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  • 99
    Name:
    Bst DNA Polymerase Full Length
    Description:
    Bst DNA Polymerase Full Length 500 units
    Catalog Number:
    m0328s
    Price:
    70
    Size:
    500 units
    Category:
    Thermostable DNA Polymerases
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    Structured Review

    New England Biolabs full length
    Bst DNA Polymerase Full Length
    Bst DNA Polymerase Full Length 500 units
    https://www.bioz.com/result/full length/product/New England Biolabs
    Average 99 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    full length - by Bioz Stars, 2020-07
    99/100 stars

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    Produced:

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis
    Article Snippet: .. 8 U of Bst DNA polymerase produced the best LAMP results under the optimal ratio of inner to outer primers and MgSO4 concentration (Figs and ). ..

    Amplification:

    Article Title: Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability
    Article Snippet: .. 2.2 Viral RNA amplified by RT-LAMP Five microliters of extracted RNA (the same amount for regular RT-PCR) was taken for RT-LAMP amplification in a 25-μL mixture consisting of Bst DNA polymerase (New England Biolab) 8 U, 10× thermal buffer 1.0 μL, AMV reverse transcriptase (Fermentas) 5 U, 5× AMV first-strand buffer 4.0 μL, 25 μM betaine (Sigma, USA) 1.0 μL, 2.5 mmol/L dNTP mixtures 5 μL, 0.1% Triton X-100 0.25 μL, and a primer mixture, including 0.2 μM F3 and B3, and 1.6 μM FIP and BIP. .. RT-LAMP primer sets were designed using the Primer Explorer V3.0 online software ( http://primerexplorer.jp/v3_manual/index.html ) and manually selected according to the principle for RT-LAMP ( and ).

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis
    Article Snippet: .. In this work, we showed that as the concentration of Bst DNA polymerase was increased from 4 U to 8 U, the amplification improved dramatically, however, the tendency of amplification efficiency improvement from 6 U to 8 U is far less than that of 4 U to 6 U (Figs and ). .. 8 U of Bst DNA polymerase produced the best LAMP results under the optimal ratio of inner to outer primers and MgSO4 concentration (Figs and ).

    Purification:

    Article Title: Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method
    Article Snippet: .. LAMP-assay The PSTVd-LAMP assay was set up in a total volume of 12.5 μL containing 1x supplied Thermopol reaction buffer (New England Biolabs, Frankfurt am Main, Germany), 1.2 mM of each dNTP (Thermo Scientific), 0.8 M betaine (Carl Roth, Karlsruhe, Germany), 0.32 U/µL Bst DNA Polymerase (New England Biolabs), 0.8 × SYBR Green I (Thermo Fisher Scientific), 1.6 µM of FIP and BIP primer, 0.4 µM of LF and LR pimer, 0.2 µM of F3 and B3 primer, 6 mM MgSO4 (Carl Roth) and purified DNA template. .. Hinge-assay Hinge-amplification assays were set up in a total volume of 12.5 µL containing 1x Thermo Pol® Reaction buffer, 8 mM magnesium chloride, 0.8 M betaine, 1.0 mM of dNTP-mix, 0.32 U/µL Bst 3.0 DNA Polymerase, 0.8x SYBR Green I.

    SYBR Green Assay:

    Article Title: Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method
    Article Snippet: .. LAMP-assay The PSTVd-LAMP assay was set up in a total volume of 12.5 μL containing 1x supplied Thermopol reaction buffer (New England Biolabs, Frankfurt am Main, Germany), 1.2 mM of each dNTP (Thermo Scientific), 0.8 M betaine (Carl Roth, Karlsruhe, Germany), 0.32 U/µL Bst DNA Polymerase (New England Biolabs), 0.8 × SYBR Green I (Thermo Fisher Scientific), 1.6 µM of FIP and BIP primer, 0.4 µM of LF and LR pimer, 0.2 µM of F3 and B3 primer, 6 mM MgSO4 (Carl Roth) and purified DNA template. .. Hinge-assay Hinge-amplification assays were set up in a total volume of 12.5 µL containing 1x Thermo Pol® Reaction buffer, 8 mM magnesium chloride, 0.8 M betaine, 1.0 mM of dNTP-mix, 0.32 U/µL Bst 3.0 DNA Polymerase, 0.8x SYBR Green I.

    Concentration Assay:

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis
    Article Snippet: .. 8 U of Bst DNA polymerase produced the best LAMP results under the optimal ratio of inner to outer primers and MgSO4 concentration (Figs and ). ..

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis
    Article Snippet: .. In this work, we showed that as the concentration of Bst DNA polymerase was increased from 4 U to 8 U, the amplification improved dramatically, however, the tendency of amplification efficiency improvement from 6 U to 8 U is far less than that of 4 U to 6 U (Figs and ). .. 8 U of Bst DNA polymerase produced the best LAMP results under the optimal ratio of inner to outer primers and MgSO4 concentration (Figs and ).

    Incubation:

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus
    Article Snippet: .. NC-DNA (108 copies) was incubated with 32 units (U) of Thermostable FEN1 in ThermoPol Buffer (New England Biolabs) at 65°C for 10 min, followed by incubation with 8 U of Bst DNA polymerase, 40 U of Taq DNA ligase, 100 μM dNTPs, and NAD+ (all from New England Biolabs). .. After further incubation at 37°C for 20 min, DNA was purified by phenol/chloroform extraction and ethanol precipitation, and subjected to cccDNA-selective qPCR or RCA, as described above.

    other:

    Article Title: Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability
    Article Snippet: Without Bst DNA polymerase or RNA templates, RT-LAMP was unable to amplify RNA ( A).

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis
    Article Snippet: The quantity of Bst DNA polymerase in the reaction mixture was set at 2.0 U, 4.0 U, 6.0 U and 8.0 U. Mg2+ concentrations were set to 5.0 mM, 6.0 mM, 7.0 mM and 8.0 mM, respectively (Fig. ).

    Lamp Assay:

    Article Title: Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method
    Article Snippet: .. LAMP-assay The PSTVd-LAMP assay was set up in a total volume of 12.5 μL containing 1x supplied Thermopol reaction buffer (New England Biolabs, Frankfurt am Main, Germany), 1.2 mM of each dNTP (Thermo Scientific), 0.8 M betaine (Carl Roth, Karlsruhe, Germany), 0.32 U/µL Bst DNA Polymerase (New England Biolabs), 0.8 × SYBR Green I (Thermo Fisher Scientific), 1.6 µM of FIP and BIP primer, 0.4 µM of LF and LR pimer, 0.2 µM of F3 and B3 primer, 6 mM MgSO4 (Carl Roth) and purified DNA template. .. Hinge-assay Hinge-amplification assays were set up in a total volume of 12.5 µL containing 1x Thermo Pol® Reaction buffer, 8 mM magnesium chloride, 0.8 M betaine, 1.0 mM of dNTP-mix, 0.32 U/µL Bst 3.0 DNA Polymerase, 0.8x SYBR Green I.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability
    Article Snippet: .. 2.2 Viral RNA amplified by RT-LAMP Five microliters of extracted RNA (the same amount for regular RT-PCR) was taken for RT-LAMP amplification in a 25-μL mixture consisting of Bst DNA polymerase (New England Biolab) 8 U, 10× thermal buffer 1.0 μL, AMV reverse transcriptase (Fermentas) 5 U, 5× AMV first-strand buffer 4.0 μL, 25 μM betaine (Sigma, USA) 1.0 μL, 2.5 mmol/L dNTP mixtures 5 μL, 0.1% Triton X-100 0.25 μL, and a primer mixture, including 0.2 μM F3 and B3, and 1.6 μM FIP and BIP. .. RT-LAMP primer sets were designed using the Primer Explorer V3.0 online software ( http://primerexplorer.jp/v3_manual/index.html ) and manually selected according to the principle for RT-LAMP ( and ).

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  • 92
    New England Biolabs nhej factors full length human xlf
    Pol λ interacts with XRCC4 family proteins via its BRCT domain in cells. a HEK293F cells were irradiated with 10 Gy X-ray or left untreated. Soluble nuclear extracts were isolated following 0–60 min post-irradiation recovery time at 37 °C. Following IP with anti-Pol λ or rabbit IgG (rIgG), Pol λ and associated proteins were resolved by SDS-PAGE and immunoblotted for the indicated <t>NHEJ</t> factors. b HEK293F cell nucleoplasmic (NP) or soluble chromatin (sol. Chr) extracts were immunoprecipitated with rIgG, anti-PAXX or <t>-XLF</t> or mouse IgG (mIgG) or anti-XRCC4. Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted for the indicated NHEJ factors. c As described in Panel A, except that soluble nuclear extracts were incubated with 0-200 μg/ml EtBr for 1 h prior to IP with anti-Pol λ or rIgG. d EMSA showing that interaction of Pol λ with DNA-bound Ku requires R57 and L60 in the BRCT domain of Pol λ. Reactions were performed with IRDye® 700-labelled 5nt-gapped dsDNA (33-mer) in the presence or absence of FLAG-Ku70/80 (20 nM) and either FLAG-Pol λ-WT or a R57A/L60A mutant (50 nM). e HEK293F cells were transiently transfected with either pCMX-LacZ (control) or pCMX-FLAG-Pol λ-WT, -ΔBRCT or a R57A/L60A mutant and anti-FLAG IPs performed
    Nhej Factors Full Length Human Xlf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nhej factors full length human xlf/product/New England Biolabs
    Average 92 stars, based on 1 article reviews
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    85
    New England Biolabs full length human dlk 1
    Regulations of <t>DLK-1</t> in human and mice are not the same. To evaluate mechanisms induced by DLK-1 in human CB MSC upon exposure to DLK-1/Pref1, Western Blot analysis of ERK1/2 and p-ERK1/2 was performed ( n = 3 experiments). CB MSC were treated with conditioned media of either the control cells or the DLK-1 overexpressing CB MSC. CB MSC (d0 control) and DLK-1 overexpressing CB MSC (d0 DLK+) were lysed and western blot analysis was performed applying full protein lysates after 30 minutes, 4 hours and 24 hours of incubation with conditioned media, respectively. ERK1/2 was already highly expressed in the non treated cells and remained expressed also in the cells treated. No DLK-1/Pref1 specific upregulation of p-ERK1/2 was detected in the CB MSC. As biological positive control, osteogenic differentiated (day 3 after induction) BM MSC were used. Internal loading control: β -actin.
    Full Length Human Dlk 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    New England Biolabs transfection full length hoxa7
    <t>HOXA7</t> regulates granulosa cell proliferation . KGN cells were transiently transfected with scrambled siRNA or HOXA7 siRNA and the effect of HOXA7 knockdown was verified at the mRNA level by real-time PCR (A) and protein level by Western blotting (B). (C) After 36 h of <t>transfection,</t> KGN cells were reseeded into a 96-well plate and the cell viability was detected by MTT assay. SVOG cells were transiently transfected with control vector or HOXA7 plasmid and the effect of HOXA7 overexpression was verified at the mRNA level by real-time PCR (D) and protein level by Western blotting (E). (F) After 36 h of transfection, SVOG cells were reseeded into a 96-well plate and the cell viability was detected by MTT assay. The data derived from at least three separate sets of experiments were standardized to the corresponding control. a, P
    Transfection Full Length Hoxa7, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    New England Biolabs full length brca1 expression plasmid
    Analysis of loss of heterozygosity in rare truncation and missense variants. ( a ) Bar plot shows individual truncations from nine genes (FDR shown) with lengths representing ratios of tumour-to-normal variant allele fractions (that is, the fraction of reads containing the variant allele). Statistically significant events, defined as FDR≤5%, are shaded boldly, while non-significant events are muted, with colours corresponding to genes. Cancer source of each truncation is shown underneath, for example, most <t>BRCA1</t> variants occur in ovarian and breast cancers and all BAP1 variants in KIRC. ( b ) Bar plot for individual missense variants from four genes having elevated frequencies of such variants that show very significant LOH, that is, at the 1% FDR level. ( c ) Dot plot shows individual missense variants where abscissa and ordinate are amino acid positions and the ratio of tumour-to-normal variant allele fraction, respectively. Blue and red indicate significant (FDR ≤5%) and non-significant events, respectively, with size of dots proportional to negative log of the FDR. Annotated domains from the PFAM database are aligned with position, while shaded areas indicate ‘hotspot' regions where variants having significant LOH cluster more than the rate explainable by chance. Plots are shown for ATM , BRCA1 , BRCA2 , FANCA and FANCM .
    Full Length Brca1 Expression Plasmid, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pol λ interacts with XRCC4 family proteins via its BRCT domain in cells. a HEK293F cells were irradiated with 10 Gy X-ray or left untreated. Soluble nuclear extracts were isolated following 0–60 min post-irradiation recovery time at 37 °C. Following IP with anti-Pol λ or rabbit IgG (rIgG), Pol λ and associated proteins were resolved by SDS-PAGE and immunoblotted for the indicated NHEJ factors. b HEK293F cell nucleoplasmic (NP) or soluble chromatin (sol. Chr) extracts were immunoprecipitated with rIgG, anti-PAXX or -XLF or mouse IgG (mIgG) or anti-XRCC4. Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted for the indicated NHEJ factors. c As described in Panel A, except that soluble nuclear extracts were incubated with 0-200 μg/ml EtBr for 1 h prior to IP with anti-Pol λ or rIgG. d EMSA showing that interaction of Pol λ with DNA-bound Ku requires R57 and L60 in the BRCT domain of Pol λ. Reactions were performed with IRDye® 700-labelled 5nt-gapped dsDNA (33-mer) in the presence or absence of FLAG-Ku70/80 (20 nM) and either FLAG-Pol λ-WT or a R57A/L60A mutant (50 nM). e HEK293F cells were transiently transfected with either pCMX-LacZ (control) or pCMX-FLAG-Pol λ-WT, -ΔBRCT or a R57A/L60A mutant and anti-FLAG IPs performed

    Journal: Nature Communications

    Article Title: PAXX and its paralogs synergistically direct DNA polymerase λ activity in DNA repair

    doi: 10.1038/s41467-018-06127-y

    Figure Lengend Snippet: Pol λ interacts with XRCC4 family proteins via its BRCT domain in cells. a HEK293F cells were irradiated with 10 Gy X-ray or left untreated. Soluble nuclear extracts were isolated following 0–60 min post-irradiation recovery time at 37 °C. Following IP with anti-Pol λ or rabbit IgG (rIgG), Pol λ and associated proteins were resolved by SDS-PAGE and immunoblotted for the indicated NHEJ factors. b HEK293F cell nucleoplasmic (NP) or soluble chromatin (sol. Chr) extracts were immunoprecipitated with rIgG, anti-PAXX or -XLF or mouse IgG (mIgG) or anti-XRCC4. Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted for the indicated NHEJ factors. c As described in Panel A, except that soluble nuclear extracts were incubated with 0-200 μg/ml EtBr for 1 h prior to IP with anti-Pol λ or rIgG. d EMSA showing that interaction of Pol λ with DNA-bound Ku requires R57 and L60 in the BRCT domain of Pol λ. Reactions were performed with IRDye® 700-labelled 5nt-gapped dsDNA (33-mer) in the presence or absence of FLAG-Ku70/80 (20 nM) and either FLAG-Pol λ-WT or a R57A/L60A mutant (50 nM). e HEK293F cells were transiently transfected with either pCMX-LacZ (control) or pCMX-FLAG-Pol λ-WT, -ΔBRCT or a R57A/L60A mutant and anti-FLAG IPs performed

    Article Snippet: Cloning and site-directed mutagenesis of NHEJ factors Full-length human XLF, XRCC4, Ku70, Ku80, Lig IV, Pol λ and Pol μ were amplified with Q5 polymerase (NEB) using oligo-dT primed cDNA template prepared from U2OS cells with primers which optionally incorporated additional 5′ sequences encoding either FLAG- or HA tags.

    Techniques: Irradiation, Isolation, SDS Page, Non-Homologous End Joining, Immunoprecipitation, Incubation, Mutagenesis, Transfection

    Regulations of DLK-1 in human and mice are not the same. To evaluate mechanisms induced by DLK-1 in human CB MSC upon exposure to DLK-1/Pref1, Western Blot analysis of ERK1/2 and p-ERK1/2 was performed ( n = 3 experiments). CB MSC were treated with conditioned media of either the control cells or the DLK-1 overexpressing CB MSC. CB MSC (d0 control) and DLK-1 overexpressing CB MSC (d0 DLK+) were lysed and western blot analysis was performed applying full protein lysates after 30 minutes, 4 hours and 24 hours of incubation with conditioned media, respectively. ERK1/2 was already highly expressed in the non treated cells and remained expressed also in the cells treated. No DLK-1/Pref1 specific upregulation of p-ERK1/2 was detected in the CB MSC. As biological positive control, osteogenic differentiated (day 3 after induction) BM MSC were used. Internal loading control: β -actin.

    Journal: Stem Cells International

    Article Title: Increased Haematopoietic Supportive Function of USSC from Umbilical Cord Blood Compared to CB MSC and Possible Role of DLK-1

    doi: 10.1155/2013/985285

    Figure Lengend Snippet: Regulations of DLK-1 in human and mice are not the same. To evaluate mechanisms induced by DLK-1 in human CB MSC upon exposure to DLK-1/Pref1, Western Blot analysis of ERK1/2 and p-ERK1/2 was performed ( n = 3 experiments). CB MSC were treated with conditioned media of either the control cells or the DLK-1 overexpressing CB MSC. CB MSC (d0 control) and DLK-1 overexpressing CB MSC (d0 DLK+) were lysed and western blot analysis was performed applying full protein lysates after 30 minutes, 4 hours and 24 hours of incubation with conditioned media, respectively. ERK1/2 was already highly expressed in the non treated cells and remained expressed also in the cells treated. No DLK-1/Pref1 specific upregulation of p-ERK1/2 was detected in the CB MSC. As biological positive control, osteogenic differentiated (day 3 after induction) BM MSC were used. Internal loading control: β -actin.

    Article Snippet: DLK-1 Overexpression As described previously [ ], full-length human DLK-1 was amplified using Phusion Taq Polymerase (New England Biolabs).

    Techniques: Mouse Assay, Western Blot, Incubation, Positive Control

    Haematopoiesis-supporting capacity of USSC and CB MSC. (a) Representative co-culture of CD34 + -cells on feeders of USSC, CB MSC, and CB MSC overexpressing DLK-1 (CB MSC DLK ) in multiple independent wells ( n = 6). With regards to total cell count, expansion of CD34 + -cells and colony-forming units (CFU), co-culture on a USSC-feeder resulted in higher expansion rates than on CB MSC-feeder. Within 14 days, total cell count increased significantly stronger on USSC-feeder (by factor 35.05 ± 3.75) than on CB MSC-feeder (factor 10.09 ± 0.37; P

    Journal: Stem Cells International

    Article Title: Increased Haematopoietic Supportive Function of USSC from Umbilical Cord Blood Compared to CB MSC and Possible Role of DLK-1

    doi: 10.1155/2013/985285

    Figure Lengend Snippet: Haematopoiesis-supporting capacity of USSC and CB MSC. (a) Representative co-culture of CD34 + -cells on feeders of USSC, CB MSC, and CB MSC overexpressing DLK-1 (CB MSC DLK ) in multiple independent wells ( n = 6). With regards to total cell count, expansion of CD34 + -cells and colony-forming units (CFU), co-culture on a USSC-feeder resulted in higher expansion rates than on CB MSC-feeder. Within 14 days, total cell count increased significantly stronger on USSC-feeder (by factor 35.05 ± 3.75) than on CB MSC-feeder (factor 10.09 ± 0.37; P

    Article Snippet: DLK-1 Overexpression As described previously [ ], full-length human DLK-1 was amplified using Phusion Taq Polymerase (New England Biolabs).

    Techniques: Co-Culture Assay, Cell Counting

    Effect of DLK-1-overexpression on the haematopoiesis-supporting capacity of CB MSC. (a) Exemplarily co-culture of CD34 + -cells on feeders of CB MSC and FACS-sorted CB MSC strongly overexpressing DLK-1 (CB MSC DLK high ) in multiple independent wells ( n = 4). Within 14 days, overexpression of DLK-1 did not have a beneficial effect on expansion rates of total cells, CD34 + -cells or CFU. In accordance to former results for native CB MSC, only a weak expansion of total cells was observed, while amounts of total CD34 + -cells as well as CFU slightly declined over time. (b) Expression levels of haematopoietic cytokines ( IGF-2 , IGFBP-1 , SCF , SDF-1 , THPO , ANGPTL-3 ) were analyzed by real time PCR exemplarily for a CB MSC line in native form, after induced overexpression of DLK-1 (CB MSC DLK ) as well as after further FACS-sorting for cells with high DLK-1 expression (CB MSC DLK high ), respectively. No significant differences in expression levels could be detected and results were in accordance with former data, as presented in Figure 4(b) .

    Journal: Stem Cells International

    Article Title: Increased Haematopoietic Supportive Function of USSC from Umbilical Cord Blood Compared to CB MSC and Possible Role of DLK-1

    doi: 10.1155/2013/985285

    Figure Lengend Snippet: Effect of DLK-1-overexpression on the haematopoiesis-supporting capacity of CB MSC. (a) Exemplarily co-culture of CD34 + -cells on feeders of CB MSC and FACS-sorted CB MSC strongly overexpressing DLK-1 (CB MSC DLK high ) in multiple independent wells ( n = 4). Within 14 days, overexpression of DLK-1 did not have a beneficial effect on expansion rates of total cells, CD34 + -cells or CFU. In accordance to former results for native CB MSC, only a weak expansion of total cells was observed, while amounts of total CD34 + -cells as well as CFU slightly declined over time. (b) Expression levels of haematopoietic cytokines ( IGF-2 , IGFBP-1 , SCF , SDF-1 , THPO , ANGPTL-3 ) were analyzed by real time PCR exemplarily for a CB MSC line in native form, after induced overexpression of DLK-1 (CB MSC DLK ) as well as after further FACS-sorting for cells with high DLK-1 expression (CB MSC DLK high ), respectively. No significant differences in expression levels could be detected and results were in accordance with former data, as presented in Figure 4(b) .

    Article Snippet: DLK-1 Overexpression As described previously [ ], full-length human DLK-1 was amplified using Phusion Taq Polymerase (New England Biolabs).

    Techniques: Over Expression, Co-Culture Assay, FACS, Expressing, Real-time Polymerase Chain Reaction

    DLK-1 is a marker to distinguish USSC from CB MSC in cord blood. (a) Differentiation capacity of CB-derived stromal cells. Basal characterization of CB-derived stromal cells revealed the adipogenic differentiation as discriminating characteristic between CB MSC ( n = 29) and USSC ( n = 61). CB MSC exhibited a high adipogenic differentiation potential, comparable to BM MSC, while USSC never formed lipid vacuoles following adipogenic induction as demonstrated by Oil Red O staining after 21 days of differentiation. (b) DLK-1 expression inversely correlated with the adipogenic differentiation potential. DLK-1 mRNA expression was significantly higher in USSC and clonal USSC lines compared to CB MSC and corresponding clonal populations. The DLK-1 expressing teratocarcinoma cell line nTERA-2 was tested as positive control, dermal fibroblasts (NHDF) served as biological negative control.

    Journal: Stem Cells International

    Article Title: Increased Haematopoietic Supportive Function of USSC from Umbilical Cord Blood Compared to CB MSC and Possible Role of DLK-1

    doi: 10.1155/2013/985285

    Figure Lengend Snippet: DLK-1 is a marker to distinguish USSC from CB MSC in cord blood. (a) Differentiation capacity of CB-derived stromal cells. Basal characterization of CB-derived stromal cells revealed the adipogenic differentiation as discriminating characteristic between CB MSC ( n = 29) and USSC ( n = 61). CB MSC exhibited a high adipogenic differentiation potential, comparable to BM MSC, while USSC never formed lipid vacuoles following adipogenic induction as demonstrated by Oil Red O staining after 21 days of differentiation. (b) DLK-1 expression inversely correlated with the adipogenic differentiation potential. DLK-1 mRNA expression was significantly higher in USSC and clonal USSC lines compared to CB MSC and corresponding clonal populations. The DLK-1 expressing teratocarcinoma cell line nTERA-2 was tested as positive control, dermal fibroblasts (NHDF) served as biological negative control.

    Article Snippet: DLK-1 Overexpression As described previously [ ], full-length human DLK-1 was amplified using Phusion Taq Polymerase (New England Biolabs).

    Techniques: Marker, Derivative Assay, Staining, Expressing, Positive Control, Negative Control

    DLK-1 in USSC: inhibitor of adipogenesis? (a) Representative immunofluorescence staining of CB MSC ( n = 3), USSC ( n = 4) and HepG2. USSC revealed a clear DLK-1 expression, detected by immunofluorescence, while CB MSC were completely negative. The hepatocarcinoma cell line HepG2 served as biological positive control. (b) DLK-1 flowcytometry analysis was applied to clearly distinguish between intracellular and extracellular expression of DLK-1 protein. No DLK-1 expression could be determined in USSC by extracellular staining. After permeabilization a small DLK-1 positive subpopulation was detected in the USSC lines by intracellular staining. DLK-1 overexpressing CB MSC were used a positive control for extracellular staining. (c) By ectodomain shedding, the soluble and adipogenic inhibitory DLK-1 protein is cleaved. To validate whether USSC express the functional DLK-1, an ELISA was performed. The supernatant of 3 days standard cultures of USSC ( n = 4) and CB MSC ( n = 3) was analyzed, maternal plasma was used as positive control. No specific DLK-1 release was detected in any of the analyzed USSC or CB MSC, questioning function of DLK-1 expression in USSC. However, a clear secretion of DLK-1 was detected in the CB MSC overexpressing DLK-1. (d) A heterogeneous expression of CEBP α (CB MSC n = 4, CB MSC-derived clones n = 3, USSC n = 2, USSC-derived clones n = 2) could be detected in different USSC and CB MSC cell lines analyzed by real time PCR analysis. (e) Real time PCR analysis revealed a heterogenous expression of PPAR γ 2 expression (CB MSC n = 3, USSC n = 4) in the USSC and CB MSC cell lines.

    Journal: Stem Cells International

    Article Title: Increased Haematopoietic Supportive Function of USSC from Umbilical Cord Blood Compared to CB MSC and Possible Role of DLK-1

    doi: 10.1155/2013/985285

    Figure Lengend Snippet: DLK-1 in USSC: inhibitor of adipogenesis? (a) Representative immunofluorescence staining of CB MSC ( n = 3), USSC ( n = 4) and HepG2. USSC revealed a clear DLK-1 expression, detected by immunofluorescence, while CB MSC were completely negative. The hepatocarcinoma cell line HepG2 served as biological positive control. (b) DLK-1 flowcytometry analysis was applied to clearly distinguish between intracellular and extracellular expression of DLK-1 protein. No DLK-1 expression could be determined in USSC by extracellular staining. After permeabilization a small DLK-1 positive subpopulation was detected in the USSC lines by intracellular staining. DLK-1 overexpressing CB MSC were used a positive control for extracellular staining. (c) By ectodomain shedding, the soluble and adipogenic inhibitory DLK-1 protein is cleaved. To validate whether USSC express the functional DLK-1, an ELISA was performed. The supernatant of 3 days standard cultures of USSC ( n = 4) and CB MSC ( n = 3) was analyzed, maternal plasma was used as positive control. No specific DLK-1 release was detected in any of the analyzed USSC or CB MSC, questioning function of DLK-1 expression in USSC. However, a clear secretion of DLK-1 was detected in the CB MSC overexpressing DLK-1. (d) A heterogeneous expression of CEBP α (CB MSC n = 4, CB MSC-derived clones n = 3, USSC n = 2, USSC-derived clones n = 2) could be detected in different USSC and CB MSC cell lines analyzed by real time PCR analysis. (e) Real time PCR analysis revealed a heterogenous expression of PPAR γ 2 expression (CB MSC n = 3, USSC n = 4) in the USSC and CB MSC cell lines.

    Article Snippet: DLK-1 Overexpression As described previously [ ], full-length human DLK-1 was amplified using Phusion Taq Polymerase (New England Biolabs).

    Techniques: Immunofluorescence, Staining, Expressing, Positive Control, Functional Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Clone Assay, Real-time Polymerase Chain Reaction

    HOXA7 regulates granulosa cell proliferation . KGN cells were transiently transfected with scrambled siRNA or HOXA7 siRNA and the effect of HOXA7 knockdown was verified at the mRNA level by real-time PCR (A) and protein level by Western blotting (B). (C) After 36 h of transfection, KGN cells were reseeded into a 96-well plate and the cell viability was detected by MTT assay. SVOG cells were transiently transfected with control vector or HOXA7 plasmid and the effect of HOXA7 overexpression was verified at the mRNA level by real-time PCR (D) and protein level by Western blotting (E). (F) After 36 h of transfection, SVOG cells were reseeded into a 96-well plate and the cell viability was detected by MTT assay. The data derived from at least three separate sets of experiments were standardized to the corresponding control. a, P

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: Homeobox A7 increases cell proliferation by up-regulation of epidermal growth factor receptor expression in human granulosa cells

    doi: 10.1186/1477-7827-8-61

    Figure Lengend Snippet: HOXA7 regulates granulosa cell proliferation . KGN cells were transiently transfected with scrambled siRNA or HOXA7 siRNA and the effect of HOXA7 knockdown was verified at the mRNA level by real-time PCR (A) and protein level by Western blotting (B). (C) After 36 h of transfection, KGN cells were reseeded into a 96-well plate and the cell viability was detected by MTT assay. SVOG cells were transiently transfected with control vector or HOXA7 plasmid and the effect of HOXA7 overexpression was verified at the mRNA level by real-time PCR (D) and protein level by Western blotting (E). (F) After 36 h of transfection, SVOG cells were reseeded into a 96-well plate and the cell viability was detected by MTT assay. The data derived from at least three separate sets of experiments were standardized to the corresponding control. a, P

    Article Snippet: Plasmid constructs and transfection Full-length HOXA7 (917 bp) was amplified by RT-PCR using the Phusion RT-PCR Kit (New England BioLabs, Ipswich, MA, USA) and the following primers (5'-3'): TCA TTC CTC CTC GTC C and ATG AGT TCT TCG TAT TAT GAA CG.

    Techniques: Transfection, Real-time Polymerase Chain Reaction, Western Blot, MTT Assay, Plasmid Preparation, Over Expression, Derivative Assay

    EGF-induced granulosa cell proliferation and activation of downstream signaling are modulated by the expression of HOXA7 . KGN cells were transiently transfected with scrambled siRNA or HOXA7 siRNA. SVOG cells were transiently transfected with control vector or HOXA7 plasmid. KGN cells (A) and SVOG cells (C) were reseeded into a 96-well plate after 36 h of transfection. Cells were serum starved for 12 h and incubated in serum-free medium containing 100 ng/mL EGF and/or AG1478, or were left untreated (Control) for 48 h. Cell viability was measured by the MTT assay. KGN cells (B) and SVOG cells (D) were serum starved for 12 h after 36 h transfection and incubated in serum free medium containing 100 ng/mL EGF and/or AG1478, or left untreated (Control) for 10 min. The phosphorylation of ERK1/2 or Akt was determined by Western blotting with specific antibodies. The data derived from at least three separate sets of experiments were standardized to the corresponding control, and the statistical results are presented in the column graphs. a, P

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: Homeobox A7 increases cell proliferation by up-regulation of epidermal growth factor receptor expression in human granulosa cells

    doi: 10.1186/1477-7827-8-61

    Figure Lengend Snippet: EGF-induced granulosa cell proliferation and activation of downstream signaling are modulated by the expression of HOXA7 . KGN cells were transiently transfected with scrambled siRNA or HOXA7 siRNA. SVOG cells were transiently transfected with control vector or HOXA7 plasmid. KGN cells (A) and SVOG cells (C) were reseeded into a 96-well plate after 36 h of transfection. Cells were serum starved for 12 h and incubated in serum-free medium containing 100 ng/mL EGF and/or AG1478, or were left untreated (Control) for 48 h. Cell viability was measured by the MTT assay. KGN cells (B) and SVOG cells (D) were serum starved for 12 h after 36 h transfection and incubated in serum free medium containing 100 ng/mL EGF and/or AG1478, or left untreated (Control) for 10 min. The phosphorylation of ERK1/2 or Akt was determined by Western blotting with specific antibodies. The data derived from at least three separate sets of experiments were standardized to the corresponding control, and the statistical results are presented in the column graphs. a, P

    Article Snippet: Plasmid constructs and transfection Full-length HOXA7 (917 bp) was amplified by RT-PCR using the Phusion RT-PCR Kit (New England BioLabs, Ipswich, MA, USA) and the following primers (5'-3'): TCA TTC CTC CTC GTC C and ATG AGT TCT TCG TAT TAT GAA CG.

    Techniques: Activation Assay, Expressing, Transfection, Plasmid Preparation, Incubation, MTT Assay, Western Blot, Derivative Assay

    Analysis of loss of heterozygosity in rare truncation and missense variants. ( a ) Bar plot shows individual truncations from nine genes (FDR shown) with lengths representing ratios of tumour-to-normal variant allele fractions (that is, the fraction of reads containing the variant allele). Statistically significant events, defined as FDR≤5%, are shaded boldly, while non-significant events are muted, with colours corresponding to genes. Cancer source of each truncation is shown underneath, for example, most BRCA1 variants occur in ovarian and breast cancers and all BAP1 variants in KIRC. ( b ) Bar plot for individual missense variants from four genes having elevated frequencies of such variants that show very significant LOH, that is, at the 1% FDR level. ( c ) Dot plot shows individual missense variants where abscissa and ordinate are amino acid positions and the ratio of tumour-to-normal variant allele fraction, respectively. Blue and red indicate significant (FDR ≤5%) and non-significant events, respectively, with size of dots proportional to negative log of the FDR. Annotated domains from the PFAM database are aligned with position, while shaded areas indicate ‘hotspot' regions where variants having significant LOH cluster more than the rate explainable by chance. Plots are shown for ATM , BRCA1 , BRCA2 , FANCA and FANCM .

    Journal: Nature Communications

    Article Title: Patterns and functional implications of rare germline variants across 12 cancer types

    doi: 10.1038/ncomms10086

    Figure Lengend Snippet: Analysis of loss of heterozygosity in rare truncation and missense variants. ( a ) Bar plot shows individual truncations from nine genes (FDR shown) with lengths representing ratios of tumour-to-normal variant allele fractions (that is, the fraction of reads containing the variant allele). Statistically significant events, defined as FDR≤5%, are shaded boldly, while non-significant events are muted, with colours corresponding to genes. Cancer source of each truncation is shown underneath, for example, most BRCA1 variants occur in ovarian and breast cancers and all BAP1 variants in KIRC. ( b ) Bar plot for individual missense variants from four genes having elevated frequencies of such variants that show very significant LOH, that is, at the 1% FDR level. ( c ) Dot plot shows individual missense variants where abscissa and ordinate are amino acid positions and the ratio of tumour-to-normal variant allele fraction, respectively. Blue and red indicate significant (FDR ≤5%) and non-significant events, respectively, with size of dots proportional to negative log of the FDR. Annotated domains from the PFAM database are aligned with position, while shaded areas indicate ‘hotspot' regions where variants having significant LOH cluster more than the rate explainable by chance. Plots are shown for ATM , BRCA1 , BRCA2 , FANCA and FANCM .

    Article Snippet: Functional validation of BRCA1 variants Variants were incorporated into a full-length BRCA1 expression plasmid, pcDNA-5′HA-BRCA1, using Q5 site-directed mutagenesis kit (New England BioLabs).

    Techniques: Variant Assay

    Burden analysis reveals distinct set of cancer susceptibility genes across 12 cancer types. A total of 34 genes-of-interest were identified by burden analysis by comparing the frequencies of rare truncation variants in Caucasian cancer cases ( n =3,125) versus their frequencies in the WHI control population ( n =1,039). Two oncogenes ( ABL2 and BCR) were omitted. ( a ) Significant genes across Pan-Cancer types. Data were analysed with the total frequency test (TFT) followed by false discovery rate (FDR) ranking. Dark horizontal line indicates the 5% FDR threshold, which is satisfied by five genes, including BRCA1, BRCA2, ATM, BRIP1 and PALB2 . Inset shows closer visual resolution. ( b ) Significant genes for specific cancer types. Each plot shows the top tested genes, by FDR, from the same TFT analysis procedure for all 12 individual cancer types. Eight genes in addition to the five shown in a are significant at the 5% FDR level from cancer-type-specific analysis. ( c ) Cohort frequencies of genes. Bubble plot shows frequency of rare truncation mutation as a percentage of cases in each cohort (all 4,034 cases included for frequency calculation). The x -axis denotes the test group of a specific cancer type, the Pan-Cancer discovery cohort (4,034) and the validation cohort (1,627). Genes found to be significant at 5% FDR using the Pan-Cancer discovery cohort are labelled in boldface. Rings indicate genes that are significant (TFT, FDR ≤5%) for a particular cohort on the x -axis. ( d ) Percentage of cases carrying rare truncation in the 34 genes-of-interest across 12 cancer types in the discovery cohort.

    Journal: Nature Communications

    Article Title: Patterns and functional implications of rare germline variants across 12 cancer types

    doi: 10.1038/ncomms10086

    Figure Lengend Snippet: Burden analysis reveals distinct set of cancer susceptibility genes across 12 cancer types. A total of 34 genes-of-interest were identified by burden analysis by comparing the frequencies of rare truncation variants in Caucasian cancer cases ( n =3,125) versus their frequencies in the WHI control population ( n =1,039). Two oncogenes ( ABL2 and BCR) were omitted. ( a ) Significant genes across Pan-Cancer types. Data were analysed with the total frequency test (TFT) followed by false discovery rate (FDR) ranking. Dark horizontal line indicates the 5% FDR threshold, which is satisfied by five genes, including BRCA1, BRCA2, ATM, BRIP1 and PALB2 . Inset shows closer visual resolution. ( b ) Significant genes for specific cancer types. Each plot shows the top tested genes, by FDR, from the same TFT analysis procedure for all 12 individual cancer types. Eight genes in addition to the five shown in a are significant at the 5% FDR level from cancer-type-specific analysis. ( c ) Cohort frequencies of genes. Bubble plot shows frequency of rare truncation mutation as a percentage of cases in each cohort (all 4,034 cases included for frequency calculation). The x -axis denotes the test group of a specific cancer type, the Pan-Cancer discovery cohort (4,034) and the validation cohort (1,627). Genes found to be significant at 5% FDR using the Pan-Cancer discovery cohort are labelled in boldface. Rings indicate genes that are significant (TFT, FDR ≤5%) for a particular cohort on the x -axis. ( d ) Percentage of cases carrying rare truncation in the 34 genes-of-interest across 12 cancer types in the discovery cohort.

    Article Snippet: Functional validation of BRCA1 variants Variants were incorporated into a full-length BRCA1 expression plasmid, pcDNA-5′HA-BRCA1, using Q5 site-directed mutagenesis kit (New England BioLabs).

    Techniques: Mutagenesis

    Germline variants correlate with somatic mutations and age at diagnosis. ( a ) Barplot illustrates the distribution of BRCA1 , BRCA2 and ATM somatic and germline mutations across cancer types. ( b , c ) Panels display genes significantly correlated with somatic mutation frequency and younger age of onset in different cancer types and in Pan-Cancer. The width of the shape indicates the density, and the horizontal line indicates the median. P value is calculated by the Wilcoxon rank-sum test and is indicated by the size of the uppermost circles.

    Journal: Nature Communications

    Article Title: Patterns and functional implications of rare germline variants across 12 cancer types

    doi: 10.1038/ncomms10086

    Figure Lengend Snippet: Germline variants correlate with somatic mutations and age at diagnosis. ( a ) Barplot illustrates the distribution of BRCA1 , BRCA2 and ATM somatic and germline mutations across cancer types. ( b , c ) Panels display genes significantly correlated with somatic mutation frequency and younger age of onset in different cancer types and in Pan-Cancer. The width of the shape indicates the density, and the horizontal line indicates the median. P value is calculated by the Wilcoxon rank-sum test and is indicated by the size of the uppermost circles.

    Article Snippet: Functional validation of BRCA1 variants Variants were incorporated into a full-length BRCA1 expression plasmid, pcDNA-5′HA-BRCA1, using Q5 site-directed mutagenesis kit (New England BioLabs).

    Techniques: Mutagenesis

    Functional validation of BRCA1 missense and truncation variants. ( a ) 68 rare missense and 4 truncation variant sites were tested by HDR assay. All samples were depleted of endogenous BRCA1 by transfection of a siRNA targeting the 3′-untranslated region. Indicated in the legend are the plasmids transfected to test for rescue of BRCA1 activity. ‘pcDNA3' is empty vector and ‘WT' represents wild-type BRCA1 plasmid. The y -axis denotes the HDR activity relative to the wild-type BRCA1 protein. Error bars depict s.d. from the mean. Dots on the x -axis represent LOH status, each dot corresponding to one case. Blue, red, dark grey and light grey denote statistical significance, non-significance, unknown LOH (due to lack of sufficient coverage) and untested, respectively. Variants in different functional domains are indicated with colours as follows: orange, RING domain; green, nuclear localization signal (NLS); blue, DNA-binding region; purple, a SQ/TQ cluster domain (SCD); and red, BRCA1 C-terminal domain (BRCT). All the HDR assays were tested in triplicate. ( b ) Crystal structure of the BRCA1 RING (left) domain in complex with the BARD1 RING domain (labelled in grey) and BRCT domain (right panel) are displayed, with HDR-defective variants labelled in red and partial HDR-defective variants tagged in orange. Variants in yellow are functional in the HDR assay.

    Journal: Nature Communications

    Article Title: Patterns and functional implications of rare germline variants across 12 cancer types

    doi: 10.1038/ncomms10086

    Figure Lengend Snippet: Functional validation of BRCA1 missense and truncation variants. ( a ) 68 rare missense and 4 truncation variant sites were tested by HDR assay. All samples were depleted of endogenous BRCA1 by transfection of a siRNA targeting the 3′-untranslated region. Indicated in the legend are the plasmids transfected to test for rescue of BRCA1 activity. ‘pcDNA3' is empty vector and ‘WT' represents wild-type BRCA1 plasmid. The y -axis denotes the HDR activity relative to the wild-type BRCA1 protein. Error bars depict s.d. from the mean. Dots on the x -axis represent LOH status, each dot corresponding to one case. Blue, red, dark grey and light grey denote statistical significance, non-significance, unknown LOH (due to lack of sufficient coverage) and untested, respectively. Variants in different functional domains are indicated with colours as follows: orange, RING domain; green, nuclear localization signal (NLS); blue, DNA-binding region; purple, a SQ/TQ cluster domain (SCD); and red, BRCA1 C-terminal domain (BRCT). All the HDR assays were tested in triplicate. ( b ) Crystal structure of the BRCA1 RING (left) domain in complex with the BARD1 RING domain (labelled in grey) and BRCT domain (right panel) are displayed, with HDR-defective variants labelled in red and partial HDR-defective variants tagged in orange. Variants in yellow are functional in the HDR assay.

    Article Snippet: Functional validation of BRCA1 variants Variants were incorporated into a full-length BRCA1 expression plasmid, pcDNA-5′HA-BRCA1, using Q5 site-directed mutagenesis kit (New England BioLabs).

    Techniques: Functional Assay, Variant Assay, Transfection, Activity Assay, Plasmid Preparation, Binding Assay