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  • huvec  (Lonza)
    99
    Lonza huvec
    CD40 ligation upregulates membrane CX3CL1 in <t>HAEC</t> and <t>HUVEC.</t> (A–D) HAEC were transduced with MIEG3-based retroviral vector that encode EGFP or EGFP plus wt CD40. CX3CL1 expression was analyzed on transduced (EGFP + ) cells. (A) Histograms show CX3CL1 expression on HAEC transduced with wt CD40-encoding retroviral vector at 24 h post-incubation with or without multimeric CD154. (B) Expression of CX3CL1 (cMFI) on gated EGFP + cells after incubation with or without multimeric CD154 or TNF-α. (C) Transduced HAEC that express wt CD40 were incubated with multimeric CD154 with either anti-CD154 or isotype control mAb. (D) Transduced HAEC that express wt CD40 were incubated with recombinant CD154 with or without enhancer used to crosslink CD154. (E) HUVEC were incubated with or without multimeric CD154. Results are shown as mean ± SEM and are representative of 3–5 experiments. ** p
    Huvec, supplied by Lonza, used in various techniques. Bioz Stars score: 99/100, based on 1586 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC human embryonic kidney hek 293 cells
    A, Overexpression of GFP and GFP-CaSR transiently transfected in <t>HEK-293</t> cells. The cells were collected 24 hours after transfection. The lysates were assessed using Western blots and an antibody against GFP ( left panel ) or CaSR ( right panel ). GAPDH was
    Human Embryonic Kidney Hek 293 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1465 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Frontier Scientific human frontier science program
    A, Overexpression of GFP and GFP-CaSR transiently transfected in <t>HEK-293</t> cells. The cells were collected 24 hours after transfection. The lysates were assessed using Western blots and an antibody against GFP ( left panel ) or CaSR ( right panel ). GAPDH was
    Human Frontier Science Program, supplied by Frontier Scientific, used in various techniques. Bioz Stars score: 92/100, based on 4656 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore egf
    <t>EGF</t> induces heparanase nucleolar localization in human BMBC cells A. and B. ). Human BMBC (231BR3) cells were serum starved for 16 hr at <t>37°C,</t> cell nucleoli were isolated as described in Materials and Methods, Nucleoli were analyzed following stimulation with EGF (0 – 150 ng/ml) for 30 min (A.), or EGF treatment for 0 – 120 min at 100 ng/ml (B.). C. and D. Human BMBC cells (231P and 231BR3) were not treated (control) or treated with EGF (100 ng/ml for 30 min following serum starvation for 16 hr). Next, immunofluorescent analyses were performed using confocal microscopy as described in Materials and Methods . Rabbit polyclonal HPSE antibody 1453 and mouse monoclonal antibody to HPSE were used as primary antibodies (Figures 2C and 2D respectively). They were diluted 1:1000 followed by secondary antibody Alexa Fluor 488 goat anti-rabbit IgG or anti-mouse IgG (green staining). Immunofluorescent staining was subsequently analyzed by confocal microscopy. Arrows indicate heparanase presence in nucleoli of 231BR3 cells. Red fluorescence indicates propidium iodide (P.I.) staining for cell nuclei. Images were taken at an original objective magnification of × 60. Representative images of four independent experiments are shown. E. Staining specificity for anti-HPSE polyclonal and monoclonal antibodies. Anti-rabbit (left panels) and anti-mouse (right panels) naive isotypic antibodies (IgG) were used as controls for staining specificity. Cell presence was determined by parallel P.I. staining. Immunofluorescence and confocal microscopy were subsequently performed, per above. Images were taken at an original objective magnification of × 60. F. Quantitation of HPSE nucleolar staining in 231P and 231BR3 cells treated or not treated with EGF (100 ng/ml for 30 min. at 37°C). Percentage of nucleolar HPSE staining was calculated from the number of nucleoli positive for HPSE staining compared to total nucleolar number obtained by fibrillarin staining. Bars represent measurements of ten fields for each treatment condition and from three independent experiments. ** P
    Egf, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7521 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    R&D Systems bmp 2
    Fabrication and morphogen loading of HMPs. HMPs were fabricated from heparin methacrylamide using the free radical initiators ammonium persulfate (APS) and N , N , N ′, N ′-tetramethylethane-1,2-diamine (TEMED) in a water-in-oil emulsion at 55°C. <t>BMP-2</t> was loaded onto HMPs by incubating BMP-2 and HMPs together at 4°C for 16 hours. BMP-2 binds to the sulfate groups on heparin.
    Bmp 2, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 2993 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher genome wide human snp array 6 0
    a Affymetrix Genome-Wide Human <t>SNP</t> Array 6.0 showed a deletion involving the PFH6 gene on chromosome Xq26.3 (green). The mutation was de novo, given by the normal sequence of the patient's father (purple, CN-state 1) and mother (blue, CN-state 2). Genes
    Genome Wide Human Snp Array 6 0, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5247 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc caspase 9
    Mechanisms of 1.9 nm gold nanoparticle-induced cytotoxicity. ( A ) Western blot analysis determining levels of proapoptotic proteins, including PARP and <t>caspase-9.</t> ( B ) Flow cytometry analysis using propidium iodide to quantify the proportion of sub G 1 cells, indicating loss of cell viability. ( C ) Quantification of reactive oxygen species production following exposure to medium containing 12 μM gold nanoparticles for either 1 hour or 24 hours. Untreated controls and H2O2 samples were collected along with the 24 hour samples 24 hours. Statistical significance was calculated using the two-tailed unpaired t -test, with a P value of ≤0.05 considered to be statistically significant. Abbreviation: GNP, gold nanoparticles.
    Caspase 9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 8293 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher human genome u133 plus 2 0 array
    Brain-specific expression patterns for hypomethylated genes in Rahman syndrome. Gene expression profiles in brain tissues extracted from Additional file 2 : Figure S2 (highlighted by the black square). Data are obtained from 65761 Affymetrix Human Genome <t>U133</t> Plus 2.0 arrays in Genevestigator; hierarchical clustering is performed using Pearson correlation as similarity measure and optimal-leaf ordering
    Human Genome U133 Plus 2 0 Array, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3950 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore human serum
    Brain-specific expression patterns for hypomethylated genes in Rahman syndrome. Gene expression profiles in brain tissues extracted from Additional file 2 : Figure S2 (highlighted by the black square). Data are obtained from 65761 Affymetrix Human Genome <t>U133</t> Plus 2.0 arrays in Genevestigator; hierarchical clustering is performed using Pearson correlation as similarity measure and optimal-leaf ordering
    Human Serum, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc cleaved parp
    VEGFA and FGF2 protect breast cancer cells from chemotherapy-induced cell death through <t>PI3K/AKT</t> activation and apoptosis inhibition. MCF-7 and Cal51 cells were stimulated with 30 ng/ml VEGFA or 20 ng/ml FGF2 for 72 h and then treated by doxorubicin or taxol for 48 h. ( a ) IC 50 values of doxorubicin and taxol in MCF-7 cells (upper panels) and Cal51 cells (lower panels) in the presence or absence of VEGFA/FGF2. ( b ) Annexin V/propidium iodide staining of cells determined by flow cytometry after 48-h taxol treatment (10 nM for MCF-7 and 2 nM for Cal51). Representative plots of three independent experiments are shown. Quantitative data show the average percentage of annexin V-positive cells (both in early apoptosis, lower right quadrants and late apoptosis, upper right quadrants) of three independent experiments (right panel). ( c ) Detection of cleaved <t>PARP</t> and cleaved caspase-3 by western blotting in Cal51 cell in the presence or absence of VEGFA/FGF2 after 2 nM taxol treatment for 48 h. ( d ) Caspase-3/7 and caspase-9 activities in MCF-7 and Cal51 cells after 48-h taxol treatment. ( e ) Expression levels of Bax and survivin in MCF-7 and Cal51 cells determined by qPCR in the presence or absence of VEGFA/FGF2. ( f ) Expression levels of pAKT, AKT, Bax and Survivin determined by western blotting in the presence or absence of VEGFA/FGF2. Numerical data represent mean±S.D. based on three independent experiments (* P
    Cleaved Parp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 7715 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Human Metabolome Technologies America human metabolome database hmdb
    Compound profiles of exosomes and plasma based off accurate mass database search. All significant m / z ’s for both plasma and plasma-derived exosomes were subjected to accurate mass database search through CEU-MassMediator, the Human <t>Metabolome</t> Database <t>(HMDB)</t> and Metlin. Potential identities for each m / z were screened for their biological relevance. Class for each potential identification was then pulled from the respective database and graphed as a pie chart to help visualize potential metabolite and lipid profile differences between ( A ) plasma and ( B ) exosomes.
    Human Metabolome Database Hmdb, supplied by Human Metabolome Technologies America, used in various techniques. Bioz Stars score: 94/100, based on 2294 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega human genomic dna
    Infrared gel electrophoresis images of DNase I footprinting products following incubation of compounds 1–6 with <t>TOPOIIα</t> promoter <t>DNA.</t> Compounds were incubated for 17 h with TOPOIIα promoter DNA IR-labelled on the sense (A) or antisense (B) strand. Each gel consists of 50 lanes: two GA marker lanes (GA) and six sets of eight lanes for compound (l–r; 0, 0.0016, 0.008, 0.04, 0.2, 1, 5 and 25 μM). The sequence positions relating to Supplementary Figure 1 are given vertically.
    Human Genomic Dna, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 7262 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore human albumin
    Infrared gel electrophoresis images of DNase I footprinting products following incubation of compounds 1–6 with <t>TOPOIIα</t> promoter <t>DNA.</t> Compounds were incubated for 17 h with TOPOIIα promoter DNA IR-labelled on the sense (A) or antisense (B) strand. Each gel consists of 50 lanes: two GA marker lanes (GA) and six sets of eight lanes for compound (l–r; 0, 0.0016, 0.008, 0.04, 0.2, 1, 5 and 25 μM). The sequence positions relating to Supplementary Figure 1 are given vertically.
    Human Albumin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 467 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore human igg
    Purification and characterization of recombinant extracellular <t>cadherin</t> proteins. The extracellular domains of HE-cadherin and C-cadherin were both expressed in CHO cells as a COOH-terminal fusion proteins with the Fc part of human <t>IgG.</t> Recombinant proteins were purified from the media on a Protein A column. (A) Coomassie staining of HEEC1–5Fc and CEC1–5Fc run under reducing and nonreducing conditions. (B) Immunoblot analysis of the recombinant HEEC1–5Fc and CEC1–5Fc proteins using anti–human Fc, anti-HE cadherin, or anti–C-cadherin antibodies. (C) Bead aggregation assay: protein-A–coated beads coupled to HEEC1–5Fc or CEC1–5Fc were allowed to aggregate in the presence of Ca 2+ or EDTA for the indicated time period.
    Human Igg, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4232 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Miltenyi Biotec cd14 microbeads
    Presence of PGE2 antagonizes the induction of CD16 expression mediated by TGF-β. (A–D) Monocytes were obtained using <t>CD14</t> <t>microbeads</t> and then incubated with IL-4 and GM-CSF (control) or with addition of TGF-β (concentrations as indicated), PGE2 (10 −7 M), or a combination of both. At 24 h, expression of CD14 and CD16 was analyzed by flow cytometry and classical, intermediate, and non-classical monocyte subsets were defined as shown. (A) Representative dot plots are shown. (B) Quantification of intermediate monocyte subset (mean ± SEM, n = 4). (C) Expression of CD14 in the whole monocyte population (mean ± SEM, n = 4). (D) Quantification of monocyte subsets ( n = 7). Results from individual donors are expressed as % of cells with median bar, obtained from cultures using TGF-β at 0.25 and 2.5 ng/ml. In all cases, * indicates p
    Cd14 Microbeads, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 99/100, based on 5325 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Miltenyi Biotec cd4 t cell isolation kit
    Co-localization of FcRγ-chain with the CD3ε subunit of the TCR/CD3 complex in effector <t>CD4</t> T cells
    Cd4 T Cell Isolation Kit, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 99/100, based on 7283 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Human Protein Atlas human protein atlas hpa
    Survival analysis of p62 mRNA expression according to the <t>TCGA</t> and <t>HPA</t> databases. (A) Liver cancer, (B) Lung cancer, (C) Breast cancer, (D) Cervical cancer, (E) Ovarian cancer, (F) Endometrial cancer. Purple lines represent high expression of p62 and blue lines represent low expression.
    Human Protein Atlas Hpa, supplied by Human Protein Atlas, used in various techniques. Bioz Stars score: 94/100, based on 1987 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore fgf 2
    Neurite ingrowth with bridge at 6 weeks post implantation. A) Neurofilament stain (brown) of bridge implanted with the highest <t>VEGF/FGF-2</t> protein dose. The red dotted line marks the contours of the bridge at the implant site. Scale bar: 500 µm.
    Fgf 2, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 927 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore human insulin
    Neurite ingrowth with bridge at 6 weeks post implantation. A) Neurofilament stain (brown) of bridge implanted with the highest <t>VEGF/FGF-2</t> protein dose. The red dotted line marks the contours of the bridge at the implant site. Scale bar: 500 µm.
    Human Insulin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2446 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher human genome u133a array
    Bootstrap box plots of the gene expression intensity of various internal controls . (A) The box plot results show the best 14 internal control candidates, all of which exhibited consistent expression intensity in the NHRI lung adenocarcinoma microarray dataset for each re-sampling process. Moreover, also included are 10 well-known Q-RT-PCR internal controls contained in 23 probe sets on the <t>HG-U133A</t> chip. These are shown as #1–23 in x-axis. The detailed probe set characteristics were shown in Table 1. Except ABCF1 , BHLHB2 and LAPTM4A , the gene expression intensities of top 12 internal control candidates, GAPDH , and ACTB from the Boston (B) and the Ann Arbor lung cancer datasets (C) were also compared. DDX5 : (DEAD (Asp-Glu-Ala-Asp) box polypeptide 5), PKM2 : (pyruvate kinase, muscle), BHLHB2 : (basic helix-loop-helix domain containing, class B, 2), GLO1 : (glyoxalase I), LAPTM4A : (lysosomal-associated protein transmembrane 4 alpha), SET : (SET translocation (myeloid leukemia-associated)), CLTC : (clathrin, heavy chain (Hc)), MSN : (MSN/ALK fusion; moesin/anaplastic lymphoma kinase fusion protein), ABCF1 : (ATP-binding cassette, sub-family F (GCN20), member 1), EPHB3 : (EPH receptor B3), CCL5 : (chemokine (C-C motif) ligand 5), PTPN21 : (protein tyrosine phosphatase, non-receptor type 21), DDR1 : (discoidin domain receptor family, member 1), 1–4: ACTB (actin, beta), 5–6: B2M (beta-2-microglobulin), 7–12: GAPDH (glyceraldehyde-3-phosphate dehydrogenase), 13: HMBS (hydroxymethylbilane synthase), 14: HPRT1 (hypoxanthine phosphoribosyltransferase 1), 15–19: RPL13A (ribosomal protein L13a), 20: RPL32 (ribosomal protein L32), 21: SDHA (succinate dehydrogenase complex, subunit A, flavoprotein (Fp)), 22: UBC (ubiquitin C), 23: YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide).
    Human Genome U133a Array, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 758 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti bcl 2
    Effect of delphinidin on the protein expression of (A) PARP and procaspase-3, 8 and 9 (B) Bax and <t>Bcl-2</t> in HCT116 cells. The cells treated with delphinidin (30–240 μM; 48 h) were harvested and nuclear lysate was prepared and protein was subjected to SDS–PAGE as detailed in Materials and Methods Section. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin. The immunoblot shown here are representative of three independent experiments with similar results. (C) The data were presented in the bar graphs as percentage of Bax/Bcl-2 ratio.
    Anti Bcl 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 3198 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems netrin 1
    LARP1 regulates <t>Netrin-1</t> microsomal translation. Huh7.5 cells were transfected with control and LARP1-specific siRNAs, and infected by HCV at MOI 0.1 over 4 d. Cells were lysed and analyzed by immunoblotting for LARP1 knockdown as shown in S3 Fig . LARP1 depletion decreases the expression of Netrin-1 protein in microsomes in Huh7.5 cells. LARP1, Netrin-1, and HSP60 proteins were monitored for their partitioning in the cytosolic (free polysomes) and microsome (ER membrane-bound polysomes) compartments, according to the infection status of the cells through sequential extractions followed by immunoblotting. HCV core was used as an infection control.
    Netrin 1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 98/100, based on 927 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Miltenyi Biotec pan t cell isolation kit
    Suppression of <t>Bcl-2</t> and Bcl-xL expression in Stat3-deficient T cells. (a) Splenic T cells were purified using the <t>Pan</t> T Cell Isolation Kit and an autoMACS Separator (Miltenyi Biotech Inc.). Total RNA was purified and cDNA was synthesized. Quantitiative
    Pan T Cell Isolation Kit, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 99/100, based on 4306 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    CD40 ligation upregulates membrane CX3CL1 in HAEC and HUVEC. (A–D) HAEC were transduced with MIEG3-based retroviral vector that encode EGFP or EGFP plus wt CD40. CX3CL1 expression was analyzed on transduced (EGFP + ) cells. (A) Histograms show CX3CL1 expression on HAEC transduced with wt CD40-encoding retroviral vector at 24 h post-incubation with or without multimeric CD154. (B) Expression of CX3CL1 (cMFI) on gated EGFP + cells after incubation with or without multimeric CD154 or TNF-α. (C) Transduced HAEC that express wt CD40 were incubated with multimeric CD154 with either anti-CD154 or isotype control mAb. (D) Transduced HAEC that express wt CD40 were incubated with recombinant CD154 with or without enhancer used to crosslink CD154. (E) HUVEC were incubated with or without multimeric CD154. Results are shown as mean ± SEM and are representative of 3–5 experiments. ** p

    Journal: PLoS ONE

    Article Title: CD40-TRAF Signaling Upregulates CX3CL1 and TNF-α in Human Aortic Endothelial Cells but Not in Retinal Endothelial Cells

    doi: 10.1371/journal.pone.0144133

    Figure Lengend Snippet: CD40 ligation upregulates membrane CX3CL1 in HAEC and HUVEC. (A–D) HAEC were transduced with MIEG3-based retroviral vector that encode EGFP or EGFP plus wt CD40. CX3CL1 expression was analyzed on transduced (EGFP + ) cells. (A) Histograms show CX3CL1 expression on HAEC transduced with wt CD40-encoding retroviral vector at 24 h post-incubation with or without multimeric CD154. (B) Expression of CX3CL1 (cMFI) on gated EGFP + cells after incubation with or without multimeric CD154 or TNF-α. (C) Transduced HAEC that express wt CD40 were incubated with multimeric CD154 with either anti-CD154 or isotype control mAb. (D) Transduced HAEC that express wt CD40 were incubated with recombinant CD154 with or without enhancer used to crosslink CD154. (E) HUVEC were incubated with or without multimeric CD154. Results are shown as mean ± SEM and are representative of 3–5 experiments. ** p

    Article Snippet: Cells and in vitro stimulation Primary HAEC and HUVEC obtained from Lonza (Allendale, NJ) and A.T.C.C. (Manassas, VA) were cultured following supplier’s recommendations.

    Techniques: Ligation, Transduction, Plasmid Preparation, Expressing, Incubation, Recombinant

    CD40 ligation stimulates CX3CL1 secretion by HAEC and HUVEC. (A) HAEC transduced with control or wt CD40 retroviral vector were incubated with or without multimeric CD154 or TNF-α for 24 h. CX3CL1 concentrations in supernatants were determined by ELISA. (B) Transduced HAEC that express wt CD40 were incubated with multimeric CD154 with either anti-CD154 or isotype control mAb. (C) HAEC transduced with control or wt CD40 retroviral vector were incubated with recombinant CD154 with or without enhancer. (D) HUVEC were incubated with or without multimeric CD154. (E) Transduced HAEC that express wt CD40 were incubated with multimeric CD154 with or without the MMP inhibitor GM 6001. CX3CL1 concentrations in supernatants were determined by ELISA. Results are shown as mean ± SEM and are representative of 3–5 experiments. *** p

    Journal: PLoS ONE

    Article Title: CD40-TRAF Signaling Upregulates CX3CL1 and TNF-α in Human Aortic Endothelial Cells but Not in Retinal Endothelial Cells

    doi: 10.1371/journal.pone.0144133

    Figure Lengend Snippet: CD40 ligation stimulates CX3CL1 secretion by HAEC and HUVEC. (A) HAEC transduced with control or wt CD40 retroviral vector were incubated with or without multimeric CD154 or TNF-α for 24 h. CX3CL1 concentrations in supernatants were determined by ELISA. (B) Transduced HAEC that express wt CD40 were incubated with multimeric CD154 with either anti-CD154 or isotype control mAb. (C) HAEC transduced with control or wt CD40 retroviral vector were incubated with recombinant CD154 with or without enhancer. (D) HUVEC were incubated with or without multimeric CD154. (E) Transduced HAEC that express wt CD40 were incubated with multimeric CD154 with or without the MMP inhibitor GM 6001. CX3CL1 concentrations in supernatants were determined by ELISA. Results are shown as mean ± SEM and are representative of 3–5 experiments. *** p

    Article Snippet: Cells and in vitro stimulation Primary HAEC and HUVEC obtained from Lonza (Allendale, NJ) and A.T.C.C. (Manassas, VA) were cultured following supplier’s recommendations.

    Techniques: Ligation, Transduction, Plasmid Preparation, Incubation, Enzyme-linked Immunosorbent Assay, Recombinant

    Immunostaining of 3D, multicellular organoids compared with adult human distal lung. (A): Confocal micrograph of cross sections of 3D multicellular lung organoids with immunofluorescence for CD31 (HUVECs), vimentin (FLFs), and pro‐SPB and pro‐SPC (type II alveolar epithelial cells) and T1a (type I alveolar epithelial cells; scale bar = 100 μm). (B): Confocal micrograph of multicellular 3D lung organoids with immunofluorescence for CD31 (HUVECs) and PanCK (SAECs). FLFs were also seeded. (C): Confocal micrograph of a cross‐section of normal adult human lung with immunofluorescence for CD31 (HUVECs) and PanCK (SAECs; scale bar = 100 μm). Abbreviations: 3D, three‐dimensional; DAPI, 4′,6‐diamidino‐2‐phenylindole; FLFs, fetal lung fibroblasts; HUVECs, human umbilical vein endothelial cells; SAECs, small airway epithelial cells; SPB, Surfactant Protein B; SPC, Surfactant Protein C.

    Journal: Stem Cells Translational Medicine

    Article Title: Development of a Three‐Dimensional Bioengineering Technology to Generate Lung Tissue for Personalized Disease Modeling

    doi: 10.5966/sctm.2016-0192

    Figure Lengend Snippet: Immunostaining of 3D, multicellular organoids compared with adult human distal lung. (A): Confocal micrograph of cross sections of 3D multicellular lung organoids with immunofluorescence for CD31 (HUVECs), vimentin (FLFs), and pro‐SPB and pro‐SPC (type II alveolar epithelial cells) and T1a (type I alveolar epithelial cells; scale bar = 100 μm). (B): Confocal micrograph of multicellular 3D lung organoids with immunofluorescence for CD31 (HUVECs) and PanCK (SAECs). FLFs were also seeded. (C): Confocal micrograph of a cross‐section of normal adult human lung with immunofluorescence for CD31 (HUVECs) and PanCK (SAECs; scale bar = 100 μm). Abbreviations: 3D, three‐dimensional; DAPI, 4′,6‐diamidino‐2‐phenylindole; FLFs, fetal lung fibroblasts; HUVECs, human umbilical vein endothelial cells; SAECs, small airway epithelial cells; SPB, Surfactant Protein B; SPC, Surfactant Protein C.

    Article Snippet: Human umbilical vein endothelial cells (HUVECs) and small airway epithelial cells (SAECs) were maintained according to the manufacturer's recommendations (Lonza, Basel, Switzerland, http://www.lonza.com ) in endothelial growth medium (EGM)‐2 (Thermo Fisher) and small airway growth medium (SAGM) (Lonza), respectively.

    Techniques: Immunostaining, Immunofluorescence

    A, Overexpression of GFP and GFP-CaSR transiently transfected in HEK-293 cells. The cells were collected 24 hours after transfection. The lysates were assessed using Western blots and an antibody against GFP ( left panel ) or CaSR ( right panel ). GAPDH was

    Journal: The Journal of Clinical Endocrinology and Metabolism

    Article Title: Localization of CaSR Antagonists in CaSR-expressing Medullary Thyroid Cancer

    doi: 10.1210/jc.2013-1756

    Figure Lengend Snippet: A, Overexpression of GFP and GFP-CaSR transiently transfected in HEK-293 cells. The cells were collected 24 hours after transfection. The lysates were assessed using Western blots and an antibody against GFP ( left panel ) or CaSR ( right panel ). GAPDH was

    Article Snippet: Human embryonic kidney (HEK)-293 cells were obtained from American Type Culture Collection.

    Techniques: Over Expression, Transfection, Western Blot

    Inhibition of CaSR-dependent activation of ERK1/2 phosphorylation by Calhex 231 analogs. HEK-293 cells were transiently transfected with GFP-CaSR (A, C) or GFP DNA (B). Cells were then incubated with the indicated concentrations of Calhex 231 (isomers

    Journal: The Journal of Clinical Endocrinology and Metabolism

    Article Title: Localization of CaSR Antagonists in CaSR-expressing Medullary Thyroid Cancer

    doi: 10.1210/jc.2013-1756

    Figure Lengend Snippet: Inhibition of CaSR-dependent activation of ERK1/2 phosphorylation by Calhex 231 analogs. HEK-293 cells were transiently transfected with GFP-CaSR (A, C) or GFP DNA (B). Cells were then incubated with the indicated concentrations of Calhex 231 (isomers

    Article Snippet: Human embryonic kidney (HEK)-293 cells were obtained from American Type Culture Collection.

    Techniques: Inhibition, Activation Assay, Transfection, Incubation

    EGF induces heparanase nucleolar localization in human BMBC cells A. and B. ). Human BMBC (231BR3) cells were serum starved for 16 hr at 37°C, cell nucleoli were isolated as described in Materials and Methods, Nucleoli were analyzed following stimulation with EGF (0 – 150 ng/ml) for 30 min (A.), or EGF treatment for 0 – 120 min at 100 ng/ml (B.). C. and D. Human BMBC cells (231P and 231BR3) were not treated (control) or treated with EGF (100 ng/ml for 30 min following serum starvation for 16 hr). Next, immunofluorescent analyses were performed using confocal microscopy as described in Materials and Methods . Rabbit polyclonal HPSE antibody 1453 and mouse monoclonal antibody to HPSE were used as primary antibodies (Figures 2C and 2D respectively). They were diluted 1:1000 followed by secondary antibody Alexa Fluor 488 goat anti-rabbit IgG or anti-mouse IgG (green staining). Immunofluorescent staining was subsequently analyzed by confocal microscopy. Arrows indicate heparanase presence in nucleoli of 231BR3 cells. Red fluorescence indicates propidium iodide (P.I.) staining for cell nuclei. Images were taken at an original objective magnification of × 60. Representative images of four independent experiments are shown. E. Staining specificity for anti-HPSE polyclonal and monoclonal antibodies. Anti-rabbit (left panels) and anti-mouse (right panels) naive isotypic antibodies (IgG) were used as controls for staining specificity. Cell presence was determined by parallel P.I. staining. Immunofluorescence and confocal microscopy were subsequently performed, per above. Images were taken at an original objective magnification of × 60. F. Quantitation of HPSE nucleolar staining in 231P and 231BR3 cells treated or not treated with EGF (100 ng/ml for 30 min. at 37°C). Percentage of nucleolar HPSE staining was calculated from the number of nucleoli positive for HPSE staining compared to total nucleolar number obtained by fibrillarin staining. Bars represent measurements of ten fields for each treatment condition and from three independent experiments. ** P

    Journal: Molecular cancer research : MCR

    Article Title: EGF-induced Heparanase Nucleolar Localization Augments DNA Topoisomerase I Activity in Brain Metastatic Breast Cancer

    doi: 10.1158/1541-7786.MCR-09-0375

    Figure Lengend Snippet: EGF induces heparanase nucleolar localization in human BMBC cells A. and B. ). Human BMBC (231BR3) cells were serum starved for 16 hr at 37°C, cell nucleoli were isolated as described in Materials and Methods, Nucleoli were analyzed following stimulation with EGF (0 – 150 ng/ml) for 30 min (A.), or EGF treatment for 0 – 120 min at 100 ng/ml (B.). C. and D. Human BMBC cells (231P and 231BR3) were not treated (control) or treated with EGF (100 ng/ml for 30 min following serum starvation for 16 hr). Next, immunofluorescent analyses were performed using confocal microscopy as described in Materials and Methods . Rabbit polyclonal HPSE antibody 1453 and mouse monoclonal antibody to HPSE were used as primary antibodies (Figures 2C and 2D respectively). They were diluted 1:1000 followed by secondary antibody Alexa Fluor 488 goat anti-rabbit IgG or anti-mouse IgG (green staining). Immunofluorescent staining was subsequently analyzed by confocal microscopy. Arrows indicate heparanase presence in nucleoli of 231BR3 cells. Red fluorescence indicates propidium iodide (P.I.) staining for cell nuclei. Images were taken at an original objective magnification of × 60. Representative images of four independent experiments are shown. E. Staining specificity for anti-HPSE polyclonal and monoclonal antibodies. Anti-rabbit (left panels) and anti-mouse (right panels) naive isotypic antibodies (IgG) were used as controls for staining specificity. Cell presence was determined by parallel P.I. staining. Immunofluorescence and confocal microscopy were subsequently performed, per above. Images were taken at an original objective magnification of × 60. F. Quantitation of HPSE nucleolar staining in 231P and 231BR3 cells treated or not treated with EGF (100 ng/ml for 30 min. at 37°C). Percentage of nucleolar HPSE staining was calculated from the number of nucleoli positive for HPSE staining compared to total nucleolar number obtained by fibrillarin staining. Bars represent measurements of ten fields for each treatment condition and from three independent experiments. ** P

    Article Snippet: Cells were serum-starved overnight (16 hrs) at 37°C, then stimulated with EGF (E) (Sigma) or serum-free medium only (S) as indicated in figures legends before being lysed using lysis buffer (Cell Signaling Technology, Danvers, MA).

    Techniques: Isolation, Confocal Microscopy, Staining, Fluorescence, Immunofluorescence, Quantitation Assay

    Effects of recombinant human HPSE on Topo I activity in 231BR3 nucleoli Representative experiments where recombinant human active HPSE (rhaHPSE) ( A. ) and inactive HPSE (rhiHPSE) ( B. ) were added to nucleoli isolated from 231BR3 cells (no EGF treatment) at indicated concentrations (0, 10, and 100 ng/ml), following incubation for 30 min at 37°C with supercoiled DNA (100 ng) provided by the TopoGen kit. Topo I activity was examined by 1% agarose gel electrophoresis with ethidium bromide and visualized using UV transilluminator, and determined by DNA status. Relaxed DNA (Rel) indicates Topo I activity while supercoiled DNA (Sc) indicates a lack of Topo I activity. Rel and Sc DNA were used as positive/negative marker controls, respectively. Lower panels were prepared upon densitometric analyses from three independent experiments. Bars represent means plus SD measurements from three independent experiments. * P

    Journal: Molecular cancer research : MCR

    Article Title: EGF-induced Heparanase Nucleolar Localization Augments DNA Topoisomerase I Activity in Brain Metastatic Breast Cancer

    doi: 10.1158/1541-7786.MCR-09-0375

    Figure Lengend Snippet: Effects of recombinant human HPSE on Topo I activity in 231BR3 nucleoli Representative experiments where recombinant human active HPSE (rhaHPSE) ( A. ) and inactive HPSE (rhiHPSE) ( B. ) were added to nucleoli isolated from 231BR3 cells (no EGF treatment) at indicated concentrations (0, 10, and 100 ng/ml), following incubation for 30 min at 37°C with supercoiled DNA (100 ng) provided by the TopoGen kit. Topo I activity was examined by 1% agarose gel electrophoresis with ethidium bromide and visualized using UV transilluminator, and determined by DNA status. Relaxed DNA (Rel) indicates Topo I activity while supercoiled DNA (Sc) indicates a lack of Topo I activity. Rel and Sc DNA were used as positive/negative marker controls, respectively. Lower panels were prepared upon densitometric analyses from three independent experiments. Bars represent means plus SD measurements from three independent experiments. * P

    Article Snippet: Cells were serum-starved overnight (16 hrs) at 37°C, then stimulated with EGF (E) (Sigma) or serum-free medium only (S) as indicated in figures legends before being lysed using lysis buffer (Cell Signaling Technology, Danvers, MA).

    Techniques: Recombinant, Activity Assay, Isolation, Incubation, Agarose Gel Electrophoresis, Marker

    Fabrication and morphogen loading of HMPs. HMPs were fabricated from heparin methacrylamide using the free radical initiators ammonium persulfate (APS) and N , N , N ′, N ′-tetramethylethane-1,2-diamine (TEMED) in a water-in-oil emulsion at 55°C. BMP-2 was loaded onto HMPs by incubating BMP-2 and HMPs together at 4°C for 16 hours. BMP-2 binds to the sulfate groups on heparin.

    Journal: Science Advances

    Article Title: Heparin-mediated delivery of bone morphogenetic protein-2 improves spatial localization of bone regeneration

    doi: 10.1126/sciadv.aay1240

    Figure Lengend Snippet: Fabrication and morphogen loading of HMPs. HMPs were fabricated from heparin methacrylamide using the free radical initiators ammonium persulfate (APS) and N , N , N ′, N ′-tetramethylethane-1,2-diamine (TEMED) in a water-in-oil emulsion at 55°C. BMP-2 was loaded onto HMPs by incubating BMP-2 and HMPs together at 4°C for 16 hours. BMP-2 binds to the sulfate groups on heparin.

    Article Snippet: HMPs (0.1 or 1 mg) were loaded with 2.5 μg of fluorescently labeled BMP-2 for subcutaneous implantation or 30 μg of BMP-2 for implantation in femoral defects.

    Techniques:

    In silico assessment of BMP-2 release from alginate/PCL tissue-engineered constructs. ( A ) BMP-2 unbinds from HMPs at a rate of k off , rebinds to HMPs at a rate of k on , and diffuses through the alginate hydrogel and surrounding tissue at diffusion rates of D BMP, Alginate and D BMP, Tissue , respectively. ( B ) Heat map depicting the sensitivity of BMP-2 release into surrounding tissue to ±2.5-, 5-, and 10-fold changes in key parameter values. ( C ) Three-dimensional representations of BMP-2 release from tissue-engineered constructs into surrounding soft tissue at 14 days after injury. ( D ) Volume fraction analysis of BMP-2 in alginate hydrogel (red line), in surrounding tissue (blue line), and bound to HMPs (green line). Treatment groups are as follows: ( i ) 30 μg of BMP-2 + 1 mg of HMPs in alginate, ( ii ) 30 μg of BMP-2 + 0.1 of mg HMPs in alginate, ( iii ) 30 μg of BMP-2 + 0.01 mg of HMPs in alginate, and ( iv ) 30 μg of BMP-2 in alginate.

    Journal: Science Advances

    Article Title: Heparin-mediated delivery of bone morphogenetic protein-2 improves spatial localization of bone regeneration

    doi: 10.1126/sciadv.aay1240

    Figure Lengend Snippet: In silico assessment of BMP-2 release from alginate/PCL tissue-engineered constructs. ( A ) BMP-2 unbinds from HMPs at a rate of k off , rebinds to HMPs at a rate of k on , and diffuses through the alginate hydrogel and surrounding tissue at diffusion rates of D BMP, Alginate and D BMP, Tissue , respectively. ( B ) Heat map depicting the sensitivity of BMP-2 release into surrounding tissue to ±2.5-, 5-, and 10-fold changes in key parameter values. ( C ) Three-dimensional representations of BMP-2 release from tissue-engineered constructs into surrounding soft tissue at 14 days after injury. ( D ) Volume fraction analysis of BMP-2 in alginate hydrogel (red line), in surrounding tissue (blue line), and bound to HMPs (green line). Treatment groups are as follows: ( i ) 30 μg of BMP-2 + 1 mg of HMPs in alginate, ( ii ) 30 μg of BMP-2 + 0.1 of mg HMPs in alginate, ( iii ) 30 μg of BMP-2 + 0.01 mg of HMPs in alginate, and ( iv ) 30 μg of BMP-2 in alginate.

    Article Snippet: HMPs (0.1 or 1 mg) were loaded with 2.5 μg of fluorescently labeled BMP-2 for subcutaneous implantation or 30 μg of BMP-2 for implantation in femoral defects.

    Techniques: In Silico, Construct, Diffusion-based Assay

    In vivo tracking of BMP-2 released from alginate/PCL tissue-engineered constructs. ( A to D ) Longitudinal IVIS images of subcutaneously implanted constructs containing 2.5 μg of fluorescently labeled BMP-2 loaded onto 0.1 or 1 mg of HMPs at (A) day 0, (B) day 1, (C) day 4, and (D) day 7. ( E ) Quantification of fluorescence within implantation sites and fit to one-phase exponential decay curves ( R 2 = 0.88 for BMP-2, R 2 = 0.78 for BMP-2 + 0.1 mg of HMPs, and R 2 = 0.68 for BMP-2 + 1 mg of HMPs.) ( F ) Decay constants obtained from BMP-2 retention curves (* P

    Journal: Science Advances

    Article Title: Heparin-mediated delivery of bone morphogenetic protein-2 improves spatial localization of bone regeneration

    doi: 10.1126/sciadv.aay1240

    Figure Lengend Snippet: In vivo tracking of BMP-2 released from alginate/PCL tissue-engineered constructs. ( A to D ) Longitudinal IVIS images of subcutaneously implanted constructs containing 2.5 μg of fluorescently labeled BMP-2 loaded onto 0.1 or 1 mg of HMPs at (A) day 0, (B) day 1, (C) day 4, and (D) day 7. ( E ) Quantification of fluorescence within implantation sites and fit to one-phase exponential decay curves ( R 2 = 0.88 for BMP-2, R 2 = 0.78 for BMP-2 + 0.1 mg of HMPs, and R 2 = 0.68 for BMP-2 + 1 mg of HMPs.) ( F ) Decay constants obtained from BMP-2 retention curves (* P

    Article Snippet: HMPs (0.1 or 1 mg) were loaded with 2.5 μg of fluorescently labeled BMP-2 for subcutaneous implantation or 30 μg of BMP-2 for implantation in femoral defects.

    Techniques: In Vivo, Construct, Labeling, Fluorescence

    Representative radiographs and micro-CT reconstructions of femoral defects treated with alginate/PCL tissue-engineered constructs. Constructs contained 30 μg of BMP-2, 30 μg of BMP-2 + 0.1 mg of HMPs, or 30 μg of BMP-2 + 1 mg of HMPs. Radiographs were taken at 4, 8, and 12 weeks. White arrows indicate heterotopic ossification. Mineral density evaluated by micro-CT at 12 weeks is depicted in sagittal sections.

    Journal: Science Advances

    Article Title: Heparin-mediated delivery of bone morphogenetic protein-2 improves spatial localization of bone regeneration

    doi: 10.1126/sciadv.aay1240

    Figure Lengend Snippet: Representative radiographs and micro-CT reconstructions of femoral defects treated with alginate/PCL tissue-engineered constructs. Constructs contained 30 μg of BMP-2, 30 μg of BMP-2 + 0.1 mg of HMPs, or 30 μg of BMP-2 + 1 mg of HMPs. Radiographs were taken at 4, 8, and 12 weeks. White arrows indicate heterotopic ossification. Mineral density evaluated by micro-CT at 12 weeks is depicted in sagittal sections.

    Article Snippet: HMPs (0.1 or 1 mg) were loaded with 2.5 μg of fluorescently labeled BMP-2 for subcutaneous implantation or 30 μg of BMP-2 for implantation in femoral defects.

    Techniques: Micro-CT, Construct

    a Affymetrix Genome-Wide Human SNP Array 6.0 showed a deletion involving the PFH6 gene on chromosome Xq26.3 (green). The mutation was de novo, given by the normal sequence of the patient's father (purple, CN-state 1) and mother (blue, CN-state 2). Genes

    Journal: Molecular Syndromology

    Article Title: PHF6 Deletions May Cause Borjeson-Forssman-Lehmann Syndrome in Females

    doi: 10.1159/000330111

    Figure Lengend Snippet: a Affymetrix Genome-Wide Human SNP Array 6.0 showed a deletion involving the PFH6 gene on chromosome Xq26.3 (green). The mutation was de novo, given by the normal sequence of the patient's father (purple, CN-state 1) and mother (blue, CN-state 2). Genes

    Article Snippet: When high-resolution genomic oligonucleotide arrays became available (Affymetrix Genome-Wide Human SNP Array 6.0), a 14–19 kb deletion on Xq26.31 was found, with no indication of a mosaicism (fig. ).

    Techniques: Genome Wide, Mutagenesis, Sequencing

    Mechanisms of 1.9 nm gold nanoparticle-induced cytotoxicity. ( A ) Western blot analysis determining levels of proapoptotic proteins, including PARP and caspase-9. ( B ) Flow cytometry analysis using propidium iodide to quantify the proportion of sub G 1 cells, indicating loss of cell viability. ( C ) Quantification of reactive oxygen species production following exposure to medium containing 12 μM gold nanoparticles for either 1 hour or 24 hours. Untreated controls and H2O2 samples were collected along with the 24 hour samples 24 hours. Statistical significance was calculated using the two-tailed unpaired t -test, with a P value of ≤0.05 considered to be statistically significant. Abbreviation: GNP, gold nanoparticles.

    Journal: International Journal of Nanomedicine

    Article Title: Cell type-dependent uptake, localization, and cytotoxicity of 1.9 nm gold nanoparticles

    doi: 10.2147/IJN.S31751

    Figure Lengend Snippet: Mechanisms of 1.9 nm gold nanoparticle-induced cytotoxicity. ( A ) Western blot analysis determining levels of proapoptotic proteins, including PARP and caspase-9. ( B ) Flow cytometry analysis using propidium iodide to quantify the proportion of sub G 1 cells, indicating loss of cell viability. ( C ) Quantification of reactive oxygen species production following exposure to medium containing 12 μM gold nanoparticles for either 1 hour or 24 hours. Untreated controls and H2O2 samples were collected along with the 24 hour samples 24 hours. Statistical significance was calculated using the two-tailed unpaired t -test, with a P value of ≤0.05 considered to be statistically significant. Abbreviation: GNP, gold nanoparticles.

    Article Snippet: Human-specific primary antibodies used were poly(ADP-ribose)polymerase (PARP), cleaved PARP, caspase-9, cleaved caspase-9 and β-actin (Cell Signaling 9542, 9541, 9502, 9501, and Sigma A3583, respectively).

    Techniques: Western Blot, Flow Cytometry, Cytometry, Two Tailed Test

    Brain-specific expression patterns for hypomethylated genes in Rahman syndrome. Gene expression profiles in brain tissues extracted from Additional file 2 : Figure S2 (highlighted by the black square). Data are obtained from 65761 Affymetrix Human Genome U133 Plus 2.0 arrays in Genevestigator; hierarchical clustering is performed using Pearson correlation as similarity measure and optimal-leaf ordering

    Journal: Clinical Epigenetics

    Article Title: Frameshift mutations at the C-terminus of HIST1H1E result in a specific DNA hypomethylation signature

    doi: 10.1186/s13148-019-0804-0

    Figure Lengend Snippet: Brain-specific expression patterns for hypomethylated genes in Rahman syndrome. Gene expression profiles in brain tissues extracted from Additional file 2 : Figure S2 (highlighted by the black square). Data are obtained from 65761 Affymetrix Human Genome U133 Plus 2.0 arrays in Genevestigator; hierarchical clustering is performed using Pearson correlation as similarity measure and optimal-leaf ordering

    Article Snippet: Functional analysis of differentially methylated regions We analyzed the expression profiles of the DMRs-associated genes in 416 tissues/organs by means of large curated dataset of 65761 Affymetrix Human Genome U133 Plus 2.0 Array in Genevestigator V.7.3.1 tool (Nebion, Switzerland), and classified them by hierarchical clustering technique using Pearson correlation as similarity measure and optimal-leaf ordering.

    Techniques: Expressing

    VEGFA and FGF2 protect breast cancer cells from chemotherapy-induced cell death through PI3K/AKT activation and apoptosis inhibition. MCF-7 and Cal51 cells were stimulated with 30 ng/ml VEGFA or 20 ng/ml FGF2 for 72 h and then treated by doxorubicin or taxol for 48 h. ( a ) IC 50 values of doxorubicin and taxol in MCF-7 cells (upper panels) and Cal51 cells (lower panels) in the presence or absence of VEGFA/FGF2. ( b ) Annexin V/propidium iodide staining of cells determined by flow cytometry after 48-h taxol treatment (10 nM for MCF-7 and 2 nM for Cal51). Representative plots of three independent experiments are shown. Quantitative data show the average percentage of annexin V-positive cells (both in early apoptosis, lower right quadrants and late apoptosis, upper right quadrants) of three independent experiments (right panel). ( c ) Detection of cleaved PARP and cleaved caspase-3 by western blotting in Cal51 cell in the presence or absence of VEGFA/FGF2 after 2 nM taxol treatment for 48 h. ( d ) Caspase-3/7 and caspase-9 activities in MCF-7 and Cal51 cells after 48-h taxol treatment. ( e ) Expression levels of Bax and survivin in MCF-7 and Cal51 cells determined by qPCR in the presence or absence of VEGFA/FGF2. ( f ) Expression levels of pAKT, AKT, Bax and Survivin determined by western blotting in the presence or absence of VEGFA/FGF2. Numerical data represent mean±S.D. based on three independent experiments (* P

    Journal: Cell Death & Disease

    Article Title: miRNA-205 targets VEGFA and FGF2 and regulates resistance to chemotherapeutics in breast cancer

    doi: 10.1038/cddis.2016.194

    Figure Lengend Snippet: VEGFA and FGF2 protect breast cancer cells from chemotherapy-induced cell death through PI3K/AKT activation and apoptosis inhibition. MCF-7 and Cal51 cells were stimulated with 30 ng/ml VEGFA or 20 ng/ml FGF2 for 72 h and then treated by doxorubicin or taxol for 48 h. ( a ) IC 50 values of doxorubicin and taxol in MCF-7 cells (upper panels) and Cal51 cells (lower panels) in the presence or absence of VEGFA/FGF2. ( b ) Annexin V/propidium iodide staining of cells determined by flow cytometry after 48-h taxol treatment (10 nM for MCF-7 and 2 nM for Cal51). Representative plots of three independent experiments are shown. Quantitative data show the average percentage of annexin V-positive cells (both in early apoptosis, lower right quadrants and late apoptosis, upper right quadrants) of three independent experiments (right panel). ( c ) Detection of cleaved PARP and cleaved caspase-3 by western blotting in Cal51 cell in the presence or absence of VEGFA/FGF2 after 2 nM taxol treatment for 48 h. ( d ) Caspase-3/7 and caspase-9 activities in MCF-7 and Cal51 cells after 48-h taxol treatment. ( e ) Expression levels of Bax and survivin in MCF-7 and Cal51 cells determined by qPCR in the presence or absence of VEGFA/FGF2. ( f ) Expression levels of pAKT, AKT, Bax and Survivin determined by western blotting in the presence or absence of VEGFA/FGF2. Numerical data represent mean±S.D. based on three independent experiments (* P

    Article Snippet: Antibodies Antibodies for immunodetection following immunoblotting procedures were phospho-AKT (D9E), AKT1 (C73H10), Bax (D2E11), cleaved PARP (D64E10), cleaved caspase 3 (5A1E) (Cell Signalling Technology), Survivin (ab76424, Abcam, Cambridge, UK) and β -actin (sc47778, Santa Cruz Biotechnology, Dallas, TX, USA).

    Techniques: Activation Assay, Inhibition, Staining, Flow Cytometry, Cytometry, Western Blot, Expressing, Real-time Polymerase Chain Reaction

    miR-205 overexpression restores drug sensitivity and promotes apoptosis in breast cancer cells. Drug-resistant MCF-7/A02 and CALDOX cells were stably transfected with a lentivirus carrying an expression vector for miR-205, generating MCF-7/A02-miR-205 and CALDOX-miR-205 cells, respectively. Control cells, MCF-7/A02-NC and CALDOX-NC were transfected with the empty vector (pGLV2-U6-Puro). ( a ) Expression of miR-205 in transfected cells determined by qPCR and normalized to RNU6 expression. ( b ) IC 50 values of doxorubicin and taxol (3-day treatments) in transfected cells. ( c ) Effect of miR-205 on drug resistance. Cells were treated with doxorubicin (15 μ M for MCF-7/A02 and 1 μ M for CALDOX) and taxol (0.25 μ M for MCF-7/A02 and 2.5 nM for CALDOX) for 7 days, and the cells were stained with crystal violet. Dye was solubilized and the optical density at 592 nm was measured. ( d ) Transfected drug-resistant cells were treated with taxol (0.25 μ M for MCF-7/A02 and 2.5 nM for CALDOX) for 48 h. Annexin V/propidium iodide staining was detected by flow cytometry. Representative plots of three independent experiments are shown, left panels. Quantitative data show the average percentage of annexin V-positive cells (both in early apoptosis, lower right quadrant, and late apoptosis, upper right quadrant) of three independent experiments, right panels. ( e ) Caspase-3/7 and caspase-9 activities in miR-205-transfected MCF-7/A02 (upper histograms) and CALDOX cells (lower histograms) after 48-h taxol treatment. ( f ) Expression levels of cleaved PARP and cleaved caspase-3 in drug-resistant CALDOX cells were determined by western blotting after 2.5 nM taxol treatment for 48 h. ( g ) Expression levels of Bax and survivin in mir-205-transfected drug-resistant cells determined by qPCR and normalized to RPS14 expression. ( h ) Expression levels of pAKT, AKT, Bax and survivin in miR-205-transfected drug-resistant cells determined by western blotting. Numerical data represent mean±S.D. based on three independent experiments (* P

    Journal: Cell Death & Disease

    Article Title: miRNA-205 targets VEGFA and FGF2 and regulates resistance to chemotherapeutics in breast cancer

    doi: 10.1038/cddis.2016.194

    Figure Lengend Snippet: miR-205 overexpression restores drug sensitivity and promotes apoptosis in breast cancer cells. Drug-resistant MCF-7/A02 and CALDOX cells were stably transfected with a lentivirus carrying an expression vector for miR-205, generating MCF-7/A02-miR-205 and CALDOX-miR-205 cells, respectively. Control cells, MCF-7/A02-NC and CALDOX-NC were transfected with the empty vector (pGLV2-U6-Puro). ( a ) Expression of miR-205 in transfected cells determined by qPCR and normalized to RNU6 expression. ( b ) IC 50 values of doxorubicin and taxol (3-day treatments) in transfected cells. ( c ) Effect of miR-205 on drug resistance. Cells were treated with doxorubicin (15 μ M for MCF-7/A02 and 1 μ M for CALDOX) and taxol (0.25 μ M for MCF-7/A02 and 2.5 nM for CALDOX) for 7 days, and the cells were stained with crystal violet. Dye was solubilized and the optical density at 592 nm was measured. ( d ) Transfected drug-resistant cells were treated with taxol (0.25 μ M for MCF-7/A02 and 2.5 nM for CALDOX) for 48 h. Annexin V/propidium iodide staining was detected by flow cytometry. Representative plots of three independent experiments are shown, left panels. Quantitative data show the average percentage of annexin V-positive cells (both in early apoptosis, lower right quadrant, and late apoptosis, upper right quadrant) of three independent experiments, right panels. ( e ) Caspase-3/7 and caspase-9 activities in miR-205-transfected MCF-7/A02 (upper histograms) and CALDOX cells (lower histograms) after 48-h taxol treatment. ( f ) Expression levels of cleaved PARP and cleaved caspase-3 in drug-resistant CALDOX cells were determined by western blotting after 2.5 nM taxol treatment for 48 h. ( g ) Expression levels of Bax and survivin in mir-205-transfected drug-resistant cells determined by qPCR and normalized to RPS14 expression. ( h ) Expression levels of pAKT, AKT, Bax and survivin in miR-205-transfected drug-resistant cells determined by western blotting. Numerical data represent mean±S.D. based on three independent experiments (* P

    Article Snippet: Antibodies Antibodies for immunodetection following immunoblotting procedures were phospho-AKT (D9E), AKT1 (C73H10), Bax (D2E11), cleaved PARP (D64E10), cleaved caspase 3 (5A1E) (Cell Signalling Technology), Survivin (ab76424, Abcam, Cambridge, UK) and β -actin (sc47778, Santa Cruz Biotechnology, Dallas, TX, USA).

    Techniques: Over Expression, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction, Staining, Flow Cytometry, Cytometry, Western Blot

    Compound profiles of exosomes and plasma based off accurate mass database search. All significant m / z ’s for both plasma and plasma-derived exosomes were subjected to accurate mass database search through CEU-MassMediator, the Human Metabolome Database (HMDB) and Metlin. Potential identities for each m / z were screened for their biological relevance. Class for each potential identification was then pulled from the respective database and graphed as a pie chart to help visualize potential metabolite and lipid profile differences between ( A ) plasma and ( B ) exosomes.

    Journal: International Journal of Molecular Sciences

    Article Title: Plasma Derived Exosomal Biomarkers of Exposure to Ionizing Radiation in Nonhuman Primates

    doi: 10.3390/ijms19113427

    Figure Lengend Snippet: Compound profiles of exosomes and plasma based off accurate mass database search. All significant m / z ’s for both plasma and plasma-derived exosomes were subjected to accurate mass database search through CEU-MassMediator, the Human Metabolome Database (HMDB) and Metlin. Potential identities for each m / z were screened for their biological relevance. Class for each potential identification was then pulled from the respective database and graphed as a pie chart to help visualize potential metabolite and lipid profile differences between ( A ) plasma and ( B ) exosomes.

    Article Snippet: To better understand the differences between plasma and plasma-derived exosomes, we subjected the significantly dysregulated m /z ’s for each matrix to accurate mass-based database search using CEU-MassMediator, the Human Metabolome Database (HMDB) and Metlin.

    Techniques: Derivative Assay

    Infrared gel electrophoresis images of DNase I footprinting products following incubation of compounds 1–6 with TOPOIIα promoter DNA. Compounds were incubated for 17 h with TOPOIIα promoter DNA IR-labelled on the sense (A) or antisense (B) strand. Each gel consists of 50 lanes: two GA marker lanes (GA) and six sets of eight lanes for compound (l–r; 0, 0.0016, 0.008, 0.04, 0.2, 1, 5 and 25 μM). The sequence positions relating to Supplementary Figure 1 are given vertically.

    Journal: Nucleic Acids Research

    Article Title: A 96-well DNase I footprinting screen for drug-DNA interactions

    doi: 10.1093/nar/gkm467

    Figure Lengend Snippet: Infrared gel electrophoresis images of DNase I footprinting products following incubation of compounds 1–6 with TOPOIIα promoter DNA. Compounds were incubated for 17 h with TOPOIIα promoter DNA IR-labelled on the sense (A) or antisense (B) strand. Each gel consists of 50 lanes: two GA marker lanes (GA) and six sets of eight lanes for compound (l–r; 0, 0.0016, 0.008, 0.04, 0.2, 1, 5 and 25 μM). The sequence positions relating to Supplementary Figure 1 are given vertically.

    Article Snippet: DNA cloning and plasmid purification The proximal 705 bp of the human Topoisomerase II alpha (TOPOIIα) promoter was amplified by PCR from human genomic DNA (Promega) using primers TIIA-F (CACCGCACACAGCCTAC) and TIIA-R (TGGTGACGGTCGTGAAG) (Supplementary Figure 1).

    Techniques: Nucleic Acid Electrophoresis, Footprinting, Incubation, Marker, Sequencing

    A comparison of footprint profiles of compounds 1–6 on the proximal region of the TOPOIIα promoter. Single footprint profiles were program-generated starting with the IR footprint gel image shown in Figure 3 A. Where no footprinting is seen, the sequence has been given an arbitrary score of −3.9 (log 125 μM). The DNA sequence is given in Supplementary Figure 1.

    Journal: Nucleic Acids Research

    Article Title: A 96-well DNase I footprinting screen for drug-DNA interactions

    doi: 10.1093/nar/gkm467

    Figure Lengend Snippet: A comparison of footprint profiles of compounds 1–6 on the proximal region of the TOPOIIα promoter. Single footprint profiles were program-generated starting with the IR footprint gel image shown in Figure 3 A. Where no footprinting is seen, the sequence has been given an arbitrary score of −3.9 (log 125 μM). The DNA sequence is given in Supplementary Figure 1.

    Article Snippet: DNA cloning and plasmid purification The proximal 705 bp of the human Topoisomerase II alpha (TOPOIIα) promoter was amplified by PCR from human genomic DNA (Promega) using primers TIIA-F (CACCGCACACAGCCTAC) and TIIA-R (TGGTGACGGTCGTGAAG) (Supplementary Figure 1).

    Techniques: Generated, Footprinting, Sequencing

    Purification and characterization of recombinant extracellular cadherin proteins. The extracellular domains of HE-cadherin and C-cadherin were both expressed in CHO cells as a COOH-terminal fusion proteins with the Fc part of human IgG. Recombinant proteins were purified from the media on a Protein A column. (A) Coomassie staining of HEEC1–5Fc and CEC1–5Fc run under reducing and nonreducing conditions. (B) Immunoblot analysis of the recombinant HEEC1–5Fc and CEC1–5Fc proteins using anti–human Fc, anti-HE cadherin, or anti–C-cadherin antibodies. (C) Bead aggregation assay: protein-A–coated beads coupled to HEEC1–5Fc or CEC1–5Fc were allowed to aggregate in the presence of Ca 2+ or EDTA for the indicated time period.

    Journal: The Journal of Cell Biology

    Article Title: Cadherin-mediated cell sorting not determined by binding or adhesion specificity

    doi: 10.1083/jcb.200108040

    Figure Lengend Snippet: Purification and characterization of recombinant extracellular cadherin proteins. The extracellular domains of HE-cadherin and C-cadherin were both expressed in CHO cells as a COOH-terminal fusion proteins with the Fc part of human IgG. Recombinant proteins were purified from the media on a Protein A column. (A) Coomassie staining of HEEC1–5Fc and CEC1–5Fc run under reducing and nonreducing conditions. (B) Immunoblot analysis of the recombinant HEEC1–5Fc and CEC1–5Fc proteins using anti–human Fc, anti-HE cadherin, or anti–C-cadherin antibodies. (C) Bead aggregation assay: protein-A–coated beads coupled to HEEC1–5Fc or CEC1–5Fc were allowed to aggregate in the presence of Ca 2+ or EDTA for the indicated time period.

    Article Snippet: The plasmid encoding the extracellular domain of human E-cadherin fused to the Fc domain of human IgG was transfected into CHO cells and selected in 10% FCS/MEM Glasgow without glutamine and in the presence of 25 μM sulfoximine (Sigma-Aldrich).

    Techniques: Purification, Recombinant, Staining

    Presence of PGE2 antagonizes the induction of CD16 expression mediated by TGF-β. (A–D) Monocytes were obtained using CD14 microbeads and then incubated with IL-4 and GM-CSF (control) or with addition of TGF-β (concentrations as indicated), PGE2 (10 −7 M), or a combination of both. At 24 h, expression of CD14 and CD16 was analyzed by flow cytometry and classical, intermediate, and non-classical monocyte subsets were defined as shown. (A) Representative dot plots are shown. (B) Quantification of intermediate monocyte subset (mean ± SEM, n = 4). (C) Expression of CD14 in the whole monocyte population (mean ± SEM, n = 4). (D) Quantification of monocyte subsets ( n = 7). Results from individual donors are expressed as % of cells with median bar, obtained from cultures using TGF-β at 0.25 and 2.5 ng/ml. In all cases, * indicates p

    Journal: Frontiers in Immunology

    Article Title: Prostaglandin E2 Antagonizes TGF-β Actions During the Differentiation of Monocytes Into Dendritic Cells

    doi: 10.3389/fimmu.2018.01441

    Figure Lengend Snippet: Presence of PGE2 antagonizes the induction of CD16 expression mediated by TGF-β. (A–D) Monocytes were obtained using CD14 microbeads and then incubated with IL-4 and GM-CSF (control) or with addition of TGF-β (concentrations as indicated), PGE2 (10 −7 M), or a combination of both. At 24 h, expression of CD14 and CD16 was analyzed by flow cytometry and classical, intermediate, and non-classical monocyte subsets were defined as shown. (A) Representative dot plots are shown. (B) Quantification of intermediate monocyte subset (mean ± SEM, n = 4). (C) Expression of CD14 in the whole monocyte population (mean ± SEM, n = 4). (D) Quantification of monocyte subsets ( n = 7). Results from individual donors are expressed as % of cells with median bar, obtained from cultures using TGF-β at 0.25 and 2.5 ng/ml. In all cases, * indicates p

    Article Snippet: Monocytes were obtained using CD14 microbeads (Miltenyi Biotec).

    Techniques: Expressing, Incubation, Flow Cytometry, Cytometry

    Co-localization of FcRγ-chain with the CD3ε subunit of the TCR/CD3 complex in effector CD4 T cells

    Journal:

    Article Title: Proximal signaling control of human effector CD4 T cell function

    doi: 10.1016/j.clim.2007.07.002

    Figure Lengend Snippet: Co-localization of FcRγ-chain with the CD3ε subunit of the TCR/CD3 complex in effector CD4 T cells

    Article Snippet: Human CD4 T cells were purified from peripheral blood mononuclear cells (PBMC) by negative selection using the CD4 T cell isolation kit and autoMACS™ as per the manufacturer’s recommendations (Miltenyi Biotec, Auburn, CA), and subsequently depleted of CD4+ CD25+ T cells using anti-CD25 conjugated microbeads (Miltenyi Biotec), yielding > 98% pure CD4+ CD25− T cells.

    Techniques:

    Generation of effector CD4 T cells with distinct functional capacity

    Journal:

    Article Title: Proximal signaling control of human effector CD4 T cell function

    doi: 10.1016/j.clim.2007.07.002

    Figure Lengend Snippet: Generation of effector CD4 T cells with distinct functional capacity

    Article Snippet: Human CD4 T cells were purified from peripheral blood mononuclear cells (PBMC) by negative selection using the CD4 T cell isolation kit and autoMACS™ as per the manufacturer’s recommendations (Miltenyi Biotec, Auburn, CA), and subsequently depleted of CD4+ CD25+ T cells using anti-CD25 conjugated microbeads (Miltenyi Biotec), yielding > 98% pure CD4+ CD25− T cells.

    Techniques: Functional Assay

    Survival analysis of p62 mRNA expression according to the TCGA and HPA databases. (A) Liver cancer, (B) Lung cancer, (C) Breast cancer, (D) Cervical cancer, (E) Ovarian cancer, (F) Endometrial cancer. Purple lines represent high expression of p62 and blue lines represent low expression.

    Journal: Journal of Cancer

    Article Title: Cytoplasmic SQSTM1/ P62 Accumulation Predicates a Poor Prognosis in Patients with Malignant Tumor

    doi: 10.7150/jca.26399

    Figure Lengend Snippet: Survival analysis of p62 mRNA expression according to the TCGA and HPA databases. (A) Liver cancer, (B) Lung cancer, (C) Breast cancer, (D) Cervical cancer, (E) Ovarian cancer, (F) Endometrial cancer. Purple lines represent high expression of p62 and blue lines represent low expression.

    Article Snippet: We also tested the association between p62 mRNA level and patients' survival based on the Cancer Genome Atlas (TCGA) and the Human Protein Atlas (HPA) databases.

    Techniques: Expressing

    Neurite ingrowth with bridge at 6 weeks post implantation. A) Neurofilament stain (brown) of bridge implanted with the highest VEGF/FGF-2 protein dose. The red dotted line marks the contours of the bridge at the implant site. Scale bar: 500 µm.

    Journal: Journal of biomedical materials research. Part A

    Article Title: VEGF and FGF-2 delivery from spinal cord bridges to enhance angiogenesis following injury

    doi: 10.1002/jbm.a.33112

    Figure Lengend Snippet: Neurite ingrowth with bridge at 6 weeks post implantation. A) Neurofilament stain (brown) of bridge implanted with the highest VEGF/FGF-2 protein dose. The red dotted line marks the contours of the bridge at the implant site. Scale bar: 500 µm.

    Article Snippet: Proteins were encapsulated into the microspheres by emulsifying a protein solution (17 µl), containing VEGF (recombinant mouse VEGF-165, Prospec, Rehovot, Israel) or FGF-2 (recombinant human FGF-2, Chemicon, Billerica, Massachusetts), 700 µg bovine serum albumin (BSA), 50 mg/mL sucrose, and MgCO3 (3% wt of BSA), with 500 µL 3 wt% PLG in dichloromethane using sonication at 40 watt for 15 s on ice.

    Techniques: Staining

    Protein release from bridge. Bridges were fabricated with VEGF (filled line) or FGF-2 (dotted line) in three different manners: mixing only (squares), encapsulation only (triangles), or a combination of both encapsulation and mixing (circles). The amount

    Journal: Journal of biomedical materials research. Part A

    Article Title: VEGF and FGF-2 delivery from spinal cord bridges to enhance angiogenesis following injury

    doi: 10.1002/jbm.a.33112

    Figure Lengend Snippet: Protein release from bridge. Bridges were fabricated with VEGF (filled line) or FGF-2 (dotted line) in three different manners: mixing only (squares), encapsulation only (triangles), or a combination of both encapsulation and mixing (circles). The amount

    Article Snippet: Proteins were encapsulated into the microspheres by emulsifying a protein solution (17 µl), containing VEGF (recombinant mouse VEGF-165, Prospec, Rehovot, Israel) or FGF-2 (recombinant human FGF-2, Chemicon, Billerica, Massachusetts), 700 µg bovine serum albumin (BSA), 50 mg/mL sucrose, and MgCO3 (3% wt of BSA), with 500 µL 3 wt% PLG in dichloromethane using sonication at 40 watt for 15 s on ice.

    Techniques:

    VEGF and FGF-2 activity during in vitro release. Phosphorylation of (A) VEGF and (B) FGF-2 receptors was assessed by western blot. Legend: 1. control (100 ng/ml VEGF or 50 ng/ml FGF-2), 2. mixing 24 hours, 3. encapsulation 24 hours, 4. mixing 5 days,

    Journal: Journal of biomedical materials research. Part A

    Article Title: VEGF and FGF-2 delivery from spinal cord bridges to enhance angiogenesis following injury

    doi: 10.1002/jbm.a.33112

    Figure Lengend Snippet: VEGF and FGF-2 activity during in vitro release. Phosphorylation of (A) VEGF and (B) FGF-2 receptors was assessed by western blot. Legend: 1. control (100 ng/ml VEGF or 50 ng/ml FGF-2), 2. mixing 24 hours, 3. encapsulation 24 hours, 4. mixing 5 days,

    Article Snippet: Proteins were encapsulated into the microspheres by emulsifying a protein solution (17 µl), containing VEGF (recombinant mouse VEGF-165, Prospec, Rehovot, Israel) or FGF-2 (recombinant human FGF-2, Chemicon, Billerica, Massachusetts), 700 µg bovine serum albumin (BSA), 50 mg/mL sucrose, and MgCO3 (3% wt of BSA), with 500 µL 3 wt% PLG in dichloromethane using sonication at 40 watt for 15 s on ice.

    Techniques: Activity Assay, In Vitro, Western Blot

    Endothelial cell infiltration in bridge at 6 weeks post implantation. A) RECA stain (brown): from left to right: bridge containing 4 µg of VEGF encapsulated within microspheres and 2 µg of FGF-2 and VEGF mixed (high protein), and bridge

    Journal: Journal of biomedical materials research. Part A

    Article Title: VEGF and FGF-2 delivery from spinal cord bridges to enhance angiogenesis following injury

    doi: 10.1002/jbm.a.33112

    Figure Lengend Snippet: Endothelial cell infiltration in bridge at 6 weeks post implantation. A) RECA stain (brown): from left to right: bridge containing 4 µg of VEGF encapsulated within microspheres and 2 µg of FGF-2 and VEGF mixed (high protein), and bridge

    Article Snippet: Proteins were encapsulated into the microspheres by emulsifying a protein solution (17 µl), containing VEGF (recombinant mouse VEGF-165, Prospec, Rehovot, Israel) or FGF-2 (recombinant human FGF-2, Chemicon, Billerica, Massachusetts), 700 µg bovine serum albumin (BSA), 50 mg/mL sucrose, and MgCO3 (3% wt of BSA), with 500 µL 3 wt% PLG in dichloromethane using sonication at 40 watt for 15 s on ice.

    Techniques: Staining

    Protein encapsulation efficiency and protein retention after leaching. A) VEGF and FGF-2 encapsulation efficiency after microsphere encapsulation and collection. B) VEGF and FGF-2 retention after leaching bridges for 1 h. Proteins were incorporated using

    Journal: Journal of biomedical materials research. Part A

    Article Title: VEGF and FGF-2 delivery from spinal cord bridges to enhance angiogenesis following injury

    doi: 10.1002/jbm.a.33112

    Figure Lengend Snippet: Protein encapsulation efficiency and protein retention after leaching. A) VEGF and FGF-2 encapsulation efficiency after microsphere encapsulation and collection. B) VEGF and FGF-2 retention after leaching bridges for 1 h. Proteins were incorporated using

    Article Snippet: Proteins were encapsulated into the microspheres by emulsifying a protein solution (17 µl), containing VEGF (recombinant mouse VEGF-165, Prospec, Rehovot, Israel) or FGF-2 (recombinant human FGF-2, Chemicon, Billerica, Massachusetts), 700 µg bovine serum albumin (BSA), 50 mg/mL sucrose, and MgCO3 (3% wt of BSA), with 500 µL 3 wt% PLG in dichloromethane using sonication at 40 watt for 15 s on ice.

    Techniques:

    Bootstrap box plots of the gene expression intensity of various internal controls . (A) The box plot results show the best 14 internal control candidates, all of which exhibited consistent expression intensity in the NHRI lung adenocarcinoma microarray dataset for each re-sampling process. Moreover, also included are 10 well-known Q-RT-PCR internal controls contained in 23 probe sets on the HG-U133A chip. These are shown as #1–23 in x-axis. The detailed probe set characteristics were shown in Table 1. Except ABCF1 , BHLHB2 and LAPTM4A , the gene expression intensities of top 12 internal control candidates, GAPDH , and ACTB from the Boston (B) and the Ann Arbor lung cancer datasets (C) were also compared. DDX5 : (DEAD (Asp-Glu-Ala-Asp) box polypeptide 5), PKM2 : (pyruvate kinase, muscle), BHLHB2 : (basic helix-loop-helix domain containing, class B, 2), GLO1 : (glyoxalase I), LAPTM4A : (lysosomal-associated protein transmembrane 4 alpha), SET : (SET translocation (myeloid leukemia-associated)), CLTC : (clathrin, heavy chain (Hc)), MSN : (MSN/ALK fusion; moesin/anaplastic lymphoma kinase fusion protein), ABCF1 : (ATP-binding cassette, sub-family F (GCN20), member 1), EPHB3 : (EPH receptor B3), CCL5 : (chemokine (C-C motif) ligand 5), PTPN21 : (protein tyrosine phosphatase, non-receptor type 21), DDR1 : (discoidin domain receptor family, member 1), 1–4: ACTB (actin, beta), 5–6: B2M (beta-2-microglobulin), 7–12: GAPDH (glyceraldehyde-3-phosphate dehydrogenase), 13: HMBS (hydroxymethylbilane synthase), 14: HPRT1 (hypoxanthine phosphoribosyltransferase 1), 15–19: RPL13A (ribosomal protein L13a), 20: RPL32 (ribosomal protein L32), 21: SDHA (succinate dehydrogenase complex, subunit A, flavoprotein (Fp)), 22: UBC (ubiquitin C), 23: YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide).

    Journal: BMC Genomics

    Article Title: Selection of DDX5 as a novel internal control for Q-RT-PCR from microarray data using a block bootstrap re-sampling scheme

    doi: 10.1186/1471-2164-8-140

    Figure Lengend Snippet: Bootstrap box plots of the gene expression intensity of various internal controls . (A) The box plot results show the best 14 internal control candidates, all of which exhibited consistent expression intensity in the NHRI lung adenocarcinoma microarray dataset for each re-sampling process. Moreover, also included are 10 well-known Q-RT-PCR internal controls contained in 23 probe sets on the HG-U133A chip. These are shown as #1–23 in x-axis. The detailed probe set characteristics were shown in Table 1. Except ABCF1 , BHLHB2 and LAPTM4A , the gene expression intensities of top 12 internal control candidates, GAPDH , and ACTB from the Boston (B) and the Ann Arbor lung cancer datasets (C) were also compared. DDX5 : (DEAD (Asp-Glu-Ala-Asp) box polypeptide 5), PKM2 : (pyruvate kinase, muscle), BHLHB2 : (basic helix-loop-helix domain containing, class B, 2), GLO1 : (glyoxalase I), LAPTM4A : (lysosomal-associated protein transmembrane 4 alpha), SET : (SET translocation (myeloid leukemia-associated)), CLTC : (clathrin, heavy chain (Hc)), MSN : (MSN/ALK fusion; moesin/anaplastic lymphoma kinase fusion protein), ABCF1 : (ATP-binding cassette, sub-family F (GCN20), member 1), EPHB3 : (EPH receptor B3), CCL5 : (chemokine (C-C motif) ligand 5), PTPN21 : (protein tyrosine phosphatase, non-receptor type 21), DDR1 : (discoidin domain receptor family, member 1), 1–4: ACTB (actin, beta), 5–6: B2M (beta-2-microglobulin), 7–12: GAPDH (glyceraldehyde-3-phosphate dehydrogenase), 13: HMBS (hydroxymethylbilane synthase), 14: HPRT1 (hypoxanthine phosphoribosyltransferase 1), 15–19: RPL13A (ribosomal protein L13a), 20: RPL32 (ribosomal protein L32), 21: SDHA (succinate dehydrogenase complex, subunit A, flavoprotein (Fp)), 22: UBC (ubiquitin C), 23: YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide).

    Article Snippet: Labeled cDNA was hybridized to the Affymetrix GeneChip Test 3 Array to verify quality prior to hybridize to the Affymetrix Human Genome U133A Array.

    Techniques: Expressing, Microarray, Sampling, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Translocation Assay, Binding Assay, Activation Assay

    Box plots of DDX5 relative expression patterns exhibit small variation across various microarray datasets . The gene expression patterns of DDX5 were obtained from other microarray databases. These datasets were from 84 cancer cell lines (NCI60), which were classified into 8 different cancer cell types and other cell lines (A). GAPDH #5 : M33197_3_at; GAPDH #10 : 212581_x_at. The box plot results indicated that DDX5 exhibited only small variation across the various NCI60 cell types. For the lung cancer cell lines, DDX5 , CLTC and MSN all gave lower variances than ACTB and GAPDH (B). Both ACTB (#1–4) and GAPDH (#5–10) are contained in 10 probe sets on the HG-U133A chip and are shown as #1–10 on x-axis and in Table 1. The variation of DDX5 was also smaller for the HeLa cell cycle dataset, lung cancer dataset and HCC dataset from the Stanford Microarray Database (C). BR: breast cancer, CO: colon cancer, GL: glioblastoma, KI: Kidney, LE: leukemia, LU: lung cancer, ME: melanoma and OV: ovarian cancer.

    Journal: BMC Genomics

    Article Title: Selection of DDX5 as a novel internal control for Q-RT-PCR from microarray data using a block bootstrap re-sampling scheme

    doi: 10.1186/1471-2164-8-140

    Figure Lengend Snippet: Box plots of DDX5 relative expression patterns exhibit small variation across various microarray datasets . The gene expression patterns of DDX5 were obtained from other microarray databases. These datasets were from 84 cancer cell lines (NCI60), which were classified into 8 different cancer cell types and other cell lines (A). GAPDH #5 : M33197_3_at; GAPDH #10 : 212581_x_at. The box plot results indicated that DDX5 exhibited only small variation across the various NCI60 cell types. For the lung cancer cell lines, DDX5 , CLTC and MSN all gave lower variances than ACTB and GAPDH (B). Both ACTB (#1–4) and GAPDH (#5–10) are contained in 10 probe sets on the HG-U133A chip and are shown as #1–10 on x-axis and in Table 1. The variation of DDX5 was also smaller for the HeLa cell cycle dataset, lung cancer dataset and HCC dataset from the Stanford Microarray Database (C). BR: breast cancer, CO: colon cancer, GL: glioblastoma, KI: Kidney, LE: leukemia, LU: lung cancer, ME: melanoma and OV: ovarian cancer.

    Article Snippet: Labeled cDNA was hybridized to the Affymetrix GeneChip Test 3 Array to verify quality prior to hybridize to the Affymetrix Human Genome U133A Array.

    Techniques: Expressing, Microarray, Chromatin Immunoprecipitation

    Effect of delphinidin on the protein expression of (A) PARP and procaspase-3, 8 and 9 (B) Bax and Bcl-2 in HCT116 cells. The cells treated with delphinidin (30–240 μM; 48 h) were harvested and nuclear lysate was prepared and protein was subjected to SDS–PAGE as detailed in Materials and Methods Section. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin. The immunoblot shown here are representative of three independent experiments with similar results. (C) The data were presented in the bar graphs as percentage of Bax/Bcl-2 ratio.

    Journal: Molecular carcinogenesis

    Article Title: Delphinidin, an Anthocyanidin in Pigmented Fruits and Vegetables, Induces Apoptosis and Cell Cycle Arrest in Human Colon Cancer HCT116 Cells

    doi: 10.1002/mc.20477

    Figure Lengend Snippet: Effect of delphinidin on the protein expression of (A) PARP and procaspase-3, 8 and 9 (B) Bax and Bcl-2 in HCT116 cells. The cells treated with delphinidin (30–240 μM; 48 h) were harvested and nuclear lysate was prepared and protein was subjected to SDS–PAGE as detailed in Materials and Methods Section. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin. The immunoblot shown here are representative of three independent experiments with similar results. (C) The data were presented in the bar graphs as percentage of Bax/Bcl-2 ratio.

    Article Snippet: Anti-cyclin B1, anti-IKKα, anti-Bcl-2, anti-cdc2, anti-phospho-NF-κB/p65 and anti-p21WAF1/Cip1 antibodies were procured from Cell Signaling Technology (Beverly, MA).

    Techniques: Expressing, SDS Page, Stripping Membranes

    LARP1 regulates Netrin-1 microsomal translation. Huh7.5 cells were transfected with control and LARP1-specific siRNAs, and infected by HCV at MOI 0.1 over 4 d. Cells were lysed and analyzed by immunoblotting for LARP1 knockdown as shown in S3 Fig . LARP1 depletion decreases the expression of Netrin-1 protein in microsomes in Huh7.5 cells. LARP1, Netrin-1, and HSP60 proteins were monitored for their partitioning in the cytosolic (free polysomes) and microsome (ER membrane-bound polysomes) compartments, according to the infection status of the cells through sequential extractions followed by immunoblotting. HCV core was used as an infection control.

    Journal: PLoS Biology

    Article Title: Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

    doi: 10.1371/journal.pbio.1002421

    Figure Lengend Snippet: LARP1 regulates Netrin-1 microsomal translation. Huh7.5 cells were transfected with control and LARP1-specific siRNAs, and infected by HCV at MOI 0.1 over 4 d. Cells were lysed and analyzed by immunoblotting for LARP1 knockdown as shown in S3 Fig . LARP1 depletion decreases the expression of Netrin-1 protein in microsomes in Huh7.5 cells. LARP1, Netrin-1, and HSP60 proteins were monitored for their partitioning in the cytosolic (free polysomes) and microsome (ER membrane-bound polysomes) compartments, according to the infection status of the cells through sequential extractions followed by immunoblotting. HCV core was used as an infection control.

    Article Snippet: Netrin-1 does not affect HCV replication per se.

    Techniques: Transfection, Infection, Expressing

    Netrin-1 mRNA and HCV NS5A are LARP1 interactants. Quantification of ribosomal protein S18 (RPS18) ( A ) and Netrin-1 ( B ) mRNA by qPCR after immunoprecipitation using LARP1 antibody (data are represented as mean ± standard deviation, n = 3, Mann-Whitney test, p

    Journal: PLoS Biology

    Article Title: Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

    doi: 10.1371/journal.pbio.1002421

    Figure Lengend Snippet: Netrin-1 mRNA and HCV NS5A are LARP1 interactants. Quantification of ribosomal protein S18 (RPS18) ( A ) and Netrin-1 ( B ) mRNA by qPCR after immunoprecipitation using LARP1 antibody (data are represented as mean ± standard deviation, n = 3, Mann-Whitney test, p

    Article Snippet: Netrin-1 does not affect HCV replication per se.

    Techniques: Real-time Polymerase Chain Reaction, Immunoprecipitation, Standard Deviation, MANN-WHITNEY

    Netrin-1 increases HCV through the UNC5A receptor. Huh7.5 cells were transfected with siRNA against each UNC5 receptor or with a nontargeting control siRNA and infected at a MOI of 0.1 24 h after seeding. Cells were then trypsinized at day five post-infection before undergoing a second siRNA transfection. Recombinant soluble Netrin-1-Fc was added to the medium 12 h after transfection. Intracellular HCV RNA was quantified by RT-qPCR at each time point (left-hand graphs; data are represented as mean ± standard deviation, n = 3, Wilcoxon test, p

    Journal: PLoS Biology

    Article Title: Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

    doi: 10.1371/journal.pbio.1002421

    Figure Lengend Snippet: Netrin-1 increases HCV through the UNC5A receptor. Huh7.5 cells were transfected with siRNA against each UNC5 receptor or with a nontargeting control siRNA and infected at a MOI of 0.1 24 h after seeding. Cells were then trypsinized at day five post-infection before undergoing a second siRNA transfection. Recombinant soluble Netrin-1-Fc was added to the medium 12 h after transfection. Intracellular HCV RNA was quantified by RT-qPCR at each time point (left-hand graphs; data are represented as mean ± standard deviation, n = 3, Wilcoxon test, p

    Article Snippet: Netrin-1 does not affect HCV replication per se.

    Techniques: Transfection, Infection, Recombinant, Quantitative RT-PCR, Standard Deviation

    Netrin-1 overexpression increases HCV RNA and specific infectivity of HCV virions in vitro whereas Netrin-1 depletion decreases HCV RNA and specific infectivity in vitro. A. Detection of VR1-HA and Netrin-1-HA in transfected Huh7.5 cells by immunoblotting with the anti-HA antibody. B . Netrin-1 overexpression enhances intracellular HCV RNA. Huh7.5 cells were transfected with the VR1-HA or the Netrin-1-HA plasmid and infected at a MOI of 0.1 the day after seeding. Intracellular HCV RNA was quantified by RT-qPCR at each time point (data are shown as mean ± standard deviation, n = 3, Wilcoxon test, p

    Journal: PLoS Biology

    Article Title: Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

    doi: 10.1371/journal.pbio.1002421

    Figure Lengend Snippet: Netrin-1 overexpression increases HCV RNA and specific infectivity of HCV virions in vitro whereas Netrin-1 depletion decreases HCV RNA and specific infectivity in vitro. A. Detection of VR1-HA and Netrin-1-HA in transfected Huh7.5 cells by immunoblotting with the anti-HA antibody. B . Netrin-1 overexpression enhances intracellular HCV RNA. Huh7.5 cells were transfected with the VR1-HA or the Netrin-1-HA plasmid and infected at a MOI of 0.1 the day after seeding. Intracellular HCV RNA was quantified by RT-qPCR at each time point (data are shown as mean ± standard deviation, n = 3, Wilcoxon test, p

    Article Snippet: Netrin-1 does not affect HCV replication per se.

    Techniques: Over Expression, Infection, In Vitro, Transfection, Plasmid Preparation, Quantitative RT-PCR, Standard Deviation

    HCV increases Netrin-1 mRNA and protein. A . Virions depletion experiment. Huh7.5 cells were infected by HCV over 3 d, and the supernatant (SN) was collected, depleted of HCV particles by ultracentrifugation, and added to naïve Huh7.5 cells. Levels of HCV RNA (left) and Netrin-1 mRNA (right) were quantified by RT-qPCR. Data are represented as mean ± standard deviation ( n = 3). B . HCV increases the association of Netrin-1 mRNA with microsomes (endoplasmic reticulum [ER] membrane)-bound polysomes in Huh7.5 cells. Netrin-1 , Glucuronidase ( GUS) , and phosphomannomutase 1 ( PMM1) mRNA were monitored for their partitioning in the cytosolic (free polysomes) and microsome (ER membrane-bound polysomes) compartments according to the infection status of the cells through sequential extractions followed by RT-qPCR. Statistical significance was determined using the Mann-Whitney test, p

    Journal: PLoS Biology

    Article Title: Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

    doi: 10.1371/journal.pbio.1002421

    Figure Lengend Snippet: HCV increases Netrin-1 mRNA and protein. A . Virions depletion experiment. Huh7.5 cells were infected by HCV over 3 d, and the supernatant (SN) was collected, depleted of HCV particles by ultracentrifugation, and added to naïve Huh7.5 cells. Levels of HCV RNA (left) and Netrin-1 mRNA (right) were quantified by RT-qPCR. Data are represented as mean ± standard deviation ( n = 3). B . HCV increases the association of Netrin-1 mRNA with microsomes (endoplasmic reticulum [ER] membrane)-bound polysomes in Huh7.5 cells. Netrin-1 , Glucuronidase ( GUS) , and phosphomannomutase 1 ( PMM1) mRNA were monitored for their partitioning in the cytosolic (free polysomes) and microsome (ER membrane-bound polysomes) compartments according to the infection status of the cells through sequential extractions followed by RT-qPCR. Statistical significance was determined using the Mann-Whitney test, p

    Article Snippet: Netrin-1 does not affect HCV replication per se.

    Techniques: Infection, Quantitative RT-PCR, Standard Deviation, MANN-WHITNEY

    HCV levels correlate with the expression of Netrin-1 in the liver biopsies of HCV-infected patients. A . HCV-positive samples exhibit the highest levels of Netrin-1 mRNA of all the chronic liver disease biopsies. The levels of Netrin-1 mRNA were quantified by RT-qPCR. Statistical significance was determined using the Mann-Whitney test. B. Positive correlation between intrahepatic levels of HCV and Netrin-1 mRNA. HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR. Statistical significance was determined using the Spearman test. An outlier test was run to confirm these results. C and D. Netrin-1 mRNA parallels HCV RNA levels upon treatment. HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR in paired biopsies, before and after treatment, of partially responding patients ( C,D left panels ) and nonresponding patients (C,D, right panels ). E. Parenchymal Netrin-1 staining is associated with HCV infection status in HCV-infected patients. Uninfected, chronic liver disease samples (non-cirrhotic sample, n = 1; alcohol-related cirrhosis samples, n = 3) and HCV genotype 1-infected cirrhosis samples ( n = 4) were analyzed. Representative images of Netrin-1 staining (upper panels) and HCV E2 staining (lower panels) are shown. F. Netrin-1 protein expression is increased in HCV-positive samples. The level of Netrin-1 was quantified by immunoblotting using recombinant Netrin-1 (rec. Net) as a control. G. The levels of Netrin-1 mRNA are higher in HCV+ versus HCV- biopsies, regardless of the histological stage, from normal liver to HCC. Intrahepatic Netrin-1 mRNA levels were quantified by RT-qPCR. Statistical significance was determined using the Mann-Whitney test, p

    Journal: PLoS Biology

    Article Title: Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

    doi: 10.1371/journal.pbio.1002421

    Figure Lengend Snippet: HCV levels correlate with the expression of Netrin-1 in the liver biopsies of HCV-infected patients. A . HCV-positive samples exhibit the highest levels of Netrin-1 mRNA of all the chronic liver disease biopsies. The levels of Netrin-1 mRNA were quantified by RT-qPCR. Statistical significance was determined using the Mann-Whitney test. B. Positive correlation between intrahepatic levels of HCV and Netrin-1 mRNA. HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR. Statistical significance was determined using the Spearman test. An outlier test was run to confirm these results. C and D. Netrin-1 mRNA parallels HCV RNA levels upon treatment. HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR in paired biopsies, before and after treatment, of partially responding patients ( C,D left panels ) and nonresponding patients (C,D, right panels ). E. Parenchymal Netrin-1 staining is associated with HCV infection status in HCV-infected patients. Uninfected, chronic liver disease samples (non-cirrhotic sample, n = 1; alcohol-related cirrhosis samples, n = 3) and HCV genotype 1-infected cirrhosis samples ( n = 4) were analyzed. Representative images of Netrin-1 staining (upper panels) and HCV E2 staining (lower panels) are shown. F. Netrin-1 protein expression is increased in HCV-positive samples. The level of Netrin-1 was quantified by immunoblotting using recombinant Netrin-1 (rec. Net) as a control. G. The levels of Netrin-1 mRNA are higher in HCV+ versus HCV- biopsies, regardless of the histological stage, from normal liver to HCC. Intrahepatic Netrin-1 mRNA levels were quantified by RT-qPCR. Statistical significance was determined using the Mann-Whitney test, p

    Article Snippet: Netrin-1 does not affect HCV replication per se.

    Techniques: Expressing, Infection, Quantitative RT-PCR, MANN-WHITNEY, Staining, Recombinant

    HCV induces the expression of Netrin-1 in vitro. A, B, C, D. HCV induces Netrin-1 mRNA in primary human hepatocytes. Cells were infected at a MOI of 1 with HCV genotype 2 strain 4 d after seeding ( n = 4 independent preparations from four different patients, Wilcoxon test, p

    Journal: PLoS Biology

    Article Title: Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

    doi: 10.1371/journal.pbio.1002421

    Figure Lengend Snippet: HCV induces the expression of Netrin-1 in vitro. A, B, C, D. HCV induces Netrin-1 mRNA in primary human hepatocytes. Cells were infected at a MOI of 1 with HCV genotype 2 strain 4 d after seeding ( n = 4 independent preparations from four different patients, Wilcoxon test, p

    Article Snippet: Netrin-1 does not affect HCV replication per se.

    Techniques: Expressing, In Vitro, Infection

    Netrin-1 enhances HCVpp entry. RNAi-mediated knockdown of EGFR. Huh7.5 cells were transfected with anti-EGFR siRNAs #3 and #4 of a previously described study [ 3 ] and infected at a MOI of 0.1. Cells were collected 3 d post-infection and analyzed by RT-qPCR ( A ) and immunoblotting using an anti-EGFR antibody targeting an extracellular epitope of the protein ( B ) (data are represented as mean ± standard deviation, n = 4, Mann-Whitney test, p

    Journal: PLoS Biology

    Article Title: Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

    doi: 10.1371/journal.pbio.1002421

    Figure Lengend Snippet: Netrin-1 enhances HCVpp entry. RNAi-mediated knockdown of EGFR. Huh7.5 cells were transfected with anti-EGFR siRNAs #3 and #4 of a previously described study [ 3 ] and infected at a MOI of 0.1. Cells were collected 3 d post-infection and analyzed by RT-qPCR ( A ) and immunoblotting using an anti-EGFR antibody targeting an extracellular epitope of the protein ( B ) (data are represented as mean ± standard deviation, n = 4, Mann-Whitney test, p

    Article Snippet: Netrin-1 does not affect HCV replication per se.

    Techniques: Transfection, Infection, Quantitative RT-PCR, Standard Deviation, MANN-WHITNEY

    Netrin-1 impedes EGFR recycling. Huh7.5 cells infected with HCV (day four p.i.) were transfected with siRNA (control or Netrin-1-specific) or with plasmids (VR1- or Netrin-1-expressing), serum-starved for 16 h, and incubated with EGF for 5 or 15 min prior to fixation. Cells were subsequently stained for EEA1 and EGFR and visualized by confocal microscopy. A . Li diagrams and Li coefficient calculations ( B ) for Netrin-1 knockdown and forced expression experiments. Pixels present on the left and right sides of the y -axis, i.e., associated with negative and positive staining amplitude values, indicate exclusion and colocalization, respectively. Li coefficients were calculated using the JACop plugin from the imageJ software ( http://rsb.info.nih.gov/ij/plugins/track/jacop.html ). Twelve to 15 random fields were acquired per biological sample, totaling 250–300 cells analyzed for each biological sample in a given experiment ( n = 2). C . Representative immunofluorescence-based localization of EEA1 and EGFR. Nuclei were counterstained with Hoechst 33342. EEA1 (green) and EGFR (red) were detected using Alexa-488 and Alexa-594, respectively. Overlays were generated by the Leica LAS AF software upon image acquisition. Open and solid arrows show partial and total colocalization, respectively. Bar = 5 μm. D . Statistical assessment of the colocalization of EEA1 and EGFR. Red (EGFR) and green (EEA1) fluorescence intensities were measured for each pixel along a 5 μm horizontal line centered around the arrow tips in ( C ) using the Plot Profile function of the ImageJ software. Spearman correlation coefficients for each couple of intensity values are shown. The underlying data for panels in this figure can be found in S1 Data .

    Journal: PLoS Biology

    Article Title: Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

    doi: 10.1371/journal.pbio.1002421

    Figure Lengend Snippet: Netrin-1 impedes EGFR recycling. Huh7.5 cells infected with HCV (day four p.i.) were transfected with siRNA (control or Netrin-1-specific) or with plasmids (VR1- or Netrin-1-expressing), serum-starved for 16 h, and incubated with EGF for 5 or 15 min prior to fixation. Cells were subsequently stained for EEA1 and EGFR and visualized by confocal microscopy. A . Li diagrams and Li coefficient calculations ( B ) for Netrin-1 knockdown and forced expression experiments. Pixels present on the left and right sides of the y -axis, i.e., associated with negative and positive staining amplitude values, indicate exclusion and colocalization, respectively. Li coefficients were calculated using the JACop plugin from the imageJ software ( http://rsb.info.nih.gov/ij/plugins/track/jacop.html ). Twelve to 15 random fields were acquired per biological sample, totaling 250–300 cells analyzed for each biological sample in a given experiment ( n = 2). C . Representative immunofluorescence-based localization of EEA1 and EGFR. Nuclei were counterstained with Hoechst 33342. EEA1 (green) and EGFR (red) were detected using Alexa-488 and Alexa-594, respectively. Overlays were generated by the Leica LAS AF software upon image acquisition. Open and solid arrows show partial and total colocalization, respectively. Bar = 5 μm. D . Statistical assessment of the colocalization of EEA1 and EGFR. Red (EGFR) and green (EEA1) fluorescence intensities were measured for each pixel along a 5 μm horizontal line centered around the arrow tips in ( C ) using the Plot Profile function of the ImageJ software. Spearman correlation coefficients for each couple of intensity values are shown. The underlying data for panels in this figure can be found in S1 Data .

    Article Snippet: Netrin-1 does not affect HCV replication per se.

    Techniques: Infection, Transfection, Expressing, Incubation, Staining, Confocal Microscopy, Software, Immunofluorescence, Generated, Fluorescence

    Netrin-1 is virion-bound and participates in the infectivity of viral particles. HCV particles produced in cells overexpressing Netrin-1 have been preincubated with Netrin-1 antagonists and TCID 50 has been performed. A . Method validation: inhibition of HCV infectivity after incubation with anti-E2 antibody. The anti-E2 antibody was used as a control of neutralization of infectivity. B . Inhibition of HCV infectivity after incubation with three distinct Netrin-1 antagonists (data are represented as mean ± standard deviation, n = 3, Mann-Whitney test, p

    Journal: PLoS Biology

    Article Title: Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

    doi: 10.1371/journal.pbio.1002421

    Figure Lengend Snippet: Netrin-1 is virion-bound and participates in the infectivity of viral particles. HCV particles produced in cells overexpressing Netrin-1 have been preincubated with Netrin-1 antagonists and TCID 50 has been performed. A . Method validation: inhibition of HCV infectivity after incubation with anti-E2 antibody. The anti-E2 antibody was used as a control of neutralization of infectivity. B . Inhibition of HCV infectivity after incubation with three distinct Netrin-1 antagonists (data are represented as mean ± standard deviation, n = 3, Mann-Whitney test, p

    Article Snippet: Netrin-1 does not affect HCV replication per se.

    Techniques: Infection, Produced, Inhibition, Incubation, Neutralization, Standard Deviation, MANN-WHITNEY

    Suppression of Bcl-2 and Bcl-xL expression in Stat3-deficient T cells. (a) Splenic T cells were purified using the Pan T Cell Isolation Kit and an autoMACS Separator (Miltenyi Biotech Inc.). Total RNA was purified and cDNA was synthesized. Quantitiative

    Journal: Immunology

    Article Title: Signal transducer and activator of transcription 3 (Stat3) contributes to T-cell homeostasis by regulating pro-survival Bcl-2 family genes

    doi: 10.1111/imm.12133

    Figure Lengend Snippet: Suppression of Bcl-2 and Bcl-xL expression in Stat3-deficient T cells. (a) Splenic T cells were purified using the Pan T Cell Isolation Kit and an autoMACS Separator (Miltenyi Biotech Inc.). Total RNA was purified and cDNA was synthesized. Quantitiative

    Article Snippet: Next, we measured the mRNA expression of Bcl-2 family genes in splenic T cells isolated using the Pan T Cell Isolation Kit (Miltenyi Biotech Inc.).

    Techniques: Expressing, Purification, Cell Isolation, Synthesized