dna methylation analysis genomic dna  (Qiagen)


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    Qiagen dna methylation analysis genomic dna
    Gene expression and hypothalamic Pomc <t>DNA</t> methylation changes in offspring at weaning. a mRNA expression levels of Pomc , Agrp , Npy , and b Ob-Rb in the <t>ARC.</t> c Relative mRNA levels of Mc4r and Npy1r in the PVN analyzed by qRT-PCR in the 3-week-old offspring of LF- or HF-fed dams (Student’s t -test, n = 8). d Map of the Pro-opiomelanocortin ( Pomc ) gene promoter and enhancer region including functional regulatory elements and CpG dinucleotides (red lines). e Methylation analyzes of hypothalamic Pomc promoter (− 150 bp to transcription start site [TSS]) (Student’s t -test, D-LF, n = 6; D-HF, n = 7) and f , g of neuronal Pomc enhancer region 1 and 2 in the offspring of LF- or HF-fed mothers at 3 weeks of age (Student’s t -test, n = 8). Data are shown as mean ± SEM. * p
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    1) Product Images from "Maternal overnutrition programs epigenetic changes in the regulatory regions of hypothalamic Pomc in the offspring of rats"

    Article Title: Maternal overnutrition programs epigenetic changes in the regulatory regions of hypothalamic Pomc in the offspring of rats

    Journal: International Journal of Obesity (2005)

    doi: 10.1038/s41366-018-0094-1

    Gene expression and hypothalamic Pomc DNA methylation changes in offspring at weaning. a mRNA expression levels of Pomc , Agrp , Npy , and b Ob-Rb in the ARC. c Relative mRNA levels of Mc4r and Npy1r in the PVN analyzed by qRT-PCR in the 3-week-old offspring of LF- or HF-fed dams (Student’s t -test, n = 8). d Map of the Pro-opiomelanocortin ( Pomc ) gene promoter and enhancer region including functional regulatory elements and CpG dinucleotides (red lines). e Methylation analyzes of hypothalamic Pomc promoter (− 150 bp to transcription start site [TSS]) (Student’s t -test, D-LF, n = 6; D-HF, n = 7) and f , g of neuronal Pomc enhancer region 1 and 2 in the offspring of LF- or HF-fed mothers at 3 weeks of age (Student’s t -test, n = 8). Data are shown as mean ± SEM. * p
    Figure Legend Snippet: Gene expression and hypothalamic Pomc DNA methylation changes in offspring at weaning. a mRNA expression levels of Pomc , Agrp , Npy , and b Ob-Rb in the ARC. c Relative mRNA levels of Mc4r and Npy1r in the PVN analyzed by qRT-PCR in the 3-week-old offspring of LF- or HF-fed dams (Student’s t -test, n = 8). d Map of the Pro-opiomelanocortin ( Pomc ) gene promoter and enhancer region including functional regulatory elements and CpG dinucleotides (red lines). e Methylation analyzes of hypothalamic Pomc promoter (− 150 bp to transcription start site [TSS]) (Student’s t -test, D-LF, n = 6; D-HF, n = 7) and f , g of neuronal Pomc enhancer region 1 and 2 in the offspring of LF- or HF-fed mothers at 3 weeks of age (Student’s t -test, n = 8). Data are shown as mean ± SEM. * p

    Techniques Used: Expressing, DNA Methylation Assay, Quantitative RT-PCR, Functional Assay, Methylation

    2) Product Images from "In vitro selection of an XNA aptamer capable of small-molecule recognition"

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky667

    TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.
    Figure Legend Snippet: TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.

    Techniques Used: Binding Assay, Amplification, Modification, Polyacrylamide Gel Electrophoresis, Selection, Incubation, Magnetic Beads, Polymerase Chain Reaction

    3) Product Images from "Detection of the MYD88 p.L265P Mutation in the CSF of a Patient With Secondary Central Nervous System Lymphoma"

    Article Title: Detection of the MYD88 p.L265P Mutation in the CSF of a Patient With Secondary Central Nervous System Lymphoma

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2018.00382

    Droplet digital PCR results from examination of the CNS tissue and the CSF-ctDNA for MYD88 mutations. Quadrant statistics scatter plot (lower left quadrant shows negative cluster; lower right quadrant shows wild-type cluster; upper left quadrant shows mutant cluster; upper right quadrant shows double positive). Both, CNS tissue and CSF-ctDNA showed positive mutant droplets for MYD88 p.L265P, while no MYD88 p.V217F mutant droplets were detected. The representative bar chart illustrates the percentage value of mutation allele frequency (MAF) for the CNS tissue and the CSF-ctDNA.
    Figure Legend Snippet: Droplet digital PCR results from examination of the CNS tissue and the CSF-ctDNA for MYD88 mutations. Quadrant statistics scatter plot (lower left quadrant shows negative cluster; lower right quadrant shows wild-type cluster; upper left quadrant shows mutant cluster; upper right quadrant shows double positive). Both, CNS tissue and CSF-ctDNA showed positive mutant droplets for MYD88 p.L265P, while no MYD88 p.V217F mutant droplets were detected. The representative bar chart illustrates the percentage value of mutation allele frequency (MAF) for the CNS tissue and the CSF-ctDNA.

    Techniques Used: Digital PCR, Mutagenesis

    4) Product Images from "NLRP1 restricts butyrate producing commensals to exacerbate inflammatory bowel disease"

    Article Title: NLRP1 restricts butyrate producing commensals to exacerbate inflammatory bowel disease

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06125-0

    Vancomycin treatment or supplementation with butyrate ablates the Nlrp1 phenotype. a Stool from WT and Nlrp1 −/− mice was harvested before and after vancomycin (50 mg/L) treatment for 4 weeks. Bacterial DNA from stool was isolated from untreated (UT) and vancomycin-treated mice, and depletion of the Coccoides group (belonging to the Clostridiales phylum) was confirmed using specific 16S primers by quantitative PCR. Results were normalized to the total bacteria present in the stool. b Vancomycin-treated mice were subjected to 3% (w/v) DSS for 6 days and disease severity was measured according to percentage weight loss and c colon length. Short chain fatty acid analysis was performed on fecal samples collected from WT and Nlrp1 −/− mice before (UT) and after vancomycin treatment. The concentration of d butyrate and e propionate was measured by gas chromatography mass spectrometry. f WT and Nlrp1 −/− mice were supplemented with 2% butyrate in drinking water ad libitum for 28 days, and then subjected to 2.5% (w/v) DSS for 6 days and disease severity measured according to percentage weight loss or g colon length. Data are representative of 3 independent experiments with 3–6 mice per group. Means ± SEM; * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001. A two-tailed unpaired t- test was used to determine statistical significance between two groups; and a two-way ANOVA with Tukey’s post-hoc comparisons was performed on data that involved more than two comparisons
    Figure Legend Snippet: Vancomycin treatment or supplementation with butyrate ablates the Nlrp1 phenotype. a Stool from WT and Nlrp1 −/− mice was harvested before and after vancomycin (50 mg/L) treatment for 4 weeks. Bacterial DNA from stool was isolated from untreated (UT) and vancomycin-treated mice, and depletion of the Coccoides group (belonging to the Clostridiales phylum) was confirmed using specific 16S primers by quantitative PCR. Results were normalized to the total bacteria present in the stool. b Vancomycin-treated mice were subjected to 3% (w/v) DSS for 6 days and disease severity was measured according to percentage weight loss and c colon length. Short chain fatty acid analysis was performed on fecal samples collected from WT and Nlrp1 −/− mice before (UT) and after vancomycin treatment. The concentration of d butyrate and e propionate was measured by gas chromatography mass spectrometry. f WT and Nlrp1 −/− mice were supplemented with 2% butyrate in drinking water ad libitum for 28 days, and then subjected to 2.5% (w/v) DSS for 6 days and disease severity measured according to percentage weight loss or g colon length. Data are representative of 3 independent experiments with 3–6 mice per group. Means ± SEM; * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001. A two-tailed unpaired t- test was used to determine statistical significance between two groups; and a two-way ANOVA with Tukey’s post-hoc comparisons was performed on data that involved more than two comparisons

    Techniques Used: Mouse Assay, Isolation, Real-time Polymerase Chain Reaction, Concentration Assay, Gas Chromatography, Mass Spectrometry, Two Tailed Test

    5) Product Images from "DNA-methylation-mediated silencing of miR-486-5p promotes colorectal cancer proliferation and migration through activation of PLAGL2/IGF2/β-catenin signal pathways"

    Article Title: DNA-methylation-mediated silencing of miR-486-5p promotes colorectal cancer proliferation and migration through activation of PLAGL2/IGF2/β-catenin signal pathways

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1105-9

    MiR-486-5p expression is regulated by DNA methylation of its promoter. a MethPrimer website predicted that a CpG island is located at the promoter region of miR-486-5p. b , c BSP analysis demonstrated that methylated CG sites in CRC cell lines were more than that in FHC. d MSP analysis uncovered hypermethylation of the miR-486-5p promoter in CRC tissues when compared to ANTs. e The expression of miR-486-5p restored in CRC cell lines using 5-aza-2′-deoxycytidine. Data are represented as means ± S.D. from at least three independent experiments. * p
    Figure Legend Snippet: MiR-486-5p expression is regulated by DNA methylation of its promoter. a MethPrimer website predicted that a CpG island is located at the promoter region of miR-486-5p. b , c BSP analysis demonstrated that methylated CG sites in CRC cell lines were more than that in FHC. d MSP analysis uncovered hypermethylation of the miR-486-5p promoter in CRC tissues when compared to ANTs. e The expression of miR-486-5p restored in CRC cell lines using 5-aza-2′-deoxycytidine. Data are represented as means ± S.D. from at least three independent experiments. * p

    Techniques Used: Expressing, DNA Methylation Assay, Methylation

    6) Product Images from "Enhanced detection of microsatellite instability using pre-PCR elimination of wild-type DNA homo-polymers in tissue and liquid biopsies"

    Article Title: Enhanced detection of microsatellite instability using pre-PCR elimination of wild-type DNA homo-polymers in tissue and liquid biopsies

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky251

    NaME-PrO application on BAT25 microsatellite using circulating DNA from colon cancer samples. ( A ) Comparison between NaME-PrO-treated and untreated screening for BAT25 microsatellites on plasma circulating DNA following HRM analysis: examples from three positive samples (#230, 236, 266) plus cfDNA from a healthy control patient (#38) are shown. ( B ) Capillary electrophoresis analysis for the same samples (red = variant, blue = WT).
    Figure Legend Snippet: NaME-PrO application on BAT25 microsatellite using circulating DNA from colon cancer samples. ( A ) Comparison between NaME-PrO-treated and untreated screening for BAT25 microsatellites on plasma circulating DNA following HRM analysis: examples from three positive samples (#230, 236, 266) plus cfDNA from a healthy control patient (#38) are shown. ( B ) Capillary electrophoresis analysis for the same samples (red = variant, blue = WT).

    Techniques Used: Electrophoresis, Variant Assay

    7) Product Images from "A hPSC-based platform to discover gene-environment interactions that impact human β-cell and dopamine neuron survival"

    Article Title: A hPSC-based platform to discover gene-environment interactions that impact human β-cell and dopamine neuron survival

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07201-1

    Midbrain dopamine neurons are hypersensitive to propargite-induced cell toxicity. a Characterization of cortical neuron and mDA neuron derived from H9 hESCs. Upper panel represents bright field images of cortical- and mDA-neurons. Lower panel shows cortical neurons stained for MAP2 (red) and CTIP2 (green) while mDA neurons were stained for TH (red) and FOXA2 (blue). Scale bars, 50 μm. b Relative cell survival rate of cortical- and mDA-neurons treated with DMSO or different doses of propargite. Relative cell survival was quantified by dividing propargite-treated cells to the DMSO control ( n = 3). c , d Representative image ( c ) and relative cell survival rate ( d ) of mDA neurons treated with DMSO or propargite (1 μM) in the presence or absence of GSH (2 mM). mDA cells were stained for TH (red) and FOXA2 (gray), and all cells were counterstained with DAPI (blue). Scale bars, 50 μm. Relative cell survival rate was analyzed by quantification of FOXA2 + (gray) cells ( n = 3). e Representative image of mDA cells treated with DMSO, DMSO+2 mM GSH, 1 μM propargite, or 1 μM propargite+2 mM GSH. White arrows indicate propargite-treated mDA cells (TH; red) co-stained with the DNA damage marker (rH2AX; green), and all cells were counterstained with DAPI (blue). Scale bars, 50 μm. f Western blotting analysis of necrosis marker (extracellular HMGB1) in DMSO or propargite (1 μM) treated mDA cells with/without GSH (2 mM). Only propargite-treated mDA cell had high extracellular HMGB1 level ( n = 3). g Relative cell survival rate, quantified by the expression of FOXA2+ cells ( n = 3), of mDA cells derived from isogenic wild type, GSTT1 −/− , and GSTM1 −/− H1 hESCs treated with DMSO and propargite (3 μM). h GSTT1 , but not GSTM1 expression, in substantia nigra region of postmortem brains is significantly downregulated in Parkinson’s disease patients compared to age-matched controls. Values for RNA expression used here are from a published gene expression data and selected values except absent its detection 38 . Values presented as mean ± S.D. p -value was calculated by unpaired two-tailed Student’s t -test were * p
    Figure Legend Snippet: Midbrain dopamine neurons are hypersensitive to propargite-induced cell toxicity. a Characterization of cortical neuron and mDA neuron derived from H9 hESCs. Upper panel represents bright field images of cortical- and mDA-neurons. Lower panel shows cortical neurons stained for MAP2 (red) and CTIP2 (green) while mDA neurons were stained for TH (red) and FOXA2 (blue). Scale bars, 50 μm. b Relative cell survival rate of cortical- and mDA-neurons treated with DMSO or different doses of propargite. Relative cell survival was quantified by dividing propargite-treated cells to the DMSO control ( n = 3). c , d Representative image ( c ) and relative cell survival rate ( d ) of mDA neurons treated with DMSO or propargite (1 μM) in the presence or absence of GSH (2 mM). mDA cells were stained for TH (red) and FOXA2 (gray), and all cells were counterstained with DAPI (blue). Scale bars, 50 μm. Relative cell survival rate was analyzed by quantification of FOXA2 + (gray) cells ( n = 3). e Representative image of mDA cells treated with DMSO, DMSO+2 mM GSH, 1 μM propargite, or 1 μM propargite+2 mM GSH. White arrows indicate propargite-treated mDA cells (TH; red) co-stained with the DNA damage marker (rH2AX; green), and all cells were counterstained with DAPI (blue). Scale bars, 50 μm. f Western blotting analysis of necrosis marker (extracellular HMGB1) in DMSO or propargite (1 μM) treated mDA cells with/without GSH (2 mM). Only propargite-treated mDA cell had high extracellular HMGB1 level ( n = 3). g Relative cell survival rate, quantified by the expression of FOXA2+ cells ( n = 3), of mDA cells derived from isogenic wild type, GSTT1 −/− , and GSTM1 −/− H1 hESCs treated with DMSO and propargite (3 μM). h GSTT1 , but not GSTM1 expression, in substantia nigra region of postmortem brains is significantly downregulated in Parkinson’s disease patients compared to age-matched controls. Values for RNA expression used here are from a published gene expression data and selected values except absent its detection 38 . Values presented as mean ± S.D. p -value was calculated by unpaired two-tailed Student’s t -test were * p

    Techniques Used: Multiple Displacement Amplification, Derivative Assay, Staining, Marker, Western Blot, Expressing, RNA Expression, Two Tailed Test

    A hPSC-based population study discovers that GSTT1 -null pancreatic β-like cells are hypersensitive to propargite-induced cell death. a Survival rate of INS + cells derived from 10 different hESC or iPSC lines cultured in the presence of 1.6 μM propargite ( n = 3), and genotype analysis of GSTM1 and GSTT1 in those hESCs and iPSCs. b , c Correlation of INS + cell survival rate in the presence of 1.6 μM propargite in cells lacking both GSTM1 and GSTT1 ( b ), or lacking only GSTM1 ( c ). n.s. indicates a non-significant difference. d Western blotting analysis of GSTT1 or GSTM1 protein expression in INS + cells derived from isogenic wild type, GSTT1 −/− or GSTM1 −/− H1 hESCs. The −/− null clones were CRSIPR-induced biallelic frameshift mutants. The two GSTT1 knockout clones were both homozygous null mutants, and the two GSTM1 knockout clones were both compound-null mutants. e Flow cytometry analysis of C-peptide + cells in isogenic GSTT1 −/− or GSTM1 −/− hESC-derived D18 cells. f Inhibition curve of propargite on INS + cells derived from GSTT1 +/+ or GSTT1 −/− H1 hESCs ( n = 3). g , h Representative images ( g ) and DNA damage rate ( h ) of GSTT1 +/+ and GSTT1 −/− β-like cells ( n = 3). Scale bars, 800 μm. γ-H2A.X + /INS + cells are highlighted with yellow arrows. i Western blot analysis of GSTT1 protein in EndoC-βH1 cells carrying sgGSTT1 . Two CRISPR gRNAs ( sgGSTT1 -1 and sgGSTT1 -2) were used for generating GSTT1 −/− EndoC-βH1 cells. j , k Representative images ( j ) and cell death rate ( k ) of GSTT1 −/− EndoC-βH1 cells treated with 1.6 μM propargite ( n = 3). Scale bars, 200 μm. Values presented as mean ± S.D. n.s. indicates a non-significant difference. p values calculated by unpaired two-tailed Student’s t -test were * p
    Figure Legend Snippet: A hPSC-based population study discovers that GSTT1 -null pancreatic β-like cells are hypersensitive to propargite-induced cell death. a Survival rate of INS + cells derived from 10 different hESC or iPSC lines cultured in the presence of 1.6 μM propargite ( n = 3), and genotype analysis of GSTM1 and GSTT1 in those hESCs and iPSCs. b , c Correlation of INS + cell survival rate in the presence of 1.6 μM propargite in cells lacking both GSTM1 and GSTT1 ( b ), or lacking only GSTM1 ( c ). n.s. indicates a non-significant difference. d Western blotting analysis of GSTT1 or GSTM1 protein expression in INS + cells derived from isogenic wild type, GSTT1 −/− or GSTM1 −/− H1 hESCs. The −/− null clones were CRSIPR-induced biallelic frameshift mutants. The two GSTT1 knockout clones were both homozygous null mutants, and the two GSTM1 knockout clones were both compound-null mutants. e Flow cytometry analysis of C-peptide + cells in isogenic GSTT1 −/− or GSTM1 −/− hESC-derived D18 cells. f Inhibition curve of propargite on INS + cells derived from GSTT1 +/+ or GSTT1 −/− H1 hESCs ( n = 3). g , h Representative images ( g ) and DNA damage rate ( h ) of GSTT1 +/+ and GSTT1 −/− β-like cells ( n = 3). Scale bars, 800 μm. γ-H2A.X + /INS + cells are highlighted with yellow arrows. i Western blot analysis of GSTT1 protein in EndoC-βH1 cells carrying sgGSTT1 . Two CRISPR gRNAs ( sgGSTT1 -1 and sgGSTT1 -2) were used for generating GSTT1 −/− EndoC-βH1 cells. j , k Representative images ( j ) and cell death rate ( k ) of GSTT1 −/− EndoC-βH1 cells treated with 1.6 μM propargite ( n = 3). Scale bars, 200 μm. Values presented as mean ± S.D. n.s. indicates a non-significant difference. p values calculated by unpaired two-tailed Student’s t -test were * p

    Techniques Used: Derivative Assay, Cell Culture, Western Blot, Expressing, Clone Assay, Knock-Out, Flow Cytometry, Cytometry, Inhibition, CRISPR, Two Tailed Test

    8) Product Images from "Towards reducing the immunogenic potential of wheat flour: omega gliadins encoded by the D genome of hexaploid wheat may also harbor epitopes for the serious food allergy WDEIA"

    Article Title: Towards reducing the immunogenic potential of wheat flour: omega gliadins encoded by the D genome of hexaploid wheat may also harbor epitopes for the serious food allergy WDEIA

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-018-1506-z

    PCR analysis with primers specific for the omega-D4 gene from Chinese Spring. Genomic DNA from Keumkang (1), Olgeuru (2), DH20 (3), Chinese Spring (4) and nullisomic tetrasomic lines of Chinese Spring N1AT1B (5), N1AT1D (6), N1BT1A (7), N1BT1D (8), N1DT1A (9) and N1DT1B (10) was amplified. The sizes of molecular weight markers in kb are shown in lane M
    Figure Legend Snippet: PCR analysis with primers specific for the omega-D4 gene from Chinese Spring. Genomic DNA from Keumkang (1), Olgeuru (2), DH20 (3), Chinese Spring (4) and nullisomic tetrasomic lines of Chinese Spring N1AT1B (5), N1AT1D (6), N1BT1A (7), N1BT1D (8), N1DT1A (9) and N1DT1B (10) was amplified. The sizes of molecular weight markers in kb are shown in lane M

    Techniques Used: Polymerase Chain Reaction, Amplification, Molecular Weight

    PCR analysis with primers specific for LMW-GS encoded at the Glu-B3 locus ( a ), omega-5 gliadins ( b ), and repeat junction primers 19S-1.3-2 ( c ) and 143E-1-600 ( d ) located at the ends of a 5.8 Mb region of chromosome 1B in Chinese Spring. In each panel, genomic DNA from Keumkang (1), Olgeuru (2), DH20 (3), N1BT1A (4) and N1BT1D (5) nullisomic tetrasomic lines of Chinese Spring or Chinese Spring (6) was amplified. The sizes of molecular weight markers in kb are shown in lane M in each panel. Primer sequences are shown in Table 2
    Figure Legend Snippet: PCR analysis with primers specific for LMW-GS encoded at the Glu-B3 locus ( a ), omega-5 gliadins ( b ), and repeat junction primers 19S-1.3-2 ( c ) and 143E-1-600 ( d ) located at the ends of a 5.8 Mb region of chromosome 1B in Chinese Spring. In each panel, genomic DNA from Keumkang (1), Olgeuru (2), DH20 (3), N1BT1A (4) and N1BT1D (5) nullisomic tetrasomic lines of Chinese Spring or Chinese Spring (6) was amplified. The sizes of molecular weight markers in kb are shown in lane M in each panel. Primer sequences are shown in Table 2

    Techniques Used: Polymerase Chain Reaction, Amplification, Molecular Weight

    9) Product Images from "Characterization of mucoid and serous middle ear effusions from patients with chronic otitis media: implication of different biological mechanisms?"

    Article Title: Characterization of mucoid and serous middle ear effusions from patients with chronic otitis media: implication of different biological mechanisms?

    Journal: Pediatric research

    doi: 10.1038/s41390-018-0060-6

    Main genera detected by 16S rRNA sequencing. DNA was purified using a column based assay and the 16S rRNA sequencing method was used to detect the genera present in MEEs. Genera with a percentage of reads higher than 5% are shown in this graph. For each sample, the total reads is added on the top of the bars. Genus NC refers to a genus that couldn’t be characterized.
    Figure Legend Snippet: Main genera detected by 16S rRNA sequencing. DNA was purified using a column based assay and the 16S rRNA sequencing method was used to detect the genera present in MEEs. Genera with a percentage of reads higher than 5% are shown in this graph. For each sample, the total reads is added on the top of the bars. Genus NC refers to a genus that couldn’t be characterized.

    Techniques Used: Sequencing, Purification

    10) Product Images from "Breed Differences in PCV2 Uptake and Disintegration in Porcine Monocytes"

    Article Title: Breed Differences in PCV2 Uptake and Disintegration in Porcine Monocytes

    Journal: Viruses

    doi: 10.3390/v10100562

    Fate of the PCV2 genome in blood monocytes. PCV2 was added to monocytes and samples were collected at different time points after virus addition. Afterwards, cells were subjected to DNA extraction. ( A ) The number of PCV2 genome copies at each time point was quantified by qPCR and expressed as a relative percentage to that at 1 h; ( B ) The agarose gel electrophoresis image of PCR amplification results of the full-length PCV2 genome at each time point. The PCR was performed with the primer set wgPCV2-fw/rev which amplifies the full length of PCV2 genome. Data were obtained from three individual pigs (hybrid Piétrain × Hypor Libra).
    Figure Legend Snippet: Fate of the PCV2 genome in blood monocytes. PCV2 was added to monocytes and samples were collected at different time points after virus addition. Afterwards, cells were subjected to DNA extraction. ( A ) The number of PCV2 genome copies at each time point was quantified by qPCR and expressed as a relative percentage to that at 1 h; ( B ) The agarose gel electrophoresis image of PCR amplification results of the full-length PCV2 genome at each time point. The PCR was performed with the primer set wgPCV2-fw/rev which amplifies the full length of PCV2 genome. Data were obtained from three individual pigs (hybrid Piétrain × Hypor Libra).

    Techniques Used: DNA Extraction, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification

    11) Product Images from "Multiple endocrine neoplasia 2 in Cyprus: evidence for a founder effect"

    Article Title: Multiple endocrine neoplasia 2 in Cyprus: evidence for a founder effect

    Journal: Journal of Endocrinological Investigation

    doi: 10.1007/s40618-018-0841-0

    Pedigree and disease-haplotype segregation of families with the RET proto-oncogene p.Cys618Arg missense mutation. Blackened symbols represent affected individuals with MEN2A phenotype. White symbols represent individuals with a normal phenotype. Circles and squares indicate females and males, respectively. Red characters represent the mutant haplotype which segregates with the p.Cys618Arg missense mutation
    Figure Legend Snippet: Pedigree and disease-haplotype segregation of families with the RET proto-oncogene p.Cys618Arg missense mutation. Blackened symbols represent affected individuals with MEN2A phenotype. White symbols represent individuals with a normal phenotype. Circles and squares indicate females and males, respectively. Red characters represent the mutant haplotype which segregates with the p.Cys618Arg missense mutation

    Techniques Used: Mutagenesis

    12) Product Images from "Genome and epigenome analysis of monozygotic twins discordant for congenital heart disease"

    Article Title: Genome and epigenome analysis of monozygotic twins discordant for congenital heart disease

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-4814-7

    DNA methylation and gene expression detection of ZIC3 and NR2F2 from clinical cases. ( a and c ) Statistical summaries about DNA methylation status of DMRs in ZIC3 and NR2F2 in 20 clinical samples, consisting of five normal providers and fifteen DORV patients. ( b and d ) Diagrams exhibiting average methylated levels of individual CpG sites in DMRs of ZIC3 and NR2F2 from the indicated groups, respectively. ( e and f ) Histograms showing relative gene expression levels of ZIC3 and NR2F2 in different groups of specimens. ( g and h ) Scatterplots showing the gene expression levels of ZIC3 ( g ) and NR2F2 ( h ) are negatively correlated with their promoter methylation status. Pearson’s correlation coefficient and p -values were listed above the plot
    Figure Legend Snippet: DNA methylation and gene expression detection of ZIC3 and NR2F2 from clinical cases. ( a and c ) Statistical summaries about DNA methylation status of DMRs in ZIC3 and NR2F2 in 20 clinical samples, consisting of five normal providers and fifteen DORV patients. ( b and d ) Diagrams exhibiting average methylated levels of individual CpG sites in DMRs of ZIC3 and NR2F2 from the indicated groups, respectively. ( e and f ) Histograms showing relative gene expression levels of ZIC3 and NR2F2 in different groups of specimens. ( g and h ) Scatterplots showing the gene expression levels of ZIC3 ( g ) and NR2F2 ( h ) are negatively correlated with their promoter methylation status. Pearson’s correlation coefficient and p -values were listed above the plot

    Techniques Used: DNA Methylation Assay, Expressing, Methylation

    13) Product Images from "Efficient production of human interferon beta in the white of eggs from ovalbumin gene–targeted hens"

    Article Title: Efficient production of human interferon beta in the white of eggs from ovalbumin gene–targeted hens

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-28438-2

    CRISPR/Cas9-mediated human IFN- β knock-in at the OVA locus in chicken PGCs. ( a ) Schematic of the knock-in strategy. The top diagram shows the WT chicken OVA locus. The target single-guide RNA (sgRNA) sequence that is part of exon 2 is denoted by the black bar above the nucleotide sequence. The protospacer adjacent-motif sequence is indicated by the red bar. The OVA initiation codon is shown in uppercase letters. The middle diagram shows the donor construct containing the 5′ and 3′ homology regions (HR), the hIFN- β -bovine growth hormone polyadenylation signal construct, and the PGK promoter that drives the puromycin resistance gene (PGK-Puro r ). The bottom diagram shows the KI allele along with PCR primers P1 to P9 that were used for 5′, 3′, and endogenous OVA assays in this study. ( b ) PCR amplification of the donor cassette knock-in at the OVA locus in the PGC genome. KI PGCs and their parental untransfected cells (UT) were subjected to nested PCR using primers P1–4 (5′ assay) and P5–8 (3′ assay). The middle lane, labeled M, contains DNA molecular mass markers (1-kbp DNA ladder, Nacalai). PCR amplicons of the expected sizes (2.8 kb for the 5′ assay, and 3.2 kb for the 3′ assay) are indicated by the arrows.
    Figure Legend Snippet: CRISPR/Cas9-mediated human IFN- β knock-in at the OVA locus in chicken PGCs. ( a ) Schematic of the knock-in strategy. The top diagram shows the WT chicken OVA locus. The target single-guide RNA (sgRNA) sequence that is part of exon 2 is denoted by the black bar above the nucleotide sequence. The protospacer adjacent-motif sequence is indicated by the red bar. The OVA initiation codon is shown in uppercase letters. The middle diagram shows the donor construct containing the 5′ and 3′ homology regions (HR), the hIFN- β -bovine growth hormone polyadenylation signal construct, and the PGK promoter that drives the puromycin resistance gene (PGK-Puro r ). The bottom diagram shows the KI allele along with PCR primers P1 to P9 that were used for 5′, 3′, and endogenous OVA assays in this study. ( b ) PCR amplification of the donor cassette knock-in at the OVA locus in the PGC genome. KI PGCs and their parental untransfected cells (UT) were subjected to nested PCR using primers P1–4 (5′ assay) and P5–8 (3′ assay). The middle lane, labeled M, contains DNA molecular mass markers (1-kbp DNA ladder, Nacalai). PCR amplicons of the expected sizes (2.8 kb for the 5′ assay, and 3.2 kb for the 3′ assay) are indicated by the arrows.

    Techniques Used: CRISPR, Knock-In, Sequencing, Construct, Polymerase Chain Reaction, Amplification, Pyrolysis Gas Chromatography, Nested PCR, Labeling

    CRISPR/Cas9-mediated knock-in of hIFN- β at the chicken OVA locus. ( a ) Schematic of the experimental procedure that generated hIFN- β KI chickens. ( b ) KI hIFN- β in semen of chimeric G0 roosters. Genomic DNA from the sperm of four chimeric roosters (411–414) and a WT rooster was PCR amplified with primer pairs P5/P8, P1/P4, and P1/P9 for the 3′ and 5′ assays and the endogenous OVA assay (O), respectively. Genomic DNA from transplanted PGCs containing hIFN- β KI cells (KI PGC) was also PCR amplified. The gels show the PCR-amplified products. ( c ) KI hIFN- β in the G1 chickens. Genomic DNA from the blood of the G1 progenies of #411 (left panel) and #412 (right panel) was PCR amplified for the 3′, 5′, and endogenous OVA assays using primer pairs P5/P8, P1/P4, and P1/P9, respectively. The genomic DNA from the blood of WT chickens and from the transplanted PGCs (KI PGC) was also PCR amplified. The gels show the PCR-amplified products. The lanes at the left of each gel panel are the DNA molecular mass markers as described in Fig. 1.
    Figure Legend Snippet: CRISPR/Cas9-mediated knock-in of hIFN- β at the chicken OVA locus. ( a ) Schematic of the experimental procedure that generated hIFN- β KI chickens. ( b ) KI hIFN- β in semen of chimeric G0 roosters. Genomic DNA from the sperm of four chimeric roosters (411–414) and a WT rooster was PCR amplified with primer pairs P5/P8, P1/P4, and P1/P9 for the 3′ and 5′ assays and the endogenous OVA assay (O), respectively. Genomic DNA from transplanted PGCs containing hIFN- β KI cells (KI PGC) was also PCR amplified. The gels show the PCR-amplified products. ( c ) KI hIFN- β in the G1 chickens. Genomic DNA from the blood of the G1 progenies of #411 (left panel) and #412 (right panel) was PCR amplified for the 3′, 5′, and endogenous OVA assays using primer pairs P5/P8, P1/P4, and P1/P9, respectively. The genomic DNA from the blood of WT chickens and from the transplanted PGCs (KI PGC) was also PCR amplified. The gels show the PCR-amplified products. The lanes at the left of each gel panel are the DNA molecular mass markers as described in Fig. 1.

    Techniques Used: CRISPR, Knock-In, Generated, Polymerase Chain Reaction, Amplification, Pyrolysis Gas Chromatography

    14) Product Images from "ETS transcription factors induce a unique UV damage signature that drives recurrent mutagenesis in melanoma"

    Article Title: ETS transcription factors induce a unique UV damage signature that drives recurrent mutagenesis in melanoma

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05064-0

    Genome-wide map of CPD lesions reveals that CPDs are elevated at active TFBS. a Schematic diagram of the CPD-seq method for mapping CPD lesions at single nucleotide resolution. ‘T = C′ indicates a CPD lesion at TC dipyrimidine. Oligonucleotide adapters are indicated in green and purple; ‘NNNNNN′ indicates a random DNA hexamer. A 3′ hydroxyl is indicated with OH, while ‘dd’ indicates a dideoxy 3′ end. The CPD lesion is cleaved with T4 endonuclease V and apurinic/apyrimidinic endonuclease (APE1) to generate a free 3′ hydroxyl immediately upstream of the CPD lesion, which is ligated to an adapter and sequenced. b Mutation density surrounding active promoter-proximal TFBS from 184 sequenced melanoma tumors 18 . Observed mutation density (i.e., in melanoma tumors) was analyzed adjacent to known TFBS located in promoter-proximal regions (up to 2500 bp upstream of transcription start site) that were considered active (i.e., overlapping with melanocyte DNase I-hypersensitivity (DHS) regions) for 82 distinct TFs. Expected mutation density was determined from the corresponding DNA sequences surrounding each active promoter-proximal TFBS, based on the trinucleotide mutation signature frequencies for all promoter-proximal regions. c Same as part ( b ), except mutations were analyzed adjacent to promoter-proximal TFBS that were considered inactive (i.e., not overlapping with melanocyte DHS regions). d Average number of CPD lesions (per TFBS) adjacent to active promoter-proximal TFBS. CPD lesions were mapped using CPD-seq from UV-irradiated NHF1 cells (100 J m −2 ) or isolated NHF1 DNA that was UV-irradiated (80 J m −2 ) in vitro (naked DNA). Cellular DNA was harvested immediately after UV irradiation, so essentially no repair was allowed to occur. e Same as in part ( d ), except CPD lesions were analyzed adjacent to inactive promoter-proximal TFBS
    Figure Legend Snippet: Genome-wide map of CPD lesions reveals that CPDs are elevated at active TFBS. a Schematic diagram of the CPD-seq method for mapping CPD lesions at single nucleotide resolution. ‘T = C′ indicates a CPD lesion at TC dipyrimidine. Oligonucleotide adapters are indicated in green and purple; ‘NNNNNN′ indicates a random DNA hexamer. A 3′ hydroxyl is indicated with OH, while ‘dd’ indicates a dideoxy 3′ end. The CPD lesion is cleaved with T4 endonuclease V and apurinic/apyrimidinic endonuclease (APE1) to generate a free 3′ hydroxyl immediately upstream of the CPD lesion, which is ligated to an adapter and sequenced. b Mutation density surrounding active promoter-proximal TFBS from 184 sequenced melanoma tumors 18 . Observed mutation density (i.e., in melanoma tumors) was analyzed adjacent to known TFBS located in promoter-proximal regions (up to 2500 bp upstream of transcription start site) that were considered active (i.e., overlapping with melanocyte DNase I-hypersensitivity (DHS) regions) for 82 distinct TFs. Expected mutation density was determined from the corresponding DNA sequences surrounding each active promoter-proximal TFBS, based on the trinucleotide mutation signature frequencies for all promoter-proximal regions. c Same as part ( b ), except mutations were analyzed adjacent to promoter-proximal TFBS that were considered inactive (i.e., not overlapping with melanocyte DHS regions). d Average number of CPD lesions (per TFBS) adjacent to active promoter-proximal TFBS. CPD lesions were mapped using CPD-seq from UV-irradiated NHF1 cells (100 J m −2 ) or isolated NHF1 DNA that was UV-irradiated (80 J m −2 ) in vitro (naked DNA). Cellular DNA was harvested immediately after UV irradiation, so essentially no repair was allowed to occur. e Same as in part ( d ), except CPD lesions were analyzed adjacent to inactive promoter-proximal TFBS

    Techniques Used: Genome Wide, Mutagenesis, Irradiation, Isolation, In Vitro

    CPDs and mutations are elevated at specific locations in ETS binding sites. a UV-induced CPD formation and mutation density is enriched at 1279 active, promoter-proximal TFBS for the ETS TFs ELK4, ETS1, and GABPA. TFBS were aligned based on DNA strand and location of ETS consensus sequence. The upper panel depicts the information content of the DNA sequences for the aligned TFBS, matching the known ETS consensus motif. Sequence logo was generated using the weblogo tool 57 . The lower panel plots the mutation density for 184 melanoma tumors relative to average CPD levels following UV-irradiation of NHF1 cells and isolated DNA in vitro (naked DNA). CPD values are plotted at half integer locations, which reflect the average number of CPD lesions forming between the two adjacent nucleotides. CPD lesion density for naked DNA was scaled so that the total CPD levels in promoter-proximal regions in cells and naked DNA were equivalent. b Analysis of CPD lesion formation and mutation density at a subset of ETS TFBS (156 sites) that have a pyrimidine nucleotide at position −4 relative to the ETS motif midpoint (indicated with *), and thus can form CPD lesions between position −4/−3. Upper and lower panels are the same as in part ( a ), plotted for this TFBS subset
    Figure Legend Snippet: CPDs and mutations are elevated at specific locations in ETS binding sites. a UV-induced CPD formation and mutation density is enriched at 1279 active, promoter-proximal TFBS for the ETS TFs ELK4, ETS1, and GABPA. TFBS were aligned based on DNA strand and location of ETS consensus sequence. The upper panel depicts the information content of the DNA sequences for the aligned TFBS, matching the known ETS consensus motif. Sequence logo was generated using the weblogo tool 57 . The lower panel plots the mutation density for 184 melanoma tumors relative to average CPD levels following UV-irradiation of NHF1 cells and isolated DNA in vitro (naked DNA). CPD values are plotted at half integer locations, which reflect the average number of CPD lesions forming between the two adjacent nucleotides. CPD lesion density for naked DNA was scaled so that the total CPD levels in promoter-proximal regions in cells and naked DNA were equivalent. b Analysis of CPD lesion formation and mutation density at a subset of ETS TFBS (156 sites) that have a pyrimidine nucleotide at position −4 relative to the ETS motif midpoint (indicated with *), and thus can form CPD lesions between position −4/−3. Upper and lower panels are the same as in part ( a ), plotted for this TFBS subset

    Techniques Used: Binding Assay, Mutagenesis, Sequencing, Generated, Irradiation, Isolation, In Vitro

    ETS family TFBS are the primary contributors to elevated CPD levels. a, b Formation of CPD ( a ) and mCPD ( b ) lesions is significantly stimulated at active, promoter-proximal ETS family TFBS (i.e., ELF1, ELK4, ETS1, and GABPA) in UV-irradiated NHF1 cells, but not when isolated NHF1 DNA was UV-irradiated in vitro (naked DNA). c Mutation density is significantly elevated at active promoter-proximal ETS family TFBS (i.e., ELF1, ELK4, ETS1, and GABPA) in melanoma tumors, correlating with the higher initial mCPD lesion density at these sites. d, e Formation of CPD ( d ) and mCPD ( e ) lesions is not significantly stimulated at active promoter-proximal TFBS after excluding ELF1, ELK4, ETS1, and GABPA binding sites. f Mutation density is only slightly elevated surrounding non-ETS family TFBS in aggregate (see inset with expanded scale). Only active promoter-proximal TFBS were analyzed. g CPD repair activity is elevated at ETS family TFBS following UV irradiation of human cells. Average CPD repair activity at 1 h repair in UV-irradiated NHF1 cells at ETS family TFBS (i.e., ELF1, ELK4, ETS1, and GABPA). CPD repair activity was calculated using the average number of XR-seq reads 5 at locations surrounding active, promoter-proximal ETS binding sites. XR-seq reads were localized to the putative dipyrimidine lesion associated with each sequencing read. h Same as in g , except repair activity for active ETS family TFBS located outside promoter regions was analyzed. i Same as ( g ), except CPD repair activity was analyzed at non-ETS family TFBS
    Figure Legend Snippet: ETS family TFBS are the primary contributors to elevated CPD levels. a, b Formation of CPD ( a ) and mCPD ( b ) lesions is significantly stimulated at active, promoter-proximal ETS family TFBS (i.e., ELF1, ELK4, ETS1, and GABPA) in UV-irradiated NHF1 cells, but not when isolated NHF1 DNA was UV-irradiated in vitro (naked DNA). c Mutation density is significantly elevated at active promoter-proximal ETS family TFBS (i.e., ELF1, ELK4, ETS1, and GABPA) in melanoma tumors, correlating with the higher initial mCPD lesion density at these sites. d, e Formation of CPD ( d ) and mCPD ( e ) lesions is not significantly stimulated at active promoter-proximal TFBS after excluding ELF1, ELK4, ETS1, and GABPA binding sites. f Mutation density is only slightly elevated surrounding non-ETS family TFBS in aggregate (see inset with expanded scale). Only active promoter-proximal TFBS were analyzed. g CPD repair activity is elevated at ETS family TFBS following UV irradiation of human cells. Average CPD repair activity at 1 h repair in UV-irradiated NHF1 cells at ETS family TFBS (i.e., ELF1, ELK4, ETS1, and GABPA). CPD repair activity was calculated using the average number of XR-seq reads 5 at locations surrounding active, promoter-proximal ETS binding sites. XR-seq reads were localized to the putative dipyrimidine lesion associated with each sequencing read. h Same as in g , except repair activity for active ETS family TFBS located outside promoter regions was analyzed. i Same as ( g ), except CPD repair activity was analyzed at non-ETS family TFBS

    Techniques Used: Irradiation, Isolation, In Vitro, Mutagenesis, Binding Assay, Activity Assay, Sequencing

    15) Product Images from "Mitochondrial DNA Leakage Caused by Streptococcus pneumoniae Hydrogen Peroxide Promotes Type I IFN Expression in Lung Cells"

    Article Title: Mitochondrial DNA Leakage Caused by Streptococcus pneumoniae Hydrogen Peroxide Promotes Type I IFN Expression in Lung Cells

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2019.00630

    S. pn -secreted H 2 O 2 caused oxidative damage of mitochondrial DNA. (A) A549 cells were stimulated with D39 with or without catalase (Cat) and D39Δ spxB (MOI = 200), as well as 1 mM H 2 O 2 for 2 h, representative images of MitoTracker red (red), 8-OHdG immunostaining (green), and DAPI (blue) in A549 cells. The orange in the merged images of green and red fluorescence indicates 8-OHdG-positive cells. (B) A549 cells were infected with D39 (MOI = 200) at indicated time points, mtDNA copy number was analyzed by real-time PCR. (C) A549 cells were stimulated with D39 with or without catalase (Cat) and D39Δ spxB (MOI = 200), as well as 1 mM H 2 O 2 for 2 h, mtDNA copy number (left panel) and mtDNA transcript level (right panel) was analyzed by real-time PCR. (D) Female C57BL/6 mice were intranasally infected with D39 and D39Δ spxB (1 × 10 8 CFU) for 24 h, catalase was given intravenously (at 6, 12, 18, 22, 23, and 24 h) in the other five mice inoculated with 1 × 10 8 CFU of D39. mtDNA copy number (left panel) and mtDNA transcript level (right panel) were analyzed by real-time PCR. mtDNA level was normalized to the internal control GAPDH. Mitochondrial genes CoxIII and Cytb were chosen to indicate mtDNA transcription. NC, negative control. All data were presented as means ± SD from three independent experiments. ∗ P
    Figure Legend Snippet: S. pn -secreted H 2 O 2 caused oxidative damage of mitochondrial DNA. (A) A549 cells were stimulated with D39 with or without catalase (Cat) and D39Δ spxB (MOI = 200), as well as 1 mM H 2 O 2 for 2 h, representative images of MitoTracker red (red), 8-OHdG immunostaining (green), and DAPI (blue) in A549 cells. The orange in the merged images of green and red fluorescence indicates 8-OHdG-positive cells. (B) A549 cells were infected with D39 (MOI = 200) at indicated time points, mtDNA copy number was analyzed by real-time PCR. (C) A549 cells were stimulated with D39 with or without catalase (Cat) and D39Δ spxB (MOI = 200), as well as 1 mM H 2 O 2 for 2 h, mtDNA copy number (left panel) and mtDNA transcript level (right panel) was analyzed by real-time PCR. (D) Female C57BL/6 mice were intranasally infected with D39 and D39Δ spxB (1 × 10 8 CFU) for 24 h, catalase was given intravenously (at 6, 12, 18, 22, 23, and 24 h) in the other five mice inoculated with 1 × 10 8 CFU of D39. mtDNA copy number (left panel) and mtDNA transcript level (right panel) were analyzed by real-time PCR. mtDNA level was normalized to the internal control GAPDH. Mitochondrial genes CoxIII and Cytb were chosen to indicate mtDNA transcription. NC, negative control. All data were presented as means ± SD from three independent experiments. ∗ P

    Techniques Used: Immunostaining, Fluorescence, Infection, Real-time Polymerase Chain Reaction, Mouse Assay, Negative Control

    mtDNA leakage caused by S. pn -secreted H 2 O 2 was involved in IFN-I induction. (A) A549 cells were infected with D39 (MOI = 200) at indicated time points, DNA in the cytosolic fraction was isolated, and the copy number of mtDNA (mtDNA sequences as primers) was measured and normalized with the copy number of nuclear DNA (nuclear DNA sequences as primers). (B) A549 cells were stimulated with D39 with or without catalase (Cat) and D39Δ spxB (MOI = 200), as well as 1 mM H 2 O 2 for 2 h, DNA in the cytosolic fraction was isolated, and the copy number of mtDNA was measured and normalized with GAPDH. (C) A549 cells were transfected with cytosolic DNA isolated from different stimulations, including D39 with or without catalase (Cat) and D39Δ spxB , as well as H 2 O 2 , IFNβ mRNA level were determined by real-time PCR. POLY (dA: dT) (2 μg/ml) was applied as positive control. (D) mtDNA in A549 cells were evaluated by real-time PCR after being treated with EtBr (300 ng/ml) for 5 days (left panel). IFNβ mRNA level in A549 cells treated with 1 mM H 2 O 2 or D39 (MOI = 200) were determined by real-time PCR (right panel). NC, negative control. All data were presented as means ± SD from three independent experiments. ∗ P
    Figure Legend Snippet: mtDNA leakage caused by S. pn -secreted H 2 O 2 was involved in IFN-I induction. (A) A549 cells were infected with D39 (MOI = 200) at indicated time points, DNA in the cytosolic fraction was isolated, and the copy number of mtDNA (mtDNA sequences as primers) was measured and normalized with the copy number of nuclear DNA (nuclear DNA sequences as primers). (B) A549 cells were stimulated with D39 with or without catalase (Cat) and D39Δ spxB (MOI = 200), as well as 1 mM H 2 O 2 for 2 h, DNA in the cytosolic fraction was isolated, and the copy number of mtDNA was measured and normalized with GAPDH. (C) A549 cells were transfected with cytosolic DNA isolated from different stimulations, including D39 with or without catalase (Cat) and D39Δ spxB , as well as H 2 O 2 , IFNβ mRNA level were determined by real-time PCR. POLY (dA: dT) (2 μg/ml) was applied as positive control. (D) mtDNA in A549 cells were evaluated by real-time PCR after being treated with EtBr (300 ng/ml) for 5 days (left panel). IFNβ mRNA level in A549 cells treated with 1 mM H 2 O 2 or D39 (MOI = 200) were determined by real-time PCR (right panel). NC, negative control. All data were presented as means ± SD from three independent experiments. ∗ P

    Techniques Used: Infection, Isolation, Transfection, Real-time Polymerase Chain Reaction, Positive Control, Negative Control

    16) Product Images from "Gene editing enables T-cell engineering to redirect antigen specificity for potent tumor rejection"

    Article Title: Gene editing enables T-cell engineering to redirect antigen specificity for potent tumor rejection

    Journal: Life Science Alliance

    doi: 10.26508/lsa.201900367

    CRISPR-Cas9- and AAV-mediated TCR replacement. (A) Flow cytometry analysis of primary human CD8 T cells electroporated with RNPs with an α- TRAC gRNA or a non-targeting (N.T.) gRNA at day 7 after electroporation (data represent three donors in two independent experiments, n = 6). (B) ddPCR quantification of the percentage of edited TRAC alleles on day 7 ( n = 3 donors) with 10 ng genomic DNA input. (C) Representative ddPCR plots are shown. x and y axes show fluorescence intensity (arbitrary units). (D) Schematic representation of the human TRAC locus (top), the recombinant AAV6 targeting construct encoding the exogenous TCR (middle) and the successfully edited TRAC locus (bottom). LHA, about 900-bp-long left homology arm; RHA, about 900-bp-long right homology arm. (E) Representative FACS plots of primary CD8 T cells electroporated with α- TRAC or N.T. gRNA and transduced with AAV (MOI = 10 6 ) or PBS or γ-retrovirally transduced on day 7 after electroporation or transduction. Axes use biexponential scaling. Graphs are 10% contour plots with outliers displayed. (F) Flow cytometry analysis of KI- TRAC -TCR cells (data represent three donors in two independent experiments, n = 6), γ-retrovirally ( n = 3 donors), or mock-transduced cells ( n = 3 donors). (G) ddPCR quantification of the targeted integration efficiency with assays spanning the left (LHA-assay) or right homology arm (RHA-assay). (H) Representative ddPCR plots are shown. y axis shows fluorescence intensity (arbitrary units). (I, F) Flow cytometry analysis as in (F) ( n = 3 donors). Asterisks indicate statistical significance as determined by two-tailed unpaired t test. See also Fig S1 .
    Figure Legend Snippet: CRISPR-Cas9- and AAV-mediated TCR replacement. (A) Flow cytometry analysis of primary human CD8 T cells electroporated with RNPs with an α- TRAC gRNA or a non-targeting (N.T.) gRNA at day 7 after electroporation (data represent three donors in two independent experiments, n = 6). (B) ddPCR quantification of the percentage of edited TRAC alleles on day 7 ( n = 3 donors) with 10 ng genomic DNA input. (C) Representative ddPCR plots are shown. x and y axes show fluorescence intensity (arbitrary units). (D) Schematic representation of the human TRAC locus (top), the recombinant AAV6 targeting construct encoding the exogenous TCR (middle) and the successfully edited TRAC locus (bottom). LHA, about 900-bp-long left homology arm; RHA, about 900-bp-long right homology arm. (E) Representative FACS plots of primary CD8 T cells electroporated with α- TRAC or N.T. gRNA and transduced with AAV (MOI = 10 6 ) or PBS or γ-retrovirally transduced on day 7 after electroporation or transduction. Axes use biexponential scaling. Graphs are 10% contour plots with outliers displayed. (F) Flow cytometry analysis of KI- TRAC -TCR cells (data represent three donors in two independent experiments, n = 6), γ-retrovirally ( n = 3 donors), or mock-transduced cells ( n = 3 donors). (G) ddPCR quantification of the targeted integration efficiency with assays spanning the left (LHA-assay) or right homology arm (RHA-assay). (H) Representative ddPCR plots are shown. y axis shows fluorescence intensity (arbitrary units). (I, F) Flow cytometry analysis as in (F) ( n = 3 donors). Asterisks indicate statistical significance as determined by two-tailed unpaired t test. See also Fig S1 .

    Techniques Used: CRISPR, Flow Cytometry, Cytometry, Electroporation, Fluorescence, Recombinant, Construct, FACS, Transduction, Two Tailed Test

    Quantification of gene-editing frequency. (A, B) ddPCR quantification of the percentage of edited TRAC alleles on day 7 ( n = 3 donors) with 100 ng genomic DNA input (B) original ddPCR plots of the data summarized in (A). Asterisks indicate statistical significance as determined by two-tailed unpaired t test.
    Figure Legend Snippet: Quantification of gene-editing frequency. (A, B) ddPCR quantification of the percentage of edited TRAC alleles on day 7 ( n = 3 donors) with 100 ng genomic DNA input (B) original ddPCR plots of the data summarized in (A). Asterisks indicate statistical significance as determined by two-tailed unpaired t test.

    Techniques Used: Two Tailed Test

    17) Product Images from "Accurate classification of BRCA1 variants with saturation genome editing"

    Article Title: Accurate classification of BRCA1 variants with saturation genome editing

    Journal: Nature

    doi: 10.1038/s41586-018-0461-z

    HAP1 cell line optimizations for saturation genome editing to assay essential genes. a , A gRNA targeting Cas9 to the coding sequence of LIG4 , a gene integral to the non-homologous end-joining pathway, was cloned into a vector co-expressing Cas9–2A-GFP 24 . WT HAP1 cells were transfected, and single GFP-expressing cells were sorted into wells of a 96-well plate. Eight monoclonal lines were grown out over a period of three weeks and screened using Sanger sequencing for frameshifting indels in LIG4 . The Sanger trace shows the frameshifting deletion present in the clonal line chosen for subsequent experiments, referred to as ‘HAP1-Lig4KO’. b , To purify HAP1 cells for haploid cells, live cells were stained for DNA content with Hoechst 34580 and sorted using a gate to select cells with the lowest DNA content, corresponding to 1N cells in G1. c , The fraction of all possible SNVs scored is shown for each exon. SNVs were excluded mainly due to proximity to the HDR marker and/or poor sampling (Methods). d , e , Measurements across replicates are plotted for exon 17 SNVs assayed in HAP1-Lig4KO cells to show correlations of day 5 frequencies ( d ) and day 11 over library ratios ( e ). f–h , Plots comparing SNV function scores across replicate experiments for exon 17 saturation genome editing experiments performed in unsorted WT HAP1 cells ( f ), HAP1-Lig4KO cells ( g ), and WT HAP1 cells sorted on 1N ploidy ( h ). i , Function scores (averaged across replicates) are plotted to compare results for exon 17 experiments performed in WT 1N-sorted HAP1 cells and HAP1-Lig4KO cells. The number of SNVs plotted and the Spearman correlation is displayed for each plot (d-i).
    Figure Legend Snippet: HAP1 cell line optimizations for saturation genome editing to assay essential genes. a , A gRNA targeting Cas9 to the coding sequence of LIG4 , a gene integral to the non-homologous end-joining pathway, was cloned into a vector co-expressing Cas9–2A-GFP 24 . WT HAP1 cells were transfected, and single GFP-expressing cells were sorted into wells of a 96-well plate. Eight monoclonal lines were grown out over a period of three weeks and screened using Sanger sequencing for frameshifting indels in LIG4 . The Sanger trace shows the frameshifting deletion present in the clonal line chosen for subsequent experiments, referred to as ‘HAP1-Lig4KO’. b , To purify HAP1 cells for haploid cells, live cells were stained for DNA content with Hoechst 34580 and sorted using a gate to select cells with the lowest DNA content, corresponding to 1N cells in G1. c , The fraction of all possible SNVs scored is shown for each exon. SNVs were excluded mainly due to proximity to the HDR marker and/or poor sampling (Methods). d , e , Measurements across replicates are plotted for exon 17 SNVs assayed in HAP1-Lig4KO cells to show correlations of day 5 frequencies ( d ) and day 11 over library ratios ( e ). f–h , Plots comparing SNV function scores across replicate experiments for exon 17 saturation genome editing experiments performed in unsorted WT HAP1 cells ( f ), HAP1-Lig4KO cells ( g ), and WT HAP1 cells sorted on 1N ploidy ( h ). i , Function scores (averaged across replicates) are plotted to compare results for exon 17 experiments performed in WT 1N-sorted HAP1 cells and HAP1-Lig4KO cells. The number of SNVs plotted and the Spearman correlation is displayed for each plot (d-i).

    Techniques Used: Sequencing, Non-Homologous End Joining, Clone Assay, Plasmid Preparation, Expressing, Transfection, Staining, Marker, Sampling

    18) Product Images from "Performance evaluation of commercial library construction kits for PCR-based targeted sequencing using a unique molecular identifier"

    Article Title: Performance evaluation of commercial library construction kits for PCR-based targeted sequencing using a unique molecular identifier

    Journal: BMC Genomics

    doi: 10.1186/s12864-019-5583-7

    Performance evaluation of Qiagen HASTP kit. a Stacked bar plot showing the fractions of filtered reads (i.e., unaligned, duplicated, and off-target reads) and reads remaining after filtering (i.e., on-target) during raw data processing for five commercial kits with and without UMIs for deduplication. b Mean depth of unique coverage after filtering with UMIs according to the initial genomic DNA amount. c Correlation between expected allele frequencies of variants in reference material and observed allele frequencies of variants from Qiagen HASTP
    Figure Legend Snippet: Performance evaluation of Qiagen HASTP kit. a Stacked bar plot showing the fractions of filtered reads (i.e., unaligned, duplicated, and off-target reads) and reads remaining after filtering (i.e., on-target) during raw data processing for five commercial kits with and without UMIs for deduplication. b Mean depth of unique coverage after filtering with UMIs according to the initial genomic DNA amount. c Correlation between expected allele frequencies of variants in reference material and observed allele frequencies of variants from Qiagen HASTP

    Techniques Used:

    19) Product Images from "ASF1a inhibition induces p53-dependent growth arrest and senescence of cancer cells"

    Article Title: ASF1a inhibition induces p53-dependent growth arrest and senescence of cancer cells

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-019-1357-z

    Silencing ASF1a triggers DNA damage response. a Immunofluorescence staining of control (nc) and ASF1a knockdown (ASF1a si1/si2) groups of HepG2 and LNCaP cells. Nuclei were stained with DAPI (blue signals). γH2AX and 53BP1 were stained with specific antibodies (green and red signals, respectively; scale bar: 50 μm). Quantification is shown at the bottom (data are presented as the mean ± SD value of three independent experiments for HepG2 and LNCaP, respectively). Senescence-associated heterochromatin foci (SAHF) were not detected by using DAPI staining. DAPI, 4-6-diamidino-2-phenylindole dihydrochloride
    Figure Legend Snippet: Silencing ASF1a triggers DNA damage response. a Immunofluorescence staining of control (nc) and ASF1a knockdown (ASF1a si1/si2) groups of HepG2 and LNCaP cells. Nuclei were stained with DAPI (blue signals). γH2AX and 53BP1 were stained with specific antibodies (green and red signals, respectively; scale bar: 50 μm). Quantification is shown at the bottom (data are presented as the mean ± SD value of three independent experiments for HepG2 and LNCaP, respectively). Senescence-associated heterochromatin foci (SAHF) were not detected by using DAPI staining. DAPI, 4-6-diamidino-2-phenylindole dihydrochloride

    Techniques Used: Immunofluorescence, Staining

    20) Product Images from "Reduced gut microbiome protects from alcohol-induced neuroinflammation and alters intestinal and brain inflammasome expression"

    Article Title: Reduced gut microbiome protects from alcohol-induced neuroinflammation and alters intestinal and brain inflammasome expression

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-018-1328-9

    Oral antibiotics significantly reduce the gut bacterial load. a Four groups of wild-type C57BL/6J female mice were treated with pair-fed diet (PF; n = 5), 5% alcohol diet (EtOH; n = 10), oral antibiotics (Abx) with PF ( n = 6), or Abx with EtOH ( n = 9). An acute sugar or alcohol binge was given 9 h before sacrifice. b Serum endotoxin was measured at sacrifice to determine translocation of gut bacterial products into systemic circulation. c DNA was isolated from the stool of PF and EtOH mice before sacrifice, and 16S DNA was measured by qPCR using universal 16S primers. d The PCR products from c were run on an agarose gel for a general comparison of the four groups. e Stools were resuspended in thioglycolate and plated on non-selective agar to measure gut bacterial load prior to antibiotic treatment (untreated), after 5 days of Abx treatment (Abx day 5), and at the end of the experiment (Abx day 15). f Colony-forming units (CFUs) were quantified from stool extracted at sacrifice on day 15. Data are mean ± SEM, n = 5–10 mice/group. * p
    Figure Legend Snippet: Oral antibiotics significantly reduce the gut bacterial load. a Four groups of wild-type C57BL/6J female mice were treated with pair-fed diet (PF; n = 5), 5% alcohol diet (EtOH; n = 10), oral antibiotics (Abx) with PF ( n = 6), or Abx with EtOH ( n = 9). An acute sugar or alcohol binge was given 9 h before sacrifice. b Serum endotoxin was measured at sacrifice to determine translocation of gut bacterial products into systemic circulation. c DNA was isolated from the stool of PF and EtOH mice before sacrifice, and 16S DNA was measured by qPCR using universal 16S primers. d The PCR products from c were run on an agarose gel for a general comparison of the four groups. e Stools were resuspended in thioglycolate and plated on non-selective agar to measure gut bacterial load prior to antibiotic treatment (untreated), after 5 days of Abx treatment (Abx day 5), and at the end of the experiment (Abx day 15). f Colony-forming units (CFUs) were quantified from stool extracted at sacrifice on day 15. Data are mean ± SEM, n = 5–10 mice/group. * p

    Techniques Used: Mouse Assay, Translocation Assay, Isolation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    21) Product Images from "IFNα Impairs Autophagic Degradation of mtDNA Promoting Autoreactivity of SLE Monocytes in a STING-Dependent Fashion"

    Article Title: IFNα Impairs Autophagic Degradation of mtDNA Promoting Autoreactivity of SLE Monocytes in a STING-Dependent Fashion

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2018.09.001

    IFNα-Mediated Lysosomal Dysfunction Impedes Mitochondrial Clearance and Leads to mtDNA Accumulation (Α) Confocal microscopy for Mitotracker CMXRos dye staining in freshly isolated CD14 + monocytes from healthy donors (n = 8) and SLE patients (n = 8). One representative result is depicted. Averages of MFI per cell are graphed. Datasets were analyzed using non-parametric Mann-Whitney U test. (Β) TaqMan qPCR analysis for mtDNA content in freshly isolated monocytes of healthy donors (n = 5) and SLE patients (n = 5), expressed as a ratio of the Cq values of genomic DNA (gDNA) (β2 m)/mtDNA (mt minArc). Datasets were analyzed using non-parametric Mann-Whitney U test. (C) Relative mRNA expression of PINK1 compared to GAPDH in CD14 + monocytes from healthy donors (n = 8) and SLE patients (n = 9). Datasets were analyzed using non-parametric Mann-Whitney U test. (D) Confocal microscopy for JC-1 dye-staining in CD14 + monocytes from healthy donors ± IFNα (n = 4). One representative result is depicted. Average of MFI of J-aggregates per cell are shown. Datasets were analyzed using paired Student’s t test. (E) Confocal Microscopy for Mitotracker CMXRos dye staining in freshly isolated CD14 + monocytes from healthy donors (n = 20) +/− IFN-α for 18h. One representative result is depicted. Αverages of MFI per cell are graphed. (F) Relative mRNA expression of PINK1 compared to GAPDH in CD14+ monocytes from healthy donors (n = 10). (G) Taqman QPCR analysis for mtDNA content in freshly isolated monocytes of healthy donors (n = 5) +/− IFN-α, rapam for 18 h expressed as a ratio of the Cq values of gDNA (β2m)/ mt DNA (mt minArc). Scale bar, 5 μM. Results are expressed as mean + SEM. ∗ p
    Figure Legend Snippet: IFNα-Mediated Lysosomal Dysfunction Impedes Mitochondrial Clearance and Leads to mtDNA Accumulation (Α) Confocal microscopy for Mitotracker CMXRos dye staining in freshly isolated CD14 + monocytes from healthy donors (n = 8) and SLE patients (n = 8). One representative result is depicted. Averages of MFI per cell are graphed. Datasets were analyzed using non-parametric Mann-Whitney U test. (Β) TaqMan qPCR analysis for mtDNA content in freshly isolated monocytes of healthy donors (n = 5) and SLE patients (n = 5), expressed as a ratio of the Cq values of genomic DNA (gDNA) (β2 m)/mtDNA (mt minArc). Datasets were analyzed using non-parametric Mann-Whitney U test. (C) Relative mRNA expression of PINK1 compared to GAPDH in CD14 + monocytes from healthy donors (n = 8) and SLE patients (n = 9). Datasets were analyzed using non-parametric Mann-Whitney U test. (D) Confocal microscopy for JC-1 dye-staining in CD14 + monocytes from healthy donors ± IFNα (n = 4). One representative result is depicted. Average of MFI of J-aggregates per cell are shown. Datasets were analyzed using paired Student’s t test. (E) Confocal Microscopy for Mitotracker CMXRos dye staining in freshly isolated CD14 + monocytes from healthy donors (n = 20) +/− IFN-α for 18h. One representative result is depicted. Αverages of MFI per cell are graphed. (F) Relative mRNA expression of PINK1 compared to GAPDH in CD14+ monocytes from healthy donors (n = 10). (G) Taqman QPCR analysis for mtDNA content in freshly isolated monocytes of healthy donors (n = 5) +/− IFN-α, rapam for 18 h expressed as a ratio of the Cq values of gDNA (β2m)/ mt DNA (mt minArc). Scale bar, 5 μM. Results are expressed as mean + SEM. ∗ p

    Techniques Used: Confocal Microscopy, Staining, Isolation, MANN-WHITNEY, Real-time Polymerase Chain Reaction, Expressing

    22) Product Images from "The non-canonical SMC protein SmcHD1 antagonises TAD formation and compartmentalisation on the inactive X chromosome"

    Article Title: The non-canonical SMC protein SmcHD1 antagonises TAD formation and compartmentalisation on the inactive X chromosome

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07907-2

    The Xi methylome in wt and SmcHD1 mutant MEFs. a WGBS reads binned into 100 kb intervals illustrating reduced level of CpG methylation on the X chromosome relative to autosomes. b Allele-specific DNA methylation profile of Xa (top) and Xi (bottom) in wt and germline SmcHD1 mutant (mut) MEFs plotted as % of CpGm averaged within 10 kb bins. Regions of low mappability are indicated with reduced colour intensity. c DNA methylation of CGIs on wt and mut Xi (left) and SmcHD1 occupancy (right). CGIs in all heatmaps sorted according to the SmcHD1 enrichment. d Metagene plots of DNA methylation in wt and mut MEFs generated for Xa and Xi as well as for maternal and paternal Chr7. e Example of a hypermethylated domain on Xi in mut cells, showing that the region of high DNA methylation extends beyond expressed genes to encompass genes that are silenced. Non-expressed genes with DNA methylation above the background level are highlighted with shadowed boxes
    Figure Legend Snippet: The Xi methylome in wt and SmcHD1 mutant MEFs. a WGBS reads binned into 100 kb intervals illustrating reduced level of CpG methylation on the X chromosome relative to autosomes. b Allele-specific DNA methylation profile of Xa (top) and Xi (bottom) in wt and germline SmcHD1 mutant (mut) MEFs plotted as % of CpGm averaged within 10 kb bins. Regions of low mappability are indicated with reduced colour intensity. c DNA methylation of CGIs on wt and mut Xi (left) and SmcHD1 occupancy (right). CGIs in all heatmaps sorted according to the SmcHD1 enrichment. d Metagene plots of DNA methylation in wt and mut MEFs generated for Xa and Xi as well as for maternal and paternal Chr7. e Example of a hypermethylated domain on Xi in mut cells, showing that the region of high DNA methylation extends beyond expressed genes to encompass genes that are silenced. Non-expressed genes with DNA methylation above the background level are highlighted with shadowed boxes

    Techniques Used: Mutagenesis, CpG Methylation Assay, DNA Methylation Assay, Generated

    23) Product Images from "Backpack PCR: A point-of-collection diagnostic platform for the rapid detection of Brugia parasites in mosquitoes"

    Article Title: Backpack PCR: A point-of-collection diagnostic platform for the rapid detection of Brugia parasites in mosquitoes

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0006962

    Test strip-based detection of amplification products generated by miniPCR. A dark purple control band is visible near the top of each test strip, indicating the proper functioning of the dipsticks. (A) Only samples containing PCR amplification products generated from B . malayi DNA produce a visible band (test band) below the location of the control band. (B) Negative control reactions do not display visible accumulation of tagged amplification product at the location of the test band, indicating that parasite DNA was not present in these samples.
    Figure Legend Snippet: Test strip-based detection of amplification products generated by miniPCR. A dark purple control band is visible near the top of each test strip, indicating the proper functioning of the dipsticks. (A) Only samples containing PCR amplification products generated from B . malayi DNA produce a visible band (test band) below the location of the control band. (B) Negative control reactions do not display visible accumulation of tagged amplification product at the location of the test band, indicating that parasite DNA was not present in these samples.

    Techniques Used: Stripping Membranes, Amplification, Generated, Polymerase Chain Reaction, Negative Control

    24) Product Images from "Embryonic Neocortical Microglia Express Toll-Like Receptor 9 and Respond to Plasmid DNA Injected into the Ventricle: Technical Considerations Regarding Microglial Distribution in Electroporated Brain Walls"

    Article Title: Embryonic Neocortical Microglia Express Toll-Like Receptor 9 and Respond to Plasmid DNA Injected into the Ventricle: Technical Considerations Regarding Microglial Distribution in Electroporated Brain Walls

    Journal: eNeuro

    doi: 10.1523/ENEURO.0312-18.2018

    Intraventricular administration of TLR9 antagonist decreases microglial infiltration induced by plasmid DNA injection. A , Relative expression of TLR9 (normalized against GAPDH ) in FACS-isolated CX3CR1 - and CX3CR1 + cells derived from the cerebral wall of E14 CX3CR1-GFP mice. Data represent mean ± SD ( n = 4 samples obtained from independent experiments; p = 0.0286, Mann–Whitney U test). B , Experimental design for ODN 2088 treatment. ODN 2088 was injected together with plasmid DNA into the lateral ventricle of E12 CX3CR1-GFP mice, and after 2 d (E14) the embryonic brains were fixed. C , Immunofluorescence with anti-GFP antibody, showing the distribution of microglia in the pallium and choroid plexus. Yellow arrowheads indicate microglia aberrantly accumulated on the apical surface of the pallium or in the choroid plexus. Cyan arrowheads show microglia which were almost homogenously distributed in the cerebral wall. Scale bar, 100 µm. D , E , Graphs indicate the number of CX3CR1-GFP + cells in each 40 µm bin of the pallium ( D ) and density of microglia directly adhered to the choroid plexus ( E ), comparing control, only ODN 2088-treated, plasmid DNA-injected, and plasmid DNA + ODN 2088 coinjected brains. F , Graph showing the total number of pallial microglia within 240 µm from the apical surface. G , Double-immunofluorescence for GFP (CX3CR1) and RFP (Lyn-mCherry) in the cortex of IUE E14 brain treated with ODN 2088. Microglia exhibited a normal distribution pattern in the Lyn-mCherry expressing region where IUE succeeded ( Movies 1 ). Scale bar, 100 µm. For statistical analyses in D – F , n = 16 samples obtained from eight embryos (2 sections, each) were quantified. Two or three littermates per dam were subjected to a series of tests. Data represent mean ± SD. *** p
    Figure Legend Snippet: Intraventricular administration of TLR9 antagonist decreases microglial infiltration induced by plasmid DNA injection. A , Relative expression of TLR9 (normalized against GAPDH ) in FACS-isolated CX3CR1 - and CX3CR1 + cells derived from the cerebral wall of E14 CX3CR1-GFP mice. Data represent mean ± SD ( n = 4 samples obtained from independent experiments; p = 0.0286, Mann–Whitney U test). B , Experimental design for ODN 2088 treatment. ODN 2088 was injected together with plasmid DNA into the lateral ventricle of E12 CX3CR1-GFP mice, and after 2 d (E14) the embryonic brains were fixed. C , Immunofluorescence with anti-GFP antibody, showing the distribution of microglia in the pallium and choroid plexus. Yellow arrowheads indicate microglia aberrantly accumulated on the apical surface of the pallium or in the choroid plexus. Cyan arrowheads show microglia which were almost homogenously distributed in the cerebral wall. Scale bar, 100 µm. D , E , Graphs indicate the number of CX3CR1-GFP + cells in each 40 µm bin of the pallium ( D ) and density of microglia directly adhered to the choroid plexus ( E ), comparing control, only ODN 2088-treated, plasmid DNA-injected, and plasmid DNA + ODN 2088 coinjected brains. F , Graph showing the total number of pallial microglia within 240 µm from the apical surface. G , Double-immunofluorescence for GFP (CX3CR1) and RFP (Lyn-mCherry) in the cortex of IUE E14 brain treated with ODN 2088. Microglia exhibited a normal distribution pattern in the Lyn-mCherry expressing region where IUE succeeded ( Movies 1 ). Scale bar, 100 µm. For statistical analyses in D – F , n = 16 samples obtained from eight embryos (2 sections, each) were quantified. Two or three littermates per dam were subjected to a series of tests. Data represent mean ± SD. *** p

    Techniques Used: Plasmid Preparation, Injection, Expressing, FACS, Isolation, Derivative Assay, Mouse Assay, MANN-WHITNEY, Immunofluorescence

    25) Product Images from "Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence the outcome of mutagenic DNA repair"

    Article Title: Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence the outcome of mutagenic DNA repair

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2005595

    Imprinted chromatin as a model system to quantify epigenetic influences on genome editing. (A) Schematic outlining the experimental workflow. Throughout the text, F1 hybrid cell lines are depicted with the maternal strain denoted before the paternal strain (i.e., In B×J: B is maternal and J paternal). sgRNAs are designed to cleave approximately 40–100 bp from a heterozygous SNP within imprinted chromatin (open and closed circles). MiSeq amplicons span both the SNP and site of mutation, which allows simultaneous assessment of genome editing outcome and parental allele at high-throughput. (B) Top: schematic showing the imprinted mouse Kcnq1 gene including H3K9me3 ChIP and DNase-I–seq data from mESCs available through EncODE (ENCSR000CFZ, GSM1014187) (bottom). Higher-resolution view of the KvDMR imprinted CpG island within Kcnq1 , showing the position of three sgRNAs used in panel E. (C) Allele-specific enrichment of H3K9me3 and H4K20me3. PCR fragments spanning the target sites of sgKvDMR#2 and #3 were amplified from input, or ChIP DNA prior to Sanger sequencing across an allelic SNP. gDNA = genomic DNA from purebred mice. (D) Example of CpG methylation data from the KvDMR locus. Bisulphite-converted gDNA was subjected to Illumina amplicon sequencing across a region spanning 13 CpG dinucleotides ( S1A Fig ), and reads were classified according to the proportion of nonconverted (methylated) CpGs. The black dashed line indicates the expected level of methylation across all alleles when imprinting is completely maintained (50%). In subsequent editing experiments, the percentage of hypermethylated ( > 80%) strands is reported together with histograms showing allele-specific mutation frequency. Quantitative data underlying panel D are provided in S1 Data , and details of MiSeq libraries including SRA accessions are provided in S2 Data . ChIP, chromatin immunoprecipitation; gDNA, genomic DNA; HDR, homology-directed repair; mESC, mouse embryonic stem cell, NHEJ, nonhomologous end joining; sgRNA, single guide RNA; SNP, single nucleotide polymorphism; SRA, Sequence Read Archive; ssODN, single-stranded oligodeoxynucleotide.
    Figure Legend Snippet: Imprinted chromatin as a model system to quantify epigenetic influences on genome editing. (A) Schematic outlining the experimental workflow. Throughout the text, F1 hybrid cell lines are depicted with the maternal strain denoted before the paternal strain (i.e., In B×J: B is maternal and J paternal). sgRNAs are designed to cleave approximately 40–100 bp from a heterozygous SNP within imprinted chromatin (open and closed circles). MiSeq amplicons span both the SNP and site of mutation, which allows simultaneous assessment of genome editing outcome and parental allele at high-throughput. (B) Top: schematic showing the imprinted mouse Kcnq1 gene including H3K9me3 ChIP and DNase-I–seq data from mESCs available through EncODE (ENCSR000CFZ, GSM1014187) (bottom). Higher-resolution view of the KvDMR imprinted CpG island within Kcnq1 , showing the position of three sgRNAs used in panel E. (C) Allele-specific enrichment of H3K9me3 and H4K20me3. PCR fragments spanning the target sites of sgKvDMR#2 and #3 were amplified from input, or ChIP DNA prior to Sanger sequencing across an allelic SNP. gDNA = genomic DNA from purebred mice. (D) Example of CpG methylation data from the KvDMR locus. Bisulphite-converted gDNA was subjected to Illumina amplicon sequencing across a region spanning 13 CpG dinucleotides ( S1A Fig ), and reads were classified according to the proportion of nonconverted (methylated) CpGs. The black dashed line indicates the expected level of methylation across all alleles when imprinting is completely maintained (50%). In subsequent editing experiments, the percentage of hypermethylated ( > 80%) strands is reported together with histograms showing allele-specific mutation frequency. Quantitative data underlying panel D are provided in S1 Data , and details of MiSeq libraries including SRA accessions are provided in S2 Data . ChIP, chromatin immunoprecipitation; gDNA, genomic DNA; HDR, homology-directed repair; mESC, mouse embryonic stem cell, NHEJ, nonhomologous end joining; sgRNA, single guide RNA; SNP, single nucleotide polymorphism; SRA, Sequence Read Archive; ssODN, single-stranded oligodeoxynucleotide.

    Techniques Used: Mutagenesis, High Throughput Screening Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Sequencing, Mouse Assay, CpG Methylation Assay, Methylation, Non-Homologous End Joining

    26) Product Images from "Molecular Signature of CAID Syndrome: Noncanonical Roles of SGO1 in Regulation of TGF-β Signaling and Epigenomics"

    Article Title: Molecular Signature of CAID Syndrome: Noncanonical Roles of SGO1 in Regulation of TGF-β Signaling and Epigenomics

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    doi: 10.1016/j.jcmgh.2018.10.011

    Epigenetic profile of CAID patients. ( A ) Heatmap representation of methylation levels in CAID patients vs controls among all tiles at early (p8) and late stage (p14) showed a global hypermethylation pattern. ( B ) Volcano plot of the methylation difference (%) at early (p8) and late stage (p14). Yellow ≥-30% and blue ≥30% methylation differences. ( C ) Tile proportions of differentially methylated regions at early (p8) and late stage (p14). For each group, N = 3 independent biological replicates. P values were corrected for multiple testing using the Benjamini–Hochberg method (q value). ( D ) Methylation percentage of LINE-1 CpG sites assessed by pyrosequencing. LINE-1 CpGs are significantly more methylated in CAID patients than in controls. Error bars signify SD. For each condition, the experiment was performed on N = 3 independent biological replicates in technical replicates. Significance was calculated by 1-way analysis of variance with the Bonferroni post-test ( • P
    Figure Legend Snippet: Epigenetic profile of CAID patients. ( A ) Heatmap representation of methylation levels in CAID patients vs controls among all tiles at early (p8) and late stage (p14) showed a global hypermethylation pattern. ( B ) Volcano plot of the methylation difference (%) at early (p8) and late stage (p14). Yellow ≥-30% and blue ≥30% methylation differences. ( C ) Tile proportions of differentially methylated regions at early (p8) and late stage (p14). For each group, N = 3 independent biological replicates. P values were corrected for multiple testing using the Benjamini–Hochberg method (q value). ( D ) Methylation percentage of LINE-1 CpG sites assessed by pyrosequencing. LINE-1 CpGs are significantly more methylated in CAID patients than in controls. Error bars signify SD. For each condition, the experiment was performed on N = 3 independent biological replicates in technical replicates. Significance was calculated by 1-way analysis of variance with the Bonferroni post-test ( • P

    Techniques Used: Methylation

    27) Product Images from "Development of a rapid and visual detection method for Rickettsia rickettsii combining recombinase polymerase assay with lateral flow test"

    Article Title: Development of a rapid and visual detection method for Rickettsia rickettsii combining recombinase polymerase assay with lateral flow test

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0207811

    Specificity of the RPA-LF method. Strips 1 to 8 used genomic DNA samples from R . rickettsii (7×10 3 copies/reaction), C . burnetii (3×10 5 copies/reaction), O . tsutsugamushi (1×10 6 copies/reaction), R . heilongjiangensis (1×10 6 copies/reaction), R . sibirica (7×10 6 copies/reaction), S . aureus (8×10 7 copies/reaction), and S . suis (6×10 7 copies/reaction), and human plasma DNA, respectively, as templates to evaluate the RPA-LF method.
    Figure Legend Snippet: Specificity of the RPA-LF method. Strips 1 to 8 used genomic DNA samples from R . rickettsii (7×10 3 copies/reaction), C . burnetii (3×10 5 copies/reaction), O . tsutsugamushi (1×10 6 copies/reaction), R . heilongjiangensis (1×10 6 copies/reaction), R . sibirica (7×10 6 copies/reaction), S . aureus (8×10 7 copies/reaction), and S . suis (6×10 7 copies/reaction), and human plasma DNA, respectively, as templates to evaluate the RPA-LF method.

    Techniques Used: Recombinase Polymerase Amplification

    28) Product Images from "Immunomodulatory Properties of DNA Hypomethylating Agents: Selecting the Optimal Epigenetic Partner for Cancer Immunotherapy"

    Article Title: Immunomodulatory Properties of DNA Hypomethylating Agents: Selecting the Optimal Epigenetic Partner for Cancer Immunotherapy

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2018.01443

    qMSP analysis of the methylation status of LINE-1 promoter in melanoma and hematological tumor cell lines treated with DHAs. Genomic DNA was extracted from 14 melanoma (A) and 10 hematological tumor (B) cell lines treated with 1 μM guadecitabine (gray), DAC (pink) or AZA (green). Real-time qMSP analyses of LINE-1 promoter were performed on bisulfite modified genomic DNA using methylated- or unmethylated-specific primer pairs. Data are reported as mean values ± SD of % of LINE-1 demethylation in DHAs-treated vs. untreated cells.
    Figure Legend Snippet: qMSP analysis of the methylation status of LINE-1 promoter in melanoma and hematological tumor cell lines treated with DHAs. Genomic DNA was extracted from 14 melanoma (A) and 10 hematological tumor (B) cell lines treated with 1 μM guadecitabine (gray), DAC (pink) or AZA (green). Real-time qMSP analyses of LINE-1 promoter were performed on bisulfite modified genomic DNA using methylated- or unmethylated-specific primer pairs. Data are reported as mean values ± SD of % of LINE-1 demethylation in DHAs-treated vs. untreated cells.

    Techniques Used: Methylation, Modification

    29) Product Images from "SOX17 overexpression sensitizes chemoradiation response in esophageal cancer by transcriptional down-regulation of DNA repair and damage response genes"

    Article Title: SOX17 overexpression sensitizes chemoradiation response in esophageal cancer by transcriptional down-regulation of DNA repair and damage response genes

    Journal: Journal of Biomedical Science

    doi: 10.1186/s12929-019-0510-4

    Model of SOX17 molecular functions in sensitization of chemoradiation. a Tumor suppressor SOX17 transcriptionally inactivates DNA repair and DNA damage responsive genes to enhance sensitivity to chemoradiation in ESCC. b Low SOX17 protein expression resulting from gene hypermethylation is associated with resistance to chemoradiation in ESCC. Low SOX17 immunoreactivity could be a biomarker for CCRT response prediction in pre-treatment endoscopic biopsies from ESCC patients
    Figure Legend Snippet: Model of SOX17 molecular functions in sensitization of chemoradiation. a Tumor suppressor SOX17 transcriptionally inactivates DNA repair and DNA damage responsive genes to enhance sensitivity to chemoradiation in ESCC. b Low SOX17 protein expression resulting from gene hypermethylation is associated with resistance to chemoradiation in ESCC. Low SOX17 immunoreactivity could be a biomarker for CCRT response prediction in pre-treatment endoscopic biopsies from ESCC patients

    Techniques Used: Expressing, Biomarker Assay

    Overexpression of SOX17 suppresses DNA repair genes of xenograft derived from radiation resistant ESCC cell line to CCRT treatment. a Xenograft tissues were extracted for RNA and examined for mRNA expression of DNA repair genes and damage response genes. The RT-qPCR results showed that expression of BRCA1 , DNAPK , p21 , and RAD51 was inhibited in KYSE510-R-SOX17 cells in comparison with KYSE510-R-EV cells with ( black bar ) or without ( black slashed bar ) CCRT treatment. SOX17 mRNA levels are shown for comparison ( right ). P -values were determined by two-tailed Student’s t -test. * P
    Figure Legend Snippet: Overexpression of SOX17 suppresses DNA repair genes of xenograft derived from radiation resistant ESCC cell line to CCRT treatment. a Xenograft tissues were extracted for RNA and examined for mRNA expression of DNA repair genes and damage response genes. The RT-qPCR results showed that expression of BRCA1 , DNAPK , p21 , and RAD51 was inhibited in KYSE510-R-SOX17 cells in comparison with KYSE510-R-EV cells with ( black bar ) or without ( black slashed bar ) CCRT treatment. SOX17 mRNA levels are shown for comparison ( right ). P -values were determined by two-tailed Student’s t -test. * P

    Techniques Used: Over Expression, Derivative Assay, Expressing, Quantitative RT-PCR, Two Tailed Test

    SOX17 overexpression suppresses DNA repair and damage response genes though transcriptional regulation. a KYSE510 radio-resistant cells (KYSE510-R-EV and KYSE510-R-SOX17) were treated with cisplatin 1 μM, radiation 2 Gy, or CCRT treatments. After 24 h, cells were harvested and analyzed for mRNA expression level by qRT-PCR. mRNA expression levels of DNA repair genes ( upper ) DNA damage response genes ( lower ) were inhibited in KYSE510-R-SOX17 cells in comparison with KYSE510-R-EV cells. b ChIP-qPCR showed that SOX17 bound to the promoter region of genes analyzed in KYSE510-R-SOX17 cells ( black bars ). ‘IgG’ used as a negative control ( gray bars ). c SOX17 downstream genes p21 and NFAT5 promoter activities were significantly inhibited in KYSE510-R-SOX17 compared to KYSE510-R-EV cells. Data represent mean ± S.D. from three independent experiments. P -values were determined by two-tailed Student’s t -test. * P
    Figure Legend Snippet: SOX17 overexpression suppresses DNA repair and damage response genes though transcriptional regulation. a KYSE510 radio-resistant cells (KYSE510-R-EV and KYSE510-R-SOX17) were treated with cisplatin 1 μM, radiation 2 Gy, or CCRT treatments. After 24 h, cells were harvested and analyzed for mRNA expression level by qRT-PCR. mRNA expression levels of DNA repair genes ( upper ) DNA damage response genes ( lower ) were inhibited in KYSE510-R-SOX17 cells in comparison with KYSE510-R-EV cells. b ChIP-qPCR showed that SOX17 bound to the promoter region of genes analyzed in KYSE510-R-SOX17 cells ( black bars ). ‘IgG’ used as a negative control ( gray bars ). c SOX17 downstream genes p21 and NFAT5 promoter activities were significantly inhibited in KYSE510-R-SOX17 compared to KYSE510-R-EV cells. Data represent mean ± S.D. from three independent experiments. P -values were determined by two-tailed Student’s t -test. * P

    Techniques Used: Over Expression, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Two Tailed Test

    SOX17 mRNA expression, DNA methylation and protein expression correlate with CCRT response in 70 ESCC patients. a Concordance analysis of low SOX17 mRNA expression and high SOX17 methylation in non-responders ESCC patients. The percentage of ESCC cases is indicated in the plot. P -value was calculated by Pearson χ2-test. b Inverse correlation between DNA methylation and mRNA expression in SOX17 gene. DNA methylation level of tumor (Log 10 (T)) tissues is indicated in the Y-axis, while tumor mRNA expression (Log 10 (T)) is plotted as the X-axis. One circle symbolizes one patient sample. Correlation coefficients (r) and P value were calculated by Pearson correlation coefficient analysis and are shown at the top. c Representative IHC of SOX17 protein in endoscopic biopsy sample from four ESCC patients. SOX17 nuclear immunoreactivity (+) was found in responder patients 1 and 2, whereas non-responder patients 3 and 4 showed SOX17 expression (−) in nuclei of tumor tissue. A 4-fold enlarged image of tumor area indicated by black box is shown in lower left inset for each patient (Original magnification: 100X). d IHC staining results showed higher SOX17 expression in CCRT responders than CCRT non-responders. The SOX17 IHC staining level is indicated in the Y-axis. One dots represented one patient slides. P values were calculated by 2-tailed t test and are shown as indicated. e Inverse correlation between DNA methylation and protein expression in SOX17. f ROC curve showing the performance of SOX17 protein expression in predicting the CCRT response, with the area under curve (AUC) being 0.664 and P values being 0.048
    Figure Legend Snippet: SOX17 mRNA expression, DNA methylation and protein expression correlate with CCRT response in 70 ESCC patients. a Concordance analysis of low SOX17 mRNA expression and high SOX17 methylation in non-responders ESCC patients. The percentage of ESCC cases is indicated in the plot. P -value was calculated by Pearson χ2-test. b Inverse correlation between DNA methylation and mRNA expression in SOX17 gene. DNA methylation level of tumor (Log 10 (T)) tissues is indicated in the Y-axis, while tumor mRNA expression (Log 10 (T)) is plotted as the X-axis. One circle symbolizes one patient sample. Correlation coefficients (r) and P value were calculated by Pearson correlation coefficient analysis and are shown at the top. c Representative IHC of SOX17 protein in endoscopic biopsy sample from four ESCC patients. SOX17 nuclear immunoreactivity (+) was found in responder patients 1 and 2, whereas non-responder patients 3 and 4 showed SOX17 expression (−) in nuclei of tumor tissue. A 4-fold enlarged image of tumor area indicated by black box is shown in lower left inset for each patient (Original magnification: 100X). d IHC staining results showed higher SOX17 expression in CCRT responders than CCRT non-responders. The SOX17 IHC staining level is indicated in the Y-axis. One dots represented one patient slides. P values were calculated by 2-tailed t test and are shown as indicated. e Inverse correlation between DNA methylation and protein expression in SOX17. f ROC curve showing the performance of SOX17 protein expression in predicting the CCRT response, with the area under curve (AUC) being 0.664 and P values being 0.048

    Techniques Used: Expressing, DNA Methylation Assay, Methylation, Immunohistochemistry, Staining

    The genomic maps of SOX17 binding sites in promoter of DNA repair genes and DNA damage response genes analyzed in the current study. Hashtags (#) indicate the SRY sites (5′-(A/T)(A/T)CAA(A/T)G-3′) in promoter region (− 1000 ~ + 1), which are the binding site for SOX17 and are predicted using PROMO software. TSS: transcription start site as indicated by (+ 1). The nucleotides relative to TSS are shown
    Figure Legend Snippet: The genomic maps of SOX17 binding sites in promoter of DNA repair genes and DNA damage response genes analyzed in the current study. Hashtags (#) indicate the SRY sites (5′-(A/T)(A/T)CAA(A/T)G-3′) in promoter region (− 1000 ~ + 1), which are the binding site for SOX17 and are predicted using PROMO software. TSS: transcription start site as indicated by (+ 1). The nucleotides relative to TSS are shown

    Techniques Used: Binding Assay, Software

    Overexpression of SOX17 sensitized ESCC radiation resistant cell line to CCRT treatment. a Cells were treated with radiation (0~2 Gy). The foci were stained and scored on day 6 after radiation. b KYSE510 radio-resistant (KYSE510-R) cells showed higher clonogenic survival under radiation condition compared with KYSE510 parental (KYSE510) cells. P values were calculated by two-tailed Student’s t test from three independent experiments. c The RT-qPCR results showed that KYSE510-R cells had lower SOX17 mRNA expression than KYSE510 parental cells ( left ). The DNA pyrosequencing methylation results indicated that KYSE510-R cells had 1.38-fold higher SOX17 DNA methylation level compared to KYSE510 cells ( right ). P values were calculated by two-tailed t test. d Western blot analysis confirmed that SOX17 protein was expressed in KYSE510-R-SOX17 cell. e Clonogenic survival was measured at day 6 after replating the KYSE510-R-SOX17 and KYSE510-R-EV cells treated with different doses of cisplatin (0~2.5 μM), radiation (0~2 Gy), or CCRT for 72 h. f The colony formation ability of KYSE510-R-SOX17 cells were inhibited compared to KYSE510-R-EV cells after CCRT treatment with 1 μM of cisplatin and 1 Gy of radition. P values were calculated by two-way ANOVA. P -values were determined by two-tailed Student’s t -test. * P
    Figure Legend Snippet: Overexpression of SOX17 sensitized ESCC radiation resistant cell line to CCRT treatment. a Cells were treated with radiation (0~2 Gy). The foci were stained and scored on day 6 after radiation. b KYSE510 radio-resistant (KYSE510-R) cells showed higher clonogenic survival under radiation condition compared with KYSE510 parental (KYSE510) cells. P values were calculated by two-tailed Student’s t test from three independent experiments. c The RT-qPCR results showed that KYSE510-R cells had lower SOX17 mRNA expression than KYSE510 parental cells ( left ). The DNA pyrosequencing methylation results indicated that KYSE510-R cells had 1.38-fold higher SOX17 DNA methylation level compared to KYSE510 cells ( right ). P values were calculated by two-tailed t test. d Western blot analysis confirmed that SOX17 protein was expressed in KYSE510-R-SOX17 cell. e Clonogenic survival was measured at day 6 after replating the KYSE510-R-SOX17 and KYSE510-R-EV cells treated with different doses of cisplatin (0~2.5 μM), radiation (0~2 Gy), or CCRT for 72 h. f The colony formation ability of KYSE510-R-SOX17 cells were inhibited compared to KYSE510-R-EV cells after CCRT treatment with 1 μM of cisplatin and 1 Gy of radition. P values were calculated by two-way ANOVA. P -values were determined by two-tailed Student’s t -test. * P

    Techniques Used: Over Expression, Staining, Two Tailed Test, Quantitative RT-PCR, Expressing, Methylation, DNA Methylation Assay, Western Blot

    30) Product Images from "Epigallocatechin-3-gallate local pre-exposure application prevents SHIV rectal infection of macaques"

    Article Title: Epigallocatechin-3-gallate local pre-exposure application prevents SHIV rectal infection of macaques

    Journal: Mucosal immunology

    doi: 10.1038/s41385-018-0025-4

    EGCG protects macaques from intra-rectal SHIV infection. (a) Experimental design of EGCG protective effect on macaques. Sixteen male macaques were administrated with 2 ml of 5 mM EGCG (8 animals) or 2 ml of PBS (8 animals) atraumatically in rectum 10 min prior to each SHIV SF162P3N challenge. All animals were rectally inoculated with SHIV SF162P3N (10 TCID 50 ) for up to 8 times or until infection occurred. All animals were biopsied at week 20 and necropsied at week 36 postinfection for the evaluation of SHIV RNA and proviral DNA in the multiple tissues. (b, c) Longitudinal assessment of the plasma SHIV RNA (copies ml -1 ) levels in the animals with intrarectal pretreatment with PBS (control) or EGCG prior to SHIV challenges (up to 8 times or till infection occurred). Duplicate plasma samples were analyzed for SHIV RNA detection. Animals were considered infected and the virus challenges were stopped following two consecutive positive plasma SHIV RNA results.
    Figure Legend Snippet: EGCG protects macaques from intra-rectal SHIV infection. (a) Experimental design of EGCG protective effect on macaques. Sixteen male macaques were administrated with 2 ml of 5 mM EGCG (8 animals) or 2 ml of PBS (8 animals) atraumatically in rectum 10 min prior to each SHIV SF162P3N challenge. All animals were rectally inoculated with SHIV SF162P3N (10 TCID 50 ) for up to 8 times or until infection occurred. All animals were biopsied at week 20 and necropsied at week 36 postinfection for the evaluation of SHIV RNA and proviral DNA in the multiple tissues. (b, c) Longitudinal assessment of the plasma SHIV RNA (copies ml -1 ) levels in the animals with intrarectal pretreatment with PBS (control) or EGCG prior to SHIV challenges (up to 8 times or till infection occurred). Duplicate plasma samples were analyzed for SHIV RNA detection. Animals were considered infected and the virus challenges were stopped following two consecutive positive plasma SHIV RNA results.

    Techniques Used: Infection, RNA Detection

    SHIV RNA and DNA detection in multiple tissues necropsied at week 36 post first SHIV challenge. SHIV RNA (a) and DNA (b) assays in the indicated tissues from the animals of PBS control (red symbols, n=6∼8) and EGCG group (green symbols, n=8) at necropsy. Log SHIV copies 2 μg -1 total genomic RNA or DNA equivalents are shown. Symbols represent individual animals and are pooled from three independent experiments. Triplication tissue samples were conducted in each independent experiment. The dot line: the detection threshold. GI: gastrointestinal tract; LN: lymph nodes; Jej-Mes: jejunal mesenteric; Col-Mes: colonal mesenteric; IEL: intraepithelial lymphocytes.
    Figure Legend Snippet: SHIV RNA and DNA detection in multiple tissues necropsied at week 36 post first SHIV challenge. SHIV RNA (a) and DNA (b) assays in the indicated tissues from the animals of PBS control (red symbols, n=6∼8) and EGCG group (green symbols, n=8) at necropsy. Log SHIV copies 2 μg -1 total genomic RNA or DNA equivalents are shown. Symbols represent individual animals and are pooled from three independent experiments. Triplication tissue samples were conducted in each independent experiment. The dot line: the detection threshold. GI: gastrointestinal tract; LN: lymph nodes; Jej-Mes: jejunal mesenteric; Col-Mes: colonal mesenteric; IEL: intraepithelial lymphocytes.

    Techniques Used:

    31) Product Images from "Dysregulation of sphingolipid metabolism contributes to bortezomib-induced neuropathic pain"

    Article Title: Dysregulation of sphingolipid metabolism contributes to bortezomib-induced neuropathic pain

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20170584

    Astrocytes are cellular targets of S1PR1 activity in the spinal cord. (A) Representative image of DNA PCR of spinal cords and DRGs from naive mice with astrocyte-specific S1pr1 deletion ( S1pr1 fl/fl ;Gfap-Cre ; n = 3) or their controls ( S1pr1 fl/fl ; n = 3). Assays were repeated twice on different days. (B) S1PR1 Western blot in the spinal cord of mice ( n = 3 per group; performed twice on different days). (C) Mechano-allodynia was measured in bortezomib-treated male mice with astrocyte-specific deletions of S1pr1 ( n = 7) and their controls ( n = 5). (D) Mechano-allodynia was measured in mice with astrocyte-specific reductions in S1pr1 and their controls treated concurrently with bortezomib and i.p. vehicle ( n = 7 per group) or FTY720 (1 mg/kg; S1pr1 fl/+ : n = 7, S1pr1 fl/+ ;Gfap-Cre : n = 6). Data are mean ± SD for n mice; *, P
    Figure Legend Snippet: Astrocytes are cellular targets of S1PR1 activity in the spinal cord. (A) Representative image of DNA PCR of spinal cords and DRGs from naive mice with astrocyte-specific S1pr1 deletion ( S1pr1 fl/fl ;Gfap-Cre ; n = 3) or their controls ( S1pr1 fl/fl ; n = 3). Assays were repeated twice on different days. (B) S1PR1 Western blot in the spinal cord of mice ( n = 3 per group; performed twice on different days). (C) Mechano-allodynia was measured in bortezomib-treated male mice with astrocyte-specific deletions of S1pr1 ( n = 7) and their controls ( n = 5). (D) Mechano-allodynia was measured in mice with astrocyte-specific reductions in S1pr1 and their controls treated concurrently with bortezomib and i.p. vehicle ( n = 7 per group) or FTY720 (1 mg/kg; S1pr1 fl/+ : n = 7, S1pr1 fl/+ ;Gfap-Cre : n = 6). Data are mean ± SD for n mice; *, P

    Techniques Used: Activity Assay, Polymerase Chain Reaction, Mouse Assay, Western Blot

    32) Product Images from "Development of transgenic male-sterile rice by using anther-specific promoters identified by comprehensive screening of the gene expression profile database ‘RiceXPro’"

    Article Title: Development of transgenic male-sterile rice by using anther-specific promoters identified by comprehensive screening of the gene expression profile database ‘RiceXPro’

    Journal: Breeding Science

    doi: 10.1270/jsbbs.18019

    Binary vector used in this study, pZH2Bi-KXB. ASP , anther-specific expressed gene promoter region; aadA , spectinomycin resistance protein; P-35S , CaMV 35S promoter; mHPT , modified hygromycin phosphotransferase; T-nos , nopaline synthase terminator; DT, 35S and nos double terminator; LB, T-DNA left border; RB, T-DNA right border.
    Figure Legend Snippet: Binary vector used in this study, pZH2Bi-KXB. ASP , anther-specific expressed gene promoter region; aadA , spectinomycin resistance protein; P-35S , CaMV 35S promoter; mHPT , modified hygromycin phosphotransferase; T-nos , nopaline synthase terminator; DT, 35S and nos double terminator; LB, T-DNA left border; RB, T-DNA right border.

    Techniques Used: Plasmid Preparation, Modification

    33) Product Images from "Autologous cell lines from circulating colon cancer cells captured from sequential liquid biopsies as model to study therapy-driven tumor changes"

    Article Title: Autologous cell lines from circulating colon cancer cells captured from sequential liquid biopsies as model to study therapy-driven tumor changes

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34365-z

    Hierarchical clustering of the nine colon CTC lines relative to the HT-29 and SW620 colorectal cancer cell lines and leukocytes. The expression of 34 mRNAs (Supplementary Table S2 ) was analyzed in the nine colon CTC lines, HT-29 cells (from a primary colorectal tumor), and SW620 cells (from a lymph node metastasis of colon cancer). These transcripts covered different properties of tumor cells: epithelial ( ECAD, CK19, EPCAM ), mesenchymal ( NCAD, VIM, FN1 ), stemness ( CD133, ALDH1 ), angiogenesis ( VEGF ), proto-oncogene ( MET ), osteomimicry ( OPG, BMP7 ), EMT ( SNAIL, TWIST , WNT, DKK1 ), immune system ( IL33 , DEFA6 , CEACAM1 ), apoptosis ( CCND2, BCL11A, TNFRS1B ), DNA repair ( GAL, SMARCA1 ), cell growth ( BST2, SEMA6A ), cell interactions ( ADAMTS6, GJB6, GATA2, TGFB2 ) and energy metabolism ( ABCB1, PTGS2 ). B2M (housekeeping gene) and CD45 (leukocyte marker) were used as reference genes. Leukocyte cDNA was used as negative control. Results were normalized to the expression level of the reference B2M gene. Cell lines were clustered based on their expression profile.
    Figure Legend Snippet: Hierarchical clustering of the nine colon CTC lines relative to the HT-29 and SW620 colorectal cancer cell lines and leukocytes. The expression of 34 mRNAs (Supplementary Table S2 ) was analyzed in the nine colon CTC lines, HT-29 cells (from a primary colorectal tumor), and SW620 cells (from a lymph node metastasis of colon cancer). These transcripts covered different properties of tumor cells: epithelial ( ECAD, CK19, EPCAM ), mesenchymal ( NCAD, VIM, FN1 ), stemness ( CD133, ALDH1 ), angiogenesis ( VEGF ), proto-oncogene ( MET ), osteomimicry ( OPG, BMP7 ), EMT ( SNAIL, TWIST , WNT, DKK1 ), immune system ( IL33 , DEFA6 , CEACAM1 ), apoptosis ( CCND2, BCL11A, TNFRS1B ), DNA repair ( GAL, SMARCA1 ), cell growth ( BST2, SEMA6A ), cell interactions ( ADAMTS6, GJB6, GATA2, TGFB2 ) and energy metabolism ( ABCB1, PTGS2 ). B2M (housekeeping gene) and CD45 (leukocyte marker) were used as reference genes. Leukocyte cDNA was used as negative control. Results were normalized to the expression level of the reference B2M gene. Cell lines were clustered based on their expression profile.

    Techniques Used: Expressing, Marker, Negative Control

    34) Product Images from "Complexity of the Genetics and Clinical Presentation of Spinocerebellar Ataxia 17"

    Article Title: Complexity of the Genetics and Clinical Presentation of Spinocerebellar Ataxia 17

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2018.00429

    Pedigrees of two case reports from the SCA17 cloned patient cohort. The first family shows two monozygotic twins (II:1, subject #13; II:2, subject #16), who have a clone sequenced mean pathogenic allele size of 47 repeats and mean normal alleles of 38 and 37 repeats, respectively (A) . Subject #13 (II:1) presented with a more severe phenotype compared to their sibling, with an age of onset 3 years prior and dementia/cognitive impairment and chorea in addition to the ataxia observed in their sibling (Subject #16, II:2). The second family shows a father (I:1, subject #5) and child (II:3; subject #4) where the father was asymptomatic at the age of 50 years old, whilst the child developed ataxic symptoms at the age of 21 years old (B) . Despite the apparent anticipation, they both had a clone sequenced mean pathogenic allele size of 51 repeats, but their normal alleles differed with the child having a slightly larger mean allele size of 39 repeats compared to 37 repeats in their father. DNA was only available for individuals who were cloned, with the given subject number in italics.
    Figure Legend Snippet: Pedigrees of two case reports from the SCA17 cloned patient cohort. The first family shows two monozygotic twins (II:1, subject #13; II:2, subject #16), who have a clone sequenced mean pathogenic allele size of 47 repeats and mean normal alleles of 38 and 37 repeats, respectively (A) . Subject #13 (II:1) presented with a more severe phenotype compared to their sibling, with an age of onset 3 years prior and dementia/cognitive impairment and chorea in addition to the ataxia observed in their sibling (Subject #16, II:2). The second family shows a father (I:1, subject #5) and child (II:3; subject #4) where the father was asymptomatic at the age of 50 years old, whilst the child developed ataxic symptoms at the age of 21 years old (B) . Despite the apparent anticipation, they both had a clone sequenced mean pathogenic allele size of 51 repeats, but their normal alleles differed with the child having a slightly larger mean allele size of 39 repeats compared to 37 repeats in their father. DNA was only available for individuals who were cloned, with the given subject number in italics.

    Techniques Used: Clone Assay

    35) Product Images from "CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions"

    Article Title: CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions

    Journal: Nature

    doi: 10.1038/s41586-018-0291-z

    Related to Figure 4a-c. Collateral loss of RNASEH2B in CLL and metastatic castration-resistant prostate cancer (CRPC). a, b, Multiplex ligation-dependent probe amplification (MLPA) analysis ( a ) and comparative genomic hybridization (CGH) array profiles for chromosome 13q ( b ) of representative CLL samples carrying two wild-type (WT) RNASEH2B alleles (top), a monoallelic RNASEH2B deletion (middle) or biallelic deletion (bottom). a , For MLPA analysis, genomic DNA from reference and experimental samples was analyzed using probes targeting control loci and individual RNASEH2B exons (Exon 1-11). MLPA ratio calculated per probe and normalised to control probes and reference samples. Error bars indicate SD of the mean from 8 control probes for each sample. Dashed lines indicate the threshold set for diploid copy number. b , For each CGH array profile the y-axes of the top and bottom plots indicate copy number probe intensity (log R ratio) and the x axes mark the position on chromosome 13 represented by the ideogram (middle). An enlargement of the frequently deleted 13q14.2-14.3 region, including the miRNA-15A/16-1 gene cluster and the RNASEH2B gene, is shown in the bottom plot. n = 1 experiment. c , RNASEH2B is frequently co-deleted with RB1 in CRPC. Copy number alterations (CNA) in the RB1-RNASEH2B region in CRPC (n = 226 cases) are shown. Horizontal lines represent the CNA profile for individual CRPC samples (dark blue, homozygous loss; light blue, heterozygous loss; grey, no change; pink, copy number gain (CNA 3-4); red, copy number amplification (CNA > 4); white, insufficient data to determine CNA). Samples are clustered based on RNASEH2B gene status. CNA frequencies for RNASEH2B and the RB1-RNASEH2B region without a copy number breakpoint are shown on the right.
    Figure Legend Snippet: Related to Figure 4a-c. Collateral loss of RNASEH2B in CLL and metastatic castration-resistant prostate cancer (CRPC). a, b, Multiplex ligation-dependent probe amplification (MLPA) analysis ( a ) and comparative genomic hybridization (CGH) array profiles for chromosome 13q ( b ) of representative CLL samples carrying two wild-type (WT) RNASEH2B alleles (top), a monoallelic RNASEH2B deletion (middle) or biallelic deletion (bottom). a , For MLPA analysis, genomic DNA from reference and experimental samples was analyzed using probes targeting control loci and individual RNASEH2B exons (Exon 1-11). MLPA ratio calculated per probe and normalised to control probes and reference samples. Error bars indicate SD of the mean from 8 control probes for each sample. Dashed lines indicate the threshold set for diploid copy number. b , For each CGH array profile the y-axes of the top and bottom plots indicate copy number probe intensity (log R ratio) and the x axes mark the position on chromosome 13 represented by the ideogram (middle). An enlargement of the frequently deleted 13q14.2-14.3 region, including the miRNA-15A/16-1 gene cluster and the RNASEH2B gene, is shown in the bottom plot. n = 1 experiment. c , RNASEH2B is frequently co-deleted with RB1 in CRPC. Copy number alterations (CNA) in the RB1-RNASEH2B region in CRPC (n = 226 cases) are shown. Horizontal lines represent the CNA profile for individual CRPC samples (dark blue, homozygous loss; light blue, heterozygous loss; grey, no change; pink, copy number gain (CNA 3-4); red, copy number amplification (CNA > 4); white, insufficient data to determine CNA). Samples are clustered based on RNASEH2B gene status. CNA frequencies for RNASEH2B and the RB1-RNASEH2B region without a copy number breakpoint are shown on the right.

    Techniques Used: Multiplex Assay, Ligation, Amplification, Multiplex Ligation-dependent Probe Amplification, Hybridization

    36) Product Images from "The polymerase L528M mutation cooperates with nucleotide binding-site mutations, increasing hepatitis B virus replication and drug resistance"

    Article Title: The polymerase L528M mutation cooperates with nucleotide binding-site mutations, increasing hepatitis B virus replication and drug resistance

    Journal: Journal of Clinical Investigation

    doi:

    Southern blot hybridization analysis of replication of wild-type HBV and five mutants. Lanes correspond to DNA extracted from viral core particles derived from HuH-7 cells that were transfected with DNA of wild-type HBV or one of five mutants. Single-stranded bands (SS) were quantified using an LAS1000 image analyzer and then normalized for transfection efficiency based on β-galactosidase activity. The relative ratio of the normalized single-stranded band is shown below each lane, assuming the single-stranded band of wild-type HBV to be 100%. OC, open circular; DS, double-stranded HBV DNA.
    Figure Legend Snippet: Southern blot hybridization analysis of replication of wild-type HBV and five mutants. Lanes correspond to DNA extracted from viral core particles derived from HuH-7 cells that were transfected with DNA of wild-type HBV or one of five mutants. Single-stranded bands (SS) were quantified using an LAS1000 image analyzer and then normalized for transfection efficiency based on β-galactosidase activity. The relative ratio of the normalized single-stranded band is shown below each lane, assuming the single-stranded band of wild-type HBV to be 100%. OC, open circular; DS, double-stranded HBV DNA.

    Techniques Used: Southern Blot, Hybridization, Derivative Assay, Transfection, Activity Assay

    37) Product Images from "A DNA prime-live vaccine boost strategy in mice can augment IFN-? responses to mycobacterial antigens but does not increase the protective efficacy of two attenuated strains of Mycobacterium bovis against bovine tuberculosis"

    Article Title: A DNA prime-live vaccine boost strategy in mice can augment IFN-? responses to mycobacterial antigens but does not increase the protective efficacy of two attenuated strains of Mycobacterium bovis against bovine tuberculosis

    Journal: Immunology

    doi: 10.1046/j.1365-2567.2003.01589.x

    Interferon-γ-secreting cells in BALB/c mice 14 weeks after first vaccination. Mice were primed either with plasmid DNA expressing ESAT-6 and Ag85A or control plasmid before boosting with BCG only (a) or WAg520 (b). Some mice were given plasmid DNA or control plasmid only (no boost) or an attenuated M. bovis only (no prime). IFN-γ responses were measured by ELISPOT in response to bovine PPD (black bars), ESAT-6 (grey bars) or Ag85A (white bars). The means and SD of replicate determinations are shown.
    Figure Legend Snippet: Interferon-γ-secreting cells in BALB/c mice 14 weeks after first vaccination. Mice were primed either with plasmid DNA expressing ESAT-6 and Ag85A or control plasmid before boosting with BCG only (a) or WAg520 (b). Some mice were given plasmid DNA or control plasmid only (no boost) or an attenuated M. bovis only (no prime). IFN-γ responses were measured by ELISPOT in response to bovine PPD (black bars), ESAT-6 (grey bars) or Ag85A (white bars). The means and SD of replicate determinations are shown.

    Techniques Used: Mouse Assay, Plasmid Preparation, Expressing, Enzyme-linked Immunospot

    Interferon-γ-secreting cells in C57BL/6 mice 14 weeks after first vaccination. Mice were primed with plasmid DNA (no boost) expressing ESAT-6 and Ag85A, or control plasmid before boosting with BCG (a) or WAg520 (b). Some mice were given plasmid DNA (no boost) or control plasmid only (no prime). IFN-γ responses were measured by ELISPOT in response to bovine PPD (black bars), ESAT-6 (grey bars) or Ag85A (white bars). The means and SD of replicate determinations are shown.
    Figure Legend Snippet: Interferon-γ-secreting cells in C57BL/6 mice 14 weeks after first vaccination. Mice were primed with plasmid DNA (no boost) expressing ESAT-6 and Ag85A, or control plasmid before boosting with BCG (a) or WAg520 (b). Some mice were given plasmid DNA (no boost) or control plasmid only (no prime). IFN-γ responses were measured by ELISPOT in response to bovine PPD (black bars), ESAT-6 (grey bars) or Ag85A (white bars). The means and SD of replicate determinations are shown.

    Techniques Used: Mouse Assay, Plasmid Preparation, Expressing, Enzyme-linked Immunospot

    Interferon-γ-secreting cells in BALB/c mice 24 weeks after first vaccination. Mice were primed with plasmid DNA expressing ESAT-6 and Ag85A before boosting with BCG. Some mice were given only plasmid DNA (no boost) or BCG only (no prime). IFN-γ responses were measured by ELISPOT in response to bovine PPD (black bars), ESAT-6 (grey bars) or Ag85A (white bars). The means and SD of replicate determination are shown.
    Figure Legend Snippet: Interferon-γ-secreting cells in BALB/c mice 24 weeks after first vaccination. Mice were primed with plasmid DNA expressing ESAT-6 and Ag85A before boosting with BCG. Some mice were given only plasmid DNA (no boost) or BCG only (no prime). IFN-γ responses were measured by ELISPOT in response to bovine PPD (black bars), ESAT-6 (grey bars) or Ag85A (white bars). The means and SD of replicate determination are shown.

    Techniques Used: Mouse Assay, Plasmid Preparation, Expressing, Enzyme-linked Immunospot

    38) Product Images from "A novel denaturing heteroduplex tracking assay for genotypic prediction of HIV-1 tropism"

    Article Title: A novel denaturing heteroduplex tracking assay for genotypic prediction of HIV-1 tropism

    Journal: Journal of virological methods

    doi: 10.1016/j.jviromet.2012.06.013

    Tropism determination of HIV-1 from patient plasma samples by the HIV-1 V3 denaturing HTA. (A) HIV-1 V3 denaturing HTA result using plasma sample of patient WC202. 1. 100ng of purified HIV-1 V3 RT PCR product from plasma sample of patient WC202. 2. 50ng V3 DNA from clone WC202PR5b. 3. 50ng V3 DNA from clone WC202PR5f. 4. 50ng V3 DNA from clone WC202PX4. p. probe only. (B) HIV-1 V3 denaturing HTA result using plasma sample of patient WC28. 1. 100ng of purified HIV-1 V3 RT PCR product from plasma sample of patient WC28. 2. 50ng V3 DNA from clone WC28PR5. 3. 50ng V3 DNA from clone WC28PX4a. 4. 50ng V3 DNA from clone WC28PX4c. 5. 50ng V3 DNA from clone WC28PX4b. p. probe only. (C) Amino acid sequence alignment of V3 clones from samples WC202 and WC28.
    Figure Legend Snippet: Tropism determination of HIV-1 from patient plasma samples by the HIV-1 V3 denaturing HTA. (A) HIV-1 V3 denaturing HTA result using plasma sample of patient WC202. 1. 100ng of purified HIV-1 V3 RT PCR product from plasma sample of patient WC202. 2. 50ng V3 DNA from clone WC202PR5b. 3. 50ng V3 DNA from clone WC202PR5f. 4. 50ng V3 DNA from clone WC202PX4. p. probe only. (B) HIV-1 V3 denaturing HTA result using plasma sample of patient WC28. 1. 100ng of purified HIV-1 V3 RT PCR product from plasma sample of patient WC28. 2. 50ng V3 DNA from clone WC28PR5. 3. 50ng V3 DNA from clone WC28PX4a. 4. 50ng V3 DNA from clone WC28PX4c. 5. 50ng V3 DNA from clone WC28PX4b. p. probe only. (C) Amino acid sequence alignment of V3 clones from samples WC202 and WC28.

    Techniques Used: Purification, Reverse Transcription Polymerase Chain Reaction, Sequencing, Clone Assay

    39) Product Images from "Quantitation of pyridyloxobutyl-DNA adducts in tissues of rats treated chronically with (R)- or (S)-N’-nitrosonornicotine (NNN) in a carcinogenicity study"

    Article Title: Quantitation of pyridyloxobutyl-DNA adducts in tissues of rats treated chronically with (R)- or (S)-N’-nitrosonornicotine (NNN) in a carcinogenicity study

    Journal: Chemical research in toxicology

    doi: 10.1021/tx400235x

    Typical SRM chromatograms obtained upon analysis of oral mucosa DNA isolated from (A) control rats; (B) ( R )-NNN-treated rats; and (C) ( S )-NNN-treated rats after 70 weeks of treatment. Individual POB-DNA adducts or internal standards were monitored as indicated on each channel.
    Figure Legend Snippet: Typical SRM chromatograms obtained upon analysis of oral mucosa DNA isolated from (A) control rats; (B) ( R )-NNN-treated rats; and (C) ( S )-NNN-treated rats after 70 weeks of treatment. Individual POB-DNA adducts or internal standards were monitored as indicated on each channel.

    Techniques Used: Isolation

    Structures of the POB-DNA adducts discussed in this study.
    Figure Legend Snippet: Structures of the POB-DNA adducts discussed in this study.

    Techniques Used:

    Chromatograms obtained upon analysis of O 6 -POB-dGuo in nasal respiratory mucosa DNA of rats treated for 70 weeks with ( S )-NNN, using (A) the TSQ Quantum Vantage triple quadrupole mass spectrometer and (B) the high resolution LTQ Orbitrap Velos mass spectrometer.
    Figure Legend Snippet: Chromatograms obtained upon analysis of O 6 -POB-dGuo in nasal respiratory mucosa DNA of rats treated for 70 weeks with ( S )-NNN, using (A) the TSQ Quantum Vantage triple quadrupole mass spectrometer and (B) the high resolution LTQ Orbitrap Velos mass spectrometer.

    Techniques Used: Mass Spectrometry

    Levels of each POB-DNA adduct in the (A) oral mucosa of ( R )-NNN-treated rats, (B) oral mucosa of ( S )-NNN-treated rats, (C) esophageal mucosa of ( R )-NNN-treated rats, (D) esophageal mucosa of ( S )-NNN-treated rats, (E) nasal respiratory mucosa of ( R )-NNN-treated rats, (F) nasal respiratory mucosa of ( S )-NNN-treated rats, (G) nasal olfactory mucosa of ( R )-NNN-treated rats, (H) nasal olfactory mucosa of ( S )-NNN-treated rats, (I) liver of ( R )-NNN-treated rats, (J) liver of ( S )-NNN-treated rats, (K) lung of ( R )-NNN-treated rats, and (L) lung of ( S )-NNN-treated rats. Adduct levels are shown in sequence at each time point, as follows: open bars 7-POB-Gua; striped bars O 2 -POB-dThd. All differences between levels of 7-POB-Gua and O 2 , Supporting Information.
    Figure Legend Snippet: Levels of each POB-DNA adduct in the (A) oral mucosa of ( R )-NNN-treated rats, (B) oral mucosa of ( S )-NNN-treated rats, (C) esophageal mucosa of ( R )-NNN-treated rats, (D) esophageal mucosa of ( S )-NNN-treated rats, (E) nasal respiratory mucosa of ( R )-NNN-treated rats, (F) nasal respiratory mucosa of ( S )-NNN-treated rats, (G) nasal olfactory mucosa of ( R )-NNN-treated rats, (H) nasal olfactory mucosa of ( S )-NNN-treated rats, (I) liver of ( R )-NNN-treated rats, (J) liver of ( S )-NNN-treated rats, (K) lung of ( R )-NNN-treated rats, and (L) lung of ( S )-NNN-treated rats. Adduct levels are shown in sequence at each time point, as follows: open bars 7-POB-Gua; striped bars O 2 -POB-dThd. All differences between levels of 7-POB-Gua and O 2 , Supporting Information.

    Techniques Used: Sequencing

    Plots of total adduct levels (fmol/mg of DNA) vs time (weeks) in (A) oral mucosa, (B) esophageal mucosa, (C) nasal respiratory mucosa, (D) nasal olfactory mucosa, (E) liver, and (F) lung DNA from ( R )- and ( S )-NNN-treated rats. Values of the total adduct levels are the sum of the amounts of all POB-DNA adducts measured in oral, esophageal, nasal respiratory, nasal olfactory, liver and lung DNA at each time point ± S. D. Symbol designations are ■, total adduct levels from ( R )-NNN treatment and □, total adduct levels from ( S , Supporting Information.
    Figure Legend Snippet: Plots of total adduct levels (fmol/mg of DNA) vs time (weeks) in (A) oral mucosa, (B) esophageal mucosa, (C) nasal respiratory mucosa, (D) nasal olfactory mucosa, (E) liver, and (F) lung DNA from ( R )- and ( S )-NNN-treated rats. Values of the total adduct levels are the sum of the amounts of all POB-DNA adducts measured in oral, esophageal, nasal respiratory, nasal olfactory, liver and lung DNA at each time point ± S. D. Symbol designations are ■, total adduct levels from ( R )-NNN treatment and □, total adduct levels from ( S , Supporting Information.

    Techniques Used:

    Comparison of total POB-DNA adduct levels (fmol/mg DNA) vs time (weeks) in oral mucosa, esophageal mucosa, nasal respiratory mucosa, nasal olfactory mucosa, liver and lung DNA from (A); ( R )-NNN-treated rats and (B); ( S )-NNN-treated rats. Symbols and colors are as follows: nasal respiratory, red closed square; liver, magenta open of the Supporting Information.
    Figure Legend Snippet: Comparison of total POB-DNA adduct levels (fmol/mg DNA) vs time (weeks) in oral mucosa, esophageal mucosa, nasal respiratory mucosa, nasal olfactory mucosa, liver and lung DNA from (A); ( R )-NNN-treated rats and (B); ( S )-NNN-treated rats. Symbols and colors are as follows: nasal respiratory, red closed square; liver, magenta open of the Supporting Information.

    Techniques Used:

    40) Product Images from "CD27 signaling on chronic myelogenous leukemia stem cells activates Wnt target genes and promotes disease progression"

    Article Title: CD27 signaling on chronic myelogenous leukemia stem cells activates Wnt target genes and promotes disease progression

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI45977

    CD27 signaling promotes CML progression. ( A ) Experimental model. ( B ) Genomic DNA was isolated from spleens of WT ( n = 5) and Cd27 –/– ( n = 3) CML mice and analyzed by real-time PCR. ΔΔC t values of human c-abl were normalized to ΔΔC t values of murine c-abl. ( C ) Expression of human c-abl mRNA in FACS-purified, pooled LSCs from WT ( n = 19) and Cd27 –/– ( n = 21) CML mice 20 days after transplantation. ( D ) Expression of BCR/ABL-GFP in lin – BM cells of WT ( n = 37) and Cd27 –/– ( n = 45) CML mice (pooled data from 7 independent experiments). ( E ) Blood smears (upper row) and cytospins (lower row) of naive BL/6, WT CML, and Cd27 –/– CML mice. Scale bars: 20 μm. ( F ) Numbers of BCR/ABL-GFP + granulocytes/μl blood ( n = 8 mice per group) and ( G ) Kaplan-Meier survival curves resulting from primary transplantations of BCR/ABL-GFP–transduced BL/6 (black line, n = 13) versus Cd27 –/– (dotted line, n = 15) BM cells into BL/6 recipients (pooled data from 2 independent experiments). ( H ) LSC numbers per mouse 15 days ( n = 5 mice per group) and 20 days ( n = 15 mice per group) after transplantation. ( I ) Numbers of lin – , BCR/ABL-GFP + cells per mouse 20 days after transplantation ( n = 15 mice per group). Data are displayed as mean ± SEM. Statistics: Student’s t test ( B – D , I ), 2-way ANOVA ( F ), log-rank test ( G ), and 1-way ANOVA ( H ). Cells/mouse = cells from both femora, tibiae, and humeri.
    Figure Legend Snippet: CD27 signaling promotes CML progression. ( A ) Experimental model. ( B ) Genomic DNA was isolated from spleens of WT ( n = 5) and Cd27 –/– ( n = 3) CML mice and analyzed by real-time PCR. ΔΔC t values of human c-abl were normalized to ΔΔC t values of murine c-abl. ( C ) Expression of human c-abl mRNA in FACS-purified, pooled LSCs from WT ( n = 19) and Cd27 –/– ( n = 21) CML mice 20 days after transplantation. ( D ) Expression of BCR/ABL-GFP in lin – BM cells of WT ( n = 37) and Cd27 –/– ( n = 45) CML mice (pooled data from 7 independent experiments). ( E ) Blood smears (upper row) and cytospins (lower row) of naive BL/6, WT CML, and Cd27 –/– CML mice. Scale bars: 20 μm. ( F ) Numbers of BCR/ABL-GFP + granulocytes/μl blood ( n = 8 mice per group) and ( G ) Kaplan-Meier survival curves resulting from primary transplantations of BCR/ABL-GFP–transduced BL/6 (black line, n = 13) versus Cd27 –/– (dotted line, n = 15) BM cells into BL/6 recipients (pooled data from 2 independent experiments). ( H ) LSC numbers per mouse 15 days ( n = 5 mice per group) and 20 days ( n = 15 mice per group) after transplantation. ( I ) Numbers of lin – , BCR/ABL-GFP + cells per mouse 20 days after transplantation ( n = 15 mice per group). Data are displayed as mean ± SEM. Statistics: Student’s t test ( B – D , I ), 2-way ANOVA ( F ), log-rank test ( G ), and 1-way ANOVA ( H ). Cells/mouse = cells from both femora, tibiae, and humeri.

    Techniques Used: Isolation, Mouse Assay, Real-time Polymerase Chain Reaction, Expressing, FACS, Purification, Transplantation Assay

    41) Product Images from "The Murine G+C-Rich Promoter Binding Protein mGPBP Is Required for Promoter-Specific Transcription"

    Article Title: The Murine G+C-Rich Promoter Binding Protein mGPBP Is Required for Promoter-Specific Transcription

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.23.8773-8785.2003

    The purified recombinant mGPBP can bind specifically to the mouse Ada gene's G+C-rich promoter in EMSA. (A) The DNA probe 4C′ is four copies of the MSPE C′ that were end ligated. Purified recombinant mGPBP (rmGPBP) bound specifically to the probe and caused a shift in probe electrophoretic mobility from the free-probe location (lane 1) to the bound-probe location (lane 2). This binding can be specifically competed out by adding 35-fold (lane 3) and 175-fold (lane 4) excess unlabeled probes but cannot be competed out by adding similar amounts of unlabeled E2F binding motifs (lanes 5 and 6) or a 200-bp plasmid sequence (lanes 7 and 8). (B) A single copy of the fragment C′ in the context of the labeled 236-bp mouse Ada gene promoter can also bind to and be electrophoretically retarded by the purified rmGPBP (lanes 1 and 2). This binding can be competed out by excess unlabeled probe (lanes 3 and 4) or the 4C′ probe used in panel A (lanes 5 and 6). This binding is again not competed out by unlabeled E2F binding sequences (lanes 7 and 8) or the 200-bp plasmid sequences (lanes 9 and 10).
    Figure Legend Snippet: The purified recombinant mGPBP can bind specifically to the mouse Ada gene's G+C-rich promoter in EMSA. (A) The DNA probe 4C′ is four copies of the MSPE C′ that were end ligated. Purified recombinant mGPBP (rmGPBP) bound specifically to the probe and caused a shift in probe electrophoretic mobility from the free-probe location (lane 1) to the bound-probe location (lane 2). This binding can be specifically competed out by adding 35-fold (lane 3) and 175-fold (lane 4) excess unlabeled probes but cannot be competed out by adding similar amounts of unlabeled E2F binding motifs (lanes 5 and 6) or a 200-bp plasmid sequence (lanes 7 and 8). (B) A single copy of the fragment C′ in the context of the labeled 236-bp mouse Ada gene promoter can also bind to and be electrophoretically retarded by the purified rmGPBP (lanes 1 and 2). This binding can be competed out by excess unlabeled probe (lanes 3 and 4) or the 4C′ probe used in panel A (lanes 5 and 6). This binding is again not competed out by unlabeled E2F binding sequences (lanes 7 and 8) or the 200-bp plasmid sequences (lanes 9 and 10).

    Techniques Used: Purification, Recombinant, Binding Assay, Plasmid Preparation, Sequencing, Labeling

    42) Product Images from "Oral Immunization with Recombinant Lactobacillus acidophilus Expressing the Adhesin Hp0410 of Helicobacter pylori Induces Mucosal and Systemic Immune Responses"

    Article Title: Oral Immunization with Recombinant Lactobacillus acidophilus Expressing the Adhesin Hp0410 of Helicobacter pylori Induces Mucosal and Systemic Immune Responses

    Journal: Clinical and Vaccine Immunology : CVI

    doi: 10.1128/CVI.00434-13

    Identification of the plasmid digested with XbaI/HindIII. Lane 1, the recombinant pMG36e-hp0410 plasmid; lane 2, the recombinant pMG36e-hp0410 plasmid digested with XbaI and HindIII, showing the 750-bp Hp0410 and the bone of the plasmid; lane M, DNA marker DL2k.
    Figure Legend Snippet: Identification of the plasmid digested with XbaI/HindIII. Lane 1, the recombinant pMG36e-hp0410 plasmid; lane 2, the recombinant pMG36e-hp0410 plasmid digested with XbaI and HindIII, showing the 750-bp Hp0410 and the bone of the plasmid; lane M, DNA marker DL2k.

    Techniques Used: Plasmid Preparation, Recombinant, Marker

    43) Product Images from "Stability of Borrelia burgdorferi bdr Loci In Vitro and In Vivo"

    Article Title: Stability of Borrelia burgdorferi bdr Loci In Vitro and In Vivo

    Journal: Infection and Immunity

    doi:

    RFLP analysis indicating stability of the bdr -flanking regions. Southern blots of total B. burgdorferi DNA digested with Hin dIII (data not shown) and Xba I were probed with a pool of PCR-derived bdr probes. Three in vitro-cultured B31 clones (5A3, ATCC, and B313) and 17 isolates obtained 1 year after infection of mice with B31-5A3 (1487 to 1503) are shown. A 1-kb ladder (Gibco) served as the size marker.
    Figure Legend Snippet: RFLP analysis indicating stability of the bdr -flanking regions. Southern blots of total B. burgdorferi DNA digested with Hin dIII (data not shown) and Xba I were probed with a pool of PCR-derived bdr probes. Three in vitro-cultured B31 clones (5A3, ATCC, and B313) and 17 isolates obtained 1 year after infection of mice with B31-5A3 (1487 to 1503) are shown. A 1-kb ladder (Gibco) served as the size marker.

    Techniques Used: Polymerase Chain Reaction, Derivative Assay, In Vitro, Cell Culture, Clone Assay, Infection, Mouse Assay, Marker

    44) Product Images from "Absence of keratins 8 and 18 expression in rodent epithelial cell lines associates with keratin gene mutation and DNA methylation: cell line selective effects on cell invasion"

    Article Title: Absence of keratins 8 and 18 expression in rodent epithelial cell lines associates with keratin gene mutation and DNA methylation: cell line selective effects on cell invasion

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2015.04.003

    Methylation-specific PCR and bisulfite sequencing analysis. (A) MSP-1, MSP-2 and control MSP (directed towards the promoter region of mouse K8) were designed to test methylation of the K8 promoter region in BNL, NMuLi and CT26 cells. MSP amplified bands were only found in CT26 genomic DNA (lanes 1 and 2 on right side of panel). (B) Rat (r) MSP-1, MSP-5 and control MSP (directed towards the promoter region of rat K18) were used in rat liver tissue and IEC-6 cells. Notably, MSP-amplified bands were only found in IEC-6 genomic DNA. (C) Bisulfite sequencing of the promoter region of K8 and K18 in the indicated tissues and cell lines showed different level of methylation in NMuLi, CT26, rat liver and IEC-6 cells, with K18 being hypermethylated in IEC-6 cells (11 of 30 in IEC-6 versus 0 of 30 potential sites in rat liver). Similarly, K8 is hypermethylated in CT26 cells (13 of 20 in CT26 versus 4 of 20 potential sites in mouse liver).
    Figure Legend Snippet: Methylation-specific PCR and bisulfite sequencing analysis. (A) MSP-1, MSP-2 and control MSP (directed towards the promoter region of mouse K8) were designed to test methylation of the K8 promoter region in BNL, NMuLi and CT26 cells. MSP amplified bands were only found in CT26 genomic DNA (lanes 1 and 2 on right side of panel). (B) Rat (r) MSP-1, MSP-5 and control MSP (directed towards the promoter region of rat K18) were used in rat liver tissue and IEC-6 cells. Notably, MSP-amplified bands were only found in IEC-6 genomic DNA. (C) Bisulfite sequencing of the promoter region of K8 and K18 in the indicated tissues and cell lines showed different level of methylation in NMuLi, CT26, rat liver and IEC-6 cells, with K18 being hypermethylated in IEC-6 cells (11 of 30 in IEC-6 versus 0 of 30 potential sites in rat liver). Similarly, K8 is hypermethylated in CT26 cells (13 of 20 in CT26 versus 4 of 20 potential sites in mouse liver).

    Techniques Used: Methylation, Polymerase Chain Reaction, Methylation Sequencing, Amplification

    45) Product Images from "Construction and Characterization of a Highly Efficient Francisella Shuttle Plasmid"

    Article Title: Construction and Characterization of a Highly Efficient Francisella Shuttle Plasmid

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.70.12.7511-7519.2004

    Efficiency of electroporation of pTOPO/FNL10 and pFNLTP1 into E. coli DH5α or Francisella spp. ( F. tularensis LVS or F. novicida U112). The transformation efficiency, expressed as log CFU per microgram of DNA (mean ± standard deviation), was determined from three individual preparations of competent cells. Competent cells of E. coli were prepared with 10% glycerol, while 0.5 M sucrose was used to prepare competent cells of F. tularensis LVS and F. novicida U112. Most electroporations were performed with 100 ng of plasmid DNA; the only exception was the electroporation of pTOPO/FNL10 into F. tularensis LVS, for which 500 ng was used. The strains indicated in parentheses are the hosts from which plasmid DNA was isolated.
    Figure Legend Snippet: Efficiency of electroporation of pTOPO/FNL10 and pFNLTP1 into E. coli DH5α or Francisella spp. ( F. tularensis LVS or F. novicida U112). The transformation efficiency, expressed as log CFU per microgram of DNA (mean ± standard deviation), was determined from three individual preparations of competent cells. Competent cells of E. coli were prepared with 10% glycerol, while 0.5 M sucrose was used to prepare competent cells of F. tularensis LVS and F. novicida U112. Most electroporations were performed with 100 ng of plasmid DNA; the only exception was the electroporation of pTOPO/FNL10 into F. tularensis LVS, for which 500 ng was used. The strains indicated in parentheses are the hosts from which plasmid DNA was isolated.

    Techniques Used: Electroporation, Transformation Assay, Standard Deviation, Plasmid Preparation, Isolation

    46) Product Images from "CTLA-4+PD-1− memory CD4+ T cells critically contribute to viral persistence in antiretroviral therapy-suppressed, SIV-infected rhesus macaques"

    Article Title: CTLA-4+PD-1− memory CD4+ T cells critically contribute to viral persistence in antiretroviral therapy-suppressed, SIV-infected rhesus macaques

    Journal: Immunity

    doi: 10.1016/j.immuni.2017.09.018

    CTLA-4 + PD-1 − memory CD4 + T cells harbor higher amounts of SIV DNA following ART-mediated, viral load suppression ( A )Study design. Ten RMs were infected i.v. with 1000 TCID50 SIVmac251 (day 0), and at 7 weeks post-infection, initiated ART (PMPA, FTC, raltegravir, and ritonavir-boosted darunavir). All animals were maintained on ART regimen until plasma viremia was undetectable for at least 3 months. Peripheral blood (WB), rectal biopsy (Gut), and lymph node (LN) biopsies were collected at the indicated time points and multiple organs were harvested at elective necropsy. Sorting of memory CD4 + T cells by Co-IR expression was performed at two time points during ART: first, at Mid ART (approximately 1 month following undetectable viremia); and second, at necropsy. ( B ) Plasma viral loads are shown for the 10 individual RMs, quantified using the standard qRT-PCR assay (limit of detection, LOD, of 60 SIV RNA copies/mL of plasma represented by the horizontal dotted line). Undetectable measurements are plotted as one-half of the LOD (30 copies/mL). ( C ) Frequencies (of live CD3 + T cells) of CD4 + T cells were longitudinally measured in WB, LN, and gut biopsies. The gray shaded area represents time on ART; Nx represents the measured values from animal necropsy. Repeated-measures analyses were performed using a means model (SAS Mixed Procedure, version 9.4) to determine statistical significance, with indicated tests of significance representing comparison to pre-SIV infection (WB, Gut) or pre-ART initiation (LN). ( D ) Representative SIV DNA quantities in the PBMCs, LN, spleen, and gut tissues for an individual RM (RLr10) after 206 days of viral load suppression (n=9). ( E ) Cell-associated SIV GAG .
    Figure Legend Snippet: CTLA-4 + PD-1 − memory CD4 + T cells harbor higher amounts of SIV DNA following ART-mediated, viral load suppression ( A )Study design. Ten RMs were infected i.v. with 1000 TCID50 SIVmac251 (day 0), and at 7 weeks post-infection, initiated ART (PMPA, FTC, raltegravir, and ritonavir-boosted darunavir). All animals were maintained on ART regimen until plasma viremia was undetectable for at least 3 months. Peripheral blood (WB), rectal biopsy (Gut), and lymph node (LN) biopsies were collected at the indicated time points and multiple organs were harvested at elective necropsy. Sorting of memory CD4 + T cells by Co-IR expression was performed at two time points during ART: first, at Mid ART (approximately 1 month following undetectable viremia); and second, at necropsy. ( B ) Plasma viral loads are shown for the 10 individual RMs, quantified using the standard qRT-PCR assay (limit of detection, LOD, of 60 SIV RNA copies/mL of plasma represented by the horizontal dotted line). Undetectable measurements are plotted as one-half of the LOD (30 copies/mL). ( C ) Frequencies (of live CD3 + T cells) of CD4 + T cells were longitudinally measured in WB, LN, and gut biopsies. The gray shaded area represents time on ART; Nx represents the measured values from animal necropsy. Repeated-measures analyses were performed using a means model (SAS Mixed Procedure, version 9.4) to determine statistical significance, with indicated tests of significance representing comparison to pre-SIV infection (WB, Gut) or pre-ART initiation (LN). ( D ) Representative SIV DNA quantities in the PBMCs, LN, spleen, and gut tissues for an individual RM (RLr10) after 206 days of viral load suppression (n=9). ( E ) Cell-associated SIV GAG .

    Techniques Used: Infection, Western Blot, Expressing, Quantitative RT-PCR

    47) Product Images from "DNA Methyltransferase 3a and Mitogen-activated Protein Kinase Signaling Regulate the Expression of Fibroblast Growth Factor-inducible 14 (Fn14) during Denervation-induced Skeletal Muscle Atrophy *"

    Article Title: DNA Methyltransferase 3a and Mitogen-activated Protein Kinase Signaling Regulate the Expression of Fibroblast Growth Factor-inducible 14 (Fn14) during Denervation-induced Skeletal Muscle Atrophy *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.568626

    Methylation status of CpG sites within 5′-flanking region of Fn14 gene. A , schematic illustration of CpG sites within conserved region of mouse Fn14 promoter. B , bisulfite sequencing results demonstrating methylated and unmethylated CpG sites in undenervated and 3-day denervated GA muscle of mice. Each row of small boxes represents an individual clone sequenced. Unmethylated ( blue boxes ) or methylated ( red boxes ) CpG sites are indicated. C , quantification of methylation on CpG sites in Fn14 promoter in undenervated and 3-day denervated GA muscle of mice ( n = 3). The data presented here demonstrate that denervation causes demethylation at specific CpG sites in Fn14 promoter. D , quantification of methylation on CpG sites in Fn14 promoter in undenervated GA muscle of mice and cultured C2C12 myoblasts. The data presented here demonstrate increased demethylation at specific CpG sites in Fn14 promoter in C2C12 myoblasts compared with naïve adult skeletal muscle of mice ( n = 3 in each group). E , relative global DNA methylation levels in undenervated and denervated muscle were calculated as percentages relative to methylated control DNA using the Imprint® methylated DNA quantification kit ( n = 4 in each group). Error bars represent the S.D. *, p
    Figure Legend Snippet: Methylation status of CpG sites within 5′-flanking region of Fn14 gene. A , schematic illustration of CpG sites within conserved region of mouse Fn14 promoter. B , bisulfite sequencing results demonstrating methylated and unmethylated CpG sites in undenervated and 3-day denervated GA muscle of mice. Each row of small boxes represents an individual clone sequenced. Unmethylated ( blue boxes ) or methylated ( red boxes ) CpG sites are indicated. C , quantification of methylation on CpG sites in Fn14 promoter in undenervated and 3-day denervated GA muscle of mice ( n = 3). The data presented here demonstrate that denervation causes demethylation at specific CpG sites in Fn14 promoter. D , quantification of methylation on CpG sites in Fn14 promoter in undenervated GA muscle of mice and cultured C2C12 myoblasts. The data presented here demonstrate increased demethylation at specific CpG sites in Fn14 promoter in C2C12 myoblasts compared with naïve adult skeletal muscle of mice ( n = 3 in each group). E , relative global DNA methylation levels in undenervated and denervated muscle were calculated as percentages relative to methylated control DNA using the Imprint® methylated DNA quantification kit ( n = 4 in each group). Error bars represent the S.D. *, p

    Techniques Used: Methylation, Methylation Sequencing, Mouse Assay, Cell Culture, DNA Methylation Assay

    SP1 and AP1 transcription factors bind to mouse Fn14 promoter in denervated skeletal muscle. A , EMSA gels presented here demonstrate that DNA binding activity of SP1 and AP1 to their consensus sequence at indicated positions upstream of ATG in Fn14 promoter is increased in GA muscle of mice after 3 days of denervation. B and C , undenervated and 3-day denervated GA muscle of mice was processed for ChIP assay to study in vivo binding of SP1 ( B ) and c-Jun ( C ) to their consensus sequence in mouse Fn14 promoter. The data presented here demonstrate that enrichment of SP1 and c-Jun to Fn14 promoter is drastically increased in denervated GA muscle of mice. PCR was performed with primer sets amplifying SP1 (−80 bp) or AP1 (−167, −391, and −504 bp) consensus sequence containing fragments within the −1-kb region of mouse Fn14 promoter. Total input (10%) was used as a positive control, whereas the isotype-matched IgG was used as a negative control. Myoblasts were transfected with control, SP1, or c-Jun siRNA for 48 h, and the protein extracts made were probed for Fn14, SP1, and c-Jun. D and E , representative immunoblots from two independent experiments each done in triplicate presented here demonstrate that knockdown of SP1 ( D ) or c-Jun ( E ) decreased the protein levels of Fn14 in myoblasts.
    Figure Legend Snippet: SP1 and AP1 transcription factors bind to mouse Fn14 promoter in denervated skeletal muscle. A , EMSA gels presented here demonstrate that DNA binding activity of SP1 and AP1 to their consensus sequence at indicated positions upstream of ATG in Fn14 promoter is increased in GA muscle of mice after 3 days of denervation. B and C , undenervated and 3-day denervated GA muscle of mice was processed for ChIP assay to study in vivo binding of SP1 ( B ) and c-Jun ( C ) to their consensus sequence in mouse Fn14 promoter. The data presented here demonstrate that enrichment of SP1 and c-Jun to Fn14 promoter is drastically increased in denervated GA muscle of mice. PCR was performed with primer sets amplifying SP1 (−80 bp) or AP1 (−167, −391, and −504 bp) consensus sequence containing fragments within the −1-kb region of mouse Fn14 promoter. Total input (10%) was used as a positive control, whereas the isotype-matched IgG was used as a negative control. Myoblasts were transfected with control, SP1, or c-Jun siRNA for 48 h, and the protein extracts made were probed for Fn14, SP1, and c-Jun. D and E , representative immunoblots from two independent experiments each done in triplicate presented here demonstrate that knockdown of SP1 ( D ) or c-Jun ( E ) decreased the protein levels of Fn14 in myoblasts.

    Techniques Used: Binding Assay, Activity Assay, Sequencing, Mouse Assay, Chromatin Immunoprecipitation, In Vivo, Polymerase Chain Reaction, Positive Control, Negative Control, Transfection, Western Blot

    48) Product Images from "Genetic Analyses of DNA-Binding Mutants in the Catalytic Core Domain of Human Immunodeficiency Virus Type 1 Integrase"

    Article Title: Genetic Analyses of DNA-Binding Mutants in the Catalytic Core Domain of Human Immunodeficiency Virus Type 1 Integrase

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.4.2493-2505.2005

    Identification and characterization of a HIV-1 Q62A revertant virus. (A) Jurkat cells (5 × 10 6 ) infected with 5 × 10 5 RT cpm of HIV-1 Q62A ) or (•) HIV-1 Q62A(r) obtained from a prior infection of Jurkat cells at 19 dpi were monitored for RT activity at the indicated time points. WT HIV-1 NL 4 - 3 was derived from transfected HeLa cells. (B) In vitro 3′ processing (upper panel) and DNA strand transfer (lower panel) activities of WT and IN mutant proteins. IN was omitted from the reaction in lane 1. The reactions in lanes 2 to 6 contained 7, 14, 28, 56, and 112 mM NaCl, respectively (the triangle represents the increase in NaCl concentration). The reactions in lanes 7 to 12, 13 to 18, and 19 to 24 were the same as in lanes 1 to 6 except for the identity of the IN protein, as indicated beneath the gel. The migration positions of the 3′ processing substrate and product are marked 30 and 28, respectively. ST, products of strand transfer. Whereas the gel in the upper panel was exposed to X-ray film for 15 min, the lower panel was exposed for 8 h. (C) Jurkat cells (2 × 10 6 ) infected with the indicated molecularly cloned viruses (10 6 RT cpm) were monitored for RT activity at the indicated times.
    Figure Legend Snippet: Identification and characterization of a HIV-1 Q62A revertant virus. (A) Jurkat cells (5 × 10 6 ) infected with 5 × 10 5 RT cpm of HIV-1 Q62A ) or (•) HIV-1 Q62A(r) obtained from a prior infection of Jurkat cells at 19 dpi were monitored for RT activity at the indicated time points. WT HIV-1 NL 4 - 3 was derived from transfected HeLa cells. (B) In vitro 3′ processing (upper panel) and DNA strand transfer (lower panel) activities of WT and IN mutant proteins. IN was omitted from the reaction in lane 1. The reactions in lanes 2 to 6 contained 7, 14, 28, 56, and 112 mM NaCl, respectively (the triangle represents the increase in NaCl concentration). The reactions in lanes 7 to 12, 13 to 18, and 19 to 24 were the same as in lanes 1 to 6 except for the identity of the IN protein, as indicated beneath the gel. The migration positions of the 3′ processing substrate and product are marked 30 and 28, respectively. ST, products of strand transfer. Whereas the gel in the upper panel was exposed to X-ray film for 15 min, the lower panel was exposed for 8 h. (C) Jurkat cells (2 × 10 6 ) infected with the indicated molecularly cloned viruses (10 6 RT cpm) were monitored for RT activity at the indicated times.

    Techniques Used: Infection, Activity Assay, Derivative Assay, Transfection, In Vitro, Mutagenesis, Concentration Assay, Migration, Clone Assay

    49) Product Images from "Disruption of the Shc/Grb2 Complex during Abelson Virus Transformation Affects Proliferation, but Not Apoptosis"

    Article Title: Disruption of the Shc/Grb2 Complex during Abelson Virus Transformation Affects Proliferation, but Not Apoptosis

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.4.2325-2334.2005

    DN Shc decreases Erk activation, an event associated with reduced cell growth. Pre-B cells fully transformed with wild-type Ab-MLV were plated at 5 × 10 5 cells/ml and treated with 70 μM PD98059 (○) or dimethyl sulfoxide (•); triplicate cultures were stained with trypan blue and counted using a hemocytometer on a regular basis. The numbers of viable cells (A) and the percentages of viability (B) were averaged for each time point; error bars represent standard deviations. These data are representative of experiments conducted with three independent cell lines that were each analyzed two to three times. (C) 293T cells were transfected with 5 μg of DNA expressing P120 and 2 μg of DNA expressing DN Ras, the cells were serum starved, and lysates prepared 48 h later were analyzed by Western blotting with the indicated antibodies. (D) 293T cells were transfected with 5 μg of DNA expressing P120 and the various forms of Shc and serum starved, and lysates prepared 48 h later were analyzed by Western blotting with the indicated antibodies.
    Figure Legend Snippet: DN Shc decreases Erk activation, an event associated with reduced cell growth. Pre-B cells fully transformed with wild-type Ab-MLV were plated at 5 × 10 5 cells/ml and treated with 70 μM PD98059 (○) or dimethyl sulfoxide (•); triplicate cultures were stained with trypan blue and counted using a hemocytometer on a regular basis. The numbers of viable cells (A) and the percentages of viability (B) were averaged for each time point; error bars represent standard deviations. These data are representative of experiments conducted with three independent cell lines that were each analyzed two to three times. (C) 293T cells were transfected with 5 μg of DNA expressing P120 and 2 μg of DNA expressing DN Ras, the cells were serum starved, and lysates prepared 48 h later were analyzed by Western blotting with the indicated antibodies. (D) 293T cells were transfected with 5 μg of DNA expressing P120 and the various forms of Shc and serum starved, and lysates prepared 48 h later were analyzed by Western blotting with the indicated antibodies.

    Techniques Used: Activation Assay, Transformation Assay, Staining, Transfection, Expressing, Western Blot

    Expression of DN Shc reduces v-Abl-mediated Ras activation. 293T cells were transfected with 5 μg of DNA expressing P120 and the various forms of Shc. Cells were serum starved and harvested 48 h posttransfection. (A) Activated Ras was recovered by binding to GST-RBD Raf and analyzed by Western blotting using α-Ras antibodies. Lysate from mock-transfected cells (Mock) was treated with GTPγS as the positive control (+Ctrl). (B) Whole-cell lysates were analyzed by Western blotting to control for the amounts of Ras and HA-Shc present in the samples.
    Figure Legend Snippet: Expression of DN Shc reduces v-Abl-mediated Ras activation. 293T cells were transfected with 5 μg of DNA expressing P120 and the various forms of Shc. Cells were serum starved and harvested 48 h posttransfection. (A) Activated Ras was recovered by binding to GST-RBD Raf and analyzed by Western blotting using α-Ras antibodies. Lysate from mock-transfected cells (Mock) was treated with GTPγS as the positive control (+Ctrl). (B) Whole-cell lysates were analyzed by Western blotting to control for the amounts of Ras and HA-Shc present in the samples.

    Techniques Used: Expressing, Activation Assay, Transfection, Binding Assay, Western Blot, Positive Control

    Raf tyrosine 340/341 phosphorylation affects v-Abl signals to Erk. (A) Transformed pre-B cells were treated with various concentrations of STI-571 for 4 h, lysed, and analyzed by Western blotting with the indicated antibodies. (B) 293T cells were transfected with 5 μg of DNA expressing P120 and 1 μg of DNA expressing the different Raf plasmids and serum starved, and lysates prepared 48 h later were analyzed by Western blotting with the indicated antibodies. The autoradiogram shown is representative of three independent experiments in which densitometry revealed that levels of p-Erk were decreased by approximately threefold in the presence of Raf Y340/341F. (C) 293T cells were transfected with 5 μg of DNA expressing P120 and the different forms of Shc and serum starved, and lysates prepared 48 h later were analyzed by Western blotting with the indicated antibodies.
    Figure Legend Snippet: Raf tyrosine 340/341 phosphorylation affects v-Abl signals to Erk. (A) Transformed pre-B cells were treated with various concentrations of STI-571 for 4 h, lysed, and analyzed by Western blotting with the indicated antibodies. (B) 293T cells were transfected with 5 μg of DNA expressing P120 and 1 μg of DNA expressing the different Raf plasmids and serum starved, and lysates prepared 48 h later were analyzed by Western blotting with the indicated antibodies. The autoradiogram shown is representative of three independent experiments in which densitometry revealed that levels of p-Erk were decreased by approximately threefold in the presence of Raf Y340/341F. (C) 293T cells were transfected with 5 μg of DNA expressing P120 and the different forms of Shc and serum starved, and lysates prepared 48 h later were analyzed by Western blotting with the indicated antibodies.

    Techniques Used: Transformation Assay, Western Blot, Transfection, Expressing

    Expression of DN Shc does not alter v-Abl-mediated Rac activation. (A) COS7 cells were transfected with the 10 μg of DNA encoding P120 and the various forms of Shc and 3 μg of pJ3 myc-Rac1. Lysates prepared 48 h posttransfection were incubated with GST-PBD, and Rac was recovered and analyzed by Western blotting with the indicated antibodies. (B) 293T cells were transfected with 5 μg of DNA expressing P120 and 1 μg of DNA expressing DN Rac and serum starved, and lysates prepared 48 h later were analyzed by Western blotting with the indicated antibodies. The autoradiogram shown is representative of three independent experiments in which densitometry revealed that levels of p-Erk were decreased by approximately twofold in the presence of DN Rac.
    Figure Legend Snippet: Expression of DN Shc does not alter v-Abl-mediated Rac activation. (A) COS7 cells were transfected with the 10 μg of DNA encoding P120 and the various forms of Shc and 3 μg of pJ3 myc-Rac1. Lysates prepared 48 h posttransfection were incubated with GST-PBD, and Rac was recovered and analyzed by Western blotting with the indicated antibodies. (B) 293T cells were transfected with 5 μg of DNA expressing P120 and 1 μg of DNA expressing DN Rac and serum starved, and lysates prepared 48 h later were analyzed by Western blotting with the indicated antibodies. The autoradiogram shown is representative of three independent experiments in which densitometry revealed that levels of p-Erk were decreased by approximately twofold in the presence of DN Rac.

    Techniques Used: Expressing, Activation Assay, Transfection, Incubation, Western Blot

    50) Product Images from "Preparing DNA Libraries for Multiplexed Paired-End Deep Sequencing for Illumina GA Sequencers"

    Article Title: Preparing DNA Libraries for Multiplexed Paired-End Deep Sequencing for Illumina GA Sequencers

    Journal: Current protocols in microbiology

    doi: 10.1002/9780471729259.mc01e04s20

    Fragmented DNA smearing patterns after ligation of adapters (Ligation Mix) and after PCR (PCR Mix). Note increase in smear intensity as well as a shift up in the size range of the smear.
    Figure Legend Snippet: Fragmented DNA smearing patterns after ligation of adapters (Ligation Mix) and after PCR (PCR Mix). Note increase in smear intensity as well as a shift up in the size range of the smear.

    Techniques Used: Ligation, Polymerase Chain Reaction

    Overview of DNA library preparation for deep sequencing. The purified genomic DNA is processed and modified as described in the basic protocols. The adapter-modified DNA fragments (library) are then enriched through PCR. Note the relative positions and characteristics (Inset) of the InPE1.0, InPE2.0 and the Index primers used during PCR. For multiplexed paired-end PCR, the same InPE1.0 and InPE2.0 primers are used, however, up to 12 different Index primers can be added to the enrichment reactions individually. Sequencing is performed in one direction through sequencing by synthesis , and then in the alternate direction as described in the background information. The complementary sequences are shaded darker.
    Figure Legend Snippet: Overview of DNA library preparation for deep sequencing. The purified genomic DNA is processed and modified as described in the basic protocols. The adapter-modified DNA fragments (library) are then enriched through PCR. Note the relative positions and characteristics (Inset) of the InPE1.0, InPE2.0 and the Index primers used during PCR. For multiplexed paired-end PCR, the same InPE1.0 and InPE2.0 primers are used, however, up to 12 different Index primers can be added to the enrichment reactions individually. Sequencing is performed in one direction through sequencing by synthesis , and then in the alternate direction as described in the background information. The complementary sequences are shaded darker.

    Techniques Used: Sequencing, Purification, Modification, Polymerase Chain Reaction

    51) Product Images from "The Human Fetal Glial Cell Line SVG p12 Contains Infectious BK Polyomavirus"

    Article Title: The Human Fetal Glial Cell Line SVG p12 Contains Infectious BK Polyomavirus

    Journal: Journal of Virology

    doi: 10.1128/JVI.00696-14

    The SVG p12 cell line produces infectious BKPyV. (A) Electron microscopy of negatively stained viral particles from supernatant harvested from SVG p12 cells 4 days postseeding. (B) Immunofluorescence staining of RPTECs 3 days following exposure to supernatant harvested from SVG p12 cells 4 days postseeding. Indirect immunofluorescence staining was performed using a combination of BKPyV agnoprotein rabbit polyclonal antiserum (green, Alexa Fluor 488) and SV40 LTag mouse monoclonal antibody, Pab 416 (red, Alexa Fluor 568). The DNA (nucleus) was stained with Draq5 (blue). Images were acquired by confocal microscopy with a 40× objective.
    Figure Legend Snippet: The SVG p12 cell line produces infectious BKPyV. (A) Electron microscopy of negatively stained viral particles from supernatant harvested from SVG p12 cells 4 days postseeding. (B) Immunofluorescence staining of RPTECs 3 days following exposure to supernatant harvested from SVG p12 cells 4 days postseeding. Indirect immunofluorescence staining was performed using a combination of BKPyV agnoprotein rabbit polyclonal antiserum (green, Alexa Fluor 488) and SV40 LTag mouse monoclonal antibody, Pab 416 (red, Alexa Fluor 568). The DNA (nucleus) was stained with Draq5 (blue). Images were acquired by confocal microscopy with a 40× objective.

    Techniques Used: Electron Microscopy, Staining, Immunofluorescence, Confocal Microscopy

    The BKPyV DNA isolated from the SVG p12 cell line consisted of a mixture of complete and defective genomes. (A) PCR products generated from long-range PCR of SVG p12 supernatants 4 days postseeding and 58 days postseeding were separated on a 0.8% agarose gel. A supernatant harvested from BKPyV-infected RPTECs 3 days postinfection was included as a positive control. (B) PCR products generated from long-range PCR of RPTEC supernatant after infection with an SVG p12 supernatant (the virus was passaged twice in RPTECs). A supernatant harvested from BKPyV-infected RPTECs 3 days postinfection was included as a positive control. (C) Defective genomes detected by long-range PCR followed by TA cloning and Sanger sequencing. The different regions of the genome are color coded: NCCR, blue; LVGR, green; intergenic region, gray; EVGR, yellow. Deletions are indicated by a thin line. The star indicates the binding sites of the partly overlapping primers used for long-range PCR. (D) Schematic display of the BKPyV UT genome with the different reading frames annotated and with the PCR-amplified regions marked in red and subsequent PCR products from EVGR and LVGR, respectively, separated on a 1% agarose gel. PCR product from the BKPyV Dunlop plasmid served as a positive control. M, 1 Kb Plus DNA ladder (Invitrogen).
    Figure Legend Snippet: The BKPyV DNA isolated from the SVG p12 cell line consisted of a mixture of complete and defective genomes. (A) PCR products generated from long-range PCR of SVG p12 supernatants 4 days postseeding and 58 days postseeding were separated on a 0.8% agarose gel. A supernatant harvested from BKPyV-infected RPTECs 3 days postinfection was included as a positive control. (B) PCR products generated from long-range PCR of RPTEC supernatant after infection with an SVG p12 supernatant (the virus was passaged twice in RPTECs). A supernatant harvested from BKPyV-infected RPTECs 3 days postinfection was included as a positive control. (C) Defective genomes detected by long-range PCR followed by TA cloning and Sanger sequencing. The different regions of the genome are color coded: NCCR, blue; LVGR, green; intergenic region, gray; EVGR, yellow. Deletions are indicated by a thin line. The star indicates the binding sites of the partly overlapping primers used for long-range PCR. (D) Schematic display of the BKPyV UT genome with the different reading frames annotated and with the PCR-amplified regions marked in red and subsequent PCR products from EVGR and LVGR, respectively, separated on a 1% agarose gel. PCR product from the BKPyV Dunlop plasmid served as a positive control. M, 1 Kb Plus DNA ladder (Invitrogen).

    Techniques Used: Isolation, Polymerase Chain Reaction, Generated, Agarose Gel Electrophoresis, Infection, Positive Control, TA Cloning, Sequencing, Binding Assay, Amplification, Plasmid Preparation

    JCPyV can infect SVG p12 cells both with and without BKPyV late protein expression. SVG p12 cells were infected with JCPyV, and indirect immunofluorescence staining was performed 4 days postinfection using a combination of the BKPyV-specific agnoprotein rabbit polyclonal antiserum (green, Alexa Fluor 488) and the JCPyV-specific JCPyV VP1 mouse monoclonal antibody (red, Alexa Fluor 568). The DNA (nucleus) was stained with Draq5 (blue). Images were acquired by confocal microscopy with a 40× objective. Cells were either infected with BKPyV or JCPyV (A) or coinfected with BKPyV and JCPyV (B).
    Figure Legend Snippet: JCPyV can infect SVG p12 cells both with and without BKPyV late protein expression. SVG p12 cells were infected with JCPyV, and indirect immunofluorescence staining was performed 4 days postinfection using a combination of the BKPyV-specific agnoprotein rabbit polyclonal antiserum (green, Alexa Fluor 488) and the JCPyV-specific JCPyV VP1 mouse monoclonal antibody (red, Alexa Fluor 568). The DNA (nucleus) was stained with Draq5 (blue). Images were acquired by confocal microscopy with a 40× objective. Cells were either infected with BKPyV or JCPyV (A) or coinfected with BKPyV and JCPyV (B).

    Techniques Used: Expressing, Infection, Immunofluorescence, Staining, Confocal Microscopy

    The BKPyV DNA isolated from the SVG p12 cell line consisted of a mixture of complete and defective genomes as detected by NGS. (A) Coverage per nucleotide of the 454 reads when aligned to the BKV UT genome. The positions of the dominant deletion as well as the BKPyV open reading frames are indicated. (B) Some defective genomes detected by NGS. Numbers of reads are indicated next to the defective genomes, and the average coverage is given in parentheses. The different regions of the genome are color coded: NCCR, blue; LVGR, green; intergenic region, gray; EVGR, yellow. Deletions are indicated by a thin line. The star indicates the binding sites of the partly overlapping primers used for long-range PCR.
    Figure Legend Snippet: The BKPyV DNA isolated from the SVG p12 cell line consisted of a mixture of complete and defective genomes as detected by NGS. (A) Coverage per nucleotide of the 454 reads when aligned to the BKV UT genome. The positions of the dominant deletion as well as the BKPyV open reading frames are indicated. (B) Some defective genomes detected by NGS. Numbers of reads are indicated next to the defective genomes, and the average coverage is given in parentheses. The different regions of the genome are color coded: NCCR, blue; LVGR, green; intergenic region, gray; EVGR, yellow. Deletions are indicated by a thin line. The star indicates the binding sites of the partly overlapping primers used for long-range PCR.

    Techniques Used: Isolation, Next-Generation Sequencing, Binding Assay, Polymerase Chain Reaction

    The SVG p12 cell line expresses BKPyV late proteins. SVG p12 cells were fixed 4 days postseeding, and indirect immunofluorescence staining was performed using different combinations of primary antibodies: SV40 VP1 rabbit polyclonal antiserum (green, Alexa Fluor 488) with the SV40-specific LTag mouse monoclonal antibody Pab419 (red, Alexa Fluor 568) (A); and BKPyV agnoprotein rabbit polyclonal antiserum (green, Alexa 488) with SV40 LTag mouse monoclonal antibody Pab416 (red, Alexa Fluor 568) (B). The DNA (nucleus) was stained with Draq5 (blue), and both images were acquired by confocal microscopy with a 40× objective. (C) Western blot of cell lysate (7.2 μg protein/lane) from Vero, COS-7, and SVG p12 cells 4 days postseeding. The membrane was labeled with SV40 LTag mouse monoclonal antibody, Pab 416, BKPyV VP1 rabbit polyclonal antiserum, BKPyV agnoprotein rabbit polyclonal antiserum, and anti-GAPDH mouse monoclonal antibody. M, molecular weight marker (MagicMark XP Western standard; Invitrogen).
    Figure Legend Snippet: The SVG p12 cell line expresses BKPyV late proteins. SVG p12 cells were fixed 4 days postseeding, and indirect immunofluorescence staining was performed using different combinations of primary antibodies: SV40 VP1 rabbit polyclonal antiserum (green, Alexa Fluor 488) with the SV40-specific LTag mouse monoclonal antibody Pab419 (red, Alexa Fluor 568) (A); and BKPyV agnoprotein rabbit polyclonal antiserum (green, Alexa 488) with SV40 LTag mouse monoclonal antibody Pab416 (red, Alexa Fluor 568) (B). The DNA (nucleus) was stained with Draq5 (blue), and both images were acquired by confocal microscopy with a 40× objective. (C) Western blot of cell lysate (7.2 μg protein/lane) from Vero, COS-7, and SVG p12 cells 4 days postseeding. The membrane was labeled with SV40 LTag mouse monoclonal antibody, Pab 416, BKPyV VP1 rabbit polyclonal antiserum, BKPyV agnoprotein rabbit polyclonal antiserum, and anti-GAPDH mouse monoclonal antibody. M, molecular weight marker (MagicMark XP Western standard; Invitrogen).

    Techniques Used: Immunofluorescence, Staining, Confocal Microscopy, Western Blot, Labeling, Molecular Weight, Marker

    SVG-A cells do not express BKPyV late proteins inherently but are permissive for BKPyV infection. Indirect immunofluorescence staining of SVG-A cells was performed using a combination of BKPyV agnoprotein rabbit polyclonal antiserum (green, Alexa Fluor 488) and SV40 LTag mouse monoclonal antibody Pab416 (red, Alexa Fluor 568). The DNA (nucleus) was stained with Draq5 (blue). Images were acquired by confocal microscopy with a 20× objective. Cells 3 days postseeding (A) and 3 days postinfection with BKPyV Dunlop (B).
    Figure Legend Snippet: SVG-A cells do not express BKPyV late proteins inherently but are permissive for BKPyV infection. Indirect immunofluorescence staining of SVG-A cells was performed using a combination of BKPyV agnoprotein rabbit polyclonal antiserum (green, Alexa Fluor 488) and SV40 LTag mouse monoclonal antibody Pab416 (red, Alexa Fluor 568). The DNA (nucleus) was stained with Draq5 (blue). Images were acquired by confocal microscopy with a 20× objective. Cells 3 days postseeding (A) and 3 days postinfection with BKPyV Dunlop (B).

    Techniques Used: Infection, Immunofluorescence, Staining, Confocal Microscopy

    52) Product Images from "Viral Adaptation to Host Immune Responses Occurs in Chronic Hepatitis B Virus (HBV) Infection, and Adaptation Is Greatest in HBV e Antigen-Negative Disease"

    Article Title: Viral Adaptation to Host Immune Responses Occurs in Chronic Hepatitis B Virus (HBV) Infection, and Adaptation Is Greatest in HBV e Antigen-Negative Disease

    Journal: Journal of Virology

    doi: 10.1128/JVI.05308-11

    Associations between the adaptation score and clinical parameters for patients infected with genotypes B (left) and C (right). The parameters assessed included HBeAg status (A), ALT level (B), and HBV DNA load (C). (A and B) Data are shown as box-and-whisker plots, where the middle line represents the median; the edges of each box represent the 25th and 75th percentiles; and the whiskers represent the range. (C) Data are plotted as the proportion of cases ( y axis) exceeding the corresponding log viral load on the x axis (Kaplan-Meier plots).
    Figure Legend Snippet: Associations between the adaptation score and clinical parameters for patients infected with genotypes B (left) and C (right). The parameters assessed included HBeAg status (A), ALT level (B), and HBV DNA load (C). (A and B) Data are shown as box-and-whisker plots, where the middle line represents the median; the edges of each box represent the 25th and 75th percentiles; and the whiskers represent the range. (C) Data are plotted as the proportion of cases ( y axis) exceeding the corresponding log viral load on the x axis (Kaplan-Meier plots).

    Techniques Used: Infection, Whisker Assay

    53) Product Images from "Nucleotide Parasitism by Simkania negevensis (Chlamydiae) ▿) ▿ †"

    Article Title: Nucleotide Parasitism by Simkania negevensis (Chlamydiae) ▿) ▿ †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00919-10

    Transcription of Sn NTT1 to Sn NTT4 during multiplication of S. negevensis within Acanthamoeba castellani UWC1. Lanes 1, amplification of cDNA synthesized from whole RNA from amoebae harboring S. negevensis ; lanes 2, PCR positive control using DNA isolated from S. negevensis growing in amoebae; lanes 3, PCR negative control (no cDNA added); lanes 4, PCR using whole RNA from amoebae containing S. negevensis (control for the absence of DNA in the purified and DNase-digested RNA); m, molecular size marker.
    Figure Legend Snippet: Transcription of Sn NTT1 to Sn NTT4 during multiplication of S. negevensis within Acanthamoeba castellani UWC1. Lanes 1, amplification of cDNA synthesized from whole RNA from amoebae harboring S. negevensis ; lanes 2, PCR positive control using DNA isolated from S. negevensis growing in amoebae; lanes 3, PCR negative control (no cDNA added); lanes 4, PCR using whole RNA from amoebae containing S. negevensis (control for the absence of DNA in the purified and DNase-digested RNA); m, molecular size marker.

    Techniques Used: Amplification, Synthesized, Polymerase Chain Reaction, Positive Control, Isolation, Negative Control, Purification, Marker

    54) Product Images from "Effects of Truncation of the Carboxy Terminus of Pseudorabies Virus Glycoprotein B on Infectivity"

    Article Title: Effects of Truncation of the Carboxy Terminus of Pseudorabies Virus Glycoprotein B on Infectivity

    Journal: Journal of Virology

    doi:

    Subcellular localization of C-terminally truncated gB forms. Normal RK13 cells (A) and cells expressing wild-type gB (B), gB-008 (C), gB-007 (D), gB-006 (E), or gB-005 (F) were grown to confluency, fixed with 3% paraformaldehyde–0.3% Triton X-100, and incubated with anti-gB MAb A20-c26. Confocal laser scanning microscopy was performed after incubation with FITC-conjugated secondary antibodies and staining of nuclear DNA with propidium iodide.
    Figure Legend Snippet: Subcellular localization of C-terminally truncated gB forms. Normal RK13 cells (A) and cells expressing wild-type gB (B), gB-008 (C), gB-007 (D), gB-006 (E), or gB-005 (F) were grown to confluency, fixed with 3% paraformaldehyde–0.3% Triton X-100, and incubated with anti-gB MAb A20-c26. Confocal laser scanning microscopy was performed after incubation with FITC-conjugated secondary antibodies and staining of nuclear DNA with propidium iodide.

    Techniques Used: Expressing, Incubation, Confocal Laser Scanning Microscopy, Staining

    55) Product Images from "Quantifying Adenovirus-Neutralizing Antibodies by Luciferase Transgene Detection: Addressing Preexisting Immunity to Vaccine and Gene Therapy Vectors"

    Article Title: Quantifying Adenovirus-Neutralizing Antibodies by Luciferase Transgene Detection: Addressing Preexisting Immunity to Vaccine and Gene Therapy Vectors

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.41.11.5046-5052.2003

    Comparison between transgene expression and the number of Ad genomes per cell. A standard neutralization was performed with human Ad5-positive serum in combination with the vectors Ad5.Luc and Ad35.Luc at 500 VP/cell. (A) Cells were analyzed for luciferase activity. Ad5-positive serum shows a serotype-specific inhibition of Ad vector transduction. (B) Packaged DNA was isolated from A549 cells used in a neutralization assay as in panel A. Isolated DNA was used as a template for Ad-specific real-time PCR. The number of Ad5 genome copies per cell is decreased due to Ad5-specific serum. Ad35 genome copies are stable irrespective of concentrations in serum.
    Figure Legend Snippet: Comparison between transgene expression and the number of Ad genomes per cell. A standard neutralization was performed with human Ad5-positive serum in combination with the vectors Ad5.Luc and Ad35.Luc at 500 VP/cell. (A) Cells were analyzed for luciferase activity. Ad5-positive serum shows a serotype-specific inhibition of Ad vector transduction. (B) Packaged DNA was isolated from A549 cells used in a neutralization assay as in panel A. Isolated DNA was used as a template for Ad-specific real-time PCR. The number of Ad5 genome copies per cell is decreased due to Ad5-specific serum. Ad35 genome copies are stable irrespective of concentrations in serum.

    Techniques Used: Expressing, Neutralization, Luciferase, Activity Assay, Inhibition, Plasmid Preparation, Transduction, Isolation, Real-time Polymerase Chain Reaction

    56) Product Images from "High mobility group A2 protein and its derivatives bind a specific region of the promoter of DNA repair gene ERCC1 and modulate its activity"

    Article Title: High mobility group A2 protein and its derivatives bind a specific region of the promoter of DNA repair gene ERCC1 and modulate its activity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkg884

    Footprinting of the HMGA2 on the end-labeled –426 to –257 fragment of the ERCC1 promoter. The ERCC1 promoter DNA that was 32 P end-labeled at the 3′ of the top strand was digested with hydroxyl-radicals in the absence (–) or presence (+) of 100 µM HMGA2. ( A ) is shown in the right lane. ( B ) Quantification of the DNA footprinting data shown in (A). The 100% cutting frequency corresponds to digestion of the DNA fragment in the absence of protein so that lower values mean protection upon HMGA2 binding. Arrows label the maxima of protection. The presented results are mean values from three independent experiments.
    Figure Legend Snippet: Footprinting of the HMGA2 on the end-labeled –426 to –257 fragment of the ERCC1 promoter. The ERCC1 promoter DNA that was 32 P end-labeled at the 3′ of the top strand was digested with hydroxyl-radicals in the absence (–) or presence (+) of 100 µM HMGA2. ( A ) is shown in the right lane. ( B ) Quantification of the DNA footprinting data shown in (A). The 100% cutting frequency corresponds to digestion of the DNA fragment in the absence of protein so that lower values mean protection upon HMGA2 binding. Arrows label the maxima of protection. The presented results are mean values from three independent experiments.

    Techniques Used: Footprinting, Labeling, DNA Footprinting, Binding Assay

    Identification of the high affinity HMGA2 binding site on the ERCC1 promoter. ( A ) Less than 1 nM 32 P-end-labeled fragments –426 to –257, –307 to –137 and –191 to –12 of the ERCC1 promoter (top) were incubated with increasing concentrations of HMGA2 and electrophoresed on 6% polyacrylamide gels in low ionic strength buffer. The gels were dried, and radioactivity was scanned by a PhosphorImager. ( B ) The end-labeled –426 to –257 fragment was partially digested with the Eam1104I restriction nuclease cutting the fragment between –351 and –350. The digestion mixture was incubated with increasing concentrations of HMGA2 and analyzed as described in (A). ( C ) Comparison of the binding affinities of HMGA2, HMGA1b and HMG1a to the –426 to –257 ERCC1 fragment using a more narrow protein concentration range as described in (A). ( D ) Quantification of the HMGA2 binding data of (C). 100% – free DNA was plotted against the protein concentration on a logarithmic scale. The line represents the theoretical curve calculated from the relationship K d = [100% – % free DNA] × [free protein]/[complexes] using SigmaPlot Hill regression. K d(app) was 1.75 ± 0.31 nM for the –426 to –257 fragment.
    Figure Legend Snippet: Identification of the high affinity HMGA2 binding site on the ERCC1 promoter. ( A ) Less than 1 nM 32 P-end-labeled fragments –426 to –257, –307 to –137 and –191 to –12 of the ERCC1 promoter (top) were incubated with increasing concentrations of HMGA2 and electrophoresed on 6% polyacrylamide gels in low ionic strength buffer. The gels were dried, and radioactivity was scanned by a PhosphorImager. ( B ) The end-labeled –426 to –257 fragment was partially digested with the Eam1104I restriction nuclease cutting the fragment between –351 and –350. The digestion mixture was incubated with increasing concentrations of HMGA2 and analyzed as described in (A). ( C ) Comparison of the binding affinities of HMGA2, HMGA1b and HMG1a to the –426 to –257 ERCC1 fragment using a more narrow protein concentration range as described in (A). ( D ) Quantification of the HMGA2 binding data of (C). 100% – free DNA was plotted against the protein concentration on a logarithmic scale. The line represents the theoretical curve calculated from the relationship K d = [100% – % free DNA] × [free protein]/[complexes] using SigmaPlot Hill regression. K d(app) was 1.75 ± 0.31 nM for the –426 to –257 fragment.

    Techniques Used: Binding Assay, Labeling, Incubation, Radioactivity, Protein Concentration

    Fine mapping of HMGA2 (black bars) and ΔHMGA2 (gray bars) binding to the –330 to –287 region of ERCC1 promoter fragment. ( A ) Either the top or the bottom strand was 5′ end-labeled with T4 polynucleotide kinase and the double-stranded DNA was digested with hydroxyl-radicals in the absence or presence of 100 nM of the proteins. Reaction products were separated on 18% acrylamide sequencing gels and dried gels were scanned by PhosphorImager. ( B ) Quantification of the DNA footprinting of the top strand (top) or bottom strand (bottom). Each bar shows relative cutting frequency at a single base. The 100% cutting frequency corresponds to digestion of the DNA fragment in the absence of protein so that lower values mean protection upon protein binding. The presented results are mean values from four independent experiments. The sequence of the fragment is shown between the two panels. Arrows label the maxima of protection of the wild-type protein with the corresponding nucleotide number relative to the transcription start.
    Figure Legend Snippet: Fine mapping of HMGA2 (black bars) and ΔHMGA2 (gray bars) binding to the –330 to –287 region of ERCC1 promoter fragment. ( A ) Either the top or the bottom strand was 5′ end-labeled with T4 polynucleotide kinase and the double-stranded DNA was digested with hydroxyl-radicals in the absence or presence of 100 nM of the proteins. Reaction products were separated on 18% acrylamide sequencing gels and dried gels were scanned by PhosphorImager. ( B ) Quantification of the DNA footprinting of the top strand (top) or bottom strand (bottom). Each bar shows relative cutting frequency at a single base. The 100% cutting frequency corresponds to digestion of the DNA fragment in the absence of protein so that lower values mean protection upon protein binding. The presented results are mean values from four independent experiments. The sequence of the fragment is shown between the two panels. Arrows label the maxima of protection of the wild-type protein with the corresponding nucleotide number relative to the transcription start.

    Techniques Used: Binding Assay, Labeling, Sequencing, DNA Footprinting, Protein Binding

    Perturbation of DNA ERCC1 promoter conformation by HMGA2 and its mutants. A series of DNA constructs comprising promoter region –316 to –294 were produced either with inserted luminescence donor [(Eu3+)DTPA-AMCA-maleimide at the 3′ of the top strand ( A – C ) or at the bottom strand ( D – F ) and the fluorescence acceptor ( X ) Cy5 at different positions within the complementary strand, respectively. The LRET measurements were performed in the absence or presence of 12.5, 37 and 100 nM of HMGA2 (triangles), ΔHMGA2 (circles) and HMG2/LPP (squares). The concentration of labeled duplex was 15 nM.
    Figure Legend Snippet: Perturbation of DNA ERCC1 promoter conformation by HMGA2 and its mutants. A series of DNA constructs comprising promoter region –316 to –294 were produced either with inserted luminescence donor [(Eu3+)DTPA-AMCA-maleimide at the 3′ of the top strand ( A – C ) or at the bottom strand ( D – F ) and the fluorescence acceptor ( X ) Cy5 at different positions within the complementary strand, respectively. The LRET measurements were performed in the absence or presence of 12.5, 37 and 100 nM of HMGA2 (triangles), ΔHMGA2 (circles) and HMG2/LPP (squares). The concentration of labeled duplex was 15 nM.

    Techniques Used: Construct, Produced, Fluorescence, Concentration Assay, Labeling

    57) Product Images from "Role of Simian Virus 40 Vp1 Cysteines in Virion Infectivity"

    Article Title: Role of Simian Virus 40 Vp1 Cysteines in Virion Infectivity

    Journal: Journal of Virology

    doi:

    DNA replication and packaging by C207S and C254L mutants. To analyze viral DNA replication, total DNA was prepared from the sonicated lysate of cells transfected with wild-type or mutant (C207S or C254L) NO-SV40 as described in the text, and identical aliquots thereof were digested with Kpn I alone or with both Kpn I and Dpn I, followed by separation on a 0.9% agarose gel, Southern transfer, and hybridization with a nick-translated 32 P-labeled SV40 DNA probe (0.1 μg, or 10 7 cpm, in 10 ml). The radioactivity of the linearized 5.2-kbp viral DNA band for the Kpn I- Dpn I double reaction, quantitated by a phosphorimager, was expressed as a percentage of the radioactivity of the same band for the Kpn I-only reaction. To analyze viral DNA packaging, identical aliquots of each transfected cell lysate were either treated or not treated with DNase I as described in the text, and the remaining DNA in the lysate was purified and digested with Kpn I and analyzed by Southern blotting as above. The radioactivity of the 5.2-kbp DNase I-resistant viral DNA band was expressed as a percentage of the radioactivity of the same band for the non-DNase I-treated sample.
    Figure Legend Snippet: DNA replication and packaging by C207S and C254L mutants. To analyze viral DNA replication, total DNA was prepared from the sonicated lysate of cells transfected with wild-type or mutant (C207S or C254L) NO-SV40 as described in the text, and identical aliquots thereof were digested with Kpn I alone or with both Kpn I and Dpn I, followed by separation on a 0.9% agarose gel, Southern transfer, and hybridization with a nick-translated 32 P-labeled SV40 DNA probe (0.1 μg, or 10 7 cpm, in 10 ml). The radioactivity of the linearized 5.2-kbp viral DNA band for the Kpn I- Dpn I double reaction, quantitated by a phosphorimager, was expressed as a percentage of the radioactivity of the same band for the Kpn I-only reaction. To analyze viral DNA packaging, identical aliquots of each transfected cell lysate were either treated or not treated with DNase I as described in the text, and the remaining DNA in the lysate was purified and digested with Kpn I and analyzed by Southern blotting as above. The radioactivity of the 5.2-kbp DNase I-resistant viral DNA band was expressed as a percentage of the radioactivity of the same band for the non-DNase I-treated sample.

    Techniques Used: Sonication, Transfection, Mutagenesis, Agarose Gel Electrophoresis, Hybridization, Labeling, Radioactivity, Purification, Southern Blot

    The C254L mutant forms virion-like particles. Sonicated lysate alignots of wild-type (Wt) or C254L mutant NO-SV40-transfected cells, estimated to contain similar amounts of DNase I-resistant viral DNA (200 μl for the wild type and 1 ml for the C254L mutant), were centrifuged at 350 × g for 5 min at 4°C to pellet cellular debris. The supernatant was treated with DNase I as described in the text and was sedimented through a 5 to 32% sucrose gradient in 50 mM HEPES (pH 7.5) at 37,000 rpm at 4°C for 80 min in an SW41 rotor. Eighteen fractions were collected from the bottom of the gradient, and the DNA in each fraction was extracted following proteinase K treatment. One-half of each DNA sample was separated on a 0.9% agarose gel, Southern transferred, and hybridized with a nick-translated 32 P-labeled SV40 DNA probe. To the left of each panel, upper and lower lines indicate the positions of 7,554- and 5,243-bp marker DNA fragments, respectively, and an arrow points to the covalently closed circular DNA band. An arrowhead above fraction 4 indicates the location of control wild-type virions sedimented in a parallel gradient.
    Figure Legend Snippet: The C254L mutant forms virion-like particles. Sonicated lysate alignots of wild-type (Wt) or C254L mutant NO-SV40-transfected cells, estimated to contain similar amounts of DNase I-resistant viral DNA (200 μl for the wild type and 1 ml for the C254L mutant), were centrifuged at 350 × g for 5 min at 4°C to pellet cellular debris. The supernatant was treated with DNase I as described in the text and was sedimented through a 5 to 32% sucrose gradient in 50 mM HEPES (pH 7.5) at 37,000 rpm at 4°C for 80 min in an SW41 rotor. Eighteen fractions were collected from the bottom of the gradient, and the DNA in each fraction was extracted following proteinase K treatment. One-half of each DNA sample was separated on a 0.9% agarose gel, Southern transferred, and hybridized with a nick-translated 32 P-labeled SV40 DNA probe. To the left of each panel, upper and lower lines indicate the positions of 7,554- and 5,243-bp marker DNA fragments, respectively, and an arrow points to the covalently closed circular DNA band. An arrowhead above fraction 4 indicates the location of control wild-type virions sedimented in a parallel gradient.

    Techniques Used: Mutagenesis, Sonication, Transfection, Agarose Gel Electrophoresis, Labeling, Marker

    58) Product Images from "The E8∧E2 Gene Product of Human Papillomavirus Type 16 Represses Early Transcription and Replication but Is Dispensable for Viral Plasmid Persistence in Keratinocytes ▿"

    Article Title: The E8∧E2 Gene Product of Human Papillomavirus Type 16 Represses Early Transcription and Replication but Is Dispensable for Viral Plasmid Persistence in Keratinocytes ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01481-08

    16-E8 ∧ E2 represses initial HPV-16 amplification. (A) SCC13 cells were transfected with HPV-16 wt or E8− mutant plasmids and the indicated quantities (ng) of pCG 16-E8 ∧ E2. BamHI and DpnI-resistant fragments (derived from total DNA harvest and digestion) were detected by Southern blotting. DNA binding by 16-E8 ∧ E2 represses replication of the SV40 origin. (B) SCC13 cells were transfected with either E2x3 SVE cat or SVE cat (where the origin of replication [ORI] and E2 binding sites are shown as open and filled boxes, respectively), pCG Tag (except lanes 1 and 6), and the indicated quantities of pCG 16-E8 ∧ E2. DNA was harvested after 3 days, and the DpnI-resistant, BamHI digestion fragments were visualized by Southern blotting. (C) SCC13 cells were cotransfected with E2x3 SVE cat and the indicated mixture of wt and E8− mutant HPV-16 DNA (in μg). Total DNA was harvested and analyzed as described for panel A with probes specific to HPV-16 or SV40 DNA. The arrows indicate the mobilities of the DpnI-resistant HPV-16 (8 kb) and SV40 (4.5 kb) fragments in each panel. (D) Complementation of HPV-16 replication, using E1 (plasmid A)- or E8 (plasmid B)-expressing constructs. Total DNA was harvested, and the replication of plasmid A and B species was monitored by Southern blotting of respective 7.9-kb and 5.4-kb DpnI-resistant fragments after appropriate digestion. A linearized HPV-16 genome (30 pg) was included as a positive blotting control.
    Figure Legend Snippet: 16-E8 ∧ E2 represses initial HPV-16 amplification. (A) SCC13 cells were transfected with HPV-16 wt or E8− mutant plasmids and the indicated quantities (ng) of pCG 16-E8 ∧ E2. BamHI and DpnI-resistant fragments (derived from total DNA harvest and digestion) were detected by Southern blotting. DNA binding by 16-E8 ∧ E2 represses replication of the SV40 origin. (B) SCC13 cells were transfected with either E2x3 SVE cat or SVE cat (where the origin of replication [ORI] and E2 binding sites are shown as open and filled boxes, respectively), pCG Tag (except lanes 1 and 6), and the indicated quantities of pCG 16-E8 ∧ E2. DNA was harvested after 3 days, and the DpnI-resistant, BamHI digestion fragments were visualized by Southern blotting. (C) SCC13 cells were cotransfected with E2x3 SVE cat and the indicated mixture of wt and E8− mutant HPV-16 DNA (in μg). Total DNA was harvested and analyzed as described for panel A with probes specific to HPV-16 or SV40 DNA. The arrows indicate the mobilities of the DpnI-resistant HPV-16 (8 kb) and SV40 (4.5 kb) fragments in each panel. (D) Complementation of HPV-16 replication, using E1 (plasmid A)- or E8 (plasmid B)-expressing constructs. Total DNA was harvested, and the replication of plasmid A and B species was monitored by Southern blotting of respective 7.9-kb and 5.4-kb DpnI-resistant fragments after appropriate digestion. A linearized HPV-16 genome (30 pg) was included as a positive blotting control.

    Techniques Used: Amplification, Transfection, Mutagenesis, Derivative Assay, Southern Blot, Binding Assay, Plasmid Preparation, Expressing, Construct

    Mapping of HPV-16 E8 ∧ E2 transcripts. (A) Schematic diagram of the 16-E8 ∧ E2 cistron and mRNA mapping schemes. (B) “Primer walk” of E8 ∧ E2 cDNA generated by competitive PCR, where “competimer” refers to the 1,052-bp PCR competitor DNA spanning the 16-E8 ∧ E2 splice junction and “M” represents a 100-bp ladder size marker. Arrows indicate approximate mobilities of cDNA relative to competimer products of various molecular weights. Representative amplification ratios (cDNA to competimer) were determined by scanning densitometry.
    Figure Legend Snippet: Mapping of HPV-16 E8 ∧ E2 transcripts. (A) Schematic diagram of the 16-E8 ∧ E2 cistron and mRNA mapping schemes. (B) “Primer walk” of E8 ∧ E2 cDNA generated by competitive PCR, where “competimer” refers to the 1,052-bp PCR competitor DNA spanning the 16-E8 ∧ E2 splice junction and “M” represents a 100-bp ladder size marker. Arrows indicate approximate mobilities of cDNA relative to competimer products of various molecular weights. Representative amplification ratios (cDNA to competimer) were determined by scanning densitometry.

    Techniques Used: Generated, Polymerase Chain Reaction, Marker, Amplification

    Functional mapping of the 16-E8 ∧ E2 cistron. (A) SCC13 cells were transfected with the indicated HPV-16 wt or mutant plasmid. Total cell DNA was harvested from each, transfected after 5 days of culture, and digested with BamHI and DpnI. Initial plasmid amplification was detected as DpnI-resistant fragments by Southern blotting. A linearized HPV-16 genome (30 pg) was included as a positive blotting control. E8−, E1−, and E2− indicate translation terminations within the respective viral genes, while SD1302− indicates the splice donor site at nt 1302. (B) Summary schematic of 16-E8 ∧ E2 cistron with locations of mutations within the E1, E2, and E8 coding regions. Termination linkers are indicated by asterisks and deletions by brackets, while the shaded boxes correspond to conserved regions of the ORFs.
    Figure Legend Snippet: Functional mapping of the 16-E8 ∧ E2 cistron. (A) SCC13 cells were transfected with the indicated HPV-16 wt or mutant plasmid. Total cell DNA was harvested from each, transfected after 5 days of culture, and digested with BamHI and DpnI. Initial plasmid amplification was detected as DpnI-resistant fragments by Southern blotting. A linearized HPV-16 genome (30 pg) was included as a positive blotting control. E8−, E1−, and E2− indicate translation terminations within the respective viral genes, while SD1302− indicates the splice donor site at nt 1302. (B) Summary schematic of 16-E8 ∧ E2 cistron with locations of mutations within the E1, E2, and E8 coding regions. Termination linkers are indicated by asterisks and deletions by brackets, while the shaded boxes correspond to conserved regions of the ORFs.

    Techniques Used: Functional Assay, Transfection, Mutagenesis, Plasmid Preparation, Amplification, Southern Blot

    A 16-E8 ∧ E2 spliced message encodes a transcriptional repressor. (A) Organization of the HPV-16 genome; the E8 and E2 coding regions are shown in black. (B) Total mRNA was isolated from HFKs that were stably transfected with HPV-16 DNA. The reverse-transcribed mRNA was amplified by reverse transcription-PCR with primers located within the E2 coding regions and upstream of the E8 coding regions (see Results). (C) Expression vectors used in this study. (D) 16-E8 ∧ E2 interferes with E2 transactivation. HeLa cells were cotransfected with pCG (16)-E2 (10 ng), E2x2-Sp1x2 tk (−38) cat (3 μg), and the indicated quantities of pCG (16)E8 ∧ E2 plasmid. Enzymatic CAT activities are expressed as a percentage of the baseline and represent averages of two or three independent experiments. (E to G) 16-E8 ∧ E2 interferes with enhancer function. Cells were transfected with tk promoter cat constructions containing E2 binding sites upstream or downstream of the SV40 enhancer or a control (G) without E2 binding sites. (H) 16-E8 ∧ E2 represses enhancer-dependent P97 transcription. HeLa cells were transfected with p(16)-6153-506 cat , the indicated amounts of pCG (16)E8 ∧ E2, and pSVNΔ13 cat as an internal transfection control. HPV-16 E2 binding sites 4 through 1 are represented by black boxes; Enh, keratinocyte-dependent enhancer. Total RNA was analyzed by RNase protection. (C to G) The total amount of pCG vector was kept constant by the addition of reciprocal amounts of the plasmid pCG- neo .
    Figure Legend Snippet: A 16-E8 ∧ E2 spliced message encodes a transcriptional repressor. (A) Organization of the HPV-16 genome; the E8 and E2 coding regions are shown in black. (B) Total mRNA was isolated from HFKs that were stably transfected with HPV-16 DNA. The reverse-transcribed mRNA was amplified by reverse transcription-PCR with primers located within the E2 coding regions and upstream of the E8 coding regions (see Results). (C) Expression vectors used in this study. (D) 16-E8 ∧ E2 interferes with E2 transactivation. HeLa cells were cotransfected with pCG (16)-E2 (10 ng), E2x2-Sp1x2 tk (−38) cat (3 μg), and the indicated quantities of pCG (16)E8 ∧ E2 plasmid. Enzymatic CAT activities are expressed as a percentage of the baseline and represent averages of two or three independent experiments. (E to G) 16-E8 ∧ E2 interferes with enhancer function. Cells were transfected with tk promoter cat constructions containing E2 binding sites upstream or downstream of the SV40 enhancer or a control (G) without E2 binding sites. (H) 16-E8 ∧ E2 represses enhancer-dependent P97 transcription. HeLa cells were transfected with p(16)-6153-506 cat , the indicated amounts of pCG (16)E8 ∧ E2, and pSVNΔ13 cat as an internal transfection control. HPV-16 E2 binding sites 4 through 1 are represented by black boxes; Enh, keratinocyte-dependent enhancer. Total RNA was analyzed by RNase protection. (C to G) The total amount of pCG vector was kept constant by the addition of reciprocal amounts of the plasmid pCG- neo .

    Techniques Used: Isolation, Stable Transfection, Transfection, Amplification, Polymerase Chain Reaction, Expressing, Plasmid Preparation, Binding Assay

    cis elements regulate 16-E8 ∧ E2 expression. Equimolar quantities of HPV-16 E8-defective (plasmid A) and E8-expressing (plasmid B) plasmid species were cotransfected into SCC13 cells as illustrated in the complementation scheme. Total DNA was harvested, and the total replication from both plasmid A and B species was monitored by Southern blotting after appropriate digestion. E1−, E8−, and E2 TAD− indicate mutations in the respective genes, while SA409 refers to the indicated splice acceptor signal at nt 409. T(65) refers to mutation of the TATAA box at nt 65, enh− refers to the mutated keratinocyte-dependent enhancer, and E2#1+2− and E2#3+4− indicate mutations in the respective E2 binding sites. A linearized HPV-16 genome (30 pg) was included as a positive blotting control.
    Figure Legend Snippet: cis elements regulate 16-E8 ∧ E2 expression. Equimolar quantities of HPV-16 E8-defective (plasmid A) and E8-expressing (plasmid B) plasmid species were cotransfected into SCC13 cells as illustrated in the complementation scheme. Total DNA was harvested, and the total replication from both plasmid A and B species was monitored by Southern blotting after appropriate digestion. E1−, E8−, and E2 TAD− indicate mutations in the respective genes, while SA409 refers to the indicated splice acceptor signal at nt 409. T(65) refers to mutation of the TATAA box at nt 65, enh− refers to the mutated keratinocyte-dependent enhancer, and E2#1+2− and E2#3+4− indicate mutations in the respective E2 binding sites. A linearized HPV-16 genome (30 pg) was included as a positive blotting control.

    Techniques Used: Expressing, Plasmid Preparation, Southern Blot, Mutagenesis, Binding Assay

    59) Product Images from "Bisulfite sequencing of chromatin immunoprecipitated DNA (BisChIP-seq) directly informs methylation status of histone-modified DNA"

    Article Title: Bisulfite sequencing of chromatin immunoprecipitated DNA (BisChIP-seq) directly informs methylation status of histone-modified DNA

    Journal: Genome Research

    doi: 10.1101/gr.132076.111

    BisChIP-seq examples showing differential methylation and allele-specific methylation in H3K27me3-enriched ChIP DNA. ( A ) UCSC Genome Browser screen shot of BisChIP-seq data showing the RCSD1 TSS and CpG island, where H3K27me3-modified histones are enriched in both PrEC and LNCaP. (Purple shading) In PrEC cells the CpG island is unmethylated, whereas in LNCaP cells the island becomes extensively DNA methylated without losing the H3K27me3 mark. Individual bisulfite methylation sequencing reads are shown with CpG sites (black circles) in yellow shading for each molecule. (Red circles) CpG DNA methylation. ( B ) Example of allele-specific methylation in PrEC cells at rs637481 on chromosome 1. UCSC Genome Browser screen shot of BisChIP-seq data indicates regions of significant H3K27me3-enrichment called by ChromaBlocks. (Purple line) Position of the A/G SNP at rs637481. Individual bisulfite molecule sequencing reads are shown with all CpG sites in the sequence (black circles) in yellow shading for each molecule. (Red circles) BisChIP-seq readout of CpG DNA methylation. The allele-specific methylation ratio is indicated by bar graphs.
    Figure Legend Snippet: BisChIP-seq examples showing differential methylation and allele-specific methylation in H3K27me3-enriched ChIP DNA. ( A ) UCSC Genome Browser screen shot of BisChIP-seq data showing the RCSD1 TSS and CpG island, where H3K27me3-modified histones are enriched in both PrEC and LNCaP. (Purple shading) In PrEC cells the CpG island is unmethylated, whereas in LNCaP cells the island becomes extensively DNA methylated without losing the H3K27me3 mark. Individual bisulfite methylation sequencing reads are shown with CpG sites (black circles) in yellow shading for each molecule. (Red circles) CpG DNA methylation. ( B ) Example of allele-specific methylation in PrEC cells at rs637481 on chromosome 1. UCSC Genome Browser screen shot of BisChIP-seq data indicates regions of significant H3K27me3-enrichment called by ChromaBlocks. (Purple line) Position of the A/G SNP at rs637481. Individual bisulfite molecule sequencing reads are shown with all CpG sites in the sequence (black circles) in yellow shading for each molecule. (Red circles) BisChIP-seq readout of CpG DNA methylation. The allele-specific methylation ratio is indicated by bar graphs.

    Techniques Used: Methylation, Chromatin Immunoprecipitation, Modification, Sequencing, DNA Methylation Assay

    60) Product Images from "Novel amplification of DNA in a hairpin structure: towards a radical elimination of PCR errors from amplified DNA"

    Article Title: Novel amplification of DNA in a hairpin structure: towards a radical elimination of PCR errors from amplified DNA

    Journal: Nucleic Acids Research

    doi:

    Outline of hairpin-PCR. ( A ) Scheme for removing PCR errors following amplification of DNA in a hairpin structure. ( B ) Expected structure and sequence of hairpin A. ( C ) Expected structure and sequence of hairpin D, an oligonucleotide encompassing both top and bottom strands of p53 exon 9.
    Figure Legend Snippet: Outline of hairpin-PCR. ( A ) Scheme for removing PCR errors following amplification of DNA in a hairpin structure. ( B ) Expected structure and sequence of hairpin A. ( C ) Expected structure and sequence of hairpin D, an oligonucleotide encompassing both top and bottom strands of p53 exon 9.

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing

    61) Product Images from "Timing of Developmentally Programmed Excision and Circularization of Paramecium Internal Eliminated Sequences"

    Article Title: Timing of Developmentally Programmed Excision and Circularization of Paramecium Internal Eliminated Sequences

    Journal: Molecular and Cellular Biology

    doi:

    Direct detection of circular forms of the excised IESs in autogamous cells. (A) Diagram of IESs 51A2591 and 51G4404, with the corresponding oligonucleotide primers represented by arrowheads. (B) PCR amplification of circular junctions from autogamous cells. Primer sets 51A1 plus 51A9 and 51G7 plus 51G8 were used for the detection of a junction between the ends of IES 51A2591 (left panel) and IES 51G4404 (right panel), respectively (see Materials and Methods). The t = 0 time point was arbitrarily defined as stated in the text, and other time points refer to later stages of autogamy (see the text for details), up to karyonidal division (kar. div.). Electrophoresis was carried out on a 3% NuSieve gel. The major amplification products are indicated by their sizes, and the 342-bp minor band corresponding to the form lacking the 28-bp IES internal to IES 51A2591 is indicated by an asterisk. (C) Southern blot analysis of uncut genomic DNA from autogamous cells. Electrophoresis was carried out on a 1.5% agarose–1.5% NuSieve gel. Chromosomal (I) and extrachromosomal (II) forms of IES 51A2591 (left panel) were revealed after hybridization with a 370-bp PCR fragment specific for the IES sequence and amplified with primers 51A3 and 51A4. A 236-bp fragment, specific for IES 51G4404 and amplified with primers 51G5 and 51G6, was used as a probe in the right panel. The central panel shows ethidium bromide staining of supercoiled (C) or linear (L) forms of a 411-bp control minicircle (see Materials and Methods), electrophoresed on the same gel. (D) Restriction maps of the micronuclear regions around IES 51A2591 (left) and IES 51G4404 (right). Chromosomal IESs are drawn as black boxes, and the 28-bp IES inside IES 51A2591 is shown as a white square. The corresponding circular IES molecules are not drawn to scale. Restriction enzymes: B, Bsa I; D, Dde I; H, Hin fI; Xm, Xmn I. (E) Dde I and exonuclease III (exo III) treatment of total genomic DNA from 100% autogamous cells. DNA extracted from autogamous cells at t = 12 h 15 min and t = 21 h was treated with Dde I and, where indicated, incubated for 2 h at 37°C with 200 U of exonuclease III. Electrophoresis and hybridization probes were as in panel C. IESs are shown as black boxes on the diagrams of their corresponding micronuclear Dde I fragments.
    Figure Legend Snippet: Direct detection of circular forms of the excised IESs in autogamous cells. (A) Diagram of IESs 51A2591 and 51G4404, with the corresponding oligonucleotide primers represented by arrowheads. (B) PCR amplification of circular junctions from autogamous cells. Primer sets 51A1 plus 51A9 and 51G7 plus 51G8 were used for the detection of a junction between the ends of IES 51A2591 (left panel) and IES 51G4404 (right panel), respectively (see Materials and Methods). The t = 0 time point was arbitrarily defined as stated in the text, and other time points refer to later stages of autogamy (see the text for details), up to karyonidal division (kar. div.). Electrophoresis was carried out on a 3% NuSieve gel. The major amplification products are indicated by their sizes, and the 342-bp minor band corresponding to the form lacking the 28-bp IES internal to IES 51A2591 is indicated by an asterisk. (C) Southern blot analysis of uncut genomic DNA from autogamous cells. Electrophoresis was carried out on a 1.5% agarose–1.5% NuSieve gel. Chromosomal (I) and extrachromosomal (II) forms of IES 51A2591 (left panel) were revealed after hybridization with a 370-bp PCR fragment specific for the IES sequence and amplified with primers 51A3 and 51A4. A 236-bp fragment, specific for IES 51G4404 and amplified with primers 51G5 and 51G6, was used as a probe in the right panel. The central panel shows ethidium bromide staining of supercoiled (C) or linear (L) forms of a 411-bp control minicircle (see Materials and Methods), electrophoresed on the same gel. (D) Restriction maps of the micronuclear regions around IES 51A2591 (left) and IES 51G4404 (right). Chromosomal IESs are drawn as black boxes, and the 28-bp IES inside IES 51A2591 is shown as a white square. The corresponding circular IES molecules are not drawn to scale. Restriction enzymes: B, Bsa I; D, Dde I; H, Hin fI; Xm, Xmn I. (E) Dde I and exonuclease III (exo III) treatment of total genomic DNA from 100% autogamous cells. DNA extracted from autogamous cells at t = 12 h 15 min and t = 21 h was treated with Dde I and, where indicated, incubated for 2 h at 37°C with 200 U of exonuclease III. Electrophoresis and hybridization probes were as in panel C. IESs are shown as black boxes on the diagrams of their corresponding micronuclear Dde I fragments.

    Techniques Used: Polymerase Chain Reaction, Amplification, Electrophoresis, Southern Blot, Hybridization, Sequencing, Staining, Incubation

    62) Product Images from "Mitochondrial DNA Maintenance Is Regulated in Human Hepatoma Cells by Glycogen Synthase Kinase 3? and p53 in Response to Tumor Necrosis Factor ?"

    Article Title: Mitochondrial DNA Maintenance Is Regulated in Human Hepatoma Cells by Glycogen Synthase Kinase 3? and p53 in Response to Tumor Necrosis Factor ?

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0040879

    TNF-α induced mtDNA depletion, lesions and repair. (A) Cells were pretreated or not (TNF-α) for 1 h with 1 µg/ml TNF-R1 antibody (TNF-R1 Ab) or with 5 mM NAC. They were then treated for 0 to 6 h with 30 ng/ml TNF-α. To evaluate mtDNA depletion, total genomic DNA was isolated and quantification of mtDNA performed by simultaneous real-time qPCR amplification of fragments encoding mitochondrial 12S rRNA and nuclear 18S rRNA used as a reference gene. Results are expressed in 12S mtDNA over 18S nDNA relative ratio (mean values ± SEM of four independent experiments with four replicates, *p
    Figure Legend Snippet: TNF-α induced mtDNA depletion, lesions and repair. (A) Cells were pretreated or not (TNF-α) for 1 h with 1 µg/ml TNF-R1 antibody (TNF-R1 Ab) or with 5 mM NAC. They were then treated for 0 to 6 h with 30 ng/ml TNF-α. To evaluate mtDNA depletion, total genomic DNA was isolated and quantification of mtDNA performed by simultaneous real-time qPCR amplification of fragments encoding mitochondrial 12S rRNA and nuclear 18S rRNA used as a reference gene. Results are expressed in 12S mtDNA over 18S nDNA relative ratio (mean values ± SEM of four independent experiments with four replicates, *p

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Amplification

    63) Product Images from "A quick and cost effective method for the diagnosis of Mycobacterium ulcerans infection"

    Article Title: A quick and cost effective method for the diagnosis of Mycobacterium ulcerans infection

    Journal: BMC Infectious Diseases

    doi: 10.1186/1471-2334-12-8

    Visualization of BU-LAMP products exposed to UV light . Tube 1 contains DNA extracted using the boil preparation method; tube 2 contains Qiagen extracted DNA; tube 3 contains raw/untreated sample; tube 4 is the positive control and tube 5 is the negative control.
    Figure Legend Snippet: Visualization of BU-LAMP products exposed to UV light . Tube 1 contains DNA extracted using the boil preparation method; tube 2 contains Qiagen extracted DNA; tube 3 contains raw/untreated sample; tube 4 is the positive control and tube 5 is the negative control.

    Techniques Used: Positive Control, Negative Control

    Gel electrophoregram of BU-LAMP products . M = 100 base pair marker, Lane 1 = raw sample, Lane 2 = Boil prepared DNA, Lane 3 = Qiagen extracted DNA, Lane 4 = Positive control, Lane 5 = M. marinum DNA, Lane 6 = Negative control.
    Figure Legend Snippet: Gel electrophoregram of BU-LAMP products . M = 100 base pair marker, Lane 1 = raw sample, Lane 2 = Boil prepared DNA, Lane 3 = Qiagen extracted DNA, Lane 4 = Positive control, Lane 5 = M. marinum DNA, Lane 6 = Negative control.

    Techniques Used: Marker, Positive Control, Negative Control

    64) Product Images from "Absence of XMRV and Closely Related Viruses in Primary Prostate Cancer Tissues Used to Derive the XMRV-Infected Cell Line 22Rv1"

    Article Title: Absence of XMRV and Closely Related Viruses in Primary Prostate Cancer Tissues Used to Derive the XMRV-Infected Cell Line 22Rv1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0036072

    The sensitivity and specificity of qPCR assays were demonstrated with linear regression curves for XMRV VP62 plasmid and mouse tail DNA. (A) Nine different dilutions of XMRV VP62 plasmid (15 to 15×10 9 copies each reaction in duplicate) were used to generate the standard curve using gag , pol and env primer probe combinations. (B–D) Serial dilution of mouse tail DNA (1 fg to 100 ng each reaction in duplicate) were used to generate the data for (B) gag , (C) env and (D) IAP. e, exponent (10 to the power of n ).
    Figure Legend Snippet: The sensitivity and specificity of qPCR assays were demonstrated with linear regression curves for XMRV VP62 plasmid and mouse tail DNA. (A) Nine different dilutions of XMRV VP62 plasmid (15 to 15×10 9 copies each reaction in duplicate) were used to generate the standard curve using gag , pol and env primer probe combinations. (B–D) Serial dilution of mouse tail DNA (1 fg to 100 ng each reaction in duplicate) were used to generate the data for (B) gag , (C) env and (D) IAP. e, exponent (10 to the power of n ).

    Techniques Used: Real-time Polymerase Chain Reaction, Plasmid Preparation, Serial Dilution

    65) Product Images from "Resistance of Trichoplusia ni to Bacillus thuringiensis Toxin Cry1Ac Is Independent of Alteration of the Cadherin-Like Receptor for Cry Toxins"

    Article Title: Resistance of Trichoplusia ni to Bacillus thuringiensis Toxin Cry1Ac Is Independent of Alteration of the Cadherin-Like Receptor for Cry Toxins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0035991

    Schematic structures of T. ni cadherin cDNA and deduced protein sequences. (A) The cDNA (5734 bp in length) contains an open reading frame of 5202 bp from position 141 to 5342, and a poly A tail at the 3′ end. Also shown in (A) are two fragments of the genomic DNA of the cadherin gene, gDNA fragment 1 and gDNA fragment 2, amplified by PCR. gDNA PCR fragment 1 corresponds to the cDNA region from base positions 1705 to 1856 and contains an intron of 367–411 bp inserted between the cDNA base positions 1822 and 1823. gDNA PCR fragment 2 corresponds to the cDNA region from base position 4911 to 5114 and contains an intron of 291 bp inserted between cDNA base positions 4969 and 4970. (B) The deduced cadherin sequence (733 aa in length) contains a 21-aa signal peptide at the N-terminus, 11 cadherin repeats (from 1 to 11), followed by a membrane-proximal region (MPR), a transmembrane domain (TMD) of 23 amino acid residues, and a cytoplasmic domain (CPD) of 128 amino acid residues.
    Figure Legend Snippet: Schematic structures of T. ni cadherin cDNA and deduced protein sequences. (A) The cDNA (5734 bp in length) contains an open reading frame of 5202 bp from position 141 to 5342, and a poly A tail at the 3′ end. Also shown in (A) are two fragments of the genomic DNA of the cadherin gene, gDNA fragment 1 and gDNA fragment 2, amplified by PCR. gDNA PCR fragment 1 corresponds to the cDNA region from base positions 1705 to 1856 and contains an intron of 367–411 bp inserted between the cDNA base positions 1822 and 1823. gDNA PCR fragment 2 corresponds to the cDNA region from base position 4911 to 5114 and contains an intron of 291 bp inserted between cDNA base positions 4969 and 4970. (B) The deduced cadherin sequence (733 aa in length) contains a 21-aa signal peptide at the N-terminus, 11 cadherin repeats (from 1 to 11), followed by a membrane-proximal region (MPR), a transmembrane domain (TMD) of 23 amino acid residues, and a cytoplasmic domain (CPD) of 128 amino acid residues.

    Techniques Used: Amplification, Polymerase Chain Reaction, Sequencing

    66) Product Images from "A Novel Role of the PrpR as a Transcription Factor Involved in the Regulation of Methylcitrate Pathway in Mycobacterium tuberculosis"

    Article Title: A Novel Role of the PrpR as a Transcription Factor Involved in the Regulation of Methylcitrate Pathway in Mycobacterium tuberculosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043651

    PrpR binds the promoter region of prpDC and icl1 genes in intact M. tuberculosis cells. Identification of intracellular PrpR-DNA complex using immunoprecipitation. PrpR-DNA complexes cross-linked with glutaraldehyde were immunoprecipitated with anti-6HisPrpRMt polyclonal antibodies (sample 1). PCR was carried out with the primer pairs, p1129_Fw and p1129_Rv (p prpDR )(A); picl_Fw and picl_Rv (p icl1 )(B); and pmtrA_Fw and pmtrA_Rv (p mtrA , negative control)(C). Negative control (2) consisted of DNA template extracted from the cells subjected to immunoprecipitation, but nucleoprotein complexes were not previously cross-linked. Positives controls (+) were also performed using template obtained from strains subjected only to cross-linking (3) or total DNA extracted from the cells (4).
    Figure Legend Snippet: PrpR binds the promoter region of prpDC and icl1 genes in intact M. tuberculosis cells. Identification of intracellular PrpR-DNA complex using immunoprecipitation. PrpR-DNA complexes cross-linked with glutaraldehyde were immunoprecipitated with anti-6HisPrpRMt polyclonal antibodies (sample 1). PCR was carried out with the primer pairs, p1129_Fw and p1129_Rv (p prpDR )(A); picl_Fw and picl_Rv (p icl1 )(B); and pmtrA_Fw and pmtrA_Rv (p mtrA , negative control)(C). Negative control (2) consisted of DNA template extracted from the cells subjected to immunoprecipitation, but nucleoprotein complexes were not previously cross-linked. Positives controls (+) were also performed using template obtained from strains subjected only to cross-linking (3) or total DNA extracted from the cells (4).

    Techniques Used: Immunoprecipitation, Polymerase Chain Reaction, Negative Control

    67) Product Images from "Characterization of Genes Encoding for Acquired Bacitracin Resistance in Clostridium perfringens"

    Article Title: Characterization of Genes Encoding for Acquired Bacitracin Resistance in Clostridium perfringens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0044449

    PFGE and hybridization analysis of I-CeuI and MluI double-digested DNA of the bacitracin resistant C. perfringens strain c1261_A. PFGE analysis of C. perfringens strain c1261_A total DNA (A). Southern blot of C. perfringens isolate c1261_A total DNA probed with rrn (B) and with bcrB (C). Sizes (in kilobases) are indicated on the left.
    Figure Legend Snippet: PFGE and hybridization analysis of I-CeuI and MluI double-digested DNA of the bacitracin resistant C. perfringens strain c1261_A. PFGE analysis of C. perfringens strain c1261_A total DNA (A). Southern blot of C. perfringens isolate c1261_A total DNA probed with rrn (B) and with bcrB (C). Sizes (in kilobases) are indicated on the left.

    Techniques Used: Hybridization, Southern Blot

    68) Product Images from "Randomized pilot trial of a synbiotic dietary supplement in chronic HIV-1 infection"

    Article Title: Randomized pilot trial of a synbiotic dietary supplement in chronic HIV-1 infection

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/1472-6882-12-84

    Changes in fecal levels of supplemented probiotic bacteria. DNA from fresh stool was amplified using primer sets specific to the supplemented bacteria and also using universal r16S primers to derive total fecal bacterial content. The number of r16S copies of each probiotic species in relation to one million total fecal r16S copies was calculated and the change in this value from baseline to day 28 shown here. ( A ) Lactobacillus plantarum 2362, ( B ) Pediococcus pentosaceus 5–33:3, ( C ) Leuconostoc mesenteroides 32–77:1, and ( D ) Lactobacillus paracasei subsp paracasei 19, ( E ) Spearman correlation between changes in P. pent and L. plant (Synbiotic group only) . Horizontal bars denote the mean. N.S., not statistically significant ( p > 0.05). r, Spearman r value.
    Figure Legend Snippet: Changes in fecal levels of supplemented probiotic bacteria. DNA from fresh stool was amplified using primer sets specific to the supplemented bacteria and also using universal r16S primers to derive total fecal bacterial content. The number of r16S copies of each probiotic species in relation to one million total fecal r16S copies was calculated and the change in this value from baseline to day 28 shown here. ( A ) Lactobacillus plantarum 2362, ( B ) Pediococcus pentosaceus 5–33:3, ( C ) Leuconostoc mesenteroides 32–77:1, and ( D ) Lactobacillus paracasei subsp paracasei 19, ( E ) Spearman correlation between changes in P. pent and L. plant (Synbiotic group only) . Horizontal bars denote the mean. N.S., not statistically significant ( p > 0.05). r, Spearman r value.

    Techniques Used: Amplification

    69) Product Images from "Demethylation of Cancer/Testis Antigens and CpG ODN Stimulation Enhance Dendritic Cell and Cytotoxic T Lymphocyte Function in a Mouse Mammary Model"

    Article Title: Demethylation of Cancer/Testis Antigens and CpG ODN Stimulation Enhance Dendritic Cell and Cytotoxic T Lymphocyte Function in a Mouse Mammary Model

    Journal: BioMed Research International

    doi: 10.1155/2013/196894

    Methylation status of the P1A gene promoter and dose- and time-dependent P1A induction with 5-aza in 4T1 cells. 4T1, CT26, and A20 cell lines were treated with or without 3 μ M 5-aza for 48 h, and (a) methylated levels were shown, −, not treated with 5-aza and +, 5-aza treated cells (M, methylated DNA; U, unmethylated DNA). (b) P1A mRNA levels detected by RT-PCR. (c) 5-aza dose response. 4T1 cells were treated for 48 h with 5-aza at concentrations of 0, 0.03, 0.1, 0.3, 1.0, 3, or 10 μ M. Blank, negative control and P, positive control. (d) Time course 5-aza treatment at a fixed concentration of 3.0 μ M. 4T1 cells were incubated with 5-aza for 0, 2, 6, 12, 24, 48, or 72 h. The P1A expression was detected with P1A-specific primers. GAPDH was amplified as a control to demonstrate cDNA integrity. The positive lane contained EL9611 tumor cells that express high level of P1A.
    Figure Legend Snippet: Methylation status of the P1A gene promoter and dose- and time-dependent P1A induction with 5-aza in 4T1 cells. 4T1, CT26, and A20 cell lines were treated with or without 3 μ M 5-aza for 48 h, and (a) methylated levels were shown, −, not treated with 5-aza and +, 5-aza treated cells (M, methylated DNA; U, unmethylated DNA). (b) P1A mRNA levels detected by RT-PCR. (c) 5-aza dose response. 4T1 cells were treated for 48 h with 5-aza at concentrations of 0, 0.03, 0.1, 0.3, 1.0, 3, or 10 μ M. Blank, negative control and P, positive control. (d) Time course 5-aza treatment at a fixed concentration of 3.0 μ M. 4T1 cells were incubated with 5-aza for 0, 2, 6, 12, 24, 48, or 72 h. The P1A expression was detected with P1A-specific primers. GAPDH was amplified as a control to demonstrate cDNA integrity. The positive lane contained EL9611 tumor cells that express high level of P1A.

    Techniques Used: Methylation, Reverse Transcription Polymerase Chain Reaction, Negative Control, Positive Control, Concentration Assay, Incubation, Expressing, Amplification

    70) Product Images from "Stringent doxycycline-dependent control of gene activities using an episomal one-vector system"

    Article Title: Stringent doxycycline-dependent control of gene activities using an episomal one-vector system

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni137

    TGR-1 cells were transfected with 6 μg pRTS-1 DNA and expanded in 200 μg/ml hygromycin B for 4 weeks either in the presence ( A ) or in the absence of Dox ( B and C ). Cells expanded in the absence of Dox were treated with 1 μg/ml Dox for 24 h (B), or left untreated (C). Cells were visualized by light microscopy (upper panels of A, B and C) and fluorescence microscopy (lower panels). In (D–F), TGR-1 cells were transfected with 6 μg of either the vector expressing luciferase (pRTS-1), or, instead of luciferase, the gene encoding the nucleolar protein Bop1 wt, or its N-terminal deletion mutant Bop1Δ. Transfected cells were expanded under hygromycin B selection (200 μg/ml) for 2 weeks and eGFP expression determined by flow cytometry 24 h after the addition of Dox ( D and E ). ( F ) Equal numbers of cells transfected with luciferase, Bop1, and Bop1Δ were plated, Dox added and cell numbers determined after 6 days. The data are presented as percentage of control cells expressing luciferase. ( G ) Episomal replication of the plasmids was visualized by gel electrophoresis in 1% agarose after lysis of the cells in the gel ( 22 ). To evaluate the copy number, different number of Raji cells were included as well as TGR-1 cells to which different amounts of pRTS-1 had been added. Lanes (a)–(e) contain TGR-1 cells transfected with pRTS-1 (a), and vector expressing the nucleolar proteins or mutants WDR12 (b), WDR12 ΔNle (c), Bop1 (d) and Bop1Δ (e).
    Figure Legend Snippet: TGR-1 cells were transfected with 6 μg pRTS-1 DNA and expanded in 200 μg/ml hygromycin B for 4 weeks either in the presence ( A ) or in the absence of Dox ( B and C ). Cells expanded in the absence of Dox were treated with 1 μg/ml Dox for 24 h (B), or left untreated (C). Cells were visualized by light microscopy (upper panels of A, B and C) and fluorescence microscopy (lower panels). In (D–F), TGR-1 cells were transfected with 6 μg of either the vector expressing luciferase (pRTS-1), or, instead of luciferase, the gene encoding the nucleolar protein Bop1 wt, or its N-terminal deletion mutant Bop1Δ. Transfected cells were expanded under hygromycin B selection (200 μg/ml) for 2 weeks and eGFP expression determined by flow cytometry 24 h after the addition of Dox ( D and E ). ( F ) Equal numbers of cells transfected with luciferase, Bop1, and Bop1Δ were plated, Dox added and cell numbers determined after 6 days. The data are presented as percentage of control cells expressing luciferase. ( G ) Episomal replication of the plasmids was visualized by gel electrophoresis in 1% agarose after lysis of the cells in the gel ( 22 ). To evaluate the copy number, different number of Raji cells were included as well as TGR-1 cells to which different amounts of pRTS-1 had been added. Lanes (a)–(e) contain TGR-1 cells transfected with pRTS-1 (a), and vector expressing the nucleolar proteins or mutants WDR12 (b), WDR12 ΔNle (c), Bop1 (d) and Bop1Δ (e).

    Techniques Used: Transfection, Light Microscopy, Fluorescence, Microscopy, Plasmid Preparation, Expressing, Luciferase, Mutagenesis, Selection, Flow Cytometry, Cytometry, Nucleic Acid Electrophoresis, Lysis

    Reduction of background by the expression of tTS KRAB . ( A ) BJAB cells (10 7 ) were transfected with 10 μg pRT-1 and pRTS-1 DNA by electroporation. Cells were left untreated or treated with 1 μg/ml Dox immediately after electroporation and luciferase activity measured in cell extracts after 48 h. The relative light units of untreated cells are given above the columns. ( B and C ) Three BJAB cell lines were transfected with pRT-1 (B) or pRTS-1 (C), expanded in the absence of Dox, and analysed ∼2 months after transfection. Cells were treated with Dox or left untreated and were analysed for luciferase and eGFP expression 48 h after Dox addition. The luciferase activities of uninduced cells and the factor of inducibility are presented in the left panels. The differences in the factor of inducibility of pRT-1- and pRTS-1-transfected cells are due to the background activity in the absence of Dox. Note that the pattern of eGFP induction is not uniform in the different BJAB cell lines, even within one cell line. This is most probably due to the fact that the cell lines arising in individual wells of 96-well plates are not of clonal origin.
    Figure Legend Snippet: Reduction of background by the expression of tTS KRAB . ( A ) BJAB cells (10 7 ) were transfected with 10 μg pRT-1 and pRTS-1 DNA by electroporation. Cells were left untreated or treated with 1 μg/ml Dox immediately after electroporation and luciferase activity measured in cell extracts after 48 h. The relative light units of untreated cells are given above the columns. ( B and C ) Three BJAB cell lines were transfected with pRT-1 (B) or pRTS-1 (C), expanded in the absence of Dox, and analysed ∼2 months after transfection. Cells were treated with Dox or left untreated and were analysed for luciferase and eGFP expression 48 h after Dox addition. The luciferase activities of uninduced cells and the factor of inducibility are presented in the left panels. The differences in the factor of inducibility of pRT-1- and pRTS-1-transfected cells are due to the background activity in the absence of Dox. Note that the pattern of eGFP induction is not uniform in the different BJAB cell lines, even within one cell line. This is most probably due to the fact that the cell lines arising in individual wells of 96-well plates are not of clonal origin.

    Techniques Used: Expressing, Transfection, Electroporation, Luciferase, Activity Assay

    71) Product Images from "Stringent doxycycline-dependent control of gene activities using an episomal one-vector system"

    Article Title: Stringent doxycycline-dependent control of gene activities using an episomal one-vector system

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni137

    TGR-1 cells were transfected with 6 μg pRTS-1 DNA and expanded in 200 μg/ml hygromycin B for 4 weeks either in the presence ( A ) or in the absence of Dox ( B and C ). Cells expanded in the absence of Dox were treated with 1 μg/ml Dox for 24 h (B), or left untreated (C). Cells were visualized by light microscopy (upper panels of A, B and C) and fluorescence microscopy (lower panels). In (D–F), TGR-1 cells were transfected with 6 μg of either the vector expressing luciferase (pRTS-1), or, instead of luciferase, the gene encoding the nucleolar protein Bop1 wt, or its N-terminal deletion mutant Bop1Δ. Transfected cells were expanded under hygromycin B selection (200 μg/ml) for 2 weeks and eGFP expression determined by flow cytometry 24 h after the addition of Dox ( D and E ). ( F ) Equal numbers of cells transfected with luciferase, Bop1, and Bop1Δ were plated, Dox added and cell numbers determined after 6 days. The data are presented as percentage of control cells expressing luciferase. ( G ) Episomal replication of the plasmids was visualized by gel electrophoresis in 1% agarose after lysis of the cells in the gel ( 22 ). To evaluate the copy number, different number of Raji cells were included as well as TGR-1 cells to which different amounts of pRTS-1 had been added. Lanes (a)–(e) contain TGR-1 cells transfected with pRTS-1 (a), and vector expressing the nucleolar proteins or mutants WDR12 (b), WDR12 ΔNle (c), Bop1 (d) and Bop1Δ (e).
    Figure Legend Snippet: TGR-1 cells were transfected with 6 μg pRTS-1 DNA and expanded in 200 μg/ml hygromycin B for 4 weeks either in the presence ( A ) or in the absence of Dox ( B and C ). Cells expanded in the absence of Dox were treated with 1 μg/ml Dox for 24 h (B), or left untreated (C). Cells were visualized by light microscopy (upper panels of A, B and C) and fluorescence microscopy (lower panels). In (D–F), TGR-1 cells were transfected with 6 μg of either the vector expressing luciferase (pRTS-1), or, instead of luciferase, the gene encoding the nucleolar protein Bop1 wt, or its N-terminal deletion mutant Bop1Δ. Transfected cells were expanded under hygromycin B selection (200 μg/ml) for 2 weeks and eGFP expression determined by flow cytometry 24 h after the addition of Dox ( D and E ). ( F ) Equal numbers of cells transfected with luciferase, Bop1, and Bop1Δ were plated, Dox added and cell numbers determined after 6 days. The data are presented as percentage of control cells expressing luciferase. ( G ) Episomal replication of the plasmids was visualized by gel electrophoresis in 1% agarose after lysis of the cells in the gel ( 22 ). To evaluate the copy number, different number of Raji cells were included as well as TGR-1 cells to which different amounts of pRTS-1 had been added. Lanes (a)–(e) contain TGR-1 cells transfected with pRTS-1 (a), and vector expressing the nucleolar proteins or mutants WDR12 (b), WDR12 ΔNle (c), Bop1 (d) and Bop1Δ (e).

    Techniques Used: Transfection, Light Microscopy, Fluorescence, Microscopy, Plasmid Preparation, Expressing, Luciferase, Mutagenesis, Selection, Flow Cytometry, Cytometry, Nucleic Acid Electrophoresis, Lysis

    Reduction of background by the expression of tTS KRAB . ( A ) BJAB cells (10 7 ) were transfected with 10 μg pRT-1 and pRTS-1 DNA by electroporation. Cells were left untreated or treated with 1 μg/ml Dox immediately after electroporation and luciferase activity measured in cell extracts after 48 h. The relative light units of untreated cells are given above the columns. ( B and C ) Three BJAB cell lines were transfected with pRT-1 (B) or pRTS-1 (C), expanded in the absence of Dox, and analysed ∼2 months after transfection. Cells were treated with Dox or left untreated and were analysed for luciferase and eGFP expression 48 h after Dox addition. The luciferase activities of uninduced cells and the factor of inducibility are presented in the left panels. The differences in the factor of inducibility of pRT-1- and pRTS-1-transfected cells are due to the background activity in the absence of Dox. Note that the pattern of eGFP induction is not uniform in the different BJAB cell lines, even within one cell line. This is most probably due to the fact that the cell lines arising in individual wells of 96-well plates are not of clonal origin.
    Figure Legend Snippet: Reduction of background by the expression of tTS KRAB . ( A ) BJAB cells (10 7 ) were transfected with 10 μg pRT-1 and pRTS-1 DNA by electroporation. Cells were left untreated or treated with 1 μg/ml Dox immediately after electroporation and luciferase activity measured in cell extracts after 48 h. The relative light units of untreated cells are given above the columns. ( B and C ) Three BJAB cell lines were transfected with pRT-1 (B) or pRTS-1 (C), expanded in the absence of Dox, and analysed ∼2 months after transfection. Cells were treated with Dox or left untreated and were analysed for luciferase and eGFP expression 48 h after Dox addition. The luciferase activities of uninduced cells and the factor of inducibility are presented in the left panels. The differences in the factor of inducibility of pRT-1- and pRTS-1-transfected cells are due to the background activity in the absence of Dox. Note that the pattern of eGFP induction is not uniform in the different BJAB cell lines, even within one cell line. This is most probably due to the fact that the cell lines arising in individual wells of 96-well plates are not of clonal origin.

    Techniques Used: Expressing, Transfection, Electroporation, Luciferase, Activity Assay

    72) Product Images from "Effects of DNMT1 and HDAC Inhibitors on Gene-Specific Methylation Reprogramming during Porcine Somatic Cell Nuclear Transfer"

    Article Title: Effects of DNMT1 and HDAC Inhibitors on Gene-Specific Methylation Reprogramming during Porcine Somatic Cell Nuclear Transfer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064705

    Overexpression of MBD3 at eight cell stage. To exclude the embryos without over-expression due to failure of injection or import of plasmid DNA into the nucleus, the same molar ratio of CMV promoter-driven MBD3 -coding plasmid and eGFP-coding plasmid were co-injected into the cytoplasmic of constructed oocytes, only those embryos with green fluorescence were picked(A).In addition, over-expressed MBD3 was also validated by quantitative PCR (B).
    Figure Legend Snippet: Overexpression of MBD3 at eight cell stage. To exclude the embryos without over-expression due to failure of injection or import of plasmid DNA into the nucleus, the same molar ratio of CMV promoter-driven MBD3 -coding plasmid and eGFP-coding plasmid were co-injected into the cytoplasmic of constructed oocytes, only those embryos with green fluorescence were picked(A).In addition, over-expressed MBD3 was also validated by quantitative PCR (B).

    Techniques Used: Over Expression, Injection, Plasmid Preparation, Construct, Fluorescence, Real-time Polymerase Chain Reaction

    73) Product Images from "Direct Cell Lysis for Single-Cell Gene Expression Profiling"

    Article Title: Direct Cell Lysis for Single-Cell Gene Expression Profiling

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2013.00274

    Evaluation of direct cell lysis protocols on RT-qPCR . (A) The RT-qPCR yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike using 17 lysis conditions. Five nanograms of purified RNA was used in all RT reactions. Relative RT yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage relative to the water control for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Lysis conditions with Cq-values below that of the water control are RT enhancing agents, while conditions with higher Cq-values are inhibitory. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are shown in Table S4 in Supplementary Material. (B) Mean RT yield for Gapdh , Vim , Dll , and Jag1 . The relative transcript yield of each transcript was averaged and compared to the optimal RT-qPCR condition (RT mix). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.
    Figure Legend Snippet: Evaluation of direct cell lysis protocols on RT-qPCR . (A) The RT-qPCR yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike using 17 lysis conditions. Five nanograms of purified RNA was used in all RT reactions. Relative RT yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage relative to the water control for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Lysis conditions with Cq-values below that of the water control are RT enhancing agents, while conditions with higher Cq-values are inhibitory. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are shown in Table S4 in Supplementary Material. (B) Mean RT yield for Gapdh , Vim , Dll , and Jag1 . The relative transcript yield of each transcript was averaged and compared to the optimal RT-qPCR condition (RT mix). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

    Techniques Used: Lysis, Quantitative RT-PCR, Purification, Polymerase Chain Reaction

    Evaluation of direct cell lysis protocols . (A) The lysis yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike compared at 17 lysis conditions. Thirty-two astrocytes were sorted for each condition. Relative cDNA yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage compared to the optimal lysis condition for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are listed in Table S3 in Supplementary Material. (B) Mean cDNA yield of the transcripts. Expressions of Gapdh , Vim , Dll , and Jag1 were averaged and are compared to the overall optimal lysis condition (1 mg/ml BSA). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.
    Figure Legend Snippet: Evaluation of direct cell lysis protocols . (A) The lysis yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike compared at 17 lysis conditions. Thirty-two astrocytes were sorted for each condition. Relative cDNA yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage compared to the optimal lysis condition for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are listed in Table S3 in Supplementary Material. (B) Mean cDNA yield of the transcripts. Expressions of Gapdh , Vim , Dll , and Jag1 were averaged and are compared to the overall optimal lysis condition (1 mg/ml BSA). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

    Techniques Used: Lysis, Polymerase Chain Reaction

    74) Product Images from "Hox genes are involved in vascular wall-resident multipotent stem cell differentiation into smooth muscle cells"

    Article Title: Hox genes are involved in vascular wall-resident multipotent stem cell differentiation into smooth muscle cells

    Journal: Scientific Reports

    doi: 10.1038/srep02178

    HOXB7, HOXC6 and HOXC8 signalling down-stream regulated genes were analyzed in human VW-MPSCs by gene expression profiling using Affymetrix® DNA chips. Results are presented as scheme depicting numbers of genes significantly altered upon siRNA silencing (mean signal control (average) compared to mean signal HOXC6 (average) as well as HOXB7 (average) and HOXC8 (average)). For each probe set three (Control, HOXC6, HOXB7) and two (HOXC8) Affymetrix® DNA chips were used. Histone genes as well as TAGLN which were represented on the chips are listened and fold induction (siRNA treatment/control) was calculated. For complete list see Supplementary Table S1 online.
    Figure Legend Snippet: HOXB7, HOXC6 and HOXC8 signalling down-stream regulated genes were analyzed in human VW-MPSCs by gene expression profiling using Affymetrix® DNA chips. Results are presented as scheme depicting numbers of genes significantly altered upon siRNA silencing (mean signal control (average) compared to mean signal HOXC6 (average) as well as HOXB7 (average) and HOXC8 (average)). For each probe set three (Control, HOXC6, HOXB7) and two (HOXC8) Affymetrix® DNA chips were used. Histone genes as well as TAGLN which were represented on the chips are listened and fold induction (siRNA treatment/control) was calculated. For complete list see Supplementary Table S1 online.

    Techniques Used: Expressing

    Cell cycle analysis after silencing over-expressed HOX genes and H1 in cultured vascular wall-derived MPSCs. VW-derived MPSCs were transfected with Control (non-silencing, HOXC8, HOXC6, HOXB7 or H1 siRNA alone or in indicated combinations and subjected for immunofluorescence staining for the proliferation marker Ki67 (A). Cell cycle distribution was analysed by Western blot analysis of Cyclin B1 and D1 expression (B) as well as by Nicoletti staining (C). Two-color flow cytometric analysis was used to quantify cells actively synthesizing DNA and their position in G1, G0/1, S, G2/M phase of the cell cycle. Representative images for three independent experiments are shown. Data are presented as mean from 3 independent experiments.
    Figure Legend Snippet: Cell cycle analysis after silencing over-expressed HOX genes and H1 in cultured vascular wall-derived MPSCs. VW-derived MPSCs were transfected with Control (non-silencing, HOXC8, HOXC6, HOXB7 or H1 siRNA alone or in indicated combinations and subjected for immunofluorescence staining for the proliferation marker Ki67 (A). Cell cycle distribution was analysed by Western blot analysis of Cyclin B1 and D1 expression (B) as well as by Nicoletti staining (C). Two-color flow cytometric analysis was used to quantify cells actively synthesizing DNA and their position in G1, G0/1, S, G2/M phase of the cell cycle. Representative images for three independent experiments are shown. Data are presented as mean from 3 independent experiments.

    Techniques Used: Cell Cycle Assay, Cell Culture, Derivative Assay, Transfection, Immunofluorescence, Staining, Marker, Western Blot, Expressing, Flow Cytometry

    HOXB7, HOXC6 and HOXC8 gene expression silencing alters methylation pattern of the TAGLN promoter sequence. Genomic DNA was isolated from control (non-silencing siRNA) and silencing siRNA (HOXC8, HOXC6, HOXB7) transfected VW-MPSC, subjected for bisulfite conversion and finally sequenced using specific primers for the modified DNA which do not contain any CpG sites in their sequence and were generated around the predicted CpG islands. Predicted promoter regions of the human gene TAGLN (ENSG00000149591) were emphasized. Org: Original sequence; BSC bisulfite modified sequence (MethPrimer results); HOXB7, HOXC6, HOXC8, Con (non-silencing control) sequence analysis after bisulfite conversion of siRNA treated genomic DNA isolates; ++ CpG sites (for display, assume all CpG sites are methylated); non-CpG 'C' converted to ‘T’; * similar to BSC (original); # C vs T; TATA box (at position 368) in bold red; promoter start at position 500 encircled. Arrows indicate positions which were altered in non silencing siRNA treated cells as compared to original sequence (BSC), whereas siRNA (HOXB7 and HOXC6) treatment results in sequences similar to original sequence (BSC). Underlined nucleotides of control sequence indicate additional CpG sites.
    Figure Legend Snippet: HOXB7, HOXC6 and HOXC8 gene expression silencing alters methylation pattern of the TAGLN promoter sequence. Genomic DNA was isolated from control (non-silencing siRNA) and silencing siRNA (HOXC8, HOXC6, HOXB7) transfected VW-MPSC, subjected for bisulfite conversion and finally sequenced using specific primers for the modified DNA which do not contain any CpG sites in their sequence and were generated around the predicted CpG islands. Predicted promoter regions of the human gene TAGLN (ENSG00000149591) were emphasized. Org: Original sequence; BSC bisulfite modified sequence (MethPrimer results); HOXB7, HOXC6, HOXC8, Con (non-silencing control) sequence analysis after bisulfite conversion of siRNA treated genomic DNA isolates; ++ CpG sites (for display, assume all CpG sites are methylated); non-CpG 'C' converted to ‘T’; * similar to BSC (original); # C vs T; TATA box (at position 368) in bold red; promoter start at position 500 encircled. Arrows indicate positions which were altered in non silencing siRNA treated cells as compared to original sequence (BSC), whereas siRNA (HOXB7 and HOXC6) treatment results in sequences similar to original sequence (BSC). Underlined nucleotides of control sequence indicate additional CpG sites.

    Techniques Used: Expressing, Methylation, Sequencing, Isolation, Transfection, Modification, Generated

    75) Product Images from "Mismatch Repair Genes Mlh1 and Mlh3 Modify CAG Instability in Huntington's Disease Mice: Genome-Wide and Candidate Approaches"

    Article Title: Mismatch Repair Genes Mlh1 and Mlh3 Modify CAG Instability in Huntington's Disease Mice: Genome-Wide and Candidate Approaches

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003930

    Mlh1 is required for striatal and liver HTT CAG repeat instability in B6. Hdh Q111/+ mice. (A) Representative GeneMapper profiles of HTT CAG repeat size distributions in the tail, striatum and liver of 22-week-old B6. Hdh Q111/+ mice on different Mlh1 genetic backgrounds. Mlh1 +/+ , CAG113; Mlh1 +/− , CAG113; Mlh1 −/− , CAG110. (B) Quantification of striatal and liver HTT CAG instability indices in these mice reveals a statistically significant decrease in HTT CAG instability in the absence of Mlh1 . Mlh1 +/+ , CAG115.3±4.9SD, n = 6; Mlh1 +/− , CAG112.0±2.1SD, n = 6; Mlh1 −/− , CAG109.3±2.6SD, n = 6. Bar graphs represent mean ±SD. ****, p
    Figure Legend Snippet: Mlh1 is required for striatal and liver HTT CAG repeat instability in B6. Hdh Q111/+ mice. (A) Representative GeneMapper profiles of HTT CAG repeat size distributions in the tail, striatum and liver of 22-week-old B6. Hdh Q111/+ mice on different Mlh1 genetic backgrounds. Mlh1 +/+ , CAG113; Mlh1 +/− , CAG113; Mlh1 −/− , CAG110. (B) Quantification of striatal and liver HTT CAG instability indices in these mice reveals a statistically significant decrease in HTT CAG instability in the absence of Mlh1 . Mlh1 +/+ , CAG115.3±4.9SD, n = 6; Mlh1 +/− , CAG112.0±2.1SD, n = 6; Mlh1 −/− , CAG109.3±2.6SD, n = 6. Bar graphs represent mean ±SD. ****, p

    Techniques Used: Mouse Assay

    Proposed model of MutS and MutL-dependent events leading to CAG•CTG somatic instability. CAG•CTG repeat structures are initially recognized by the MutSβ (MSH2-MSH3) complex [25] , [98] . The loop in the CAG•CTG repeat tract represents a short slip-out, previously identified as the main substrate for MMR protein-dependent repair of CAG•CTG structures in cell free systems [50] , [51] . However, the nature of the putative CAG•CTG structure(s) that leads to MutS and MutL-dependent somatic instability in vivo is unknown. Following ATP hydrolysis by DNA-bound MutSβ [27] , a MutLγ (MLH1–MLH3) heterodimer is preferentially recruited to the complex (thick arrow) over the MutLα (MLH1-PMS2) heterodimer (thin arrow). The total absence of HTT CAG expansion in Mlh3 −/− mice suggests that PMS2 plays no role at all in this process. However, PMS2 has been shown to play a role in the expansion of CTG repeats in a DM1 mouse model [24] , suggesting that these events may be genetic locus and/or mouse strain dependent. Following MutLγ binding, various pathways, e.g. canonical mismatch repair (MMR), noncanonical mismatch repair (ncMMR) and/or other DNA repair processes may be engaged and process the repeats such that they ultimately undergo expansion. Other members of alternative DNA repair pathways, namely OGG1, XPA and NEIL1 have been directly implicated in CAG/CTG somatic instability in mice [76] – [78] , however, how these proteins intersect with MMR protein-dependent pathways has yet to be demonstrated.
    Figure Legend Snippet: Proposed model of MutS and MutL-dependent events leading to CAG•CTG somatic instability. CAG•CTG repeat structures are initially recognized by the MutSβ (MSH2-MSH3) complex [25] , [98] . The loop in the CAG•CTG repeat tract represents a short slip-out, previously identified as the main substrate for MMR protein-dependent repair of CAG•CTG structures in cell free systems [50] , [51] . However, the nature of the putative CAG•CTG structure(s) that leads to MutS and MutL-dependent somatic instability in vivo is unknown. Following ATP hydrolysis by DNA-bound MutSβ [27] , a MutLγ (MLH1–MLH3) heterodimer is preferentially recruited to the complex (thick arrow) over the MutLα (MLH1-PMS2) heterodimer (thin arrow). The total absence of HTT CAG expansion in Mlh3 −/− mice suggests that PMS2 plays no role at all in this process. However, PMS2 has been shown to play a role in the expansion of CTG repeats in a DM1 mouse model [24] , suggesting that these events may be genetic locus and/or mouse strain dependent. Following MutLγ binding, various pathways, e.g. canonical mismatch repair (MMR), noncanonical mismatch repair (ncMMR) and/or other DNA repair processes may be engaged and process the repeats such that they ultimately undergo expansion. Other members of alternative DNA repair pathways, namely OGG1, XPA and NEIL1 have been directly implicated in CAG/CTG somatic instability in mice [76] – [78] , however, how these proteins intersect with MMR protein-dependent pathways has yet to be demonstrated.

    Techniques Used: In Vivo, Mouse Assay, CTG Assay, Binding Assay

    Identification of a quantitative trait locus (QTL) associated with striatal HTT CAG instability. Linkage analysis in 10-week-old (B6x129). Hdh Q111/+ F2 mice ( n = 69) identified a single QTL on chromosome 9, with a maximum LOD score of approximately 14 and a 2-LOD-dropoff interval of 5 Mb (chr9:107,982,655–113,057,967; GRCm38/mm10) ( Figure S6 ). Note that the 2 markers positioned within the Mlh1 gene (dbSNP rs30131926 and rs30174694) define the QTL peak. The red dashed line represents the threshold (LOD = 4.3) considered for the identification of significant QTLs [85] . The coordinates (cM) of the 147 genetic markers used are represented by open triangles.
    Figure Legend Snippet: Identification of a quantitative trait locus (QTL) associated with striatal HTT CAG instability. Linkage analysis in 10-week-old (B6x129). Hdh Q111/+ F2 mice ( n = 69) identified a single QTL on chromosome 9, with a maximum LOD score of approximately 14 and a 2-LOD-dropoff interval of 5 Mb (chr9:107,982,655–113,057,967; GRCm38/mm10) ( Figure S6 ). Note that the 2 markers positioned within the Mlh1 gene (dbSNP rs30131926 and rs30174694) define the QTL peak. The red dashed line represents the threshold (LOD = 4.3) considered for the identification of significant QTLs [85] . The coordinates (cM) of the 147 genetic markers used are represented by open triangles.

    Techniques Used: Mouse Assay

    Somatic HTT CAG instability differs between B6. Hdh Q111 /+ and 129. Hdh Q111 /+ mice. (A) Representative GeneMapper profiles of HTT CAG repeat size distributions in the tail, striatum and liver of 10-week-old B6. Hdh Q111/+ and 129. Hdh Q111/+ mice, highlighting the altered contribution of B6 and 129 genetic background to somatic HTT CAG repeat expansion, as previously described [17] . Tail and striatum: B6. Hdh Q111/+ , CAG116; 129. Hdh Q111/+ , CAG112. Liver: B6. Hdh Q111/+ , CAG113; 129. Hdh Q111/+ , CAG111 (B) Quantification of CAG instability index reveals a statistically significant decrease in somatic HTT CAG instability in the striatum and liver of 129. Hdh Q111 /+ mice compared to B6. Hdh Q111 /+ mice. B6. Hdh Q111/+ striatum, n = 10, CAG116.9±1.2SD; B6. Hdh Q111/+ liver, n = 10, CAG114.3±1.2SD; 129. Hdh Q111/+ striatum, n = 12, CAG110.9±1.2SD; 129. Hdh Q111/+ liver, n = 9, CAG109.5±1.4SD; Bar graphs represent mean ±SD; ****, p
    Figure Legend Snippet: Somatic HTT CAG instability differs between B6. Hdh Q111 /+ and 129. Hdh Q111 /+ mice. (A) Representative GeneMapper profiles of HTT CAG repeat size distributions in the tail, striatum and liver of 10-week-old B6. Hdh Q111/+ and 129. Hdh Q111/+ mice, highlighting the altered contribution of B6 and 129 genetic background to somatic HTT CAG repeat expansion, as previously described [17] . Tail and striatum: B6. Hdh Q111/+ , CAG116; 129. Hdh Q111/+ , CAG112. Liver: B6. Hdh Q111/+ , CAG113; 129. Hdh Q111/+ , CAG111 (B) Quantification of CAG instability index reveals a statistically significant decrease in somatic HTT CAG instability in the striatum and liver of 129. Hdh Q111 /+ mice compared to B6. Hdh Q111 /+ mice. B6. Hdh Q111/+ striatum, n = 10, CAG116.9±1.2SD; B6. Hdh Q111/+ liver, n = 10, CAG114.3±1.2SD; 129. Hdh Q111/+ striatum, n = 12, CAG110.9±1.2SD; 129. Hdh Q111/+ liver, n = 9, CAG109.5±1.4SD; Bar graphs represent mean ±SD; ****, p

    Techniques Used: Mouse Assay

    Mlh3 is required for striatal and liver HTT CAG repeat instability in B6. Hdh Q111/+ mice. (A) Representative GeneMapper profiles of HTT CAG repeat size distributions in the tail, striatum and liver of 24-week-old B6. Hdh Q111/+ mice on different Mlh3 genetic backgrounds. Mlh3 +/+ , CAG103; Mlh3 +/− , CAG101; Mlh3 −/− , CAG102. (B) Quantification of striatal and liver HTT CAG instability indices in these animals reveals a statistically significant suppression of HTT CAG instability in the absence of Mlh3 . Mlh3 +/+ , CAG103.3±1.5SD, n = 3; Mlh3 +/− , CAG101.3±0.5SD, n = 4; Mlh3 −/− , CAG101.3±0.6SD, n = 3. Bar graphs represent mean ±SD. *, p
    Figure Legend Snippet: Mlh3 is required for striatal and liver HTT CAG repeat instability in B6. Hdh Q111/+ mice. (A) Representative GeneMapper profiles of HTT CAG repeat size distributions in the tail, striatum and liver of 24-week-old B6. Hdh Q111/+ mice on different Mlh3 genetic backgrounds. Mlh3 +/+ , CAG103; Mlh3 +/− , CAG101; Mlh3 −/− , CAG102. (B) Quantification of striatal and liver HTT CAG instability indices in these animals reveals a statistically significant suppression of HTT CAG instability in the absence of Mlh3 . Mlh3 +/+ , CAG103.3±1.5SD, n = 3; Mlh3 +/− , CAG101.3±0.5SD, n = 4; Mlh3 −/− , CAG101.3±0.6SD, n = 3. Bar graphs represent mean ±SD. *, p

    Techniques Used: Mouse Assay

    Striatal HTT CAG instability in 10-week-old Hdh Q111/+ mice on different genetic backgrounds. Graphical representation of striatal CAG instability indices from individual (A) B6, 129, (B6x129).F1 and (B6x129).F2 mice, color-coded based on strain genetic background; and from (B) (B6x129).F2 mice color-coded by genotype at the Mlh1 , Msh3 and Msh2 genes (“undetermined” indicates failed genotype). F2 mice homozygous or heterozygous for B6 Mlh1 alleles display significantly higher levels of striatal somatic CAG instability than F2 mice homozygous for 129 Mlh1 alleles ( p
    Figure Legend Snippet: Striatal HTT CAG instability in 10-week-old Hdh Q111/+ mice on different genetic backgrounds. Graphical representation of striatal CAG instability indices from individual (A) B6, 129, (B6x129).F1 and (B6x129).F2 mice, color-coded based on strain genetic background; and from (B) (B6x129).F2 mice color-coded by genotype at the Mlh1 , Msh3 and Msh2 genes (“undetermined” indicates failed genotype). F2 mice homozygous or heterozygous for B6 Mlh1 alleles display significantly higher levels of striatal somatic CAG instability than F2 mice homozygous for 129 Mlh1 alleles ( p

    Techniques Used: Mouse Assay

    Related Articles

    Clone Assay:

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    Centrifugation:

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    Amplification:

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    Filtration:

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    Real-time Polymerase Chain Reaction:

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    Incubation:

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    Transformation Assay:

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    Sequencing:

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    Genomic Sequencing:

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    Cell Culture:

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    Generated:

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    Polymerase Chain Reaction:

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    Sonication:

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    Recombinant:

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    Pyromark Assay:

    Article Title: Genome-wide placental DNA methylation analysis of severely growth-discordant monochorionic twins reveals novel epigenetic targets for intrauterine growth restriction
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    DNA Extraction:

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    Article Title: Genome data on the extinct Bison schoetensacki establish it as a sister species of the extant European bison (Bison bonasus)
    Article Snippet: Paragraph title: DNA extraction ... It was purified further using a silica-membrane-based method designed for small DNA fragments (Qiagen purification kit #28004; Venlo, Netherlands).

    Article Title: Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy
    Article Snippet: Genomic DNA extraction and quantitative-PCR . .. Animals were sacrificed at indicated time points and tissues were harvested in a manner to avoid cross-contamination, snap frozen in liquid nitrogen and stored at −80 °C. gDNA was extracted using a DNeasy blood and tissue kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. gDNA concentrations were determined using an Eppendorf Biophotometer (Eppendorf, Hamburg, Germany). rAAV genome copies in the gDNA from heart, liver, tibialis anterior, and brown fat were quantified by quantitative-PCR with an ABI 7900 HT sequence detection system (Applied Biosystems, Carlsbad, CA) according to the manufacturer's instructions and results were analyzed using SDS 2.3 software.

    Article Title: Characterization of Pit, a Streptococcus pneumoniae Iron Uptake ABC Transporter
    Article Snippet: .. Nucleic acids were isolated with the indicated kits: S. pneumoniae chromosomal DNA, Wizard genomic DNA isolation kits (Promega); plasmid DNA from E. coli , Qiagen plasmid kits (Qiagen); and S. pneumoniae RNA, SV Total RNA Isolation System (Promega). .. Prepared RNA samples were protected from degradation by addition of 0.5% RNasin (Promega) and storage as single-use aliquots at −70°C.

    RNA Sequencing Assay:

    Article Title: Evolution of structural diversity of trichothecenes, a family of toxins produced by plant pathogenic and entomopathogenic fungi
    Article Snippet: RNA was isolated with the RNeasy method (Qiagen), and cDNA libraries were prepared with the MinElute Reaction Cleanup Kit (Qiagen). cDNA libraries were then sonicated for five cycles with Diagenode Bioruptor system (Diagenode) as specified by the manufacturer to obtain 100- to 300-bp fragments. .. The resulting sequence reads were analyzed using the RNA-Seq Analysis function in CLC Genomics Workbench.

    Isolation:

    Article Title: Evolution of structural diversity of trichothecenes, a family of toxins produced by plant pathogenic and entomopathogenic fungi
    Article Snippet: .. RNA was isolated with the RNeasy method (Qiagen), and cDNA libraries were prepared with the MinElute Reaction Cleanup Kit (Qiagen). cDNA libraries were then sonicated for five cycles with Diagenode Bioruptor system (Diagenode) as specified by the manufacturer to obtain 100- to 300-bp fragments. .. Sequencing libraries were prepared from the sonicated DNA using a NEBNext Fast DNA Library Prep Set for Ion Torrent (New England BioLabs).

    Article Title: phgABC, a Three-Gene Operon Required for Growth of Streptococcus pneumoniae in Hyperosmotic Medium and In Vivo
    Article Snippet: .. The following kits were used for the isolation of nucleic acids: for S. pneumoniae chromosomal DNA, Wizard genomic DNA isolation kits (Promega); for plasmid DNA from E. coli , QIAGEN plasmid kits; and for S. pneumoniae RNA, the SV Total RNA Isolation System (Promega). .. Prepared RNA samples were stored as single-use aliquots at −70°C and protected from degradation by the addition of 0.5% RNasin (Promega).

    Article Title: Concerted copy number variation balances ribosomal DNA dosage in human and mouse genomes
    Article Snippet: .. Cells were harvested 24 h after the treatment with BPA, and gDNA was isolated using DNeasy Blood and Tissue kit following the method described by the manufacturer (Qiagen). gDNA samples were treated with RNase A, and concentration was estimated with NanoDrop (Thermo Scientific). ..

    Article Title: Developmental Expression of CYP2B6: A Comprehensive Analysis of mRNA Expression, Protein Content and Bupropion Hydroxylase Activity and the Impact of Genetic Variation
    Article Snippet: .. gDNA was isolated from ∼25 mg of tissue using a DNeasy Tissue Kit (Qiagen) or an Illustra Tissue and Cells Genomic Prep Kit (GE Healthcare). gDNA was assessed by agarose gel electrophoresis for quality; concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo-Scientific, Waltham, MA). gDNA samples were diluted to 15 ng/ µ l. Genotyping was carried out on an Applied Biosystems 7900 HT Real-Time PCR System using predesigned Life Technologies TaqMan assays for CYP2B6 rs34223104 (−82T > C); rs3745274 (516G > T, Q172H), rs28399499 (983T > C, I328T), and rs3211371 (1459C > T R487C). .. Reactions were scaled to 8 µ l and contained 4 µ l KAPA PROBE FAST Universal 2X qPCR Master Mix (KAPA Biosystems, Woburn, MA), and 0.2 or 0.4 µ l TaqMan assay mix, respectively, depending on whether it was a 20× or 40× mix, and 12–18 ng gDNA.

    Article Title: Characterization of Pit, a Streptococcus pneumoniae Iron Uptake ABC Transporter
    Article Snippet: .. Nucleic acids were isolated with the indicated kits: S. pneumoniae chromosomal DNA, Wizard genomic DNA isolation kits (Promega); plasmid DNA from E. coli , Qiagen plasmid kits (Qiagen); and S. pneumoniae RNA, SV Total RNA Isolation System (Promega). .. Prepared RNA samples were protected from degradation by addition of 0.5% RNasin (Promega) and storage as single-use aliquots at −70°C.

    Mouse Assay:

    Article Title: Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy
    Article Snippet: For cold fast challenge, after the 18-hour fast, mice were singly housed in minimal bedding cages and placed in a 4 °C cold room for 150 minutes or until rectal temperatures were below 20 °C. .. Animals were sacrificed at indicated time points and tissues were harvested in a manner to avoid cross-contamination, snap frozen in liquid nitrogen and stored at −80 °C. gDNA was extracted using a DNeasy blood and tissue kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. gDNA concentrations were determined using an Eppendorf Biophotometer (Eppendorf, Hamburg, Germany). rAAV genome copies in the gDNA from heart, liver, tibialis anterior, and brown fat were quantified by quantitative-PCR with an ABI 7900 HT sequence detection system (Applied Biosystems, Carlsbad, CA) according to the manufacturer's instructions and results were analyzed using SDS 2.3 software.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: phgABC, a Three-Gene Operon Required for Growth of Streptococcus pneumoniae in Hyperosmotic Medium and In Vivo
    Article Snippet: The following kits were used for the isolation of nucleic acids: for S. pneumoniae chromosomal DNA, Wizard genomic DNA isolation kits (Promega); for plasmid DNA from E. coli , QIAGEN plasmid kits; and for S. pneumoniae RNA, the SV Total RNA Isolation System (Promega). .. The Access reverse transcription (RT)-PCR system (Promega) was used to amplify cDNA from RNA by using 400 pmol of target-specific primers.

    Article Title: Characterization of Pit, a Streptococcus pneumoniae Iron Uptake ABC Transporter
    Article Snippet: Nucleic acids were isolated with the indicated kits: S. pneumoniae chromosomal DNA, Wizard genomic DNA isolation kits (Promega); plasmid DNA from E. coli , Qiagen plasmid kits (Qiagen); and S. pneumoniae RNA, SV Total RNA Isolation System (Promega). .. The Access RT-PCR System (Promega) and target-specific primers were used to derive and amplify cDNA from RNA.

    Software:

    Article Title: Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy
    Article Snippet: .. Animals were sacrificed at indicated time points and tissues were harvested in a manner to avoid cross-contamination, snap frozen in liquid nitrogen and stored at −80 °C. gDNA was extracted using a DNeasy blood and tissue kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. gDNA concentrations were determined using an Eppendorf Biophotometer (Eppendorf, Hamburg, Germany). rAAV genome copies in the gDNA from heart, liver, tibialis anterior, and brown fat were quantified by quantitative-PCR with an ABI 7900 HT sequence detection system (Applied Biosystems, Carlsbad, CA) according to the manufacturer's instructions and results were analyzed using SDS 2.3 software. .. Briefly, primers and probe were designed to amplify SV40 poly-A tail of the rAAV9-CBA-VLCAD vector.

    Purification:

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples
    Article Snippet: The 50 μL reaction was diluted with 450 μL sterile water and purified with Vivaspin 500 MWCO 100,000 Protein Concentrator Spin Columns (GE Healthcare Life Sciences) which reduced the reaction volume to 10 μL. .. Plasmid DNA was extracted from TRACA clones using the Qiagen HiSpeed Plasmid Midi kit and visualized on a 1% agarose gel stained with GelRed, run at 70 V for 60 min. Bands of plasmid DNA (B1 and B2) were harvested from a 1% agarose gel stained with SYBR Safe using the Cleaver Scientific runVIEW system run at the same conditions as before.

    Article Title: Genome data on the extinct Bison schoetensacki establish it as a sister species of the extant European bison (Bison bonasus)
    Article Snippet: .. It was purified further using a silica-membrane-based method designed for small DNA fragments (Qiagen purification kit #28004; Venlo, Netherlands). .. PCR and Sanger sequencing DNA analysis PCR reactions were performed in a 50-μl reaction volume containing 0.2–0.4 μl of mock or ancient DNA extracts, 300 nM of sense and antisense primers, 200 μM dNTP, 2.5 mM MgCl2 , 5 μl of GeneAmp 10X PCR buffer II, and 2.5 U of AmpliTaq Gold DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA).

    Article Title: A Novel A3 Group Aconitase Tolerates Oxidation and Nitric Oxide *
    Article Snippet: .. Total DNA from bacterial strains was purified using DNeasy blood and tissue kits (Qiagen, Hilden, Germany). acnA3 (accession number ), acnA4 , and acnB ), and total DNA as a template, were cloned into pET28a(+) (Novagen, Madison, WI). .. AcnA3, AcnA4, and AcnB were produced using plasmids pAcnA3, pAcnA4, and pAcnB, respectively.

    Article Title: Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy
    Article Snippet: All animal procedures were approved by University of Massachusetts Medical School Institutional Animal Care and Use Committee as well as University of Florida Institutional Animal Care and Use Committee in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care International specifications. rAAV vectors . rAAV9/2 pseudotyped vectors were generated to express human VLCAD under the transcriptional control of the cytomegalovirus enhancer/chicken β-actin promoter. rAAV vectors were produced, purified and tittered as previously described (UMMS Gene Therapy Center, Worcester, MA). .. Animals were sacrificed at indicated time points and tissues were harvested in a manner to avoid cross-contamination, snap frozen in liquid nitrogen and stored at −80 °C. gDNA was extracted using a DNeasy blood and tissue kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. gDNA concentrations were determined using an Eppendorf Biophotometer (Eppendorf, Hamburg, Germany). rAAV genome copies in the gDNA from heart, liver, tibialis anterior, and brown fat were quantified by quantitative-PCR with an ABI 7900 HT sequence detection system (Applied Biosystems, Carlsbad, CA) according to the manufacturer's instructions and results were analyzed using SDS 2.3 software.

    Plasmid Preparation:

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples
    Article Snippet: .. Plasmid DNA was extracted from TRACA clones using the Qiagen HiSpeed Plasmid Midi kit and visualized on a 1% agarose gel stained with GelRed, run at 70 V for 60 min. Bands of plasmid DNA (B1 and B2) were harvested from a 1% agarose gel stained with SYBR Safe using the Cleaver Scientific runVIEW system run at the same conditions as before. .. The harvested DNA bands were electroporated into E. coli DH5α, selected on ampicillin (32 mg/L), tetracycline (16 mg/L), kanamycin (25 mg/L), colistin (16 mg/L), or ciprofloxacin (4 mg/L), and incubated at 37°C overnight.

    Article Title: phgABC, a Three-Gene Operon Required for Growth of Streptococcus pneumoniae in Hyperosmotic Medium and In Vivo
    Article Snippet: .. The following kits were used for the isolation of nucleic acids: for S. pneumoniae chromosomal DNA, Wizard genomic DNA isolation kits (Promega); for plasmid DNA from E. coli , QIAGEN plasmid kits; and for S. pneumoniae RNA, the SV Total RNA Isolation System (Promega). .. Prepared RNA samples were stored as single-use aliquots at −70°C and protected from degradation by the addition of 0.5% RNasin (Promega).

    Article Title: Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy
    Article Snippet: Animals were sacrificed at indicated time points and tissues were harvested in a manner to avoid cross-contamination, snap frozen in liquid nitrogen and stored at −80 °C. gDNA was extracted using a DNeasy blood and tissue kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. gDNA concentrations were determined using an Eppendorf Biophotometer (Eppendorf, Hamburg, Germany). rAAV genome copies in the gDNA from heart, liver, tibialis anterior, and brown fat were quantified by quantitative-PCR with an ABI 7900 HT sequence detection system (Applied Biosystems, Carlsbad, CA) according to the manufacturer's instructions and results were analyzed using SDS 2.3 software. .. Briefly, primers and probe were designed to amplify SV40 poly-A tail of the rAAV9-CBA-VLCAD vector.

    Article Title: Characterization of Pit, a Streptococcus pneumoniae Iron Uptake ABC Transporter
    Article Snippet: .. Nucleic acids were isolated with the indicated kits: S. pneumoniae chromosomal DNA, Wizard genomic DNA isolation kits (Promega); plasmid DNA from E. coli , Qiagen plasmid kits (Qiagen); and S. pneumoniae RNA, SV Total RNA Isolation System (Promega). .. Prepared RNA samples were protected from degradation by addition of 0.5% RNasin (Promega) and storage as single-use aliquots at −70°C.

    TaqMan Assay:

    Article Title: Developmental Expression of CYP2B6: A Comprehensive Analysis of mRNA Expression, Protein Content and Bupropion Hydroxylase Activity and the Impact of Genetic Variation
    Article Snippet: gDNA was isolated from ∼25 mg of tissue using a DNeasy Tissue Kit (Qiagen) or an Illustra Tissue and Cells Genomic Prep Kit (GE Healthcare). gDNA was assessed by agarose gel electrophoresis for quality; concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo-Scientific, Waltham, MA). gDNA samples were diluted to 15 ng/ µ l. Genotyping was carried out on an Applied Biosystems 7900 HT Real-Time PCR System using predesigned Life Technologies TaqMan assays for CYP2B6 rs34223104 (−82T > C); rs3745274 (516G > T, Q172H), rs28399499 (983T > C, I328T), and rs3211371 (1459C > T R487C). .. Reactions were scaled to 8 µ l and contained 4 µ l KAPA PROBE FAST Universal 2X qPCR Master Mix (KAPA Biosystems, Woburn, MA), and 0.2 or 0.4 µ l TaqMan assay mix, respectively, depending on whether it was a 20× or 40× mix, and 12–18 ng gDNA.

    SYBR Green Assay:

    Article Title: Age related changes in microglial phenotype vary between CNS regions: Grey versus white matter differences
    Article Snippet: Total RNA was extracted from brain tissue using RNeasy mini kits (Qiagen, Crawley, UK) and treated with DNAse I to remove any contaminating gDNA (Qiagen). cDNA was synthesised using reverse transcription reagents from Applied Biosystems (Warrington, UK). .. SYBR green super mix (BioRad, Hemel Hempstead, UK) was used to detect amplification of primer products.

    Agarose Gel Electrophoresis:

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples
    Article Snippet: .. Plasmid DNA was extracted from TRACA clones using the Qiagen HiSpeed Plasmid Midi kit and visualized on a 1% agarose gel stained with GelRed, run at 70 V for 60 min. Bands of plasmid DNA (B1 and B2) were harvested from a 1% agarose gel stained with SYBR Safe using the Cleaver Scientific runVIEW system run at the same conditions as before. .. The harvested DNA bands were electroporated into E. coli DH5α, selected on ampicillin (32 mg/L), tetracycline (16 mg/L), kanamycin (25 mg/L), colistin (16 mg/L), or ciprofloxacin (4 mg/L), and incubated at 37°C overnight.

    Article Title: Developmental Expression of CYP2B6: A Comprehensive Analysis of mRNA Expression, Protein Content and Bupropion Hydroxylase Activity and the Impact of Genetic Variation
    Article Snippet: .. gDNA was isolated from ∼25 mg of tissue using a DNeasy Tissue Kit (Qiagen) or an Illustra Tissue and Cells Genomic Prep Kit (GE Healthcare). gDNA was assessed by agarose gel electrophoresis for quality; concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo-Scientific, Waltham, MA). gDNA samples were diluted to 15 ng/ µ l. Genotyping was carried out on an Applied Biosystems 7900 HT Real-Time PCR System using predesigned Life Technologies TaqMan assays for CYP2B6 rs34223104 (−82T > C); rs3745274 (516G > T, Q172H), rs28399499 (983T > C, I328T), and rs3211371 (1459C > T R487C). .. Reactions were scaled to 8 µ l and contained 4 µ l KAPA PROBE FAST Universal 2X qPCR Master Mix (KAPA Biosystems, Woburn, MA), and 0.2 or 0.4 µ l TaqMan assay mix, respectively, depending on whether it was a 20× or 40× mix, and 12–18 ng gDNA.

    Spectrophotometry:

    Article Title: Developmental Expression of CYP2B6: A Comprehensive Analysis of mRNA Expression, Protein Content and Bupropion Hydroxylase Activity and the Impact of Genetic Variation
    Article Snippet: .. gDNA was isolated from ∼25 mg of tissue using a DNeasy Tissue Kit (Qiagen) or an Illustra Tissue and Cells Genomic Prep Kit (GE Healthcare). gDNA was assessed by agarose gel electrophoresis for quality; concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo-Scientific, Waltham, MA). gDNA samples were diluted to 15 ng/ µ l. Genotyping was carried out on an Applied Biosystems 7900 HT Real-Time PCR System using predesigned Life Technologies TaqMan assays for CYP2B6 rs34223104 (−82T > C); rs3745274 (516G > T, Q172H), rs28399499 (983T > C, I328T), and rs3211371 (1459C > T R487C). .. Reactions were scaled to 8 µ l and contained 4 µ l KAPA PROBE FAST Universal 2X qPCR Master Mix (KAPA Biosystems, Woburn, MA), and 0.2 or 0.4 µ l TaqMan assay mix, respectively, depending on whether it was a 20× or 40× mix, and 12–18 ng gDNA.

    DNA Methylation Assay:

    Article Title: Genome-wide placental DNA methylation analysis of severely growth-discordant monochorionic twins reveals novel epigenetic targets for intrauterine growth restriction
    Article Snippet: .. Validation analysis of DNA methylation using bisulfite pyrosequencing Results of top candidate CpG sites (cg01971612 and cg18485485 for DECR1 ; cg04675542, cg02343823, and cg08580836 for ZNF300 ; and cg08234308 for LEPR ) were validated using pyrosequencing of bisulfite converted DNA (PyroMark Q24; Qiagen). .. Polymerase chain reaction (PCR) and sequencing primers were designed using PyroMark Assay Design 2.0.

    Produced:

    Article Title: A Novel A3 Group Aconitase Tolerates Oxidation and Nitric Oxide *
    Article Snippet: Total DNA from bacterial strains was purified using DNeasy blood and tissue kits (Qiagen, Hilden, Germany). acnA3 (accession number ), acnA4 , and acnB ), and total DNA as a template, were cloned into pET28a(+) (Novagen, Madison, WI). .. AcnA3, AcnA4, and AcnB were produced using plasmids pAcnA3, pAcnA4, and pAcnB, respectively.

    Article Title: Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy
    Article Snippet: All animal procedures were approved by University of Massachusetts Medical School Institutional Animal Care and Use Committee as well as University of Florida Institutional Animal Care and Use Committee in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care International specifications. rAAV vectors . rAAV9/2 pseudotyped vectors were generated to express human VLCAD under the transcriptional control of the cytomegalovirus enhancer/chicken β-actin promoter. rAAV vectors were produced, purified and tittered as previously described (UMMS Gene Therapy Center, Worcester, MA). .. Animals were sacrificed at indicated time points and tissues were harvested in a manner to avoid cross-contamination, snap frozen in liquid nitrogen and stored at −80 °C. gDNA was extracted using a DNeasy blood and tissue kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. gDNA concentrations were determined using an Eppendorf Biophotometer (Eppendorf, Hamburg, Germany). rAAV genome copies in the gDNA from heart, liver, tibialis anterior, and brown fat were quantified by quantitative-PCR with an ABI 7900 HT sequence detection system (Applied Biosystems, Carlsbad, CA) according to the manufacturer's instructions and results were analyzed using SDS 2.3 software.

    Concentration Assay:

    Article Title: Concerted copy number variation balances ribosomal DNA dosage in human and mouse genomes
    Article Snippet: .. Cells were harvested 24 h after the treatment with BPA, and gDNA was isolated using DNeasy Blood and Tissue kit following the method described by the manufacturer (Qiagen). gDNA samples were treated with RNase A, and concentration was estimated with NanoDrop (Thermo Scientific). ..

    Article Title: Developmental Expression of CYP2B6: A Comprehensive Analysis of mRNA Expression, Protein Content and Bupropion Hydroxylase Activity and the Impact of Genetic Variation
    Article Snippet: .. gDNA was isolated from ∼25 mg of tissue using a DNeasy Tissue Kit (Qiagen) or an Illustra Tissue and Cells Genomic Prep Kit (GE Healthcare). gDNA was assessed by agarose gel electrophoresis for quality; concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo-Scientific, Waltham, MA). gDNA samples were diluted to 15 ng/ µ l. Genotyping was carried out on an Applied Biosystems 7900 HT Real-Time PCR System using predesigned Life Technologies TaqMan assays for CYP2B6 rs34223104 (−82T > C); rs3745274 (516G > T, Q172H), rs28399499 (983T > C, I328T), and rs3211371 (1459C > T R487C). .. Reactions were scaled to 8 µ l and contained 4 µ l KAPA PROBE FAST Universal 2X qPCR Master Mix (KAPA Biosystems, Woburn, MA), and 0.2 or 0.4 µ l TaqMan assay mix, respectively, depending on whether it was a 20× or 40× mix, and 12–18 ng gDNA.

    Article Title: Characterization of Pit, a Streptococcus pneumoniae Iron Uptake ABC Transporter
    Article Snippet: Nucleic acids were isolated with the indicated kits: S. pneumoniae chromosomal DNA, Wizard genomic DNA isolation kits (Promega); plasmid DNA from E. coli , Qiagen plasmid kits (Qiagen); and S. pneumoniae RNA, SV Total RNA Isolation System (Promega). .. The primer concentration for reverse transcription-PCR (RT-PCR) used for assessing operon structure was 400 pmol, and that for assessing the relative abundance of gene transcripts was 80 pmol.

    Staining:

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples
    Article Snippet: .. Plasmid DNA was extracted from TRACA clones using the Qiagen HiSpeed Plasmid Midi kit and visualized on a 1% agarose gel stained with GelRed, run at 70 V for 60 min. Bands of plasmid DNA (B1 and B2) were harvested from a 1% agarose gel stained with SYBR Safe using the Cleaver Scientific runVIEW system run at the same conditions as before. .. The harvested DNA bands were electroporated into E. coli DH5α, selected on ampicillin (32 mg/L), tetracycline (16 mg/L), kanamycin (25 mg/L), colistin (16 mg/L), or ciprofloxacin (4 mg/L), and incubated at 37°C overnight.

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    For use with QIAxcel instruments for DNA fragment analysis Kit contents DNA size marker with fragments of 72 118 194 234 271 281 310 603 872 1078 and 1353 bp
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    Qiagen dna methylation analysis genomic dna
    Gene expression and hypothalamic Pomc <t>DNA</t> methylation changes in offspring at weaning. a mRNA expression levels of Pomc , Agrp , Npy , and b Ob-Rb in the <t>ARC.</t> c Relative mRNA levels of Mc4r and Npy1r in the PVN analyzed by qRT-PCR in the 3-week-old offspring of LF- or HF-fed dams (Student’s t -test, n = 8). d Map of the Pro-opiomelanocortin ( Pomc ) gene promoter and enhancer region including functional regulatory elements and CpG dinucleotides (red lines). e Methylation analyzes of hypothalamic Pomc promoter (− 150 bp to transcription start site [TSS]) (Student’s t -test, D-LF, n = 6; D-HF, n = 7) and f , g of neuronal Pomc enhancer region 1 and 2 in the offspring of LF- or HF-fed mothers at 3 weeks of age (Student’s t -test, n = 8). Data are shown as mean ± SEM. * p
    Dna Methylation Analysis Genomic Dna, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen stranded dna
    TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated <t>DNA</t> template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and <t>PCR</t> amplified for the next round of selection.
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    Qiagen csf derived circulating tumor dna ctdna analysis formalin fixed paraffin embedded
    Droplet digital PCR results from examination of the CNS tissue and the <t>CSF-ctDNA</t> for MYD88 mutations. Quadrant statistics scatter plot (lower left quadrant shows negative cluster; lower right quadrant shows wild-type cluster; upper left quadrant shows mutant cluster; upper right quadrant shows double positive). Both, CNS tissue and CSF-ctDNA showed positive mutant droplets for MYD88 p.L265P, while no MYD88 p.V217F mutant droplets were detected. The representative bar chart illustrates the percentage value of mutation allele frequency (MAF) for the CNS tissue and the CSF-ctDNA.
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    Gene expression and hypothalamic Pomc DNA methylation changes in offspring at weaning. a mRNA expression levels of Pomc , Agrp , Npy , and b Ob-Rb in the ARC. c Relative mRNA levels of Mc4r and Npy1r in the PVN analyzed by qRT-PCR in the 3-week-old offspring of LF- or HF-fed dams (Student’s t -test, n = 8). d Map of the Pro-opiomelanocortin ( Pomc ) gene promoter and enhancer region including functional regulatory elements and CpG dinucleotides (red lines). e Methylation analyzes of hypothalamic Pomc promoter (− 150 bp to transcription start site [TSS]) (Student’s t -test, D-LF, n = 6; D-HF, n = 7) and f , g of neuronal Pomc enhancer region 1 and 2 in the offspring of LF- or HF-fed mothers at 3 weeks of age (Student’s t -test, n = 8). Data are shown as mean ± SEM. * p

    Journal: International Journal of Obesity (2005)

    Article Title: Maternal overnutrition programs epigenetic changes in the regulatory regions of hypothalamic Pomc in the offspring of rats

    doi: 10.1038/s41366-018-0094-1

    Figure Lengend Snippet: Gene expression and hypothalamic Pomc DNA methylation changes in offspring at weaning. a mRNA expression levels of Pomc , Agrp , Npy , and b Ob-Rb in the ARC. c Relative mRNA levels of Mc4r and Npy1r in the PVN analyzed by qRT-PCR in the 3-week-old offspring of LF- or HF-fed dams (Student’s t -test, n = 8). d Map of the Pro-opiomelanocortin ( Pomc ) gene promoter and enhancer region including functional regulatory elements and CpG dinucleotides (red lines). e Methylation analyzes of hypothalamic Pomc promoter (− 150 bp to transcription start site [TSS]) (Student’s t -test, D-LF, n = 6; D-HF, n = 7) and f , g of neuronal Pomc enhancer region 1 and 2 in the offspring of LF- or HF-fed mothers at 3 weeks of age (Student’s t -test, n = 8). Data are shown as mean ± SEM. * p

    Article Snippet: DNA methylation analysis Genomic DNA from ARC punches were isolated using Qiagen AllPrep DNA/RNA mini kit (80204) according to the manufacturer’s instructions.

    Techniques: Expressing, DNA Methylation Assay, Quantitative RT-PCR, Functional Assay, Methylation

    TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.

    Journal: Nucleic Acids Research

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition

    doi: 10.1093/nar/gky667

    Figure Lengend Snippet: TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.

    Article Snippet: The amplified double stranded DNA was purified using a PCR cleanup column (Qiagen) and the PEG-functionalized strand was separated from the FAM labeled strand on a denaturing 10% polyacrylamide gel.

    Techniques: Binding Assay, Amplification, Modification, Polyacrylamide Gel Electrophoresis, Selection, Incubation, Magnetic Beads, Polymerase Chain Reaction

    Droplet digital PCR results from examination of the CNS tissue and the CSF-ctDNA for MYD88 mutations. Quadrant statistics scatter plot (lower left quadrant shows negative cluster; lower right quadrant shows wild-type cluster; upper left quadrant shows mutant cluster; upper right quadrant shows double positive). Both, CNS tissue and CSF-ctDNA showed positive mutant droplets for MYD88 p.L265P, while no MYD88 p.V217F mutant droplets were detected. The representative bar chart illustrates the percentage value of mutation allele frequency (MAF) for the CNS tissue and the CSF-ctDNA.

    Journal: Frontiers in Oncology

    Article Title: Detection of the MYD88 p.L265P Mutation in the CSF of a Patient With Secondary Central Nervous System Lymphoma

    doi: 10.3389/fonc.2018.00382

    Figure Lengend Snippet: Droplet digital PCR results from examination of the CNS tissue and the CSF-ctDNA for MYD88 mutations. Quadrant statistics scatter plot (lower left quadrant shows negative cluster; lower right quadrant shows wild-type cluster; upper left quadrant shows mutant cluster; upper right quadrant shows double positive). Both, CNS tissue and CSF-ctDNA showed positive mutant droplets for MYD88 p.L265P, while no MYD88 p.V217F mutant droplets were detected. The representative bar chart illustrates the percentage value of mutation allele frequency (MAF) for the CNS tissue and the CSF-ctDNA.

    Article Snippet: CSF-derived circulating tumor DNA (CtDNA) analysis Formalin-fixed paraffin-embedded (FFPE) tissue was used to extract DNA using QIAamp DNA FFPE Tissue Kit (Qiagen, Valencia, CA, USA).

    Techniques: Digital PCR, Mutagenesis