippet: The human EGFL6 recombinant protein was obtained from Sinobiological (Beijing, China).

Techniques: Injection, Ligation, Immunofluorescence, Staining, Expressing

Journal: Cell reports

Article Title: Differential effects of EGFL6 on tumor versus wound angiogenesis

doi: 10.1016/j.celrep.2017.11.020

Figure Lengend Snippet: a, EGFL6 promoter reporter analysis under normoxia and hypoxic conditions. b, TWIST1 ectopic expression increases EGFL6 transcription activity. c, Increase in TWIST1 and EGFL6 expression in hypoxia and CoCl2 treatment. d, Ectopic expression of TWIST1 increases EGFL6 expression in RF24 cells. e, ChIP analysis of TWIST1 binding to EGFL6 promoter region in hypoxia compared with normoxia. Cross-linked chromatin from RF24 cells incubated in hypoxia chamber for 48 h and immunoprecipitated with TWIST1 or IgG control antibodies. The input and immunoprecipitated DNA was subjected to PCR using primers corresponding to the base pairs upstream of EGFL6 transcription start site. f, EGFL6 gene silencing using siRNA leads to increased cell death in hypoxia condition. (n = 3) **p<0.005, *p<0.05 See also Figure S3.

Article Snippet: The human EGFL6 recombinant protein was obtained from Sinobiological (Beijing, China).

Techniques: Expressing, Activity Assay, Binding Assay, Incubation, Immunoprecipitation

Journal: Cell reports

Article Title: Differential effects of EGFL6 on tumor versus wound angiogenesis

doi: 10.1016/j.celrep.2017.11.020

Figure Lengend Snippet: a, Expression level change in selected proteins after normalization by RPPA analysis. b and c, Western blotting of EGFL6-mediated activation of PI3K/AKT signaling, Tie2 and EGFR signaling. d, RF24 cells treated with Control (PBS) or EGFL6. Extracts were subjected to anti-Tie2 immunoprecipitation (IP) and integrin α5, αV, β1, and β3 detected by immunoblotting. e, Expression level of pTie2 and pAKT in cytosol and membrane fractions with Control (PBS) and EGFL6 treatment. αβ-tubulin was used as internal control of cytosolic fraction; NaK ATPase was used as membrane marker. f, Silencing of Integrin β1 (ITGB1) and Tie2 using specific siRNAs decreases Tie2 and AKT signaling. g–h, Silencing of Integrin β1 (ITGB1) and Tie2 decreases EGFL6-mediated tube formation (g) and migration (h) in endothelial cells. *p<0.05 vs. Control siRNA. In i–k, RGD blocking peptide decreases Tie2/AKT activation (i), tube formation (j) and migration (k). (n=3) *p<0.05 vs. Control. See also Figure S4.

Article Snippet: The human EGFL6 recombinant protein was obtained from Sinobiological (Beijing, China).

Techniques: Expressing, Western Blot, Activation Assay, Immunoprecipitation, Marker, Migration, Blocking Assay

Journal: Cell reports

Article Title: Differential effects of EGFL6 on tumor versus wound angiogenesis

doi: 10.1016/j.celrep.2017.11.020

Figure Lengend Snippet: a, The binding affinity of recombinant EGFL6 to monoclonal antibody #93 and #135 was measured by Biacore. The dissociation constant (Kd) value of the monoclonal antibodies were calculated to be 1.89 nM (#93) and 2.19 nM (#135). b, Effect of EGFL6 blocking antibodies on Tie2/AKT activation in RF24 cells (n=3). c, Effect of EGFL6 blocking antibodies on wound healing in vivo (n=5 mice/group, #135; 5 mg/kg). d and e, EGFL6 antibodies decreased tube formation and migration of RF24 cells. f, Effect of EGFL6 blocking antibodies on tumor weight and tumor nodules in SKOV3ip1 tumor-bearing mice. Seven days after tumor cell injection, mice were randomly divided into three groups (10 mice/group) to receive therapy: (1) Control IgG Ab, (2) EGFL6 #93, and (3) EGFL6 Ab #135 (5 mg/kg). Antibody treatment was given once a week. g, Effect of targeted EGFL6 on proliferation (Ki67) and microvessel density (CD31). Scale bar = 100µm. The bars in the graphs correspond sequentially to the labeled columns of images on the left. Error bars indicates SEM. *p<0.05 vs. Control IgG. See also Figure S5.

Article Snippet: The human EGFL6 recombinant protein was obtained from Sinobiological (Beijing, China).

Techniques: Binding Assay, Recombinant, Blocking Assay, Activation Assay, In Vivo, Migration, Injection, Labeling

Journal: Cell reports

Article Title: Differential effects of EGFL6 on tumor versus wound angiogenesis

doi: 10.1016/j.celrep.2017.11.020

Figure Lengend Snippet: a, Tumor growth in Tie2;EGFL6 KO mice (KO) and WT mice. ID8 murine ovarian cancer cells were injected into KO and WT mice (n=10 mice per group). b, Double immunofluorescence staining of CD31 and EGFL6 in ID8 tumor from WT and KO mice. Scale bar = 100 µm. c, Bar graph represents tumor weight. d and e, Proliferation (Ki67) and microvessel density (CD31) counted in WT and KO mice tumor sections. Error bars indicate SEM. Scale bar = 100 µm. *p<0.05 vs. WT mice. f, Representative images of human ovarian cancer vasculature with low or high immunohistochemical staining for EGFL6. Scale bar =200 µm. Representative images were taken from different samples. g, Kaplan-Meier curves of disease-specific mortality of patients whose ovarian vasculature expressed low versus high EGFL6. See also Figure S6 and Table S1.

Article Snippet: The human EGFL6 recombinant protein was obtained from Sinobiological (Beijing, China).

Techniques: Injection, Double Immunofluorescence Staining, Immunohistochemical staining, Staining