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  • 94
    Thermo Fisher pbr322 plasmid dna
    GSH potentiates kinamycin F-induced <t>DNA</t> nicking. (A) This fluorescent image of the ethidium bromide-stained gel shows that when GSH (5 mM) was added to reaction mixtures containing various amounts of kinamycin F, the amount of DNA nicking was increased. In the absence of GSH a slight increase in nicking was seen at 400 μM kinamycin F. (B) In this image it is shown that deferoxamine (100 μM) and DMSO (0.5% (v/v)) pre-treatment of the DNA reduced the amount of DNA nicking induced by kinamycin F (200 μM) and GSH (5 mM). (C) In this image it is shown that catalase (50 μg/ml) decreased the amount of DNA nicking, but boiled catalase (b, 50 μg/ml) did not. Catalase was added immediately prior to the kinamycin F (200 μM) and GSH (5 mM). All lanes contained 80 ng of <t>pBR322</t> plasmid DNA, except for those with linear DNA which contained 80 ng of linear pBR322 plasmid DNA. Linear DNA was present in lane 7 and lanes 4 and 11 in (A) and (B), respectively. NC, nicked circular DNA; LIN, linear DNA; SC, supercoiled DNA. All incubations for the DNA nicking experiments were for 5 h at 37°C.
    Pbr322 Plasmid Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore plasmid dna
    Human UBL5 is required for cell proliferation and sister chromatid cohesion maintenance A Whole-cell extracts (WCE) of HeLa cells transfected with non-targeting (CTRL) or UBL5 siRNAs for 48 h were immunoblotted with anti-UBL5 antibody. B HeLa cells were counted at indicated times after transfection with control, UBL5, or SGO1 siRNAs [mean ± SD (error bars); N = 3]. C Sub-G1 <t>DNA</t> content of HeLa cells transfected with indicated siRNAs for 72 h was measured by flow cytometry analysis of propidium iodide-labeled cells [mean ± SD (error bars); N = 3]. D Mitotic indices of HeLa cells transfected with indicated siRNAs for 48 h were quantified by flow cytometry analysis of the proportion of phospho-H3(Ser10)-positive cells [mean ± SD (error bars); N = 3]. E Mitotic duration from nuclear envelope breakdown to chromatin decondensation in single live HeLa/H2B-mCherry cells transfected with control ( n = 20) or UBL5 <t>siRNA</t> ( n = 80) was monitored by time-lapse microscopy and classified as normal (45–60 min), delayed (longer than 60 min), or arrested (no decondensation). F Time-lapse analysis of mitotic chromosome dynamics in live HeLa/H2B-mCherry cells transfected with control or UBL5 siRNA. Selected time frames of representative mitotic progression patterns and their frequencies are shown. . H Quantification of precocious sister chromatid separation in HeLa cells after knockdown of UBL5 [mean ± SD (error bars); N = 3]. At least 100 metaphase spreads were counted. I HeLa cells inducibly expressing siRNA-resistant (si R ) Strep-HA-UBL5 were transfected with control or UBL5 siRNA in the absence or presence of doxycycline (DOX). Two days later, cells were treated with nocodazole for 3 h and harvested for chromosome analysis.
    Plasmid Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7013 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Promega plasmid dna pdna
    <t>PsiCHECK</t> ™ -2 vector map. It is a specifically designed 6.3kb <t>pDNA</t> for RNAi experiments. It has two different reporter genes, firefly (hluc+) luciferase with HSV-TK promoter and Renilla (hRluc) luciferase with SV40 promoter, and a synthetic poly(A)
    Plasmid Dna Pdna, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore plasmid dna pdna
    The effect of the addition of citrate in different concentrations to <t>CaP-DNA</t> particles on the fluorescence of MC3T3-E1 cell line transfected with GFP using these particles (a) and <t>pDNA-CaP</t> binding efficiency as a function of the concentration of citric
    Plasmid Dna Pdna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Becton Dickinson plasmid dna plasmid dna pires2 egfp pdna
    The effect of the addition of citrate in different concentrations to <t>CaP-DNA</t> particles on the fluorescence of MC3T3-E1 cell line transfected with GFP using these particles (a) and <t>pDNA-CaP</t> binding efficiency as a function of the concentration of citric
    Plasmid Dna Plasmid Dna Pires2 Egfp Pdna, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Eton Bioscience plasmid dna pdna
    Plasmid <t>DNA</t> containing human CD59 on a high-yield promoter <t>(pDNA,</t> A) was incorporated into chitosan microparticles (pCsM, B). Both pCsM and microparticles without pDNA (CsM, C) were precipitated from chitosan polymers (Cs, D). All chitosan treatments were prepared and delivered in mildly acidic Vehicle (pH 5.6, E).
    Plasmid Dna Pdna, supplied by Eton Bioscience, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    NanoVector plasmid dna pdna
    Plasmid <t>DNA</t> containing human CD59 on a high-yield promoter <t>(pDNA,</t> A) was incorporated into chitosan microparticles (pCsM, B). Both pCsM and microparticles without pDNA (CsM, C) were precipitated from chitosan polymers (Cs, D). All chitosan treatments were prepared and delivered in mildly acidic Vehicle (pH 5.6, E).
    Plasmid Dna Pdna, supplied by NanoVector, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Aldevron plasmid dna pdna
    Scatterplots showing the relative number of up- (▲) and downregulated (■) genes for the jetPEI ™ and <t>GFP</t> <t>pDNA</t> (CMV) polyplex treatments compared to the untreated control at 24 h following transfection. (a) and (b) Cell cycle array.
    Plasmid Dna Pdna, supplied by Aldevron, used in various techniques. Bioz Stars score: 91/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    PlasmidFactory plasmid dna pdna
    Scatterplots showing the relative number of up- (▲) and downregulated (■) genes for the jetPEI ™ and <t>GFP</t> <t>pDNA</t> (CMV) polyplex treatments compared to the untreated control at 24 h following transfection. (a) and (b) Cell cycle array.
    Plasmid Dna Pdna, supplied by PlasmidFactory, used in various techniques. Bioz Stars score: 90/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Kaneka Corp plasmid dna
    HDAC1 promoter activity is repressed by a dominant-negative mutant of NF-YA. U2OS cells were transiently <t>transfected</t> with 0.5 μg of HDAC1 pP721 wild-type reporter plasmid (wt) or the corresponding CCAAT mutant reporter plasmid (CCAAT mut) without (−) or together with 0.5 or 1 μg of expression vector encoding dominant-negative NF-YA (NFY dn) and a β-galactosidase reference vector. As a control, the reporter plasmids were cotransfected with 1 μg of the expression plasmid pNF-YA13 encoding wild-type NF-YA (NFY wt). pCIneo empty vector was used to bring the total amount of the <t>DNA</t> mixture to 2 μg. The activity (in relative light units [RLU]) of luciferase relative to that of β-galactosidase is presented. The means ± standard deviations of three independent experiments are shown.
    Plasmid Dna, supplied by Kaneka Corp, used in various techniques. Bioz Stars score: 92/100, based on 117 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    tiangen biotech co plasmid dna pdna preparation
    TEM, particle size, zeta potential, agarose gel electrophoresis, and NMR images. Notes: All nanoparticles, PEI ( A ), PV 10 ( B ), PV 20 ( C ), and PV 40 ( D ), were made at an N/P of 10 (scale bar: 100 nm). The detailed data of particle size and zeta potential are shown in ( E ). ( F ) shows the <t>DNA</t> ladder marker (a) and that PEI complexed DNA completely at an N/P of 2, PV 10 at 3, PV 20 at 5, and PV 40 at 6 (b). The different results from gel electrophoresis of PEI (c), PV 10 (d), PV 20 (e), and PV 40 (f) complexed with <t>pDNA</t> at various N/P ratios are shown. ( G ) shows the characterization of PV polymer by 1H NMR spectra. The PEI was activated into PEI-PDT by SPDP, and then the PEI-PDT was conjugated with VTW peptide and made into PV. Abbreviations: NMR, nuclear magnetic resonance; N/P, nitrogen in PEI amino residues to phosphates of DNA; PDT, pyridyldithio; PEI, polyethylenimine; PV, VTW-modified PEI copolymers; SPDP, N-succinimidyl 3-(2-pyridyldithio)-propionate; TEM, transmission electron microscopy; VTW, VTWTPQAWFQWV; pDNA, plasmid DNA.
    Plasmid Dna Pdna Preparation, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 85/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    TaKaRa plasmid dna
    <t>DNA</t> replication activities of E1 mutants. (A) Relative DNA replication (percentage of signal obtained with 5 ng of WT E1) of the indicated HPV31 <t>EYFP-E1</t> WT or mutants were tested using three different amounts of expression vector (1.25, 2.5, and 5 ng) as described in the text. The MFI mutation changes the three hydrophobic residues of the E1 MxxFI motif to three alanines. (B) Western blot showing the expression of the different EYFP-tagged E1 mutants. Tubulin was used as a loading control. α-GFP, antibody against green fluorescent protein. (C) Fluorescence confocal microscopy images showing the intracellular localization of the indicated E1 mutant proteins fused to EYFP in C33A cells. Nuclei (DNA) were stained with TO-PRO 3.
    Plasmid Dna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 2633 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    JenKem plasmid dna pdna pegfpluc
    <t>DNA</t> replication activities of E1 mutants. (A) Relative DNA replication (percentage of signal obtained with 5 ng of WT E1) of the indicated HPV31 <t>EYFP-E1</t> WT or mutants were tested using three different amounts of expression vector (1.25, 2.5, and 5 ng) as described in the text. The MFI mutation changes the three hydrophobic residues of the E1 MxxFI motif to three alanines. (B) Western blot showing the expression of the different EYFP-tagged E1 mutants. Tubulin was used as a loading control. α-GFP, antibody against green fluorescent protein. (C) Fluorescence confocal microscopy images showing the intracellular localization of the indicated E1 mutant proteins fused to EYFP in C33A cells. Nuclei (DNA) were stained with TO-PRO 3.
    Plasmid Dna Pdna Pegfpluc, supplied by JenKem, 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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    88
    MACHEREY NAGEL endotoxin free plasmid dna pdna purification kits
    <t>DNA</t> replication activities of E1 mutants. (A) Relative DNA replication (percentage of signal obtained with 5 ng of WT E1) of the indicated HPV31 <t>EYFP-E1</t> WT or mutants were tested using three different amounts of expression vector (1.25, 2.5, and 5 ng) as described in the text. The MFI mutation changes the three hydrophobic residues of the E1 MxxFI motif to three alanines. (B) Western blot showing the expression of the different EYFP-tagged E1 mutants. Tubulin was used as a loading control. α-GFP, antibody against green fluorescent protein. (C) Fluorescence confocal microscopy images showing the intracellular localization of the indicated E1 mutant proteins fused to EYFP in C33A cells. Nuclei (DNA) were stained with TO-PRO 3.
    Endotoxin Free Plasmid Dna Pdna Purification Kits, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 88/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bioneer Corporation pbt7 plasmid dna pdna
    <t>DNA</t> replication activities of E1 mutants. (A) Relative DNA replication (percentage of signal obtained with 5 ng of WT E1) of the indicated HPV31 <t>EYFP-E1</t> WT or mutants were tested using three different amounts of expression vector (1.25, 2.5, and 5 ng) as described in the text. The MFI mutation changes the three hydrophobic residues of the E1 MxxFI motif to three alanines. (B) Western blot showing the expression of the different EYFP-tagged E1 mutants. Tubulin was used as a loading control. α-GFP, antibody against green fluorescent protein. (C) Fluorescence confocal microscopy images showing the intracellular localization of the indicated E1 mutant proteins fused to EYFP in C33A cells. Nuclei (DNA) were stained with TO-PRO 3.
    Pbt7 Plasmid Dna Pdna, supplied by Bioneer Corporation, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore circular plasmid dna
    Effect of pH or temperature on <t>Rv0888</t> enzymatic activity. ( A ) Analysis of the effect of temperature on Rv0888 enzymatic activity by agarose gel electrophoresis. The reaction was performed in 20 mM Tris-HCl pH 6.5, 5 mM CaCl 2 and 5 mM MnCl 2 for 1 h. Line M: DL5000 <t>DNA</t> Marker; Line 1: circular plasmid DNA; Lines 2-11: circular plasmid DNA and Rv0888 ranging between 33 °C and 51 °C at 2 °C intervals. ( B ) Quantification of DNase activity by spectrophotometry and error bars are given as standard deviations. ( C ) Analysis of the effect of the pH on Rv0888 enzymatic activity by agarose gel electrophoresis. The reaction was performed in 20 mM Tris-HCl, 5 mM CaCl 2 and 5 mM MnCl 2 for 1 h at 37 °C. Line M: DL5000 DNA Marker; Lines 1, 3, 5, 7, 9: circular plasmid DNA ranging between pH 6.0 and pH 8.0 at 0.5 intervals; Lines 2, 4, 6, 8, 10: circular plasmid DNA and Rv0888 ranging between pH 6.0 and pH 8.0 at 0.5 intervals. ( D ) DNase activity was quantified by spectrophotometry and error bars are given as standard deviations.
    Circular Plasmid Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    GSH potentiates kinamycin F-induced DNA nicking. (A) This fluorescent image of the ethidium bromide-stained gel shows that when GSH (5 mM) was added to reaction mixtures containing various amounts of kinamycin F, the amount of DNA nicking was increased. In the absence of GSH a slight increase in nicking was seen at 400 μM kinamycin F. (B) In this image it is shown that deferoxamine (100 μM) and DMSO (0.5% (v/v)) pre-treatment of the DNA reduced the amount of DNA nicking induced by kinamycin F (200 μM) and GSH (5 mM). (C) In this image it is shown that catalase (50 μg/ml) decreased the amount of DNA nicking, but boiled catalase (b, 50 μg/ml) did not. Catalase was added immediately prior to the kinamycin F (200 μM) and GSH (5 mM). All lanes contained 80 ng of pBR322 plasmid DNA, except for those with linear DNA which contained 80 ng of linear pBR322 plasmid DNA. Linear DNA was present in lane 7 and lanes 4 and 11 in (A) and (B), respectively. NC, nicked circular DNA; LIN, linear DNA; SC, supercoiled DNA. All incubations for the DNA nicking experiments were for 5 h at 37°C.

    Journal: Free radical biology & medicine

    Article Title: Mechanism of the cytotoxicity of the diazoparaquinone antitumor antibiotic kinamycin F

    doi: 10.1016/j.freeradbiomed.2007.07.005

    Figure Lengend Snippet: GSH potentiates kinamycin F-induced DNA nicking. (A) This fluorescent image of the ethidium bromide-stained gel shows that when GSH (5 mM) was added to reaction mixtures containing various amounts of kinamycin F, the amount of DNA nicking was increased. In the absence of GSH a slight increase in nicking was seen at 400 μM kinamycin F. (B) In this image it is shown that deferoxamine (100 μM) and DMSO (0.5% (v/v)) pre-treatment of the DNA reduced the amount of DNA nicking induced by kinamycin F (200 μM) and GSH (5 mM). (C) In this image it is shown that catalase (50 μg/ml) decreased the amount of DNA nicking, but boiled catalase (b, 50 μg/ml) did not. Catalase was added immediately prior to the kinamycin F (200 μM) and GSH (5 mM). All lanes contained 80 ng of pBR322 plasmid DNA, except for those with linear DNA which contained 80 ng of linear pBR322 plasmid DNA. Linear DNA was present in lane 7 and lanes 4 and 11 in (A) and (B), respectively. NC, nicked circular DNA; LIN, linear DNA; SC, supercoiled DNA. All incubations for the DNA nicking experiments were for 5 h at 37°C.

    Article Snippet: Thioglo-1 was from EMD Biosciences (Mississauga, Canada), kDNA was obtained from TopoGEN (Columbus, OH) and pBR322 plasmid DNA was obtained from MBI Fermentas (Burlington, ON, Canada).

    Techniques: Staining, Plasmid Preparation

    Human UBL5 is required for cell proliferation and sister chromatid cohesion maintenance A Whole-cell extracts (WCE) of HeLa cells transfected with non-targeting (CTRL) or UBL5 siRNAs for 48 h were immunoblotted with anti-UBL5 antibody. B HeLa cells were counted at indicated times after transfection with control, UBL5, or SGO1 siRNAs [mean ± SD (error bars); N = 3]. C Sub-G1 DNA content of HeLa cells transfected with indicated siRNAs for 72 h was measured by flow cytometry analysis of propidium iodide-labeled cells [mean ± SD (error bars); N = 3]. D Mitotic indices of HeLa cells transfected with indicated siRNAs for 48 h were quantified by flow cytometry analysis of the proportion of phospho-H3(Ser10)-positive cells [mean ± SD (error bars); N = 3]. E Mitotic duration from nuclear envelope breakdown to chromatin decondensation in single live HeLa/H2B-mCherry cells transfected with control ( n = 20) or UBL5 siRNA ( n = 80) was monitored by time-lapse microscopy and classified as normal (45–60 min), delayed (longer than 60 min), or arrested (no decondensation). F Time-lapse analysis of mitotic chromosome dynamics in live HeLa/H2B-mCherry cells transfected with control or UBL5 siRNA. Selected time frames of representative mitotic progression patterns and their frequencies are shown. . H Quantification of precocious sister chromatid separation in HeLa cells after knockdown of UBL5 [mean ± SD (error bars); N = 3]. At least 100 metaphase spreads were counted. I HeLa cells inducibly expressing siRNA-resistant (si R ) Strep-HA-UBL5 were transfected with control or UBL5 siRNA in the absence or presence of doxycycline (DOX). Two days later, cells were treated with nocodazole for 3 h and harvested for chromosome analysis.

    Journal: EMBO Reports

    Article Title: UBL5 is essential for pre-mRNA splicing and sister chromatid cohesion in human cells

    doi: 10.15252/embr.201438679

    Figure Lengend Snippet: Human UBL5 is required for cell proliferation and sister chromatid cohesion maintenance A Whole-cell extracts (WCE) of HeLa cells transfected with non-targeting (CTRL) or UBL5 siRNAs for 48 h were immunoblotted with anti-UBL5 antibody. B HeLa cells were counted at indicated times after transfection with control, UBL5, or SGO1 siRNAs [mean ± SD (error bars); N = 3]. C Sub-G1 DNA content of HeLa cells transfected with indicated siRNAs for 72 h was measured by flow cytometry analysis of propidium iodide-labeled cells [mean ± SD (error bars); N = 3]. D Mitotic indices of HeLa cells transfected with indicated siRNAs for 48 h were quantified by flow cytometry analysis of the proportion of phospho-H3(Ser10)-positive cells [mean ± SD (error bars); N = 3]. E Mitotic duration from nuclear envelope breakdown to chromatin decondensation in single live HeLa/H2B-mCherry cells transfected with control ( n = 20) or UBL5 siRNA ( n = 80) was monitored by time-lapse microscopy and classified as normal (45–60 min), delayed (longer than 60 min), or arrested (no decondensation). F Time-lapse analysis of mitotic chromosome dynamics in live HeLa/H2B-mCherry cells transfected with control or UBL5 siRNA. Selected time frames of representative mitotic progression patterns and their frequencies are shown. . H Quantification of precocious sister chromatid separation in HeLa cells after knockdown of UBL5 [mean ± SD (error bars); N = 3]. At least 100 metaphase spreads were counted. I HeLa cells inducibly expressing siRNA-resistant (si R ) Strep-HA-UBL5 were transfected with control or UBL5 siRNA in the absence or presence of doxycycline (DOX). Two days later, cells were treated with nocodazole for 3 h and harvested for chromosome analysis.

    Article Snippet: Plasmid DNA and siRNA transfections were performed using GeneJuice (Novagen) and Lipofectamine RNAiMAX (Invitrogen), respectively, according to the manufacturer’s instructions.

    Techniques: Transfection, Flow Cytometry, Cytometry, Labeling, Time-lapse Microscopy, Expressing

    PsiCHECK ™ -2 vector map. It is a specifically designed 6.3kb pDNA for RNAi experiments. It has two different reporter genes, firefly (hluc+) luciferase with HSV-TK promoter and Renilla (hRluc) luciferase with SV40 promoter, and a synthetic poly(A)

    Journal: International Journal of Pharmaceutics

    Article Title: Synthesis and characterization of mannosylated pegylated polyethylenimine as a carrier for siRNA

    doi: 10.1016/j.ijpharm.2011.08.014

    Figure Lengend Snippet: PsiCHECK ™ -2 vector map. It is a specifically designed 6.3kb pDNA for RNAi experiments. It has two different reporter genes, firefly (hluc+) luciferase with HSV-TK promoter and Renilla (hRluc) luciferase with SV40 promoter, and a synthetic poly(A)

    Article Snippet: All the siRNAs (DS scrambled negative control, RLuc-S1 DS positive control, Cy-3™ , NC1, and HPRT) and pDNA (psiCHECK™ -2, Promega, Madison, WI) were kindly provided by Integrated DNA Technologies (Coralville, IA).

    Techniques: Plasmid Preparation, Luciferase

    The effect of the addition of citrate in different concentrations to CaP-DNA particles on the fluorescence of MC3T3-E1 cell line transfected with GFP using these particles (a) and pDNA-CaP binding efficiency as a function of the concentration of citric

    Journal: Journal of colloid and interface science

    Article Title: Gene delivery using calcium phosphate nanoparticles: Optimization of the transfection process and the effects of citrate and poly(l-lysine) as additives

    doi: 10.1016/j.jcis.2016.03.007

    Figure Lengend Snippet: The effect of the addition of citrate in different concentrations to CaP-DNA particles on the fluorescence of MC3T3-E1 cell line transfected with GFP using these particles (a) and pDNA-CaP binding efficiency as a function of the concentration of citric

    Article Snippet: Two precursor solutions were made in identical volumes, one containing 250 mM CaCl2 (Sigma-Aldrich) and 50 μg/ml plasmid DNA (pDNA) encoding for enhanced green fluorescent protein (eGFP) and the other one containing varying amounts of NaH2 PO4 (Sigma-Aldrich), 140 mM NaCl (Sigma-Aldrich) and 50 mM HEPES (pKa =7.5, Life Technologies). pH of the NaH2 PO4 solution was set to 7 unless noted otherwise. pDNA did not affect pH in the system.

    Techniques: Fluorescence, Transfection, Binding Assay, Concentration Assay

    Zeta potential of pure CaP, pure DNA and CaP-DNA complexes at pH 7 (a). The effect of varying the concentration of PLL (-●-) and citrate (-○-) during the precipitation of CaP-pDNA particles on their zeta potential (b). Data are shown as

    Journal: Journal of colloid and interface science

    Article Title: Gene delivery using calcium phosphate nanoparticles: Optimization of the transfection process and the effects of citrate and poly(l-lysine) as additives

    doi: 10.1016/j.jcis.2016.03.007

    Figure Lengend Snippet: Zeta potential of pure CaP, pure DNA and CaP-DNA complexes at pH 7 (a). The effect of varying the concentration of PLL (-●-) and citrate (-○-) during the precipitation of CaP-pDNA particles on their zeta potential (b). Data are shown as

    Article Snippet: Two precursor solutions were made in identical volumes, one containing 250 mM CaCl2 (Sigma-Aldrich) and 50 μg/ml plasmid DNA (pDNA) encoding for enhanced green fluorescent protein (eGFP) and the other one containing varying amounts of NaH2 PO4 (Sigma-Aldrich), 140 mM NaCl (Sigma-Aldrich) and 50 mM HEPES (pKa =7.5, Life Technologies). pH of the NaH2 PO4 solution was set to 7 unless noted otherwise. pDNA did not affect pH in the system.

    Techniques: Concentration Assay

    Plasmid DNA containing human CD59 on a high-yield promoter (pDNA, A) was incorporated into chitosan microparticles (pCsM, B). Both pCsM and microparticles without pDNA (CsM, C) were precipitated from chitosan polymers (Cs, D). All chitosan treatments were prepared and delivered in mildly acidic Vehicle (pH 5.6, E).

    Journal: Carbohydrate polymers

    Article Title: Stability and Bioactivity of Chitosan as a Transfection Agent in Primary Human Cell Cultures: A Case for Chitosan-only Controls

    doi: 10.1016/j.carbpol.2017.10.021

    Figure Lengend Snippet: Plasmid DNA containing human CD59 on a high-yield promoter (pDNA, A) was incorporated into chitosan microparticles (pCsM, B). Both pCsM and microparticles without pDNA (CsM, C) were precipitated from chitosan polymers (Cs, D). All chitosan treatments were prepared and delivered in mildly acidic Vehicle (pH 5.6, E).

    Article Snippet: Plasmid yield was determined by spectrophotometry (Nanodrop 1000, Thermo Fisher, Delaware) and plasmid DNA (pDNA) was commercially sequenced ( ) by Eton Bioscience (San Diego, California) with forward (VP1.5) and reverse (XL39) primers from Origene.

    Techniques: Plasmid Preparation

    Scatterplots showing the relative number of up- (▲) and downregulated (■) genes for the jetPEI ™ and GFP pDNA (CMV) polyplex treatments compared to the untreated control at 24 h following transfection. (a) and (b) Cell cycle array.

    Journal: Molecular pharmaceutics

    Article Title: Polyplex Exposure Inhibits Cell Cycle, Increases Inflammatory Response, and Can Cause Protein Expression Without Cell Division

    doi: 10.1021/mp300470d

    Figure Lengend Snippet: Scatterplots showing the relative number of up- (▲) and downregulated (■) genes for the jetPEI ™ and GFP pDNA (CMV) polyplex treatments compared to the untreated control at 24 h following transfection. (a) and (b) Cell cycle array.

    Article Snippet: The pDNAs used were 4.1 kb CFP-encoding pDNA (pAmCyan1-C1; Cat. No. 632441; Clontech; Mountain View, CA), 5.1 kb blank pDNA that encoded for no fluorescent reporter protein (gWIZ™ -blank; Cat. No. 5008; Aldevron; Fargo, ND), and 5.8 kb green fluorescent protein (GFP)-encoding pDNA (gWIZ™ -GFP; Cat. No. 5006; Aldevron).

    Techniques: Transfection

    HDAC1 promoter activity is repressed by a dominant-negative mutant of NF-YA. U2OS cells were transiently transfected with 0.5 μg of HDAC1 pP721 wild-type reporter plasmid (wt) or the corresponding CCAAT mutant reporter plasmid (CCAAT mut) without (−) or together with 0.5 or 1 μg of expression vector encoding dominant-negative NF-YA (NFY dn) and a β-galactosidase reference vector. As a control, the reporter plasmids were cotransfected with 1 μg of the expression plasmid pNF-YA13 encoding wild-type NF-YA (NFY wt). pCIneo empty vector was used to bring the total amount of the DNA mixture to 2 μg. The activity (in relative light units [RLU]) of luciferase relative to that of β-galactosidase is presented. The means ± standard deviations of three independent experiments are shown.

    Journal: Molecular and Cellular Biology

    Article Title: Autoregulation of Mouse Histone Deacetylase 1 Expression

    doi: 10.1128/MCB.23.19.6993-7004.2003

    Figure Lengend Snippet: HDAC1 promoter activity is repressed by a dominant-negative mutant of NF-YA. U2OS cells were transiently transfected with 0.5 μg of HDAC1 pP721 wild-type reporter plasmid (wt) or the corresponding CCAAT mutant reporter plasmid (CCAAT mut) without (−) or together with 0.5 or 1 μg of expression vector encoding dominant-negative NF-YA (NFY dn) and a β-galactosidase reference vector. As a control, the reporter plasmids were cotransfected with 1 μg of the expression plasmid pNF-YA13 encoding wild-type NF-YA (NFY wt). pCIneo empty vector was used to bring the total amount of the DNA mixture to 2 μg. The activity (in relative light units [RLU]) of luciferase relative to that of β-galactosidase is presented. The means ± standard deviations of three independent experiments are shown.

    Article Snippet: To generate stable cell lines, 5 μg of linearized plasmid DNA was transfected into Swiss 3T3 fibroblasts using DAC 30 transfection reagent (Eurogentec) as recommended by the supplier.

    Techniques: Activity Assay, Dominant Negative Mutation, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Luciferase

    The distal SP1 binding site and the CCAAT box are crucial for TSA-mediated activation and recruitment of HDAC1 to its promoter region. (A) Schematic representations of wild-type and mutated promoter regions of the mouse HDAC1 gene are on the left. Site-specific disruption of individual elements is indicated by crossed boxes. Stably transfected Swiss 3T3 cells containing the HDAC1 5′ deletion mutants were serum arrested for 48 h and treated with TSA. After a further 20 h, the cells were harvested and assayed for luciferase activity. Enzyme activity was normalized to the corresponding protein concentration and plotted relative to the untreated value set as 1. The means ± standard deviations of three independent experiments are shown. (B) Stably transfected Swiss 3T3 cells containing the HDAC1 point mutants were treated as described for panel A and examined for luciferase activity. (C) Chromatin immunoprecipitation analysis of stably transfected HDAC1 promoter P721 for the presence of HDAC1. Cross-linked chromatin of resting (rest) or TSA-treated (TSA) stable cell lines carrying the wild-type HDAC1 promoter (wt) or the double-mutated HDAC1 promoter (mut) was immunoprecipitated with an HDAC1 antibody (HDAC1) or a nonspecific antibody (un AB). DNA from the antibody-bound fraction and total input DNA isolated from chromatin used for the immunoprecipitation were analyzed by quantitative PCR using a primer specific for the transfected promoter (HD1/Luci).

    Journal: Molecular and Cellular Biology

    Article Title: Autoregulation of Mouse Histone Deacetylase 1 Expression

    doi: 10.1128/MCB.23.19.6993-7004.2003

    Figure Lengend Snippet: The distal SP1 binding site and the CCAAT box are crucial for TSA-mediated activation and recruitment of HDAC1 to its promoter region. (A) Schematic representations of wild-type and mutated promoter regions of the mouse HDAC1 gene are on the left. Site-specific disruption of individual elements is indicated by crossed boxes. Stably transfected Swiss 3T3 cells containing the HDAC1 5′ deletion mutants were serum arrested for 48 h and treated with TSA. After a further 20 h, the cells were harvested and assayed for luciferase activity. Enzyme activity was normalized to the corresponding protein concentration and plotted relative to the untreated value set as 1. The means ± standard deviations of three independent experiments are shown. (B) Stably transfected Swiss 3T3 cells containing the HDAC1 point mutants were treated as described for panel A and examined for luciferase activity. (C) Chromatin immunoprecipitation analysis of stably transfected HDAC1 promoter P721 for the presence of HDAC1. Cross-linked chromatin of resting (rest) or TSA-treated (TSA) stable cell lines carrying the wild-type HDAC1 promoter (wt) or the double-mutated HDAC1 promoter (mut) was immunoprecipitated with an HDAC1 antibody (HDAC1) or a nonspecific antibody (un AB). DNA from the antibody-bound fraction and total input DNA isolated from chromatin used for the immunoprecipitation were analyzed by quantitative PCR using a primer specific for the transfected promoter (HD1/Luci).

    Article Snippet: To generate stable cell lines, 5 μg of linearized plasmid DNA was transfected into Swiss 3T3 fibroblasts using DAC 30 transfection reagent (Eurogentec) as recommended by the supplier.

    Techniques: Binding Assay, Activation Assay, Stable Transfection, Transfection, Luciferase, Activity Assay, Protein Concentration, Chromatin Immunoprecipitation, Immunoprecipitation, Isolation, Real-time Polymerase Chain Reaction

    Involvement of histone acetyltransferases in the activation of the HDAC1 gene. (A) U2OS cells were transiently cotransfected with 0.5 μg of HDAC1 wild-type reporter plasmid (pP721) or with 0.5 or 1 μg of expression vectors encoding p300 or P/CAF, together with a β-galactosidase reference vector. As a control, the reporter plasmid was cotransfected with 1 μg of expression plasmids encoding the TK protein. pCIneo empty vector was used to bring the total amount of the DNA mixture to 2 μg. The activity of luciferase (in relative light units [RLU]) to relative to that of β-galactosidase is presented. The means ± standard deviations of three independent experiments are shown. (B) Cotransfection was carried out as described for panel A using HDAC1 point mutants (mut) as reporter constructs. (C) Chromatin immunoprecipitation performed with antibodies against acetylated histone H3 and H4 using chromatin isolated from stably transfected Swiss 3T3 cells carrying either the wild-type HDAC1 promoter (wt) or the double-mutated HDAC1 promoter (mut) before (rest) and after (TSA) TSA treatment. DNA from the antibody-bound fraction and total input DNA isolated from chromatin used for the immunoprecipitation were analyzed by quantitative PCR using a primer specific for the transfected promoter (HD1/Luci; see Materials and Methods). PCR products were quantified using the ImageQuant program, and relative signal intensities (-fold) are indicated.

    Journal: Molecular and Cellular Biology

    Article Title: Autoregulation of Mouse Histone Deacetylase 1 Expression

    doi: 10.1128/MCB.23.19.6993-7004.2003

    Figure Lengend Snippet: Involvement of histone acetyltransferases in the activation of the HDAC1 gene. (A) U2OS cells were transiently cotransfected with 0.5 μg of HDAC1 wild-type reporter plasmid (pP721) or with 0.5 or 1 μg of expression vectors encoding p300 or P/CAF, together with a β-galactosidase reference vector. As a control, the reporter plasmid was cotransfected with 1 μg of expression plasmids encoding the TK protein. pCIneo empty vector was used to bring the total amount of the DNA mixture to 2 μg. The activity of luciferase (in relative light units [RLU]) to relative to that of β-galactosidase is presented. The means ± standard deviations of three independent experiments are shown. (B) Cotransfection was carried out as described for panel A using HDAC1 point mutants (mut) as reporter constructs. (C) Chromatin immunoprecipitation performed with antibodies against acetylated histone H3 and H4 using chromatin isolated from stably transfected Swiss 3T3 cells carrying either the wild-type HDAC1 promoter (wt) or the double-mutated HDAC1 promoter (mut) before (rest) and after (TSA) TSA treatment. DNA from the antibody-bound fraction and total input DNA isolated from chromatin used for the immunoprecipitation were analyzed by quantitative PCR using a primer specific for the transfected promoter (HD1/Luci; see Materials and Methods). PCR products were quantified using the ImageQuant program, and relative signal intensities (-fold) are indicated.

    Article Snippet: To generate stable cell lines, 5 μg of linearized plasmid DNA was transfected into Swiss 3T3 fibroblasts using DAC 30 transfection reagent (Eurogentec) as recommended by the supplier.

    Techniques: Activation Assay, Plasmid Preparation, Expressing, Activity Assay, Luciferase, Cotransfection, Construct, Chromatin Immunoprecipitation, Isolation, Stable Transfection, Transfection, Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Protein complexes interacting with binding sites in the HDAC1 promoter. (A) Electrophoretic mobility shift assays were performed by incubating protein extracts from exponentially growing Swiss 3T3 cells with the indicated oligonucleotide probes (Oligo). Antibodies (AB) and double-stranded competitor oligonucleotides (Comp; 10- and 100-fold molar excess) were added as indicated (−, not added). Specific complexes are marked by arrows. (B) NF-Y and SP1 are associated with the proximal HDAC1 promoter. Formaldehyde-cross-linked chromatin was prepared from proliferating Swiss 3T3 cells and precipitated with NF-YB antibodies (NFY), SP1 antibodies (SP1), or nonspecific antibodies (unAB). DNA from the antibody-bound fraction and total input DNA isolated from chromatin used for the immunoprecipitation were analyzed by quantitative PCR using primers specific for the HDAC1 promoter or the 3′ part of the mouse HDAC1 gene (exon 12).

    Journal: Molecular and Cellular Biology

    Article Title: Autoregulation of Mouse Histone Deacetylase 1 Expression

    doi: 10.1128/MCB.23.19.6993-7004.2003

    Figure Lengend Snippet: Protein complexes interacting with binding sites in the HDAC1 promoter. (A) Electrophoretic mobility shift assays were performed by incubating protein extracts from exponentially growing Swiss 3T3 cells with the indicated oligonucleotide probes (Oligo). Antibodies (AB) and double-stranded competitor oligonucleotides (Comp; 10- and 100-fold molar excess) were added as indicated (−, not added). Specific complexes are marked by arrows. (B) NF-Y and SP1 are associated with the proximal HDAC1 promoter. Formaldehyde-cross-linked chromatin was prepared from proliferating Swiss 3T3 cells and precipitated with NF-YB antibodies (NFY), SP1 antibodies (SP1), or nonspecific antibodies (unAB). DNA from the antibody-bound fraction and total input DNA isolated from chromatin used for the immunoprecipitation were analyzed by quantitative PCR using primers specific for the HDAC1 promoter or the 3′ part of the mouse HDAC1 gene (exon 12).

    Article Snippet: To generate stable cell lines, 5 μg of linearized plasmid DNA was transfected into Swiss 3T3 fibroblasts using DAC 30 transfection reagent (Eurogentec) as recommended by the supplier.

    Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Isolation, Immunoprecipitation, Real-time Polymerase Chain Reaction

    Autoregulation of the HDAC1 promoter. (A) Activation of stably integrated HDAC1 promoter P721 by TSA. Stably transfected Swiss 3T3 cells were serum arrested for 48 h and treated with TSA for the indicated periods. The cells were harvested, and luciferase activity (in relative light units [RLU]) was determined and normalized to the corresponding amount of protein. (B) HDAC1 is associated with its own promoter region. Formaldehyde-cross-linked chromatin was prepared from undifferentiated proliferating wild-type (wt) and HDAC1 −/− (−/−) ES cells and immunoprecipitated with an HDAC1 antibody (HDAC1) or a nonspecific antibody (unAB). DNA from the antibody-bound fraction and total input DNA isolated from chromatin used for the immunoprecipitation were analyzed by quantitative PCR for the presence of the HDAC1 promoter region and the histone H4 control gene. (C) Specific recruitment of HDAC1 to the HDAC1 promoter region in resting Swiss 3T3 fibroblasts. Cross-linked chromatin was prepared from resting fibroblasts before (rest) and after (ind) restimulation with 20% FCS for 18 h and precipitated with the monoclonal HDAC1 antibody or an unrelated control antibody (unAB). The precipitated DNA was analyzed as described for panel B.

    Journal: Molecular and Cellular Biology

    Article Title: Autoregulation of Mouse Histone Deacetylase 1 Expression

    doi: 10.1128/MCB.23.19.6993-7004.2003

    Figure Lengend Snippet: Autoregulation of the HDAC1 promoter. (A) Activation of stably integrated HDAC1 promoter P721 by TSA. Stably transfected Swiss 3T3 cells were serum arrested for 48 h and treated with TSA for the indicated periods. The cells were harvested, and luciferase activity (in relative light units [RLU]) was determined and normalized to the corresponding amount of protein. (B) HDAC1 is associated with its own promoter region. Formaldehyde-cross-linked chromatin was prepared from undifferentiated proliferating wild-type (wt) and HDAC1 −/− (−/−) ES cells and immunoprecipitated with an HDAC1 antibody (HDAC1) or a nonspecific antibody (unAB). DNA from the antibody-bound fraction and total input DNA isolated from chromatin used for the immunoprecipitation were analyzed by quantitative PCR for the presence of the HDAC1 promoter region and the histone H4 control gene. (C) Specific recruitment of HDAC1 to the HDAC1 promoter region in resting Swiss 3T3 fibroblasts. Cross-linked chromatin was prepared from resting fibroblasts before (rest) and after (ind) restimulation with 20% FCS for 18 h and precipitated with the monoclonal HDAC1 antibody or an unrelated control antibody (unAB). The precipitated DNA was analyzed as described for panel B.

    Article Snippet: To generate stable cell lines, 5 μg of linearized plasmid DNA was transfected into Swiss 3T3 fibroblasts using DAC 30 transfection reagent (Eurogentec) as recommended by the supplier.

    Techniques: Activation Assay, Stable Transfection, Transfection, Luciferase, Activity Assay, Immunoprecipitation, Isolation, Real-time Polymerase Chain Reaction

    TEM, particle size, zeta potential, agarose gel electrophoresis, and NMR images. Notes: All nanoparticles, PEI ( A ), PV 10 ( B ), PV 20 ( C ), and PV 40 ( D ), were made at an N/P of 10 (scale bar: 100 nm). The detailed data of particle size and zeta potential are shown in ( E ). ( F ) shows the DNA ladder marker (a) and that PEI complexed DNA completely at an N/P of 2, PV 10 at 3, PV 20 at 5, and PV 40 at 6 (b). The different results from gel electrophoresis of PEI (c), PV 10 (d), PV 20 (e), and PV 40 (f) complexed with pDNA at various N/P ratios are shown. ( G ) shows the characterization of PV polymer by 1H NMR spectra. The PEI was activated into PEI-PDT by SPDP, and then the PEI-PDT was conjugated with VTW peptide and made into PV. Abbreviations: NMR, nuclear magnetic resonance; N/P, nitrogen in PEI amino residues to phosphates of DNA; PDT, pyridyldithio; PEI, polyethylenimine; PV, VTW-modified PEI copolymers; SPDP, N-succinimidyl 3-(2-pyridyldithio)-propionate; TEM, transmission electron microscopy; VTW, VTWTPQAWFQWV; pDNA, plasmid DNA.

    Journal: International Journal of Nanomedicine

    Article Title: A peptide-mediated targeting gene delivery system for malignant glioma cells

    doi: 10.2147/IJN.S44990

    Figure Lengend Snippet: TEM, particle size, zeta potential, agarose gel electrophoresis, and NMR images. Notes: All nanoparticles, PEI ( A ), PV 10 ( B ), PV 20 ( C ), and PV 40 ( D ), were made at an N/P of 10 (scale bar: 100 nm). The detailed data of particle size and zeta potential are shown in ( E ). ( F ) shows the DNA ladder marker (a) and that PEI complexed DNA completely at an N/P of 2, PV 10 at 3, PV 20 at 5, and PV 40 at 6 (b). The different results from gel electrophoresis of PEI (c), PV 10 (d), PV 20 (e), and PV 40 (f) complexed with pDNA at various N/P ratios are shown. ( G ) shows the characterization of PV polymer by 1H NMR spectra. The PEI was activated into PEI-PDT by SPDP, and then the PEI-PDT was conjugated with VTW peptide and made into PV. Abbreviations: NMR, nuclear magnetic resonance; N/P, nitrogen in PEI amino residues to phosphates of DNA; PDT, pyridyldithio; PEI, polyethylenimine; PV, VTW-modified PEI copolymers; SPDP, N-succinimidyl 3-(2-pyridyldithio)-propionate; TEM, transmission electron microscopy; VTW, VTWTPQAWFQWV; pDNA, plasmid DNA.

    Article Snippet: Plasmid DNA (pDNA) preparation The enhanced green fluorescent protein plasmid (pEGFP) was transfected into Escherichia coli DH5α competent cells (Tiangen, Beijing, People’s Republic of China) and amplified.

    Techniques: Transmission Electron Microscopy, Agarose Gel Electrophoresis, Nuclear Magnetic Resonance, Marker, Nucleic Acid Electrophoresis, Modification, Transmission Assay, Electron Microscopy, Plasmid Preparation

    Transfection efficiency of nanoparticles for different kinds of cells. Notes: Fluorescent micrographs and flow cytometry results of U-87 cells transfected with PV 10 , PV 20 , and PV 40 nanoparticles at the N/P of 10 are presented. The transfection results of T98G and U251 are also shown. The fluorescent images were merged with optical ones. Negative control: naked pDNA; positive control: Lipofectamine ® (Life Technologies, Carlsbad, CA, USA)–pDNA complexes. The PV 20 nanoparticles showed very low transfection effect for PC12 and A549 cells and absolutely no effect for neural cells. Competition assays for glioma cells are shown. Scale bar: 100 μm. Abbreviations: N/P, nitrogen in polyethylenimine amino residues to phosphates of DNA; pDNA, plasmid DNA; PEI, polyethylenimine; PV, VTWTPQAWFQWV-modified PEI copolymers.

    Journal: International Journal of Nanomedicine

    Article Title: A peptide-mediated targeting gene delivery system for malignant glioma cells

    doi: 10.2147/IJN.S44990

    Figure Lengend Snippet: Transfection efficiency of nanoparticles for different kinds of cells. Notes: Fluorescent micrographs and flow cytometry results of U-87 cells transfected with PV 10 , PV 20 , and PV 40 nanoparticles at the N/P of 10 are presented. The transfection results of T98G and U251 are also shown. The fluorescent images were merged with optical ones. Negative control: naked pDNA; positive control: Lipofectamine ® (Life Technologies, Carlsbad, CA, USA)–pDNA complexes. The PV 20 nanoparticles showed very low transfection effect for PC12 and A549 cells and absolutely no effect for neural cells. Competition assays for glioma cells are shown. Scale bar: 100 μm. Abbreviations: N/P, nitrogen in polyethylenimine amino residues to phosphates of DNA; pDNA, plasmid DNA; PEI, polyethylenimine; PV, VTWTPQAWFQWV-modified PEI copolymers.

    Article Snippet: Plasmid DNA (pDNA) preparation The enhanced green fluorescent protein plasmid (pEGFP) was transfected into Escherichia coli DH5α competent cells (Tiangen, Beijing, People’s Republic of China) and amplified.

    Techniques: Transfection, Flow Cytometry, Cytometry, Negative Control, Positive Control, Plasmid Preparation, Modification

    Internalization of nanoparticles. Notes: U-87 cell was incubated with the PV 20 nanoparticles containing the FITC-labeled pDNA. Lysosome was marked with Lyso-Tracker Red (Beyotime, Haimen, People’s Republic of China) to investigate the location of nanoparticles in cytoplasm. The nucleus was stained with Hoechst 33342 (Beyotime, Haimen, People’s Republic of China). Internalization assays showed that PV 20 nanoparticles penetrated into U-87 cells, were mostly co-localized with lysosomes at 1 hour after the beginning of transfection, and escaped successfully from lysosomes at 4 hours. Competition assay showed there was very little internalization of PV 20 nanoparticles in U-87 cells due to the block effect by free VTW peptide. Scale bar: 50 μm. Abbreviations: FITC, fluorescein isothiocyanate; pDNA, plasmid DNA; PV, VTW-modified PEI copolymers; VTW, VTWTPQAWFQWV.

    Journal: International Journal of Nanomedicine

    Article Title: A peptide-mediated targeting gene delivery system for malignant glioma cells

    doi: 10.2147/IJN.S44990

    Figure Lengend Snippet: Internalization of nanoparticles. Notes: U-87 cell was incubated with the PV 20 nanoparticles containing the FITC-labeled pDNA. Lysosome was marked with Lyso-Tracker Red (Beyotime, Haimen, People’s Republic of China) to investigate the location of nanoparticles in cytoplasm. The nucleus was stained with Hoechst 33342 (Beyotime, Haimen, People’s Republic of China). Internalization assays showed that PV 20 nanoparticles penetrated into U-87 cells, were mostly co-localized with lysosomes at 1 hour after the beginning of transfection, and escaped successfully from lysosomes at 4 hours. Competition assay showed there was very little internalization of PV 20 nanoparticles in U-87 cells due to the block effect by free VTW peptide. Scale bar: 50 μm. Abbreviations: FITC, fluorescein isothiocyanate; pDNA, plasmid DNA; PV, VTW-modified PEI copolymers; VTW, VTWTPQAWFQWV.

    Article Snippet: Plasmid DNA (pDNA) preparation The enhanced green fluorescent protein plasmid (pEGFP) was transfected into Escherichia coli DH5α competent cells (Tiangen, Beijing, People’s Republic of China) and amplified.

    Techniques: Incubation, Labeling, Staining, Transfection, Competitive Binding Assay, Blocking Assay, Plasmid Preparation, Modification

    The synthesis of nanoparticles with plasmid DNA made of PEI or PV and their internalization. Abbreviations: PEI, polyethylenimine; PV, VTW-modified PEI copolymers; VTW, VTWTPQAWFQWV.

    Journal: International Journal of Nanomedicine

    Article Title: A peptide-mediated targeting gene delivery system for malignant glioma cells

    doi: 10.2147/IJN.S44990

    Figure Lengend Snippet: The synthesis of nanoparticles with plasmid DNA made of PEI or PV and their internalization. Abbreviations: PEI, polyethylenimine; PV, VTW-modified PEI copolymers; VTW, VTWTPQAWFQWV.

    Article Snippet: Plasmid DNA (pDNA) preparation The enhanced green fluorescent protein plasmid (pEGFP) was transfected into Escherichia coli DH5α competent cells (Tiangen, Beijing, People’s Republic of China) and amplified.

    Techniques: Plasmid Preparation, Modification

    DNA replication activities of E1 mutants. (A) Relative DNA replication (percentage of signal obtained with 5 ng of WT E1) of the indicated HPV31 EYFP-E1 WT or mutants were tested using three different amounts of expression vector (1.25, 2.5, and 5 ng) as described in the text. The MFI mutation changes the three hydrophobic residues of the E1 MxxFI motif to three alanines. (B) Western blot showing the expression of the different EYFP-tagged E1 mutants. Tubulin was used as a loading control. α-GFP, antibody against green fluorescent protein. (C) Fluorescence confocal microscopy images showing the intracellular localization of the indicated E1 mutant proteins fused to EYFP in C33A cells. Nuclei (DNA) were stained with TO-PRO 3.

    Journal: Journal of Virology

    Article Title: A Conserved Amphipathic Helix in the N-Terminal Regulatory Region of the Papillomavirus E1 Helicase Is Required for Efficient Viral DNA Replication ▿A Conserved Amphipathic Helix in the N-Terminal Regulatory Region of the Papillomavirus E1 Helicase Is Required for Efficient Viral DNA Replication ▿ †

    doi: 10.1128/JVI.01829-10

    Figure Lengend Snippet: DNA replication activities of E1 mutants. (A) Relative DNA replication (percentage of signal obtained with 5 ng of WT E1) of the indicated HPV31 EYFP-E1 WT or mutants were tested using three different amounts of expression vector (1.25, 2.5, and 5 ng) as described in the text. The MFI mutation changes the three hydrophobic residues of the E1 MxxFI motif to three alanines. (B) Western blot showing the expression of the different EYFP-tagged E1 mutants. Tubulin was used as a loading control. α-GFP, antibody against green fluorescent protein. (C) Fluorescence confocal microscopy images showing the intracellular localization of the indicated E1 mutant proteins fused to EYFP in C33A cells. Nuclei (DNA) were stained with TO-PRO 3.

    Article Snippet: When different amounts of EYFP-E1 expression plasmid (pEYFP-HPV31 E1) were used, the quantity of plasmid DNA was adjusted to 50 ng with the empty vector (pEYFP-C1; Clontech).

    Techniques: Expressing, Plasmid Preparation, Mutagenesis, Western Blot, Fluorescence, Confocal Microscopy, Staining

    Transactivation activity of the E1 N-terminal region (NTR) in mammalian cells. (A) Transactivation activity of the HPV31 NTR compared to that of the complete (full-length) p53 TAD (p53 FL), p53 TAD2, and HPV31 E2 (31 E2) TAD fused to the Gal4 DNA-binding domain (DBD). Luciferase activities were measured from C33A cervical carcinoma cells transfected with increasing amounts of a plasmid encoding the indicated Gal4 fusion protein and a constant amount of a firefly luciferase reporter gene under the control of five Gal4-binding sites (pG5luc). Plasmid pRL, encoding Renilla luciferase, was used as an internal control. Luciferase activity values are presented as the fold activation of the reporter gene above that measured with the empty Gal4 DBD vector, which was set at 1. (B) Transactivation activities of the E1 NTRs from different HPV types. Transactivation activities of the Gal4-E1 NTR from the indicated papillomavirus types were measured as described above for panel A. (C) Effects of mutations in the E1 α-helix (AH) on transactivation activity. Transactivation activities of the Gal4-E1 NTR mutant derivatives were measured as described above for panel A. The MFI mutation changes the three hydrophobic residues of the E1 MxxFI motif to three alanines. Western blots showing the expression of the different Gal4 fusion proteins are shown to the right of each graph. The position of the full-length Gal4-E2 TAD protein, which was consistently found to show evidence of degradation products, is indicated by an asterisk. Tubulin was used as a loading control.

    Journal: Journal of Virology

    Article Title: A Conserved Amphipathic Helix in the N-Terminal Regulatory Region of the Papillomavirus E1 Helicase Is Required for Efficient Viral DNA Replication ▿A Conserved Amphipathic Helix in the N-Terminal Regulatory Region of the Papillomavirus E1 Helicase Is Required for Efficient Viral DNA Replication ▿ †

    doi: 10.1128/JVI.01829-10

    Figure Lengend Snippet: Transactivation activity of the E1 N-terminal region (NTR) in mammalian cells. (A) Transactivation activity of the HPV31 NTR compared to that of the complete (full-length) p53 TAD (p53 FL), p53 TAD2, and HPV31 E2 (31 E2) TAD fused to the Gal4 DNA-binding domain (DBD). Luciferase activities were measured from C33A cervical carcinoma cells transfected with increasing amounts of a plasmid encoding the indicated Gal4 fusion protein and a constant amount of a firefly luciferase reporter gene under the control of five Gal4-binding sites (pG5luc). Plasmid pRL, encoding Renilla luciferase, was used as an internal control. Luciferase activity values are presented as the fold activation of the reporter gene above that measured with the empty Gal4 DBD vector, which was set at 1. (B) Transactivation activities of the E1 NTRs from different HPV types. Transactivation activities of the Gal4-E1 NTR from the indicated papillomavirus types were measured as described above for panel A. (C) Effects of mutations in the E1 α-helix (AH) on transactivation activity. Transactivation activities of the Gal4-E1 NTR mutant derivatives were measured as described above for panel A. The MFI mutation changes the three hydrophobic residues of the E1 MxxFI motif to three alanines. Western blots showing the expression of the different Gal4 fusion proteins are shown to the right of each graph. The position of the full-length Gal4-E2 TAD protein, which was consistently found to show evidence of degradation products, is indicated by an asterisk. Tubulin was used as a loading control.

    Article Snippet: When different amounts of EYFP-E1 expression plasmid (pEYFP-HPV31 E1) were used, the quantity of plasmid DNA was adjusted to 50 ng with the empty vector (pEYFP-C1; Clontech).

    Techniques: Activity Assay, Binding Assay, Luciferase, Transfection, Plasmid Preparation, Activation Assay, Mutagenesis, Western Blot, Expressing

    The E1 N-terminal region activates transcription in yeast. (A) β-Galactosidase activities measured in yeast strain EGY48 expressing the indicated LexA fusion proteins, either WT or mutants, are reported as fold activation of the reporter gene relative to that of the LexA DNA-binding domain alone (control [ctl]), which was set at a value of 1. The MFI mutation (LFI mutation for HPV16) changes the three hydrophobic residues of the E1 MxxFI motif (LxxFI motif for HPV16) to three alanines. E1 proteins of HPV6, HPV11, HPV16, HPV18, and HPV31 are shown (e.g., 31 E1, HPV31 E1). (B) Pictures of yeast colonies grown on solid selective medium 4 days after their transformation with plasmids coding for the indicated LexA-DBD fusion proteins. (C) Transactivation activity of the indicated LexA fusion proteins measured in yeast strain BY4741 or its isogenic derivatives lacking the GCN5, ADA1, or SPT7 gene.

    Journal: Journal of Virology

    Article Title: A Conserved Amphipathic Helix in the N-Terminal Regulatory Region of the Papillomavirus E1 Helicase Is Required for Efficient Viral DNA Replication ▿A Conserved Amphipathic Helix in the N-Terminal Regulatory Region of the Papillomavirus E1 Helicase Is Required for Efficient Viral DNA Replication ▿ †

    doi: 10.1128/JVI.01829-10

    Figure Lengend Snippet: The E1 N-terminal region activates transcription in yeast. (A) β-Galactosidase activities measured in yeast strain EGY48 expressing the indicated LexA fusion proteins, either WT or mutants, are reported as fold activation of the reporter gene relative to that of the LexA DNA-binding domain alone (control [ctl]), which was set at a value of 1. The MFI mutation (LFI mutation for HPV16) changes the three hydrophobic residues of the E1 MxxFI motif (LxxFI motif for HPV16) to three alanines. E1 proteins of HPV6, HPV11, HPV16, HPV18, and HPV31 are shown (e.g., 31 E1, HPV31 E1). (B) Pictures of yeast colonies grown on solid selective medium 4 days after their transformation with plasmids coding for the indicated LexA-DBD fusion proteins. (C) Transactivation activity of the indicated LexA fusion proteins measured in yeast strain BY4741 or its isogenic derivatives lacking the GCN5, ADA1, or SPT7 gene.

    Article Snippet: When different amounts of EYFP-E1 expression plasmid (pEYFP-HPV31 E1) were used, the quantity of plasmid DNA was adjusted to 50 ng with the empty vector (pEYFP-C1; Clontech).

    Techniques: Expressing, Activation Assay, Binding Assay, CTL Assay, Mutagenesis, Transformation Assay, Activity Assay

    Deletion analysis of the E1 N-terminal region. (A) (Top) Schematic representation of full-length HPV31 E1 highlighting the locations of the p80-binding domain (amino acids [aa] 10 to 40), caspase 3/caspase 7 cleavage sites, nucleocytoplasmic shuttling module (aa 83 to 125), origin-binding domain (OBD) (aa 160 to 332), and the helicase domain, which encompasses regions required for oligomerization (O) (aa 332 to 414) and ATPase activity (aa 414 to 629). (Bottom) Sequence alignment of the first 60 amino acids of E1 from five different HPV types. Conserved residues of the caspase cleavage sites are indicated by white letters on a black background. The arrows point to the amino acids at the beginning of each E1 truncation (C10, C20, C30, C40, C50, and C53). (B) Relative DNA replication (percentage of signal obtained with 10 ng of wild-type [WT] E1) of Flag-tagged HPV31 E1 and truncated derivatives. Each E1 protein was tested using three different amounts of expression vector (2.5, 5, and 10 ng; indicated by the height of the triangle below each bar) in duplicate experiments. (C) Anti-Flag Western blot showing the expression of the different E1 proteins. Tubulin was used as a loading control. α-Flag- E1, anti-Flag-E1.

    Journal: Journal of Virology

    Article Title: A Conserved Amphipathic Helix in the N-Terminal Regulatory Region of the Papillomavirus E1 Helicase Is Required for Efficient Viral DNA Replication ▿A Conserved Amphipathic Helix in the N-Terminal Regulatory Region of the Papillomavirus E1 Helicase Is Required for Efficient Viral DNA Replication ▿ †

    doi: 10.1128/JVI.01829-10

    Figure Lengend Snippet: Deletion analysis of the E1 N-terminal region. (A) (Top) Schematic representation of full-length HPV31 E1 highlighting the locations of the p80-binding domain (amino acids [aa] 10 to 40), caspase 3/caspase 7 cleavage sites, nucleocytoplasmic shuttling module (aa 83 to 125), origin-binding domain (OBD) (aa 160 to 332), and the helicase domain, which encompasses regions required for oligomerization (O) (aa 332 to 414) and ATPase activity (aa 414 to 629). (Bottom) Sequence alignment of the first 60 amino acids of E1 from five different HPV types. Conserved residues of the caspase cleavage sites are indicated by white letters on a black background. The arrows point to the amino acids at the beginning of each E1 truncation (C10, C20, C30, C40, C50, and C53). (B) Relative DNA replication (percentage of signal obtained with 10 ng of wild-type [WT] E1) of Flag-tagged HPV31 E1 and truncated derivatives. Each E1 protein was tested using three different amounts of expression vector (2.5, 5, and 10 ng; indicated by the height of the triangle below each bar) in duplicate experiments. (C) Anti-Flag Western blot showing the expression of the different E1 proteins. Tubulin was used as a loading control. α-Flag- E1, anti-Flag-E1.

    Article Snippet: When different amounts of EYFP-E1 expression plasmid (pEYFP-HPV31 E1) were used, the quantity of plasmid DNA was adjusted to 50 ng with the empty vector (pEYFP-C1; Clontech).

    Techniques: Binding Assay, Activity Assay, Sequencing, Expressing, Plasmid Preparation, Western Blot

    The E1 MFI mutation affects DNA replication at a step following assembly of the E1-E2-ori preinitiation complex. (A) Interaction of HPV31 E1 with HPV31 E2 in vivo . EYFP alone (No E1), EYFP-HPV31 E1 (wild type [WT]), or EYFP-HPV31 E1 MFI and ATPase (K463A) mutants were cotransfected with Flag-tagged HPV31 E2. Coimmunoprecipitated proteins were separated by SDS-PAGE and analyzed by Western blotting using anti-GFP and anti-Flag antibodies as indicated. Tubulin was used as a loading control. The MFI mutation changes the three hydrophobic residues of the E1 MxxFI motif to three alanines. (B) Inhibition of HPV31 DNA replication (as a percentage) by increasing amounts of EYFP-E1 K463A and MFI/K463A double mutant proteins. The inhibition curves were obtained by nonlinear regression analysis. (C) DNA-binding activity of recombinant HPV31 E1(2-332). Fluorescence polarization binding isotherms were performed with a 10 nM concentration of a probe containing two E1-binding sites (2E1BS) or with a probe containing no E1-binding site (mutant [Mut]) and with increasing concentrations of the indicated WT or MFI E1 protein fragments. Each isotherm was performed in triplicate; error bars are barely visible on the graph, as they are smaller than the symbols. Polarization values (in millipolarization levels [mP]) were determined as described in Materials and Methods. The apparent dissociation constants ( K D ) determined from these binding isotherms are indicated below the graph.

    Journal: Journal of Virology

    Article Title: A Conserved Amphipathic Helix in the N-Terminal Regulatory Region of the Papillomavirus E1 Helicase Is Required for Efficient Viral DNA Replication ▿A Conserved Amphipathic Helix in the N-Terminal Regulatory Region of the Papillomavirus E1 Helicase Is Required for Efficient Viral DNA Replication ▿ †

    doi: 10.1128/JVI.01829-10

    Figure Lengend Snippet: The E1 MFI mutation affects DNA replication at a step following assembly of the E1-E2-ori preinitiation complex. (A) Interaction of HPV31 E1 with HPV31 E2 in vivo . EYFP alone (No E1), EYFP-HPV31 E1 (wild type [WT]), or EYFP-HPV31 E1 MFI and ATPase (K463A) mutants were cotransfected with Flag-tagged HPV31 E2. Coimmunoprecipitated proteins were separated by SDS-PAGE and analyzed by Western blotting using anti-GFP and anti-Flag antibodies as indicated. Tubulin was used as a loading control. The MFI mutation changes the three hydrophobic residues of the E1 MxxFI motif to three alanines. (B) Inhibition of HPV31 DNA replication (as a percentage) by increasing amounts of EYFP-E1 K463A and MFI/K463A double mutant proteins. The inhibition curves were obtained by nonlinear regression analysis. (C) DNA-binding activity of recombinant HPV31 E1(2-332). Fluorescence polarization binding isotherms were performed with a 10 nM concentration of a probe containing two E1-binding sites (2E1BS) or with a probe containing no E1-binding site (mutant [Mut]) and with increasing concentrations of the indicated WT or MFI E1 protein fragments. Each isotherm was performed in triplicate; error bars are barely visible on the graph, as they are smaller than the symbols. Polarization values (in millipolarization levels [mP]) were determined as described in Materials and Methods. The apparent dissociation constants ( K D ) determined from these binding isotherms are indicated below the graph.

    Article Snippet: When different amounts of EYFP-E1 expression plasmid (pEYFP-HPV31 E1) were used, the quantity of plasmid DNA was adjusted to 50 ng with the empty vector (pEYFP-C1; Clontech).

    Techniques: Mutagenesis, In Vivo, SDS Page, Western Blot, Inhibition, Binding Assay, Activity Assay, Recombinant, Fluorescence, Concentration Assay

    The DNA replication defect of the E1 MFI mutant is not altered by lengthening the time of the assay or by overexpression of the protein. (A) DNA replication levels (firefly luciferase/ Renilla luciferase [Fluc/Rluc] ratio) obtained using 1.25, 2.5, and 5 ng of HPV31 EYFP-E1 WT or MFI mutant expression vector as indicated below the bars at 24, 48, 72, 96, and 120 h posttransfection. The MFI mutation changes the three hydrophobic residues of the E1 MxxFI motif to three alanines. (B) DNA replication levels obtained using increasing amounts (in nanograms) of EYFP-E1 WT and MFI mutant expression vector 72 h posttransfection. (C) As in panel B but using triple-Flag-tagged E1 rather than EYFP-E1.

    Journal: Journal of Virology

    Article Title: A Conserved Amphipathic Helix in the N-Terminal Regulatory Region of the Papillomavirus E1 Helicase Is Required for Efficient Viral DNA Replication ▿A Conserved Amphipathic Helix in the N-Terminal Regulatory Region of the Papillomavirus E1 Helicase Is Required for Efficient Viral DNA Replication ▿ †

    doi: 10.1128/JVI.01829-10

    Figure Lengend Snippet: The DNA replication defect of the E1 MFI mutant is not altered by lengthening the time of the assay or by overexpression of the protein. (A) DNA replication levels (firefly luciferase/ Renilla luciferase [Fluc/Rluc] ratio) obtained using 1.25, 2.5, and 5 ng of HPV31 EYFP-E1 WT or MFI mutant expression vector as indicated below the bars at 24, 48, 72, 96, and 120 h posttransfection. The MFI mutation changes the three hydrophobic residues of the E1 MxxFI motif to three alanines. (B) DNA replication levels obtained using increasing amounts (in nanograms) of EYFP-E1 WT and MFI mutant expression vector 72 h posttransfection. (C) As in panel B but using triple-Flag-tagged E1 rather than EYFP-E1.

    Article Snippet: When different amounts of EYFP-E1 expression plasmid (pEYFP-HPV31 E1) were used, the quantity of plasmid DNA was adjusted to 50 ng with the empty vector (pEYFP-C1; Clontech).

    Techniques: Mutagenesis, Over Expression, Luciferase, Expressing, Plasmid Preparation

    Effect of pH or temperature on Rv0888 enzymatic activity. ( A ) Analysis of the effect of temperature on Rv0888 enzymatic activity by agarose gel electrophoresis. The reaction was performed in 20 mM Tris-HCl pH 6.5, 5 mM CaCl 2 and 5 mM MnCl 2 for 1 h. Line M: DL5000 DNA Marker; Line 1: circular plasmid DNA; Lines 2-11: circular plasmid DNA and Rv0888 ranging between 33 °C and 51 °C at 2 °C intervals. ( B ) Quantification of DNase activity by spectrophotometry and error bars are given as standard deviations. ( C ) Analysis of the effect of the pH on Rv0888 enzymatic activity by agarose gel electrophoresis. The reaction was performed in 20 mM Tris-HCl, 5 mM CaCl 2 and 5 mM MnCl 2 for 1 h at 37 °C. Line M: DL5000 DNA Marker; Lines 1, 3, 5, 7, 9: circular plasmid DNA ranging between pH 6.0 and pH 8.0 at 0.5 intervals; Lines 2, 4, 6, 8, 10: circular plasmid DNA and Rv0888 ranging between pH 6.0 and pH 8.0 at 0.5 intervals. ( D ) DNase activity was quantified by spectrophotometry and error bars are given as standard deviations.

    Journal: Scientific Reports

    Article Title: Characterization of Rv0888, a Novel Extracellular Nuclease from Mycobacterium tuberculosis

    doi: 10.1038/srep19033

    Figure Lengend Snippet: Effect of pH or temperature on Rv0888 enzymatic activity. ( A ) Analysis of the effect of temperature on Rv0888 enzymatic activity by agarose gel electrophoresis. The reaction was performed in 20 mM Tris-HCl pH 6.5, 5 mM CaCl 2 and 5 mM MnCl 2 for 1 h. Line M: DL5000 DNA Marker; Line 1: circular plasmid DNA; Lines 2-11: circular plasmid DNA and Rv0888 ranging between 33 °C and 51 °C at 2 °C intervals. ( B ) Quantification of DNase activity by spectrophotometry and error bars are given as standard deviations. ( C ) Analysis of the effect of the pH on Rv0888 enzymatic activity by agarose gel electrophoresis. The reaction was performed in 20 mM Tris-HCl, 5 mM CaCl 2 and 5 mM MnCl 2 for 1 h at 37 °C. Line M: DL5000 DNA Marker; Lines 1, 3, 5, 7, 9: circular plasmid DNA ranging between pH 6.0 and pH 8.0 at 0.5 intervals; Lines 2, 4, 6, 8, 10: circular plasmid DNA and Rv0888 ranging between pH 6.0 and pH 8.0 at 0.5 intervals. ( D ) DNase activity was quantified by spectrophotometry and error bars are given as standard deviations.

    Article Snippet: Substrates for Rv0888 nuclease To measure the substrate specificity for the recombinant Rv0888 nuclease, 0.2 μg of linear dsDNA (PCR amplified), circular plasmid DNA (pGEX-6p-1 vector), chromosomal DNA (E. coli DNA) or RNA from baker’s yeast (Sigma-Aldrich) were incubated with 3 μg of the purified Rv0888 protein in 10 μL of reaction buffer (20 mM Tris-HCl, 5 mM MgCl2, pH 7.5) at 37 °C.

    Techniques: Activity Assay, Agarose Gel Electrophoresis, Marker, Plasmid Preparation, Spectrophotometry

    Digestion of various nucleic acids by purified Rv0888. The reaction was performed in 20 mM Tris-HCl pH 7.5 and 5 mM MgCl 2 for 1 h at 37 °C. ( A ) Digestion of various DNA with purified Rv0888. Line M: DL5000 DNA Marker; Line 1: chromosomal DNA in 20 mM Tris-HCl (pH 7.5); Line 2: chromosomal DNA and purified Rv0888; Line 3: circular plasmid DNA in 20 mM Tris-HCl (pH 7.5); Line 4: circular plasmid DNA and purified Rv0888; Line 5: linear dsDNA in 20 mM Tris-HCl (pH 7.5); Line 6: linear dsDNA and purified Rv0888. ( B ) Digestion of RNA with purified Rv0888. Line M: DL5000 DNA Marker; Line 1: baker’s yeast RNA in 20 mM Tris-HCl (pH 7.5); Line 2: baker’s yeast RNA and purified Rv0888. (C) DNase activity requires cations. Line M: DL5000 DNA Marker; Line 1: circular plasmid DNA in 20 mM Tris-HCl (pH 7.5); Line 2: circular plasmid DNA and purified Rv0888 with 5 mM CaCl 2 and 5 mM MnCl 2 ; Line 3: circular plasmid DNA and purified Rv0888 with 5 mM CaCl 2 , 5 mM MnCl 2 and 20 mM EDTA.

    Journal: Scientific Reports

    Article Title: Characterization of Rv0888, a Novel Extracellular Nuclease from Mycobacterium tuberculosis

    doi: 10.1038/srep19033

    Figure Lengend Snippet: Digestion of various nucleic acids by purified Rv0888. The reaction was performed in 20 mM Tris-HCl pH 7.5 and 5 mM MgCl 2 for 1 h at 37 °C. ( A ) Digestion of various DNA with purified Rv0888. Line M: DL5000 DNA Marker; Line 1: chromosomal DNA in 20 mM Tris-HCl (pH 7.5); Line 2: chromosomal DNA and purified Rv0888; Line 3: circular plasmid DNA in 20 mM Tris-HCl (pH 7.5); Line 4: circular plasmid DNA and purified Rv0888; Line 5: linear dsDNA in 20 mM Tris-HCl (pH 7.5); Line 6: linear dsDNA and purified Rv0888. ( B ) Digestion of RNA with purified Rv0888. Line M: DL5000 DNA Marker; Line 1: baker’s yeast RNA in 20 mM Tris-HCl (pH 7.5); Line 2: baker’s yeast RNA and purified Rv0888. (C) DNase activity requires cations. Line M: DL5000 DNA Marker; Line 1: circular plasmid DNA in 20 mM Tris-HCl (pH 7.5); Line 2: circular plasmid DNA and purified Rv0888 with 5 mM CaCl 2 and 5 mM MnCl 2 ; Line 3: circular plasmid DNA and purified Rv0888 with 5 mM CaCl 2 , 5 mM MnCl 2 and 20 mM EDTA.

    Article Snippet: Substrates for Rv0888 nuclease To measure the substrate specificity for the recombinant Rv0888 nuclease, 0.2 μg of linear dsDNA (PCR amplified), circular plasmid DNA (pGEX-6p-1 vector), chromosomal DNA (E. coli DNA) or RNA from baker’s yeast (Sigma-Aldrich) were incubated with 3 μg of the purified Rv0888 protein in 10 μL of reaction buffer (20 mM Tris-HCl, 5 mM MgCl2, pH 7.5) at 37 °C.

    Techniques: Purification, Marker, Plasmid Preparation, Activity Assay

    Oleuropein, 6-Gingerol, Corylifolinin, and Acteoside inhibit Rv0888 activity against circular plasmid DNA. The reaction was performed in 20 mM Tris-HCl pH 6.5, 5 mM CaCl 2 and 5 mM MnCl 2 for 1 h at 41 °C. (A) Line M: DL5000 DNA Marker; Line 1: circular plasmid DNA; Line 2: circular plasm id DNA and Rv0888; Line 3: circular plasmid DNA and Rv0888 in 50% ethanol; Line 4: circular plasmid DNA and Rv0888 in 50% dimethyl sulfoxide; Lines 5–7: circular plasmid DNA, Rv0888 and Oleuropein (0.5 mM, 1 mM, 2 mM, respectively); Lines 8–10: circular plasmid DNA, Rv0888 and 6-Gingerol (0.5 mM, 1 mM, 2 mM, respectively); Lines 11–13: circular plasmid DNA, Rv0888 and Corylifolinin (0.5 mM, 1 mM, 2 mM, respectively); Lines 14–16: circular plasmid DNA, Rv0888 and Acteoside (0.5 mM, 1 mM, 2 mM, respectively). (B) Quantification of DNase activity by spectrophotometry and error bars are given as standard deviations.

    Journal: Scientific Reports

    Article Title: Characterization of Rv0888, a Novel Extracellular Nuclease from Mycobacterium tuberculosis

    doi: 10.1038/srep19033

    Figure Lengend Snippet: Oleuropein, 6-Gingerol, Corylifolinin, and Acteoside inhibit Rv0888 activity against circular plasmid DNA. The reaction was performed in 20 mM Tris-HCl pH 6.5, 5 mM CaCl 2 and 5 mM MnCl 2 for 1 h at 41 °C. (A) Line M: DL5000 DNA Marker; Line 1: circular plasmid DNA; Line 2: circular plasm id DNA and Rv0888; Line 3: circular plasmid DNA and Rv0888 in 50% ethanol; Line 4: circular plasmid DNA and Rv0888 in 50% dimethyl sulfoxide; Lines 5–7: circular plasmid DNA, Rv0888 and Oleuropein (0.5 mM, 1 mM, 2 mM, respectively); Lines 8–10: circular plasmid DNA, Rv0888 and 6-Gingerol (0.5 mM, 1 mM, 2 mM, respectively); Lines 11–13: circular plasmid DNA, Rv0888 and Corylifolinin (0.5 mM, 1 mM, 2 mM, respectively); Lines 14–16: circular plasmid DNA, Rv0888 and Acteoside (0.5 mM, 1 mM, 2 mM, respectively). (B) Quantification of DNase activity by spectrophotometry and error bars are given as standard deviations.

    Article Snippet: Substrates for Rv0888 nuclease To measure the substrate specificity for the recombinant Rv0888 nuclease, 0.2 μg of linear dsDNA (PCR amplified), circular plasmid DNA (pGEX-6p-1 vector), chromosomal DNA (E. coli DNA) or RNA from baker’s yeast (Sigma-Aldrich) were incubated with 3 μg of the purified Rv0888 protein in 10 μL of reaction buffer (20 mM Tris-HCl, 5 mM MgCl2, pH 7.5) at 37 °C.

    Techniques: Activity Assay, Plasmid Preparation, Marker, Spectrophotometry

    Michaelis-Menton kinetics assays of the DNase ( A ) and the RNase ( B ) activity of Rv0888. The reaction was performed in 20 mM Tris-HCl pH 6.5, 5 mM CaCl 2 and 5 mM MnCl 2 for 1 h at 41 °C. K m values for DNA and RNA were 0.306 ± 0.04 mg/mL and 0.012 ± 0.01 mg/mL, respectively; the V max values for DNase and RNase activity were 600.56 U/mg/min and 241.11 U/mg/min, respectively.

    Journal: Scientific Reports

    Article Title: Characterization of Rv0888, a Novel Extracellular Nuclease from Mycobacterium tuberculosis

    doi: 10.1038/srep19033

    Figure Lengend Snippet: Michaelis-Menton kinetics assays of the DNase ( A ) and the RNase ( B ) activity of Rv0888. The reaction was performed in 20 mM Tris-HCl pH 6.5, 5 mM CaCl 2 and 5 mM MnCl 2 for 1 h at 41 °C. K m values for DNA and RNA were 0.306 ± 0.04 mg/mL and 0.012 ± 0.01 mg/mL, respectively; the V max values for DNase and RNase activity were 600.56 U/mg/min and 241.11 U/mg/min, respectively.

    Article Snippet: Substrates for Rv0888 nuclease To measure the substrate specificity for the recombinant Rv0888 nuclease, 0.2 μg of linear dsDNA (PCR amplified), circular plasmid DNA (pGEX-6p-1 vector), chromosomal DNA (E. coli DNA) or RNA from baker’s yeast (Sigma-Aldrich) were incubated with 3 μg of the purified Rv0888 protein in 10 μL of reaction buffer (20 mM Tris-HCl, 5 mM MgCl2, pH 7.5) at 37 °C.

    Techniques: Activity Assay