double-stranded dna Search Results


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  • 99
    Millipore deoxyribonucleic acid dna
    Autoimmunity in Btn2a2 −/− mice. (A) EAE was induced by immunization with MOG 35–55 + CFA in Btn2a2 −/− (KO) and WT mice or in KO→WT and WT→KO chimeras. (Top left) Mean clinical scores, disease incidence, and maximum scores are shown as shading highlighting time points analyzed in B and C. (B) Maximum disease scores and SC-infiltrating cells (total cells, CD4 + T cells, and MOG-specific IFN-γ + , IL-17 + , or Foxp3 + CD4 + T cells) were quantified by flow cytometry in WT and Btn2a2 −/− mice at disease onset and peak disease. (C) Ratios between SC-infiltrating Th1 or Th17 cells and T reg cells were determined at peak disease in WT and Btn2a2 −/− mice. (A–C) Results are pooled from two experiments, each with five mice per group. (D) IFN-γ + , IL-17 + , and Ki67 + Foxp3 + CD4 + T cells were quantified by flow cytometry in LN cells harvested at pre-onset, onset, and peak disease. Results are derived from three experiments, each with six mice per group. (E) Serum <t>anti-DNA</t> Ab titers were measured by <t>ELISA</t> in age-matched 1-yr-old Btn2a2 −/− and WT mice. A representative of two experiments is shown, each with eight mice per group. *, P
    Deoxyribonucleic Acid Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 491 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore dna synthesis
    Mean intracellular reactive oxygen species (ROS) levels and <t>DNA</t> damage in all- trans retinoic acid (ATRA)-treated and control ARPE-19 cells exposed to tert -butyl hydroperoxide (tBH). Reactive oxygen species (ROS) are expressed as the relative fluorescence of 2′,7′-dichlorofluorescein (DCF) normalized to controls after 30 min incubation with tBH at 37 °C. DNA damage was measured as a percentage of tail DNA in the comets in alkaline versions of the comet assay following exposure to tBH for 15 min at 37 °C. The induction of DNA double strand breaks was assessed by the extent of phosphorylation of H2AX and <t>ATM</t> (ataxia teleangiectasia mutated) (γH2AX and pATM) after γ-ray irradiation (2 Gy/min) of cells. Results are presented as mean ± SEM, n = 12 for ROS level, n = 100 for comet assay, n = 8 for H2AX/ATM phosphorylation, *** p
    Dna Synthesis, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 165 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore double stranded dna ctdna
    Mean intracellular reactive oxygen species (ROS) levels and <t>DNA</t> damage in all- trans retinoic acid (ATRA)-treated and control ARPE-19 cells exposed to tert -butyl hydroperoxide (tBH). Reactive oxygen species (ROS) are expressed as the relative fluorescence of 2′,7′-dichlorofluorescein (DCF) normalized to controls after 30 min incubation with tBH at 37 °C. DNA damage was measured as a percentage of tail DNA in the comets in alkaline versions of the comet assay following exposure to tBH for 15 min at 37 °C. The induction of DNA double strand breaks was assessed by the extent of phosphorylation of H2AX and <t>ATM</t> (ataxia teleangiectasia mutated) (γH2AX and pATM) after γ-ray irradiation (2 Gy/min) of cells. Results are presented as mean ± SEM, n = 12 for ROS level, n = 100 for comet assay, n = 8 for H2AX/ATM phosphorylation, *** p
    Double Stranded Dna Ctdna, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher double stranded dna
    Nanopore detection of small-molecule binding to single-stranded <t>DNA.</t> (a) TEM image of a 2.2 nm pore, and the molecules used in this study. Since SYBR Green II <t>(SGII)</t> binds to poly(dA) with very low affinity, dA 60 was used as a negative control and dA 50 dN 50 as a positive control. (b) Typical translocation events for dA 60 , before and after the addition of SGII to the cis chamber (side histogram is an all-point histogram of the current data). Typical translocation events for dA 50 dN 50 , before and after the addition of SGII, are shown in (c) and (d), respectively. The two-step events prevalent after SGII binding were characterized by amplitudes of 0.58 and 0.96 nA, corresponding to dA 50 and SGII-bound dN 50 , respectively (see text).
    Double Stranded Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 2863 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    ATUM double stranded dna
    Nanopore detection of small-molecule binding to single-stranded <t>DNA.</t> (a) TEM image of a 2.2 nm pore, and the molecules used in this study. Since SYBR Green II <t>(SGII)</t> binds to poly(dA) with very low affinity, dA 60 was used as a negative control and dA 50 dN 50 as a positive control. (b) Typical translocation events for dA 60 , before and after the addition of SGII to the cis chamber (side histogram is an all-point histogram of the current data). Typical translocation events for dA 50 dN 50 , before and after the addition of SGII, are shown in (c) and (d), respectively. The two-step events prevalent after SGII binding were characterized by amplitudes of 0.58 and 0.96 nA, corresponding to dA 50 and SGII-bound dN 50 , respectively (see text).
    Double Stranded Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 92/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Avantor double stranded dna
    Nanopore detection of small-molecule binding to single-stranded <t>DNA.</t> (a) TEM image of a 2.2 nm pore, and the molecules used in this study. Since SYBR Green II <t>(SGII)</t> binds to poly(dA) with very low affinity, dA 60 was used as a negative control and dA 50 dN 50 as a positive control. (b) Typical translocation events for dA 60 , before and after the addition of SGII to the cis chamber (side histogram is an all-point histogram of the current data). Typical translocation events for dA 50 dN 50 , before and after the addition of SGII, are shown in (c) and (d), respectively. The two-step events prevalent after SGII binding were characterized by amplitudes of 0.58 and 0.96 nA, corresponding to dA 50 and SGII-bound dN 50 , respectively (see text).
    Double Stranded Dna, supplied by Avantor, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GenScript double stranded dna
    Genome-wide identification of SsrB palindrome sequences. (A) A position weight matrix was generated from all naturally-evolved palindromes in SPI-2 and used to search the genome for similar sequences. Palindromes identified in regulatory <t>DNA</t> that co-occurred with ChIP peaks upstream of SsrB-regulated genes were selected, aligned and binned according to those scoring > 0.8 against the position weight matrix (top box) and those scoring between 0.7–0.8 (lower box). The left (7′) and right (7″) heptamers and the 4-bp spacer are displayed as a heat-map to show bases of high conservation (dark blue) from degenerate regions (light blue/white). The genes controlled by these promoters are indicated to the left of the sequences and coloured according to function: structural components of the T3SS (orange), effectors (blue), regulatory elements (red), T3SS chaperones and translocon (green), and SsrB-regulated genes of unknown function (black). (B) The high-scoring palindrome in the ssaR IGR is functional. A single-copy ssaR reporter was integrated on the chromosome and tested for functional activity in wild-type cells and in Δ ssrB . Promoter activity is shown as the mean with standard error from three separate experiments. (C) Functional validation of the intragenic high-scoring palindrome in the ssaE coding sequence. A single-copy transcriptional reporter that either contained (WT P <t>sseA</t> ) or lacked (P sseA del) the single heptamer site in the sseA IGR was integrated on the chromosome in wild-type cells, or mutants lacking either ssrB or the ssaE coding sequence that removed the high-scoring intragenic palindrome (P sseA '). Transcriptional activity at 6 h is shown as the mean of triplicate determinations with standard error.
    Double Stranded Dna, supplied by GenScript, used in various techniques. Bioz Stars score: 92/100, based on 91 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Autoimmunity in Btn2a2 −/− mice. (A) EAE was induced by immunization with MOG 35–55 + CFA in Btn2a2 −/− (KO) and WT mice or in KO→WT and WT→KO chimeras. (Top left) Mean clinical scores, disease incidence, and maximum scores are shown as shading highlighting time points analyzed in B and C. (B) Maximum disease scores and SC-infiltrating cells (total cells, CD4 + T cells, and MOG-specific IFN-γ + , IL-17 + , or Foxp3 + CD4 + T cells) were quantified by flow cytometry in WT and Btn2a2 −/− mice at disease onset and peak disease. (C) Ratios between SC-infiltrating Th1 or Th17 cells and T reg cells were determined at peak disease in WT and Btn2a2 −/− mice. (A–C) Results are pooled from two experiments, each with five mice per group. (D) IFN-γ + , IL-17 + , and Ki67 + Foxp3 + CD4 + T cells were quantified by flow cytometry in LN cells harvested at pre-onset, onset, and peak disease. Results are derived from three experiments, each with six mice per group. (E) Serum anti-DNA Ab titers were measured by ELISA in age-matched 1-yr-old Btn2a2 −/− and WT mice. A representative of two experiments is shown, each with eight mice per group. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Btn2a2, a T cell immunomodulatory molecule coregulated with MHC class II genes

    doi: 10.1084/jem.20150435

    Figure Lengend Snippet: Autoimmunity in Btn2a2 −/− mice. (A) EAE was induced by immunization with MOG 35–55 + CFA in Btn2a2 −/− (KO) and WT mice or in KO→WT and WT→KO chimeras. (Top left) Mean clinical scores, disease incidence, and maximum scores are shown as shading highlighting time points analyzed in B and C. (B) Maximum disease scores and SC-infiltrating cells (total cells, CD4 + T cells, and MOG-specific IFN-γ + , IL-17 + , or Foxp3 + CD4 + T cells) were quantified by flow cytometry in WT and Btn2a2 −/− mice at disease onset and peak disease. (C) Ratios between SC-infiltrating Th1 or Th17 cells and T reg cells were determined at peak disease in WT and Btn2a2 −/− mice. (A–C) Results are pooled from two experiments, each with five mice per group. (D) IFN-γ + , IL-17 + , and Ki67 + Foxp3 + CD4 + T cells were quantified by flow cytometry in LN cells harvested at pre-onset, onset, and peak disease. Results are derived from three experiments, each with six mice per group. (E) Serum anti-DNA Ab titers were measured by ELISA in age-matched 1-yr-old Btn2a2 −/− and WT mice. A representative of two experiments is shown, each with eight mice per group. *, P

    Article Snippet: Double-stranded DNA was coated onto ELISA plates precoated with poly-l -lysine (Sigma-Aldrich).

    Techniques: Mouse Assay, Flow Cytometry, Cytometry, Derivative Assay, Enzyme-linked Immunosorbent Assay

    Nonradioactive EMSA of Cra and its binding site in aceBAK . EMSA experiment using 0.75 μM of the Cra site-containing DNA fragment and increasing concentrations of wild type Cra or the Cra mutant proteins in the presence of FBP ( a ) and the gray value of free DNA determined using Quantity one ( b ) and in the absence of FBP ( c ) and the gray value of free DNA determined using Quantity one ( d ). Lane 1, no protein; lane 2–5, 1, 2, 3, 4 μM Cra; lanes 6–9, 1, 2, 3, 4 μM Cra mutant (Tang1541); lanes 10–13, 1, 2, 3, 4 μM Cra mutant (Tang1544), respectively.

    Journal: Scientific Reports

    Article Title: Enhancing succinic acid biosynthesis in Escherichia coli by engineering its global transcription factor, catabolite repressor/activator (Cra)

    doi: 10.1038/srep36526

    Figure Lengend Snippet: Nonradioactive EMSA of Cra and its binding site in aceBAK . EMSA experiment using 0.75 μM of the Cra site-containing DNA fragment and increasing concentrations of wild type Cra or the Cra mutant proteins in the presence of FBP ( a ) and the gray value of free DNA determined using Quantity one ( b ) and in the absence of FBP ( c ) and the gray value of free DNA determined using Quantity one ( d ). Lane 1, no protein; lane 2–5, 1, 2, 3, 4 μM Cra; lanes 6–9, 1, 2, 3, 4 μM Cra mutant (Tang1541); lanes 10–13, 1, 2, 3, 4 μM Cra mutant (Tang1544), respectively.

    Article Snippet: Cra was incubated with the DNA fragment in the presence of 1 mM FBP (Sigma, 98% purity).

    Techniques: Binding Assay, Mutagenesis

    Transfected MT-COI DNA upregulates type I interferon and A3A expression. ( A ) Interferon α and β production following transfection of THP-1 cells by dT containing PCR DNA fragments. MT-COI * indicates incubation with DNA but no transfection. ( B ) APOBEC3 transcription profiling of transfected THP-1 cells. Data were normalized to the expression levels of RPL13A housekeeping reference gene. Profiling was performed in duplicate and normalized to JetPRIME to facilitate comparison. ( C ) Western blot of A3A isoforms p1 and p2 from transfected THP-1 and compared to β-actin. Lanes 2, 3 and lanes 4, 5 represent duplicates. ( D ) Agarose gel of MT-COI PCR products (511 bp) amplified with Taq or Pfu polymerase in the presence of dTTP, dUTP, dTTP+dUTP. M, molecular weight markers. ( E ) Interferon α and β production following transfection of THP-1 cells by dU or 50:50 mixture dT+dU containing PCR DNA fragments. ( F and G ) Transcriptomes were established for DNA containing dU (F) or a 50:50 mixture of dT and dU (G). Duplicates were normalized to the expression levels of RPL13A housekeeping gene and normalized to JetPRIME to facilitate comparison.

    Journal: Nucleic Acids Research

    Article Title: Self-cytoplasmic DNA upregulates the mutator enzyme APOBEC3A leading to chromosomal DNA damage

    doi: 10.1093/nar/gkx001

    Figure Lengend Snippet: Transfected MT-COI DNA upregulates type I interferon and A3A expression. ( A ) Interferon α and β production following transfection of THP-1 cells by dT containing PCR DNA fragments. MT-COI * indicates incubation with DNA but no transfection. ( B ) APOBEC3 transcription profiling of transfected THP-1 cells. Data were normalized to the expression levels of RPL13A housekeeping reference gene. Profiling was performed in duplicate and normalized to JetPRIME to facilitate comparison. ( C ) Western blot of A3A isoforms p1 and p2 from transfected THP-1 and compared to β-actin. Lanes 2, 3 and lanes 4, 5 represent duplicates. ( D ) Agarose gel of MT-COI PCR products (511 bp) amplified with Taq or Pfu polymerase in the presence of dTTP, dUTP, dTTP+dUTP. M, molecular weight markers. ( E ) Interferon α and β production following transfection of THP-1 cells by dU or 50:50 mixture dT+dU containing PCR DNA fragments. ( F and G ) Transcriptomes were established for DNA containing dU (F) or a 50:50 mixture of dT and dU (G). Duplicates were normalized to the expression levels of RPL13A housekeeping gene and normalized to JetPRIME to facilitate comparison.

    Article Snippet: FACS analysis for DNA double stranded breaks Twenty-four hours post-transfection, THP-1 cells were washed with PBS, fixed in 2% ice-cold paraformaldehyde (Electron Microscopy Sciences) for 15 min and permeabilized in 90% ice-cold methanol (Sigma) for 30 min. After two washes with PBS, cells were incubated for 1 h with 1:100 diluted Alexa Fluor 488-conjugated mouse monoclonal anti-γH2AX (N1-431) antibody (BD Pharmingen).

    Techniques: Transfection, Expressing, Polymerase Chain Reaction, Incubation, Western Blot, Agarose Gel Electrophoresis, Amplification, Molecular Weight

    Transfected dsDNA triggers the RIG-I pathway via RNA polymerase III. ( A ) Western blots of TBK1, TBK1-P (phosphorylated), IRF3, IRF3-P (phosphorylated), RIG-I, MDA5, MAVS, STING, IKKε at 24 h post DNA transfection of THP-1 cells. ( B ) Interferon alpha production by DNA transfected THP-1 cells along with RNA Polymerase III inhibitor (ML-60218). Peptidoglycans (PGN) were used as control. ( C ) A3-edited MT-CYB DNA recovered by 3DPCR in presence or absence of 25–75 μM of RNA polymerase III inhibitor. The white line indicates the threshold between edited and unedited 3DPCR products. M: molecular weight markers. ( D and E ) Transcription profiling of A3A and quantification of V1V2 DNA upon V1V2 DNA transfected THP-1 cells along with RNA polymerase III inhibitor (ML-60218).

    Journal: Nucleic Acids Research

    Article Title: Self-cytoplasmic DNA upregulates the mutator enzyme APOBEC3A leading to chromosomal DNA damage

    doi: 10.1093/nar/gkx001

    Figure Lengend Snippet: Transfected dsDNA triggers the RIG-I pathway via RNA polymerase III. ( A ) Western blots of TBK1, TBK1-P (phosphorylated), IRF3, IRF3-P (phosphorylated), RIG-I, MDA5, MAVS, STING, IKKε at 24 h post DNA transfection of THP-1 cells. ( B ) Interferon alpha production by DNA transfected THP-1 cells along with RNA Polymerase III inhibitor (ML-60218). Peptidoglycans (PGN) were used as control. ( C ) A3-edited MT-CYB DNA recovered by 3DPCR in presence or absence of 25–75 μM of RNA polymerase III inhibitor. The white line indicates the threshold between edited and unedited 3DPCR products. M: molecular weight markers. ( D and E ) Transcription profiling of A3A and quantification of V1V2 DNA upon V1V2 DNA transfected THP-1 cells along with RNA polymerase III inhibitor (ML-60218).

    Article Snippet: FACS analysis for DNA double stranded breaks Twenty-four hours post-transfection, THP-1 cells were washed with PBS, fixed in 2% ice-cold paraformaldehyde (Electron Microscopy Sciences) for 15 min and permeabilized in 90% ice-cold methanol (Sigma) for 30 min. After two washes with PBS, cells were incubated for 1 h with 1:100 diluted Alexa Fluor 488-conjugated mouse monoclonal anti-γH2AX (N1-431) antibody (BD Pharmingen).

    Techniques: Transfection, Western Blot, Molecular Weight

    Cytoplasmic RNA synthesis by DNA-dependent RNA polymerase III binds to RIG-I. ( A ) THP-1 cells were transfected with control siRNA or 2 RNA polymerase III siRNA (siRNA polIII 1 and siRNA polIII 2 ) or cGAS siRNA. Six hours post-transfection with siRNA, cells were transfecte with 500 ng of MT-COI DNA. At 24 h post-transfection, total RNA was extracted and quantitative PCR was performed on A3A. ( B ) WB control of the experiment performed in 5A, using cGAS, pol III 1 and pol III 2 antibodies. The β-actin loading controls are shown below. ( C ) Interferon alpha and beta production by THP-1 following transfection by 500 ng V1V2 HIV-1 DNA. JetPRIME was used as control. ( D ) A3A quantification by THP-1 following transfection by 500 ng inMT-COI 0 mut. and inMT-COI 32 mut. DNA. ( E ) V1V2 RNA transcripts from DNA transfected THP-1 cells. Total RNA was extracted at 24 h and a cDNA corresponding to V1V2 was produced in presence or absence of RT and amplified by PCR. T1 and T2 refer to independent transfections. ( F ) Immunoprecipitation with an anti-RIG-I monoclonal antibody performed at 8 h along with an anti-HA as control. V1V2 specific RT-PCR products were recovered only from anti-RIG-I immunoprecipitation. RT, T1 and T2 as for 5E. ‘*’ indicates statistically significant difference between two observed values ( P

    Journal: Nucleic Acids Research

    Article Title: Self-cytoplasmic DNA upregulates the mutator enzyme APOBEC3A leading to chromosomal DNA damage

    doi: 10.1093/nar/gkx001

    Figure Lengend Snippet: Cytoplasmic RNA synthesis by DNA-dependent RNA polymerase III binds to RIG-I. ( A ) THP-1 cells were transfected with control siRNA or 2 RNA polymerase III siRNA (siRNA polIII 1 and siRNA polIII 2 ) or cGAS siRNA. Six hours post-transfection with siRNA, cells were transfecte with 500 ng of MT-COI DNA. At 24 h post-transfection, total RNA was extracted and quantitative PCR was performed on A3A. ( B ) WB control of the experiment performed in 5A, using cGAS, pol III 1 and pol III 2 antibodies. The β-actin loading controls are shown below. ( C ) Interferon alpha and beta production by THP-1 following transfection by 500 ng V1V2 HIV-1 DNA. JetPRIME was used as control. ( D ) A3A quantification by THP-1 following transfection by 500 ng inMT-COI 0 mut. and inMT-COI 32 mut. DNA. ( E ) V1V2 RNA transcripts from DNA transfected THP-1 cells. Total RNA was extracted at 24 h and a cDNA corresponding to V1V2 was produced in presence or absence of RT and amplified by PCR. T1 and T2 refer to independent transfections. ( F ) Immunoprecipitation with an anti-RIG-I monoclonal antibody performed at 8 h along with an anti-HA as control. V1V2 specific RT-PCR products were recovered only from anti-RIG-I immunoprecipitation. RT, T1 and T2 as for 5E. ‘*’ indicates statistically significant difference between two observed values ( P

    Article Snippet: FACS analysis for DNA double stranded breaks Twenty-four hours post-transfection, THP-1 cells were washed with PBS, fixed in 2% ice-cold paraformaldehyde (Electron Microscopy Sciences) for 15 min and permeabilized in 90% ice-cold methanol (Sigma) for 30 min. After two washes with PBS, cells were incubated for 1 h with 1:100 diluted Alexa Fluor 488-conjugated mouse monoclonal anti-γH2AX (N1-431) antibody (BD Pharmingen).

    Techniques: Transfection, Real-time Polymerase Chain Reaction, Western Blot, Produced, Amplification, Polymerase Chain Reaction, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Transfected MT-COI DNA induces double-stranded DNA breaks. ( A ) Interferon α production following transfection of THP-1 cells by 500 ng MT-COI T or MT-COI U , 500 ng of WT 1+2 , WT 2+3 , Hyp 1+2 , Hyp 3+4 , WT 1+3 and WT 2+4 ( Supplementary Table S1 ). ( B ) FACS analysis of γH2AX-positive THP-1 cells transfected with MT-COI . Means and standard deviation of γH2AX-positive cell frequencies for duplicate transfection. ( C ) FACS analysis of γH2AX-positive DSBs in THP-1 transfected with WT 3+2 , WT 3+2FAM , WT 1+2 and W 1+2FAM respectively. Means and standard deviation of γH2AX-positive cell frequencies for duplicate transfection. ( D ) FACS analysis of γH2AX-positive DSBs in THP-1 transfected with cells gated on FAM-positive cells after transfection with WT 3+2FAM and W 1+2FAM respectively. Means and standard deviation of γH2AX-positive cell frequencies for duplicate transfection. ( E ) Transcription profiling of A3A in THP-1 transfected cells with WT 1+2FAM (dsDNA) or WT 3+2FAM (ssDNA) respectively, in presence or absence of JetPRIME. ‘*’ indicates statistically significant difference between two observed percentages ( P

    Journal: Nucleic Acids Research

    Article Title: Self-cytoplasmic DNA upregulates the mutator enzyme APOBEC3A leading to chromosomal DNA damage

    doi: 10.1093/nar/gkx001

    Figure Lengend Snippet: Transfected MT-COI DNA induces double-stranded DNA breaks. ( A ) Interferon α production following transfection of THP-1 cells by 500 ng MT-COI T or MT-COI U , 500 ng of WT 1+2 , WT 2+3 , Hyp 1+2 , Hyp 3+4 , WT 1+3 and WT 2+4 ( Supplementary Table S1 ). ( B ) FACS analysis of γH2AX-positive THP-1 cells transfected with MT-COI . Means and standard deviation of γH2AX-positive cell frequencies for duplicate transfection. ( C ) FACS analysis of γH2AX-positive DSBs in THP-1 transfected with WT 3+2 , WT 3+2FAM , WT 1+2 and W 1+2FAM respectively. Means and standard deviation of γH2AX-positive cell frequencies for duplicate transfection. ( D ) FACS analysis of γH2AX-positive DSBs in THP-1 transfected with cells gated on FAM-positive cells after transfection with WT 3+2FAM and W 1+2FAM respectively. Means and standard deviation of γH2AX-positive cell frequencies for duplicate transfection. ( E ) Transcription profiling of A3A in THP-1 transfected cells with WT 1+2FAM (dsDNA) or WT 3+2FAM (ssDNA) respectively, in presence or absence of JetPRIME. ‘*’ indicates statistically significant difference between two observed percentages ( P

    Article Snippet: FACS analysis for DNA double stranded breaks Twenty-four hours post-transfection, THP-1 cells were washed with PBS, fixed in 2% ice-cold paraformaldehyde (Electron Microscopy Sciences) for 15 min and permeabilized in 90% ice-cold methanol (Sigma) for 30 min. After two washes with PBS, cells were incubated for 1 h with 1:100 diluted Alexa Fluor 488-conjugated mouse monoclonal anti-γH2AX (N1-431) antibody (BD Pharmingen).

    Techniques: Transfection, FACS, Standard Deviation

    Paracrine effect of interferon β and editing of endogenous MT-CYB . ( A ) A3A relative expression was amplified by paracrine IFNβ induction in THP-1 cells and analyzed with only JetPRIME or transfected with 500 ng of MT-COI T DNA. Cell supernatant (SN- MT-COI T ) was incubated with 2 or 10 μg of antibody against IFNα, IFNβ or IFNγ for 1 h and added to 1.5 × 10 6 fresh THP-1 cells for 24 h. ND, not detectable. ( B ) THP-1 DNA transfection increases A3-editing of endogenous cytoplasmic MT-CYB DNA recovered by 3DPCR. The white line indicates the threshold between edited and unedited 3DPCR products. Asterisks refer to the samples cloned and sequenced. M: molecular weight markers. ( C ) Frequency distribution of C→T editing per clone as a function of the quantity of DNA transfected.

    Journal: Nucleic Acids Research

    Article Title: Self-cytoplasmic DNA upregulates the mutator enzyme APOBEC3A leading to chromosomal DNA damage

    doi: 10.1093/nar/gkx001

    Figure Lengend Snippet: Paracrine effect of interferon β and editing of endogenous MT-CYB . ( A ) A3A relative expression was amplified by paracrine IFNβ induction in THP-1 cells and analyzed with only JetPRIME or transfected with 500 ng of MT-COI T DNA. Cell supernatant (SN- MT-COI T ) was incubated with 2 or 10 μg of antibody against IFNα, IFNβ or IFNγ for 1 h and added to 1.5 × 10 6 fresh THP-1 cells for 24 h. ND, not detectable. ( B ) THP-1 DNA transfection increases A3-editing of endogenous cytoplasmic MT-CYB DNA recovered by 3DPCR. The white line indicates the threshold between edited and unedited 3DPCR products. Asterisks refer to the samples cloned and sequenced. M: molecular weight markers. ( C ) Frequency distribution of C→T editing per clone as a function of the quantity of DNA transfected.

    Article Snippet: FACS analysis for DNA double stranded breaks Twenty-four hours post-transfection, THP-1 cells were washed with PBS, fixed in 2% ice-cold paraformaldehyde (Electron Microscopy Sciences) for 15 min and permeabilized in 90% ice-cold methanol (Sigma) for 30 min. After two washes with PBS, cells were incubated for 1 h with 1:100 diluted Alexa Fluor 488-conjugated mouse monoclonal anti-γH2AX (N1-431) antibody (BD Pharmingen).

    Techniques: Expressing, Amplification, Transfection, Incubation, Clone Assay, Molecular Weight

    Effects of (A) pH, (B) applied potential, and (C) temperature. These are effects on amperometric response of the GOD/PtAuNP/ss-DNA/GR modified electrode to 0.1 mM glucose in 0.1 M PBS (pH 7.0).

    Journal: Nanoscale Research Letters

    Article Title: DNA-templated synthesis of PtAu bimetallic nanoparticle/graphene nanocomposites and their application in glucose biosensor

    doi: 10.1186/1556-276X-9-99

    Figure Lengend Snippet: Effects of (A) pH, (B) applied potential, and (C) temperature. These are effects on amperometric response of the GOD/PtAuNP/ss-DNA/GR modified electrode to 0.1 mM glucose in 0.1 M PBS (pH 7.0).

    Article Snippet: Native double-stranded DNA (ds-DNA) from calf thymus and GOD were purchased from Sigma Chemical (St. Louis, MO, USA).

    Techniques: Modification

    Impedance spectrum of various electrodes in 5.0 mM K 3 Fe(CN) 6 /K 4 Fe(CN) 6 (1:1) containing 0.1 M KCl. Bare electrode (curve a), ss-DNA/GR modified electrode (curve b), PtAuNP/ss-DNA/GR modified electrode (curve c), and GOD/PtAuNP/ss-DNA/GR modified electrode (curve d).

    Journal: Nanoscale Research Letters

    Article Title: DNA-templated synthesis of PtAu bimetallic nanoparticle/graphene nanocomposites and their application in glucose biosensor

    doi: 10.1186/1556-276X-9-99

    Figure Lengend Snippet: Impedance spectrum of various electrodes in 5.0 mM K 3 Fe(CN) 6 /K 4 Fe(CN) 6 (1:1) containing 0.1 M KCl. Bare electrode (curve a), ss-DNA/GR modified electrode (curve b), PtAuNP/ss-DNA/GR modified electrode (curve c), and GOD/PtAuNP/ss-DNA/GR modified electrode (curve d).

    Article Snippet: Native double-stranded DNA (ds-DNA) from calf thymus and GOD were purchased from Sigma Chemical (St. Louis, MO, USA).

    Techniques: Modification

    Cyclic voltammograms of GOD/PtAuNP/ss-DNA/GR modified electrode. They are in (curve a) N 2 -saturated and O 2 -saturated PBS (pH 7.0) in the (curve b) absence and (curve c) presence of 1.0 mM glucose at 100 mV s -1 .

    Journal: Nanoscale Research Letters

    Article Title: DNA-templated synthesis of PtAu bimetallic nanoparticle/graphene nanocomposites and their application in glucose biosensor

    doi: 10.1186/1556-276X-9-99

    Figure Lengend Snippet: Cyclic voltammograms of GOD/PtAuNP/ss-DNA/GR modified electrode. They are in (curve a) N 2 -saturated and O 2 -saturated PBS (pH 7.0) in the (curve b) absence and (curve c) presence of 1.0 mM glucose at 100 mV s -1 .

    Article Snippet: Native double-stranded DNA (ds-DNA) from calf thymus and GOD were purchased from Sigma Chemical (St. Louis, MO, USA).

    Techniques: Modification

    Amperometric responses of modified electrodes to additions of 0.1 mM glucose in 10-mL PBS at -0.2 V. GOD/ss-DNA/GR (curve a), GOD/PtNP/ss-DNA/GR (curve b), GOD/AuNP/ss-DNA/GR (curve c), and GOD/PtAuNP/ss-DNA/GR (curve d) modified electrodes. Left inset is the calibration curve of the biosensor.

    Journal: Nanoscale Research Letters

    Article Title: DNA-templated synthesis of PtAu bimetallic nanoparticle/graphene nanocomposites and their application in glucose biosensor

    doi: 10.1186/1556-276X-9-99

    Figure Lengend Snippet: Amperometric responses of modified electrodes to additions of 0.1 mM glucose in 10-mL PBS at -0.2 V. GOD/ss-DNA/GR (curve a), GOD/PtNP/ss-DNA/GR (curve b), GOD/AuNP/ss-DNA/GR (curve c), and GOD/PtAuNP/ss-DNA/GR (curve d) modified electrodes. Left inset is the calibration curve of the biosensor.

    Article Snippet: Native double-stranded DNA (ds-DNA) from calf thymus and GOD were purchased from Sigma Chemical (St. Louis, MO, USA).

    Techniques: Modification

    The formation procedures of GOD/PtAuNP/ss-DNA/GR nanocomposites.

    Journal: Nanoscale Research Letters

    Article Title: DNA-templated synthesis of PtAu bimetallic nanoparticle/graphene nanocomposites and their application in glucose biosensor

    doi: 10.1186/1556-276X-9-99

    Figure Lengend Snippet: The formation procedures of GOD/PtAuNP/ss-DNA/GR nanocomposites.

    Article Snippet: Native double-stranded DNA (ds-DNA) from calf thymus and GOD were purchased from Sigma Chemical (St. Louis, MO, USA).

    Techniques:

    Photomicrographs (Stain, Safranin O ( A ) and DAB primary antibody staining with Fast Green counter stain ( B – E ); original magnification, ×10) compare ( A ) the injured zone and the control zone in ( B ) TUNEL, ( C ) ssDNA, ( D ) anti-active caspase-3, and ( E ) ISOL. The break in the cartilage is to the right of the injured zone images. TUNEL = terminal deoxynucleotidyl transferase end labeling; ssDNA = DNA denaturation analysis using anti single-stranded DNA antibody; ISOL = in situ oligonucleotide ligation.

    Journal: Clinical Orthopaedics and Related Research

    Article Title: Chondrocyte Apoptosis after Simulated Intraarticular Fracture: A Comparison of Histologic Detection Methods

    doi: 10.1007/s11999-009-0829-3

    Figure Lengend Snippet: Photomicrographs (Stain, Safranin O ( A ) and DAB primary antibody staining with Fast Green counter stain ( B – E ); original magnification, ×10) compare ( A ) the injured zone and the control zone in ( B ) TUNEL, ( C ) ssDNA, ( D ) anti-active caspase-3, and ( E ) ISOL. The break in the cartilage is to the right of the injured zone images. TUNEL = terminal deoxynucleotidyl transferase end labeling; ssDNA = DNA denaturation analysis using anti single-stranded DNA antibody; ISOL = in situ oligonucleotide ligation.

    Article Snippet: Detection of double-stranded DNA breaks by TUNEL was performed using direct incorporation of fluorescein-labeled nucleotides with modification of the protocol provided with the ApopTag® Fluorescein Direct (Millipore Corp, Bedford, MA).

    Techniques: Staining, TUNEL Assay, End Labeling, In Situ, Ligation

    A graph compares the apoptotic indices in the injured zone and control zone using TUNEL, ssDNA, anti-active caspase-3, ISOL A (single 3′ base overhang), and ISOL B (blunt DNA ends). Error bars represent standard errors of the mean. The apoptotic indices in the control versus the injured zones were statistically different in all test groups; the representative p values are above each paired control and injured group. TUNEL = terminal deoxynucleotidyl transferase end labeling; ssDNA = DNA denaturation analysis using anti single-stranded DNA antibody; ISOL = in situ oligonucleotide ligation.

    Journal: Clinical Orthopaedics and Related Research

    Article Title: Chondrocyte Apoptosis after Simulated Intraarticular Fracture: A Comparison of Histologic Detection Methods

    doi: 10.1007/s11999-009-0829-3

    Figure Lengend Snippet: A graph compares the apoptotic indices in the injured zone and control zone using TUNEL, ssDNA, anti-active caspase-3, ISOL A (single 3′ base overhang), and ISOL B (blunt DNA ends). Error bars represent standard errors of the mean. The apoptotic indices in the control versus the injured zones were statistically different in all test groups; the representative p values are above each paired control and injured group. TUNEL = terminal deoxynucleotidyl transferase end labeling; ssDNA = DNA denaturation analysis using anti single-stranded DNA antibody; ISOL = in situ oligonucleotide ligation.

    Article Snippet: Detection of double-stranded DNA breaks by TUNEL was performed using direct incorporation of fluorescein-labeled nucleotides with modification of the protocol provided with the ApopTag® Fluorescein Direct (Millipore Corp, Bedford, MA).

    Techniques: TUNEL Assay, End Labeling, In Situ, Ligation

    Photomicrographs (Stain, A fluorescein with DAPI counterstain, B – D DAB for primary antibody with DAPI counterstain; original magnification, ×20) show growth plate cartilage stained with ( A ) TUNEL, ( B ) ssDNA, ( C ) anti-active caspase-3, and ( D ) ISOL A. In all techniques, positive cells were observed only in the hypertrophic zone of the growth plate cartilage, showing good spatial distribution for these techniques. TUNEL = terminal deoxynucleotidyl transferase end labeling; ISOL = in situ oligonucleotide ligation; ssDNA = DNA denaturation analysis using anti-single-stranded DNA antibody.

    Journal: Clinical Orthopaedics and Related Research

    Article Title: Chondrocyte Apoptosis after Simulated Intraarticular Fracture: A Comparison of Histologic Detection Methods

    doi: 10.1007/s11999-009-0829-3

    Figure Lengend Snippet: Photomicrographs (Stain, A fluorescein with DAPI counterstain, B – D DAB for primary antibody with DAPI counterstain; original magnification, ×20) show growth plate cartilage stained with ( A ) TUNEL, ( B ) ssDNA, ( C ) anti-active caspase-3, and ( D ) ISOL A. In all techniques, positive cells were observed only in the hypertrophic zone of the growth plate cartilage, showing good spatial distribution for these techniques. TUNEL = terminal deoxynucleotidyl transferase end labeling; ISOL = in situ oligonucleotide ligation; ssDNA = DNA denaturation analysis using anti-single-stranded DNA antibody.

    Article Snippet: Detection of double-stranded DNA breaks by TUNEL was performed using direct incorporation of fluorescein-labeled nucleotides with modification of the protocol provided with the ApopTag® Fluorescein Direct (Millipore Corp, Bedford, MA).

    Techniques: Staining, TUNEL Assay, End Labeling, In Situ, Ligation

    Polyreactivity of monoclonal antibodies (mAbs) cloned from circulating plasmablasts of control and chronically HIV-infected individuals. (A) ELISA results of known bnAbs (PG9, PG16, 4E10, and 2F5) against double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), insulin, and lipopolysaccharide (LPS). (B) ELISA results of each recombinant mAbs derived from plasmablasts of control and HIV-infected individuals against dsDNA, ssDNA, insulin, and LPS. Red lines represent 4E10, as the positive control. Green lines represent PG16, as the negative control. Dashed lines represent the cut-off OD450 nm value for positive reactivity, which is 3 SEM above the average reactivity of control Abs. Pie charts summarize the frequency of polyreactive (black) and non-polyreactive (white) Abs. The percentages of polyreactive Abs in control and HIV-positive group are summarized in the bottom panel. P -values are in comparison with pooled Abs derived from the control donors. P -value was determined by Fisher’s exact test. (C–G) Correlations of the anti-dsDNA reactivities of the 72 recombinant mAbs from HIV-infected individuals with their reactivities against ssDNA (C) , insulin (D) , LPS (E) , and gp140 (F) . (G) Correlations of the anti-dsDNA reactivities with gp140-binding Abs only. rho and P -values were determined by Spearman’s ranked correlation test. (H) Numbers of non-polyreactive Abs among non-anti-gp140 and anti-gp140 Abs compared with polyreactive Abs. P -value was determined by chi-squared test.

    Journal: Frontiers in Immunology

    Article Title: Circulating Plasmablasts from Chronically Human Immunodeficiency Virus-Infected Individuals Predominantly Produce Polyreactive/Autoreactive Antibodies

    doi: 10.3389/fimmu.2017.01691

    Figure Lengend Snippet: Polyreactivity of monoclonal antibodies (mAbs) cloned from circulating plasmablasts of control and chronically HIV-infected individuals. (A) ELISA results of known bnAbs (PG9, PG16, 4E10, and 2F5) against double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), insulin, and lipopolysaccharide (LPS). (B) ELISA results of each recombinant mAbs derived from plasmablasts of control and HIV-infected individuals against dsDNA, ssDNA, insulin, and LPS. Red lines represent 4E10, as the positive control. Green lines represent PG16, as the negative control. Dashed lines represent the cut-off OD450 nm value for positive reactivity, which is 3 SEM above the average reactivity of control Abs. Pie charts summarize the frequency of polyreactive (black) and non-polyreactive (white) Abs. The percentages of polyreactive Abs in control and HIV-positive group are summarized in the bottom panel. P -values are in comparison with pooled Abs derived from the control donors. P -value was determined by Fisher’s exact test. (C–G) Correlations of the anti-dsDNA reactivities of the 72 recombinant mAbs from HIV-infected individuals with their reactivities against ssDNA (C) , insulin (D) , LPS (E) , and gp140 (F) . (G) Correlations of the anti-dsDNA reactivities with gp140-binding Abs only. rho and P -values were determined by Spearman’s ranked correlation test. (H) Numbers of non-polyreactive Abs among non-anti-gp140 and anti-gp140 Abs compared with polyreactive Abs. P -value was determined by chi-squared test.

    Article Snippet: ELISA plates were coated with double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) in PBS, lipopolysaccharide (LPS), insulin (Sigma-Aldrich, St. Louis, MO, USA), DWEYS in coating buffer (pH = 7.6) at 10 µg/mL, and YU2 gp140 and gp140 trimer (HIV-1 clade B) (Immune Tech, Suzhou, Jiangsu, China) in PBS at 1 µg/mL at 4°C overnight.

    Techniques: Infection, Enzyme-linked Immunosorbent Assay, Recombinant, Derivative Assay, Positive Control, Negative Control, Binding Assay

    The Nyquist diagram obtained on the glassy carbon electrode covered with poly(Azure B–proflavine) and DNA previously mixed with doxorubicin solution (2 μL of the mixture containing 1.0 mg/mL DNA in 0.1 M HEPES + 0.03 M NaCl, pH = 7.0). Measurements in 0.025 M PB + 0.1 M KCl, pH = 7.0, in the presence of 10 mM [Fe(CN) 6 ] 3−/4− .

    Journal: Nanomaterials

    Article Title: Electrochemical DNA Sensor Based on the Copolymer of Proflavine and Azure B for Doxorubicin Determination

    doi: 10.3390/nano10050924

    Figure Lengend Snippet: The Nyquist diagram obtained on the glassy carbon electrode covered with poly(Azure B–proflavine) and DNA previously mixed with doxorubicin solution (2 μL of the mixture containing 1.0 mg/mL DNA in 0.1 M HEPES + 0.03 M NaCl, pH = 7.0). Measurements in 0.025 M PB + 0.1 M KCl, pH = 7.0, in the presence of 10 mM [Fe(CN) 6 ] 3−/4− .

    Article Snippet: Reagents Azure B (3-(dimethylamino)-7-(methylamino)phenothiazin-5-ium chloride, 97%), proflavine hydrochloride (3,6-Diamino-10-methylacridinium chloride, 95%), doxorubicin hydrochloride ((8S,10S)-6,8,10,11-tetrahydroxy-8-(hydroxyacetyl)-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione, 97%), low molecular double-stranded DNA from salmon sperm (average mol. mass 4.6 kDa [ ]) HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), hydroquinone, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich, Dortmund, Germany ( https://www.sigmaaldrich.com/catalog ).

    Techniques:

    The Nyquist diagram obtained with the glassy carbon electrode (1) covered with poly(Azure B–proflavine) prior to (2) and after (3) application of DNA (2 μL of 1.0 mg/mL solution in 0.1 M HEPES + 0.03 M NaCl, pH = 7.0) or DNA-1.0 nM doxorubicin aliquot (4). Measurements in 0.025 M PB + 0.1 M KCl, pH = 7.0, in the presence of 10 mM [Fe(CN) 6 ] 3−/4− . Inset: equivalent circuit, C —constant phase element, R —charge transfer resistance.

    Journal: Nanomaterials

    Article Title: Electrochemical DNA Sensor Based on the Copolymer of Proflavine and Azure B for Doxorubicin Determination

    doi: 10.3390/nano10050924

    Figure Lengend Snippet: The Nyquist diagram obtained with the glassy carbon electrode (1) covered with poly(Azure B–proflavine) prior to (2) and after (3) application of DNA (2 μL of 1.0 mg/mL solution in 0.1 M HEPES + 0.03 M NaCl, pH = 7.0) or DNA-1.0 nM doxorubicin aliquot (4). Measurements in 0.025 M PB + 0.1 M KCl, pH = 7.0, in the presence of 10 mM [Fe(CN) 6 ] 3−/4− . Inset: equivalent circuit, C —constant phase element, R —charge transfer resistance.

    Article Snippet: Reagents Azure B (3-(dimethylamino)-7-(methylamino)phenothiazin-5-ium chloride, 97%), proflavine hydrochloride (3,6-Diamino-10-methylacridinium chloride, 95%), doxorubicin hydrochloride ((8S,10S)-6,8,10,11-tetrahydroxy-8-(hydroxyacetyl)-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione, 97%), low molecular double-stranded DNA from salmon sperm (average mol. mass 4.6 kDa [ ]) HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), hydroquinone, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich, Dortmund, Germany ( https://www.sigmaaldrich.com/catalog ).

    Techniques:

    Chromatin Mg 2+ -dependent self-association is independent of the NRL, DNA topology, and unconstrained supercoiling. A , DNA electrophoresis showing separation of covalently closed ( longer gray arrow ) and nicked ( shorter white arrow ) DNA from circular plasmids. B , DNA from a covalently closed ( cc ) band ( gray arrow ) and nicked band ( white arrow ) was extracted from the gel ( A ) and separated on an agarose gels run in the presence of 1.5 μg/ml CQ in the gel and electrode buffer. C , rates of Mg 2+ -dependent self-association in linear and circular (relaxed and supercoiled) nucleosome arrays. D , graphs showing the concentration of Mg 2+ at which 50% of the material is precipitated. Error bars , S.D.

    Journal: The Journal of Biological Chemistry

    Article Title: Nucleosome spacing periodically modulates nucleosome chain folding and DNA topology in circular nucleosome arrays

    doi: 10.1074/jbc.RA118.006412

    Figure Lengend Snippet: Chromatin Mg 2+ -dependent self-association is independent of the NRL, DNA topology, and unconstrained supercoiling. A , DNA electrophoresis showing separation of covalently closed ( longer gray arrow ) and nicked ( shorter white arrow ) DNA from circular plasmids. B , DNA from a covalently closed ( cc ) band ( gray arrow ) and nicked band ( white arrow ) was extracted from the gel ( A ) and separated on an agarose gels run in the presence of 1.5 μg/ml CQ in the gel and electrode buffer. C , rates of Mg 2+ -dependent self-association in linear and circular (relaxed and supercoiled) nucleosome arrays. D , graphs showing the concentration of Mg 2+ at which 50% of the material is precipitated. Error bars , S.D.

    Article Snippet: Linear and circular nucleosome arrays dissolved in 10 μg/ml HNE buffer at DNA concentration 25 μg/ml were mixed with increasing concentrations of MgCl2 (Sigma-Aldrich, catalog no. M1028 1 ml) and incubated for 20 min on ice.

    Techniques: Nucleic Acid Electrophoresis, Concentration Assay

    Mean intracellular reactive oxygen species (ROS) levels and DNA damage in all- trans retinoic acid (ATRA)-treated and control ARPE-19 cells exposed to tert -butyl hydroperoxide (tBH). Reactive oxygen species (ROS) are expressed as the relative fluorescence of 2′,7′-dichlorofluorescein (DCF) normalized to controls after 30 min incubation with tBH at 37 °C. DNA damage was measured as a percentage of tail DNA in the comets in alkaline versions of the comet assay following exposure to tBH for 15 min at 37 °C. The induction of DNA double strand breaks was assessed by the extent of phosphorylation of H2AX and ATM (ataxia teleangiectasia mutated) (γH2AX and pATM) after γ-ray irradiation (2 Gy/min) of cells. Results are presented as mean ± SEM, n = 12 for ROS level, n = 100 for comet assay, n = 8 for H2AX/ATM phosphorylation, *** p

    Journal: International Journal of Molecular Sciences

    Article Title: All-Trans Retinoic Acid Modulates DNA Damage Response and the Expression of the VEGF-A and MKI67 Genes in ARPE-19 Cells Subjected to Oxidative Stress

    doi: 10.3390/ijms17060898

    Figure Lengend Snippet: Mean intracellular reactive oxygen species (ROS) levels and DNA damage in all- trans retinoic acid (ATRA)-treated and control ARPE-19 cells exposed to tert -butyl hydroperoxide (tBH). Reactive oxygen species (ROS) are expressed as the relative fluorescence of 2′,7′-dichlorofluorescein (DCF) normalized to controls after 30 min incubation with tBH at 37 °C. DNA damage was measured as a percentage of tail DNA in the comets in alkaline versions of the comet assay following exposure to tBH for 15 min at 37 °C. The induction of DNA double strand breaks was assessed by the extent of phosphorylation of H2AX and ATM (ataxia teleangiectasia mutated) (γH2AX and pATM) after γ-ray irradiation (2 Gy/min) of cells. Results are presented as mean ± SEM, n = 12 for ROS level, n = 100 for comet assay, n = 8 for H2AX/ATM phosphorylation, *** p

    Article Snippet: The events for ATM activated cells, H2AX activated cells, cells with DNA double-strand breaks determined as dual activation of both ATM and H2AX, and negative cells, were counted with the Muse Cell Analyzer (Millipore, Hayward, CA, USA) and analyzed with MuseSoft 1.4.0.0 (Millipore, Hayward, CA, USA).

    Techniques: Fluorescence, Incubation, Single Cell Gel Electrophoresis, Irradiation

    Nanopore detection of small-molecule binding to single-stranded DNA. (a) TEM image of a 2.2 nm pore, and the molecules used in this study. Since SYBR Green II (SGII) binds to poly(dA) with very low affinity, dA 60 was used as a negative control and dA 50 dN 50 as a positive control. (b) Typical translocation events for dA 60 , before and after the addition of SGII to the cis chamber (side histogram is an all-point histogram of the current data). Typical translocation events for dA 50 dN 50 , before and after the addition of SGII, are shown in (c) and (d), respectively. The two-step events prevalent after SGII binding were characterized by amplitudes of 0.58 and 0.96 nA, corresponding to dA 50 and SGII-bound dN 50 , respectively (see text).

    Journal: Nano letters

    Article Title: DNA Profiling Using Solid-State Nanopores: Detection of DNA-Binding Molecules

    doi: 10.1021/nl901691v

    Figure Lengend Snippet: Nanopore detection of small-molecule binding to single-stranded DNA. (a) TEM image of a 2.2 nm pore, and the molecules used in this study. Since SYBR Green II (SGII) binds to poly(dA) with very low affinity, dA 60 was used as a negative control and dA 50 dN 50 as a positive control. (b) Typical translocation events for dA 60 , before and after the addition of SGII to the cis chamber (side histogram is an all-point histogram of the current data). Typical translocation events for dA 50 dN 50 , before and after the addition of SGII, are shown in (c) and (d), respectively. The two-step events prevalent after SGII binding were characterized by amplitudes of 0.58 and 0.96 nA, corresponding to dA 50 and SGII-bound dN 50 , respectively (see text).

    Article Snippet: In this part of our study we have used a 2.2 nm pore (see ) to probe the interaction of single - stranded DNA molecules with the cyanine dye SYBR Green II (SGII), an RNA-selective stain that preferentially intercalates into single-stranded DNA over double-stranded DNA (Invitrogen Corp., Carlsbad, CA).

    Techniques: Binding Assay, Transmission Electron Microscopy, SYBR Green Assay, Negative Control, Positive Control, Translocation Assay

    Genome-wide identification of SsrB palindrome sequences. (A) A position weight matrix was generated from all naturally-evolved palindromes in SPI-2 and used to search the genome for similar sequences. Palindromes identified in regulatory DNA that co-occurred with ChIP peaks upstream of SsrB-regulated genes were selected, aligned and binned according to those scoring > 0.8 against the position weight matrix (top box) and those scoring between 0.7–0.8 (lower box). The left (7′) and right (7″) heptamers and the 4-bp spacer are displayed as a heat-map to show bases of high conservation (dark blue) from degenerate regions (light blue/white). The genes controlled by these promoters are indicated to the left of the sequences and coloured according to function: structural components of the T3SS (orange), effectors (blue), regulatory elements (red), T3SS chaperones and translocon (green), and SsrB-regulated genes of unknown function (black). (B) The high-scoring palindrome in the ssaR IGR is functional. A single-copy ssaR reporter was integrated on the chromosome and tested for functional activity in wild-type cells and in Δ ssrB . Promoter activity is shown as the mean with standard error from three separate experiments. (C) Functional validation of the intragenic high-scoring palindrome in the ssaE coding sequence. A single-copy transcriptional reporter that either contained (WT P sseA ) or lacked (P sseA del) the single heptamer site in the sseA IGR was integrated on the chromosome in wild-type cells, or mutants lacking either ssrB or the ssaE coding sequence that removed the high-scoring intragenic palindrome (P sseA '). Transcriptional activity at 6 h is shown as the mean of triplicate determinations with standard error.

    Journal: PLoS Genetics

    Article Title: Identification of the Regulatory Logic Controlling Salmonella Pathoadaptation by the SsrA-SsrB Two-Component System

    doi: 10.1371/journal.pgen.1000875

    Figure Lengend Snippet: Genome-wide identification of SsrB palindrome sequences. (A) A position weight matrix was generated from all naturally-evolved palindromes in SPI-2 and used to search the genome for similar sequences. Palindromes identified in regulatory DNA that co-occurred with ChIP peaks upstream of SsrB-regulated genes were selected, aligned and binned according to those scoring > 0.8 against the position weight matrix (top box) and those scoring between 0.7–0.8 (lower box). The left (7′) and right (7″) heptamers and the 4-bp spacer are displayed as a heat-map to show bases of high conservation (dark blue) from degenerate regions (light blue/white). The genes controlled by these promoters are indicated to the left of the sequences and coloured according to function: structural components of the T3SS (orange), effectors (blue), regulatory elements (red), T3SS chaperones and translocon (green), and SsrB-regulated genes of unknown function (black). (B) The high-scoring palindrome in the ssaR IGR is functional. A single-copy ssaR reporter was integrated on the chromosome and tested for functional activity in wild-type cells and in Δ ssrB . Promoter activity is shown as the mean with standard error from three separate experiments. (C) Functional validation of the intragenic high-scoring palindrome in the ssaE coding sequence. A single-copy transcriptional reporter that either contained (WT P sseA ) or lacked (P sseA del) the single heptamer site in the sseA IGR was integrated on the chromosome in wild-type cells, or mutants lacking either ssrB or the ssaE coding sequence that removed the high-scoring intragenic palindrome (P sseA '). Transcriptional activity at 6 h is shown as the mean of triplicate determinations with standard error.

    Article Snippet: Transcriptional reporter experiments Transcriptional fusions to lacZ for the ssaG and sseA promoter palindrome analysis were generated using chemically synthesized double-stranded DNA (Genscript Corp).

    Techniques: Genome Wide, Generated, Chromatin Immunoprecipitation, Functional Assay, Activity Assay, Sequencing