Journal:

Article Title: ER71 acts downstream of BMP, Notch and Wnt signaling in blood and vessel progenitor specification

doi: 10.1016/j.stem.2008.03.008

Figure Lengend Snippet: ES cells were differentiated in serum (FCS), inhibitors were added on day 1, and day 3 EB cells were analyzed for FLK1 expression (A) and subjected to gene expression (B–D). *P<0.05 versus FCS. Genes were normalized against Gapdh and then the ratio of the gene quantity (FCS) to gene quantity (FCS + inhibitors) was determined to yield fold change shown on the Y-axis.

Article Snippet: Antibodies used were anti- PECAM1 (BD), anti-FLK1 (BD) and HRP-conjugated goat anti-rat IgG (Jackson ImmunoResearch).

Techniques: Expressing

Journal:

Article Title: ER71 acts downstream of BMP, Notch and Wnt signaling in blood and vessel progenitor specification

doi: 10.1016/j.stem.2008.03.008

Figure Lengend Snippet: (A) Schematic diagram of the inducible ER71 (iER71) ES cells used, with indicated loci carrying alterations allowing for production of the rtTA, expression of the Er71 cDNA and generation of hCD4 as a surrogate marker for Scl. (B, C) Er71 induction upon Dox treatment and the ER71-mediated transcriptional program. iER71 ES cells were differentiated for 1 day in serum or for 2 days in SR and then treated with 1 μg/ml Dox for an additional 2 days. RNA was prepared and used for qRT-PCR. Genes were normalized against Gapdh and the ratio of the gene quantity (+Dox) to gene quantity (−Dox) was determined to yield normalized fold change. (D) iER71 ES cells were differentiated as described. BMP4 was added on day 2. The resulting cells were FACS analyzed for FLK1. Numbers in insets indicate the percentage of FLK1+ cells. (E) iER71 ES cells were differentiated in serum as described. Noggin, DAPT and DKK1, singularly or in combination, were added on day 1 of differentiation. Dox was added on day 1 and FLK1+ cells were analyzed on day 3. Numbers in insets indicate the percentage of FLK1+ cells. (F) (upper) Schematic diagram of the Flk1 promoter used for luciferase assay. Diamonds indicate potential Ets binding sites (red in sense and blue in anti-sense). For mutagenesis, potential Ets sites GGAA/T (sense) and A/TTCC (anti-sense) were mutated to TTAA/T and A/TTTT, respectively. (lower) 293T cells were transfected with pGL3 or pGL3-Flk1 promoter-luciferase reporter plasmid with or without pCS3-Myc-Er71(WT or MT). Fire fly luciferase activity was normalized by Renilla luciferase activity. (G) iER71 ES cells differentiated in serum in the presence of Dox were cross-linked, sonicated and subjected to ChIP assay. Numbers on the X-axis indicate the locations of amplicons of each qPCR primer set on Flk1. The value normalized against IgG IP values is shown on the Y-axis.

Article Snippet: Antibodies used were anti- PECAM1 (BD), anti-FLK1 (BD) and HRP-conjugated goat anti-rat IgG (Jackson ImmunoResearch).

Techniques: Expressing, Marker, Quantitative RT-PCR, Luciferase, Binding Assay, Mutagenesis, Transfection, Plasmid Preparation, Activity Assay, Sonication

Journal:

Article Title: ER71 acts downstream of BMP, Notch and Wnt signaling in blood and vessel progenitor specification

doi: 10.1016/j.stem.2008.03.008

Figure Lengend Snippet: (A) iER71 ES cells were differentiated in SR or serum and Dox (1 μg/ml) was added on day 3. BMP4 and VEGFA165 were added on day 2 and day 3, respectively. In all cases, cells were FACS analyzed for FLK1, hCD4 or VE-Cadherin (VE-Cad) expression on day 5 (FCS) or day 6 (SR). (left panels) Numbers in a given box indicate the percentage of cells that are FLK1−hCD4− (lower left) FLK1+hCD4− (lower right), FLK1+hCD4+ (upper right), or FLK1−hCD4+ (upper left). (right panels) Numbers in a given box indicate the percentage of VE-Cad+ cells. (B) iER71 cells differentiated for 6 days in serum free conditions with indicated factors were replated in methylcellulose medium containing hematopoietic cytokines. Colonies were counted 5–7 days after replating. EB cells generated in SR alone gave rise to few erythoid colonies (average=10.7) with no Mac or E/M colonies. Error bars indicate standard deviations from three independent experiments. Ery; Erythrocytes, Mac; Macrophages, E/M; Erythroid/Macrophage colonies. (C) Transient expression of ER71 increases the generation of primitive erythroid progenitors. Dox was added to the culture at the indicated day and kept until day 4 or washed out (WO) the next day. On day 4, iER71 EB cells were replated for primitive erythroid colonies. Colonies were counted 4 days later. Data represent four independent experiments. (D) Hematopoietic and endothelial cell development upon ER71 overexpression using embryo-derived cell culture. Pooled embryos between E8.0–E8.5 were dissociated into single cells and infected with MSCV or MSCV-ER71-IRES-GFP virus. Subsequently, cells cultured on OP9 cells were subjected to hematopoietic colony replating (upper, values are mean±s.e.m. from two independent experiments) or PECAM1 staining (lower, representative images are shown).

Article Snippet: Antibodies used were anti- PECAM1 (BD), anti-FLK1 (BD) and HRP-conjugated goat anti-rat IgG (Jackson ImmunoResearch).

Techniques: Expressing, Generated, Over Expression, Derivative Assay, Cell Culture, Infection, Staining

Journal:

Article Title: ER71 acts downstream of BMP, Notch and Wnt signaling in blood and vessel progenitor specification

doi: 10.1016/j.stem.2008.03.008

Figure Lengend Snippet: (A) (left) Benzidine staining. Scale bars; 200 μm. (right) Hematopoietic progenitor assay. Cells prepared from E8.5 YS were replated into methycellulose containing hematopoietic cytokines. (B) FLK1 expression in E7.5 embryos. FLK1 is expressed in primitive streak (PS), blood islands (Bl), allantois (Al) and amnion (Am) in the wild type but not in the mutants. Scale bars; 50 μm. (C) FLK1 expression in E8.5 embryos. (upper) Whole mount FLK1 staining of the embryo proper. Scale bars; 200 μm. (lower) Immunofluorescent FLK1 (green) and PECAM1 (red) staining of the yolk sac. Nuclei were stained with DAPI (blue). Em, embryo proper. Scale bars; 50 μm. (D) Whole-mount PECAM1 or FLK1 staining of E9.5 embryos. The boxed area is the vasculature of the yolk sac, brain, heart and intersomitic regions at higher magnification. Scale bars; 200 μm.

Article Snippet: Antibodies used were anti- PECAM1 (BD), anti-FLK1 (BD) and HRP-conjugated goat anti-rat IgG (Jackson ImmunoResearch).

Techniques: Staining, Expressing

Journal: BMC Cancer

Article Title: 3- O -Acetyloleanolic acid inhibits VEGF-A-induced lymphangiogenesis and lymph node metastasis in an oral cancer sentinel lymph node animal model

doi: 10.1186/s12885-018-4630-0

Figure Lengend Snippet: Effects of 3AOA on expressions of VEGFR-1 and VEGFR-2, and activation of VEGFR-1, VEGFR-2 and lymphangiogenesis related downstream signaling factors in rhVEGF-A-treated HLMECs. a-b Expression levels of VEGFR-1 and -2 proteins were determined using Western blot analysis. Amounts of VEGFR-1 and -2 obtained in three independent experiments were quantified and represented as a bar diagram. Levels of VEGFR-1 and -2 in 3AOA- and rhVEGF-A-untreated cells were estimated as 100%. c-d , Cell lysates were immunoprecipitated with anti-phospho-Tyr (anti-p-Tyr). The level of phosphorylated VEGFR-1 and -2 in immunoprecipitates was detected using Western blot analysis with anti-VEGFR-1 and anti-VEGFR-2. Phosphorylation levels of VEGFR-1 and -2 obtained in three independent experiments were quantified and represented as a bar diagram. Phosphorylation levels of VEGFR-1 and -2 in 3AOA- and rhVEGF-A-untreated cells were estimated as 100%. e , HLMECs were serum starved for 6 h, then were treated with different concentrations of 3AOA (0, 2.5, 5 μM) in the presence of rhVEGF-A (20 ng/mL) for 60 min. The phosphorylation levels of FAK, PI3K, AKT, and ERK1/2 were determined using Western blot analysis with anti-p-FAK, anti-p-PI3K, anti-p-AKT, and anti-p-ERK1/2. Data are presented as a mean ± S.D. of three independent experiments ( * p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Immunoprecipitated proteins were subjected to SDS-PAGE (6%) and Western blotting using mouse anti-VEGFR-1 and goat anti-VEGFR-2 antibodies (Santa Cruz Biotech.

Techniques: Activation Assay, Expressing, Western Blot, Immunoprecipitation

Journal: Journal of Angiogenesis Research

Article Title: A role for Egfl7 during endothelial organization in the embryoid body model system

doi: 10.1186/2040-2384-2-4

Figure Lengend Snippet: Effect of Egfl7 knock-down on in vitro endothelial development . Cryosections of EBs at day 7 (a, b, f, g, k, l, p, q) and day 14 (c, d, h, i, m, n, r, s) were subjected to indirect IF using antibodies against CD31 plus collagen IV (a-d), CD31 plus VE-cadherin (f-i), CD31 plus claudin 5 (k-n), or CD31 plus Flk1 (p-s). Magnification used; (a-d, f-i, k-n) inserts show whole EBs at 20×, and large panels show detail at 63×, (p-s) panels show EBs at 20×, (e, j, o, t) panels show whole EB IgG controls at 20× (arrows, CD31+ cords; arrowheads, CD31+ sheets). Images were acquired using a confocal laser microscope (Leica Microsystems; a-o) or an Axioplan 2 imaging microscope (Carl Zeiss; p-t).

Article Snippet: Sequential double-staining was carried out with the anti-CD31 antibody first, and antibodies were used as follows; rat anti-mouse CD31, 5 μg/ml (BD Biosciences), goat anti-mouse Flk1, 4 μg/ml (Santa Cruz), rabbit anti-mouse Collagen IV, 5 μg/ml (Chemicon), goat anti-mouse VE-Cadherin, 5 μg/ml (R&D Systems), rabbit anti-mouse Claudin-5, 2.5 μg/ml (Invitrogen), rabbit anti-mouse Ki67, 1.5 μg/ml (Abcam), rabbit anti-mouse Annexin-V, 2.5 μg/ml (Abcam).

Techniques: Knockdown, In Vitro, Microscopy, Imaging