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
![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](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2215/pmc07962215/pmc07962215__13578_2021_561_Fig2_HTML.jpg)
![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](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2215/pmc07962215/pmc07962215__13578_2021_561_Fig5_HTML.jpg)
