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  • 99
    New England Biolabs t7 endonuclease i
    SMD is sensitive to T7 endonuclease I. Logarithmically growing  apn1/2  cells were arrested in G2/M with nocodazole, treated with MMS (0.1%, 15 min) in PBS and returned to the YPDA+nocodazole medium. Cells were collected at the indicated times and processed for DNA plug preparation. The plugs were then incubated with T7 endonuclease I as described in the   Materials and Methods  or in buffer only (mock-treated). The SMD was estimated in the ethidium bromide stained gel by comparing the amount of DNA material in the SMD region to DNA in the small chromosomes that experienced little breakage, “SMD / Chr (I+VI)”.
    T7 Endonuclease I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2927 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dnase i enzyme
    <t>DNase</t> I treatment is effective against C. jejuni biofilms on stainless steel surfaces and in the presence of organic materials in aerobic conditions . The ability of DNase I to inhibit biofilm formation of C. jejuni NCTC 11168 on sterile, stainless steel coupons (A) or in the presence of chicken juice, mimicking a conditioned surface (B) . TTC staining was used to measure biofilm formation in the presence of chicken juice (B) . DNase I is able to significantly decrease biofilm formation in both conditions. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P
    Dnase I Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 284 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dnase i
    Perlecan deficiency does not affect cell turnover in white adipose tissue. ( a ) TUNEL staining in the VAT of the WT-Tg (upper panel) and the  Hspg2 −/− -Tg (lower panel) mice fed either ND or HFD. The areas containing positive nuclei of adipocytes are selected and shown. The positive control was made using DNase I. Note that TUNEL-positive nuclei are brown and negative nuclei are blue. ( b ) The percentage of TUNEL-positive nuclei of adipocytes. The nuclei from at least 100 adipocytes per mouse were evaluated. Data points and error bars represent the mean ± S.D. (n = 5). ( c ) Representative immunohistochemical staining of Ki67 in the VAT of the WT-Tg (upper panel) and the  Hspg2 −/− -Tg (lower panel) mice fed either ND or HFD. The tissue from human abdominal cancer was used as positive control. No nuclei were positive for Ki67 in any groups. Data were analyzed by two-way ANOVA with Tukey’s multiple comparison ( b ). Scale bar, 50  μm.
    Dnase I, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 26477 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dnase i
    Effects of noncomplementary dNTPs on <t>DNase</t> I footprints and stable complex formation by HIV-1 RT
    Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 56770 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rnase free dnase i
    Consensus GGUG-containing RNA oligonucleotide promotes the inhibitory effect of TLS on CBP/p300 HAT activities a , Co-immunoprecipitation (IP) of p300 and TLS from <t>RNase</t> A-treated HeLa cells. b, P300 HAT activity was measured using micrococcal nuclease (MNase) or <t>DNase</t> I pre-treated GST and GST-TLS in the presence of GGUG- or CCUC-oligonucleotide. * p
    Rnase Free Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8524 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Boehringer Mannheim dnase i
    Exonuclease activity of rLiEndoG. 500′-labeled with FAM were digested with rLiEndoG or DNase I. The amount of enzyme used for digestion is indicated. Digestion products were processed by both agarose gel (1%) and capillary electrophoresis.  A ) Agarose gel of the DNA fragments generated after 1 h of digestion with rLiEndoG or DNase I.  B ) Capillary electrophoresis of the DNA probe digested for 1 h with 0.1 µg of rLiEndoG.  C ) Capillary electrophoresis results obtained for the DNA probe digested for 1 hour with 0.01 units of DNase I. Digested DNA was heat-denatured prior to capillary electrophoresis. Fluorescence intensities (arbitrary units) of the single-stranded DNA fragments generated after digestion and denaturation are shown on the y axis. Sizes of the ssDNA fragments (in nucleotides) are shown on the x axis. Fragment sizes were analyzed with the Peak Scanner (Applied Biosystems) software. Accurate sizes can only be predicted for fragments longer than 50 nucleotides.
    Dnase I, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 93/100, based on 1869 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche dnase i
    Impact of <t>DNase</t> I treatment on B . pseudomallei biofilm formation. Static biofilms of B . pseudomallei strains L1, P1 and H777 in LB were treated with DNase I (0.01, 0.1 and 1 U/mL) at 0 h, 24 h or 45 h after inoculation and maintained for up to 48 h. Biofilm formation and eDNA concentration of the 2-day biofilms were assessed using crystal-violet absorbance (OD 620 ) and the QuantiFluor dsDNA System, respectively. DNase I buffer acted as control. Biofilm formation of each strain was examined in eight replicates and eDNA was quantified in duplicates, on three independent occasions. Data represents mean ± SD. * p
    Dnase I, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 26508 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    FUJIFILM dnase i
    Impact of <t>DNase</t> I treatment on B . pseudomallei biofilm formation. Static biofilms of B . pseudomallei strains L1, P1 and H777 in LB were treated with DNase I (0.01, 0.1 and 1 U/mL) at 0 h, 24 h or 45 h after inoculation and maintained for up to 48 h. Biofilm formation and eDNA concentration of the 2-day biofilms were assessed using crystal-violet absorbance (OD 620 ) and the QuantiFluor dsDNA System, respectively. DNase I buffer acted as control. Biofilm formation of each strain was examined in eight replicates and eDNA was quantified in duplicates, on three independent occasions. Data represents mean ± SD. * p
    Dnase I, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 95/100, based on 103 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Applichem dnase i
    <t>DNase</t> I footprinting demonstrates HilD binding to the flhDC P5 promoter region. (A) DNase I footprinting of an flhDC P5 promoter DNA fragment. A DNA fragment covering a region of nucleotides from position −668 to −388 upstream from the flhD start codon was DIG labeled on the noncoding strand and incubated alone (lane P) and with increasing amounts of purified HilD protein (lane 1, 4.23 pmol; lane 2, 8.45 pmol; lane 3, 12.68 pmol; lane 4, 16.9 pmol; lane 5, 21.13 pmol; lane 6, 42.25 pmol) and digested with DNase I before being loaded on a sequencing gel. The vertical line indicates the region protected from DNase I digestion. Lanes C, T, A, and G show the specific nucleotides of the noncoding strand. Exposed nucleotides are highlighted by dots. (B) Partial nucleotide sequence of the P5 promoter of flhDC that is relevant for HilD binding. A horizontal line marks the protected region, and the four most sensitive nucleotides are highlighted by dots. The transcriptional start site (marked as +1) and the −10 element of the P5 flhDC promoter are indicated. (C) Comparison of HilD binding sites in the flhDC , rtsA , hilC , hilD , and hilA ). The proposed consensus is displayed at the bottom; uppercase letters indicate predominant nucleotides ( > 80% conserved), and lowercase letters indicate conserved nucleotides ( > 60% conserved).
    Dnase I, supplied by Applichem, used in various techniques. Bioz Stars score: 95/100, based on 480 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega dnase i
    MdcY-mediated protection of the  mdc  operator against digestion by DNase I. Lanes 1 to 4, target DNA (0.1 pmol, 2 × 10 5  cpm) was incubated in the absence or in the presence of MdcY (the number above each lane indicates the nanomolar concentration of MdcY protein). In lanes 5 to 7, target DNA and 2.4 nM MdcY were incubated with 50, 100, and 500 μM malonate, respectively. The vertical arrows beside the nucleotide sequence indicate a palindromic structure.
    Dnase I, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 10850 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa dnase i
    Variations in expression levels of fungus-responsive genes in the hemolymph of silkworms, Bombyx mori , infected with Beauveria bassiana . The third-day larvae of the fifth instar of Dazao strains were infected with B. bassiana . Total RNA was extracted from the hemolymph at the indicated time points after infection and subjected to <t>DNase</t> I treatment and reverse transcription. Two microliters of each 10-fold diluted first strand cDNA (20 ng) was analyzed in each real-time qPCR reaction. The reaction was performed with specific primers for amplifying each of the six genes. The relative expression level of each gene at each time point was normalized using the Ct values obtained for the Bm GAPDH amplifications run in the same plate. In each assay, the expression level is shown relative to the lowest expression level, which was set to one. All samples were tested in triplicate. The mean value ± SD was used for the analysis of the relative transcript levels for each time point using the △△Ct method. The B. bassiana injected and water-treated individuals are shown on the left (blue) and right (purple), respectively. A. Chemosensory protein 11; B. Muscle LIM protein isoform 1; C. Transferrin; D. Sex-specific storage-protein SP1 precursor; E. Arylphorin; F. Low molecular lipoprotein 30K pBmHPC-6 (Lp-c6); G. lysozyme; H. Moricin. High quality figures are available online.
    Dnase I, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 8161 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    US Biological Life Sciences dnase i
    Variations in expression levels of fungus-responsive genes in the hemolymph of silkworms, Bombyx mori , infected with Beauveria bassiana . The third-day larvae of the fifth instar of Dazao strains were infected with B. bassiana . Total RNA was extracted from the hemolymph at the indicated time points after infection and subjected to <t>DNase</t> I treatment and reverse transcription. Two microliters of each 10-fold diluted first strand cDNA (20 ng) was analyzed in each real-time qPCR reaction. The reaction was performed with specific primers for amplifying each of the six genes. The relative expression level of each gene at each time point was normalized using the Ct values obtained for the Bm GAPDH amplifications run in the same plate. In each assay, the expression level is shown relative to the lowest expression level, which was set to one. All samples were tested in triplicate. The mean value ± SD was used for the analysis of the relative transcript levels for each time point using the △△Ct method. The B. bassiana injected and water-treated individuals are shown on the left (blue) and right (purple), respectively. A. Chemosensory protein 11; B. Muscle LIM protein isoform 1; C. Transferrin; D. Sex-specific storage-protein SP1 precursor; E. Arylphorin; F. Low molecular lipoprotein 30K pBmHPC-6 (Lp-c6); G. lysozyme; H. Moricin. High quality figures are available online.
    Dnase I, supplied by US Biological Life Sciences, used in various techniques. Bioz Stars score: 86/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs dnase i rnase free
    Variations in expression levels of fungus-responsive genes in the hemolymph of silkworms, Bombyx mori , infected with Beauveria bassiana . The third-day larvae of the fifth instar of Dazao strains were infected with B. bassiana . Total RNA was extracted from the hemolymph at the indicated time points after infection and subjected to <t>DNase</t> I treatment and reverse transcription. Two microliters of each 10-fold diluted first strand cDNA (20 ng) was analyzed in each real-time qPCR reaction. The reaction was performed with specific primers for amplifying each of the six genes. The relative expression level of each gene at each time point was normalized using the Ct values obtained for the Bm GAPDH amplifications run in the same plate. In each assay, the expression level is shown relative to the lowest expression level, which was set to one. All samples were tested in triplicate. The mean value ± SD was used for the analysis of the relative transcript levels for each time point using the △△Ct method. The B. bassiana injected and water-treated individuals are shown on the left (blue) and right (purple), respectively. A. Chemosensory protein 11; B. Muscle LIM protein isoform 1; C. Transferrin; D. Sex-specific storage-protein SP1 precursor; E. Arylphorin; F. Low molecular lipoprotein 30K pBmHPC-6 (Lp-c6); G. lysozyme; H. Moricin. High quality figures are available online.
    Dnase I Rnase Free, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 712 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dnase i dnase i
    Nonspecific stimulation by unfractionated B. bovis merozoite antigen is DNase sensitive. PBMC from two calves never exposed to babesial parasites were cultured for 3 (A and C) or 6 (B and D) days with medium alone (0) or 12.5 or 25 μg of B. bovis CM antigen per ml that was untreated (solid bars) or <t>DNase</t> I treated (striped bars), and proliferation was determined. Results are expressed as the mean cpm ± 1 SD of triplicate cultures.
    Dnase I Dnase I, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 161 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad dnase i
    Confirmatory real-time PCR analysis ( a–f ) DNase-chip–identified regions were confirmed by real-time PCR for CD4 + T cells ( a,c,e ) and GM06990 cells ( b,d,f (MPSS cluster). Real-time PCR using primer sets flanking DNase-chip peaks that are present with all three <t>DNase</t> I concentrations ( a,b ). Real-time PCR using primers sets flanking DNase-chip peaks that are present with two out of three DNase I concentrations ( c,d ). Real-time PCR using primer sets flanking DNase-chip peaks that are present with only a single DNase I concentration ( e,f ).
    Dnase I, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 682 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher a594 dnaase i
    Confirmatory real-time PCR analysis ( a–f ) DNase-chip–identified regions were confirmed by real-time PCR for CD4 + T cells ( a,c,e ) and GM06990 cells ( b,d,f (MPSS cluster). Real-time PCR using primer sets flanking DNase-chip peaks that are present with all three <t>DNase</t> I concentrations ( a,b ). Real-time PCR using primers sets flanking DNase-chip peaks that are present with two out of three DNase I concentrations ( c,d ). Real-time PCR using primer sets flanking DNase-chip peaks that are present with only a single DNase I concentration ( e,f ).
    A594 Dnaase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega dnaase i digestion
    Confirmatory real-time PCR analysis ( a–f ) DNase-chip–identified regions were confirmed by real-time PCR for CD4 + T cells ( a,c,e ) and GM06990 cells ( b,d,f (MPSS cluster). Real-time PCR using primer sets flanking DNase-chip peaks that are present with all three <t>DNase</t> I concentrations ( a,b ). Real-time PCR using primers sets flanking DNase-chip peaks that are present with two out of three DNase I concentrations ( c,d ). Real-time PCR using primer sets flanking DNase-chip peaks that are present with only a single DNase I concentration ( e,f ).
    Dnaase I Digestion, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche pancreatic dnaase i
    Confirmatory real-time PCR analysis ( a–f ) DNase-chip–identified regions were confirmed by real-time PCR for CD4 + T cells ( a,c,e ) and GM06990 cells ( b,d,f (MPSS cluster). Real-time PCR using primer sets flanking DNase-chip peaks that are present with all three <t>DNase</t> I concentrations ( a,b ). Real-time PCR using primers sets flanking DNase-chip peaks that are present with two out of three DNase I concentrations ( c,d ). Real-time PCR using primer sets flanking DNase-chip peaks that are present with only a single DNase I concentration ( e,f ).
    Pancreatic Dnaase I, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ambion dnase i
    Confirmatory real-time PCR analysis ( a–f ) DNase-chip–identified regions were confirmed by real-time PCR for CD4 + T cells ( a,c,e ) and GM06990 cells ( b,d,f (MPSS cluster). Real-time PCR using primer sets flanking DNase-chip peaks that are present with all three <t>DNase</t> I concentrations ( a,b ). Real-time PCR using primers sets flanking DNase-chip peaks that are present with two out of three DNase I concentrations ( c,d ). Real-time PCR using primer sets flanking DNase-chip peaks that are present with only a single DNase I concentration ( e,f ).
    Ambion Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dnase i buffer
    Analysis of the Mur34 binding site by <t>DNase</t> I footprinting assay. (A) Analysis of antisense strand γ- 32 P labeled DNA (left) and the sense strand γ- 32 P labeled DNA (right) upstream of mur33 . Lanes G (1), A (2), T (3) and C (4) are sequencing ladder. Samples from lands 5–10 contain the same amount of the binding DNA with an increasing amount (0–3.2 µg µl -1 ) of purified His 6 Mur34. The complexes from the samples were digested by DNase I (0.004U per10 µl) at 30°C for 1 min. The vertical sequences to the right of each gel picture indicate the DNA regions protected from the cleavage of DNase I. The transcription start point (TSP) was shown for each DNA strand. (B) “G” indicates the TSP. The sequences underlined were the protected regions by His 6 Mur34 under DNase I, “CAC” indicates the translation initiation codon (TIC), the bold regions upstream of TSP are -10 “TGATAT” and -35 “GTAAAACAG” regions. The bases in the boxes found are palindromes, and the bold and underlined bases near the TIC are supposed to be the Shine-Dalgarno consensus.
    Dnase I Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 444 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher turbo dnase i
    Effect of the K297R mutation on progeny virus release. iSLK-BAC16 and iSLK-BAC-K297R cells were induced with Dox and butyrate for indicated time. The extracellular virions were collected from culture media and treated with Turbo DNase I. Viral DNAs were extracted and KSHV genomic DNA copy numbers were estimated by qPCR along with external standards of known concentrations of the viral DNA with primers against the ORF73 gene (A). Intracellular KSHV genomic DNAs were extracted from harvested cells and quantitated by qPCR as above (B). (*,  p
    Turbo Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc cas9 nickase
    Majority of indels is present between both cleavage sites after editing with <t>Cas9</t> <t>nickase.</t> Distance of the start position of each insertion or deletion to the theoretical cleavage site (=3 bp upstream of the PAM sequence) is presented on the x-axis, while the relative frequency of the indel is presented on the y-axis after editing with the Cas9 nickase. Length of indels are represented by the different colors. Only 1 replicate is shown. The majority of indels is present between the two theoretical cleavage sites (indicated by the black lines at position 0 (sgRNA1) and at positions 44 bp ( TUNA ), 25 bp ( EMX1 ) and 38 bp ( MEG3 ). Percentages of indels 1) within or spanning both the two cleavage sites and 2) indels spanning at least one of the two sites are shown below the graph. Replicate 2 of EMX1 has an editing efficiency of 2.7% (compared to 5% for replicate 1), which can explain the lower percentage of indels located between the two cleavage sites.
    Cas9 Nickase, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Addgene inc nickase cas9d10a
    Majority of indels is present between both cleavage sites after editing with <t>Cas9</t> <t>nickase.</t> Distance of the start position of each insertion or deletion to the theoretical cleavage site (=3 bp upstream of the PAM sequence) is presented on the x-axis, while the relative frequency of the indel is presented on the y-axis after editing with the Cas9 nickase. Length of indels are represented by the different colors. Only 1 replicate is shown. The majority of indels is present between the two theoretical cleavage sites (indicated by the black lines at position 0 (sgRNA1) and at positions 44 bp ( TUNA ), 25 bp ( EMX1 ) and 38 bp ( MEG3 ). Percentages of indels 1) within or spanning both the two cleavage sites and 2) indels spanning at least one of the two sites are shown below the graph. Replicate 2 of EMX1 has an editing efficiency of 2.7% (compared to 5% for replicate 1), which can explain the lower percentage of indels located between the two cleavage sites.
    Nickase Cas9d10a, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa rnaase free dnaase i
    Majority of indels is present between both cleavage sites after editing with <t>Cas9</t> <t>nickase.</t> Distance of the start position of each insertion or deletion to the theoretical cleavage site (=3 bp upstream of the PAM sequence) is presented on the x-axis, while the relative frequency of the indel is presented on the y-axis after editing with the Cas9 nickase. Length of indels are represented by the different colors. Only 1 replicate is shown. The majority of indels is present between the two theoretical cleavage sites (indicated by the black lines at position 0 (sgRNA1) and at positions 44 bp ( TUNA ), 25 bp ( EMX1 ) and 38 bp ( MEG3 ). Percentages of indels 1) within or spanning both the two cleavage sites and 2) indels spanning at least one of the two sites are shown below the graph. Replicate 2 of EMX1 has an editing efficiency of 2.7% (compared to 5% for replicate 1), which can explain the lower percentage of indels located between the two cleavage sites.
    Rnaase Free Dnaase I, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega rnaase free dnaase i
    Majority of indels is present between both cleavage sites after editing with <t>Cas9</t> <t>nickase.</t> Distance of the start position of each insertion or deletion to the theoretical cleavage site (=3 bp upstream of the PAM sequence) is presented on the x-axis, while the relative frequency of the indel is presented on the y-axis after editing with the Cas9 nickase. Length of indels are represented by the different colors. Only 1 replicate is shown. The majority of indels is present between the two theoretical cleavage sites (indicated by the black lines at position 0 (sgRNA1) and at positions 44 bp ( TUNA ), 25 bp ( EMX1 ) and 38 bp ( MEG3 ). Percentages of indels 1) within or spanning both the two cleavage sites and 2) indels spanning at least one of the two sites are shown below the graph. Replicate 2 of EMX1 has an editing efficiency of 2.7% (compared to 5% for replicate 1), which can explain the lower percentage of indels located between the two cleavage sites.
    Rnaase Free Dnaase I, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega rq 1 dnaase i
    Majority of indels is present between both cleavage sites after editing with <t>Cas9</t> <t>nickase.</t> Distance of the start position of each insertion or deletion to the theoretical cleavage site (=3 bp upstream of the PAM sequence) is presented on the x-axis, while the relative frequency of the indel is presented on the y-axis after editing with the Cas9 nickase. Length of indels are represented by the different colors. Only 1 replicate is shown. The majority of indels is present between the two theoretical cleavage sites (indicated by the black lines at position 0 (sgRNA1) and at positions 44 bp ( TUNA ), 25 bp ( EMX1 ) and 38 bp ( MEG3 ). Percentages of indels 1) within or spanning both the two cleavage sites and 2) indels spanning at least one of the two sites are shown below the graph. Replicate 2 of EMX1 has an editing efficiency of 2.7% (compared to 5% for replicate 1), which can explain the lower percentage of indels located between the two cleavage sites.
    Rq 1 Dnaase I, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa rnas free dnaase i
    Majority of indels is present between both cleavage sites after editing with <t>Cas9</t> <t>nickase.</t> Distance of the start position of each insertion or deletion to the theoretical cleavage site (=3 bp upstream of the PAM sequence) is presented on the x-axis, while the relative frequency of the indel is presented on the y-axis after editing with the Cas9 nickase. Length of indels are represented by the different colors. Only 1 replicate is shown. The majority of indels is present between the two theoretical cleavage sites (indicated by the black lines at position 0 (sgRNA1) and at positions 44 bp ( TUNA ), 25 bp ( EMX1 ) and 38 bp ( MEG3 ). Percentages of indels 1) within or spanning both the two cleavage sites and 2) indels spanning at least one of the two sites are shown below the graph. Replicate 2 of EMX1 has an editing efficiency of 2.7% (compared to 5% for replicate 1), which can explain the lower percentage of indels located between the two cleavage sites.
    Rnas Free Dnaase I, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    SMD is sensitive to T7 endonuclease I. Logarithmically growing  apn1/2  cells were arrested in G2/M with nocodazole, treated with MMS (0.1%, 15 min) in PBS and returned to the YPDA+nocodazole medium. Cells were collected at the indicated times and processed for DNA plug preparation. The plugs were then incubated with T7 endonuclease I as described in the   Materials and Methods  or in buffer only (mock-treated). The SMD was estimated in the ethidium bromide stained gel by comparing the amount of DNA material in the SMD region to DNA in the small chromosomes that experienced little breakage, “SMD / Chr (I+VI)”.

    Journal: PLoS Genetics

    Article Title: Alkylation Base Damage Is Converted into Repairable Double-Strand Breaks and Complex Intermediates in G2 Cells Lacking AP Endonuclease

    doi: 10.1371/journal.pgen.1002059

    Figure Lengend Snippet: SMD is sensitive to T7 endonuclease I. Logarithmically growing apn1/2 cells were arrested in G2/M with nocodazole, treated with MMS (0.1%, 15 min) in PBS and returned to the YPDA+nocodazole medium. Cells were collected at the indicated times and processed for DNA plug preparation. The plugs were then incubated with T7 endonuclease I as described in the Materials and Methods or in buffer only (mock-treated). The SMD was estimated in the ethidium bromide stained gel by comparing the amount of DNA material in the SMD region to DNA in the small chromosomes that experienced little breakage, “SMD / Chr (I+VI)”.

    Article Snippet: Resolution of the slow moving DNA (SMD) intermediate with T7 endonuclease I DNA was digested in agarose plugs with T7 endonuclease I (New England Biolabs, Beverly, MA).

    Techniques: Incubation, Staining

    DNase I treatment is effective against C. jejuni biofilms on stainless steel surfaces and in the presence of organic materials in aerobic conditions . The ability of DNase I to inhibit biofilm formation of C. jejuni NCTC 11168 on sterile, stainless steel coupons (A) or in the presence of chicken juice, mimicking a conditioned surface (B) . TTC staining was used to measure biofilm formation in the presence of chicken juice (B) . DNase I is able to significantly decrease biofilm formation in both conditions. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Journal: Frontiers in Microbiology

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment

    doi: 10.3389/fmicb.2015.00699

    Figure Lengend Snippet: DNase I treatment is effective against C. jejuni biofilms on stainless steel surfaces and in the presence of organic materials in aerobic conditions . The ability of DNase I to inhibit biofilm formation of C. jejuni NCTC 11168 on sterile, stainless steel coupons (A) or in the presence of chicken juice, mimicking a conditioned surface (B) . TTC staining was used to measure biofilm formation in the presence of chicken juice (B) . DNase I is able to significantly decrease biofilm formation in both conditions. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Article Snippet: For the assays assessing the time required for DNase I activity, biofilms were allowed to form for 48 h before addition of 4 U/ml v/v DNase I enzyme (1 U/μl, Fermentas) and 4 μl of DNase I buffer to the samples, followed by incubation for up to 2 h. During the incubation with enzyme the samples were placed in 37°C, aerobic conditions.

    Techniques: Staining, Standard Deviation, MANN-WHITNEY

    DNase I is able to rapidly degrade C. jejuni NCTC 11168 biofilms . (A) DNase I (4 units/ml) was added at defined intervals to aerobically incubated NCTC 11168 cultures over a 48 h static incubation and biofilm degradation assessed by crystal violet staining. (B) Following a 48 h static incubation to allow biofilm formation, DNase I was added to biofilms for between 5 and 120 min before biofilm degradation was assessed. (C) The concentration of DNase I required for biofilm control was also assessed using DNase I concentrations of between 0.01 and 5 U/ml. In each graph, “11168” represents an untreated biofilm culture of C. jejuni NCTC 11168 and “control” represents a tube containing sterile Brucella medium only. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Journal: Frontiers in Microbiology

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment

    doi: 10.3389/fmicb.2015.00699

    Figure Lengend Snippet: DNase I is able to rapidly degrade C. jejuni NCTC 11168 biofilms . (A) DNase I (4 units/ml) was added at defined intervals to aerobically incubated NCTC 11168 cultures over a 48 h static incubation and biofilm degradation assessed by crystal violet staining. (B) Following a 48 h static incubation to allow biofilm formation, DNase I was added to biofilms for between 5 and 120 min before biofilm degradation was assessed. (C) The concentration of DNase I required for biofilm control was also assessed using DNase I concentrations of between 0.01 and 5 U/ml. In each graph, “11168” represents an untreated biofilm culture of C. jejuni NCTC 11168 and “control” represents a tube containing sterile Brucella medium only. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Article Snippet: For the assays assessing the time required for DNase I activity, biofilms were allowed to form for 48 h before addition of 4 U/ml v/v DNase I enzyme (1 U/μl, Fermentas) and 4 μl of DNase I buffer to the samples, followed by incubation for up to 2 h. During the incubation with enzyme the samples were placed in 37°C, aerobic conditions.

    Techniques: Incubation, Staining, Concentration Assay, Standard Deviation, MANN-WHITNEY

    Treatment of pre-existing biofilms with DNase I leads to inhibition of biofilm regrowth . C. jejuni NCTC 11168 biofilms were allowed to form for 48 h in sterile borosilicate glass test tubes. To assess biofilm re-growth following DNase I treatment, two sets of tubes were treated with 4 U/ml DNase I for 15 min then washed with sterile PBS. Tubes were then supplemented with either fresh Brucella media (fifth bar) or fresh C. jejuni NCTC 11168 culture (sixth bar) and incubated for a further 48 h. The following controls were also prepared: C. jejuni NCTC 11168 biofilm formation following primary culture (first bar, white), tubes supplemented with sterile Brucella media (second bar, black), C. jejuni NCTC 11168 biofilm formation following only secondary culture (third bar, light gray), and 48 h-old C. jejuni NCTC 11168 biofilm, washed with PBS, then supplemented with fresh C. jejuni NCTC 11168 culture (fourth bar, dark gray). Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Journal: Frontiers in Microbiology

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment

    doi: 10.3389/fmicb.2015.00699

    Figure Lengend Snippet: Treatment of pre-existing biofilms with DNase I leads to inhibition of biofilm regrowth . C. jejuni NCTC 11168 biofilms were allowed to form for 48 h in sterile borosilicate glass test tubes. To assess biofilm re-growth following DNase I treatment, two sets of tubes were treated with 4 U/ml DNase I for 15 min then washed with sterile PBS. Tubes were then supplemented with either fresh Brucella media (fifth bar) or fresh C. jejuni NCTC 11168 culture (sixth bar) and incubated for a further 48 h. The following controls were also prepared: C. jejuni NCTC 11168 biofilm formation following primary culture (first bar, white), tubes supplemented with sterile Brucella media (second bar, black), C. jejuni NCTC 11168 biofilm formation following only secondary culture (third bar, light gray), and 48 h-old C. jejuni NCTC 11168 biofilm, washed with PBS, then supplemented with fresh C. jejuni NCTC 11168 culture (fourth bar, dark gray). Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Article Snippet: For the assays assessing the time required for DNase I activity, biofilms were allowed to form for 48 h before addition of 4 U/ml v/v DNase I enzyme (1 U/μl, Fermentas) and 4 μl of DNase I buffer to the samples, followed by incubation for up to 2 h. During the incubation with enzyme the samples were placed in 37°C, aerobic conditions.

    Techniques: Inhibition, Incubation, Standard Deviation, MANN-WHITNEY

    Restriction endonuclease treatment of C. jejuni biofilms reduces biofilm formation . Static cultures of C. jejuni NCTC 11168 (A,B) and 81116 (C,D) were prepared then supplemented with either DNase I, RNase, or a single restriction endonuclease. Cultures were incubated for 48 h at 37°C in aerobic conditions. A range of restriction enzymes was selected, based on varying levels of DNA fragmentation following digestion of C. jejuni NCTC 11168 (B) and 81116 (D) genomic DNA. Restriction enzyme and DNase I treatment of NCTC 11168 biofilms lead to a reduction in biofilm formation. The same trend was observed for C. jejuni 81116, although only DNase I and Hae III digestion were significantly different from the control. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Journal: Frontiers in Microbiology

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment

    doi: 10.3389/fmicb.2015.00699

    Figure Lengend Snippet: Restriction endonuclease treatment of C. jejuni biofilms reduces biofilm formation . Static cultures of C. jejuni NCTC 11168 (A,B) and 81116 (C,D) were prepared then supplemented with either DNase I, RNase, or a single restriction endonuclease. Cultures were incubated for 48 h at 37°C in aerobic conditions. A range of restriction enzymes was selected, based on varying levels of DNA fragmentation following digestion of C. jejuni NCTC 11168 (B) and 81116 (D) genomic DNA. Restriction enzyme and DNase I treatment of NCTC 11168 biofilms lead to a reduction in biofilm formation. The same trend was observed for C. jejuni 81116, although only DNase I and Hae III digestion were significantly different from the control. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Article Snippet: For the assays assessing the time required for DNase I activity, biofilms were allowed to form for 48 h before addition of 4 U/ml v/v DNase I enzyme (1 U/μl, Fermentas) and 4 μl of DNase I buffer to the samples, followed by incubation for up to 2 h. During the incubation with enzyme the samples were placed in 37°C, aerobic conditions.

    Techniques: Incubation, Standard Deviation, MANN-WHITNEY

    Perlecan deficiency does not affect cell turnover in white adipose tissue. ( a ) TUNEL staining in the VAT of the WT-Tg (upper panel) and the  Hspg2 −/− -Tg (lower panel) mice fed either ND or HFD. The areas containing positive nuclei of adipocytes are selected and shown. The positive control was made using DNase I. Note that TUNEL-positive nuclei are brown and negative nuclei are blue. ( b ) The percentage of TUNEL-positive nuclei of adipocytes. The nuclei from at least 100 adipocytes per mouse were evaluated. Data points and error bars represent the mean ± S.D. (n = 5). ( c ) Representative immunohistochemical staining of Ki67 in the VAT of the WT-Tg (upper panel) and the  Hspg2 −/− -Tg (lower panel) mice fed either ND or HFD. The tissue from human abdominal cancer was used as positive control. No nuclei were positive for Ki67 in any groups. Data were analyzed by two-way ANOVA with Tukey’s multiple comparison ( b ). Scale bar, 50  μm.

    Journal: Scientific Reports

    Article Title: Perlecan, a heparan sulfate proteoglycan, regulates systemic metabolism with dynamic changes in adipose tissue and skeletal muscle

    doi: 10.1038/s41598-018-25635-x

    Figure Lengend Snippet: Perlecan deficiency does not affect cell turnover in white adipose tissue. ( a ) TUNEL staining in the VAT of the WT-Tg (upper panel) and the Hspg2 −/− -Tg (lower panel) mice fed either ND or HFD. The areas containing positive nuclei of adipocytes are selected and shown. The positive control was made using DNase I. Note that TUNEL-positive nuclei are brown and negative nuclei are blue. ( b ) The percentage of TUNEL-positive nuclei of adipocytes. The nuclei from at least 100 adipocytes per mouse were evaluated. Data points and error bars represent the mean ± S.D. (n = 5). ( c ) Representative immunohistochemical staining of Ki67 in the VAT of the WT-Tg (upper panel) and the Hspg2 −/− -Tg (lower panel) mice fed either ND or HFD. The tissue from human abdominal cancer was used as positive control. No nuclei were positive for Ki67 in any groups. Data were analyzed by two-way ANOVA with Tukey’s multiple comparison ( b ). Scale bar, 50  μm.

    Article Snippet: A positive control for the TUNEL assay was made by incubating sections for 15 min at 37 °C with 1 μg/mL DNase I (D4263-1VL, SIGMA) in 50 mM Tris-HCl, pH 7.5, containing 10 mM MgCl2 and 1 mg/mL BSA.

    Techniques: TUNEL Assay, Staining, Mouse Assay, Positive Control, Immunohistochemistry

    Effects of noncomplementary dNTPs on DNase I footprints and stable complex formation by HIV-1 RT

    Journal:

    Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet

    doi: 10.1016/j.jmb.2007.03.006

    Figure Lengend Snippet: Effects of noncomplementary dNTPs on DNase I footprints and stable complex formation by HIV-1 RT

    Article Snippet: After incubation on ice for 5 min, 0.03 U DNase I (USB corp.), in RB buffer, was added and the samples were incubated at room temperature for 3 min.

    Techniques:

    Effects of foscarnet on DNase I protection and stable complex formation by HIV-1 RT

    Journal:

    Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet

    doi: 10.1016/j.jmb.2007.03.006

    Figure Lengend Snippet: Effects of foscarnet on DNase I protection and stable complex formation by HIV-1 RT

    Article Snippet: After incubation on ice for 5 min, 0.03 U DNase I (USB corp.), in RB buffer, was added and the samples were incubated at room temperature for 3 min.

    Techniques:

    DNase I protection on P/Ts terminated with dT analogues

    Journal:

    Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet

    doi: 10.1016/j.jmb.2007.03.006

    Figure Lengend Snippet: DNase I protection on P/Ts terminated with dT analogues

    Article Snippet: After incubation on ice for 5 min, 0.03 U DNase I (USB corp.), in RB buffer, was added and the samples were incubated at room temperature for 3 min.

    Techniques:

    Effects of the next complementary dNTP on DNase I protection and stable complex formation by HIV-1 RT

    Journal:

    Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet

    doi: 10.1016/j.jmb.2007.03.006

    Figure Lengend Snippet: Effects of the next complementary dNTP on DNase I protection and stable complex formation by HIV-1 RT

    Article Snippet: After incubation on ice for 5 min, 0.03 U DNase I (USB corp.), in RB buffer, was added and the samples were incubated at room temperature for 3 min.

    Techniques:

    DNase I cleavage patterns and footprint distribution for overrepresented footprints surrounding TSSs. ( A ) Mean per-nucleotide DNase I cleavage profile from aligning the annotated TSSs of 5050 genes (+/− 1 kb regions). ( B ) Top heat map plotted for DNase I cleavage patterns of 5050 genes at +/− 1 kb TSS flanking regions by K-means clustering, which were subsequently divided into four distinct clusters, marked with red, blue, green and purple bars. The bottom mean DNase I cleavage patterns derived from four distinct clusters, where the line colors correspond to the marked colors of the heatmap. ( C ) Distribution of digital footprints (FDR

    Journal: Nucleic Acids Research

    Article Title: Survey of protein–DNA interactions in Aspergillus oryzae on a genomic scale

    doi: 10.1093/nar/gkv334

    Figure Lengend Snippet: DNase I cleavage patterns and footprint distribution for overrepresented footprints surrounding TSSs. ( A ) Mean per-nucleotide DNase I cleavage profile from aligning the annotated TSSs of 5050 genes (+/− 1 kb regions). ( B ) Top heat map plotted for DNase I cleavage patterns of 5050 genes at +/− 1 kb TSS flanking regions by K-means clustering, which were subsequently divided into four distinct clusters, marked with red, blue, green and purple bars. The bottom mean DNase I cleavage patterns derived from four distinct clusters, where the line colors correspond to the marked colors of the heatmap. ( C ) Distribution of digital footprints (FDR

    Article Snippet: RT-qPCR reactions, including 10 μl of reaction mixture with 50 ng DNase I digestion product, 2 × 0.4 μl primers (forward and reverse, 10 μM), 0.2 μl ROX reference dye II (50×), 5 μl SYBR Premix Ex Taq II (2×) and dH2 O, were amplified using an Applied Biosystems 7500 Real-time PCR System for 1 min at 95°C, followed by 40 cycles of 95°C for 5 s, 55°C for 20 s and 72°C for 34 s. The degree of DNase I digestion was determined based on changes in Ct values.

    Techniques: Derivative Assay

    Diversity of DNase I cleavage patterns and function annotation of target genes for the overrepresented motifs in genomic footprints. ( A ) DNase I cleavage density per nucleotide calculated for footprint instances from two culture conditions. Shaded regions delineate the overrepresented motifs derived from the footprint region. The MEME logo of overrepresented motifs derived from footprints is shown below the graph. ( B ) GO function enrichment for the target genes under the DPY_motif 3 and DPY_motif 7. The genes containing at least one motif instance inside the 1-kb region of the annotated TSSs were selected. The genes under the same motif were analyzed using ClueGo. Functional group networks are represented by nodes linked with each other based on their kappa score level ( > 0.3). The node size represents the percentage of associated genes with the enrichment significance of the term (Term P -value

    Journal: Nucleic Acids Research

    Article Title: Survey of protein–DNA interactions in Aspergillus oryzae on a genomic scale

    doi: 10.1093/nar/gkv334

    Figure Lengend Snippet: Diversity of DNase I cleavage patterns and function annotation of target genes for the overrepresented motifs in genomic footprints. ( A ) DNase I cleavage density per nucleotide calculated for footprint instances from two culture conditions. Shaded regions delineate the overrepresented motifs derived from the footprint region. The MEME logo of overrepresented motifs derived from footprints is shown below the graph. ( B ) GO function enrichment for the target genes under the DPY_motif 3 and DPY_motif 7. The genes containing at least one motif instance inside the 1-kb region of the annotated TSSs were selected. The genes under the same motif were analyzed using ClueGo. Functional group networks are represented by nodes linked with each other based on their kappa score level ( > 0.3). The node size represents the percentage of associated genes with the enrichment significance of the term (Term P -value

    Article Snippet: RT-qPCR reactions, including 10 μl of reaction mixture with 50 ng DNase I digestion product, 2 × 0.4 μl primers (forward and reverse, 10 μM), 0.2 μl ROX reference dye II (50×), 5 μl SYBR Premix Ex Taq II (2×) and dH2 O, were amplified using an Applied Biosystems 7500 Real-time PCR System for 1 min at 95°C, followed by 40 cycles of 95°C for 5 s, 55°C for 20 s and 72°C for 34 s. The degree of DNase I digestion was determined based on changes in Ct values.

    Techniques: Derivative Assay, Functional Assay

    The DNase I cleavage patterns of five family types of TFs parallel the co-crystal structures of protein and DNA interaction. ( A ) Strand-specific DNase-seq signal for DNase I cleavage imbalance between the plus and minus motif sequences of five family types of the TFs independent of strand orientation. The upper panels show the heat maps of per-nucleotide DNase I cleavage derived from all instances of plus (red) and minus (blue) TFBS motifs within DHSs under DPY conditions ranked according to the probability of MILLIPEDE (FIMO P

    Journal: Nucleic Acids Research

    Article Title: Survey of protein–DNA interactions in Aspergillus oryzae on a genomic scale

    doi: 10.1093/nar/gkv334

    Figure Lengend Snippet: The DNase I cleavage patterns of five family types of TFs parallel the co-crystal structures of protein and DNA interaction. ( A ) Strand-specific DNase-seq signal for DNase I cleavage imbalance between the plus and minus motif sequences of five family types of the TFs independent of strand orientation. The upper panels show the heat maps of per-nucleotide DNase I cleavage derived from all instances of plus (red) and minus (blue) TFBS motifs within DHSs under DPY conditions ranked according to the probability of MILLIPEDE (FIMO P

    Article Snippet: RT-qPCR reactions, including 10 μl of reaction mixture with 50 ng DNase I digestion product, 2 × 0.4 μl primers (forward and reverse, 10 μM), 0.2 μl ROX reference dye II (50×), 5 μl SYBR Premix Ex Taq II (2×) and dH2 O, were amplified using an Applied Biosystems 7500 Real-time PCR System for 1 min at 95°C, followed by 40 cycles of 95°C for 5 s, 55°C for 20 s and 72°C for 34 s. The degree of DNase I digestion was determined based on changes in Ct values.

    Techniques: Derivative Assay

    Analysis of RNA isolated from purified virions and dense bodies (DBs). Virions and DBs were purified from RNase-ONE-treated virus stocks, dialysed and treated with micrococcal nuclease; RNA was extracted, and during the RNA extraction the column was treated with DNase-I. (a) RNA concentrations of virions and DBs. Presence of (b) viral IE RNA transcripts and (c) lncRNA2.7 transcripts determined by TaqMan PCR assays. Values are mean± sem of three experiments performed in triplicate. RT, reverse transcriptase.

    Journal: The Journal of General Virology

    Article Title: Human cytomegalovirus microRNAs are carried by virions and dense bodies and are delivered to target cells

    doi: 10.1099/jgv.0.000736

    Figure Lengend Snippet: Analysis of RNA isolated from purified virions and dense bodies (DBs). Virions and DBs were purified from RNase-ONE-treated virus stocks, dialysed and treated with micrococcal nuclease; RNA was extracted, and during the RNA extraction the column was treated with DNase-I. (a) RNA concentrations of virions and DBs. Presence of (b) viral IE RNA transcripts and (c) lncRNA2.7 transcripts determined by TaqMan PCR assays. Values are mean± sem of three experiments performed in triplicate. RT, reverse transcriptase.

    Article Snippet: RNA was isolated with the miRNeasy mini kit (Qiagen), including the DNase-I treatment or isolated with Trizol-LS reagent (Life Technologies).

    Techniques: Isolation, Purification, RNA Extraction, Polymerase Chain Reaction

    Procedure to study protein-nucleic acid interactions. The oligonucleotide is designed to contain a 5'-biotin tag, a 3'-fluorescent tag and a UV sensitive group in the middle. (A) Crosslink initiated by exposing to UV (305+16 nm). (B) After three hours of UV activation, denatured samples were subjected to SDS-PAGE analysis, where crosslinked species were confirmed by fluorescent imaging and all bands were visualized by Commassie staining technique. (C) Interesting spots were picked for protease enzymatic in-gel digestion to yield peptide mixtures. (D) Crosslinked peptides were extracted by magnetic streptavidin beads and uncrosslinked peptides were washed away. (E) The crosslinked peptides were subjected to DNase I degradation to minimize the attaching oligonucleotide moieties. (F) Crosslinked peptides with the remaining nucleic acid attached were extracted by reverse phase ZipTip C18 cartridge and analyzed by Q-tof ESI. (G) Raw data were collected and processed by Protein-Lynx to generate a PKL file, which was used as input to CLPM to identify matches with theoretical peptides.

    Journal: BMC Bioinformatics

    Article Title: CLPM: A Cross-Linked Peptide Mapping Algorithm for Mass Spectrometric Analysis

    doi: 10.1186/1471-2105-6-S2-S9

    Figure Lengend Snippet: Procedure to study protein-nucleic acid interactions. The oligonucleotide is designed to contain a 5'-biotin tag, a 3'-fluorescent tag and a UV sensitive group in the middle. (A) Crosslink initiated by exposing to UV (305+16 nm). (B) After three hours of UV activation, denatured samples were subjected to SDS-PAGE analysis, where crosslinked species were confirmed by fluorescent imaging and all bands were visualized by Commassie staining technique. (C) Interesting spots were picked for protease enzymatic in-gel digestion to yield peptide mixtures. (D) Crosslinked peptides were extracted by magnetic streptavidin beads and uncrosslinked peptides were washed away. (E) The crosslinked peptides were subjected to DNase I degradation to minimize the attaching oligonucleotide moieties. (F) Crosslinked peptides with the remaining nucleic acid attached were extracted by reverse phase ZipTip C18 cartridge and analyzed by Q-tof ESI. (G) Raw data were collected and processed by Protein-Lynx to generate a PKL file, which was used as input to CLPM to identify matches with theoretical peptides.

    Article Snippet: • DNase I (Ambion).

    Techniques: Activation Assay, SDS Page, Imaging, Staining

    Possible ion structures for the fragmentation of tryptic peptide crosslinked to the dinucleotide (dGdU) after DNase I digestion as proposed by Golden et al [31] . The blue line on the right of each diagram represents the peptide moiety in the heteroconjugate.

    Journal: BMC Bioinformatics

    Article Title: CLPM: A Cross-Linked Peptide Mapping Algorithm for Mass Spectrometric Analysis

    doi: 10.1186/1471-2105-6-S2-S9

    Figure Lengend Snippet: Possible ion structures for the fragmentation of tryptic peptide crosslinked to the dinucleotide (dGdU) after DNase I digestion as proposed by Golden et al [31] . The blue line on the right of each diagram represents the peptide moiety in the heteroconjugate.

    Article Snippet: • DNase I (Ambion).

    Techniques:

    Developing surface-tethered nuclease sensor (SNS) for the in situ mapping of membrane-bound nuclease (MN) activity on the cell membrane. (A) SNS is a fluorophore (here Cy3) conjugated with a biotin and a quencher-labeled dsDNA. The fluorophore is freed from quenching when the dsDNA is degraded by MN, thus reporting the MN activity by fluorescence on site. (B) SNS-coated surface reported DNase I in solution at a series of concentrations. (C) The detection limit for soluble DNase I by SNS is calibrated to be 0.01 unit/ml (rounding 0.05/8.3 to 0.01). (D) An SNS-coated surface reported highly organized MN activity on the ventral membrane of adherent MDA-MB-231 cells. (E) The structure feature of the SNS pattern is finer than 1 μm.

    Journal: Journal of biophotonics

    Article Title: Optical sensor revealed abnormal nuclease spatial activity on cancer cell membrane

    doi: 10.1002/jbio.201800351

    Figure Lengend Snippet: Developing surface-tethered nuclease sensor (SNS) for the in situ mapping of membrane-bound nuclease (MN) activity on the cell membrane. (A) SNS is a fluorophore (here Cy3) conjugated with a biotin and a quencher-labeled dsDNA. The fluorophore is freed from quenching when the dsDNA is degraded by MN, thus reporting the MN activity by fluorescence on site. (B) SNS-coated surface reported DNase I in solution at a series of concentrations. (C) The detection limit for soluble DNase I by SNS is calibrated to be 0.01 unit/ml (rounding 0.05/8.3 to 0.01). (D) An SNS-coated surface reported highly organized MN activity on the ventral membrane of adherent MDA-MB-231 cells. (E) The structure feature of the SNS pattern is finer than 1 μm.

    Article Snippet: Soluble nuclease DNase I (89836, ThermoFisher Scientific) solutions at a series of concentrations were added to SNS coated petridishes.

    Techniques: In Situ, Activity Assay, Labeling, Fluorescence, Multiple Displacement Amplification

    DNase I expression and activity. (A) The protein expression pattern of DNase I in kidney sections from (NZBxNZW)F1 mice as revealed by IHC, IF, IEM, and confocal microscopy (DNase I in red and Trap1 in green as a cytoplasmic marker). (B) DNase I endonuclease activity as revealed by zymography on samples from whole kidney lysates of representative Group 1, 2, and 3 (NZBxNZW)F1 mice, as well as a control (R, recombinant DNase I) and a serum (S) sample. (C) Urinary DNase I endonuclease activity in different groups of (NZBxNZW)F1 mice as revealed by zymography.

    Journal: The Journal of Pathology: Clinical Research

    Article Title: Lupus nephritis: low urinary DNase I levels reflect loss of renal DNase I and may be utilized as a biomarker of disease progression

    doi: 10.1002/cjp2.99

    Figure Lengend Snippet: DNase I expression and activity. (A) The protein expression pattern of DNase I in kidney sections from (NZBxNZW)F1 mice as revealed by IHC, IF, IEM, and confocal microscopy (DNase I in red and Trap1 in green as a cytoplasmic marker). (B) DNase I endonuclease activity as revealed by zymography on samples from whole kidney lysates of representative Group 1, 2, and 3 (NZBxNZW)F1 mice, as well as a control (R, recombinant DNase I) and a serum (S) sample. (C) Urinary DNase I endonuclease activity in different groups of (NZBxNZW)F1 mice as revealed by zymography.

    Article Snippet: In addition, a radial diffusion assay was used to measure DNase I activity in 1% agarose gel with 30 µg/ml heat‐denatured salmon sperm DNA (Invitrogen) in the DNase I reaction buffer.

    Techniques: Expressing, Activity Assay, Mouse Assay, Immunohistochemistry, Confocal Microscopy, Marker, Zymography, Recombinant

    Kinetics of loop DNA digestion with DNase I in nucleoids from P0, P7, P80 and P540 rat neurons. Nucleoids were treated with DNase I at 0.92 U/ml. Each time-point value corresponds to the average of independent experiments using separate animals as the source of nucleoids (P0 n = 4; P7 n = 5; P80 n = 5; P540 n = 4). The data sets for P7 and P540 were taken from our previous work [21] . The topological zones relative to the NM for the corresponding kinetics were established considering the local slopes between pairs of time points ( Table 3 ) and the corresponding S.D.

    Journal: PLoS ONE

    Article Title: Continued Stabilization of the Nuclear Higher-Order Structure of Post-Mitotic Neurons In Vivo

    doi: 10.1371/journal.pone.0021360

    Figure Lengend Snippet: Kinetics of loop DNA digestion with DNase I in nucleoids from P0, P7, P80 and P540 rat neurons. Nucleoids were treated with DNase I at 0.92 U/ml. Each time-point value corresponds to the average of independent experiments using separate animals as the source of nucleoids (P0 n = 4; P7 n = 5; P80 n = 5; P540 n = 4). The data sets for P7 and P540 were taken from our previous work [21] . The topological zones relative to the NM for the corresponding kinetics were established considering the local slopes between pairs of time points ( Table 3 ) and the corresponding S.D.

    Article Snippet: Standard PCR was carried out using 1.25 U GoTaq Flexi DNA polymerase (Promega) and 60 ng of nuclear matrix-bound DNA obtained from each DNase I digestion point, using an Applied Biosystems 2720 thermal cycler, and the same amplification program was used for all pairs of primers ( ): initial denaturising step at 94°C for 5 min, denaturising step at 94°C for 45 s, annealing at 56°C for 30 s, and extension at 72°C for 1 min for 35 cycles, with a final extension at 72°C for 10 min.

    Techniques:

    Luciferase gene expression and DNA accessibility as a function of r charge using pre-casted RNA gels. (A) G4 dendrimers and (B) CTAB. The synthesized amounts of RNA are displayed and samples were not pretreated with Dnase I. References are displayed in B where lane 1 shows the sample consisting only of DNA and without any compacting agent or transcriptional activity. Lane 2 shows the control sample containing DNA and the in vitro transcription mixture in the absence of compacting agents. Gels were post-stained using GelStar.

    Journal: PLoS ONE

    Article Title: DNA Compaction Induced by a Cationic Polymer or Surfactant Impact Gene Expression and DNA Degradation

    doi: 10.1371/journal.pone.0092692

    Figure Lengend Snippet: Luciferase gene expression and DNA accessibility as a function of r charge using pre-casted RNA gels. (A) G4 dendrimers and (B) CTAB. The synthesized amounts of RNA are displayed and samples were not pretreated with Dnase I. References are displayed in B where lane 1 shows the sample consisting only of DNA and without any compacting agent or transcriptional activity. Lane 2 shows the control sample containing DNA and the in vitro transcription mixture in the absence of compacting agents. Gels were post-stained using GelStar.

    Article Snippet: Degradation of DNA Dnase I (Turbo Dnase I, Ambion) was used to elucidate how the degree of compaction affects the protection against enzymatic digestion of DNA.

    Techniques: Luciferase, Expressing, Synthesized, Activity Assay, In Vitro, Staining

    Protection of DNA against Dnase I digestion by CTAB using a gel stained with EtBr. A gel electrophoresis gel where samples in lanes 1 and 6 contain linearized plasmid DNA only (control). The remaining lanes contain CTAB/DNA of the r charge values indicated. The samples loaded onto lanes 6–10 were incubated with Dnase I for 20 min following DNA condensation. At r charge ≤1.0, the DNA is completely degraded. At higher concentrations of CTAB, DNA degradation is inhibited.

    Journal: PLoS ONE

    Article Title: DNA Compaction Induced by a Cationic Polymer or Surfactant Impact Gene Expression and DNA Degradation

    doi: 10.1371/journal.pone.0092692

    Figure Lengend Snippet: Protection of DNA against Dnase I digestion by CTAB using a gel stained with EtBr. A gel electrophoresis gel where samples in lanes 1 and 6 contain linearized plasmid DNA only (control). The remaining lanes contain CTAB/DNA of the r charge values indicated. The samples loaded onto lanes 6–10 were incubated with Dnase I for 20 min following DNA condensation. At r charge ≤1.0, the DNA is completely degraded. At higher concentrations of CTAB, DNA degradation is inhibited.

    Article Snippet: Degradation of DNA Dnase I (Turbo Dnase I, Ambion) was used to elucidate how the degree of compaction affects the protection against enzymatic digestion of DNA.

    Techniques: Staining, Nucleic Acid Electrophoresis, Plasmid Preparation, Incubation

    Protection of DNA against Dnase I digestion using gels stained with GelStar. (A) G4/DNA complexes with r charge = 0.4 are used and the untreated complex is loaded on lane 1. The 2 nd lane displays the dissociated complex after treatment with 10 μg mL −1 heparin for 30 min. All other samples (lanes 3–7) are treated with 1 unit of Dnase I for the indicated time periods. To the samples in lanes 4–7, heparin was added after the Dnase I enzyme was heat inactivated. (B) Linearized plasmid DNA only is loaded onto lane 1 and the sample loaded onto lane 2 contains DNA, treated with Dnase I for 30 min. Samples loaded onto lanes 3–7 contain DNA and CTAB ( r charge = 7.5). Samples loaded onto lanes 4–7 are treated with Dnase I for the time periods indicated.

    Journal: PLoS ONE

    Article Title: DNA Compaction Induced by a Cationic Polymer or Surfactant Impact Gene Expression and DNA Degradation

    doi: 10.1371/journal.pone.0092692

    Figure Lengend Snippet: Protection of DNA against Dnase I digestion using gels stained with GelStar. (A) G4/DNA complexes with r charge = 0.4 are used and the untreated complex is loaded on lane 1. The 2 nd lane displays the dissociated complex after treatment with 10 μg mL −1 heparin for 30 min. All other samples (lanes 3–7) are treated with 1 unit of Dnase I for the indicated time periods. To the samples in lanes 4–7, heparin was added after the Dnase I enzyme was heat inactivated. (B) Linearized plasmid DNA only is loaded onto lane 1 and the sample loaded onto lane 2 contains DNA, treated with Dnase I for 30 min. Samples loaded onto lanes 3–7 contain DNA and CTAB ( r charge = 7.5). Samples loaded onto lanes 4–7 are treated with Dnase I for the time periods indicated.

    Article Snippet: Degradation of DNA Dnase I (Turbo Dnase I, Ambion) was used to elucidate how the degree of compaction affects the protection against enzymatic digestion of DNA.

    Techniques: Staining, Plasmid Preparation

    Expression of CES genes in opossum. Liver and intestinal cDNAs were reverse transcribed from DNase I-treated RNA, and they were used as templates in RT-PCR to analyze CES gene expression. Lanes 2 and 3 are RT-PCR products amplified from liver (L) and intestine (I) cDNAs for the CES1 gene; lanes 4 and 5, RT-PCR products from liver (L) and intestine (I) cDNAs for the CES2.1 gene; lanes 6 and 7, RT-PCR products from liver (L) and intestine (I) for cDNAs for the CES2.2 gene; and lanes 8 and 9, RT-PCR products from liver (L) and intestine (I) for cDNAs for the CES2.3 gene. M shows the DNA size ladder.

    Journal: BMC Evolutionary Biology

    Article Title: Opossum carboxylesterases: sequences, phylogeny and evidence for CES gene duplication events predating the marsupial-eutherian common ancestor

    doi: 10.1186/1471-2148-8-54

    Figure Lengend Snippet: Expression of CES genes in opossum. Liver and intestinal cDNAs were reverse transcribed from DNase I-treated RNA, and they were used as templates in RT-PCR to analyze CES gene expression. Lanes 2 and 3 are RT-PCR products amplified from liver (L) and intestine (I) cDNAs for the CES1 gene; lanes 4 and 5, RT-PCR products from liver (L) and intestine (I) cDNAs for the CES2.1 gene; lanes 6 and 7, RT-PCR products from liver (L) and intestine (I) for cDNAs for the CES2.2 gene; and lanes 8 and 9, RT-PCR products from liver (L) and intestine (I) for cDNAs for the CES2.3 gene. M shows the DNA size ladder.

    Article Snippet: DNase I-treated RNA was reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification

    Consensus GGUG-containing RNA oligonucleotide promotes the inhibitory effect of TLS on CBP/p300 HAT activities a , Co-immunoprecipitation (IP) of p300 and TLS from RNase A-treated HeLa cells. b, P300 HAT activity was measured using micrococcal nuclease (MNase) or DNase I pre-treated GST and GST-TLS in the presence of GGUG- or CCUC-oligonucleotide. * p

    Journal: Nature

    Article Title: Induced ncRNAs Allosterically Modify RNA Binding Proteins in cis to Inhibit Transcription

    doi: 10.1038/nature06992

    Figure Lengend Snippet: Consensus GGUG-containing RNA oligonucleotide promotes the inhibitory effect of TLS on CBP/p300 HAT activities a , Co-immunoprecipitation (IP) of p300 and TLS from RNase A-treated HeLa cells. b, P300 HAT activity was measured using micrococcal nuclease (MNase) or DNase I pre-treated GST and GST-TLS in the presence of GGUG- or CCUC-oligonucleotide. * p

    Article Snippet: The soluble DNA-bound RNA fraction was collected after centrifugation at 4,000 g for 15 min. RNA was extracted using Trizol (Invitrogen) and treated with RNase-free DNase I (DNA-free; Ambion).

    Techniques: HAT Assay, Immunoprecipitation, Activity Assay

    Exonuclease activity of rLiEndoG. 500′-labeled with FAM were digested with rLiEndoG or DNase I. The amount of enzyme used for digestion is indicated. Digestion products were processed by both agarose gel (1%) and capillary electrophoresis.  A ) Agarose gel of the DNA fragments generated after 1 h of digestion with rLiEndoG or DNase I.  B ) Capillary electrophoresis of the DNA probe digested for 1 h with 0.1 µg of rLiEndoG.  C ) Capillary electrophoresis results obtained for the DNA probe digested for 1 hour with 0.01 units of DNase I. Digested DNA was heat-denatured prior to capillary electrophoresis. Fluorescence intensities (arbitrary units) of the single-stranded DNA fragments generated after digestion and denaturation are shown on the y axis. Sizes of the ssDNA fragments (in nucleotides) are shown on the x axis. Fragment sizes were analyzed with the Peak Scanner (Applied Biosystems) software. Accurate sizes can only be predicted for fragments longer than 50 nucleotides.

    Journal: PLoS ONE

    Article Title: Leishmania infantum EndoG Is an Endo/Exo-Nuclease Essential for Parasite Survival

    doi: 10.1371/journal.pone.0089526

    Figure Lengend Snippet: Exonuclease activity of rLiEndoG. 500′-labeled with FAM were digested with rLiEndoG or DNase I. The amount of enzyme used for digestion is indicated. Digestion products were processed by both agarose gel (1%) and capillary electrophoresis. A ) Agarose gel of the DNA fragments generated after 1 h of digestion with rLiEndoG or DNase I. B ) Capillary electrophoresis of the DNA probe digested for 1 h with 0.1 µg of rLiEndoG. C ) Capillary electrophoresis results obtained for the DNA probe digested for 1 hour with 0.01 units of DNase I. Digested DNA was heat-denatured prior to capillary electrophoresis. Fluorescence intensities (arbitrary units) of the single-stranded DNA fragments generated after digestion and denaturation are shown on the y axis. Sizes of the ssDNA fragments (in nucleotides) are shown on the x axis. Fragment sizes were analyzed with the Peak Scanner (Applied Biosystems) software. Accurate sizes can only be predicted for fragments longer than 50 nucleotides.

    Article Snippet: By contrast, the peak pattern observed after digestion with DNase I is much more dispersed along the x axis and the smallest fragments are not the most abundant ( ).

    Techniques: Activity Assay, Labeling, Agarose Gel Electrophoresis, Electrophoresis, Generated, Fluorescence, Software

    Impact of DNase I treatment on B . pseudomallei biofilm formation. Static biofilms of B . pseudomallei strains L1, P1 and H777 in LB were treated with DNase I (0.01, 0.1 and 1 U/mL) at 0 h, 24 h or 45 h after inoculation and maintained for up to 48 h. Biofilm formation and eDNA concentration of the 2-day biofilms were assessed using crystal-violet absorbance (OD 620 ) and the QuantiFluor dsDNA System, respectively. DNase I buffer acted as control. Biofilm formation of each strain was examined in eight replicates and eDNA was quantified in duplicates, on three independent occasions. Data represents mean ± SD. * p

    Journal: PLoS ONE

    Article Title: Extracellular DNA facilitates bacterial adhesion during Burkholderia pseudomallei biofilm formation

    doi: 10.1371/journal.pone.0213288

    Figure Lengend Snippet: Impact of DNase I treatment on B . pseudomallei biofilm formation. Static biofilms of B . pseudomallei strains L1, P1 and H777 in LB were treated with DNase I (0.01, 0.1 and 1 U/mL) at 0 h, 24 h or 45 h after inoculation and maintained for up to 48 h. Biofilm formation and eDNA concentration of the 2-day biofilms were assessed using crystal-violet absorbance (OD 620 ) and the QuantiFluor dsDNA System, respectively. DNase I buffer acted as control. Biofilm formation of each strain was examined in eight replicates and eDNA was quantified in duplicates, on three independent occasions. Data represents mean ± SD. * p

    Article Snippet: DNase I noticeably lowered eDNA concentrations in biofilm if the enzyme was added into the starting inoculum (0 h).

    Techniques: Concentration Assay

    Exogenous chromosomal DNA did not alter either untreated biofilm or DNase I-treated biofilm of B . pseudomallei H777. (A) Amount of 2-day B . pseudomallei H777 biofilm formed in LB, treated with DNase I, supplemented with either salmon sperm DNA (SS DNA) or B . pseudomallei genomic DNA (Bp DNA) compared to the controls. Data represents mean ± SD from three independent experiments. (B) Amount of 2-day B . pseudomallei H777 biofilm formed in LB after treatment with 0.01 U/mL DNase I for 3 h, followed by washing steps to remove DNase, and then supplemented with exogenous salmon sperm DNA or B . pseudomallei genomic DNA. Data represents mean ± SD from three independent experiments. ** p

    Journal: PLoS ONE

    Article Title: Extracellular DNA facilitates bacterial adhesion during Burkholderia pseudomallei biofilm formation

    doi: 10.1371/journal.pone.0213288

    Figure Lengend Snippet: Exogenous chromosomal DNA did not alter either untreated biofilm or DNase I-treated biofilm of B . pseudomallei H777. (A) Amount of 2-day B . pseudomallei H777 biofilm formed in LB, treated with DNase I, supplemented with either salmon sperm DNA (SS DNA) or B . pseudomallei genomic DNA (Bp DNA) compared to the controls. Data represents mean ± SD from three independent experiments. (B) Amount of 2-day B . pseudomallei H777 biofilm formed in LB after treatment with 0.01 U/mL DNase I for 3 h, followed by washing steps to remove DNase, and then supplemented with exogenous salmon sperm DNA or B . pseudomallei genomic DNA. Data represents mean ± SD from three independent experiments. ** p

    Article Snippet: DNase I noticeably lowered eDNA concentrations in biofilm if the enzyme was added into the starting inoculum (0 h).

    Techniques:

    DNase I treatment affects initial attachment and biofilm formation of B . pseudomallei . B . pseudomallei L1, P1 and H777 biofilms were grown in LB at 37°C. The biofilms were treated with DNase I (0.01 U/mL) at 0 h and 24 h post-seeding and maintained until 48 h. (A) CLSM images of DNase I treated biofilm structure and eDNA on coverslips. The 2-day biofilm architecture and quantity of eDNA were examined after staining with FITC-ConA (green) and TOTO-3 (red), respectively. The scale bars indicate 10 μm. The images were taken using a Zeiss 800 CLSM microscope (63× magnification). (B) COMSTAT image analysis of DNase I-treated B . pseudomallei biofilms and eDNA. Data represents mean ± SD of 18 images from three independent experiments. * p

    Journal: PLoS ONE

    Article Title: Extracellular DNA facilitates bacterial adhesion during Burkholderia pseudomallei biofilm formation

    doi: 10.1371/journal.pone.0213288

    Figure Lengend Snippet: DNase I treatment affects initial attachment and biofilm formation of B . pseudomallei . B . pseudomallei L1, P1 and H777 biofilms were grown in LB at 37°C. The biofilms were treated with DNase I (0.01 U/mL) at 0 h and 24 h post-seeding and maintained until 48 h. (A) CLSM images of DNase I treated biofilm structure and eDNA on coverslips. The 2-day biofilm architecture and quantity of eDNA were examined after staining with FITC-ConA (green) and TOTO-3 (red), respectively. The scale bars indicate 10 μm. The images were taken using a Zeiss 800 CLSM microscope (63× magnification). (B) COMSTAT image analysis of DNase I-treated B . pseudomallei biofilms and eDNA. Data represents mean ± SD of 18 images from three independent experiments. * p

    Article Snippet: DNase I noticeably lowered eDNA concentrations in biofilm if the enzyme was added into the starting inoculum (0 h).

    Techniques: Confocal Laser Scanning Microscopy, Staining, Microscopy

    DNase I footprinting demonstrates HilD binding to the flhDC P5 promoter region. (A) DNase I footprinting of an flhDC P5 promoter DNA fragment. A DNA fragment covering a region of nucleotides from position −668 to −388 upstream from the flhD start codon was DIG labeled on the noncoding strand and incubated alone (lane P) and with increasing amounts of purified HilD protein (lane 1, 4.23 pmol; lane 2, 8.45 pmol; lane 3, 12.68 pmol; lane 4, 16.9 pmol; lane 5, 21.13 pmol; lane 6, 42.25 pmol) and digested with DNase I before being loaded on a sequencing gel. The vertical line indicates the region protected from DNase I digestion. Lanes C, T, A, and G show the specific nucleotides of the noncoding strand. Exposed nucleotides are highlighted by dots. (B) Partial nucleotide sequence of the P5 promoter of flhDC that is relevant for HilD binding. A horizontal line marks the protected region, and the four most sensitive nucleotides are highlighted by dots. The transcriptional start site (marked as +1) and the −10 element of the P5 flhDC promoter are indicated. (C) Comparison of HilD binding sites in the flhDC , rtsA , hilC , hilD , and hilA ). The proposed consensus is displayed at the bottom; uppercase letters indicate predominant nucleotides ( > 80% conserved), and lowercase letters indicate conserved nucleotides ( > 60% conserved).

    Journal: Journal of Bacteriology

    Article Title: The Salmonella Spi1 Virulence Regulatory Protein HilD Directly Activates Transcription of the Flagellar Master Operon flhDC

    doi: 10.1128/JB.01438-13

    Figure Lengend Snippet: DNase I footprinting demonstrates HilD binding to the flhDC P5 promoter region. (A) DNase I footprinting of an flhDC P5 promoter DNA fragment. A DNA fragment covering a region of nucleotides from position −668 to −388 upstream from the flhD start codon was DIG labeled on the noncoding strand and incubated alone (lane P) and with increasing amounts of purified HilD protein (lane 1, 4.23 pmol; lane 2, 8.45 pmol; lane 3, 12.68 pmol; lane 4, 16.9 pmol; lane 5, 21.13 pmol; lane 6, 42.25 pmol) and digested with DNase I before being loaded on a sequencing gel. The vertical line indicates the region protected from DNase I digestion. Lanes C, T, A, and G show the specific nucleotides of the noncoding strand. Exposed nucleotides are highlighted by dots. (B) Partial nucleotide sequence of the P5 promoter of flhDC that is relevant for HilD binding. A horizontal line marks the protected region, and the four most sensitive nucleotides are highlighted by dots. The transcriptional start site (marked as +1) and the −10 element of the P5 flhDC promoter are indicated. (C) Comparison of HilD binding sites in the flhDC , rtsA , hilC , hilD , and hilA ). The proposed consensus is displayed at the bottom; uppercase letters indicate predominant nucleotides ( > 80% conserved), and lowercase letters indicate conserved nucleotides ( > 60% conserved).

    Article Snippet: An flhDC promoter fragment comprising nucleotides −668 to −388 upstream from the flhDC coding region was incubated with increasing concentrations of purified HilD protein and, after partial digestion with DNase I, the resulting fragments were subjected to denaturing gel electrophoresis ( ).

    Techniques: Footprinting, Binding Assay, Labeling, Incubation, Purification, Sequencing

    MdcY-mediated protection of the  mdc  operator against digestion by DNase I. Lanes 1 to 4, target DNA (0.1 pmol, 2 × 10 5  cpm) was incubated in the absence or in the presence of MdcY (the number above each lane indicates the nanomolar concentration of MdcY protein). In lanes 5 to 7, target DNA and 2.4 nM MdcY were incubated with 50, 100, and 500 μM malonate, respectively. The vertical arrows beside the nucleotide sequence indicate a palindromic structure.

    Journal: Journal of Bacteriology

    Article Title: The Malonate Decarboxylase Operon of Acinetobacter calcoaceticus KCCM 40902 Is Regulated by Malonate and the Transcriptional Repressor MdcY

    doi:

    Figure Lengend Snippet: MdcY-mediated protection of the mdc operator against digestion by DNase I. Lanes 1 to 4, target DNA (0.1 pmol, 2 × 10 5 cpm) was incubated in the absence or in the presence of MdcY (the number above each lane indicates the nanomolar concentration of MdcY protein). In lanes 5 to 7, target DNA and 2.4 nM MdcY were incubated with 50, 100, and 500 μM malonate, respectively. The vertical arrows beside the nucleotide sequence indicate a palindromic structure.

    Article Snippet: The reaction was stopped by the addition 36 μl of DNase I stop solution (200 mM NaCl, 30 mM EDTA, 1% sodium dodecyl sulfate [SDS], 100 μl of yeast RNA per ml).

    Techniques: Incubation, Concentration Assay, Sequencing

    Variations in expression levels of fungus-responsive genes in the hemolymph of silkworms, Bombyx mori , infected with Beauveria bassiana . The third-day larvae of the fifth instar of Dazao strains were infected with B. bassiana . Total RNA was extracted from the hemolymph at the indicated time points after infection and subjected to DNase I treatment and reverse transcription. Two microliters of each 10-fold diluted first strand cDNA (20 ng) was analyzed in each real-time qPCR reaction. The reaction was performed with specific primers for amplifying each of the six genes. The relative expression level of each gene at each time point was normalized using the Ct values obtained for the Bm GAPDH amplifications run in the same plate. In each assay, the expression level is shown relative to the lowest expression level, which was set to one. All samples were tested in triplicate. The mean value ± SD was used for the analysis of the relative transcript levels for each time point using the △△Ct method. The B. bassiana injected and water-treated individuals are shown on the left (blue) and right (purple), respectively. A. Chemosensory protein 11; B. Muscle LIM protein isoform 1; C. Transferrin; D. Sex-specific storage-protein SP1 precursor; E. Arylphorin; F. Low molecular lipoprotein 30K pBmHPC-6 (Lp-c6); G. lysozyme; H. Moricin. High quality figures are available online.

    Journal: Journal of Insect Science

    Article Title: Differentially Expressed Genes in the Cuticle and Hemolymph of the Silkworm, Bombyx mori, Injected with the Fungus Beauveria bassiana

    doi: 10.1673/031.013.13801

    Figure Lengend Snippet: Variations in expression levels of fungus-responsive genes in the hemolymph of silkworms, Bombyx mori , infected with Beauveria bassiana . The third-day larvae of the fifth instar of Dazao strains were infected with B. bassiana . Total RNA was extracted from the hemolymph at the indicated time points after infection and subjected to DNase I treatment and reverse transcription. Two microliters of each 10-fold diluted first strand cDNA (20 ng) was analyzed in each real-time qPCR reaction. The reaction was performed with specific primers for amplifying each of the six genes. The relative expression level of each gene at each time point was normalized using the Ct values obtained for the Bm GAPDH amplifications run in the same plate. In each assay, the expression level is shown relative to the lowest expression level, which was set to one. All samples were tested in triplicate. The mean value ± SD was used for the analysis of the relative transcript levels for each time point using the △△Ct method. The B. bassiana injected and water-treated individuals are shown on the left (blue) and right (purple), respectively. A. Chemosensory protein 11; B. Muscle LIM protein isoform 1; C. Transferrin; D. Sex-specific storage-protein SP1 precursor; E. Arylphorin; F. Low molecular lipoprotein 30K pBmHPC-6 (Lp-c6); G. lysozyme; H. Moricin. High quality figures are available online.

    Article Snippet: The RNA was treated with DNase I following the manufacturer' s instructions.

    Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction, Injection

    Nonspecific stimulation by unfractionated B. bovis merozoite antigen is DNase sensitive. PBMC from two calves never exposed to babesial parasites were cultured for 3 (A and C) or 6 (B and D) days with medium alone (0) or 12.5 or 25 μg of B. bovis CM antigen per ml that was untreated (solid bars) or DNase I treated (striped bars), and proliferation was determined. Results are expressed as the mean cpm ± 1 SD of triplicate cultures.

    Journal: Infection and Immunity

    Article Title: DNA and a CpG Oligonucleotide Derived from Babesia bovis Are Mitogenic for Bovine B Cells

    doi:

    Figure Lengend Snippet: Nonspecific stimulation by unfractionated B. bovis merozoite antigen is DNase sensitive. PBMC from two calves never exposed to babesial parasites were cultured for 3 (A and C) or 6 (B and D) days with medium alone (0) or 12.5 or 25 μg of B. bovis CM antigen per ml that was untreated (solid bars) or DNase I treated (striped bars), and proliferation was determined. Results are expressed as the mean cpm ± 1 SD of triplicate cultures.

    Article Snippet: CM antigen was incubated for 2 h at 37°C at a concentration of 1 mg of protein per ml of DNase I buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , and 0.5 mg of BSA per ml with a final concentration of 1 mg of DNase I (Sigma) per ml.

    Techniques: Cell Culture

    Fig. 3. Displacement of mcm4 from chromatin requires progression through S phase or DNase I digestion. ( A ) Experimental procedure. ( B ) mcm4–GFP chromatin association and DNA staining (DAPI) were determined by fluorescence microscopy during a time course after addition of hydroxyurea. Bar = 10 μm. ( C ) Proportion of uninucleate cells with mcm4-positive nuclei before and after extraction with a Triton X-100-containing buffer. ( D ) Flow cytometric analysis of DNA contents of the cells shown in (B). ( E ) Cells from the ‘2h + hydroxyurea’ time point were digested with DNase I (for details, see Materials and methods). mcm4–GFP localization (left) and DNA staining (DAPI, right) were determined by fluorescence microscopy after Triton extraction. Bar = 10 μm. ( F ) Proportion of Triton-extracted cells with mcm4-positive nuclei with and without digestion with DNase I.

    Journal: The EMBO Journal

    Article Title: Chromatin binding of the fission yeast replication factor mcm4 occurs during anaphase and requires ORC and cdc18

    doi: 10.1093/emboj/19.7.1681

    Figure Lengend Snippet: Fig. 3. Displacement of mcm4 from chromatin requires progression through S phase or DNase I digestion. ( A ) Experimental procedure. ( B ) mcm4–GFP chromatin association and DNA staining (DAPI) were determined by fluorescence microscopy during a time course after addition of hydroxyurea. Bar = 10 μm. ( C ) Proportion of uninucleate cells with mcm4-positive nuclei before and after extraction with a Triton X-100-containing buffer. ( D ) Flow cytometric analysis of DNA contents of the cells shown in (B). ( E ) Cells from the ‘2h + hydroxyurea’ time point were digested with DNase I (for details, see Materials and methods). mcm4–GFP localization (left) and DNA staining (DAPI, right) were determined by fluorescence microscopy after Triton extraction. Bar = 10 μm. ( F ) Proportion of Triton-extracted cells with mcm4-positive nuclei with and without digestion with DNase I.

    Article Snippet: For DNase I digestion, following the STOP and EB washes, cells were resuspended in EB containing 5 mM MgAc, 1/1000 volume of protease inhibitor cocktail (Sigma P-8215) and 1% (w/v) Triton X-100.

    Techniques: Staining, Fluorescence, Microscopy, Flow Cytometry

    Analysis of ciprofloxacin-induced release of DNA associated with extracellular vesicles (EVs). (a) DNA content of size-based EV fractions determined with/without DNase I digestion of EVs. Concentration values represent the amount of EV-associated DNA (eluted in 3 0 µL) isolated from the conditioned medium of 2.5 × 10 7 Jurkat cells. Plotted values are presented as the mean+/− S.D. (error bars) of 8 independent experiments. (**P

    Journal: Scientific Reports

    Article Title: Antibiotic-induced release of small extracellular vesicles (exosomes) with surface-associated DNA

    doi: 10.1038/s41598-017-08392-1

    Figure Lengend Snippet: Analysis of ciprofloxacin-induced release of DNA associated with extracellular vesicles (EVs). (a) DNA content of size-based EV fractions determined with/without DNase I digestion of EVs. Concentration values represent the amount of EV-associated DNA (eluted in 3 0 µL) isolated from the conditioned medium of 2.5 × 10 7 Jurkat cells. Plotted values are presented as the mean+/− S.D. (error bars) of 8 independent experiments. (**P

    Article Snippet: Analysis of vesicular DNA DNA content of size-based EV fractions was analyzed with/without DNase I (Sigma) digestion.

    Techniques: Concentration Assay, Isolation

    Assessment of DNA and DNA-binding proteins in extracellular vesicle (EV) preparations.  (a-b)  DNA was purified from apoptotic bodies (APOs), microvesicles (MVs) or exosomes (EXOs) released by ciprofloxacin-exposed control, activated or apoptotic Jurkat cells.  (a)  Nuclear (GAPDH, p53) and  (b)  mitochondrial (control region, RNR1) DNA sequences of DNase I-digested/non-digested EVs were amplified by PCR and analyzed by agarose gel electrophoreses. The figure displays cropped gels. Full-length, uncropped gels are shown in Supplementary Figures   S11 –  S13. (c )  Detection of exosomal DNA derived from ciprofloxacin-exposed control, activated or apoptotic Jurkat cells using an Agilent 2100 Bioanalyzer (DNA 12,000 Kit). The electropherograms show the size distribution of purified exosomal DNA in base pairs (bp) with DNA markers at 50 bp and 17,000 bp. FU: fluorescence units.  (d)  Semi-quantitative mass spectrometry analysis of DNA-binding histones in EV samples. Values in the table are proportional to the amount of histones found in EVs. The presence of f lap endonuclease 1 (FEN1, also known as a mitochondrial DNA-binding nucleoid protein) was also identified by mass spectrometry.

    Journal: Scientific Reports

    Article Title: Antibiotic-induced release of small extracellular vesicles (exosomes) with surface-associated DNA

    doi: 10.1038/s41598-017-08392-1

    Figure Lengend Snippet: Assessment of DNA and DNA-binding proteins in extracellular vesicle (EV) preparations. (a-b) DNA was purified from apoptotic bodies (APOs), microvesicles (MVs) or exosomes (EXOs) released by ciprofloxacin-exposed control, activated or apoptotic Jurkat cells. (a) Nuclear (GAPDH, p53) and (b) mitochondrial (control region, RNR1) DNA sequences of DNase I-digested/non-digested EVs were amplified by PCR and analyzed by agarose gel electrophoreses. The figure displays cropped gels. Full-length, uncropped gels are shown in Supplementary Figures  S11 – S13. (c ) Detection of exosomal DNA derived from ciprofloxacin-exposed control, activated or apoptotic Jurkat cells using an Agilent 2100 Bioanalyzer (DNA 12,000 Kit). The electropherograms show the size distribution of purified exosomal DNA in base pairs (bp) with DNA markers at 50 bp and 17,000 bp. FU: fluorescence units. (d) Semi-quantitative mass spectrometry analysis of DNA-binding histones in EV samples. Values in the table are proportional to the amount of histones found in EVs. The presence of f lap endonuclease 1 (FEN1, also known as a mitochondrial DNA-binding nucleoid protein) was also identified by mass spectrometry.

    Article Snippet: Analysis of vesicular DNA DNA content of size-based EV fractions was analyzed with/without DNase I (Sigma) digestion.

    Techniques: DNA Binding Assay, Purification, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Derivative Assay, Fluorescence, Mass Spectrometry, Binding Assay

    ( A ) Binding of TieA to dsDNA: electrophoretic mobility shift assays were carried out by incubating different concentrations of TieA (0.1, 0.5, 1 and 2 μg) with 0.5 nM 32 P-labeled DNA substrates. Samples were subjected to electrophoresis on native PAGE and visualized by autoradiography as mentioned in materials and methods section. ( B ) TieA binds to DNA non-specifically: electrophoretic mobility shift assays were carried out by incubating 1 μg of TieA with mutated oligos 1–5 (see Supplementary Table S1). ( C ) Nuclease activity of TieA: different concentrations of TieA (0.01, 0.1, 0.2, 0.5, 1 and 2 μg corresponding to lanes 7-12, respectively) were incubated with 1 μg of pUC19 DNA for 1 h at 37 °C. The reaction was stopped by addition of 10 mM EDTA and samples were deprotonized by adding proteinase K (10 μg/sample) in presence of 0.05% SDS for 15 min at 65°C. The digested products were separated on 1.2% agarose gel. Rv3131 (0.5 μg) was used as a negative control in lane 6. MboII (1 unit/reaction) and DNase I (1 unit/reaction) served as positive controls in lanes 3 and 5, respectively. Lane 4 represents heat inactivated TieA. ( D ) TieA cleaves both pUC19 (circular) and Lambda DNA (linear): pUC19 and Lambda DNA were incubated with TieA (lanes 5, 6, 14 and 15) for 1 h at 37°C and processed as described above. MboII (lanes 3 and 12) and DNase I (lanes 4 and 13) were used as positive controls. Rv3131 protein was used as a negative control (lanes 7 and 16). Ca 2+ –Mg 2+ dependent nuclease activity of TieA was confirmed by pre-incubating pUC19/Lambda DNA with either SDS (0.05%) or EDTA (10 mM) for 10 min (lanes 8, 9, 17 and 18) and later 1 μg of TieA was added and further processed as described above. Data are representative of three independent experiments. HI: heat inactivated.

    Journal: Nucleic Acids Research

    Article Title: Multipronged regulatory functions of a novel endonuclease (TieA) from Helicobacter pylori

    doi: 10.1093/nar/gkw730

    Figure Lengend Snippet: ( A ) Binding of TieA to dsDNA: electrophoretic mobility shift assays were carried out by incubating different concentrations of TieA (0.1, 0.5, 1 and 2 μg) with 0.5 nM 32 P-labeled DNA substrates. Samples were subjected to electrophoresis on native PAGE and visualized by autoradiography as mentioned in materials and methods section. ( B ) TieA binds to DNA non-specifically: electrophoretic mobility shift assays were carried out by incubating 1 μg of TieA with mutated oligos 1–5 (see Supplementary Table S1). ( C ) Nuclease activity of TieA: different concentrations of TieA (0.01, 0.1, 0.2, 0.5, 1 and 2 μg corresponding to lanes 7-12, respectively) were incubated with 1 μg of pUC19 DNA for 1 h at 37 °C. The reaction was stopped by addition of 10 mM EDTA and samples were deprotonized by adding proteinase K (10 μg/sample) in presence of 0.05% SDS for 15 min at 65°C. The digested products were separated on 1.2% agarose gel. Rv3131 (0.5 μg) was used as a negative control in lane 6. MboII (1 unit/reaction) and DNase I (1 unit/reaction) served as positive controls in lanes 3 and 5, respectively. Lane 4 represents heat inactivated TieA. ( D ) TieA cleaves both pUC19 (circular) and Lambda DNA (linear): pUC19 and Lambda DNA were incubated with TieA (lanes 5, 6, 14 and 15) for 1 h at 37°C and processed as described above. MboII (lanes 3 and 12) and DNase I (lanes 4 and 13) were used as positive controls. Rv3131 protein was used as a negative control (lanes 7 and 16). Ca 2+ –Mg 2+ dependent nuclease activity of TieA was confirmed by pre-incubating pUC19/Lambda DNA with either SDS (0.05%) or EDTA (10 mM) for 10 min (lanes 8, 9, 17 and 18) and later 1 μg of TieA was added and further processed as described above. Data are representative of three independent experiments. HI: heat inactivated.

    Article Snippet: RNA samples (1 μg) were subjected to RNase free DNase digestion by using amplification grade DNase I (Sigma-Aldrich) enzyme. cDNA synthesis was performed with 1 μg of purified RNA using the superscript III cDNA synthesis kit (Invitrogen Life Technologies).

    Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Electrophoresis, Clear Native PAGE, Autoradiography, Activity Assay, Incubation, Agarose Gel Electrophoresis, Negative Control, Lambda DNA Preparation

    ( A ) Measurement of cell death by Annexin V-FITC/PI staining. AGS cells were infected (WT and KO strains of H. pylori ) or treated (5 μg of TieA protein) for 24 h as indicated and were examined for apoptotic cells using Annexin V-FITC apoptosis detection kit as described in materials and methods section. Staurosporine and DNase I treated AGS cells were used as positive controls. ( B ) Annexin V-FITC/PI staining to analyze apoptosis in AGS cell line induced by TieA using flow cytometry. ( C ) AGS cells were infected with H. pylori (WT or KO) at an MOI of 100 for 24 h, and cell death was measured as fold change in histone release. Data are mean ± SD of three independent experiments; two-tailed Student's t test was performed for statistical analysis, * P ≤ 0.05. ( D ) Flow cytometry analysis showing expression of Fas receptors on AGS cells upon treatment with TieA (5 μg) after 24 h. The shift in the histogram peak for TieA and staurosporine as compared to the untreated AGS cells indicates an enhanced expression of Fas receptors on AGS cells. HI: heat inactivated.

    Journal: Nucleic Acids Research

    Article Title: Multipronged regulatory functions of a novel endonuclease (TieA) from Helicobacter pylori

    doi: 10.1093/nar/gkw730

    Figure Lengend Snippet: ( A ) Measurement of cell death by Annexin V-FITC/PI staining. AGS cells were infected (WT and KO strains of H. pylori ) or treated (5 μg of TieA protein) for 24 h as indicated and were examined for apoptotic cells using Annexin V-FITC apoptosis detection kit as described in materials and methods section. Staurosporine and DNase I treated AGS cells were used as positive controls. ( B ) Annexin V-FITC/PI staining to analyze apoptosis in AGS cell line induced by TieA using flow cytometry. ( C ) AGS cells were infected with H. pylori (WT or KO) at an MOI of 100 for 24 h, and cell death was measured as fold change in histone release. Data are mean ± SD of three independent experiments; two-tailed Student's t test was performed for statistical analysis, * P ≤ 0.05. ( D ) Flow cytometry analysis showing expression of Fas receptors on AGS cells upon treatment with TieA (5 μg) after 24 h. The shift in the histogram peak for TieA and staurosporine as compared to the untreated AGS cells indicates an enhanced expression of Fas receptors on AGS cells. HI: heat inactivated.

    Article Snippet: RNA samples (1 μg) were subjected to RNase free DNase digestion by using amplification grade DNase I (Sigma-Aldrich) enzyme. cDNA synthesis was performed with 1 μg of purified RNA using the superscript III cDNA synthesis kit (Invitrogen Life Technologies).

    Techniques: Staining, Infection, Flow Cytometry, Cytometry, Two Tailed Test, Expressing

    Identification of AphB binding site using DNase I protection analysis (A) and sequence analysis of the cadC upstream region (B). (A) The 32 P-labeled 298-bp cadC regulatory region was incubated with increasing amounts of AphB and then digested with DNase

    Journal: Journal of Bacteriology

    Article Title: AphB Influences Acid Tolerance of Vibrio vulnificus by Activating Expression of the Positive Regulator CadC

    doi: 10.1128/JB.00533-06

    Figure Lengend Snippet: Identification of AphB binding site using DNase I protection analysis (A) and sequence analysis of the cadC upstream region (B). (A) The 32 P-labeled 298-bp cadC regulatory region was incubated with increasing amounts of AphB and then digested with DNase

    Article Snippet: The protein-DNA binding reactions with AphB were carried out as described above, except that 20 μl of 10 mM MgCl2 and 5 mM CaCl2 mix was added to the reaction mixtures, along with 1 μl of a DNase I solution (10 ng/μl; Sigma) ( ).

    Techniques: Binding Assay, Sequencing, Labeling, Incubation

    Chromatin binding activity of Mrc1. (A) KT2791 cells (Mrc1-13myc) or HZ109 cells (Mrc1-3SA-13myc) were grown in minimal medium overnight and then either mock treated or treated with 15 mM HU for 3 h. Cells were harvested and extracted with 1% Triton X-100 for an in situ chromatin binding assay (see Materials and Methods). (B) Mrc1 chromatin association is sensitive to DNase I treatment. KT2791 cells (Mrc1-13myc) were grown in the presence of 15 mM HU and harvested for a chromatin binding assay. Cells were incubated with DNase I together with Triton X-100 and then subjected to an indirect immunofluorescence (IF) assay. The same cells are shown on the left side. (C) KT2791 cells (Mrc1-13myc), HZ109 cells (3SA-13myc), or HZ110 cells ( rad3 Δ tel1 Δ Mrc1-13myc) were treated with 15 mM HU. Cells were subjected to Triton X-100 extraction for in situ chromatin binding assays. In each case, the number of cells with Mrc1 nuclear signal versus the total number of cells is represented on the y axis. At least 200 cells were counted from at least two different experiments, and the standard error is was 15% of the average. WT, wild type.

    Journal: Molecular and Cellular Biology

    Article Title: Replication Checkpoint Protein Mrc1 Is Regulated by Rad3 and Tel1 in Fission Yeast

    doi: 10.1128/MCB.23.22.8395-8403.2003

    Figure Lengend Snippet: Chromatin binding activity of Mrc1. (A) KT2791 cells (Mrc1-13myc) or HZ109 cells (Mrc1-3SA-13myc) were grown in minimal medium overnight and then either mock treated or treated with 15 mM HU for 3 h. Cells were harvested and extracted with 1% Triton X-100 for an in situ chromatin binding assay (see Materials and Methods). (B) Mrc1 chromatin association is sensitive to DNase I treatment. KT2791 cells (Mrc1-13myc) were grown in the presence of 15 mM HU and harvested for a chromatin binding assay. Cells were incubated with DNase I together with Triton X-100 and then subjected to an indirect immunofluorescence (IF) assay. The same cells are shown on the left side. (C) KT2791 cells (Mrc1-13myc), HZ109 cells (3SA-13myc), or HZ110 cells ( rad3 Δ tel1 Δ Mrc1-13myc) were treated with 15 mM HU. Cells were subjected to Triton X-100 extraction for in situ chromatin binding assays. In each case, the number of cells with Mrc1 nuclear signal versus the total number of cells is represented on the y axis. At least 200 cells were counted from at least two different experiments, and the standard error is was 15% of the average. WT, wild type.

    Article Snippet: For DNase I treatment, cells were incubated with 5 U of DNase I (Sigma) together with Triton as previously described ( ).

    Techniques: Binding Assay, Activity Assay, In Situ, Incubation, Immunofluorescence

    Curcumin activates p38MAPK and phosphorylates HSP25 in cultured Pods . 5.5 mM glucose (NG), 5.5 mM glucose+30 μM curcumin (NG+Cur), 30 mM glucose (HG), 30 mM glucose+30 μM curcumin (HG+Cur), 5.5 mM glucose+24.5 mM mannitol (NG+M). (a) Representative Western blots of phospho-specific p38MAPK (pp38MAPK) and quantitative evaluation of pp38MAPK relative to total p38MAPK (p38MAPK) by densitometric analysis. (b) Representative IEF separating total HSP25 into its nonphosphorylated isoform (P0), mono- (P1), and bi-phoshorylated (P2) isoforms, Western blots of HSP25, and quantitative evaluation of relative phosphorylated HSP25 isoforms to total HSP25 ((P1 + P2)/(P0 + P1 + P2)) by densitometry analysis. Mannitol values (n = 2) are not displayed but were similar to NG. (c) DNAse I assay of F-actin/G-actin ratios. All data expressed as mean ± SEM (n = 3). *P

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Curcumin activates the p38MPAK-HSP25 pathway in vitro but fails to attenuate diabetic nephropathy in DBA2J mice despite urinary clearance documented by HPLC

    doi: 10.1186/1472-6882-10-67

    Figure Lengend Snippet: Curcumin activates p38MAPK and phosphorylates HSP25 in cultured Pods . 5.5 mM glucose (NG), 5.5 mM glucose+30 μM curcumin (NG+Cur), 30 mM glucose (HG), 30 mM glucose+30 μM curcumin (HG+Cur), 5.5 mM glucose+24.5 mM mannitol (NG+M). (a) Representative Western blots of phospho-specific p38MAPK (pp38MAPK) and quantitative evaluation of pp38MAPK relative to total p38MAPK (p38MAPK) by densitometric analysis. (b) Representative IEF separating total HSP25 into its nonphosphorylated isoform (P0), mono- (P1), and bi-phoshorylated (P2) isoforms, Western blots of HSP25, and quantitative evaluation of relative phosphorylated HSP25 isoforms to total HSP25 ((P1 + P2)/(P0 + P1 + P2)) by densitometry analysis. Mannitol values (n = 2) are not displayed but were similar to NG. (c) DNAse I assay of F-actin/G-actin ratios. All data expressed as mean ± SEM (n = 3). *P

    Article Snippet: Actin was measured using a standard curve for inhibition of DNase I activity using rabbit skeletal muscle G-actin (Sigma-Aldrich).

    Techniques: Cell Culture, Western Blot, Electrofocusing

    DNase I disrupts established biofilms of B. bronchiseptica and B. pertussis formed in the mouse respiratory tract. CLSM images of biofilms harvested from mouse nose 15 and 19 days postinoculation with RB50 (top) or Bp536 (bottom), respectively. The harvested nasal septum was excised into two equal parts and incubated either with DNase I buffer (Mock, left panels) or with DNase I (right panels) before processing for staining as described in the Materials and Methods . Green staining depicts Bordetella biofilms formed on top of the host epithelium, which is stained red.

    Journal: PLoS ONE

    Article Title: Extracellular DNA Is Essential for Maintaining Bordetella Biofilm Integrity on Abiotic Surfaces and in the Upper Respiratory Tract of Mice

    doi: 10.1371/journal.pone.0016861

    Figure Lengend Snippet: DNase I disrupts established biofilms of B. bronchiseptica and B. pertussis formed in the mouse respiratory tract. CLSM images of biofilms harvested from mouse nose 15 and 19 days postinoculation with RB50 (top) or Bp536 (bottom), respectively. The harvested nasal septum was excised into two equal parts and incubated either with DNase I buffer (Mock, left panels) or with DNase I (right panels) before processing for staining as described in the Materials and Methods . Green staining depicts Bordetella biofilms formed on top of the host epithelium, which is stained red.

    Article Snippet: Briefly overnight grown culture of B. bronchiseptica was inoculated in the wells of a 96 well microtitre plate at OD600 of 0.05 followed by addition of either DNase I reaction buffer (10mM Tris-HCl, pH 7.5, 50% glycerol, 10mM MgCl2 ), or with DNase I (40 Kuntz units/ml)(Sigma or Promega) resuspended in the DNase I reaction buffer.

    Techniques: Confocal Laser Scanning Microscopy, Incubation, Staining

    Scanning electron microscopy of mock treated (left) or DNase I treated Bordetella biofilms formed in the mouse nose. Nasal septa were harvested from mice 15 days post-inoculation, excised into two equal parts, treated with either the DNase I buffer (Mock, left panels) or DNase I (right) followed by processing for SEM as described in the Materials and Methods .

    Journal: PLoS ONE

    Article Title: Extracellular DNA Is Essential for Maintaining Bordetella Biofilm Integrity on Abiotic Surfaces and in the Upper Respiratory Tract of Mice

    doi: 10.1371/journal.pone.0016861

    Figure Lengend Snippet: Scanning electron microscopy of mock treated (left) or DNase I treated Bordetella biofilms formed in the mouse nose. Nasal septa were harvested from mice 15 days post-inoculation, excised into two equal parts, treated with either the DNase I buffer (Mock, left panels) or DNase I (right) followed by processing for SEM as described in the Materials and Methods .

    Article Snippet: Briefly overnight grown culture of B. bronchiseptica was inoculated in the wells of a 96 well microtitre plate at OD600 of 0.05 followed by addition of either DNase I reaction buffer (10mM Tris-HCl, pH 7.5, 50% glycerol, 10mM MgCl2 ), or with DNase I (40 Kuntz units/ml)(Sigma or Promega) resuspended in the DNase I reaction buffer.

    Techniques: Electron Microscopy, Mouse Assay

    DNase I leads to the disruption of established Bordetella biofilms grown in microtitre plates. Preformed 48h RB50 biofilms grown in 96 well plates were rinsed with PBS followed by incubation with PBS, PBS and reaction buffer, PBS and DNase I, PBS and heat inactivated (HI) DNase I, or PBS with reaction buffer and DNase I. Biofilm formation was then quantitated via crystal violet staining. Each point is an average of at least 6 wells, and error bars indicate the standard deviation. Asterisks designate a value of P

    Journal: PLoS ONE

    Article Title: Extracellular DNA Is Essential for Maintaining Bordetella Biofilm Integrity on Abiotic Surfaces and in the Upper Respiratory Tract of Mice

    doi: 10.1371/journal.pone.0016861

    Figure Lengend Snippet: DNase I leads to the disruption of established Bordetella biofilms grown in microtitre plates. Preformed 48h RB50 biofilms grown in 96 well plates were rinsed with PBS followed by incubation with PBS, PBS and reaction buffer, PBS and DNase I, PBS and heat inactivated (HI) DNase I, or PBS with reaction buffer and DNase I. Biofilm formation was then quantitated via crystal violet staining. Each point is an average of at least 6 wells, and error bars indicate the standard deviation. Asterisks designate a value of P

    Article Snippet: Briefly overnight grown culture of B. bronchiseptica was inoculated in the wells of a 96 well microtitre plate at OD600 of 0.05 followed by addition of either DNase I reaction buffer (10mM Tris-HCl, pH 7.5, 50% glycerol, 10mM MgCl2 ), or with DNase I (40 Kuntz units/ml)(Sigma or Promega) resuspended in the DNase I reaction buffer.

    Techniques: Incubation, Staining, Standard Deviation

    DNase I inhibits Bordetella biofilm formation. The indicated strains were grown in 96 well microtitre plates for RB50 or 12 well plates for Bp536 for designated time points in SS medium supplemented with either DNase I resuspended in the reaction buffer or the reaction buffer alone. Wells were rinsed and stained with crystal violet followed by quantification of bound crystal violet as described in Materials and Methods . Each data point is the average of six wells, and error bars indicate the standard deviation. Representative data from one of at least three independent experiments are shown. Asterisks designate a value of P

    Journal: PLoS ONE

    Article Title: Extracellular DNA Is Essential for Maintaining Bordetella Biofilm Integrity on Abiotic Surfaces and in the Upper Respiratory Tract of Mice

    doi: 10.1371/journal.pone.0016861

    Figure Lengend Snippet: DNase I inhibits Bordetella biofilm formation. The indicated strains were grown in 96 well microtitre plates for RB50 or 12 well plates for Bp536 for designated time points in SS medium supplemented with either DNase I resuspended in the reaction buffer or the reaction buffer alone. Wells were rinsed and stained with crystal violet followed by quantification of bound crystal violet as described in Materials and Methods . Each data point is the average of six wells, and error bars indicate the standard deviation. Representative data from one of at least three independent experiments are shown. Asterisks designate a value of P

    Article Snippet: Briefly overnight grown culture of B. bronchiseptica was inoculated in the wells of a 96 well microtitre plate at OD600 of 0.05 followed by addition of either DNase I reaction buffer (10mM Tris-HCl, pH 7.5, 50% glycerol, 10mM MgCl2 ), or with DNase I (40 Kuntz units/ml)(Sigma or Promega) resuspended in the DNase I reaction buffer.

    Techniques: Staining, Standard Deviation

    Susceptibility of flow cell biofilms to DNase I. Representative z-reconstructions of RB50 biofilms grown under flow conditions for 6, 72, or 120h and imaged using CLSM for live GFP expressing cells (green) and eDNA stained with DDAO (red or yellow with co-localization). The image of untreated biofilms (left panels) were taken immediately prior to incubation with DNase I and the images of same biofilms treated with DNase I for 1.5h (left panels). Images shown here are representative of two independent experiments.

    Journal: PLoS ONE

    Article Title: Extracellular DNA Is Essential for Maintaining Bordetella Biofilm Integrity on Abiotic Surfaces and in the Upper Respiratory Tract of Mice

    doi: 10.1371/journal.pone.0016861

    Figure Lengend Snippet: Susceptibility of flow cell biofilms to DNase I. Representative z-reconstructions of RB50 biofilms grown under flow conditions for 6, 72, or 120h and imaged using CLSM for live GFP expressing cells (green) and eDNA stained with DDAO (red or yellow with co-localization). The image of untreated biofilms (left panels) were taken immediately prior to incubation with DNase I and the images of same biofilms treated with DNase I for 1.5h (left panels). Images shown here are representative of two independent experiments.

    Article Snippet: Briefly overnight grown culture of B. bronchiseptica was inoculated in the wells of a 96 well microtitre plate at OD600 of 0.05 followed by addition of either DNase I reaction buffer (10mM Tris-HCl, pH 7.5, 50% glycerol, 10mM MgCl2 ), or with DNase I (40 Kuntz units/ml)(Sigma or Promega) resuspended in the DNase I reaction buffer.

    Techniques: Flow Cytometry, Confocal Laser Scanning Microscopy, Expressing, Staining, Incubation

    DNase I leads to the disruption of established Bordetella biofilms grown on glass coverslips under static conditions. Biofilms were grown on glass coverslips for 48h for RB50 (A) and 96 h for Bp536 (B). The coverslips were gently rinsed followed by treatment with DNase I for either 30min or 90min. The cells were tagged with GFP and thus are green. For each micrograph, the middle panel represents the x-y plane, and the adjacent top and side panels represent the x-z and y-z planes, respectively. The images of a biofilm not treated with DNase I and treated only with DNase I buffer are also depicted. CLSM was utilized to image the biofilms.

    Journal: PLoS ONE

    Article Title: Extracellular DNA Is Essential for Maintaining Bordetella Biofilm Integrity on Abiotic Surfaces and in the Upper Respiratory Tract of Mice

    doi: 10.1371/journal.pone.0016861

    Figure Lengend Snippet: DNase I leads to the disruption of established Bordetella biofilms grown on glass coverslips under static conditions. Biofilms were grown on glass coverslips for 48h for RB50 (A) and 96 h for Bp536 (B). The coverslips were gently rinsed followed by treatment with DNase I for either 30min or 90min. The cells were tagged with GFP and thus are green. For each micrograph, the middle panel represents the x-y plane, and the adjacent top and side panels represent the x-z and y-z planes, respectively. The images of a biofilm not treated with DNase I and treated only with DNase I buffer are also depicted. CLSM was utilized to image the biofilms.

    Article Snippet: Briefly overnight grown culture of B. bronchiseptica was inoculated in the wells of a 96 well microtitre plate at OD600 of 0.05 followed by addition of either DNase I reaction buffer (10mM Tris-HCl, pH 7.5, 50% glycerol, 10mM MgCl2 ), or with DNase I (40 Kuntz units/ml)(Sigma or Promega) resuspended in the DNase I reaction buffer.

    Techniques: Confocal Laser Scanning Microscopy

    WASH promotes AHR expression in NKp46 +  ILC3s. ( a ) Expression levels of the indicated genes were examined in the indicated cells sorted from the indicated mice by RT-PCR analysis. ( b ) AHR expression levels were examined in the indicated ILC3 subsets from the indicated mice. For  a  and  b ,  n =5. ( c ) Sorted NKp46 +  ILC3 cells were subjected to ChIP assay with antibody against WASH, followed by detection of  Ahr  promoter (right panel) with different primers shown as in the left panel. ( d ) The indicated ILC3 subsets were subjected to ChIP assay with anti-WASH antibody, followed by detection of  Ahr  promoter through PCR. ( e ) The indicated ILC3 subsets were subjected to nuclear run-on assay, followed by RT-PCR analysis of  Ahr . ( f ) Vector or WASH overexpressing ILC3 subsets were subjected to nuclear run-on assay, followed by RT-PCR analysis of  Ahr . For  c – f ,  n =5. ( g ) pGL3-AHR promoter expressing NK92 cells were transfected with the indicated WASH variants with or without cytochalasin D, followed by luciferase assays (upper panel). Endogenous expression levels of AHR were examined by immunoblotting with anti-AHR antibody (lower panel). ( h )  In situ  hybridization of  Ahr  promoter and WASH in sorted NKp46 +  ILC3 cells (left panel). White arrow head denotes  Ahr  promoters colocalized with WASH. Percentages of cells with WASH colocalized with  Ahr  promoter were calculated (right panel). At least 200 NKp46 +  ILC3 cells were counted. Scale bar, 5 μm. ( i , j ) ChIP analysis of  Ahr  promoter in NKp46 +  ILC3 cells sorted from the indicated mice with antibodies against H3K9K14ac ( i ) or H3K4me3 ( j ). ( k ) NKp46 +  ILC3 nuclei of the indicated mice were treated with indicated units of DNase I. DNA was extracted and examined by PCR (lower panel). Intensities of PCR products were calculated (upper panel). ( l ) ChIP analysis of  Ahr  promoter in NKp46 +  ILC3 cells sorted from the indicated mice with anti-H3K27me3 antibody. ( m ) NKp46 +  ILC3 cells sorted from the indicated mice were subjected to nuclear run-on assay, followed by RT-PCR analysis of  Ahr . For ( i – m ),  n =9. Data are shown as means±s.d. * P

    Journal: Nature Communications

    Article Title: WASH maintains NKp46+ ILC3 cells by promoting AHR expression

    doi: 10.1038/ncomms15685

    Figure Lengend Snippet: WASH promotes AHR expression in NKp46 + ILC3s. ( a ) Expression levels of the indicated genes were examined in the indicated cells sorted from the indicated mice by RT-PCR analysis. ( b ) AHR expression levels were examined in the indicated ILC3 subsets from the indicated mice. For a and b , n =5. ( c ) Sorted NKp46 + ILC3 cells were subjected to ChIP assay with antibody against WASH, followed by detection of Ahr promoter (right panel) with different primers shown as in the left panel. ( d ) The indicated ILC3 subsets were subjected to ChIP assay with anti-WASH antibody, followed by detection of Ahr promoter through PCR. ( e ) The indicated ILC3 subsets were subjected to nuclear run-on assay, followed by RT-PCR analysis of Ahr . ( f ) Vector or WASH overexpressing ILC3 subsets were subjected to nuclear run-on assay, followed by RT-PCR analysis of Ahr . For c – f , n =5. ( g ) pGL3-AHR promoter expressing NK92 cells were transfected with the indicated WASH variants with or without cytochalasin D, followed by luciferase assays (upper panel). Endogenous expression levels of AHR were examined by immunoblotting with anti-AHR antibody (lower panel). ( h ) In situ hybridization of Ahr promoter and WASH in sorted NKp46 + ILC3 cells (left panel). White arrow head denotes Ahr promoters colocalized with WASH. Percentages of cells with WASH colocalized with Ahr promoter were calculated (right panel). At least 200 NKp46 + ILC3 cells were counted. Scale bar, 5 μm. ( i , j ) ChIP analysis of Ahr promoter in NKp46 + ILC3 cells sorted from the indicated mice with antibodies against H3K9K14ac ( i ) or H3K4me3 ( j ). ( k ) NKp46 + ILC3 nuclei of the indicated mice were treated with indicated units of DNase I. DNA was extracted and examined by PCR (lower panel). Intensities of PCR products were calculated (upper panel). ( l ) ChIP analysis of Ahr promoter in NKp46 + ILC3 cells sorted from the indicated mice with anti-H3K27me3 antibody. ( m ) NKp46 + ILC3 cells sorted from the indicated mice were subjected to nuclear run-on assay, followed by RT-PCR analysis of Ahr . For ( i – m ), n =9. Data are shown as means±s.d. * P

    Article Snippet: Then nuclei were resuspended with DNase I digestion buffer and treated with indicated units of DNase I (Sigma-Aldrich) at 37°C for 5 min. 2 × DNase I stop buffer (20 mM Tris Ph 8.0, 4 mM EDTA, 2 mM EGTA) was added to stop reactions.

    Techniques: Expressing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Nuclear Run-on Assay, Plasmid Preparation, Transfection, Luciferase, In Situ Hybridization

    WASH is required for Arid1a to activate AHR expression. ( a ) Arid1a mRNA levels in the indicated cell subsets were examined through RT-PCR. ( b , c ) ChIP analysis of Ahr promoters in NKp46 + ILC3 cells from Arid1a flox/flox and Arid1a flox/flox Rorc -Cre mice with antibodies against H3K9K14ac ( b ) or H3K4me3 ( c ). ( d ) Nuclei isolated from Arid1a flox/flox and Arid1a flox/flox Rorc -Cre NKp46 + ILC3 cells were digested by 1 unit of DNase I for the indicated times, followed by DNA extraction for PCR analysis of Ahr promoter. For b – d , n =6. ( e ) WASH silenced NK92 cells were transfected with full-length (FL) or truncated Arid1a (Δ968-1484 and Δ1935-2283), followed by luciferase assay (upper panel). Endogenous expression levels of AHR were examined by immunoblotting with anti-AHR antibody (lower panel). ( f ) NKp46 + ILC3 cells from Wash flox/flox and Wash flox/flox Ncr1 -Cre mice were in situ hybridized with probes against Ahr promoter, followed by staining with antibodies against WASH and Arid1a (upper panel). White arrow head indicates Ahr promoters colocalized with Arid1a. Percentages of cells with Arid1a colocalized Ahr promoter were calculated (lower panel). At least 200 NKp46 + ILC3 cells were counted. Scale bar, 5 μm. ( g ) NKp46 + ILC3 cells from Wash flox/flox and Wash flox/flox Ncr1 -Cre mice were subjected to ChIP assay with antibody against Arid1a, followed by PCR assay of Ahr promoter. n =5. ( h ) NKp46 + ILC3 cells from Wash flox/flox and Wash flox/flox Ncr1 -Cre mice were subjected to ChIP assay with antibodies against the indicated BAF components, followed by PCR assay of Ahr promoter. n =5. ( i ) Control and WASH silenced NK92 cells were transfected with the indicated Arid1a plasmids, followed by ChIP of Ahr promoter with antibody against Flag. Data are shown as means±s.d. * P

    Journal: Nature Communications

    Article Title: WASH maintains NKp46+ ILC3 cells by promoting AHR expression

    doi: 10.1038/ncomms15685

    Figure Lengend Snippet: WASH is required for Arid1a to activate AHR expression. ( a ) Arid1a mRNA levels in the indicated cell subsets were examined through RT-PCR. ( b , c ) ChIP analysis of Ahr promoters in NKp46 + ILC3 cells from Arid1a flox/flox and Arid1a flox/flox Rorc -Cre mice with antibodies against H3K9K14ac ( b ) or H3K4me3 ( c ). ( d ) Nuclei isolated from Arid1a flox/flox and Arid1a flox/flox Rorc -Cre NKp46 + ILC3 cells were digested by 1 unit of DNase I for the indicated times, followed by DNA extraction for PCR analysis of Ahr promoter. For b – d , n =6. ( e ) WASH silenced NK92 cells were transfected with full-length (FL) or truncated Arid1a (Δ968-1484 and Δ1935-2283), followed by luciferase assay (upper panel). Endogenous expression levels of AHR were examined by immunoblotting with anti-AHR antibody (lower panel). ( f ) NKp46 + ILC3 cells from Wash flox/flox and Wash flox/flox Ncr1 -Cre mice were in situ hybridized with probes against Ahr promoter, followed by staining with antibodies against WASH and Arid1a (upper panel). White arrow head indicates Ahr promoters colocalized with Arid1a. Percentages of cells with Arid1a colocalized Ahr promoter were calculated (lower panel). At least 200 NKp46 + ILC3 cells were counted. Scale bar, 5 μm. ( g ) NKp46 + ILC3 cells from Wash flox/flox and Wash flox/flox Ncr1 -Cre mice were subjected to ChIP assay with antibody against Arid1a, followed by PCR assay of Ahr promoter. n =5. ( h ) NKp46 + ILC3 cells from Wash flox/flox and Wash flox/flox Ncr1 -Cre mice were subjected to ChIP assay with antibodies against the indicated BAF components, followed by PCR assay of Ahr promoter. n =5. ( i ) Control and WASH silenced NK92 cells were transfected with the indicated Arid1a plasmids, followed by ChIP of Ahr promoter with antibody against Flag. Data are shown as means±s.d. * P

    Article Snippet: Then nuclei were resuspended with DNase I digestion buffer and treated with indicated units of DNase I (Sigma-Aldrich) at 37°C for 5 min. 2 × DNase I stop buffer (20 mM Tris Ph 8.0, 4 mM EDTA, 2 mM EGTA) was added to stop reactions.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Mouse Assay, Isolation, DNA Extraction, Polymerase Chain Reaction, Transfection, Luciferase, In Situ, Staining

    WASH associates with Arid1a. ( a ) Yeast strain AH109 was co-transfected with Gal4 DNA-binding domain (BD)-fused WASH and Gal4 activating domain (AD)-fused Arid1a. p53 and large T antigen were introduced as a positive control. ( b ) GST-WASH was incubated with LPL lysates, followed by a GST pulldown assay. ( c ) 4 × 10 5 NKp46 + ILC3 cells (pooled from RORγt-GFP reporter mice) were lysed and immunoprecipitated with anti-WASH antibody, followed by immunoblotting with the indicated antibodies. Lysates were treated by DNase I before incubation with antibodies. 4 × 10 5 Arid1a deleted ILC3 cells sorted from Arid1a flox/flox Rorc -Cre mice were also lysed and immunoprecipitated with anti-WASH antibody for co-IP assay as a control. Experiment was repeated for two times. ( d ) Flag-tagged WT and truncated Arid1a (upper panel) were co-transfected with HA-tagged WASH into NK92 cells, followed by immunoprecipitation with antibody against Flag. Immunoprecipitates were immunoblotted with the indicated antibodies (lower panel). ( e ) NK92 cells with or without Arid1a knockdown were subjected to immunoprecipitation with antibody against WASH, followed by immunoblotting with the indicated antibodies. ( f ) Sorted NKp46 + ILC3 cells were subjected to ChIP assay with antibody against Arid1a, followed by PCR analysis with the indicated fragment primers of Ahr . ( g ) Sorted NKp46 + ILC3 cells were subjected to ChIP assay with antibodies against the indicated BAF components, followed by PCR examination with primers specific to Ahr promoter. ( h ) ILC3s were sorted from Arid1a flox/flox and Arid1a flox/flox Rorc -Cre mice, followed by immunoblotting. ( i ) NKp46 + ILC3 cells from Arid1a flox/flox and Arid1a flox/flox Rorc -Cre mice were subjected to nuclear run-on assay, followed by RT-PCR analysis of Ahr . ( j ) Arid1a overexpressing BM cells together with equal numbers of recipient BM cells were transplanted into lethally irradiated recipient mice. Vector or Arid1a overexpressing ILC3 cells were sorted from above reconstituted mice and subjected to nuclear run-on assay, followed by RT-PCR analysis of Ahr . For i and j ), n =5. Data are shown as means±s.d. * P

    Journal: Nature Communications

    Article Title: WASH maintains NKp46+ ILC3 cells by promoting AHR expression

    doi: 10.1038/ncomms15685

    Figure Lengend Snippet: WASH associates with Arid1a. ( a ) Yeast strain AH109 was co-transfected with Gal4 DNA-binding domain (BD)-fused WASH and Gal4 activating domain (AD)-fused Arid1a. p53 and large T antigen were introduced as a positive control. ( b ) GST-WASH was incubated with LPL lysates, followed by a GST pulldown assay. ( c ) 4 × 10 5 NKp46 + ILC3 cells (pooled from RORγt-GFP reporter mice) were lysed and immunoprecipitated with anti-WASH antibody, followed by immunoblotting with the indicated antibodies. Lysates were treated by DNase I before incubation with antibodies. 4 × 10 5 Arid1a deleted ILC3 cells sorted from Arid1a flox/flox Rorc -Cre mice were also lysed and immunoprecipitated with anti-WASH antibody for co-IP assay as a control. Experiment was repeated for two times. ( d ) Flag-tagged WT and truncated Arid1a (upper panel) were co-transfected with HA-tagged WASH into NK92 cells, followed by immunoprecipitation with antibody against Flag. Immunoprecipitates were immunoblotted with the indicated antibodies (lower panel). ( e ) NK92 cells with or without Arid1a knockdown were subjected to immunoprecipitation with antibody against WASH, followed by immunoblotting with the indicated antibodies. ( f ) Sorted NKp46 + ILC3 cells were subjected to ChIP assay with antibody against Arid1a, followed by PCR analysis with the indicated fragment primers of Ahr . ( g ) Sorted NKp46 + ILC3 cells were subjected to ChIP assay with antibodies against the indicated BAF components, followed by PCR examination with primers specific to Ahr promoter. ( h ) ILC3s were sorted from Arid1a flox/flox and Arid1a flox/flox Rorc -Cre mice, followed by immunoblotting. ( i ) NKp46 + ILC3 cells from Arid1a flox/flox and Arid1a flox/flox Rorc -Cre mice were subjected to nuclear run-on assay, followed by RT-PCR analysis of Ahr . ( j ) Arid1a overexpressing BM cells together with equal numbers of recipient BM cells were transplanted into lethally irradiated recipient mice. Vector or Arid1a overexpressing ILC3 cells were sorted from above reconstituted mice and subjected to nuclear run-on assay, followed by RT-PCR analysis of Ahr . For i and j ), n =5. Data are shown as means±s.d. * P

    Article Snippet: Then nuclei were resuspended with DNase I digestion buffer and treated with indicated units of DNase I (Sigma-Aldrich) at 37°C for 5 min. 2 × DNase I stop buffer (20 mM Tris Ph 8.0, 4 mM EDTA, 2 mM EGTA) was added to stop reactions.

    Techniques: Transfection, Binding Assay, Positive Control, Incubation, GST Pulldown Assay, Mouse Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Nuclear Run-on Assay, Reverse Transcription Polymerase Chain Reaction, Irradiation, Plasmid Preparation

    The amount of DNA present in the specimen was represented by the PicoGreen fluorescence intensity. Fluorescence intensities were normalized against the initial intensity and fit to a single exponential function ( R 2 > 0.98 in all cases). ( A ) The level of fluorescence intensity from the isolated nuclei exposed to 100, 300, and 700 mOsm/kg, as being digested by DNase I over time. The differences between the osmotic groups are statistically significant from t = 40 min onwards. ( B ) The DNase I digestion profile for λ -DNA in 100, 300, and 700 mOsm/kg. Only at the first 5.5 min was the fluorescence intensity of λ -DNA exposed to 100 mOsm/kg statistically higher than the other groups. Error bars show standard error.

    Journal: Biophysical Journal

    Article Title: Osmotic Challenge Drives Rapid and Reversible Chromatin Condensation in Chondrocytes

    doi: 10.1016/j.bpj.2013.01.006

    Figure Lengend Snippet: The amount of DNA present in the specimen was represented by the PicoGreen fluorescence intensity. Fluorescence intensities were normalized against the initial intensity and fit to a single exponential function ( R 2 > 0.98 in all cases). ( A ) The level of fluorescence intensity from the isolated nuclei exposed to 100, 300, and 700 mOsm/kg, as being digested by DNase I over time. The differences between the osmotic groups are statistically significant from t = 40 min onwards. ( B ) The DNase I digestion profile for λ -DNA in 100, 300, and 700 mOsm/kg. Only at the first 5.5 min was the fluorescence intensity of λ -DNA exposed to 100 mOsm/kg statistically higher than the other groups. Error bars show standard error.

    Article Snippet: To control for changes in DNase I activity due to different osmolality, 3.5 μ g λ -DNA (Sigma-Aldrich) was digested by 1 U of DNase I, at different osmolality conditions (100, 300, and 700 mOsm/kg).

    Techniques: Fluorescence, Isolation

    Confirmatory real-time PCR analysis ( a–f ) DNase-chip–identified regions were confirmed by real-time PCR for CD4 + T cells ( a,c,e ) and GM06990 cells ( b,d,f (MPSS cluster). Real-time PCR using primer sets flanking DNase-chip peaks that are present with all three DNase I concentrations ( a,b ). Real-time PCR using primers sets flanking DNase-chip peaks that are present with two out of three DNase I concentrations ( c,d ). Real-time PCR using primer sets flanking DNase-chip peaks that are present with only a single DNase I concentration ( e,f ).

    Journal: Nature methods

    Article Title: DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays

    doi: 10.1038/NMETH888

    Figure Lengend Snippet: Confirmatory real-time PCR analysis ( a–f ) DNase-chip–identified regions were confirmed by real-time PCR for CD4 + T cells ( a,c,e ) and GM06990 cells ( b,d,f (MPSS cluster). Real-time PCR using primer sets flanking DNase-chip peaks that are present with all three DNase I concentrations ( a,b ). Real-time PCR using primers sets flanking DNase-chip peaks that are present with two out of three DNase I concentrations ( c,d ). Real-time PCR using primer sets flanking DNase-chip peaks that are present with only a single DNase I concentration ( e,f ).

    Article Snippet: We used pulsed field gel electrophoresis (pulse time, 20–60 s for 18 h) to identify the three concentrations of DNase I to be used for DNase-chip (Bio-Rad).

    Techniques: Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Concentration Assay

    Location of DNase I hypersensitive sites relative to the annotated genome ( a,b ) DNase-chip peaks were mapped to ENCODE regions stratified by gene density and human-mouse sequence conservation for both CD4 + T cells ( a ) and the GM06990 lymphoblastoid cell line ( b ). ( c ) The genomic locations of DNase I hypersensitive sites (detected with at least two concentrations of DNase I) and computationally generated random controls ( n = 1,000) were compared to Gencode transcription start and end sites (within a 2-kb window), CpG islands, first introns, non-first introns, first exons, non-first exons, conserved sequences (MCS), conserved sequences minus coding exons (MCS-no-CDS). The number of DNase I hypersensitive sites at different distances (0 kb, 2 kb, 10 kb, and 25 kb) from Gencode genes was also determined. Error bars represent the entire range of values seen randomly generated mock datasets ( n = 1,000). Compared to the random controls, the locations of the DNase-chip peaks are significantly (Monte Carlo P

    Journal: Nature methods

    Article Title: DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays

    doi: 10.1038/NMETH888

    Figure Lengend Snippet: Location of DNase I hypersensitive sites relative to the annotated genome ( a,b ) DNase-chip peaks were mapped to ENCODE regions stratified by gene density and human-mouse sequence conservation for both CD4 + T cells ( a ) and the GM06990 lymphoblastoid cell line ( b ). ( c ) The genomic locations of DNase I hypersensitive sites (detected with at least two concentrations of DNase I) and computationally generated random controls ( n = 1,000) were compared to Gencode transcription start and end sites (within a 2-kb window), CpG islands, first introns, non-first introns, first exons, non-first exons, conserved sequences (MCS), conserved sequences minus coding exons (MCS-no-CDS). The number of DNase I hypersensitive sites at different distances (0 kb, 2 kb, 10 kb, and 25 kb) from Gencode genes was also determined. Error bars represent the entire range of values seen randomly generated mock datasets ( n = 1,000). Compared to the random controls, the locations of the DNase-chip peaks are significantly (Monte Carlo P

    Article Snippet: We used pulsed field gel electrophoresis (pulse time, 20–60 s for 18 h) to identify the three concentrations of DNase I to be used for DNase-chip (Bio-Rad).

    Techniques: Chromatin Immunoprecipitation, Sequencing, Generated

    Identification of cell type–specific DNase I hypersensitive sites ( a,b ) Real-time PCR was performed on both CD4 + T cells and GM06990 cells. Real-time PCR using primer sets that flank random regions of the genome or DNase-chip peaks that are present for both cell types ( a ). Real-time PCR using primer sets that flank DNase-chip peaks that are present in only CD4 + T cells (CD4) or GM06990 cells (GM; b ).

    Journal: Nature methods

    Article Title: DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays

    doi: 10.1038/NMETH888

    Figure Lengend Snippet: Identification of cell type–specific DNase I hypersensitive sites ( a,b ) Real-time PCR was performed on both CD4 + T cells and GM06990 cells. Real-time PCR using primer sets that flank random regions of the genome or DNase-chip peaks that are present for both cell types ( a ). Real-time PCR using primer sets that flank DNase-chip peaks that are present in only CD4 + T cells (CD4) or GM06990 cells (GM; b ).

    Article Snippet: We used pulsed field gel electrophoresis (pulse time, 20–60 s for 18 h) to identify the three concentrations of DNase I to be used for DNase-chip (Bio-Rad).

    Techniques: Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

    Expression of genes relative to proximity to DNase I hypersensitive sites ( a,b ) The distance of each transcription start site (blue dots) to the nearest DNase I hypersensitive site was compared to the gene expression values of each transcript for both CD4 + T cells ( a ) and the GM06990 lymphoblastoid cell line ( b ). Horizontal red lines mark the expression level that separates most genes that have a DNase I hypersensitive site nearby (

    Journal: Nature methods

    Article Title: DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays

    doi: 10.1038/NMETH888

    Figure Lengend Snippet: Expression of genes relative to proximity to DNase I hypersensitive sites ( a,b ) The distance of each transcription start site (blue dots) to the nearest DNase I hypersensitive site was compared to the gene expression values of each transcript for both CD4 + T cells ( a ) and the GM06990 lymphoblastoid cell line ( b ). Horizontal red lines mark the expression level that separates most genes that have a DNase I hypersensitive site nearby (

    Article Snippet: We used pulsed field gel electrophoresis (pulse time, 20–60 s for 18 h) to identify the three concentrations of DNase I to be used for DNase-chip (Bio-Rad).

    Techniques: Expressing

    DNase-chip protocol ( a ) Pulsed field gel electrophoresis of DNase I–digested nuclear DNA. The concentrations of DNase I used for DNase-chip are labeled as A, B and C. ( b ) Outline of DNase-chip protocol. ( c ) Histogram of signal ratios of DNase I–treated versus random-sheared DNA. Tiled oligos that displayed the top 5% ratios are located to the right of the red bar. ( d ) Identification of regions with significant P values. The raw ratio data are plotted in gray, with the y-axis label on the right; the top 5% cutoff is displayed as a dotted horizontal gray line. The P value data for sliding 500-bp windows are plotted in red, with the y -axis label on the left.

    Journal: Nature methods

    Article Title: DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays

    doi: 10.1038/NMETH888

    Figure Lengend Snippet: DNase-chip protocol ( a ) Pulsed field gel electrophoresis of DNase I–digested nuclear DNA. The concentrations of DNase I used for DNase-chip are labeled as A, B and C. ( b ) Outline of DNase-chip protocol. ( c ) Histogram of signal ratios of DNase I–treated versus random-sheared DNA. Tiled oligos that displayed the top 5% ratios are located to the right of the red bar. ( d ) Identification of regions with significant P values. The raw ratio data are plotted in gray, with the y-axis label on the right; the top 5% cutoff is displayed as a dotted horizontal gray line. The P value data for sliding 500-bp windows are plotted in red, with the y -axis label on the left.

    Article Snippet: We used pulsed field gel electrophoresis (pulse time, 20–60 s for 18 h) to identify the three concentrations of DNase I to be used for DNase-chip (Bio-Rad).

    Techniques: Chromatin Immunoprecipitation, Pulsed-Field Gel, Electrophoresis, Labeling

    TALEs facilitate displacement of a positioned nucleosome on the IL-2 promoter. (A) Proposed TALE mechanism upon binding to the IL-2 promoter (not drawn to scale). Top panel is an illustration of the proposed chromatin structure of the IL-2 promoter in the absence of TALE activators. The approximate location of response elements adapted from previously published data [31] . Circle represents a positioned nucleosome located approximately 60 to 200 bp upstream of the TSS [31] . Boxed enclosed ‘T’ represents the TATA box. Bottom panel indicates location of TALE activators relative to TSS and proposed mechanism of action on the IL-2 promoter. IL-2 specific primers were used to probe various regions across the IL-2 promoter. Arrows indicate the approximate location of each region amplified by their corresponding primer set. All TALE activators were fused to VP64 (activation domain colored green) with the exception of IL2D’, which was fused to TBP (activation domain colored red). (B) Results of chromatin analysis using CHART-PCR. Percent accessibility was calculated as described in Methods and plotted for both empty vector control (black bars) and TALE activators (shaded bars). GAPDH used as internal control to monitor DNase I digestion. Results shown are of three independent experiments (error bars, mean +/− SD, n = 3). Statistical analysis was determined using one tailed, Welch’s t -test (P

    Journal: PLoS ONE

    Article Title: Activation of Silenced Cytokine Gene Promoters by the Synergistic Effect of TBP-TALE and VP64-TALE Activators

    doi: 10.1371/journal.pone.0095790

    Figure Lengend Snippet: TALEs facilitate displacement of a positioned nucleosome on the IL-2 promoter. (A) Proposed TALE mechanism upon binding to the IL-2 promoter (not drawn to scale). Top panel is an illustration of the proposed chromatin structure of the IL-2 promoter in the absence of TALE activators. The approximate location of response elements adapted from previously published data [31] . Circle represents a positioned nucleosome located approximately 60 to 200 bp upstream of the TSS [31] . Boxed enclosed ‘T’ represents the TATA box. Bottom panel indicates location of TALE activators relative to TSS and proposed mechanism of action on the IL-2 promoter. IL-2 specific primers were used to probe various regions across the IL-2 promoter. Arrows indicate the approximate location of each region amplified by their corresponding primer set. All TALE activators were fused to VP64 (activation domain colored green) with the exception of IL2D’, which was fused to TBP (activation domain colored red). (B) Results of chromatin analysis using CHART-PCR. Percent accessibility was calculated as described in Methods and plotted for both empty vector control (black bars) and TALE activators (shaded bars). GAPDH used as internal control to monitor DNase I digestion. Results shown are of three independent experiments (error bars, mean +/− SD, n = 3). Statistical analysis was determined using one tailed, Welch’s t -test (P

    Article Snippet: To determine percent accessibility to DNase I digestion, the EpiQ chromatin analysis tool (Bio-Rad) was used to analyze each sample using the following equation: % Accessibility to DNase I = (1 − 2ΔCt ) × 100.

    Techniques: Binding Assay, Amplification, Activation Assay, Polymerase Chain Reaction, Plasmid Preparation, One-tailed Test

    Analysis of the Mur34 binding site by DNase I footprinting assay. (A) Analysis of antisense strand γ- 32 P labeled DNA (left) and the sense strand γ- 32 P labeled DNA (right) upstream of mur33 . Lanes G (1), A (2), T (3) and C (4) are sequencing ladder. Samples from lands 5–10 contain the same amount of the binding DNA with an increasing amount (0–3.2 µg µl -1 ) of purified His 6 Mur34. The complexes from the samples were digested by DNase I (0.004U per10 µl) at 30°C for 1 min. The vertical sequences to the right of each gel picture indicate the DNA regions protected from the cleavage of DNase I. The transcription start point (TSP) was shown for each DNA strand. (B) “G” indicates the TSP. The sequences underlined were the protected regions by His 6 Mur34 under DNase I, “CAC” indicates the translation initiation codon (TIC), the bold regions upstream of TSP are -10 “TGATAT” and -35 “GTAAAACAG” regions. The bases in the boxes found are palindromes, and the bold and underlined bases near the TIC are supposed to be the Shine-Dalgarno consensus.

    Journal: PLoS ONE

    Article Title: Identification of Mur34 as the Novel Negative Regulator Responsible for the Biosynthesis of Muraymycin in Streptomyces sp. NRRL30471

    doi: 10.1371/journal.pone.0076068

    Figure Lengend Snippet: Analysis of the Mur34 binding site by DNase I footprinting assay. (A) Analysis of antisense strand γ- 32 P labeled DNA (left) and the sense strand γ- 32 P labeled DNA (right) upstream of mur33 . Lanes G (1), A (2), T (3) and C (4) are sequencing ladder. Samples from lands 5–10 contain the same amount of the binding DNA with an increasing amount (0–3.2 µg µl -1 ) of purified His 6 Mur34. The complexes from the samples were digested by DNase I (0.004U per10 µl) at 30°C for 1 min. The vertical sequences to the right of each gel picture indicate the DNA regions protected from the cleavage of DNase I. The transcription start point (TSP) was shown for each DNA strand. (B) “G” indicates the TSP. The sequences underlined were the protected regions by His 6 Mur34 under DNase I, “CAC” indicates the translation initiation codon (TIC), the bold regions upstream of TSP are -10 “TGATAT” and -35 “GTAAAACAG” regions. The bases in the boxes found are palindromes, and the bold and underlined bases near the TIC are supposed to be the Shine-Dalgarno consensus.

    Article Snippet: For binding site analysis, the reaction mixture contained 500 cps 32 P-lablelled DNA fragments (50 nM), after the binding of protein with DNA, the reaction mixture was incubated in ice bath for 5 min prior to addition of 2.5 µl DNase I buffer and 0.3 U of DNase I (Fermentas), then was carried out for further incubation at 30°C for 1 min.

    Techniques: Binding Assay, Footprinting, Labeling, Sequencing, Purification

    Gene expression analysis of the  mur  genes. (A) Transcription analysis of intergenic region of the selected  mur  genes. Top, ethidium bromide-stained agarose gels showing RT-PCR fragments from intergenic regions.  mur10 ← mur11  means that the detected region between  mur10  and  mur11 , and the arrows showed the possible orientation of transcription. In each gel, the left band was positive control using genomic DNA as template, the middle band showed the PCR sample using cDNA as template, the right band is negative control using template from total RNA sample digested with DNase I. (B) Time course of the transcription difference of  mur11  and  mur27  for DM-5 and the wild type strain. (C). The transcription difference of DM-5 and the wild type strain for 96 h incubation was used for the comparative analysis.

    Journal: PLoS ONE

    Article Title: Identification of Mur34 as the Novel Negative Regulator Responsible for the Biosynthesis of Muraymycin in Streptomyces sp. NRRL30471

    doi: 10.1371/journal.pone.0076068

    Figure Lengend Snippet: Gene expression analysis of the mur genes. (A) Transcription analysis of intergenic region of the selected mur genes. Top, ethidium bromide-stained agarose gels showing RT-PCR fragments from intergenic regions. mur10 ← mur11 means that the detected region between mur10 and mur11 , and the arrows showed the possible orientation of transcription. In each gel, the left band was positive control using genomic DNA as template, the middle band showed the PCR sample using cDNA as template, the right band is negative control using template from total RNA sample digested with DNase I. (B) Time course of the transcription difference of mur11 and mur27 for DM-5 and the wild type strain. (C). The transcription difference of DM-5 and the wild type strain for 96 h incubation was used for the comparative analysis.

    Article Snippet: For binding site analysis, the reaction mixture contained 500 cps 32 P-lablelled DNA fragments (50 nM), after the binding of protein with DNA, the reaction mixture was incubated in ice bath for 5 min prior to addition of 2.5 µl DNase I buffer and 0.3 U of DNase I (Fermentas), then was carried out for further incubation at 30°C for 1 min.

    Techniques: Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Positive Control, Polymerase Chain Reaction, Negative Control, Incubation

    Effect of the K297R mutation on progeny virus release. iSLK-BAC16 and iSLK-BAC-K297R cells were induced with Dox and butyrate for indicated time. The extracellular virions were collected from culture media and treated with Turbo DNase I. Viral DNAs were extracted and KSHV genomic DNA copy numbers were estimated by qPCR along with external standards of known concentrations of the viral DNA with primers against the ORF73 gene (A). Intracellular KSHV genomic DNAs were extracted from harvested cells and quantitated by qPCR as above (B). (*,  p

    Journal: PLoS Pathogens

    Article Title: Mono-ubiquitylated ORF45 Mediates Association of KSHV Particles with Internal Lipid Rafts for Viral Assembly and Egress

    doi: 10.1371/journal.ppat.1005332

    Figure Lengend Snippet: Effect of the K297R mutation on progeny virus release. iSLK-BAC16 and iSLK-BAC-K297R cells were induced with Dox and butyrate for indicated time. The extracellular virions were collected from culture media and treated with Turbo DNase I. Viral DNAs were extracted and KSHV genomic DNA copy numbers were estimated by qPCR along with external standards of known concentrations of the viral DNA with primers against the ORF73 gene (A). Intracellular KSHV genomic DNAs were extracted from harvested cells and quantitated by qPCR as above (B). (*, p

    Article Snippet: For the preparation of DNA from intact virions, 200 μl of virus stocks was pretreated with 2 μl of Turbo DNase I (Ambion) for 1 h at 37°C.

    Techniques: Mutagenesis, BAC Assay, Real-time Polymerase Chain Reaction

    Majority of indels is present between both cleavage sites after editing with Cas9 nickase. Distance of the start position of each insertion or deletion to the theoretical cleavage site (=3 bp upstream of the PAM sequence) is presented on the x-axis, while the relative frequency of the indel is presented on the y-axis after editing with the Cas9 nickase. Length of indels are represented by the different colors. Only 1 replicate is shown. The majority of indels is present between the two theoretical cleavage sites (indicated by the black lines at position 0 (sgRNA1) and at positions 44 bp ( TUNA ), 25 bp ( EMX1 ) and 38 bp ( MEG3 ). Percentages of indels 1) within or spanning both the two cleavage sites and 2) indels spanning at least one of the two sites are shown below the graph. Replicate 2 of EMX1 has an editing efficiency of 2.7% (compared to 5% for replicate 1), which can explain the lower percentage of indels located between the two cleavage sites.

    Journal: Scientific Reports

    Article Title: CRISPR/Cas9-mediated genome editing in naïve human embryonic stem cells

    doi: 10.1038/s41598-017-16932-y

    Figure Lengend Snippet: Majority of indels is present between both cleavage sites after editing with Cas9 nickase. Distance of the start position of each insertion or deletion to the theoretical cleavage site (=3 bp upstream of the PAM sequence) is presented on the x-axis, while the relative frequency of the indel is presented on the y-axis after editing with the Cas9 nickase. Length of indels are represented by the different colors. Only 1 replicate is shown. The majority of indels is present between the two theoretical cleavage sites (indicated by the black lines at position 0 (sgRNA1) and at positions 44 bp ( TUNA ), 25 bp ( EMX1 ) and 38 bp ( MEG3 ). Percentages of indels 1) within or spanning both the two cleavage sites and 2) indels spanning at least one of the two sites are shown below the graph. Replicate 2 of EMX1 has an editing efficiency of 2.7% (compared to 5% for replicate 1), which can explain the lower percentage of indels located between the two cleavage sites.

    Article Snippet: Cas9 nuclease The two sgRNAs used with the Cas9 nickase were individually cloned into the pX330 plasmid (Addgene Plasmid #42230).

    Techniques: Sequencing