genomic dna Search Results


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  • 92
    Zymo Research gdna
    Gdna, supplied by Zymo Research, used in various techniques. Bioz Stars score: 92/100, based on 1349 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher genomic dna
    Genomic Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 48727 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen genomic dna
    Genomic Dna, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 127055 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega wizard genomic dna purification kit
    Neutralizing the cation chelating activity of the <t>DNA</t> backbone of NETs protects bacteria. ( A ) Percent survival of P. <t>aeruginosa</t> PAO1 and E. coli DH5α as determined by direct plate counts (CFU/ml) before and after 4 hour incubation with PMA-activated neutrophils or combined treatment of NETS with DNase, PTase or Mg 2+ . Error bars are SEM from 6 replicates. ** or *** denotes a statistically significant difference (P
    Wizard Genomic Dna Purification Kit, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 32734 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa gdna eraser
    Neutralizing the cation chelating activity of the <t>DNA</t> backbone of NETs protects bacteria. ( A ) Percent survival of P. <t>aeruginosa</t> PAO1 and E. coli DH5α as determined by direct plate counts (CFU/ml) before and after 4 hour incubation with PMA-activated neutrophils or combined treatment of NETS with DNase, PTase or Mg 2+ . Error bars are SEM from 6 replicates. ** or *** denotes a statistically significant difference (P
    Gdna Eraser, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 27257 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc genomic dna
    <t>Paupar</t> functions in trans to modulate the activity of neurodevelopmental gene transcriptional regulatory elements. A Paupar interacts with PAX6 protein in N2A cells. Nuclear extracts were prepared from UV cross-linked cells and immuno-precipitated using either anti-PAX6 or control IgG antibodies. Associated RNAs were purified and the levels of Paupar and control U1 snRNA detected in each UV-RIP using qRT-PCR. Results are expressed as fold enrichment relative to an isotype IgG control antibody. B PAX6 and Paupar co-occupy a specific set of genomic binding sites. ChIP assays were performed in N2A cells using either an antibody against PAX6 or an isotype-specific control. The indicated <t>DNA</t> fragments were amplified using qPCR. Fold enrichment was calculated as 2 −ΔΔ Ct (IP/IgG). C, D Paupar binding sites act as transcriptional regulatory elements. N2A cells were transfected with the indicated reporter constructs in a luciferase assay. Luciferase activity was compared to that of the empty SV40 promoter construct. E–G Paupar transcript modulates the transcriptional activity of its binding sites in trans . Luciferase reporters were co-transfected into N2A cells together with either a non-targeting control or two independent Paupar targeting shRNA expression vectors. Paupar depletion was confirmed using qRT-PCR. For these reporter assays, a Renilla expression vector was used as a transfection control and the total amount of DNA transfected in each case was made equal. Data information: Results are presented as mean values ± s.e., n = 3 (A–D) or n = 4 (E–G); *** P
    Genomic Dna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 93/100, based on 12613 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore gdna
    <t>Paupar</t> functions in trans to modulate the activity of neurodevelopmental gene transcriptional regulatory elements. A Paupar interacts with PAX6 protein in N2A cells. Nuclear extracts were prepared from UV cross-linked cells and immuno-precipitated using either anti-PAX6 or control IgG antibodies. Associated RNAs were purified and the levels of Paupar and control U1 snRNA detected in each UV-RIP using qRT-PCR. Results are expressed as fold enrichment relative to an isotype IgG control antibody. B PAX6 and Paupar co-occupy a specific set of genomic binding sites. ChIP assays were performed in N2A cells using either an antibody against PAX6 or an isotype-specific control. The indicated <t>DNA</t> fragments were amplified using qPCR. Fold enrichment was calculated as 2 −ΔΔ Ct (IP/IgG). C, D Paupar binding sites act as transcriptional regulatory elements. N2A cells were transfected with the indicated reporter constructs in a luciferase assay. Luciferase activity was compared to that of the empty SV40 promoter construct. E–G Paupar transcript modulates the transcriptional activity of its binding sites in trans . Luciferase reporters were co-transfected into N2A cells together with either a non-targeting control or two independent Paupar targeting shRNA expression vectors. Paupar depletion was confirmed using qRT-PCR. For these reporter assays, a Renilla expression vector was used as a transfection control and the total amount of DNA transfected in each case was made equal. Data information: Results are presented as mean values ± s.e., n = 3 (A–D) or n = 4 (E–G); *** P
    Gdna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1013 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Gentra Systems genomic dna
    The Foxl2 promoter is hypomethylated in purified murine pituitary cells. <t>DNA</t> was extracted from genetically labeled gonadotropes (YFP+) or non-gonadotropes (YFP-) of adult male and female mice. The data show percent methylation of the indicated CpGs in the Foxl2 promoter as assessed by <t>pyrosequencing.</t>
    Genomic Dna, supplied by Gentra Systems, used in various techniques. Bioz Stars score: 92/100, based on 7521 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co genomic dna
    miR-29c-3p directly targets <t>DNA</t> methyltransferase 3B (DNMT3B) and miR-29c-3p levels were inversely correlated with DNMT3B protein levels. a Venn diagram displaying miR-29c-3p computationally predicted to target DNMT3B by four different prediction algorithms: TargetScan, MiRanda, Oncomir, and miRWalk. b miR-29c-3p expression was negatively correlated with DNMT3B expression in hepatocellular carcinoma (HCC) tissues. Spearman's rank test ( r = −0.751, p
    Genomic Dna, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 94/100, based on 7944 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher purelink genomic dna mini kit
    miR-29c-3p directly targets <t>DNA</t> methyltransferase 3B (DNMT3B) and miR-29c-3p levels were inversely correlated with DNMT3B protein levels. a Venn diagram displaying miR-29c-3p computationally predicted to target DNMT3B by four different prediction algorithms: TargetScan, MiRanda, Oncomir, and miRWalk. b miR-29c-3p expression was negatively correlated with DNMT3B expression in hepatocellular carcinoma (HCC) tissues. Spearman's rank test ( r = −0.751, p
    Purelink Genomic Dna Mini Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4585 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co tianamp genomic dna kit
    miR-29c-3p directly targets <t>DNA</t> methyltransferase 3B (DNMT3B) and miR-29c-3p levels were inversely correlated with DNMT3B protein levels. a Venn diagram displaying miR-29c-3p computationally predicted to target DNMT3B by four different prediction algorithms: TargetScan, MiRanda, Oncomir, and miRWalk. b miR-29c-3p expression was negatively correlated with DNMT3B expression in hepatocellular carcinoma (HCC) tissues. Spearman's rank test ( r = −0.751, p
    Tianamp Genomic Dna Kit, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 94/100, based on 4305 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega human genomic dna
    miR-29c-3p directly targets <t>DNA</t> methyltransferase 3B (DNMT3B) and miR-29c-3p levels were inversely correlated with DNMT3B protein levels. a Venn diagram displaying miR-29c-3p computationally predicted to target DNMT3B by four different prediction algorithms: TargetScan, MiRanda, Oncomir, and miRWalk. b miR-29c-3p expression was negatively correlated with DNMT3B expression in hepatocellular carcinoma (HCC) tissues. Spearman's rank test ( r = −0.751, p
    Human Genomic Dna, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 7262 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MACHEREY NAGEL genomic dna
    miR-29c-3p directly targets <t>DNA</t> methyltransferase 3B (DNMT3B) and miR-29c-3p levels were inversely correlated with DNMT3B protein levels. a Venn diagram displaying miR-29c-3p computationally predicted to target DNMT3B by four different prediction algorithms: TargetScan, MiRanda, Oncomir, and miRWalk. b miR-29c-3p expression was negatively correlated with DNMT3B expression in hepatocellular carcinoma (HCC) tissues. Spearman's rank test ( r = −0.751, p
    Genomic Dna, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 92/100, based on 4748 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore genelute bacterial genomic dna kit
    miR-29c-3p directly targets <t>DNA</t> methyltransferase 3B (DNMT3B) and miR-29c-3p levels were inversely correlated with DNMT3B protein levels. a Venn diagram displaying miR-29c-3p computationally predicted to target DNMT3B by four different prediction algorithms: TargetScan, MiRanda, Oncomir, and miRWalk. b miR-29c-3p expression was negatively correlated with DNMT3B expression in hepatocellular carcinoma (HCC) tissues. Spearman's rank test ( r = −0.751, p
    Genelute Bacterial Genomic Dna Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3476 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo gdna remover
    Genomic imprinting recapitulated in LCb118 knock-in mice. a Breeding scheme. In order to distinguish parental origin of the alleles by using SNPs between inbred mouse strains, endo-LCb118 homo-knock-in mice (LCb118/LCb118; C57BL/6 J [B6] background) were mated with wild-type mice ( H19 ICR/ H19 ICR; JF1/Msf [JF1]), and offspring was obtained. b – d Livers from two pairs of E18.5 embryos, each inheriting the knock-in allele either paternally (pat.; P) or maternally (mat.; M) were used for genomic DNA, total RNA, and chromatin preparations, as in Fig. 6 . b DNA methylation status of LCb118 region (the same position as in Fig. 7 a) was analyzed by bisulfite sequencing. c ChIP analysis of CTCF occupancy at the LCb118 sequence. Chromatin was immunoprecipitated using either control IgG or anti-CTCF antibodies. Following <t>qPCR</t> analyses of three distinct genomic regions ( Necdin ; negative control, endogenous H19 ICR; positive control, and LCb118), relative enrichment values (CTCF/IgG signal ratio) were calculated. The average and standard deviation (S.D.), determined by three reactions, are depicted. Statistical differences were determined using an unpaired t test (N.S., not significant). d The allele-specific expression of the Igf2 and H19 genes was examined by RFLP analysis. Igf2 and H19 gene transcripts were amplified by RT-PCR followed by BstUI or Cac8I digestions, respectively. Parental origin of transcripts was discriminated by allele-specific restriction sites. The sites were also introduced into primer sequence so that complete digestion of PCR products can be concomitantly monitored. e Schematic representation of the genomic imprinting recapitulated in the LCb118 knock-in allele. f Hypothetical model for post-fertilization methylation maintenance mechanism at the Igf2/H19 locus
    Gdna Remover, supplied by Toyobo, used in various techniques. Bioz Stars score: 93/100, based on 3213 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad genomic dna
    Pbp1 PolyQ expansions induce retrotransposon-inhibiting protein aggregation and shorten replicative lifespan. a SDD-AGE of Pbp1 variants. b SDD-AGE of Gag in Pbp1-polyQ cells. Shown below blots is multimer quantification relative to Pbp1-1Q cells. c SDD-AGE of Gag in rad27 ∆ cells. Shown below blots is multimer quantification relative to Pbp1-1Q cells. d CoIP examining interactions between Pbp1 variants and Ty1 Gag ( n = 2). e SDD-AGE of Gag in Pbp1-polyQ-expanded cells in the absence/presence of Hsp104 over-expression. Shown below blots are multimer quantification relative to Pbp1-1Q cells. f Semi-quantitative PCR products reflecting Ty1 integration upstream of 12 tRNA GLY loci in cells with Pbp1 variants and Hsp104 over-expression. TEL1 amplification served as control. g Effects of Hsp104 over-expression on Ty1 mRNA levels in cells with Pbp1 variants. Relative mRNA levels shown were detected by <t>RT-qPCR</t> (Mean ± SD; n = 3). Statistics are relative to levels detected in Pbp1-1Q cells with/without Hsp104 over-expression (one-way ANOVA and Dunnett’s post hoc). h ChIP analysis examining the localization of RNA Pol II at Ty1 in cells with Pbp1 variants and Hsp104 over-expression. Presented are relative levels of Ty1 <t>DNA</t> co-immunoprecipitating with RNA Pol II (phosphorylated serine 5) relative to input, as detected by qPCR (Mean ± SD; n = 3). Statistics are relative to levels detected in Pbp1-1Q cells with/without Hsp104 over-expression (one-way ANOVA and Dunnett’s post hoc). i Effect of Pbp1-polyQ expansion on replicative lifespan. Values are plotted as a survival curve, and mean lifespans are indicated in parentheses. Statistics are relative to Pbp1-1Q (Mann–Whitney U -test). j Effect of decreased retromobility on replicative lifespan. Values are plotted as a survival curve, and mean lifespans are indicated in parentheses. Statistics are relative to Pbp1-1Q (Mann–Whitney U -test). a – j * p
    Genomic Dna, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 3921 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher genejet genomic dna purification kit
    Pbp1 PolyQ expansions induce retrotransposon-inhibiting protein aggregation and shorten replicative lifespan. a SDD-AGE of Pbp1 variants. b SDD-AGE of Gag in Pbp1-polyQ cells. Shown below blots is multimer quantification relative to Pbp1-1Q cells. c SDD-AGE of Gag in rad27 ∆ cells. Shown below blots is multimer quantification relative to Pbp1-1Q cells. d CoIP examining interactions between Pbp1 variants and Ty1 Gag ( n = 2). e SDD-AGE of Gag in Pbp1-polyQ-expanded cells in the absence/presence of Hsp104 over-expression. Shown below blots are multimer quantification relative to Pbp1-1Q cells. f Semi-quantitative PCR products reflecting Ty1 integration upstream of 12 tRNA GLY loci in cells with Pbp1 variants and Hsp104 over-expression. TEL1 amplification served as control. g Effects of Hsp104 over-expression on Ty1 mRNA levels in cells with Pbp1 variants. Relative mRNA levels shown were detected by <t>RT-qPCR</t> (Mean ± SD; n = 3). Statistics are relative to levels detected in Pbp1-1Q cells with/without Hsp104 over-expression (one-way ANOVA and Dunnett’s post hoc). h ChIP analysis examining the localization of RNA Pol II at Ty1 in cells with Pbp1 variants and Hsp104 over-expression. Presented are relative levels of Ty1 <t>DNA</t> co-immunoprecipitating with RNA Pol II (phosphorylated serine 5) relative to input, as detected by qPCR (Mean ± SD; n = 3). Statistics are relative to levels detected in Pbp1-1Q cells with/without Hsp104 over-expression (one-way ANOVA and Dunnett’s post hoc). i Effect of Pbp1-polyQ expansion on replicative lifespan. Values are plotted as a survival curve, and mean lifespans are indicated in parentheses. Statistics are relative to Pbp1-1Q (Mann–Whitney U -test). j Effect of decreased retromobility on replicative lifespan. Values are plotted as a survival curve, and mean lifespans are indicated in parentheses. Statistics are relative to Pbp1-1Q (Mann–Whitney U -test). a – j * p
    Genejet Genomic Dna Purification Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2328 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies genomic dna
    REF-1 assay. ( A ) HCT116 nuclear extract was incubated with 32 P-labeled consensus (CON) or mutant (MUT) AP-1 oligonucleotide substrates, and binding reactions were resolved on a non-denaturing polyacrylamide gel. Control reactions without nuclear extract (no extract) are shown. The arrow designates the position of the AP-1-specific consensus binding complex, not seen with the MUT double-stranded <t>DNA.</t> Higher molecular weight non-specific complexes are observed. ( B ) Reduced wild-type (WT) or variant <t>APE1</t> protein was incubated with HCT116 nuclear extract in the presence of the 32 P-labeled AP-1 CON DNA substrate. Shown is the AP-1-specific complex in the absence (no protein) or presence of the indicated reduced APE1 protein after phosphorimager analysis. Plotted is the relative AP-1 DNA binding activity, in comparison with reduced WT protein. Values represent the average and standard deviation of 3 independent experimental points.
    Genomic Dna, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 3133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore genelute mammalian genomic dna miniprep kit
    REF-1 assay. ( A ) HCT116 nuclear extract was incubated with 32 P-labeled consensus (CON) or mutant (MUT) AP-1 oligonucleotide substrates, and binding reactions were resolved on a non-denaturing polyacrylamide gel. Control reactions without nuclear extract (no extract) are shown. The arrow designates the position of the AP-1-specific consensus binding complex, not seen with the MUT double-stranded <t>DNA.</t> Higher molecular weight non-specific complexes are observed. ( B ) Reduced wild-type (WT) or variant <t>APE1</t> protein was incubated with HCT116 nuclear extract in the presence of the 32 P-labeled AP-1 CON DNA substrate. Shown is the AP-1-specific complex in the absence (no protein) or presence of the indicated reduced APE1 protein after phosphorimager analysis. Plotted is the relative AP-1 DNA binding activity, in comparison with reduced WT protein. Values represent the average and standard deviation of 3 independent experimental points.
    Genelute Mammalian Genomic Dna Miniprep Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2601 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co plant genomic dna kit
    Retention of <t>TYLCV</t> <t>DNA</t> by B. tabaci Q females after transfer to nonhost (cotton) plants as affected by previous exposure of the whiteflies to TSWV. TSWV + or TSWV - : B. tabaci Q females that had a 72-h AAP on TSWV-infected pepper plants or on noninfected pepper plants, respectively, before they fed on TYLCV-infected tomato plants. The B. tabaci Q used for TSWV + were assumed to contain TSWV based on Table 1 . (A) Relative quantity of TYLCV retained. (B) Rate at which TYLCV titer decreased in B. tabaci Q females. Values are means ± SE. Asterisks indicate significant differences between TSWV + vs. TSWV - treatments (one-way analysis of variance; ∗ P
    Plant Genomic Dna Kit, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 93/100, based on 1882 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gdna  (ATUM)
    92
    ATUM gdna
    Changes in the amount of proliferation markers Ki-67 and PCNA in haMSCs exposed to <t>gDNA</t> or <t>GC-DNA</t> at final concentration 50 ng/mL (FACS). (a) (A) fixed cells stained with anti-Ki-67 (FITC) antibodies. Distribution of fluorescence intensities of the cells exposed to DNAs . Background fluorescence was quantified using FITC-conjugated secondary antibodies (grey color); (B) the average of the median signal intensity of FL1 (Ki-67) in haMSC. (b) (A) fixed cells stained with anti-PCNA (FITC) antibodies. Distribution of fluorescence intensities of haMSCs exposed to DNAs . Background fluorescence was quantified using FITC-conjugated secondary antibodies (grey color). (B) The average of the median signal intensity of FL1 (PCNA). (c) The ratio of the levels of mRNA CCND1, CDKN2, and CDKN1A in cells exposed to gDNA, pBR322, or GC-DNA. The incubation time is indicated on the histogram. Data points were averaged and represented as mean ± SD for three biological replicates. ( * ) P
    Gdna, supplied by ATUM, used in various techniques. Bioz Stars score: 92/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega wizard sv genomic dna purification system
    Changes in the amount of proliferation markers Ki-67 and PCNA in haMSCs exposed to <t>gDNA</t> or <t>GC-DNA</t> at final concentration 50 ng/mL (FACS). (a) (A) fixed cells stained with anti-Ki-67 (FITC) antibodies. Distribution of fluorescence intensities of the cells exposed to DNAs . Background fluorescence was quantified using FITC-conjugated secondary antibodies (grey color); (B) the average of the median signal intensity of FL1 (Ki-67) in haMSC. (b) (A) fixed cells stained with anti-PCNA (FITC) antibodies. Distribution of fluorescence intensities of haMSCs exposed to DNAs . Background fluorescence was quantified using FITC-conjugated secondary antibodies (grey color). (B) The average of the median signal intensity of FL1 (PCNA). (c) The ratio of the levels of mRNA CCND1, CDKN2, and CDKN1A in cells exposed to gDNA, pBR322, or GC-DNA. The incubation time is indicated on the histogram. Data points were averaged and represented as mean ± SD for three biological replicates. ( * ) P
    Wizard Sv Genomic Dna Purification System, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1916 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Valiant genomic dna
    PCR confirmation of ICE Apl2 in MIDG2331ΔureC::nadV and detection of extrachromosomal ICE Apl2 in <t>MIDG3553</t> and MIDG2331ΔureC::nadV. (a) PCR products from amplification of traG (530 bp) and nadV (1.5 kb) sequences from donor MIDG3553 (lanes 2 and 6), recipient MIDG2331ΔureC::nadV prior to conjugation (lanes 3 and 7) and selected transconjugants (lanes 4, 5, 8 and 9). Lane 1, 100 bp Plus <t>DNA</t> ladder. (b) Detection of a circular intermediate form by nested PCR in MIDG3553 (lanes 2 and 5) and selected transconjugants (lanes 3, 4, 6 and 7). Nested-PCR products generated by primers IceFlo_L1_out/IceFlo_R1_out and IceFlo_L2_out/IceFlo_R2_out are labelled as product 1 (983 bp) and product 2 (360 bp), respectively. Lane 1, 100 bp Plus DNA ladder (Invitrogen).
    Genomic Dna, supplied by Valiant, used in various techniques. Bioz Stars score: 94/100, based on 1949 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Neutralizing the cation chelating activity of the DNA backbone of NETs protects bacteria. ( A ) Percent survival of P. aeruginosa PAO1 and E. coli DH5α as determined by direct plate counts (CFU/ml) before and after 4 hour incubation with PMA-activated neutrophils or combined treatment of NETS with DNase, PTase or Mg 2+ . Error bars are SEM from 6 replicates. ** or *** denotes a statistically significant difference (P

    Journal: PLoS Pathogens

    Article Title: DNA Is an Antimicrobial Component of Neutrophil Extracellular Traps

    doi: 10.1371/journal.ppat.1004593

    Figure Lengend Snippet: Neutralizing the cation chelating activity of the DNA backbone of NETs protects bacteria. ( A ) Percent survival of P. aeruginosa PAO1 and E. coli DH5α as determined by direct plate counts (CFU/ml) before and after 4 hour incubation with PMA-activated neutrophils or combined treatment of NETS with DNase, PTase or Mg 2+ . Error bars are SEM from 6 replicates. ** or *** denotes a statistically significant difference (P

    Article Snippet: Pseudomonas aeruginosa genomic DNA was purified using the Wizard Genomic DNA purification kit (Promega).

    Techniques: Activity Assay, Incubation

    Bacterial species differ in their susceptibility to killing by DNA and histones. ( A ) P. aeruginosa PAO1, S. aureus and E. coli killing assay in the presence of 0.25% DNA (w/v). PAO1 was significantly more sensitive at both eDNA concentrations relative to the highly tolerant S. aureus . ( B ) P. aeruginosa PAO1, S. aureus and E. coli killing assay in presence of 1.25 µg/mL histones. Bacterial survival was quantified after 1 and 2 hours by colony count, and statistical significance assessed by 2-tailed student t-tests. * denotes statistical differences (P

    Journal: PLoS Pathogens

    Article Title: DNA Is an Antimicrobial Component of Neutrophil Extracellular Traps

    doi: 10.1371/journal.ppat.1004593

    Figure Lengend Snippet: Bacterial species differ in their susceptibility to killing by DNA and histones. ( A ) P. aeruginosa PAO1, S. aureus and E. coli killing assay in the presence of 0.25% DNA (w/v). PAO1 was significantly more sensitive at both eDNA concentrations relative to the highly tolerant S. aureus . ( B ) P. aeruginosa PAO1, S. aureus and E. coli killing assay in presence of 1.25 µg/mL histones. Bacterial survival was quantified after 1 and 2 hours by colony count, and statistical significance assessed by 2-tailed student t-tests. * denotes statistical differences (P

    Article Snippet: Pseudomonas aeruginosa genomic DNA was purified using the Wizard Genomic DNA purification kit (Promega).

    Techniques:

    Quantification of NETosis in human neutrophils ex vivo and mouse neutrophils during an infection. ( A ) Human neutrophils were stimulated with PMA, P. aeruginosa , E. coli and S. aureus and Sytox green fluorescence was measured as an indicator of DNA release by NETosis after 1 hr stimulation. Exogenous DNase was added as a control to confirm extracellular DNA presence in NETs, indicated by a plus sign (+). Asterisks denote a significant difference in extracellular DNA release between stimulated and unstimulated neutrophils (white bar) (**P

    Journal: PLoS Pathogens

    Article Title: DNA Is an Antimicrobial Component of Neutrophil Extracellular Traps

    doi: 10.1371/journal.ppat.1004593

    Figure Lengend Snippet: Quantification of NETosis in human neutrophils ex vivo and mouse neutrophils during an infection. ( A ) Human neutrophils were stimulated with PMA, P. aeruginosa , E. coli and S. aureus and Sytox green fluorescence was measured as an indicator of DNA release by NETosis after 1 hr stimulation. Exogenous DNase was added as a control to confirm extracellular DNA presence in NETs, indicated by a plus sign (+). Asterisks denote a significant difference in extracellular DNA release between stimulated and unstimulated neutrophils (white bar) (**P

    Article Snippet: Pseudomonas aeruginosa genomic DNA was purified using the Wizard Genomic DNA purification kit (Promega).

    Techniques: Ex Vivo, Infection, Fluorescence

    Extracellular DNA exerts bactericidal activity through cation chelation-mediated disruption of the bacterial outer membrane. ( A ) Survival analysis of 1 × 10 7 CFU P. aeruginosa PAO1 coincubated with 0.125% (w/v) DNA or DNA pretreated with DNase I, PTase or 5 mM Mg 2+ . Bacterial counts were performed before (0) and after two hours treatment with DNA (2). Results are representative of three independent experiments. Error bars represent the standard deviation (SD) from eight replicates. ( B ) High, medium and low concentrations of DNase (430kU, 43kU, 4.3kU), PTase (50 U, 10 U, 1 U) or excess Mg 2+ (5 mM, 500 μM, 5 μM) leads to increased levels of protection from killing with 0.15% DNA (w/v). ( C ) Visualization of the outer membrane integrity of P. aeruginosa PAO1::OM-lipoChFP expressing an outer membrane-localized mCherry fluorescent lipoprotein [ 38 ] immediately after 2% (w/v) DNA-exposure. Insets represent increased magnification of presented micrographs. Scale bar: 10 μM. ( D ) Quantification of ChFP-rich OMV generation in the field of view from 6 representative images generated from bacteria-DNA coincubation as described in ( C ) or DNA pretreated with DNase, PTase and Mg 2+ . Error bars represent SD from 6 fields of view. ( E ) Flow cytometry of DNA-exposed P. aeruginosa PAO1 using SYTO9-PI dual staining as a measure of membrane-compromised bacteria [ 28 , 32 ]. 2.5 × 10 7 CFU P. aeruginosa PAO1 were exposed to 0.0125% DNA alone or pretreated as in ( A ) then immediately analyzed by the collection of positive events (N = 50 000) by BD LSRII. Numbers in corners represent the % of 50 000 events that fall into each quadrant gate. ( F ) Quantification of membrane-compromised, PI-stained P. aeruginosa PAO1 as measured by flow cytometry. Mean percent PI stained was derived from the average of three replicates (each with N = 50 000 for each plot) in each exposure condition as in ( E ). *** denotes a significant difference between the control and 0.125% DNA sample. ### and # denote a statistically significant difference, P

    Journal: PLoS Pathogens

    Article Title: DNA Is an Antimicrobial Component of Neutrophil Extracellular Traps

    doi: 10.1371/journal.ppat.1004593

    Figure Lengend Snippet: Extracellular DNA exerts bactericidal activity through cation chelation-mediated disruption of the bacterial outer membrane. ( A ) Survival analysis of 1 × 10 7 CFU P. aeruginosa PAO1 coincubated with 0.125% (w/v) DNA or DNA pretreated with DNase I, PTase or 5 mM Mg 2+ . Bacterial counts were performed before (0) and after two hours treatment with DNA (2). Results are representative of three independent experiments. Error bars represent the standard deviation (SD) from eight replicates. ( B ) High, medium and low concentrations of DNase (430kU, 43kU, 4.3kU), PTase (50 U, 10 U, 1 U) or excess Mg 2+ (5 mM, 500 μM, 5 μM) leads to increased levels of protection from killing with 0.15% DNA (w/v). ( C ) Visualization of the outer membrane integrity of P. aeruginosa PAO1::OM-lipoChFP expressing an outer membrane-localized mCherry fluorescent lipoprotein [ 38 ] immediately after 2% (w/v) DNA-exposure. Insets represent increased magnification of presented micrographs. Scale bar: 10 μM. ( D ) Quantification of ChFP-rich OMV generation in the field of view from 6 representative images generated from bacteria-DNA coincubation as described in ( C ) or DNA pretreated with DNase, PTase and Mg 2+ . Error bars represent SD from 6 fields of view. ( E ) Flow cytometry of DNA-exposed P. aeruginosa PAO1 using SYTO9-PI dual staining as a measure of membrane-compromised bacteria [ 28 , 32 ]. 2.5 × 10 7 CFU P. aeruginosa PAO1 were exposed to 0.0125% DNA alone or pretreated as in ( A ) then immediately analyzed by the collection of positive events (N = 50 000) by BD LSRII. Numbers in corners represent the % of 50 000 events that fall into each quadrant gate. ( F ) Quantification of membrane-compromised, PI-stained P. aeruginosa PAO1 as measured by flow cytometry. Mean percent PI stained was derived from the average of three replicates (each with N = 50 000 for each plot) in each exposure condition as in ( E ). *** denotes a significant difference between the control and 0.125% DNA sample. ### and # denote a statistically significant difference, P

    Article Snippet: Pseudomonas aeruginosa genomic DNA was purified using the Wizard Genomic DNA purification kit (Promega).

    Techniques: Activity Assay, Standard Deviation, Expressing, Generated, Flow Cytometry, Cytometry, Staining, Derivative Assay

    Pseudomonas aeruginosa responds to DNA in neutrophil extracellular traps and induces protective bacterial surface modifications. ( A ) Survival analysis of 1 × 10 7 CFU wild-type P. aeruginosa PAO1 or mutants with defects in the aminoarabinose-LPS modification ( PA3553::lux ) or in spermidine synthesis ( PA4774::lux ) after coincubation with 0.125% DNA. *** denotes a statistically significant difference between time 0 and 2 hours post coincubation with 0.125% DNA. ### denotes a statistically significant difference of P

    Journal: PLoS Pathogens

    Article Title: DNA Is an Antimicrobial Component of Neutrophil Extracellular Traps

    doi: 10.1371/journal.ppat.1004593

    Figure Lengend Snippet: Pseudomonas aeruginosa responds to DNA in neutrophil extracellular traps and induces protective bacterial surface modifications. ( A ) Survival analysis of 1 × 10 7 CFU wild-type P. aeruginosa PAO1 or mutants with defects in the aminoarabinose-LPS modification ( PA3553::lux ) or in spermidine synthesis ( PA4774::lux ) after coincubation with 0.125% DNA. *** denotes a statistically significant difference between time 0 and 2 hours post coincubation with 0.125% DNA. ### denotes a statistically significant difference of P

    Article Snippet: Pseudomonas aeruginosa genomic DNA was purified using the Wizard Genomic DNA purification kit (Promega).

    Techniques: Modification

    P. aeruginosa PAO1 is trapped by human and mouse neutrophil extracellular traps. ( A ) PMA-induced NETs trapped P. aeruginosa Tn 7 :: gfp and contained myeloperoxidase (MPO), DNA and histones when visualized by immunofluorescence with antibodies from autoimmune patient sera (see Methods ). Representative images of the NET components (left), Gfp-tagged PAO1 (middle), and the merged (right) are presented. ( B ) NETosis in the skin of mice infected with ChFP-labeled P. aeruginosa as visualized by Sytox green stained extracellular DNA structures. Scale bar: 40 μm. ( C ) Arrows indicate ChFP-labeled P. aeruginosa trapped by Sytox green-stained NETs in vivo . ( D ) ChFP-labeled P. aeruginosa is phagocytosed by neutrophils during a skin infection model. Neutrophils (blue) are visualized with anti-mouse GR-1 antibody. Scale bar: 25 μm.

    Journal: PLoS Pathogens

    Article Title: DNA Is an Antimicrobial Component of Neutrophil Extracellular Traps

    doi: 10.1371/journal.ppat.1004593

    Figure Lengend Snippet: P. aeruginosa PAO1 is trapped by human and mouse neutrophil extracellular traps. ( A ) PMA-induced NETs trapped P. aeruginosa Tn 7 :: gfp and contained myeloperoxidase (MPO), DNA and histones when visualized by immunofluorescence with antibodies from autoimmune patient sera (see Methods ). Representative images of the NET components (left), Gfp-tagged PAO1 (middle), and the merged (right) are presented. ( B ) NETosis in the skin of mice infected with ChFP-labeled P. aeruginosa as visualized by Sytox green stained extracellular DNA structures. Scale bar: 40 μm. ( C ) Arrows indicate ChFP-labeled P. aeruginosa trapped by Sytox green-stained NETs in vivo . ( D ) ChFP-labeled P. aeruginosa is phagocytosed by neutrophils during a skin infection model. Neutrophils (blue) are visualized with anti-mouse GR-1 antibody. Scale bar: 25 μm.

    Article Snippet: Pseudomonas aeruginosa genomic DNA was purified using the Wizard Genomic DNA purification kit (Promega).

    Techniques: Immunofluorescence, Mouse Assay, Infection, Labeling, Staining, In Vivo

    Paupar functions in trans to modulate the activity of neurodevelopmental gene transcriptional regulatory elements. A Paupar interacts with PAX6 protein in N2A cells. Nuclear extracts were prepared from UV cross-linked cells and immuno-precipitated using either anti-PAX6 or control IgG antibodies. Associated RNAs were purified and the levels of Paupar and control U1 snRNA detected in each UV-RIP using qRT-PCR. Results are expressed as fold enrichment relative to an isotype IgG control antibody. B PAX6 and Paupar co-occupy a specific set of genomic binding sites. ChIP assays were performed in N2A cells using either an antibody against PAX6 or an isotype-specific control. The indicated DNA fragments were amplified using qPCR. Fold enrichment was calculated as 2 −ΔΔ Ct (IP/IgG). C, D Paupar binding sites act as transcriptional regulatory elements. N2A cells were transfected with the indicated reporter constructs in a luciferase assay. Luciferase activity was compared to that of the empty SV40 promoter construct. E–G Paupar transcript modulates the transcriptional activity of its binding sites in trans . Luciferase reporters were co-transfected into N2A cells together with either a non-targeting control or two independent Paupar targeting shRNA expression vectors. Paupar depletion was confirmed using qRT-PCR. For these reporter assays, a Renilla expression vector was used as a transfection control and the total amount of DNA transfected in each case was made equal. Data information: Results are presented as mean values ± s.e., n = 3 (A–D) or n = 4 (E–G); *** P

    Journal: The EMBO Journal

    Article Title: The long non-coding RNA Paupar regulates the expression of both local and distal genes

    doi: 10.1002/embj.201386225

    Figure Lengend Snippet: Paupar functions in trans to modulate the activity of neurodevelopmental gene transcriptional regulatory elements. A Paupar interacts with PAX6 protein in N2A cells. Nuclear extracts were prepared from UV cross-linked cells and immuno-precipitated using either anti-PAX6 or control IgG antibodies. Associated RNAs were purified and the levels of Paupar and control U1 snRNA detected in each UV-RIP using qRT-PCR. Results are expressed as fold enrichment relative to an isotype IgG control antibody. B PAX6 and Paupar co-occupy a specific set of genomic binding sites. ChIP assays were performed in N2A cells using either an antibody against PAX6 or an isotype-specific control. The indicated DNA fragments were amplified using qPCR. Fold enrichment was calculated as 2 −ΔΔ Ct (IP/IgG). C, D Paupar binding sites act as transcriptional regulatory elements. N2A cells were transfected with the indicated reporter constructs in a luciferase assay. Luciferase activity was compared to that of the empty SV40 promoter construct. E–G Paupar transcript modulates the transcriptional activity of its binding sites in trans . Luciferase reporters were co-transfected into N2A cells together with either a non-targeting control or two independent Paupar targeting shRNA expression vectors. Paupar depletion was confirmed using qRT-PCR. For these reporter assays, a Renilla expression vector was used as a transfection control and the total amount of DNA transfected in each case was made equal. Data information: Results are presented as mean values ± s.e., n = 3 (A–D) or n = 4 (E–G); *** P

    Article Snippet: Following the CHART-seq protocol, we used RNase H elution to recover genomic DNA associated with endogenous Paupar transcripts and genomic DNA associated with the control oligonucleotide, sequencing replicate samples using the Illumina HiSeq system.

    Techniques: Activity Assay, Purification, Quantitative RT-PCR, Binding Assay, Chromatin Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Activated Clotting Time Assay, Transfection, Construct, Luciferase, shRNA, Expressing, Plasmid Preparation

    Conservation and expression of Paupar. A Schematic illustration of the mouse Pax6 genomic territory displaying coding and non-coding transcript structures (NCBI37/mm9). B A detailed view of the mouse Paupar locus (red) indicating regions of vertebrate DNA sequence conservation and the location of sequence (blue) that, in human and quail, is a Pax6 neuroretina enhancer (Plaza et al , 1999 ). C Conservation and relative sizes of identified Paupar transcripts in vertebrates. For human and mouse Paupar , transcript start sites (arrows) and transcript ends were confirmed by RACE (primer sequences in Supplementary Table 1). The identified orthologous ESTs from dog (DN871729), frog (CX414799, DN054151 and DN054152), and zebrafish (CT684153 and CT684154) are unlikely to represent full-length transcripts. Each of these Paupar orthologues displays conserved genomic location and transcriptional orientation relative to Pax6 . D, E Paupar is a brain-expressed lncRNA. Paupar (D) and Pax6 (E) expression levels were measured across a panel of adult mouse tissues using quantitative RT-PCR (qRT-PCR). Results are presented relative to the average value of Gapdh and Tbp reference genes. Mean values ± standard error (s.e.) shown, n = 3 replicates. F, G Similarly to Pax6 , Paupar is up-regulated during neuronal differentiation of mouse ES cells. Neuronal differentiation of mouse ES cells was induced using RA. We determined the levels of Paupar (F) and Pax6 (G) using qRT-PCR. Results are expressed relative to an Idh1 control which does not change significantly during differentiation. Mean ± s.e., n = 3. H, I Paupar is a chromatin-associated transcript that functions to regulate Pax6 expression. N2A cells were biochemically separated into cytoplasmic, nucleoplasm, 420 mM salt and chromatin fractions. The relative levels of Paupar (H) and a control mRNA ( Tbp ) (I) in each fraction were determined by qRT-PCR. Mean values ± s.e. of three independent experiments. RT, reverse transcriptase.

    Journal: The EMBO Journal

    Article Title: The long non-coding RNA Paupar regulates the expression of both local and distal genes

    doi: 10.1002/embj.201386225

    Figure Lengend Snippet: Conservation and expression of Paupar. A Schematic illustration of the mouse Pax6 genomic territory displaying coding and non-coding transcript structures (NCBI37/mm9). B A detailed view of the mouse Paupar locus (red) indicating regions of vertebrate DNA sequence conservation and the location of sequence (blue) that, in human and quail, is a Pax6 neuroretina enhancer (Plaza et al , 1999 ). C Conservation and relative sizes of identified Paupar transcripts in vertebrates. For human and mouse Paupar , transcript start sites (arrows) and transcript ends were confirmed by RACE (primer sequences in Supplementary Table 1). The identified orthologous ESTs from dog (DN871729), frog (CX414799, DN054151 and DN054152), and zebrafish (CT684153 and CT684154) are unlikely to represent full-length transcripts. Each of these Paupar orthologues displays conserved genomic location and transcriptional orientation relative to Pax6 . D, E Paupar is a brain-expressed lncRNA. Paupar (D) and Pax6 (E) expression levels were measured across a panel of adult mouse tissues using quantitative RT-PCR (qRT-PCR). Results are presented relative to the average value of Gapdh and Tbp reference genes. Mean values ± standard error (s.e.) shown, n = 3 replicates. F, G Similarly to Pax6 , Paupar is up-regulated during neuronal differentiation of mouse ES cells. Neuronal differentiation of mouse ES cells was induced using RA. We determined the levels of Paupar (F) and Pax6 (G) using qRT-PCR. Results are expressed relative to an Idh1 control which does not change significantly during differentiation. Mean ± s.e., n = 3. H, I Paupar is a chromatin-associated transcript that functions to regulate Pax6 expression. N2A cells were biochemically separated into cytoplasmic, nucleoplasm, 420 mM salt and chromatin fractions. The relative levels of Paupar (H) and a control mRNA ( Tbp ) (I) in each fraction were determined by qRT-PCR. Mean values ± s.e. of three independent experiments. RT, reverse transcriptase.

    Article Snippet: Following the CHART-seq protocol, we used RNase H elution to recover genomic DNA associated with endogenous Paupar transcripts and genomic DNA associated with the control oligonucleotide, sequencing replicate samples using the Illumina HiSeq system.

    Techniques: Expressing, Sequencing, Quantitative RT-PCR

    CHART-Seq analysis of Paupar genomic binding sites. A Specific enrichment of Paupar RNA using oligonucleotides complementary to accessible regions of Paupar , as determined by RNase H mapping (see Supplementary Fig S4), compared to the LacZ control. Mean value ± s.e., n = 4. B Sequencing of Paupar -bound DNA (RNase H elution) reveals peaks of Paupar binding, including those at the promoter of E2f2 , upstream of Sox2 and downstream of Hes1 . C–E Peaks were called by comparing with sequences both from control CHART-seq experiments and from input DNA. Here we only consider the 2,849 peaks common to both comparisons (C, and Supplementary Table 4). Paupar peaks are broadly distributed across the mouse genome (D) but are particularly enriched in 5′ UTRs and gene promoters (E). Red arrowheads in (D) indicate the position of the Paupar locus. Asterisks in (E) indicate significance at 5% FDR (Benjamini-Hochberg). F The width distribution of Paupar binding peaks. G Representative categories from Gene Ontology analysis of genes associated with Paupar binding sites reveal enrichments for stem cell and neuronal categories amongst others (Supplementary Table 6).

    Journal: The EMBO Journal

    Article Title: The long non-coding RNA Paupar regulates the expression of both local and distal genes

    doi: 10.1002/embj.201386225

    Figure Lengend Snippet: CHART-Seq analysis of Paupar genomic binding sites. A Specific enrichment of Paupar RNA using oligonucleotides complementary to accessible regions of Paupar , as determined by RNase H mapping (see Supplementary Fig S4), compared to the LacZ control. Mean value ± s.e., n = 4. B Sequencing of Paupar -bound DNA (RNase H elution) reveals peaks of Paupar binding, including those at the promoter of E2f2 , upstream of Sox2 and downstream of Hes1 . C–E Peaks were called by comparing with sequences both from control CHART-seq experiments and from input DNA. Here we only consider the 2,849 peaks common to both comparisons (C, and Supplementary Table 4). Paupar peaks are broadly distributed across the mouse genome (D) but are particularly enriched in 5′ UTRs and gene promoters (E). Red arrowheads in (D) indicate the position of the Paupar locus. Asterisks in (E) indicate significance at 5% FDR (Benjamini-Hochberg). F The width distribution of Paupar binding peaks. G Representative categories from Gene Ontology analysis of genes associated with Paupar binding sites reveal enrichments for stem cell and neuronal categories amongst others (Supplementary Table 6).

    Article Snippet: Following the CHART-seq protocol, we used RNase H elution to recover genomic DNA associated with endogenous Paupar transcripts and genomic DNA associated with the control oligonucleotide, sequencing replicate samples using the Illumina HiSeq system.

    Techniques: Binding Assay, Sequencing

    Paupar functions to regulate genes involved in cell cycle control and synaptic function. A N2A cells were transfected with either a non-targeting control or a Paupar -targeting shRNA expression vector (sh408) and Paupar levels were determined by qRT-PCR 3 days later. B Paupar knockdown induces statistically significant changes in the expression of 942 genes in N2A cells (5% FDR; Supplementary Table 2). C Significant Gene Ontology annotation enrichments of Paupar -regulated genes (5% FDR, Supplementary Table 3). D Paupar is important for normal S-phase progression and entry into mitosis. Mouse N2A cells were transfected with either a control or a Paupar -targeting shRNA expression vector. Three days later cells were fixed, stained with propidium iodide and the DNA content measured using flow cytometry. E Paupar loss-of-function cell lines were generated by stable co-transfection of shRNA expression plasmids against either Paupar or a non-targeting control and a hygromycin expression vector for selection. qRT-PCR analysis confirms the generation of two clonal cell lines expressing reduced levels of Paupar . Mean values ± s.e. F Paupar knockdown cells display increased neurite outgrowth. Control and Paupar knockdown cells were imaged using bright field microscopy. Scale bar, 50 μm. G Quantification of neurite outgrowth. Cells with one or more neurites of length greater than twice the cell body diameter were scored as positive. Average values ± s.e., n = 3. A total of 100–200 cells were counted in each case. H, I The relative levels of the neuronal differentiation marker Tubb3 (H) and Pax6 (I) were quantified in Paupar knockdown and control cells using qRT-PCR. Samples were normalised using Gapdh and are presented relative to expression in control cells (set arbitrarily to 1). Mean values ± s.e., n = 3.

    Journal: The EMBO Journal

    Article Title: The long non-coding RNA Paupar regulates the expression of both local and distal genes

    doi: 10.1002/embj.201386225

    Figure Lengend Snippet: Paupar functions to regulate genes involved in cell cycle control and synaptic function. A N2A cells were transfected with either a non-targeting control or a Paupar -targeting shRNA expression vector (sh408) and Paupar levels were determined by qRT-PCR 3 days later. B Paupar knockdown induces statistically significant changes in the expression of 942 genes in N2A cells (5% FDR; Supplementary Table 2). C Significant Gene Ontology annotation enrichments of Paupar -regulated genes (5% FDR, Supplementary Table 3). D Paupar is important for normal S-phase progression and entry into mitosis. Mouse N2A cells were transfected with either a control or a Paupar -targeting shRNA expression vector. Three days later cells were fixed, stained with propidium iodide and the DNA content measured using flow cytometry. E Paupar loss-of-function cell lines were generated by stable co-transfection of shRNA expression plasmids against either Paupar or a non-targeting control and a hygromycin expression vector for selection. qRT-PCR analysis confirms the generation of two clonal cell lines expressing reduced levels of Paupar . Mean values ± s.e. F Paupar knockdown cells display increased neurite outgrowth. Control and Paupar knockdown cells were imaged using bright field microscopy. Scale bar, 50 μm. G Quantification of neurite outgrowth. Cells with one or more neurites of length greater than twice the cell body diameter were scored as positive. Average values ± s.e., n = 3. A total of 100–200 cells were counted in each case. H, I The relative levels of the neuronal differentiation marker Tubb3 (H) and Pax6 (I) were quantified in Paupar knockdown and control cells using qRT-PCR. Samples were normalised using Gapdh and are presented relative to expression in control cells (set arbitrarily to 1). Mean values ± s.e., n = 3.

    Article Snippet: Following the CHART-seq protocol, we used RNase H elution to recover genomic DNA associated with endogenous Paupar transcripts and genomic DNA associated with the control oligonucleotide, sequencing replicate samples using the Illumina HiSeq system.

    Techniques: Transfection, shRNA, Expressing, Plasmid Preparation, Quantitative RT-PCR, Staining, Flow Cytometry, Cytometry, Generated, Cotransfection, Selection, Microscopy, Marker

    The Foxl2 promoter is hypomethylated in purified murine pituitary cells. DNA was extracted from genetically labeled gonadotropes (YFP+) or non-gonadotropes (YFP-) of adult male and female mice. The data show percent methylation of the indicated CpGs in the Foxl2 promoter as assessed by pyrosequencing.

    Journal: PLoS ONE

    Article Title: The CpG Island in the Murine Foxl2 Proximal Promoter Is Differentially Methylated in Primary and Immortalized Cells

    doi: 10.1371/journal.pone.0076642

    Figure Lengend Snippet: The Foxl2 promoter is hypomethylated in purified murine pituitary cells. DNA was extracted from genetically labeled gonadotropes (YFP+) or non-gonadotropes (YFP-) of adult male and female mice. The data show percent methylation of the indicated CpGs in the Foxl2 promoter as assessed by pyrosequencing.

    Article Snippet: CpG methylation analysis by pyrosequencing Genomic DNA from LβT2, TαT-1, and NIH3T3 cells was extracted using the Gentra Systems DNA Isolation kit and samples sent to EpigenDx (Hopkinton, MA) for CpG methylation analysis.

    Techniques: Purification, Labeling, Mouse Assay, Methylation

    The Foxl2 promoter is hypomethylated in homologous cells and tissues. A) Analysis of percent methylation of two regions of the Foxl2 promoter in genomic DNA from LβT2 and NIH3T3 cells as assessed by qAMP (white bars) and pyrosequencing (black bars). Data are from a representative of a 2 replicate experiments, which yielded comparable results. B) Percent methylation of -81/+43 of the Foxl2 promoter in genomic DNA from the indicated murine cell lines. Data are from a single experiment. C) Percent methylation of -81/+43 of the Foxl2 promoter in genomic DNA from the indicated murine tissues Data are from a representative of a 2 replicate experiments, which yielded comparable results.

    Journal: PLoS ONE

    Article Title: The CpG Island in the Murine Foxl2 Proximal Promoter Is Differentially Methylated in Primary and Immortalized Cells

    doi: 10.1371/journal.pone.0076642

    Figure Lengend Snippet: The Foxl2 promoter is hypomethylated in homologous cells and tissues. A) Analysis of percent methylation of two regions of the Foxl2 promoter in genomic DNA from LβT2 and NIH3T3 cells as assessed by qAMP (white bars) and pyrosequencing (black bars). Data are from a representative of a 2 replicate experiments, which yielded comparable results. B) Percent methylation of -81/+43 of the Foxl2 promoter in genomic DNA from the indicated murine cell lines. Data are from a single experiment. C) Percent methylation of -81/+43 of the Foxl2 promoter in genomic DNA from the indicated murine tissues Data are from a representative of a 2 replicate experiments, which yielded comparable results.

    Article Snippet: CpG methylation analysis by pyrosequencing Genomic DNA from LβT2, TαT-1, and NIH3T3 cells was extracted using the Gentra Systems DNA Isolation kit and samples sent to EpigenDx (Hopkinton, MA) for CpG methylation analysis.

    Techniques: Methylation

    The murine Foxl2 promoter is differentially methylated in homologous and heterologous cells. A) DNA sequence (-600 to +165) of the murine Foxl2 promoter. Of 62 CpG dinucleotides pictured, 51 were analyzed by pyrosequencing (shaded in grey). The transcriptional start site (+1) mapped here by 5’ RACE is indicated with an arrow. The ATG translation initiation codon is underlined. The first nucleotides of the -432 and -187 reporters are marked above the sequence with * and **, respectively. B) Percent methylation of the CpGs at the indicated positions in genomic DNA from LβT2 (white bars), TαT1 (grey bars), and NIH3T3 (black bars) cell lines as assessed by pyrosequencing.

    Journal: PLoS ONE

    Article Title: The CpG Island in the Murine Foxl2 Proximal Promoter Is Differentially Methylated in Primary and Immortalized Cells

    doi: 10.1371/journal.pone.0076642

    Figure Lengend Snippet: The murine Foxl2 promoter is differentially methylated in homologous and heterologous cells. A) DNA sequence (-600 to +165) of the murine Foxl2 promoter. Of 62 CpG dinucleotides pictured, 51 were analyzed by pyrosequencing (shaded in grey). The transcriptional start site (+1) mapped here by 5’ RACE is indicated with an arrow. The ATG translation initiation codon is underlined. The first nucleotides of the -432 and -187 reporters are marked above the sequence with * and **, respectively. B) Percent methylation of the CpGs at the indicated positions in genomic DNA from LβT2 (white bars), TαT1 (grey bars), and NIH3T3 (black bars) cell lines as assessed by pyrosequencing.

    Article Snippet: CpG methylation analysis by pyrosequencing Genomic DNA from LβT2, TαT-1, and NIH3T3 cells was extracted using the Gentra Systems DNA Isolation kit and samples sent to EpigenDx (Hopkinton, MA) for CpG methylation analysis.

    Techniques: Methylation, Sequencing

    miR-29c-3p directly targets DNA methyltransferase 3B (DNMT3B) and miR-29c-3p levels were inversely correlated with DNMT3B protein levels. a Venn diagram displaying miR-29c-3p computationally predicted to target DNMT3B by four different prediction algorithms: TargetScan, MiRanda, Oncomir, and miRWalk. b miR-29c-3p expression was negatively correlated with DNMT3B expression in hepatocellular carcinoma (HCC) tissues. Spearman's rank test ( r = −0.751, p

    Journal: Cell Death & Disease

    Article Title: miR-29c-3p regulates DNMT3B and LATS1 methylation to inhibit tumor progression in hepatocellular carcinoma

    doi: 10.1038/s41419-018-1281-7

    Figure Lengend Snippet: miR-29c-3p directly targets DNA methyltransferase 3B (DNMT3B) and miR-29c-3p levels were inversely correlated with DNMT3B protein levels. a Venn diagram displaying miR-29c-3p computationally predicted to target DNMT3B by four different prediction algorithms: TargetScan, MiRanda, Oncomir, and miRWalk. b miR-29c-3p expression was negatively correlated with DNMT3B expression in hepatocellular carcinoma (HCC) tissues. Spearman's rank test ( r = −0.751, p

    Article Snippet: Genomic DNA was isolated from HCC tumor tissues, paired normal adjacent tissues, and HCC cells using the DNA Isolation kit (Tiangen, Beijing, China) according to the manufacturer’s protocol.

    Techniques: Expressing

    DNA methyltransferase 3B (DNMT3B) is upregulated and large tumor suppressor gene 1 (LATS1) is downregulated in hepatocellular carcinoma (HCC). a Quantitative real-time PCR (qRT-PCR) analysis of DNMT3B expression in 150 pairs of HCC tissues and paired normal adjacent tissues. b qRT-PCR analysis of LATS1 expression in 150 pairs of HCC tissues and paired normal adjacent tissues. c Immunohistochemical staining analysis of DNMT3B protein expression levels in HCC tissues. d Immunohistochemical staining analysis of LATS1 protein expression levels in HCC tissues. e Kaplan–Meier analysis of overall survival between HCC patients with high and low DNMT3B expression. f Kaplan–Meier analysis of overall survival between high and low LATS1 expression in HCC patients. g Kaplan–Meier analysis of overall survival between the high miR-29c-3p/low DNMT3B/high LATS1 expression group and low miR-29c-3p/high DNMT3B/low LATS1 expression group; ** p

    Journal: Cell Death & Disease

    Article Title: miR-29c-3p regulates DNMT3B and LATS1 methylation to inhibit tumor progression in hepatocellular carcinoma

    doi: 10.1038/s41419-018-1281-7

    Figure Lengend Snippet: DNA methyltransferase 3B (DNMT3B) is upregulated and large tumor suppressor gene 1 (LATS1) is downregulated in hepatocellular carcinoma (HCC). a Quantitative real-time PCR (qRT-PCR) analysis of DNMT3B expression in 150 pairs of HCC tissues and paired normal adjacent tissues. b qRT-PCR analysis of LATS1 expression in 150 pairs of HCC tissues and paired normal adjacent tissues. c Immunohistochemical staining analysis of DNMT3B protein expression levels in HCC tissues. d Immunohistochemical staining analysis of LATS1 protein expression levels in HCC tissues. e Kaplan–Meier analysis of overall survival between HCC patients with high and low DNMT3B expression. f Kaplan–Meier analysis of overall survival between high and low LATS1 expression in HCC patients. g Kaplan–Meier analysis of overall survival between the high miR-29c-3p/low DNMT3B/high LATS1 expression group and low miR-29c-3p/high DNMT3B/low LATS1 expression group; ** p

    Article Snippet: Genomic DNA was isolated from HCC tumor tissues, paired normal adjacent tissues, and HCC cells using the DNA Isolation kit (Tiangen, Beijing, China) according to the manufacturer’s protocol.

    Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Immunohistochemistry, Staining

    Rescue experiments are performed to confirm that DNA methyltransferase 3B (DNMT3B) is the functional target of miR-29c-3p in hepatocellular carcinoma (HCC) progression. a Western blot revealed DNMT3B protein expression in MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p that were transfected with DNMT3B vector and NC. b Proliferation of MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p cells that were transfected with DNMT3B vector and negative control (NC) was determined by CCK-8 assay. c Wound healing assay was performed to determine the effects of DNMT3B on HCC cell migration. d Colony formation assays assessed the effects of DNMT3B on HCC cell proliferation. e The methylation status of large tumor suppressor gene 1 (LATS1) was detected in MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p cells that were transfected with DNMT3B vector and NC. f Western blot revealed the expression of Hippo signaling pathway components, including proliferation- and apoptosis-related indicators in MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p cells that were transfected with DNMT3B vector and NC; * p

    Journal: Cell Death & Disease

    Article Title: miR-29c-3p regulates DNMT3B and LATS1 methylation to inhibit tumor progression in hepatocellular carcinoma

    doi: 10.1038/s41419-018-1281-7

    Figure Lengend Snippet: Rescue experiments are performed to confirm that DNA methyltransferase 3B (DNMT3B) is the functional target of miR-29c-3p in hepatocellular carcinoma (HCC) progression. a Western blot revealed DNMT3B protein expression in MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p that were transfected with DNMT3B vector and NC. b Proliferation of MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p cells that were transfected with DNMT3B vector and negative control (NC) was determined by CCK-8 assay. c Wound healing assay was performed to determine the effects of DNMT3B on HCC cell migration. d Colony formation assays assessed the effects of DNMT3B on HCC cell proliferation. e The methylation status of large tumor suppressor gene 1 (LATS1) was detected in MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p cells that were transfected with DNMT3B vector and NC. f Western blot revealed the expression of Hippo signaling pathway components, including proliferation- and apoptosis-related indicators in MHCC-97H-miR-29c-3p cells and HepG2-miR-29c-3p cells that were transfected with DNMT3B vector and NC; * p

    Article Snippet: Genomic DNA was isolated from HCC tumor tissues, paired normal adjacent tissues, and HCC cells using the DNA Isolation kit (Tiangen, Beijing, China) according to the manufacturer’s protocol.

    Techniques: Functional Assay, Western Blot, Expressing, Transfection, Plasmid Preparation, Negative Control, CCK-8 Assay, Wound Healing Assay, Migration, Methylation

    Aberrant DNA hypermethylation and expression of large tumor suppressor gene 1 (LATS1) in hepatocellular carcinoma (HCC) and HCC cell lines. a The methylation status of LATS1 was randomly detected in 7 HCC and paired normal adjacent tissues. b The methylation status of LATS1 was detected in LO2, MHCC-97H, HepG2, SMMC-7721, and Huh7cell lines. c Bisulfite sequencing analysis was performed on LATS1 promoter methylation in HCC tissues compared with paired normal adjacent tissues. d The relative mRNA expression of miR-29c-3p in 7 HCC and paired normal adjacent tissues. e The relative mRNA expression of DNA methyltransferase 3B (DNMT3B) in 7 HCC and paired normal adjacent tissues. f The relative mRNA expression of LATS1 in 7 HCC and paired normal adjacent tissues. g The CpG islands (shaded area) of LATS1 promoter region. h YAP and LATS1 protein expression in HCC and paired normal adjacent tissues. i YAP and LATS1 protein expression in HCC and LO2 cells; * p

    Journal: Cell Death & Disease

    Article Title: miR-29c-3p regulates DNMT3B and LATS1 methylation to inhibit tumor progression in hepatocellular carcinoma

    doi: 10.1038/s41419-018-1281-7

    Figure Lengend Snippet: Aberrant DNA hypermethylation and expression of large tumor suppressor gene 1 (LATS1) in hepatocellular carcinoma (HCC) and HCC cell lines. a The methylation status of LATS1 was randomly detected in 7 HCC and paired normal adjacent tissues. b The methylation status of LATS1 was detected in LO2, MHCC-97H, HepG2, SMMC-7721, and Huh7cell lines. c Bisulfite sequencing analysis was performed on LATS1 promoter methylation in HCC tissues compared with paired normal adjacent tissues. d The relative mRNA expression of miR-29c-3p in 7 HCC and paired normal adjacent tissues. e The relative mRNA expression of DNA methyltransferase 3B (DNMT3B) in 7 HCC and paired normal adjacent tissues. f The relative mRNA expression of LATS1 in 7 HCC and paired normal adjacent tissues. g The CpG islands (shaded area) of LATS1 promoter region. h YAP and LATS1 protein expression in HCC and paired normal adjacent tissues. i YAP and LATS1 protein expression in HCC and LO2 cells; * p

    Article Snippet: Genomic DNA was isolated from HCC tumor tissues, paired normal adjacent tissues, and HCC cells using the DNA Isolation kit (Tiangen, Beijing, China) according to the manufacturer’s protocol.

    Techniques: Expressing, Methylation, Methylation Sequencing

    Genomic imprinting recapitulated in LCb118 knock-in mice. a Breeding scheme. In order to distinguish parental origin of the alleles by using SNPs between inbred mouse strains, endo-LCb118 homo-knock-in mice (LCb118/LCb118; C57BL/6 J [B6] background) were mated with wild-type mice ( H19 ICR/ H19 ICR; JF1/Msf [JF1]), and offspring was obtained. b – d Livers from two pairs of E18.5 embryos, each inheriting the knock-in allele either paternally (pat.; P) or maternally (mat.; M) were used for genomic DNA, total RNA, and chromatin preparations, as in Fig. 6 . b DNA methylation status of LCb118 region (the same position as in Fig. 7 a) was analyzed by bisulfite sequencing. c ChIP analysis of CTCF occupancy at the LCb118 sequence. Chromatin was immunoprecipitated using either control IgG or anti-CTCF antibodies. Following qPCR analyses of three distinct genomic regions ( Necdin ; negative control, endogenous H19 ICR; positive control, and LCb118), relative enrichment values (CTCF/IgG signal ratio) were calculated. The average and standard deviation (S.D.), determined by three reactions, are depicted. Statistical differences were determined using an unpaired t test (N.S., not significant). d The allele-specific expression of the Igf2 and H19 genes was examined by RFLP analysis. Igf2 and H19 gene transcripts were amplified by RT-PCR followed by BstUI or Cac8I digestions, respectively. Parental origin of transcripts was discriminated by allele-specific restriction sites. The sites were also introduced into primer sequence so that complete digestion of PCR products can be concomitantly monitored. e Schematic representation of the genomic imprinting recapitulated in the LCb118 knock-in allele. f Hypothetical model for post-fertilization methylation maintenance mechanism at the Igf2/H19 locus

    Journal: Epigenetics & Chromatin

    Article Title: Recapitulation of gametic DNA methylation and its post-fertilization maintenance with reassembled DNA elements at the mouse Igf2/H19 locus

    doi: 10.1186/s13072-019-0326-1

    Figure Lengend Snippet: Genomic imprinting recapitulated in LCb118 knock-in mice. a Breeding scheme. In order to distinguish parental origin of the alleles by using SNPs between inbred mouse strains, endo-LCb118 homo-knock-in mice (LCb118/LCb118; C57BL/6 J [B6] background) were mated with wild-type mice ( H19 ICR/ H19 ICR; JF1/Msf [JF1]), and offspring was obtained. b – d Livers from two pairs of E18.5 embryos, each inheriting the knock-in allele either paternally (pat.; P) or maternally (mat.; M) were used for genomic DNA, total RNA, and chromatin preparations, as in Fig. 6 . b DNA methylation status of LCb118 region (the same position as in Fig. 7 a) was analyzed by bisulfite sequencing. c ChIP analysis of CTCF occupancy at the LCb118 sequence. Chromatin was immunoprecipitated using either control IgG or anti-CTCF antibodies. Following qPCR analyses of three distinct genomic regions ( Necdin ; negative control, endogenous H19 ICR; positive control, and LCb118), relative enrichment values (CTCF/IgG signal ratio) were calculated. The average and standard deviation (S.D.), determined by three reactions, are depicted. Statistical differences were determined using an unpaired t test (N.S., not significant). d The allele-specific expression of the Igf2 and H19 genes was examined by RFLP analysis. Igf2 and H19 gene transcripts were amplified by RT-PCR followed by BstUI or Cac8I digestions, respectively. Parental origin of transcripts was discriminated by allele-specific restriction sites. The sites were also introduced into primer sequence so that complete digestion of PCR products can be concomitantly monitored. e Schematic representation of the genomic imprinting recapitulated in the LCb118 knock-in allele. f Hypothetical model for post-fertilization methylation maintenance mechanism at the Igf2/H19 locus

    Article Snippet: RT-qPCR Total RNA was recovered from phenylhydrazine treated anaemic adult spleens (1–2 months old) of LCb118 YAC TgM using ISOGEN (Nippon Gene) and converted to cDNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO).

    Techniques: Knock-In, Mouse Assay, DNA Methylation Assay, Methylation Sequencing, Chromatin Immunoprecipitation, Sequencing, Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Positive Control, Standard Deviation, Expressing, Amplification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Methylation

    Genomic imprinting recapitulated in the LCb118 YAC-TgM. a – c Two pairs of LCb118-TgM (ID No. 2601/2602 and 2633/2640), each inheriting the transgene either paternally (P) or maternally (M) were made anaemic and spleens were removed, from which one-quarter each was used for genomic DNA or total RNA preparation with the remaining half used for chromatin preparation. a DNA methylation status of the transgene was determined by Southern blot analysis using BamHI with (+) or without (−) BstUI (vertical lines) and a λ probe. *: methylated, uncut fragments in BstUI (+) lanes. b ChIP analysis of CTCF occupancy at the transgene. Chromatin was immunoprecipitated using either control IgG or anti-CTCF antibodies. Following qPCR analyses of three distinct genomic regions ( Necdin ; negative control, endogenous H19 ICR; positive control, and LCb118 transgene), relative enrichment values (CTCF/IgG signal ratio) were calculated. The average and standard deviation (S.D.), determined by three reactions, are depicted, as a signal for Necdin (M) was arbitrary set at 1.0. Statistical differences were determined using an unpaired t test (N.S., not significant). c The relative expression levels of the human β-globin gene, after normalization to that of the endogenous mouse α-globin gene were determined by RT-qPCR analysis. The average and standard deviation (S.D.), determined by three reactions, are depicted, as a value of No. 2640 animal was arbitrary set at 1.0

    Journal: Epigenetics & Chromatin

    Article Title: Recapitulation of gametic DNA methylation and its post-fertilization maintenance with reassembled DNA elements at the mouse Igf2/H19 locus

    doi: 10.1186/s13072-019-0326-1

    Figure Lengend Snippet: Genomic imprinting recapitulated in the LCb118 YAC-TgM. a – c Two pairs of LCb118-TgM (ID No. 2601/2602 and 2633/2640), each inheriting the transgene either paternally (P) or maternally (M) were made anaemic and spleens were removed, from which one-quarter each was used for genomic DNA or total RNA preparation with the remaining half used for chromatin preparation. a DNA methylation status of the transgene was determined by Southern blot analysis using BamHI with (+) or without (−) BstUI (vertical lines) and a λ probe. *: methylated, uncut fragments in BstUI (+) lanes. b ChIP analysis of CTCF occupancy at the transgene. Chromatin was immunoprecipitated using either control IgG or anti-CTCF antibodies. Following qPCR analyses of three distinct genomic regions ( Necdin ; negative control, endogenous H19 ICR; positive control, and LCb118 transgene), relative enrichment values (CTCF/IgG signal ratio) were calculated. The average and standard deviation (S.D.), determined by three reactions, are depicted, as a signal for Necdin (M) was arbitrary set at 1.0. Statistical differences were determined using an unpaired t test (N.S., not significant). c The relative expression levels of the human β-globin gene, after normalization to that of the endogenous mouse α-globin gene were determined by RT-qPCR analysis. The average and standard deviation (S.D.), determined by three reactions, are depicted, as a value of No. 2640 animal was arbitrary set at 1.0

    Article Snippet: RT-qPCR Total RNA was recovered from phenylhydrazine treated anaemic adult spleens (1–2 months old) of LCb118 YAC TgM using ISOGEN (Nippon Gene) and converted to cDNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO).

    Techniques: DNA Methylation Assay, Southern Blot, Methylation, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Positive Control, Standard Deviation, Expressing, Quantitative RT-PCR

    Pbp1 PolyQ expansions induce retrotransposon-inhibiting protein aggregation and shorten replicative lifespan. a SDD-AGE of Pbp1 variants. b SDD-AGE of Gag in Pbp1-polyQ cells. Shown below blots is multimer quantification relative to Pbp1-1Q cells. c SDD-AGE of Gag in rad27 ∆ cells. Shown below blots is multimer quantification relative to Pbp1-1Q cells. d CoIP examining interactions between Pbp1 variants and Ty1 Gag ( n = 2). e SDD-AGE of Gag in Pbp1-polyQ-expanded cells in the absence/presence of Hsp104 over-expression. Shown below blots are multimer quantification relative to Pbp1-1Q cells. f Semi-quantitative PCR products reflecting Ty1 integration upstream of 12 tRNA GLY loci in cells with Pbp1 variants and Hsp104 over-expression. TEL1 amplification served as control. g Effects of Hsp104 over-expression on Ty1 mRNA levels in cells with Pbp1 variants. Relative mRNA levels shown were detected by RT-qPCR (Mean ± SD; n = 3). Statistics are relative to levels detected in Pbp1-1Q cells with/without Hsp104 over-expression (one-way ANOVA and Dunnett’s post hoc). h ChIP analysis examining the localization of RNA Pol II at Ty1 in cells with Pbp1 variants and Hsp104 over-expression. Presented are relative levels of Ty1 DNA co-immunoprecipitating with RNA Pol II (phosphorylated serine 5) relative to input, as detected by qPCR (Mean ± SD; n = 3). Statistics are relative to levels detected in Pbp1-1Q cells with/without Hsp104 over-expression (one-way ANOVA and Dunnett’s post hoc). i Effect of Pbp1-polyQ expansion on replicative lifespan. Values are plotted as a survival curve, and mean lifespans are indicated in parentheses. Statistics are relative to Pbp1-1Q (Mann–Whitney U -test). j Effect of decreased retromobility on replicative lifespan. Values are plotted as a survival curve, and mean lifespans are indicated in parentheses. Statistics are relative to Pbp1-1Q (Mann–Whitney U -test). a – j * p

    Journal: Communications Biology

    Article Title: Conserved Pbp1/Ataxin-2 regulates retrotransposon activity and connects polyglutamine expansion-driven protein aggregation to lifespan-controlling rDNA repeats

    doi: 10.1038/s42003-018-0187-3

    Figure Lengend Snippet: Pbp1 PolyQ expansions induce retrotransposon-inhibiting protein aggregation and shorten replicative lifespan. a SDD-AGE of Pbp1 variants. b SDD-AGE of Gag in Pbp1-polyQ cells. Shown below blots is multimer quantification relative to Pbp1-1Q cells. c SDD-AGE of Gag in rad27 ∆ cells. Shown below blots is multimer quantification relative to Pbp1-1Q cells. d CoIP examining interactions between Pbp1 variants and Ty1 Gag ( n = 2). e SDD-AGE of Gag in Pbp1-polyQ-expanded cells in the absence/presence of Hsp104 over-expression. Shown below blots are multimer quantification relative to Pbp1-1Q cells. f Semi-quantitative PCR products reflecting Ty1 integration upstream of 12 tRNA GLY loci in cells with Pbp1 variants and Hsp104 over-expression. TEL1 amplification served as control. g Effects of Hsp104 over-expression on Ty1 mRNA levels in cells with Pbp1 variants. Relative mRNA levels shown were detected by RT-qPCR (Mean ± SD; n = 3). Statistics are relative to levels detected in Pbp1-1Q cells with/without Hsp104 over-expression (one-way ANOVA and Dunnett’s post hoc). h ChIP analysis examining the localization of RNA Pol II at Ty1 in cells with Pbp1 variants and Hsp104 over-expression. Presented are relative levels of Ty1 DNA co-immunoprecipitating with RNA Pol II (phosphorylated serine 5) relative to input, as detected by qPCR (Mean ± SD; n = 3). Statistics are relative to levels detected in Pbp1-1Q cells with/without Hsp104 over-expression (one-way ANOVA and Dunnett’s post hoc). i Effect of Pbp1-polyQ expansion on replicative lifespan. Values are plotted as a survival curve, and mean lifespans are indicated in parentheses. Statistics are relative to Pbp1-1Q (Mann–Whitney U -test). j Effect of decreased retromobility on replicative lifespan. Values are plotted as a survival curve, and mean lifespans are indicated in parentheses. Statistics are relative to Pbp1-1Q (Mann–Whitney U -test). a – j * p

    Article Snippet: For copy number determination, genomic DNA was serially diluted to 0.05 ng/μL. qPCR was performed using the Biorad CFX Connect Real-Time system.

    Techniques: Co-Immunoprecipitation Assay, Over Expression, Real-time Polymerase Chain Reaction, Amplification, Quantitative RT-PCR, Chromatin Immunoprecipitation, MANN-WHITNEY

    Pbp1 polyQ expansions destabilize rDNA repeats independently of R-loops and chromatin. a rDNA repeats on Chr. XII. Expression of rRNA genes (black arrowhead) and IGS ncRNAs (magenta arrowheads) is shown. Rightward red fork = replication fork block; red rectangle = E-pro promoter; red circle = replication origin; blue arrows = R-loop accumulation-prone sites. Primers amplifying IGS regions P1-P5 are shown. b rDNA copy number analysis of Pbp1-polyQ-expanded cells detected by qPCR (Mean ± SD; n = 7). Variance is presented relative to Pbp1-1Q. c CHEF coupled with southern blotting of Chr. XII in Pbp1-polyQ-expanded cells. Bands on EtBr-stained gel served as loading control. d ChIP examining RNA–DNA hybrid levels at the IGS using the S9.6 antibody (Mean ± SD; n = 3; one-way ANOVA and Dunnett’s post hoc). e ChIP examining the enrichment of diAcH3K9/K14 open chromatin marks relative to that of total H3 at the rDNA IGS (Mean ± SD; n = 3). Statistics are relative to levels detected in Pbp1-1Q cells (one-way ANOVA and Dunnett’s post hoc). f ChIP examining the enrichment of RNA Pol II (phosphorylated serine 5) across the rDNA IGS (Mean ± SD; n = 3). Statistics are relative to levels detected in Pbp1-1Q cells (one-way ANOVA and Dunnett’s post hoc). g Interactions between Pbp1 forms and IGS ncRNAs. Presented are levels of IGS ncRNAs immunoprecipitated relative to input, as detected by RT-qPCR (Mean ± SD; n = 3). Values are normalized to pull-down from untagged cells. Statistics are relative to levels detected in untagged cells (one-way ANOVA and Dunnett’s post hoc). P1-P5 primers pair locations are in a . h Effects of Pbp1-polyQ expansions on the levels of IGS ncRNAs as detected by RT-qPCR (Mean ± SD; n = 3; Log2 scale). Statistics are presented relative to levels detected in Pbp1-1Q cells (one-way ANOVA and Dunnett’s post hoc). i Effects of human ATXN2-polyQ expansion on the levels of ncRNAs detected by RT-qPCR at three IGS sites (Mean ± SD; Student’s t -test). a – i * p

    Journal: Communications Biology

    Article Title: Conserved Pbp1/Ataxin-2 regulates retrotransposon activity and connects polyglutamine expansion-driven protein aggregation to lifespan-controlling rDNA repeats

    doi: 10.1038/s42003-018-0187-3

    Figure Lengend Snippet: Pbp1 polyQ expansions destabilize rDNA repeats independently of R-loops and chromatin. a rDNA repeats on Chr. XII. Expression of rRNA genes (black arrowhead) and IGS ncRNAs (magenta arrowheads) is shown. Rightward red fork = replication fork block; red rectangle = E-pro promoter; red circle = replication origin; blue arrows = R-loop accumulation-prone sites. Primers amplifying IGS regions P1-P5 are shown. b rDNA copy number analysis of Pbp1-polyQ-expanded cells detected by qPCR (Mean ± SD; n = 7). Variance is presented relative to Pbp1-1Q. c CHEF coupled with southern blotting of Chr. XII in Pbp1-polyQ-expanded cells. Bands on EtBr-stained gel served as loading control. d ChIP examining RNA–DNA hybrid levels at the IGS using the S9.6 antibody (Mean ± SD; n = 3; one-way ANOVA and Dunnett’s post hoc). e ChIP examining the enrichment of diAcH3K9/K14 open chromatin marks relative to that of total H3 at the rDNA IGS (Mean ± SD; n = 3). Statistics are relative to levels detected in Pbp1-1Q cells (one-way ANOVA and Dunnett’s post hoc). f ChIP examining the enrichment of RNA Pol II (phosphorylated serine 5) across the rDNA IGS (Mean ± SD; n = 3). Statistics are relative to levels detected in Pbp1-1Q cells (one-way ANOVA and Dunnett’s post hoc). g Interactions between Pbp1 forms and IGS ncRNAs. Presented are levels of IGS ncRNAs immunoprecipitated relative to input, as detected by RT-qPCR (Mean ± SD; n = 3). Values are normalized to pull-down from untagged cells. Statistics are relative to levels detected in untagged cells (one-way ANOVA and Dunnett’s post hoc). P1-P5 primers pair locations are in a . h Effects of Pbp1-polyQ expansions on the levels of IGS ncRNAs as detected by RT-qPCR (Mean ± SD; n = 3; Log2 scale). Statistics are presented relative to levels detected in Pbp1-1Q cells (one-way ANOVA and Dunnett’s post hoc). i Effects of human ATXN2-polyQ expansion on the levels of ncRNAs detected by RT-qPCR at three IGS sites (Mean ± SD; Student’s t -test). a – i * p

    Article Snippet: For copy number determination, genomic DNA was serially diluted to 0.05 ng/μL. qPCR was performed using the Biorad CFX Connect Real-Time system.

    Techniques: Expressing, Blocking Assay, Real-time Polymerase Chain Reaction, Southern Blot, Staining, Chromatin Immunoprecipitation, Immunoprecipitation, Quantitative RT-PCR

    Pbp1 represses Trf4-dependent Ty1 mRNA turnover. a ChIP examining the localization of RNA Pol II at Ty1 using an α-RNA Pol II (phosphorylated serine 5) antibody. (One-way ANOVA and Dunnett’s post hoc for left panel, Student’s t -test for right panel). b , c Effects of pbp1∆ on the levels of total Ty1 and Ty1his3AI mRNA as determined by RT-qPCR (Student’s t -test). d ChIP examining RNA–DNA hybrid levels at Ty1 using the S9.6 antibody (One-way ANOVA and Sidak’s post hoc). e Confirmation of ATXN2 knockdown by RT-qPCR in siRNA-transfected HEK293T cells. Values are normalized to GAPDH (Student’s t -test). f Effect of ATXN2 knockdown on levels of retroelement RNAs detected by RT-qPCR. Values are normalized to GAPDH and statistics are relative to siRNA control (siCTL; Student’s t -test). g Interactions between Pbp1-MYC and Ty1 mRNA. Presented are levels of Ty1 mRNA, immunoprecipitated using an α-MYC antibody, relative to input as detected by RT-qPCR (Student’s t -test). h Schematic illustrating RNA stability assay. Transcription is chemically inhibited, and relative abundance of Ty1 mRNA in 1 μg of total RNA is quantified at various time-points by RT-qPCR. More stable RNA displays a greater slope. i Stability of Ty1 mRNA as assessed with RT-qPCR at several time-points following RNA Pol II inhibition. Line of best fit is plotted (Student’s t -test). j Quantification of slopes in i . k Effect of pbp1∆ on the level of Ty1 mRNA in the absence of TRF4 as detected by RT-qPCR (One-way ANOVA and Tukey’s post hoc). l Interactions between Trf4 and Ty1 mRNA. Presented are levels of Ty1 mRNA co-immunoprecipitating with Trf4 relative to input as detected by RT-qPCR. Values are normalized to mock pull-down from trf4∆ cells (Student’s t -test). m , n Western blot examining the levels ( n = 2) of Ty1 Gag forms p49 (unprocessed), p45 (processed) and p22/18 (truncated). In control spt3 ∆ cells, p22/18 levels are induced while p49/45 levels are decreased. a – g , i , k , l Mean ± SD; n = 3. a – d , g Statistics are relative to levels in wild-type cells. a – n * p

    Journal: Communications Biology

    Article Title: Conserved Pbp1/Ataxin-2 regulates retrotransposon activity and connects polyglutamine expansion-driven protein aggregation to lifespan-controlling rDNA repeats

    doi: 10.1038/s42003-018-0187-3

    Figure Lengend Snippet: Pbp1 represses Trf4-dependent Ty1 mRNA turnover. a ChIP examining the localization of RNA Pol II at Ty1 using an α-RNA Pol II (phosphorylated serine 5) antibody. (One-way ANOVA and Dunnett’s post hoc for left panel, Student’s t -test for right panel). b , c Effects of pbp1∆ on the levels of total Ty1 and Ty1his3AI mRNA as determined by RT-qPCR (Student’s t -test). d ChIP examining RNA–DNA hybrid levels at Ty1 using the S9.6 antibody (One-way ANOVA and Sidak’s post hoc). e Confirmation of ATXN2 knockdown by RT-qPCR in siRNA-transfected HEK293T cells. Values are normalized to GAPDH (Student’s t -test). f Effect of ATXN2 knockdown on levels of retroelement RNAs detected by RT-qPCR. Values are normalized to GAPDH and statistics are relative to siRNA control (siCTL; Student’s t -test). g Interactions between Pbp1-MYC and Ty1 mRNA. Presented are levels of Ty1 mRNA, immunoprecipitated using an α-MYC antibody, relative to input as detected by RT-qPCR (Student’s t -test). h Schematic illustrating RNA stability assay. Transcription is chemically inhibited, and relative abundance of Ty1 mRNA in 1 μg of total RNA is quantified at various time-points by RT-qPCR. More stable RNA displays a greater slope. i Stability of Ty1 mRNA as assessed with RT-qPCR at several time-points following RNA Pol II inhibition. Line of best fit is plotted (Student’s t -test). j Quantification of slopes in i . k Effect of pbp1∆ on the level of Ty1 mRNA in the absence of TRF4 as detected by RT-qPCR (One-way ANOVA and Tukey’s post hoc). l Interactions between Trf4 and Ty1 mRNA. Presented are levels of Ty1 mRNA co-immunoprecipitating with Trf4 relative to input as detected by RT-qPCR. Values are normalized to mock pull-down from trf4∆ cells (Student’s t -test). m , n Western blot examining the levels ( n = 2) of Ty1 Gag forms p49 (unprocessed), p45 (processed) and p22/18 (truncated). In control spt3 ∆ cells, p22/18 levels are induced while p49/45 levels are decreased. a – g , i , k , l Mean ± SD; n = 3. a – d , g Statistics are relative to levels in wild-type cells. a – n * p

    Article Snippet: For copy number determination, genomic DNA was serially diluted to 0.05 ng/μL. qPCR was performed using the Biorad CFX Connect Real-Time system.

    Techniques: Chromatin Immunoprecipitation, Quantitative RT-PCR, Transfection, Immunoprecipitation, Stability Assay, Inhibition, Western Blot

    REF-1 assay. ( A ) HCT116 nuclear extract was incubated with 32 P-labeled consensus (CON) or mutant (MUT) AP-1 oligonucleotide substrates, and binding reactions were resolved on a non-denaturing polyacrylamide gel. Control reactions without nuclear extract (no extract) are shown. The arrow designates the position of the AP-1-specific consensus binding complex, not seen with the MUT double-stranded DNA. Higher molecular weight non-specific complexes are observed. ( B ) Reduced wild-type (WT) or variant APE1 protein was incubated with HCT116 nuclear extract in the presence of the 32 P-labeled AP-1 CON DNA substrate. Shown is the AP-1-specific complex in the absence (no protein) or presence of the indicated reduced APE1 protein after phosphorimager analysis. Plotted is the relative AP-1 DNA binding activity, in comparison with reduced WT protein. Values represent the average and standard deviation of 3 independent experimental points.

    Journal: PLoS ONE

    Article Title: Functional Assessment of Population and Tumor-Associated APE1 Protein Variants

    doi: 10.1371/journal.pone.0065922

    Figure Lengend Snippet: REF-1 assay. ( A ) HCT116 nuclear extract was incubated with 32 P-labeled consensus (CON) or mutant (MUT) AP-1 oligonucleotide substrates, and binding reactions were resolved on a non-denaturing polyacrylamide gel. Control reactions without nuclear extract (no extract) are shown. The arrow designates the position of the AP-1-specific consensus binding complex, not seen with the MUT double-stranded DNA. Higher molecular weight non-specific complexes are observed. ( B ) Reduced wild-type (WT) or variant APE1 protein was incubated with HCT116 nuclear extract in the presence of the 32 P-labeled AP-1 CON DNA substrate. Shown is the AP-1-specific complex in the absence (no protein) or presence of the indicated reduced APE1 protein after phosphorimager analysis. Plotted is the relative AP-1 DNA binding activity, in comparison with reduced WT protein. Values represent the average and standard deviation of 3 independent experimental points.

    Article Snippet: APE1 Re-sequencing Amplification of the APE1 exons was performed by mixing 200 ng genomic DNA (obtained from the National Cancer Institute), 250 nM of each of the paired amplification primers (Table S2 in ), 1 mM dNTPs, and 1 µL Herculase II Fusion DNA polymerase in 1X Herculase II reaction buffer (Agilent Technologies).

    Techniques: Incubation, Labeling, Mutagenesis, Binding Assay, Molecular Weight, Variant Assay, Activity Assay, Standard Deviation

    Reconstitution assay using purified BER proteins. ( A ) Wild-type (WT) and variant APE1 proteins were incubated with UDG and POLβ with 32 P-labeled 34U DNA substrate (1 pmol), and the reactions were resolved on a urea-polyacrylamide denaturing sequencing gel. The non-incised substrate (S), AP site incision product (P1), and gap-filling extension product (P2) were visualized and quantified using standard phosphorimaging analysis. NE = no enzyme. ( B ) Relative AP site cleavage efficiency. Shown are the averages and standard deviations of 4 independent reactions. ( C ) Relative gap-filling activity. Shown are the average and standard deviation of 4 independent assays.

    Journal: PLoS ONE

    Article Title: Functional Assessment of Population and Tumor-Associated APE1 Protein Variants

    doi: 10.1371/journal.pone.0065922

    Figure Lengend Snippet: Reconstitution assay using purified BER proteins. ( A ) Wild-type (WT) and variant APE1 proteins were incubated with UDG and POLβ with 32 P-labeled 34U DNA substrate (1 pmol), and the reactions were resolved on a urea-polyacrylamide denaturing sequencing gel. The non-incised substrate (S), AP site incision product (P1), and gap-filling extension product (P2) were visualized and quantified using standard phosphorimaging analysis. NE = no enzyme. ( B ) Relative AP site cleavage efficiency. Shown are the averages and standard deviations of 4 independent reactions. ( C ) Relative gap-filling activity. Shown are the average and standard deviation of 4 independent assays.

    Article Snippet: APE1 Re-sequencing Amplification of the APE1 exons was performed by mixing 200 ng genomic DNA (obtained from the National Cancer Institute), 250 nM of each of the paired amplification primers (Table S2 in ), 1 mM dNTPs, and 1 µL Herculase II Fusion DNA polymerase in 1X Herculase II reaction buffer (Agilent Technologies).

    Techniques: Reconstitution Assay, Purification, Variant Assay, Incubation, Labeling, Sequencing, Activity Assay, Standard Deviation

    AP endonuclease incision and AP-DNA binding. ( A ) Wild-type (WT) and variant APE1 proteins (30 pg) were incubated with 32 P-labeled 18F NMR DNA substrate (0.5 pmol), and the reactions were resolved on a urea-polyacrylamide denaturing gel. The non-incised substrate (S) and incision product (P) bands were visualized and quantified using standard phosphorimaging analysis. NE = no enzyme. ( B ) Relative AP site incision efficiency. Shown are the average and standard deviations of at least 6 independent reactions. ( C ) Wild-type (WT) and variant APE1 proteins (2 ng) were incubated with 32 P-labeled 18F NMR DNA (100 fmol), and the binding reactions were resolved on a non-denaturing polyacrylamide gel. Standard phosphorimager analysis was employed to visualize unbound substrate (DNA) and the APE1-substrate complex (C). NE = no enzyme. ( D ) Relative AP-DNA binding affinity. Shown are the average and standard deviations of relative complex formation for at least 4 independent assays. *, p = 0.002.

    Journal: PLoS ONE

    Article Title: Functional Assessment of Population and Tumor-Associated APE1 Protein Variants

    doi: 10.1371/journal.pone.0065922

    Figure Lengend Snippet: AP endonuclease incision and AP-DNA binding. ( A ) Wild-type (WT) and variant APE1 proteins (30 pg) were incubated with 32 P-labeled 18F NMR DNA substrate (0.5 pmol), and the reactions were resolved on a urea-polyacrylamide denaturing gel. The non-incised substrate (S) and incision product (P) bands were visualized and quantified using standard phosphorimaging analysis. NE = no enzyme. ( B ) Relative AP site incision efficiency. Shown are the average and standard deviations of at least 6 independent reactions. ( C ) Wild-type (WT) and variant APE1 proteins (2 ng) were incubated with 32 P-labeled 18F NMR DNA (100 fmol), and the binding reactions were resolved on a non-denaturing polyacrylamide gel. Standard phosphorimager analysis was employed to visualize unbound substrate (DNA) and the APE1-substrate complex (C). NE = no enzyme. ( D ) Relative AP-DNA binding affinity. Shown are the average and standard deviations of relative complex formation for at least 4 independent assays. *, p = 0.002.

    Article Snippet: APE1 Re-sequencing Amplification of the APE1 exons was performed by mixing 200 ng genomic DNA (obtained from the National Cancer Institute), 250 nM of each of the paired amplification primers (Table S2 in ), 1 mM dNTPs, and 1 µL Herculase II Fusion DNA polymerase in 1X Herculase II reaction buffer (Agilent Technologies).

    Techniques: Binding Assay, Variant Assay, Incubation, Labeling, Nuclear Magnetic Resonance

    Exonuclease and 3′-repair assay. ( A ) Wild-type (WT) and variant APE1 proteins (250 ng) were incubated with 32 P-labeled partially duplex 15P/34G DNA substrate (0.5 pmol), and the reactions were resolved on a high resolution denaturing sequencing gel (top). Substrate (S) and exonuclease degraded product (P) were visualized and quantified using standard phosphorimaging analysis. NE = no enzyme. Graph of relative 3′ to 5′ exonuclease efficiency is shown (bottom), depicting the average and standard deviations of 4 independent experiments. *, p = 0.01. ( B ) Partial duplex 17P/34G substrate (0.5 pmol) was incubated with APE1 proteins (250 ng) and products were resolved on a high resolution denaturing sequencing gel. Products were detected using phosphorimaging analysis. Densitometry results are graphed below from 3 independent assays. *, p = 0.0009. ( C ) The 3′-phosphate partial duplex 15-p/34G DNA substrate (0.5 pmol) was incubated with APE1 proteins (1 ng) and products were resolved on a high resolution denaturing sequencing gel. Densitometry results are graphed below from 3 independent assays. *, p = 0.01.

    Journal: PLoS ONE

    Article Title: Functional Assessment of Population and Tumor-Associated APE1 Protein Variants

    doi: 10.1371/journal.pone.0065922

    Figure Lengend Snippet: Exonuclease and 3′-repair assay. ( A ) Wild-type (WT) and variant APE1 proteins (250 ng) were incubated with 32 P-labeled partially duplex 15P/34G DNA substrate (0.5 pmol), and the reactions were resolved on a high resolution denaturing sequencing gel (top). Substrate (S) and exonuclease degraded product (P) were visualized and quantified using standard phosphorimaging analysis. NE = no enzyme. Graph of relative 3′ to 5′ exonuclease efficiency is shown (bottom), depicting the average and standard deviations of 4 independent experiments. *, p = 0.01. ( B ) Partial duplex 17P/34G substrate (0.5 pmol) was incubated with APE1 proteins (250 ng) and products were resolved on a high resolution denaturing sequencing gel. Products were detected using phosphorimaging analysis. Densitometry results are graphed below from 3 independent assays. *, p = 0.0009. ( C ) The 3′-phosphate partial duplex 15-p/34G DNA substrate (0.5 pmol) was incubated with APE1 proteins (1 ng) and products were resolved on a high resolution denaturing sequencing gel. Densitometry results are graphed below from 3 independent assays. *, p = 0.01.

    Article Snippet: APE1 Re-sequencing Amplification of the APE1 exons was performed by mixing 200 ng genomic DNA (obtained from the National Cancer Institute), 250 nM of each of the paired amplification primers (Table S2 in ), 1 mM dNTPs, and 1 µL Herculase II Fusion DNA polymerase in 1X Herculase II reaction buffer (Agilent Technologies).

    Techniques: Variant Assay, Incubation, Labeling, Sequencing

    Retention of TYLCV DNA by B. tabaci Q females after transfer to nonhost (cotton) plants as affected by previous exposure of the whiteflies to TSWV. TSWV + or TSWV - : B. tabaci Q females that had a 72-h AAP on TSWV-infected pepper plants or on noninfected pepper plants, respectively, before they fed on TYLCV-infected tomato plants. The B. tabaci Q used for TSWV + were assumed to contain TSWV based on Table 1 . (A) Relative quantity of TYLCV retained. (B) Rate at which TYLCV titer decreased in B. tabaci Q females. Values are means ± SE. Asterisks indicate significant differences between TSWV + vs. TSWV - treatments (one-way analysis of variance; ∗ P

    Journal: Frontiers in Physiology

    Article Title: Persistently Transmitted Viruses Restrict the Transmission of Other Viruses by Affecting Their Vectors

    doi: 10.3389/fphys.2018.01261

    Figure Lengend Snippet: Retention of TYLCV DNA by B. tabaci Q females after transfer to nonhost (cotton) plants as affected by previous exposure of the whiteflies to TSWV. TSWV + or TSWV - : B. tabaci Q females that had a 72-h AAP on TSWV-infected pepper plants or on noninfected pepper plants, respectively, before they fed on TYLCV-infected tomato plants. The B. tabaci Q used for TSWV + were assumed to contain TSWV based on Table 1 . (A) Relative quantity of TYLCV retained. (B) Rate at which TYLCV titer decreased in B. tabaci Q females. Values are means ± SE. Asterisks indicate significant differences between TSWV + vs. TSWV - treatments (one-way analysis of variance; ∗ P

    Article Snippet: To quantify TYLCV in tomato plants, a plant genomic DNA kit (Tiangen Biotech, Beijing, Co., Ltd.) was used to extract the DNA from tomato leaves.

    Techniques: Infection

    Acquisition of TYLCV DNA by B. tabaci Q females from tomato plants inoculated in different order with both TSWV and TYLCV. TSWV-TYLCV: plants were inoculated with TSWV and 2 weeks later with TYLCV. TYLCV-TSWV: plants were inoculated with TYLCV and 2 weeks later with TSWV. Concurrent: plants were simultaneously inoculated with both viruses. Control: plants were inoculated with TYLCV and were mock-inoculated 2 weeks later. (A) Acquisition ability of TYLCV DNA by B. tabaci Q females from TSWV-TYLCV plants. (B) Acquisition ability of TYLCV DNA by B. tabaci Q females from concurrent plants. (C) Acquisition ability of TYLCV DNA by B. tabaci Q females from TYLCV-TSWV plants. Values are means ± SE. Asterisks indicate significant differences between treatments vs. control (one-way analysis of variance; ∗ P

    Journal: Frontiers in Physiology

    Article Title: Persistently Transmitted Viruses Restrict the Transmission of Other Viruses by Affecting Their Vectors

    doi: 10.3389/fphys.2018.01261

    Figure Lengend Snippet: Acquisition of TYLCV DNA by B. tabaci Q females from tomato plants inoculated in different order with both TSWV and TYLCV. TSWV-TYLCV: plants were inoculated with TSWV and 2 weeks later with TYLCV. TYLCV-TSWV: plants were inoculated with TYLCV and 2 weeks later with TSWV. Concurrent: plants were simultaneously inoculated with both viruses. Control: plants were inoculated with TYLCV and were mock-inoculated 2 weeks later. (A) Acquisition ability of TYLCV DNA by B. tabaci Q females from TSWV-TYLCV plants. (B) Acquisition ability of TYLCV DNA by B. tabaci Q females from concurrent plants. (C) Acquisition ability of TYLCV DNA by B. tabaci Q females from TYLCV-TSWV plants. Values are means ± SE. Asterisks indicate significant differences between treatments vs. control (one-way analysis of variance; ∗ P

    Article Snippet: To quantify TYLCV in tomato plants, a plant genomic DNA kit (Tiangen Biotech, Beijing, Co., Ltd.) was used to extract the DNA from tomato leaves.

    Techniques:

    Transmission of TYLCV DNA to tomato plants by Bemisia tabaci Q females as affected by previous infection of the tomato plants by TSWV. TSWV + or TSWV - : Tomato plants inoculated with TSWV or mock-inoculated, respectively, before they were exposed to Bemisia tabaci Q females that contained TYLCV. TYLCV + : Females that acquired TYLCV from another TYLCV-infected plant. Values are means ± SE. Asterisks indicate significant differences between TSWV + vs. TSWV - treatments (one-way analysis of variance, ∗∗ P

    Journal: Frontiers in Physiology

    Article Title: Persistently Transmitted Viruses Restrict the Transmission of Other Viruses by Affecting Their Vectors

    doi: 10.3389/fphys.2018.01261

    Figure Lengend Snippet: Transmission of TYLCV DNA to tomato plants by Bemisia tabaci Q females as affected by previous infection of the tomato plants by TSWV. TSWV + or TSWV - : Tomato plants inoculated with TSWV or mock-inoculated, respectively, before they were exposed to Bemisia tabaci Q females that contained TYLCV. TYLCV + : Females that acquired TYLCV from another TYLCV-infected plant. Values are means ± SE. Asterisks indicate significant differences between TSWV + vs. TSWV - treatments (one-way analysis of variance, ∗∗ P

    Article Snippet: To quantify TYLCV in tomato plants, a plant genomic DNA kit (Tiangen Biotech, Beijing, Co., Ltd.) was used to extract the DNA from tomato leaves.

    Techniques: Transmission Assay, Infection

    Transmission of TYLCV DNA to tomato plants by Bemisia tabaci Q females as affected by previous exposure of the whiteflies to TSWV. TSWV + or TSWV - : B. tabaci Q females that had a 72-h AAP on TSWV-infected pepper plants or on noninfected pepper plants, respectively, before they fed on tomato plants. The B. tabaci Q used for TSWV + were assumed to contain TSWV based on Table 1 . Values are means ± SE. Asterisks indicate significant differences between TSWV + vs. TSWV - treatments (one-way analysis of variance, ∗∗ P

    Journal: Frontiers in Physiology

    Article Title: Persistently Transmitted Viruses Restrict the Transmission of Other Viruses by Affecting Their Vectors

    doi: 10.3389/fphys.2018.01261

    Figure Lengend Snippet: Transmission of TYLCV DNA to tomato plants by Bemisia tabaci Q females as affected by previous exposure of the whiteflies to TSWV. TSWV + or TSWV - : B. tabaci Q females that had a 72-h AAP on TSWV-infected pepper plants or on noninfected pepper plants, respectively, before they fed on tomato plants. The B. tabaci Q used for TSWV + were assumed to contain TSWV based on Table 1 . Values are means ± SE. Asterisks indicate significant differences between TSWV + vs. TSWV - treatments (one-way analysis of variance, ∗∗ P

    Article Snippet: To quantify TYLCV in tomato plants, a plant genomic DNA kit (Tiangen Biotech, Beijing, Co., Ltd.) was used to extract the DNA from tomato leaves.

    Techniques: Transmission Assay, Infection

    Acquisition of TYLCV by Frankliniella occidentalis (A) , fecundity of F. occidentalis as affected by TYLCV (B) , and transmission of TSWV by F. occidentalis as affected by previous exposure of the thrips to TYLCV (C) . (A) PCR amplification of TYLCV was performed with the DNA extracted from F. occidentalis second-instar nymphs (lanes 3–7; one nymph per lane) and newly emerged adults (lanes 8–12; one adult per lane) obtained from a TYLCV-infected tomato plant. Amplification products were visualized by agarose gel electrophoresis with Gelview staining. Lane 1: positive control; lane 2: negative control; lane M: Marker II (Tiangen). (B) Number of eggs laid by each female placed in a clip-cage attached to a leaf of a healthy or a TYLCV-infected tomato plant. Values are means ± SE of 10 replicate plants (three adults per plant). (C) The OD value of TSWV analysis obtained by DAS-ELISA indicated the difference between F. occidentalis that were exposed and not exposed to TYLCV. The number in blue indicates the mean OD value of healthy tomato plants. Values are means ± SE. For (B,C) , different letters indicate significant differences between treatments ( P

    Journal: Frontiers in Physiology

    Article Title: Persistently Transmitted Viruses Restrict the Transmission of Other Viruses by Affecting Their Vectors

    doi: 10.3389/fphys.2018.01261

    Figure Lengend Snippet: Acquisition of TYLCV by Frankliniella occidentalis (A) , fecundity of F. occidentalis as affected by TYLCV (B) , and transmission of TSWV by F. occidentalis as affected by previous exposure of the thrips to TYLCV (C) . (A) PCR amplification of TYLCV was performed with the DNA extracted from F. occidentalis second-instar nymphs (lanes 3–7; one nymph per lane) and newly emerged adults (lanes 8–12; one adult per lane) obtained from a TYLCV-infected tomato plant. Amplification products were visualized by agarose gel electrophoresis with Gelview staining. Lane 1: positive control; lane 2: negative control; lane M: Marker II (Tiangen). (B) Number of eggs laid by each female placed in a clip-cage attached to a leaf of a healthy or a TYLCV-infected tomato plant. Values are means ± SE of 10 replicate plants (three adults per plant). (C) The OD value of TSWV analysis obtained by DAS-ELISA indicated the difference between F. occidentalis that were exposed and not exposed to TYLCV. The number in blue indicates the mean OD value of healthy tomato plants. Values are means ± SE. For (B,C) , different letters indicate significant differences between treatments ( P

    Article Snippet: To quantify TYLCV in tomato plants, a plant genomic DNA kit (Tiangen Biotech, Beijing, Co., Ltd.) was used to extract the DNA from tomato leaves.

    Techniques: Transmission Assay, Polymerase Chain Reaction, Amplification, Infection, Agarose Gel Electrophoresis, Staining, Positive Control, Negative Control, Marker, Cross-linking Immunoprecipitation, Enzyme-linked Immunosorbent Assay

    Acquisition of TYLCV DNA by Bemisia tabaci Q females as affected by previous exposure of the whiteflies to TSWV. TSWV + or TSWV - : B. tabaci Q females that had a 72-h acquisition access period (AAP) on TSWV-infected pepper plants or on noninfected pepper plants, respectively, before they fed on TYLCV-infected tomato plants. The B. tabaci Q used for TSWV + were assumed to contain TSWV based on Table 1 . Values are means ± SE. Asterisks indicate significant differences between TSWV + vs. TSWV - treatments (one-way analysis of variance, ∗ P

    Journal: Frontiers in Physiology

    Article Title: Persistently Transmitted Viruses Restrict the Transmission of Other Viruses by Affecting Their Vectors

    doi: 10.3389/fphys.2018.01261

    Figure Lengend Snippet: Acquisition of TYLCV DNA by Bemisia tabaci Q females as affected by previous exposure of the whiteflies to TSWV. TSWV + or TSWV - : B. tabaci Q females that had a 72-h acquisition access period (AAP) on TSWV-infected pepper plants or on noninfected pepper plants, respectively, before they fed on TYLCV-infected tomato plants. The B. tabaci Q used for TSWV + were assumed to contain TSWV based on Table 1 . Values are means ± SE. Asterisks indicate significant differences between TSWV + vs. TSWV - treatments (one-way analysis of variance, ∗ P

    Article Snippet: To quantify TYLCV in tomato plants, a plant genomic DNA kit (Tiangen Biotech, Beijing, Co., Ltd.) was used to extract the DNA from tomato leaves.

    Techniques: Infection

    Changes in the amount of proliferation markers Ki-67 and PCNA in haMSCs exposed to gDNA or GC-DNA at final concentration 50 ng/mL (FACS). (a) (A) fixed cells stained with anti-Ki-67 (FITC) antibodies. Distribution of fluorescence intensities of the cells exposed to DNAs . Background fluorescence was quantified using FITC-conjugated secondary antibodies (grey color); (B) the average of the median signal intensity of FL1 (Ki-67) in haMSC. (b) (A) fixed cells stained with anti-PCNA (FITC) antibodies. Distribution of fluorescence intensities of haMSCs exposed to DNAs . Background fluorescence was quantified using FITC-conjugated secondary antibodies (grey color). (B) The average of the median signal intensity of FL1 (PCNA). (c) The ratio of the levels of mRNA CCND1, CDKN2, and CDKN1A in cells exposed to gDNA, pBR322, or GC-DNA. The incubation time is indicated on the histogram. Data points were averaged and represented as mean ± SD for three biological replicates. ( * ) P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells

    doi: 10.1155/2015/782123

    Figure Lengend Snippet: Changes in the amount of proliferation markers Ki-67 and PCNA in haMSCs exposed to gDNA or GC-DNA at final concentration 50 ng/mL (FACS). (a) (A) fixed cells stained with anti-Ki-67 (FITC) antibodies. Distribution of fluorescence intensities of the cells exposed to DNAs . Background fluorescence was quantified using FITC-conjugated secondary antibodies (grey color); (B) the average of the median signal intensity of FL1 (Ki-67) in haMSC. (b) (A) fixed cells stained with anti-PCNA (FITC) antibodies. Distribution of fluorescence intensities of haMSCs exposed to DNAs . Background fluorescence was quantified using FITC-conjugated secondary antibodies (grey color). (B) The average of the median signal intensity of FL1 (PCNA). (c) The ratio of the levels of mRNA CCND1, CDKN2, and CDKN1A in cells exposed to gDNA, pBR322, or GC-DNA. The incubation time is indicated on the histogram. Data points were averaged and represented as mean ± SD for three biological replicates. ( * ) P

    Article Snippet: The measurements were carried out for gDNA and for GC-DNA (20 ng/mL, ).

    Techniques: Concentration Assay, FACS, Staining, Fluorescence, Incubation

    Exposure to either gDNA or GC-DNA supports cell survival. (a) Detection of haMSCs with sings of early apoptosis (FACS). HaMSCs were exposed to DNA samples (50 ng/mL, 3 h). (A) The distribution of fluorescence intensities of the cells stained with Annexin V-FITC; (B) the proportion of Annexin V-positive cells in total cell population. (b) The ratio of the levels of mRNA BCL2 , BCL2A1 (Bfl-1/A1), BCL2L1 (BCL-X), BIRC3 (c-IAP1), and BIRC2 in cells exposed to 50 ng/mL gDNA, pBR322, or GC-DNA. The incubation time is indicated on the histogram. (c) Detection of BCL2 protein level in haMSCs exposed to DNA samples (50 ng/mL, 14 days). Cells were fixed with 3% PFA, treated with 0.1% Triton X-100 and stained with anti-BCL2 (FITC) antibodies. (A) FACS analysis showing the stained cellular fractions (SSC-FL1 (BCL2) diagram). Gate R encircles the fraction of haMSCs that express large amounts of protein BCL2; (B) the proportion of cells with large amounts of protein BCL2 (gate R); (C) the average of the median signal intensity of FL1 (BCL2) in haMSCs. Horizontal bars reflect relative expression levels in the control cells. Data points were averaged and represented as mean ± SD for three biological replicates. Asterisk ( * ) depicts the differences between exposed cells and control cells that were statistically significant by the Mann-Whitney U test ( P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells

    doi: 10.1155/2015/782123

    Figure Lengend Snippet: Exposure to either gDNA or GC-DNA supports cell survival. (a) Detection of haMSCs with sings of early apoptosis (FACS). HaMSCs were exposed to DNA samples (50 ng/mL, 3 h). (A) The distribution of fluorescence intensities of the cells stained with Annexin V-FITC; (B) the proportion of Annexin V-positive cells in total cell population. (b) The ratio of the levels of mRNA BCL2 , BCL2A1 (Bfl-1/A1), BCL2L1 (BCL-X), BIRC3 (c-IAP1), and BIRC2 in cells exposed to 50 ng/mL gDNA, pBR322, or GC-DNA. The incubation time is indicated on the histogram. (c) Detection of BCL2 protein level in haMSCs exposed to DNA samples (50 ng/mL, 14 days). Cells were fixed with 3% PFA, treated with 0.1% Triton X-100 and stained with anti-BCL2 (FITC) antibodies. (A) FACS analysis showing the stained cellular fractions (SSC-FL1 (BCL2) diagram). Gate R encircles the fraction of haMSCs that express large amounts of protein BCL2; (B) the proportion of cells with large amounts of protein BCL2 (gate R); (C) the average of the median signal intensity of FL1 (BCL2) in haMSCs. Horizontal bars reflect relative expression levels in the control cells. Data points were averaged and represented as mean ± SD for three biological replicates. Asterisk ( * ) depicts the differences between exposed cells and control cells that were statistically significant by the Mann-Whitney U test ( P

    Article Snippet: The measurements were carried out for gDNA and for GC-DNA (20 ng/mL, ).

    Techniques: FACS, Fluorescence, Staining, Incubation, Expressing, MANN-WHITNEY

    Nuclear DNA damage and repair in haMSCs exposed to DNA samples. (a) γ H2AX in cells exposed to gDNA and GC-DNA (50 ng/mL, 30 min and 180 min). Cells were fixed with 3% PFA, treated with 0.1% Triton X-100 and processed for immunofluorescence staining with anti- γ H2AX antibody (FITC) (magnification 40x); rhodamine phalloidin was used to label cytoskeletal F-actin. (b) FACS analysis of γ -foci. (A) Their main fractions of the cells as evident in gating areas R1 and R2; (B) the kinetics of changes in the relative proportions of cells with γ -foci (gate R1). Data points were averaged and represented as mean ± SD for three biological replicates. ( * ) P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells

    doi: 10.1155/2015/782123

    Figure Lengend Snippet: Nuclear DNA damage and repair in haMSCs exposed to DNA samples. (a) γ H2AX in cells exposed to gDNA and GC-DNA (50 ng/mL, 30 min and 180 min). Cells were fixed with 3% PFA, treated with 0.1% Triton X-100 and processed for immunofluorescence staining with anti- γ H2AX antibody (FITC) (magnification 40x); rhodamine phalloidin was used to label cytoskeletal F-actin. (b) FACS analysis of γ -foci. (A) Their main fractions of the cells as evident in gating areas R1 and R2; (B) the kinetics of changes in the relative proportions of cells with γ -foci (gate R1). Data points were averaged and represented as mean ± SD for three biological replicates. ( * ) P

    Article Snippet: The measurements were carried out for gDNA and for GC-DNA (20 ng/mL, ).

    Techniques: Immunofluorescence, Staining, FACS

    Distribution of CpG-motifs and Gn-motifs ( n = 3) within the model DNA samples and within rDNA (transcribed region of human ribosomal repeat) that accumulates in circulating DNA of blood plasma. The digits indicate the nucleotide order number, while the vertical bar shows the motif location. For comparison, the figure also presents the distribution of the motifs within a randomly chosen fragment of genomic DNA with a total GC-content of 42%.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells

    doi: 10.1155/2015/782123

    Figure Lengend Snippet: Distribution of CpG-motifs and Gn-motifs ( n = 3) within the model DNA samples and within rDNA (transcribed region of human ribosomal repeat) that accumulates in circulating DNA of blood plasma. The digits indicate the nucleotide order number, while the vertical bar shows the motif location. For comparison, the figure also presents the distribution of the motifs within a randomly chosen fragment of genomic DNA with a total GC-content of 42%.

    Article Snippet: The measurements were carried out for gDNA and for GC-DNA (20 ng/mL, ).

    Techniques:

    PCR confirmation of ICE Apl2 in MIDG2331ΔureC::nadV and detection of extrachromosomal ICE Apl2 in MIDG3553 and MIDG2331ΔureC::nadV. (a) PCR products from amplification of traG (530 bp) and nadV (1.5 kb) sequences from donor MIDG3553 (lanes 2 and 6), recipient MIDG2331ΔureC::nadV prior to conjugation (lanes 3 and 7) and selected transconjugants (lanes 4, 5, 8 and 9). Lane 1, 100 bp Plus DNA ladder. (b) Detection of a circular intermediate form by nested PCR in MIDG3553 (lanes 2 and 5) and selected transconjugants (lanes 3, 4, 6 and 7). Nested-PCR products generated by primers IceFlo_L1_out/IceFlo_R1_out and IceFlo_L2_out/IceFlo_R2_out are labelled as product 1 (983 bp) and product 2 (360 bp), respectively. Lane 1, 100 bp Plus DNA ladder (Invitrogen).

    Journal: Journal of Antimicrobial Chemotherapy

    Article Title: Characterization of the Actinobacillus pleuropneumoniae SXT-related integrative and conjugative element ICEApl2 and analysis of the encoded FloR protein: hydrophobic residues in transmembrane domains contribute dynamically to florfenicol and chloramphenicol efflux

    doi: 10.1093/jac/dkx342

    Figure Lengend Snippet: PCR confirmation of ICE Apl2 in MIDG2331ΔureC::nadV and detection of extrachromosomal ICE Apl2 in MIDG3553 and MIDG2331ΔureC::nadV. (a) PCR products from amplification of traG (530 bp) and nadV (1.5 kb) sequences from donor MIDG3553 (lanes 2 and 6), recipient MIDG2331ΔureC::nadV prior to conjugation (lanes 3 and 7) and selected transconjugants (lanes 4, 5, 8 and 9). Lane 1, 100 bp Plus DNA ladder. (b) Detection of a circular intermediate form by nested PCR in MIDG3553 (lanes 2 and 5) and selected transconjugants (lanes 3, 4, 6 and 7). Nested-PCR products generated by primers IceFlo_L1_out/IceFlo_R1_out and IceFlo_L2_out/IceFlo_R2_out are labelled as product 1 (983 bp) and product 2 (360 bp), respectively. Lane 1, 100 bp Plus DNA ladder (Invitrogen).

    Article Snippet: Genomic DNA was isolated from MIDG3553 using the FastDNA Spin Kit (MP Biomedicals) and shotgun WGS data were generated and assembled by the MicrobesNG Sequencing Facility ( www.microbesng.uk ).

    Techniques: Polymerase Chain Reaction, Amplification, Conjugation Assay, Nested PCR, Generated

    Expression of floR in MIDG3553 (lanes 2 and 3) and MIDG3446 (lanes 4 and 5) as determined by RT–PCR amplification of a 510 bp sequence. Lanes 2 and 4 show results for samples grown in broth without chloramphenicol and lanes 3 and 5 show results for samples grown in broth with 2 mg/L chloramphenicol. Lane 1, 100 bp Plus DNA ladder (Invitrogen). (a) Normal 2D gel image. (b) Gel image shown in 3D.

    Journal: Journal of Antimicrobial Chemotherapy

    Article Title: Characterization of the Actinobacillus pleuropneumoniae SXT-related integrative and conjugative element ICEApl2 and analysis of the encoded FloR protein: hydrophobic residues in transmembrane domains contribute dynamically to florfenicol and chloramphenicol efflux

    doi: 10.1093/jac/dkx342

    Figure Lengend Snippet: Expression of floR in MIDG3553 (lanes 2 and 3) and MIDG3446 (lanes 4 and 5) as determined by RT–PCR amplification of a 510 bp sequence. Lanes 2 and 4 show results for samples grown in broth without chloramphenicol and lanes 3 and 5 show results for samples grown in broth with 2 mg/L chloramphenicol. Lane 1, 100 bp Plus DNA ladder (Invitrogen). (a) Normal 2D gel image. (b) Gel image shown in 3D.

    Article Snippet: Genomic DNA was isolated from MIDG3553 using the FastDNA Spin Kit (MP Biomedicals) and shotgun WGS data were generated and assembled by the MicrobesNG Sequencing Facility ( www.microbesng.uk ).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Two-Dimensional Gel Electrophoresis