Structured Review

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Midbrain dopamine neurons are hypersensitive to propargite-induced cell toxicity. a Characterization of cortical neuron and mDA neuron derived from H9 <t>hESCs.</t> 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 <t>DNA</t> 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
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1) 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

2) 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:

3) 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

4) 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

5) 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

6) 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

7) 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

8) 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

9) Product Images from "Enrichment of sequencing targets from the human genome by solution hybridization"

Article Title: Enrichment of sequencing targets from the human genome by solution hybridization

Journal: Genome Biology

doi: 10.1186/gb-2009-10-10-r116

Experimental design. Genomic DNA fragment libraries were generated from two samples, Coriell (NA15510) and Wellderly (HE00069). Technical replicates of the target-enrichment steps for both samples NA15510 and HE00069 were performed (Capture 1 and Capture 2). The four target-enriched samples were loaded in separate lanes of a flow cell and sequenced by using the Illumina GAII.
Figure Legend Snippet: Experimental design. Genomic DNA fragment libraries were generated from two samples, Coriell (NA15510) and Wellderly (HE00069). Technical replicates of the target-enrichment steps for both samples NA15510 and HE00069 were performed (Capture 1 and Capture 2). The four target-enriched samples were loaded in separate lanes of a flow cell and sequenced by using the Illumina GAII.

Techniques Used: Generated, Flow Cytometry

10) Product Images from "ADAR1 is essential for maintenance of hematopoiesis and suppression of interferon signaling"

Article Title: ADAR1 is essential for maintenance of hematopoiesis and suppression of interferon signaling

Journal: Nature immunology

doi: 10.1038/ni.1680

Induced deletion of ADAR1 in HSCs of adult mice leads to hyperproliferation and apoptosis ( a ) Absolute numbers of total bone marrow cells (grey bars), and frequencies of HSCs (LKS + ) and progenitors (LKS − ) in induced (Tam) and uninduced (No tam) adult mice. ( b ) Numbers of methylcellulose colonies grown from 12,500 bone-marrow cells of induced (Tam) and uninduced (No tam) Adar .sc mice, and representative Adar allele and scl-Cre transgene specific genomic PCR analysis of individual colonies from Adar f/+ .sc (upper panel) and Adar f/− .sc (lower panel) bone marrow. DNA size markers (M) and no-template controls (N) are indicated. ( c ) Representative flow cytometry profiles of bone-marrow LKS + HSCs (red ellipse) analyzed for apoptotic and necrotic cell death by Annexin V / 7-amino-actinomycin D (7-AAD) staining. Frequencies of apoptotic LKS + HSCs (red frame): 3.2 ± 3.6 % (f/−, no tam); 13.3 ± 5.1 % (f/+, tam); 6.1 ± 0.8 % (f/+.sc, tam); 68.0 ± 26.2 % (f/− .sc, tam). ( d ) Representative cell cycle analysis of LKS + CD34 lo long-term (LT-) HSCs, LKS + CD34 hi short-term (ST-) HSCs, and LKS − multipotent progenitors (MPP). Numbers represent the frequency of cells in S-phase. All data presented in ( a-d ) are expressed as mean ± s.d. (3 mice per experimental group and 2 mice in each control group).
Figure Legend Snippet: Induced deletion of ADAR1 in HSCs of adult mice leads to hyperproliferation and apoptosis ( a ) Absolute numbers of total bone marrow cells (grey bars), and frequencies of HSCs (LKS + ) and progenitors (LKS − ) in induced (Tam) and uninduced (No tam) adult mice. ( b ) Numbers of methylcellulose colonies grown from 12,500 bone-marrow cells of induced (Tam) and uninduced (No tam) Adar .sc mice, and representative Adar allele and scl-Cre transgene specific genomic PCR analysis of individual colonies from Adar f/+ .sc (upper panel) and Adar f/− .sc (lower panel) bone marrow. DNA size markers (M) and no-template controls (N) are indicated. ( c ) Representative flow cytometry profiles of bone-marrow LKS + HSCs (red ellipse) analyzed for apoptotic and necrotic cell death by Annexin V / 7-amino-actinomycin D (7-AAD) staining. Frequencies of apoptotic LKS + HSCs (red frame): 3.2 ± 3.6 % (f/−, no tam); 13.3 ± 5.1 % (f/+, tam); 6.1 ± 0.8 % (f/+.sc, tam); 68.0 ± 26.2 % (f/− .sc, tam). ( d ) Representative cell cycle analysis of LKS + CD34 lo long-term (LT-) HSCs, LKS + CD34 hi short-term (ST-) HSCs, and LKS − multipotent progenitors (MPP). Numbers represent the frequency of cells in S-phase. All data presented in ( a-d ) are expressed as mean ± s.d. (3 mice per experimental group and 2 mice in each control group).

Techniques Used: Mouse Assay, Polymerase Chain Reaction, Flow Cytometry, Cytometry, Staining, Cell Cycle Assay

ADAR1 deficiency in HSCs leads to a global upregulation of interferon-inducible transcripts ( a ) Schematic drawing of two murine ADAR1 mRNA and protein species. ADAR1 p150 protein is encoded by an mRNA transcribed from an interferon-inducible promoter (P i ), and ADAR1 p110 protein is encoded by an mRNA transcribed from a constitutively active promoter (P C ). Translation start sites (AUG) within transcripts are indicated. White boxes, 5’-untranslated region (UTR); thick black horizontal lines, regions amplified by real-time PCR, broken horizontal lines, 3’-UTR; dark-grey boxes, putative Z-DNA binding domains (α and β); black boxes, RNA binding domains (I-III); closed circles, conserved residues within deaminase domain. ( b ) Real-time PCR analysis of ADAR1 mRNA expression in ES cells (ESC), long-term (LKS + CD34 lo ) and short-term (LKS + CD34 hi ) HSCs, total HSCs (LKS + ), and multipotent progenitors (LKS − ) using oligonucleotides specific for the IFN-inducible ADAR1 p150 (dark grey bars) and the constitutive ADAR1 p110 (light grey bars) transcripts. ADAR1 mRNA expression was normalized to YWHAZ (GenBank™/EBI accession number 22631) mRNA expression using the ΔΔC t method. Data are expressed as mean ± s.d. ( c ) Gene set enrichment analysis of genome-wide transcriptome expression data from ADAR1 −/− (KO) relative to control HSCs. Among 1247 curated gene sets (represented by green dots), those that were annotated as upregulated by interferon signaling are colored in red, whereas those downregulated by interferon signaling are represented by blue dots. Positive net enrichment scores denote gene sets that were upregulated in KO cells. Negative scores reflect downregulated gene sets. The yellow area contains significantly altered gene sets with a false-discovery rate (FDR) of
Figure Legend Snippet: ADAR1 deficiency in HSCs leads to a global upregulation of interferon-inducible transcripts ( a ) Schematic drawing of two murine ADAR1 mRNA and protein species. ADAR1 p150 protein is encoded by an mRNA transcribed from an interferon-inducible promoter (P i ), and ADAR1 p110 protein is encoded by an mRNA transcribed from a constitutively active promoter (P C ). Translation start sites (AUG) within transcripts are indicated. White boxes, 5’-untranslated region (UTR); thick black horizontal lines, regions amplified by real-time PCR, broken horizontal lines, 3’-UTR; dark-grey boxes, putative Z-DNA binding domains (α and β); black boxes, RNA binding domains (I-III); closed circles, conserved residues within deaminase domain. ( b ) Real-time PCR analysis of ADAR1 mRNA expression in ES cells (ESC), long-term (LKS + CD34 lo ) and short-term (LKS + CD34 hi ) HSCs, total HSCs (LKS + ), and multipotent progenitors (LKS − ) using oligonucleotides specific for the IFN-inducible ADAR1 p150 (dark grey bars) and the constitutive ADAR1 p110 (light grey bars) transcripts. ADAR1 mRNA expression was normalized to YWHAZ (GenBank™/EBI accession number 22631) mRNA expression using the ΔΔC t method. Data are expressed as mean ± s.d. ( c ) Gene set enrichment analysis of genome-wide transcriptome expression data from ADAR1 −/− (KO) relative to control HSCs. Among 1247 curated gene sets (represented by green dots), those that were annotated as upregulated by interferon signaling are colored in red, whereas those downregulated by interferon signaling are represented by blue dots. Positive net enrichment scores denote gene sets that were upregulated in KO cells. Negative scores reflect downregulated gene sets. The yellow area contains significantly altered gene sets with a false-discovery rate (FDR) of

Techniques Used: Amplification, Real-time Polymerase Chain Reaction, Binding Assay, RNA Binding Assay, Expressing, Genome Wide

11) Product Images from "Melting Curve Analysis after T Allele Enrichment (MelcaTle) as a Highly Sensitive and Reliable Method for Detecting the JAK2V617F Mutation"

Article Title: Melting Curve Analysis after T Allele Enrichment (MelcaTle) as a Highly Sensitive and Reliable Method for Detecting the JAK2V617F Mutation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0122003

Schematic illustration of MelcaTle. MelcaTle comprises the following steps: 1) PCR amplification of genomic DNA that includes the G1849 region, followed by Bsa XI treatment to digest the JAK2 wild-type allele (G allele), 2) a second nested-PCR amplification and Bsa XI digestion to decrease the wild-type allele concentration, and 3) a third PCR using a blocking probe (BNA probe) and subsequent melting curve analysis with a mutant detection probe (Q-probe). The BNA probe inhibits the amplification of the residual G allele that is not digested by Bsa XI. The BNA clamping probe also blocks Q-probe annealing to the wild-type (G) allele. The thickness of the bar represents the extent of T allele enrichment during the process.
Figure Legend Snippet: Schematic illustration of MelcaTle. MelcaTle comprises the following steps: 1) PCR amplification of genomic DNA that includes the G1849 region, followed by Bsa XI treatment to digest the JAK2 wild-type allele (G allele), 2) a second nested-PCR amplification and Bsa XI digestion to decrease the wild-type allele concentration, and 3) a third PCR using a blocking probe (BNA probe) and subsequent melting curve analysis with a mutant detection probe (Q-probe). The BNA probe inhibits the amplification of the residual G allele that is not digested by Bsa XI. The BNA clamping probe also blocks Q-probe annealing to the wild-type (G) allele. The thickness of the bar represents the extent of T allele enrichment during the process.

Techniques Used: Polymerase Chain Reaction, Amplification, Nested PCR, Concentration Assay, Blocking Assay, Mutagenesis

12) Product Images from "Dppa3 expression is critical for generation of fully reprogrammed iPS cells and maintenance of Dlk1-Dio3 imprinting"

Article Title: Dppa3 expression is critical for generation of fully reprogrammed iPS cells and maintenance of Dlk1-Dio3 imprinting

Journal: Nature Communications

doi: 10.1038/ncomms7008

Dppa3 enhances reprogramming and generates fully pluripotent iPSCs. ( a ) Time scale showing the start of viral transduction followed by the appearance of ESC-like and Nanog -EGFP-positive colonies during iPSC generation using either OSKM or OSKM in combination with Dppa3 (OSKM+D). ( b ) Bright-field image showing the appearance of ESC-like colonies by day 8 of reprogramming using OSKM+D (left panel). The same colony was positive for Nanog -driven EGFP on day 14 (right panel). Scale bars correspond to 100 μm. ( c ) Line graph showing the number of AP-positive (AP +ve ) and EGFP-positive (EGFP +ve ) colonies during the reprogramming time course in the presence of OSKM or OSKM+D. ( d ) Gtl2 RNA expression analysis in iPSCs generated using either OSKM (grey) or OSKM+D (purple). Gtl2 RNA expression and associated error bars, representing mean±s.d. ( n =3), were normalized to housekeeping genes, Gapdh and Hprt , and presented as percentage of expression. ( e ) DNA methylation analysis of Dlk1-Dio3 IG-DMR in OSKM-only (grey) and OSKM+D (purple) iPSCs. ESC genomic DNA (green) was used as a control. ( f ) Light micrographs of hematoxylin and eosin-stained sections of teratomas obtained from OSKM+D iPSCs showing the presence of cell type derivatives of all three germ layers. Arrowheads indicate presence of representative tissue/cell type in respective germ layer. Scale bars correspond to 200 μm. ( g ) Images of chimeras obtained from OSKM+D iPSCs (upper panel) and their F1 progeny resulting from germline transmission (lower panel). *, pup derived from the germline-competent OSKM+D iPSCs.
Figure Legend Snippet: Dppa3 enhances reprogramming and generates fully pluripotent iPSCs. ( a ) Time scale showing the start of viral transduction followed by the appearance of ESC-like and Nanog -EGFP-positive colonies during iPSC generation using either OSKM or OSKM in combination with Dppa3 (OSKM+D). ( b ) Bright-field image showing the appearance of ESC-like colonies by day 8 of reprogramming using OSKM+D (left panel). The same colony was positive for Nanog -driven EGFP on day 14 (right panel). Scale bars correspond to 100 μm. ( c ) Line graph showing the number of AP-positive (AP +ve ) and EGFP-positive (EGFP +ve ) colonies during the reprogramming time course in the presence of OSKM or OSKM+D. ( d ) Gtl2 RNA expression analysis in iPSCs generated using either OSKM (grey) or OSKM+D (purple). Gtl2 RNA expression and associated error bars, representing mean±s.d. ( n =3), were normalized to housekeeping genes, Gapdh and Hprt , and presented as percentage of expression. ( e ) DNA methylation analysis of Dlk1-Dio3 IG-DMR in OSKM-only (grey) and OSKM+D (purple) iPSCs. ESC genomic DNA (green) was used as a control. ( f ) Light micrographs of hematoxylin and eosin-stained sections of teratomas obtained from OSKM+D iPSCs showing the presence of cell type derivatives of all three germ layers. Arrowheads indicate presence of representative tissue/cell type in respective germ layer. Scale bars correspond to 200 μm. ( g ) Images of chimeras obtained from OSKM+D iPSCs (upper panel) and their F1 progeny resulting from germline transmission (lower panel). *, pup derived from the germline-competent OSKM+D iPSCs.

Techniques Used: Transduction, RNA Expression, Generated, Expressing, DNA Methylation Assay, Staining, Transmission Assay, Derivative Assay

Vitamin C mediates global chromatin relaxation and early activation of pluripotency-related genes. ( a ) Western blots showing expression of histone modifications during OSKM reprogramming on days 5 and 7 in standard ESC culture medium in the presence and absence of Vc. MEFs supplemented with no OSKM or Vc (lanes 1 and 5), either Vc only (lanes 2 and 6), or OSKM only (lanes 3 and 7) served as controls. All blots were reprobed with an anti-total Histone 3 (H3) antibody, and one representative blot shows loading control. ( b ) Quantification of densitometric intensity of H3K4me3 and H3K27me3 bands shown in ( a ). ( c ) qRT–PCR data showing the expression of Oct3/4 (left panel), Sox2 (middle panel), and Dppa3 (right panel) mRNAs during the OSKM reprogramming time course in standard ESC culture medium in the presence (+Vc) or absence (−Vc) of Vc. Gene expression and associated error bars, representing mean ±s.d. ( n =3), were normalized to expression level in ESCs (dotted line). ( d ) Publicly available ChIP-Seq data showing the binding of Oct3/4 and Sox2 (black peaks) in the Dppa3 promoter region (red box). ( e ) ChIP–qPCR analysis showing the binding of Oct3/4 (green) and Sox2 (maroon) at the Dppa3 promoter region (amplicons Dppa3-P1 and Dppa3-P2) in ESCs. ChIP with IgG (blue) served as a negative control. The ChIP data, representing mean±s.d. ( n =2), is presented as percent of input DNA. a.u., arbitrary units.
Figure Legend Snippet: Vitamin C mediates global chromatin relaxation and early activation of pluripotency-related genes. ( a ) Western blots showing expression of histone modifications during OSKM reprogramming on days 5 and 7 in standard ESC culture medium in the presence and absence of Vc. MEFs supplemented with no OSKM or Vc (lanes 1 and 5), either Vc only (lanes 2 and 6), or OSKM only (lanes 3 and 7) served as controls. All blots were reprobed with an anti-total Histone 3 (H3) antibody, and one representative blot shows loading control. ( b ) Quantification of densitometric intensity of H3K4me3 and H3K27me3 bands shown in ( a ). ( c ) qRT–PCR data showing the expression of Oct3/4 (left panel), Sox2 (middle panel), and Dppa3 (right panel) mRNAs during the OSKM reprogramming time course in standard ESC culture medium in the presence (+Vc) or absence (−Vc) of Vc. Gene expression and associated error bars, representing mean ±s.d. ( n =3), were normalized to expression level in ESCs (dotted line). ( d ) Publicly available ChIP-Seq data showing the binding of Oct3/4 and Sox2 (black peaks) in the Dppa3 promoter region (red box). ( e ) ChIP–qPCR analysis showing the binding of Oct3/4 (green) and Sox2 (maroon) at the Dppa3 promoter region (amplicons Dppa3-P1 and Dppa3-P2) in ESCs. ChIP with IgG (blue) served as a negative control. The ChIP data, representing mean±s.d. ( n =2), is presented as percent of input DNA. a.u., arbitrary units.

Techniques Used: Activation Assay, Western Blot, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Negative Control

Dppa3 -knockout (KO) fibroblasts are arrested in pre-iPSC state during reprogramming. ( a ) Immunofluorescence images showing Oct3/4-positivity and activation of Oct3/4 -driven EGFP in Dppa3 -KO fibroblasts reprogrammed either with OSKM (upper panel), OSKM+Vc (middle panel) or OSKM+Dppa3 (lower panel). Nuclei were counterstained with DAPI. Scale bars correspond to 50 μm. ( b ) qRT–PCR data showing the expression of various pluripotency marker genes in the indicated Dppa3 -KO-derived iPSCs generated with OSKM+Vc and OSKM+D, but not in OSKM-only-generated iPSCs. Gene expression and associated error bars, representing mean±s.d. ( n =3), were normalized to expression level in Gtl2 on iPSC clone 6 (R21-4-ON). ( c ) qRT–PCR data showing expression of Gtl2 RNA in Dppa3 -KO-derived iPSCs in presence of OSKM (blue), OSKM+Vc (purple) or OSKM+Dppa3 (red). Gtl2 RNA expression and associated error bars, representing mean±s.d. ( n =3), were normalized to expression level in Gtl2 on iPSC clone 6 (R21-4-ON). ( d ) DNA methylation analysis of the Gtl2 IG-DMR in iPSCs generated from Dppa3 -KO fibroblasts with OSKM-only (blue), OSKM+Vc (purple) and OSKM+Dppa3 (red). Error bars represent mean±s.d. ( n =2). ( e ) qRT–PCR data showing expression of retrotransposons, intracisternal A-particles (IAP gag, black), short interspersed nuclear elements (SINE B1, grey), and long interspersed nuclear elements (LINE 1, white), in Dppa3 -KO-derived iPSCs in presence of OSKM, OSKM+Vc or OSKM+Dppa3. Retrotransposons expression and associated error bars, representing mean ±s.d. ( n =3), were normalized to expression level in Gtl2 on iPSC clone 6 (R21-4-ON). ( f ) Bright-field image (left panel) showing colony morphology and fluorescence image (right panel) showing the activation of Oct3/4 -driven EGFP in iPSCs derived from Dppa3 -KO fibroblasts with OSKM (upper panel) and the same clones treated with Vc (iPSC_K4+Vc) (lower panel). Scale bars correspond to 200 μm.
Figure Legend Snippet: Dppa3 -knockout (KO) fibroblasts are arrested in pre-iPSC state during reprogramming. ( a ) Immunofluorescence images showing Oct3/4-positivity and activation of Oct3/4 -driven EGFP in Dppa3 -KO fibroblasts reprogrammed either with OSKM (upper panel), OSKM+Vc (middle panel) or OSKM+Dppa3 (lower panel). Nuclei were counterstained with DAPI. Scale bars correspond to 50 μm. ( b ) qRT–PCR data showing the expression of various pluripotency marker genes in the indicated Dppa3 -KO-derived iPSCs generated with OSKM+Vc and OSKM+D, but not in OSKM-only-generated iPSCs. Gene expression and associated error bars, representing mean±s.d. ( n =3), were normalized to expression level in Gtl2 on iPSC clone 6 (R21-4-ON). ( c ) qRT–PCR data showing expression of Gtl2 RNA in Dppa3 -KO-derived iPSCs in presence of OSKM (blue), OSKM+Vc (purple) or OSKM+Dppa3 (red). Gtl2 RNA expression and associated error bars, representing mean±s.d. ( n =3), were normalized to expression level in Gtl2 on iPSC clone 6 (R21-4-ON). ( d ) DNA methylation analysis of the Gtl2 IG-DMR in iPSCs generated from Dppa3 -KO fibroblasts with OSKM-only (blue), OSKM+Vc (purple) and OSKM+Dppa3 (red). Error bars represent mean±s.d. ( n =2). ( e ) qRT–PCR data showing expression of retrotransposons, intracisternal A-particles (IAP gag, black), short interspersed nuclear elements (SINE B1, grey), and long interspersed nuclear elements (LINE 1, white), in Dppa3 -KO-derived iPSCs in presence of OSKM, OSKM+Vc or OSKM+Dppa3. Retrotransposons expression and associated error bars, representing mean ±s.d. ( n =3), were normalized to expression level in Gtl2 on iPSC clone 6 (R21-4-ON). ( f ) Bright-field image (left panel) showing colony morphology and fluorescence image (right panel) showing the activation of Oct3/4 -driven EGFP in iPSCs derived from Dppa3 -KO fibroblasts with OSKM (upper panel) and the same clones treated with Vc (iPSC_K4+Vc) (lower panel). Scale bars correspond to 200 μm.

Techniques Used: Knock-Out, Immunofluorescence, Activation Assay, Quantitative RT-PCR, Expressing, Marker, Derivative Assay, Generated, RNA Expression, DNA Methylation Assay, Fluorescence, Clone Assay

Dppa3 binds to a specific region within the IG-DMR of the Dlk1-Dio3 imprinted cluster and prevents the recruitment of Dnmt3a. ( a ) Simplified schematic diagram showing the Dlk1-Dio3 imprinted cluster together with paternally expressed genes (dark grey boxes) and maternally expressed genes (light grey boxes). The ~4.2 kb long IG-DMR (black circle), representing the imprinted control region, and the eight ChIP–qPCR amplicons (P1–P8) spanning the entire IG-DMR are indicated. ( b ) ChIP–qPCR analysis of Dppa3-binding sites (grey) across the eight PCR amplicons of the IG-DMR in ESCs. ChIP with IgG (black) was used as a negative control. The ChIP data, representing mean±s.d. ( n =2), is presented as percent of input DNA. ( c , d ) ChIP–qPCR of Dppa3 (grey) and Dnmt3a (black) binding at the P2, P3 and P8 amplicons of the IG-DMR in OSKM and OSKM+D on day 9 ( c ) and day 12 ( d ) of reprogramming. ChIP with IgG (white) was used as a negative control. The ChIP data, representing mean±s.d. ( n =3), is presented as percent of input DNA.
Figure Legend Snippet: Dppa3 binds to a specific region within the IG-DMR of the Dlk1-Dio3 imprinted cluster and prevents the recruitment of Dnmt3a. ( a ) Simplified schematic diagram showing the Dlk1-Dio3 imprinted cluster together with paternally expressed genes (dark grey boxes) and maternally expressed genes (light grey boxes). The ~4.2 kb long IG-DMR (black circle), representing the imprinted control region, and the eight ChIP–qPCR amplicons (P1–P8) spanning the entire IG-DMR are indicated. ( b ) ChIP–qPCR analysis of Dppa3-binding sites (grey) across the eight PCR amplicons of the IG-DMR in ESCs. ChIP with IgG (black) was used as a negative control. The ChIP data, representing mean±s.d. ( n =2), is presented as percent of input DNA. ( c , d ) ChIP–qPCR of Dppa3 (grey) and Dnmt3a (black) binding at the P2, P3 and P8 amplicons of the IG-DMR in OSKM and OSKM+D on day 9 ( c ) and day 12 ( d ) of reprogramming. ChIP with IgG (white) was used as a negative control. The ChIP data, representing mean±s.d. ( n =3), is presented as percent of input DNA.

Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Polymerase Chain Reaction, Negative Control

Stages of somatic cell reprogramming and Dppa3 expression status. ( a ) Schematic representation of somatic cell reprogramming, in which somatic cell (mouse embryonic fibroblast, MEF) transduced with OSKM reprogramming factors passes through initiation, maturation and stabilization phases to establish fully reprogrammed iPSC. Failure in proceeding to maturation and stabilization phases results in pre-iPSC. Similarly, defects occurring during maturation and stabilization phases result in lg-iPSC. Finally, faithful progression through all these phases results in establishment of hg-iPSC. The factor(s) responsible for conversion from pre-iPSC to either lg-iPSC or hg-iPSC, as well as from lg-iPSC to hg-iPSC are not known (?). Expression of several marker genes are indicated below each stage and pluripotency capabilities of various stages are indicated in a table. MET, mesenchymal-epithelial transition. ( b ) qRT–PCR data showing expression of Gtl2 RNA above the threshold (dotted line) of expression typically found in ESCs, in only two iPSC clones generated with classical Yamanaka factors (OSKM). Gtl2 RNA expression and associated error bars, representing mean±s.d. ( n =3), were normalized to expression level in ESCs (green). ( c ) DNA methylation analysis of the Dlk1-Dio3 IG-DMR in OSKM-derived iPSC clones showed normal methylation levels of 40–60% only in iPSC-1 and -2, whereas iPSC-3, -4, -5 and -6 displayed hypermethylation. Genomic DNA from ESCs (green) served as a control. Error bars represent mean±s.d. ( n =2). ( d ) qRT–PCR data showing expression of various pluripotency marker genes only in iPSC-1, -2, -5 and -6, but not in iPSC-3 and -4. Gene expression and associated error bars, representing mean±s.d. ( n =3), were normalized to expression level in iPS-2. ( e ) Western blot analysis showing expression of Dppa3 in all iPSCs, with the exception of iPSC-3 and -4. ESC protein extract was used as a control.
Figure Legend Snippet: Stages of somatic cell reprogramming and Dppa3 expression status. ( a ) Schematic representation of somatic cell reprogramming, in which somatic cell (mouse embryonic fibroblast, MEF) transduced with OSKM reprogramming factors passes through initiation, maturation and stabilization phases to establish fully reprogrammed iPSC. Failure in proceeding to maturation and stabilization phases results in pre-iPSC. Similarly, defects occurring during maturation and stabilization phases result in lg-iPSC. Finally, faithful progression through all these phases results in establishment of hg-iPSC. The factor(s) responsible for conversion from pre-iPSC to either lg-iPSC or hg-iPSC, as well as from lg-iPSC to hg-iPSC are not known (?). Expression of several marker genes are indicated below each stage and pluripotency capabilities of various stages are indicated in a table. MET, mesenchymal-epithelial transition. ( b ) qRT–PCR data showing expression of Gtl2 RNA above the threshold (dotted line) of expression typically found in ESCs, in only two iPSC clones generated with classical Yamanaka factors (OSKM). Gtl2 RNA expression and associated error bars, representing mean±s.d. ( n =3), were normalized to expression level in ESCs (green). ( c ) DNA methylation analysis of the Dlk1-Dio3 IG-DMR in OSKM-derived iPSC clones showed normal methylation levels of 40–60% only in iPSC-1 and -2, whereas iPSC-3, -4, -5 and -6 displayed hypermethylation. Genomic DNA from ESCs (green) served as a control. Error bars represent mean±s.d. ( n =2). ( d ) qRT–PCR data showing expression of various pluripotency marker genes only in iPSC-1, -2, -5 and -6, but not in iPSC-3 and -4. Gene expression and associated error bars, representing mean±s.d. ( n =3), were normalized to expression level in iPS-2. ( e ) Western blot analysis showing expression of Dppa3 in all iPSCs, with the exception of iPSC-3 and -4. ESC protein extract was used as a control.

Techniques Used: Expressing, Transduction, Marker, Quantitative RT-PCR, Clone Assay, Generated, RNA Expression, DNA Methylation Assay, Derivative Assay, Methylation, Western Blot

13) Product Images from "SUVH1, a Su(var)3–9 family member, promotes the expression of genes targeted by DNA methylation"

Article Title: SUVH1, a Su(var)3–9 family member, promotes the expression of genes targeted by DNA methylation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv958

The suvh1–1 mutation does not affect DNA methylation. ( A and B ) McrBC-qPCR analysis of DNA methylation levels at the d35S promoter and the LUC coding region in YJ (A) and LUCH (B). qPCR was performed using genomic DNA treated with or without McrBC. The relative levels of amplified DNA for UBQ5, LUC and d35S in samples treated with McrBC compared to untreated samples were shown. Error bars were from three technical replicates. Two biological replicates were performed and gave similar results. ( C and D ) The levels of CG, CHG and CHH DNA methylation at the d35S promoter (C) and LUC coding region (D) in YJ and YJ suvh1–1 as determined through MethylC sequencing. Results from two biological replicates (rep) are shown. (E) Total genomic CG, CHG and CHH DNA methylation in YJ and YJ suvh1–1 as determined through MethylC-seq. Results from two biological replicates (rep) are shown.
Figure Legend Snippet: The suvh1–1 mutation does not affect DNA methylation. ( A and B ) McrBC-qPCR analysis of DNA methylation levels at the d35S promoter and the LUC coding region in YJ (A) and LUCH (B). qPCR was performed using genomic DNA treated with or without McrBC. The relative levels of amplified DNA for UBQ5, LUC and d35S in samples treated with McrBC compared to untreated samples were shown. Error bars were from three technical replicates. Two biological replicates were performed and gave similar results. ( C and D ) The levels of CG, CHG and CHH DNA methylation at the d35S promoter (C) and LUC coding region (D) in YJ and YJ suvh1–1 as determined through MethylC sequencing. Results from two biological replicates (rep) are shown. (E) Total genomic CG, CHG and CHH DNA methylation in YJ and YJ suvh1–1 as determined through MethylC-seq. Results from two biological replicates (rep) are shown.

Techniques Used: Mutagenesis, DNA Methylation Assay, Real-time Polymerase Chain Reaction, Amplification, Sequencing

Plots of DNA methylation levels at 1 kb gene promoter regions versus gene expression levels in YJ and YJ suvh1–1 . The x-axis represents the level of DNA methylation, and the y-axis represents the natural logarithm of the RPKM (reads per kilobase per million) value for genes from mRNA-seq. ( A and B ) Correlation plot of CG methylation level with gene expression in YJ (A) and YJ suvh1–1 (B). ( C and D ) Correlation plot of CHG methylation level with gene expression in YJ (C) and YJ suvh1–1 (D). ( E and F ) Correlation plot of CHH methylation level with gene expression in YJ (E) and YJ suvh1–1 (F) DNA methylation and gene expression levels were determined from MethylC-seq and mRNA-seq, respectively, in this study.
Figure Legend Snippet: Plots of DNA methylation levels at 1 kb gene promoter regions versus gene expression levels in YJ and YJ suvh1–1 . The x-axis represents the level of DNA methylation, and the y-axis represents the natural logarithm of the RPKM (reads per kilobase per million) value for genes from mRNA-seq. ( A and B ) Correlation plot of CG methylation level with gene expression in YJ (A) and YJ suvh1–1 (B). ( C and D ) Correlation plot of CHG methylation level with gene expression in YJ (C) and YJ suvh1–1 (D). ( E and F ) Correlation plot of CHH methylation level with gene expression in YJ (E) and YJ suvh1–1 (F) DNA methylation and gene expression levels were determined from MethylC-seq and mRNA-seq, respectively, in this study.

Techniques Used: DNA Methylation Assay, Expressing, Methylation

14) Product Images from "Induction of proto-oncogene BRF2 in breast cancer cells by the dietary soybean isoflavone daidzein"

Article Title: Induction of proto-oncogene BRF2 in breast cancer cells by the dietary soybean isoflavone daidzein

Journal: BMC Cancer

doi: 10.1186/s12885-015-1914-5

Daidzein changes DNA methylation at the BRF2 promoter specifically. CpG methylation- sensitive restriction enzymes cut sites are shown. Transcription start site (TSS) is indicated by +1. MethPrimer program was used to predict the location of CpG islands within the BRF2 (402–556 bp) and BRF1 (50–910 bp) promoters, as indicated by black arrow. Black bars denote the binding sites for primers used in the methylation profile analysis. a-d MCF-7 and ( e ) MDA-MB-231 cells were treated with 0, 3, 10 μM daidzein for 48 h. The genomic DNA was harvested and digested with methylation-sensitive restriction enzymes with cut sites within the promoter, noted on BRF2 and BRF1 promoter schematic, and as a negative control one methylation sensitive enzyme with no recognition sites in the promoter was used. BspEI and BsRFI do not have recognition sites in BRF2 and BRF1 promoters, respectively. The digestion profile was then analyzed by qPCR using primers spanning the BRF2 ( a , e, f ) and BRF1 ( b ) promoter regions, U6 ( c ) and tRNA i Met ( d ) genes. DNA methylation levels were calculated using ΔΔCt method with RPS13 expression levels used as a reference for normalization. Data presented are average of three independent experiments. Statistical analysis was performed using one-way ANOVA a Tukey’s post-test with a 95 % confidence interval (Graphpad Prism 3.03); * = p
Figure Legend Snippet: Daidzein changes DNA methylation at the BRF2 promoter specifically. CpG methylation- sensitive restriction enzymes cut sites are shown. Transcription start site (TSS) is indicated by +1. MethPrimer program was used to predict the location of CpG islands within the BRF2 (402–556 bp) and BRF1 (50–910 bp) promoters, as indicated by black arrow. Black bars denote the binding sites for primers used in the methylation profile analysis. a-d MCF-7 and ( e ) MDA-MB-231 cells were treated with 0, 3, 10 μM daidzein for 48 h. The genomic DNA was harvested and digested with methylation-sensitive restriction enzymes with cut sites within the promoter, noted on BRF2 and BRF1 promoter schematic, and as a negative control one methylation sensitive enzyme with no recognition sites in the promoter was used. BspEI and BsRFI do not have recognition sites in BRF2 and BRF1 promoters, respectively. The digestion profile was then analyzed by qPCR using primers spanning the BRF2 ( a , e, f ) and BRF1 ( b ) promoter regions, U6 ( c ) and tRNA i Met ( d ) genes. DNA methylation levels were calculated using ΔΔCt method with RPS13 expression levels used as a reference for normalization. Data presented are average of three independent experiments. Statistical analysis was performed using one-way ANOVA a Tukey’s post-test with a 95 % confidence interval (Graphpad Prism 3.03); * = p

Techniques Used: DNA Methylation Assay, CpG Methylation Assay, Binding Assay, Methylation, Multiple Displacement Amplification, Negative Control, Real-time Polymerase Chain Reaction, Expressing

15) Product Images from "Oestrogen receptor β regulates epigenetic patterns at specific genomic loci through interaction with thymine DNA glycosylase"

Article Title: Oestrogen receptor β regulates epigenetic patterns at specific genomic loci through interaction with thymine DNA glycosylase

Journal: Epigenetics & Chromatin

doi: 10.1186/s13072-016-0055-7

ERβ deficiency leads to altered DNA methylation patterns. a Histogram showing the distribution of methylation at the sequenced cytosines in wt and βerko MEFs. b Scatterplot of percentage (%) methylation in wt vs. βerko MEFs at cytosines covered in both cell types. c Pie chart presenting the genomic distribution of hypo- and hypermethylated positions. A position was considered hypermethylated if more than 80 % of the reads indicated methylation and hypomethylated if less than 20 % indicated methylation in βerko MEFs. d Enrichment (log2 ratios of observed over random) of hypo- and hypermethylated positions at different genomic features. e Comparison of regions identified by RRBS with datasets for histone modifications in MEFs [ 36 ] using GenomeInspector (Genomatix). Bar plots indicate percentages of hypomethylated (hypo) and hypermethylated (hyper) CpGs either marked by H3K4m3 ( red ), H3K27me3 ( orange ), both marks ( yellow ), or none of them (negative, white ), or H3K4m1 ( blue ), H3K4m1 plus H3K27ac ( green ), or none of them (negative, white ). Odds ratios (ORs) and p values according to Fisher exact test. f Enrichment (log2 ratios of observed over random) of histone modifications at hypo- and hypermethylated positions
Figure Legend Snippet: ERβ deficiency leads to altered DNA methylation patterns. a Histogram showing the distribution of methylation at the sequenced cytosines in wt and βerko MEFs. b Scatterplot of percentage (%) methylation in wt vs. βerko MEFs at cytosines covered in both cell types. c Pie chart presenting the genomic distribution of hypo- and hypermethylated positions. A position was considered hypermethylated if more than 80 % of the reads indicated methylation and hypomethylated if less than 20 % indicated methylation in βerko MEFs. d Enrichment (log2 ratios of observed over random) of hypo- and hypermethylated positions at different genomic features. e Comparison of regions identified by RRBS with datasets for histone modifications in MEFs [ 36 ] using GenomeInspector (Genomatix). Bar plots indicate percentages of hypomethylated (hypo) and hypermethylated (hyper) CpGs either marked by H3K4m3 ( red ), H3K27me3 ( orange ), both marks ( yellow ), or none of them (negative, white ), or H3K4m1 ( blue ), H3K4m1 plus H3K27ac ( green ), or none of them (negative, white ). Odds ratios (ORs) and p values according to Fisher exact test. f Enrichment (log2 ratios of observed over random) of histone modifications at hypo- and hypermethylated positions

Techniques Used: DNA Methylation Assay, Methylation

Hyper- but not hypomethylation in βerko MEFs is reversible by re-introduction of ERβ into βerko MEFs. a DNA methylation analysis of ten hypo- and eight hypermethylated positions. DNA methylation was assessed by methylation-specific enzymatic digest followed by qPCR. Positions with gene names in brackets were chosen for further analysis. b DNA methylation ( left panel ) and histone modifications ( right panel ) of differentially methylated genes in wt, βerko, and βerkohERβ MEFs. DNA methylation was assessed by pyrosequencing of bisulfite-treated DNA; black arrows mark DMPs identified by RRBS. Triple Asterisk indicates significant differences ( p
Figure Legend Snippet: Hyper- but not hypomethylation in βerko MEFs is reversible by re-introduction of ERβ into βerko MEFs. a DNA methylation analysis of ten hypo- and eight hypermethylated positions. DNA methylation was assessed by methylation-specific enzymatic digest followed by qPCR. Positions with gene names in brackets were chosen for further analysis. b DNA methylation ( left panel ) and histone modifications ( right panel ) of differentially methylated genes in wt, βerko, and βerkohERβ MEFs. DNA methylation was assessed by pyrosequencing of bisulfite-treated DNA; black arrows mark DMPs identified by RRBS. Triple Asterisk indicates significant differences ( p

Techniques Used: DNA Methylation Assay, Methylation, Real-time Polymerase Chain Reaction

ERβ-dependent transcription of differentially methylated genes in MEFs and ESCs. a Venn diagram visualising overlaps between differentially methylated (identified by RRBS) and differentially expressed (identified by microarray expression analysis) genes in wt and βerko cells. b Gene expression analysis of hypomethylated ( Dyx1c1 , HoxD9 ), hypermethylated complementable ( HoxA9, HoxA10, and Tnfaip2 ), and hypermethylated non-complementable genes in wt, βerko, and βerkohERβ MEFs. Gene expression was analysed by RT-qPCR (mean + SD; n ≥ 3). c DNA methylation of differentially methylated genes in wt MEFs and ESCs, assessed by methylation-specific enzymatic digest followed by qPCR. d ERβ-dependent expression of differentially methylated genes in ESCs. Gene expression was assessed by qRT-PCR 4 days after transfection with plasmid encoding for shRNA against ERβ or non-targeting control (means + SD; n ≥ 3). All the genes showed significantly decreased expression compared to shcontrol (** p
Figure Legend Snippet: ERβ-dependent transcription of differentially methylated genes in MEFs and ESCs. a Venn diagram visualising overlaps between differentially methylated (identified by RRBS) and differentially expressed (identified by microarray expression analysis) genes in wt and βerko cells. b Gene expression analysis of hypomethylated ( Dyx1c1 , HoxD9 ), hypermethylated complementable ( HoxA9, HoxA10, and Tnfaip2 ), and hypermethylated non-complementable genes in wt, βerko, and βerkohERβ MEFs. Gene expression was analysed by RT-qPCR (mean + SD; n ≥ 3). c DNA methylation of differentially methylated genes in wt MEFs and ESCs, assessed by methylation-specific enzymatic digest followed by qPCR. d ERβ-dependent expression of differentially methylated genes in ESCs. Gene expression was assessed by qRT-PCR 4 days after transfection with plasmid encoding for shRNA against ERβ or non-targeting control (means + SD; n ≥ 3). All the genes showed significantly decreased expression compared to shcontrol (** p

Techniques Used: Methylation, Microarray, Expressing, Quantitative RT-PCR, DNA Methylation Assay, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, shRNA

16) Product Images from "Immunization with Lipopolysaccharide-Deficient Whole Cells Provides Protective Immunity in an Experimental Mouse Model of Acinetobacter baumannii Infection"

Article Title: Immunization with Lipopolysaccharide-Deficient Whole Cells Provides Protective Immunity in an Experimental Mouse Model of Acinetobacter baumannii Infection

Journal: PLoS ONE

doi: 10.1371/journal.pone.0114410

Mutation and endotoxin content of IB010. (A) Genomic DNA from three independent cultures of ATCC 19606 and IB010 was extracted and amplified using primers specific for the lpxD gene. The band corresponding to approximately 1000 Kb corresponds to the intact lpxD gene, whereas the faster migrating band corresponds to the lpx D gene with a deletion of 462 nucleotides. (B) Endotoxin levels of ATCC 19606 and IB010 determined by the Limulus Amebocyte Assay. Bars represent the median values of three independent cultures, and error bars represent the standard error of the mean. EU; endotoxin units.
Figure Legend Snippet: Mutation and endotoxin content of IB010. (A) Genomic DNA from three independent cultures of ATCC 19606 and IB010 was extracted and amplified using primers specific for the lpxD gene. The band corresponding to approximately 1000 Kb corresponds to the intact lpxD gene, whereas the faster migrating band corresponds to the lpx D gene with a deletion of 462 nucleotides. (B) Endotoxin levels of ATCC 19606 and IB010 determined by the Limulus Amebocyte Assay. Bars represent the median values of three independent cultures, and error bars represent the standard error of the mean. EU; endotoxin units.

Techniques Used: Mutagenesis, Amplification

17) Product Images from "Evaluation of Glutathione-S-Transferase P1 Polymorphism and its Relation to Bone Mineral Density in Egyptian Children and Adolescents with Beta-Thalassemia Major"

Article Title: Evaluation of Glutathione-S-Transferase P1 Polymorphism and its Relation to Bone Mineral Density in Egyptian Children and Adolescents with Beta-Thalassemia Major

Journal: Mediterranean Journal of Hematology and Infectious Diseases

doi: 10.4084/MJHID.2016.004

The agarose gel electrophoresis for Ile/Val polymorphism after digestion by Alw261;Lane 1 indicates DNA ladder (50 bp);Lanes 2, 3 and 6 indicate GSTP1 Ile/Val (AG) polymorphism (329bp, 216bp and 113 bp);Lanes 4,5 indicate GSTP1 Val/Val(GG) polymorphism (216bp and113 bp). Lane 7 indicates GSTP1 Ile/Ile (AA) polymorphism (329bp and 113 bp).
Figure Legend Snippet: The agarose gel electrophoresis for Ile/Val polymorphism after digestion by Alw261;Lane 1 indicates DNA ladder (50 bp);Lanes 2, 3 and 6 indicate GSTP1 Ile/Val (AG) polymorphism (329bp, 216bp and 113 bp);Lanes 4,5 indicate GSTP1 Val/Val(GG) polymorphism (216bp and113 bp). Lane 7 indicates GSTP1 Ile/Ile (AA) polymorphism (329bp and 113 bp).

Techniques Used: Agarose Gel Electrophoresis

18) Product Images from "Inducible Expression of the De-Novo Designed Antimicrobial Peptide SP1-1 in Tomato Confers Resistance to Xanthomonas campestris pv. vesicatoria"

Article Title: Inducible Expression of the De-Novo Designed Antimicrobial Peptide SP1-1 in Tomato Confers Resistance to Xanthomonas campestris pv. vesicatoria

Journal: PLoS ONE

doi: 10.1371/journal.pone.0164097

Transgenic tomato Micro Tom lines expressing the AMP SP1-1. (A) Schematic illustration of PMIGW-4XW2/4XS::SP1-1 vector construct. The vector contains a CaMV35S promoter-driven phosphomannose isomerase (PMI) gene for mannose selection. B1, B2, B3 and B4 represent attB Gateway recombination sites. SP, signal peptide RsAFP1 from radish; SP1-1, synthetic antimicrobial peptide; TNos, nopaline synthase gene terminator; RB, right border; LB, left border; W2 cis -acting elements from the parsley PR1 gene; S, cis -elements from the parsley EL17 gene; CaMV35S, minimal promoter containing the sequence -46 to +8 from the cauliflower mosaic virus 35S promoter; T35S, terminator from the cauliflower mosaic virus 35S. (B) Molecular characterization of transformed plants. PCR was done using DNA from young leaves as template and RsAFP1-TNos-specific primers. PCR fragments were obtained for T583-4, T583-5 and T583-6. PMIGW-4XW2/4XS::SP1-1 transformation vectors and WT Micro Tom tomato plants were used as positive and negative controls respectively. (C and D) Morphological phenotypes of six weeks old WT and transgenic T1 Micro Tom tomato plants. Transgenic and WT plants were grown in climate chambers for 6 weeks. (E) Detached leaves of line T583 were infiltrated with water (H) or pep25 peptide (P). Total RNA was extracted before induction (BI) and 0, 3, and 22 h after induction. RT-PCR was carried out with gene-specific primers for RsAFP1-SP1-1 and actin and fragments of 163 bp and 586 bp were expected, respectively. Genomic DNA of the transgenic line T583 was used as control (+). Total RNA from WT plants and water (-) was used as negative control. The SP1-1 band is marked with an arrow. M, 100 bp DNA ladder.
Figure Legend Snippet: Transgenic tomato Micro Tom lines expressing the AMP SP1-1. (A) Schematic illustration of PMIGW-4XW2/4XS::SP1-1 vector construct. The vector contains a CaMV35S promoter-driven phosphomannose isomerase (PMI) gene for mannose selection. B1, B2, B3 and B4 represent attB Gateway recombination sites. SP, signal peptide RsAFP1 from radish; SP1-1, synthetic antimicrobial peptide; TNos, nopaline synthase gene terminator; RB, right border; LB, left border; W2 cis -acting elements from the parsley PR1 gene; S, cis -elements from the parsley EL17 gene; CaMV35S, minimal promoter containing the sequence -46 to +8 from the cauliflower mosaic virus 35S promoter; T35S, terminator from the cauliflower mosaic virus 35S. (B) Molecular characterization of transformed plants. PCR was done using DNA from young leaves as template and RsAFP1-TNos-specific primers. PCR fragments were obtained for T583-4, T583-5 and T583-6. PMIGW-4XW2/4XS::SP1-1 transformation vectors and WT Micro Tom tomato plants were used as positive and negative controls respectively. (C and D) Morphological phenotypes of six weeks old WT and transgenic T1 Micro Tom tomato plants. Transgenic and WT plants were grown in climate chambers for 6 weeks. (E) Detached leaves of line T583 were infiltrated with water (H) or pep25 peptide (P). Total RNA was extracted before induction (BI) and 0, 3, and 22 h after induction. RT-PCR was carried out with gene-specific primers for RsAFP1-SP1-1 and actin and fragments of 163 bp and 586 bp were expected, respectively. Genomic DNA of the transgenic line T583 was used as control (+). Total RNA from WT plants and water (-) was used as negative control. The SP1-1 band is marked with an arrow. M, 100 bp DNA ladder.

Techniques Used: Transgenic Assay, Expressing, Plasmid Preparation, Construct, Selection, Sequencing, Transformation Assay, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Negative Control

19) Product Images from "COMPARISON OF 16S rRNA-PCR-RFLP, LipL32-PCR AND OmpL1-PCR METHODS IN THE DIAGNOSIS OF LEPTOSPIROSIS"

Article Title: COMPARISON OF 16S rRNA-PCR-RFLP, LipL32-PCR AND OmpL1-PCR METHODS IN THE DIAGNOSIS OF LEPTOSPIROSIS

Journal: Revista do Instituto de Medicina Tropical de São Paulo

doi: 10.1590/S1678-9946201658064

(A) 16S rRNA-PCR products. Lane 6: 100 bp DNA ladder; Lane 1-10: 289 bp product. (B) 16S rRNA-PCR-RFLP products. Lane 5: 100 bp DNA ladder; Lane 1-2-4-6-8: RFLP with Apo I for L. interrogans (289 bp); Lane 3-7-9-10: RFLP with Apo I for reference L. biflexa (200 bp and 89 bp products). (C) LipL32-PCR products. Lane 7: 100 bp DNA ladder; Lane 1-12: 497 bp product. (D) OmpL1-PCR products. Lane 6: 100 bp DNA ladder; Lane 1-10: 406 bp product.
Figure Legend Snippet: (A) 16S rRNA-PCR products. Lane 6: 100 bp DNA ladder; Lane 1-10: 289 bp product. (B) 16S rRNA-PCR-RFLP products. Lane 5: 100 bp DNA ladder; Lane 1-2-4-6-8: RFLP with Apo I for L. interrogans (289 bp); Lane 3-7-9-10: RFLP with Apo I for reference L. biflexa (200 bp and 89 bp products). (C) LipL32-PCR products. Lane 7: 100 bp DNA ladder; Lane 1-12: 497 bp product. (D) OmpL1-PCR products. Lane 6: 100 bp DNA ladder; Lane 1-10: 406 bp product.

Techniques Used: Polymerase Chain Reaction

20) Product Images from "Retrotransposition of Long Interspersed Element 1 Induced by Methamphetamine or Cocaine *"

Article Title: Retrotransposition of Long Interspersed Element 1 Induced by Methamphetamine or Cocaine *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.559419

METH induces L1-RTP. A , schematics of the constructs used for the assay of L1-RTP and the rationale for the PCR-based assay (see details under “Experimental Procedures”). Arrows indicate primers for the PCR-based assay. SD and SA indicate splicing donors and splicing acceptors, respectively. B , the experimental protocol for the PCR-based assay and colony formation assay (see details under “Experimental Procedures”). C , results of the PCR-based assays. HT-1080, SH-SY5Y, and PC12 cells showed the amplified 340-bp band within 3 days after METH treatment ( arrowheads ). Mr , DNA molecular weight markers. B , Hepes-buffered saline (HBS) buffer; M , METH ( lanes 2 and 5 , 0.13 m m ; lanes 3 and 6 , 0.06 m m ; lane 10 , 0.5 m m ; D , PCR water control. D , the dose response of METH in the PCR-based assays. HT-1080, SH-SY5Y, and PC12 cells showed the 340-bp amplicon 3 days after METH treatment ( arrowheads ). B , HBS buffer; M , METH ( lanes 2 , 11 , and 17 , 1 m m ; lanes 3 , 12 , and 18 , 0.5 m m ; lanes 4 , 13 , and 19 , 0.25 m m ; lanes 5 , 14 , and 20 , 0.13 m m ; lanes 6 , 15 , and 21 , 0.06 m m ); D , PCR water control; N , a lane without a sample. E , METH induced L1-RTP in SH-SY5Y cells. F , colony formation assay of METH-induced L1-RTP. Cells were incubated with either HBS buffer ( B , lane 1) or METH at 0.25, 0.13, 0.06, 0.03, or 0.015 m m ( M , lanes 2- 6). At least 2 independent experiments were performed, and representative results are shown. The numbers of colonies are presented as the mean ± S.D. Asterisks indicate statistical significance ( p
Figure Legend Snippet: METH induces L1-RTP. A , schematics of the constructs used for the assay of L1-RTP and the rationale for the PCR-based assay (see details under “Experimental Procedures”). Arrows indicate primers for the PCR-based assay. SD and SA indicate splicing donors and splicing acceptors, respectively. B , the experimental protocol for the PCR-based assay and colony formation assay (see details under “Experimental Procedures”). C , results of the PCR-based assays. HT-1080, SH-SY5Y, and PC12 cells showed the amplified 340-bp band within 3 days after METH treatment ( arrowheads ). Mr , DNA molecular weight markers. B , Hepes-buffered saline (HBS) buffer; M , METH ( lanes 2 and 5 , 0.13 m m ; lanes 3 and 6 , 0.06 m m ; lane 10 , 0.5 m m ; D , PCR water control. D , the dose response of METH in the PCR-based assays. HT-1080, SH-SY5Y, and PC12 cells showed the 340-bp amplicon 3 days after METH treatment ( arrowheads ). B , HBS buffer; M , METH ( lanes 2 , 11 , and 17 , 1 m m ; lanes 3 , 12 , and 18 , 0.5 m m ; lanes 4 , 13 , and 19 , 0.25 m m ; lanes 5 , 14 , and 20 , 0.13 m m ; lanes 6 , 15 , and 21 , 0.06 m m ); D , PCR water control; N , a lane without a sample. E , METH induced L1-RTP in SH-SY5Y cells. F , colony formation assay of METH-induced L1-RTP. Cells were incubated with either HBS buffer ( B , lane 1) or METH at 0.25, 0.13, 0.06, 0.03, or 0.015 m m ( M , lanes 2- 6). At least 2 independent experiments were performed, and representative results are shown. The numbers of colonies are presented as the mean ± S.D. Asterisks indicate statistical significance ( p

Techniques Used: Construct, Polymerase Chain Reaction, Colony Assay, Amplification, Molecular Weight, Incubation

21) Product Images from "Rif2 Promotes a Telomere Fold-Back Structure through Rpd3L Recruitment in Budding Yeast"

Article Title: Rif2 Promotes a Telomere Fold-Back Structure through Rpd3L Recruitment in Budding Yeast

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1002960

Rpd3 promotes telomere end protection. (A) Strains with the indicated genotypes were spotted onto YPD media following an overnight culture at 23°C and incubated at various temperatures for 3–4 days before being imaged. (B) cdc13-1 , cdc13-1 sin3Δ and cdc13-1 sin3Δ exo1Δ cells were arrested in nocodazole at 23°C for three hours before being shifted to 26°C, after which DNA was extracted at 30 minute intervals. Non-denatured and denatured DNA was dot blotted onto a membrane and incubated with a DIG-labeled probe (oBL 207) to recognize the telomeric 3′ ssDNA overhang. The amount of ssDNA is represented as non-denatured DNA as a fraction of the total (as determined by the amount of denatured telomeric DNA). Error bars represent SD from three independent experiments. Pre = before the 26°C temperature shift. (C) The indicated genotypes were derived via tetrad dissection of the heterozygous diploid strain (yMD 1146) and diluted to an OD 600 0.01. Cells were grown for 24-hour intervals before being measured and re-diluted. The rate of senescence was increased in est2Δ rad52Δ cells when SIN3 was subsequently deleted, n = 8 for each genotype. The growth rates of rad52Δ (n = 3) and rad52Δ sin3Δ (n = 3) were similar. Population doubling refers to the number of doublings post spore germination. (D) Genomic DNA was isolated in both non-denaturing and denaturing conditions from the indicated genotypes (each n = 3) using samples generated in (C) at the specified population doubling (PD). Single-stranded telomeric DNA was detected upon hybridization with DIG labeled oBL 207 and normalized to total telomeric DNA following a denaturation step.
Figure Legend Snippet: Rpd3 promotes telomere end protection. (A) Strains with the indicated genotypes were spotted onto YPD media following an overnight culture at 23°C and incubated at various temperatures for 3–4 days before being imaged. (B) cdc13-1 , cdc13-1 sin3Δ and cdc13-1 sin3Δ exo1Δ cells were arrested in nocodazole at 23°C for three hours before being shifted to 26°C, after which DNA was extracted at 30 minute intervals. Non-denatured and denatured DNA was dot blotted onto a membrane and incubated with a DIG-labeled probe (oBL 207) to recognize the telomeric 3′ ssDNA overhang. The amount of ssDNA is represented as non-denatured DNA as a fraction of the total (as determined by the amount of denatured telomeric DNA). Error bars represent SD from three independent experiments. Pre = before the 26°C temperature shift. (C) The indicated genotypes were derived via tetrad dissection of the heterozygous diploid strain (yMD 1146) and diluted to an OD 600 0.01. Cells were grown for 24-hour intervals before being measured and re-diluted. The rate of senescence was increased in est2Δ rad52Δ cells when SIN3 was subsequently deleted, n = 8 for each genotype. The growth rates of rad52Δ (n = 3) and rad52Δ sin3Δ (n = 3) were similar. Population doubling refers to the number of doublings post spore germination. (D) Genomic DNA was isolated in both non-denaturing and denaturing conditions from the indicated genotypes (each n = 3) using samples generated in (C) at the specified population doubling (PD). Single-stranded telomeric DNA was detected upon hybridization with DIG labeled oBL 207 and normalized to total telomeric DNA following a denaturation step.

Techniques Used: Incubation, Labeling, Derivative Assay, Dissection, Isolation, Generated, Hybridization

Chromatin immunoprecipitation confirms structural defects. (A) The immunoprecipitation of Rap1 following cross-linking should be associated with subtelomeric sequences at natural telomere 6R if the fold-back structure is intact (upper diagram); however the subtelomeric ChIP will be lost upon loop opening (lower diagram). (B) Upon Rap1 ChIP from exponentially growing cells, a subtelomeric signal was detected up to 1 kb away from the base of the telomeric repeats in wild type cells, whereas the signal was largely diminished in hda1Δ , sin3Δ and sir4Δ mutants. DNA stemming from the actin locus ( ACT1 ) was not detected following Rap1 ChIP and was used as a background control. Error bars represent SD from three independent experiments. (C) Cdc13-TAP (13) was also able to precipitate subtelomeric DNA up to 1 kb away from the start of the telomeric sequence at telomere 6R following cross-linking (n = 3, error as SD) in comparison to wild type (non-tagged controls). This ChIP signal at -1000 was reduced to that of non-tagged controls in the sin3Δ strain. The difference in ChIP signal distribution between Rap1 (B) and Cdc13-TAP (C) is likely due to the different positioning of the two proteins on the telomere (compare diagrams in A and C for explanation). For all experiments above error bars represent SD of the mean from at least 3 independent experiments and * indicates statistically significant differences as determined through unpaired student's t-tests whereby * = p
Figure Legend Snippet: Chromatin immunoprecipitation confirms structural defects. (A) The immunoprecipitation of Rap1 following cross-linking should be associated with subtelomeric sequences at natural telomere 6R if the fold-back structure is intact (upper diagram); however the subtelomeric ChIP will be lost upon loop opening (lower diagram). (B) Upon Rap1 ChIP from exponentially growing cells, a subtelomeric signal was detected up to 1 kb away from the base of the telomeric repeats in wild type cells, whereas the signal was largely diminished in hda1Δ , sin3Δ and sir4Δ mutants. DNA stemming from the actin locus ( ACT1 ) was not detected following Rap1 ChIP and was used as a background control. Error bars represent SD from three independent experiments. (C) Cdc13-TAP (13) was also able to precipitate subtelomeric DNA up to 1 kb away from the start of the telomeric sequence at telomere 6R following cross-linking (n = 3, error as SD) in comparison to wild type (non-tagged controls). This ChIP signal at -1000 was reduced to that of non-tagged controls in the sin3Δ strain. The difference in ChIP signal distribution between Rap1 (B) and Cdc13-TAP (C) is likely due to the different positioning of the two proteins on the telomere (compare diagrams in A and C for explanation). For all experiments above error bars represent SD of the mean from at least 3 independent experiments and * indicates statistically significant differences as determined through unpaired student's t-tests whereby * = p

Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Sequencing

The fold-back may contribute to telomere protection. The maintenance of telomere structure requires the telomere-bound Rif2 protein to ensure that the Rpd3L complex gets properly loaded/maintained at chromosome ends. The presence of the Rpd3L KDAC (as well as Rpd3S, Sir2 and Hda1) promotes a protective structure at telomeres, which likely eminates in a fold-back of the telomeric DNA onto the subtelomeric region (1.). In the absence of this structure, telomeres remain protected due to a combination of telomerase-mediated elongation and capping via the CST complex (2.). When both capping and the fold-back structure are simultaneously compromised (3.) chromosome ends undergo accelerated nucleolytic degradation, and experience an accelerated rate of senescence in cells lacking a telomere maintenance mechanism due to the fact that rapidly resected uncapped telomeres do not get re-elongated.
Figure Legend Snippet: The fold-back may contribute to telomere protection. The maintenance of telomere structure requires the telomere-bound Rif2 protein to ensure that the Rpd3L complex gets properly loaded/maintained at chromosome ends. The presence of the Rpd3L KDAC (as well as Rpd3S, Sir2 and Hda1) promotes a protective structure at telomeres, which likely eminates in a fold-back of the telomeric DNA onto the subtelomeric region (1.). In the absence of this structure, telomeres remain protected due to a combination of telomerase-mediated elongation and capping via the CST complex (2.). When both capping and the fold-back structure are simultaneously compromised (3.) chromosome ends undergo accelerated nucleolytic degradation, and experience an accelerated rate of senescence in cells lacking a telomere maintenance mechanism due to the fact that rapidly resected uncapped telomeres do not get re-elongated.

Techniques Used:

The Rif proteins promote Rpd3L recruitment to telomeres. (A) Cells with the indicated genotypes and harboring construct 2 were spotted onto the galactose media (+/− FOA) (B) Rap1 ChIP was performed as described for Figure 2B . The defect in looping of the rif1Δ and rif2Δ strains is reflected in the loss of Rap1 association to subtelomeric DNA following cross-linking (n = 3, error as SD). (C) Looping defects of the indicated mutants were assayed as in Figure 1D in order to assess genetic interactions between rif2Δ and the Rpd3L and Rpd3S complexes. All colonies were replicated from +FOA onto SD-URA plates to ensure that construct 2 was intact. (D) The looping defect in Rpd3L ( rxt2Δ ) mutants is additive when combined with Rpd3S mutations ( eaf3Δ ), and not further exacerbated by deletion of RIF2 . (E) Both hda1Δ rif2Δ and hda2Δ rif2Δ double mutants have more severe looping defects than either of the single mutants as seen by their increased resistance to 5-FOA. (F and G) Cells expressing a TAP (tandem affinity purification) tagged version of either Rxt2-TAP (Rpd3L) or Rco1-TAP (Rpd3S) were cross-linked and DNA was precipitated with IgG beads. -6 bp and -2000 bp refer to the position of the reverse primer with respect to the beginning of the telomeric tract on telomere 6R, amplicons being on average approximately 100 bp. The Rpd3L complex is lost at telomeres in a rif2Δ mutant (F) whereas Rpd3S association is not affected (G). For all experiments above error bars represent SD of the mean from at least 3 independent experiments and * indicates statistically significant differences as determined through unpaired student's t-tests whereby * = p
Figure Legend Snippet: The Rif proteins promote Rpd3L recruitment to telomeres. (A) Cells with the indicated genotypes and harboring construct 2 were spotted onto the galactose media (+/− FOA) (B) Rap1 ChIP was performed as described for Figure 2B . The defect in looping of the rif1Δ and rif2Δ strains is reflected in the loss of Rap1 association to subtelomeric DNA following cross-linking (n = 3, error as SD). (C) Looping defects of the indicated mutants were assayed as in Figure 1D in order to assess genetic interactions between rif2Δ and the Rpd3L and Rpd3S complexes. All colonies were replicated from +FOA onto SD-URA plates to ensure that construct 2 was intact. (D) The looping defect in Rpd3L ( rxt2Δ ) mutants is additive when combined with Rpd3S mutations ( eaf3Δ ), and not further exacerbated by deletion of RIF2 . (E) Both hda1Δ rif2Δ and hda2Δ rif2Δ double mutants have more severe looping defects than either of the single mutants as seen by their increased resistance to 5-FOA. (F and G) Cells expressing a TAP (tandem affinity purification) tagged version of either Rxt2-TAP (Rpd3L) or Rco1-TAP (Rpd3S) were cross-linked and DNA was precipitated with IgG beads. -6 bp and -2000 bp refer to the position of the reverse primer with respect to the beginning of the telomeric tract on telomere 6R, amplicons being on average approximately 100 bp. The Rpd3L complex is lost at telomeres in a rif2Δ mutant (F) whereas Rpd3S association is not affected (G). For all experiments above error bars represent SD of the mean from at least 3 independent experiments and * indicates statistically significant differences as determined through unpaired student's t-tests whereby * = p

Techniques Used: Construct, Chromatin Immunoprecipitation, Expressing, Affinity Purification, Mutagenesis

22) Product Images from "A comprehensive platform for highly multiplexed mammalian functional genetic screens"

Article Title: A comprehensive platform for highly multiplexed mammalian functional genetic screens

Journal: BMC Genomics

doi: 10.1186/1471-2164-12-213

Comparison of sequencing and GMAP performance with high complexity shRNA pools . (a) Scatter plot of GMAP array signal intensity (X-axis) versus sequencing read number (Y-axis) for shRNA clones from the human Even shRNA pool. (b) Distribution plots of Illumina sequencing data for a dilution series of shRNAs in separate pools. 4x, 16x, and 64x curves are plotted as the distribution of log 2 difference between the number of sequencing reads for shRNAs in the dilution series and their read count in the reference (Even) pool. The 1x curve is plotted as the log 2 difference between a group of undiluted shRNAs in the 4x pool and the same shRNAs in the Even pool. (c) Distribution plots of Illumina sequencing data for a dilution series of shRNAs within the same pool. 2x, 5x, 10x, and 20x curves are plotted as the distribution of log 2 difference between the number of sequencing reads for groups of diluted shRNAs and their read count in the reference (Even) pool. The 1x curve is plotted as the log 2 difference between a group of undiluted shRNAs in the 2x-20x pool and the same shRNAs in the Even pool. (d) Distribution plots of GMAP features data for a dilution series of shRNAs contained within sub fractions of a ~90,000 shRNA pool where the probe was amplified from shRNA plasmid template DNA. (e) Distribution plots of GMAP features data for a dilution series of shRNAs contained within sub fractions of a ~90,000 shRNA pool where the probe was amplified from genomic DNA of A549 cells infected with lentiviral pools. 2x, 5x, 10x, and 20x curves are plotted as the distribution of log 2 difference between the array signal for groups of diluted shRNAs in the 90 k Dilution pool and their signal in the 90 k Reference pool. The 1x curve is plotted as the log 2 difference between a group of undiluted shRNAs in the 90 k Dilution pool and the same shRNAs in the 90 k Reference pool.
Figure Legend Snippet: Comparison of sequencing and GMAP performance with high complexity shRNA pools . (a) Scatter plot of GMAP array signal intensity (X-axis) versus sequencing read number (Y-axis) for shRNA clones from the human Even shRNA pool. (b) Distribution plots of Illumina sequencing data for a dilution series of shRNAs in separate pools. 4x, 16x, and 64x curves are plotted as the distribution of log 2 difference between the number of sequencing reads for shRNAs in the dilution series and their read count in the reference (Even) pool. The 1x curve is plotted as the log 2 difference between a group of undiluted shRNAs in the 4x pool and the same shRNAs in the Even pool. (c) Distribution plots of Illumina sequencing data for a dilution series of shRNAs within the same pool. 2x, 5x, 10x, and 20x curves are plotted as the distribution of log 2 difference between the number of sequencing reads for groups of diluted shRNAs and their read count in the reference (Even) pool. The 1x curve is plotted as the log 2 difference between a group of undiluted shRNAs in the 2x-20x pool and the same shRNAs in the Even pool. (d) Distribution plots of GMAP features data for a dilution series of shRNAs contained within sub fractions of a ~90,000 shRNA pool where the probe was amplified from shRNA plasmid template DNA. (e) Distribution plots of GMAP features data for a dilution series of shRNAs contained within sub fractions of a ~90,000 shRNA pool where the probe was amplified from genomic DNA of A549 cells infected with lentiviral pools. 2x, 5x, 10x, and 20x curves are plotted as the distribution of log 2 difference between the array signal for groups of diluted shRNAs in the 90 k Dilution pool and their signal in the 90 k Reference pool. The 1x curve is plotted as the log 2 difference between a group of undiluted shRNAs in the 90 k Dilution pool and the same shRNAs in the 90 k Reference pool.

Techniques Used: Sequencing, shRNA, Clone Assay, Amplification, Plasmid Preparation, Infection

23) Product Images from "Genome Sequencing Reveals a Phage in Helicobacter pylori"

Article Title: Genome Sequencing Reveals a Phage in Helicobacter pylori

Journal: mBio

doi: 10.1128/mBio.00239-11

Transmission electron microscopy (TEM) images obtained after induction of the B45 prophage. (A) Examples of TEM images obtained using negative staining: numerous phage-like particles with an eggshell structure (size, 100 ± 30 nm). (B) (1) TEM images obtained after fixation embedding and ultrathin sectioning of the bacteria. Two phage particles on the top of the bacterial cell are visualized in the cytoplasm. The dark zone in the lower part of the cell corresponds to the condensed genomic DNA. On each side of the cell, two other bacterial cells are partially visible (one with genomic DNA). Some flagellar remnants can also be seen in between the cells. (2 and 3) Higher magnification focused on these 2 particles showed that they were recognizable by the high-density mature head, with a rounded appearance, to which a tail was attached. The phage head was ~62.5 nm (±7.3 nm) in diameter, and the tail was ~92.4 nm (±2.97 nm) long and 5 to 6 nm in diameter. The total length of the phage was ~150 nm.
Figure Legend Snippet: Transmission electron microscopy (TEM) images obtained after induction of the B45 prophage. (A) Examples of TEM images obtained using negative staining: numerous phage-like particles with an eggshell structure (size, 100 ± 30 nm). (B) (1) TEM images obtained after fixation embedding and ultrathin sectioning of the bacteria. Two phage particles on the top of the bacterial cell are visualized in the cytoplasm. The dark zone in the lower part of the cell corresponds to the condensed genomic DNA. On each side of the cell, two other bacterial cells are partially visible (one with genomic DNA). Some flagellar remnants can also be seen in between the cells. (2 and 3) Higher magnification focused on these 2 particles showed that they were recognizable by the high-density mature head, with a rounded appearance, to which a tail was attached. The phage head was ~62.5 nm (±7.3 nm) in diameter, and the tail was ~92.4 nm (±2.97 nm) long and 5 to 6 nm in diameter. The total length of the phage was ~150 nm.

Techniques Used: Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Negative Staining

PCR detection of free circular phage DNA. To ensure complete elimination of bacterial genomic DNA, the DNA extracted from concentrated phage particles was treated twice with exonucleases (for 4 or 24 h) in order to digest any linear bacterial genomic DNA, leaving the circular DNA (i.e., phage DNA), which cannot be degraded by these enzymes. The extracted DNA was then tested for the presence of phage DNA and bacterial genomic DNA by PCR amplification of the phage integrase gene (using the primers F1, AAGYTTTTTAGMGTTTTGYG , and R1, CGCCCTGGCTTAGCATC , generating a 529-bp amplicon) and the cagA gene (750-bp amplicon) as already described ( 67 , 70 ). Lane M corresponds to the 1-kb DNA ladder (Promega). Lanes 1 and 7, B45 extracted phage DNA plus exonucleases, 24 hours; lanes 2 and 8, B45 extracted phage DNA plus exonuclease buffer only, 24 hours; lanes 3 and 9, B45 extracted phage DNA plus exonucleases, 4 hours; lanes 4 and 10, B45 extracted phage DNA plus exonuclease buffer only, 4 hours; lanes 5 and 11, B45 DNA (positive control); lanes 6 and 12, H 2 O (negative control).
Figure Legend Snippet: PCR detection of free circular phage DNA. To ensure complete elimination of bacterial genomic DNA, the DNA extracted from concentrated phage particles was treated twice with exonucleases (for 4 or 24 h) in order to digest any linear bacterial genomic DNA, leaving the circular DNA (i.e., phage DNA), which cannot be degraded by these enzymes. The extracted DNA was then tested for the presence of phage DNA and bacterial genomic DNA by PCR amplification of the phage integrase gene (using the primers F1, AAGYTTTTTAGMGTTTTGYG , and R1, CGCCCTGGCTTAGCATC , generating a 529-bp amplicon) and the cagA gene (750-bp amplicon) as already described ( 67 , 70 ). Lane M corresponds to the 1-kb DNA ladder (Promega). Lanes 1 and 7, B45 extracted phage DNA plus exonucleases, 24 hours; lanes 2 and 8, B45 extracted phage DNA plus exonuclease buffer only, 24 hours; lanes 3 and 9, B45 extracted phage DNA plus exonucleases, 4 hours; lanes 4 and 10, B45 extracted phage DNA plus exonuclease buffer only, 4 hours; lanes 5 and 11, B45 DNA (positive control); lanes 6 and 12, H 2 O (negative control).

Techniques Used: Polymerase Chain Reaction, Amplification, Positive Control, Negative Control

24) Product Images from "Digital karyotyping reveals probable target genes at 7q21.3 locus in hepatocellular carcinoma"

Article Title: Digital karyotyping reveals probable target genes at 7q21.3 locus in hepatocellular carcinoma

Journal: BMC Medical Genomics

doi: 10.1186/1755-8794-4-60

Real-time quantitative PCR validation of digital karyotyping data . Totally 52 normal individuals and 52 HCC samples were examined for genomic DNA level of (A) SGCE, (B) PEG10, (C) DYNC1I1 and (D) SLC25A13 respectively by real-time quantitative PCR. The purple horizontal line represented an average of genomic DNA level of each group. HCC samples which gained genomic amplification of examined genes were shown by red squares.
Figure Legend Snippet: Real-time quantitative PCR validation of digital karyotyping data . Totally 52 normal individuals and 52 HCC samples were examined for genomic DNA level of (A) SGCE, (B) PEG10, (C) DYNC1I1 and (D) SLC25A13 respectively by real-time quantitative PCR. The purple horizontal line represented an average of genomic DNA level of each group. HCC samples which gained genomic amplification of examined genes were shown by red squares.

Techniques Used: Real-time Polymerase Chain Reaction, Amplification

25) Product Images from "Peperomin E reactivates silenced tumor suppressor genes in lung cancer cells by inhibition of DNA methyltransferase"

Article Title: Peperomin E reactivates silenced tumor suppressor genes in lung cancer cells by inhibition of DNA methyltransferase

Journal: Cancer Science

doi: 10.1111/cas.13029

Effect of peperomin E (PepE) on global DNA methylation in non‐small‐cell lung cancer cells in vitro and in vivo . (a) Effects of PepE and 5‐Aza‐dC on global DNA methylation levels in NSCLC cells. DMSO served as control; and (b) Effects of PepE and 5‐Aza‐dC on the global DNA methylation levels in A549 tumor tissues extracted from nude mice. Data are presented as means ± SD ( n = 3 for a; n = 6 for b). * P
Figure Legend Snippet: Effect of peperomin E (PepE) on global DNA methylation in non‐small‐cell lung cancer cells in vitro and in vivo . (a) Effects of PepE and 5‐Aza‐dC on global DNA methylation levels in NSCLC cells. DMSO served as control; and (b) Effects of PepE and 5‐Aza‐dC on the global DNA methylation levels in A549 tumor tissues extracted from nude mice. Data are presented as means ± SD ( n = 3 for a; n = 6 for b). * P

Techniques Used: DNA Methylation Assay, In Vitro, In Vivo, Mouse Assay

Effects of peperomin E (PepE) on changes of activity and expression of DNA methyltransferase 1 ( DNMT 1). (a) Dose‐response plots of PepE and 5‐Aza‐dC against DNMT. The IC50 concentrations were determined by biochemical DNMT assays under identical conditions; (b) Western‐blot bands for DNMT1 protein expression in A549 cells before and after PepE treatment. The intensity of the bands was quantified by optical density (OD) and normalized to the OD of GAPDH; (c) Representative immunostaining for DNMT1 in the paraffin section (original magnification ×200). 3 slides per mouse were reviewed. The positive rate was quantified by IOD values and normalized to the IOD values of control group; and (d) Western‐blot bands for Sp1 and NF‐kB (p65) proteins expression in A549 cells before and after PepE treatment. The intensity of the bands was quantified by optical density (OD) and normalized to the OD of β‐actin. All data are presented as means ± SD ( n = 3 for a, b, d and e; n = 6 for c). ** P
Figure Legend Snippet: Effects of peperomin E (PepE) on changes of activity and expression of DNA methyltransferase 1 ( DNMT 1). (a) Dose‐response plots of PepE and 5‐Aza‐dC against DNMT. The IC50 concentrations were determined by biochemical DNMT assays under identical conditions; (b) Western‐blot bands for DNMT1 protein expression in A549 cells before and after PepE treatment. The intensity of the bands was quantified by optical density (OD) and normalized to the OD of GAPDH; (c) Representative immunostaining for DNMT1 in the paraffin section (original magnification ×200). 3 slides per mouse were reviewed. The positive rate was quantified by IOD values and normalized to the IOD values of control group; and (d) Western‐blot bands for Sp1 and NF‐kB (p65) proteins expression in A549 cells before and after PepE treatment. The intensity of the bands was quantified by optical density (OD) and normalized to the OD of β‐actin. All data are presented as means ± SD ( n = 3 for a, b, d and e; n = 6 for c). ** P

Techniques Used: Activity Assay, Expressing, Western Blot, Immunostaining, Paraffin Section

Molecular interactions between peperomin E and DNA methyltransferase 1. (a) Structure model of DNMT1 (PDB: 3SWR) with co‐crystallized ligand Sinefugin binding site (yellow color). (b) Superimposition of the best conformation of 5‐Aza‐dC (in red), Sinefugin (in green), S‐adenosylmethionine (SAM, in blue) and PepE (in yellow) docked in the active site pocket of DNMT1 by Discovery Studio 4.0 Libdock protocol. 2‐D diagrams illustrating protein‐ligand interactions of (c) SAM, (d) Sinefugin, (e) 5‐Aza‐dC and (f) PepE.
Figure Legend Snippet: Molecular interactions between peperomin E and DNA methyltransferase 1. (a) Structure model of DNMT1 (PDB: 3SWR) with co‐crystallized ligand Sinefugin binding site (yellow color). (b) Superimposition of the best conformation of 5‐Aza‐dC (in red), Sinefugin (in green), S‐adenosylmethionine (SAM, in blue) and PepE (in yellow) docked in the active site pocket of DNMT1 by Discovery Studio 4.0 Libdock protocol. 2‐D diagrams illustrating protein‐ligand interactions of (c) SAM, (d) Sinefugin, (e) 5‐Aza‐dC and (f) PepE.

Techniques Used: Binding Assay

26) Product Images from "CTCF loss mediates unique DNA hypermethylation landscapes in human cancers"

Article Title: CTCF loss mediates unique DNA hypermethylation landscapes in human cancers

Journal: Clinical Epigenetics

doi: 10.1186/s13148-020-00869-7

Prostate and breast tumors of the TCGA harboring CTCF copy number loss demonstrate hypermethylation events. Alterations exhibit a distinct DNA methylation profile. a Primary prostate tumors from TCGA ( n = 333) segregated by CTCF CN status, boxplots of RNA-Seq for CTCF mRNA demonstrating significantly altered expression in diploid versus deletion cancers ( P
Figure Legend Snippet: Prostate and breast tumors of the TCGA harboring CTCF copy number loss demonstrate hypermethylation events. Alterations exhibit a distinct DNA methylation profile. a Primary prostate tumors from TCGA ( n = 333) segregated by CTCF CN status, boxplots of RNA-Seq for CTCF mRNA demonstrating significantly altered expression in diploid versus deletion cancers ( P

Techniques Used: DNA Methylation Assay, RNA Sequencing Assay, Expressing

27) Product Images from "Reduced Heterochromatin Formation on the pFAR4 Miniplasmid Allows Sustained Transgene Expression in the Mouse Liver"

Article Title: Reduced Heterochromatin Formation on the pFAR4 Miniplasmid Allows Sustained Transgene Expression in the Mouse Liver

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2020.05.014

Comparative Analysis of the CpG Methylation Status of the hAAT Promoter Region Carried by the pFAR4 or the pKAR4 Plasmid Constructs The region upstream of the Sgsh cDNA sequence is represented with CpG dinucleotides (●). Gray rectangles show the location of HNF1α and HNF4 binding sites within the hAAT promoter region. For each plasmid construct, genomic DNA from three infused mice was treated with bisulfite. The represented DNA region was amplified by PCR. After cloning of the amplicons, 21–25 independent sequences were analyzed for each mouse. Data represent the mean of independent methylated sequences as determined by CpG retention after bisulfite treatment for each plasmid. Asterisk (∗) indicates the positions where the methylated CpG appears to be present in a plasmid sequence and absent in the other.
Figure Legend Snippet: Comparative Analysis of the CpG Methylation Status of the hAAT Promoter Region Carried by the pFAR4 or the pKAR4 Plasmid Constructs The region upstream of the Sgsh cDNA sequence is represented with CpG dinucleotides (●). Gray rectangles show the location of HNF1α and HNF4 binding sites within the hAAT promoter region. For each plasmid construct, genomic DNA from three infused mice was treated with bisulfite. The represented DNA region was amplified by PCR. After cloning of the amplicons, 21–25 independent sequences were analyzed for each mouse. Data represent the mean of independent methylated sequences as determined by CpG retention after bisulfite treatment for each plasmid. Asterisk (∗) indicates the positions where the methylated CpG appears to be present in a plasmid sequence and absent in the other.

Techniques Used: CpG Methylation Assay, Plasmid Preparation, Construct, Sequencing, Binding Assay, Mouse Assay, Amplification, Polymerase Chain Reaction, Clone Assay, Methylation

28) Product Images from "Recent evolution of a TET-controlled and DPPA3/STELLA-driven pathway of passive demethylation in mammals"

Article Title: Recent evolution of a TET-controlled and DPPA3/STELLA-driven pathway of passive demethylation in mammals

Journal: bioRxiv

doi: 10.1101/321604

TET1 and TET2 prevent hypermethylation of the naïve genome a , Loss of TET catalytic activity leads to global DNA hypermethylation. Percentage of total 5mC as measured by RRBS. b , Loss of TET catalytic activity leads to widespread DNA hypermethylation especially at repetitive elements. Relative proportion of DNA hypermethylation (q value
Figure Legend Snippet: TET1 and TET2 prevent hypermethylation of the naïve genome a , Loss of TET catalytic activity leads to global DNA hypermethylation. Percentage of total 5mC as measured by RRBS. b , Loss of TET catalytic activity leads to widespread DNA hypermethylation especially at repetitive elements. Relative proportion of DNA hypermethylation (q value

Techniques Used: Activity Assay

DPPA3 acts downstream of TET1 and TET2 to establish and preserve global hypomethylation a , Dppa3 loss results in global hypermethylation. Percentage of total 5mC as measured by RRBS. b , Dppa3 prevents the premature acquisition of a primed methylome. Principal component (PC) analysis of RRBS data from wt, T1CM, T2CM and T12CM ESCs, wt EpiLCs and Dppa3KO ESCs. c , DPPA3 and TET proteins promote demethylation of largely similar targets. Venn Diagrams depicting the overlap of hypermethylated sites among T1CM, T2CM, T12CM, and Dppa3KO ESCs. d , Dppa3 protects mostly repeats from hypermethylation. Fraction of hypermethylated genomic elements classified as TET-specific (only hypermethylated in TET mutant ESCs), DPPA3-specific (only hypermethylated in Dppa3KO ESCs), or common (hypermethylated in TET mutant and Dppa3KO ESCs). e , Gene ontology (GO) terms associated with promoters specifically dependent on TET activity; adjusted p-values calculated using Fisher’s exact test followed by Benjamini-Hochberg correction for multiple testing. f , TET activity remains unaffected in Dppa3KO ESCs. Relative DNA modification levels for 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) as measured by mass spectrometry (LC-MS/MS). Error bars indicate mean ± SD calculated from n > 3 biological replicates. g , Dppa3 expression can rescue the hypermethylation in TET mutant ESCs. DNA methylation levels at LINE-1 elements (%) as measured by bisulfite sequencing 0, 3, or 6 days after doxycycline (dox) induction of Dppa3 expression. In the boxplots in a, g , horizontal black lines within boxes represent median values, boxes indicate the upper and lower quartiles, and whiskers indicate the 1.5 interquartile range. The dashed red line indicates the median methylation level of wt ESCs. In a, f , and g , P-values were calculated using Welch’s two-sided t-test: ** P
Figure Legend Snippet: DPPA3 acts downstream of TET1 and TET2 to establish and preserve global hypomethylation a , Dppa3 loss results in global hypermethylation. Percentage of total 5mC as measured by RRBS. b , Dppa3 prevents the premature acquisition of a primed methylome. Principal component (PC) analysis of RRBS data from wt, T1CM, T2CM and T12CM ESCs, wt EpiLCs and Dppa3KO ESCs. c , DPPA3 and TET proteins promote demethylation of largely similar targets. Venn Diagrams depicting the overlap of hypermethylated sites among T1CM, T2CM, T12CM, and Dppa3KO ESCs. d , Dppa3 protects mostly repeats from hypermethylation. Fraction of hypermethylated genomic elements classified as TET-specific (only hypermethylated in TET mutant ESCs), DPPA3-specific (only hypermethylated in Dppa3KO ESCs), or common (hypermethylated in TET mutant and Dppa3KO ESCs). e , Gene ontology (GO) terms associated with promoters specifically dependent on TET activity; adjusted p-values calculated using Fisher’s exact test followed by Benjamini-Hochberg correction for multiple testing. f , TET activity remains unaffected in Dppa3KO ESCs. Relative DNA modification levels for 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) as measured by mass spectrometry (LC-MS/MS). Error bars indicate mean ± SD calculated from n > 3 biological replicates. g , Dppa3 expression can rescue the hypermethylation in TET mutant ESCs. DNA methylation levels at LINE-1 elements (%) as measured by bisulfite sequencing 0, 3, or 6 days after doxycycline (dox) induction of Dppa3 expression. In the boxplots in a, g , horizontal black lines within boxes represent median values, boxes indicate the upper and lower quartiles, and whiskers indicate the 1.5 interquartile range. The dashed red line indicates the median methylation level of wt ESCs. In a, f , and g , P-values were calculated using Welch’s two-sided t-test: ** P

Techniques Used: Mutagenesis, Activity Assay, Modification, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Expressing, DNA Methylation Assay, Methylation Sequencing, Methylation

29) Product Images from "Precision engineering for PRRSV resistance in pigs: Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function"

Article Title: Precision engineering for PRRSV resistance in pigs: Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006206

Excision of exon 7 results in an SRCR5 CD163 deletion in pigs. A) Representative photos of the male sibling pigs with three different ΔSRCR5 genotypes at 5 months of age. Left, wild type pig 628, middle, heterozygous pig 627, and right, biallelic pig 629. B) Genotyping of pulmonary alveolar macrophages (PAMs). DNA was extracted from PAMs and genotype assessed by PCR across Intron 6 to Exon 8. The unmodified genome PCR is predicted to result in a 900 bp product, whilst exon 7 deletion should result in a 450 bp PCR product. C) RNA phenotype of pulmonary alveolar macrophages. RNA was extracted from PAMs, converted into cDNA using oligo(dT) primer, and analyzed by PCR across Exons 4–9. The unmodified cDNA should result in a 1686 bp product, whilst the exon 7 deletion is expected to yield a 1371 bp product. D) Protein phenotype of CD163 from PAMs. PAM cells were lysed with reducing SDS sample buffer and CD163 expression analyzed by western blot. E) CD163 mRNA levels in PAMs. RNA was extracted from the same number of PAM cells, DNA removed by DNase treatment, and RNA quantified by 1-step RT-qPCR. Expression levels were normalized using β-Actin expression levels and to the highest CD163-expressing animal. Error bars represent SEM, n = 3*2.
Figure Legend Snippet: Excision of exon 7 results in an SRCR5 CD163 deletion in pigs. A) Representative photos of the male sibling pigs with three different ΔSRCR5 genotypes at 5 months of age. Left, wild type pig 628, middle, heterozygous pig 627, and right, biallelic pig 629. B) Genotyping of pulmonary alveolar macrophages (PAMs). DNA was extracted from PAMs and genotype assessed by PCR across Intron 6 to Exon 8. The unmodified genome PCR is predicted to result in a 900 bp product, whilst exon 7 deletion should result in a 450 bp PCR product. C) RNA phenotype of pulmonary alveolar macrophages. RNA was extracted from PAMs, converted into cDNA using oligo(dT) primer, and analyzed by PCR across Exons 4–9. The unmodified cDNA should result in a 1686 bp product, whilst the exon 7 deletion is expected to yield a 1371 bp product. D) Protein phenotype of CD163 from PAMs. PAM cells were lysed with reducing SDS sample buffer and CD163 expression analyzed by western blot. E) CD163 mRNA levels in PAMs. RNA was extracted from the same number of PAM cells, DNA removed by DNase treatment, and RNA quantified by 1-step RT-qPCR. Expression levels were normalized using β-Actin expression levels and to the highest CD163-expressing animal. Error bars represent SEM, n = 3*2.

Techniques Used: Polymerase Chain Reaction, Expressing, Western Blot, Quantitative RT-PCR

30) Product Images from "Regulation of PI-2b Pilus Expression in Hypervirulent Streptococcus agalactiae ST-17 BM110"

Article Title: Regulation of PI-2b Pilus Expression in Hypervirulent Streptococcus agalactiae ST-17 BM110

Journal: PLoS ONE

doi: 10.1371/journal.pone.0169840

Transcriptional start site of the PI-2b operon. (A) Characterization of PI-2b transcription start site in S . agalactiae strains A909 and BM110 by dRNA-seq. The sequence reads corresponding to transcript 5' ends generated after (TAP+) or without (TAP-) TAP treatment or obtained from strand-specific RNA-seq (RNA-seq) were aligned to the genomes of strains A909 and BM110. A significantly higher number of reads under TAP+ conditions as compared with TAP- is indicative of 5' -triphosphate ends of transcripts characteristic of transcription start sites (TSS). Identical TSS upstream PI-2b locus are detected in strains A909 and BM110. In addition, coverage of the intergenic regions between sak1445 (or san1522) and orf under conditions of RNA-seq experiments reveals a transcriptional read-through originating from sak1445 , in A909 only, that could participate to the global level of PI-2b transcription in A909. (B) Primer extension analysis of the PI-2b mRNA. Primer elongation product obtained with oligonucleotide E3 and 15 μg of total RNA from A909 (lane1) or BM110 (lane 2). Lanes T, G, C, A, results of sequencing reactions performed with primer E3. Arrow indicated the transcriptional start site. (C) Schematic representation and genomic DNA sequence, from nucleotide positions -281 to +3 (numbering from the A of the ATG start codon of orf , negative in the -3'-to-5' direction and positive in the 5' to-3' direction) of the region upstream from the PI-2b locus. Transcription start site is indicated in boldface and arrow. Consensus -10 and -35 sequences are indicated by grey boxes. The 43-bp sequence, present in BM110 and absent in A909, is underlined. The ATG start codon of orf is indicated in boldface.
Figure Legend Snippet: Transcriptional start site of the PI-2b operon. (A) Characterization of PI-2b transcription start site in S . agalactiae strains A909 and BM110 by dRNA-seq. The sequence reads corresponding to transcript 5' ends generated after (TAP+) or without (TAP-) TAP treatment or obtained from strand-specific RNA-seq (RNA-seq) were aligned to the genomes of strains A909 and BM110. A significantly higher number of reads under TAP+ conditions as compared with TAP- is indicative of 5' -triphosphate ends of transcripts characteristic of transcription start sites (TSS). Identical TSS upstream PI-2b locus are detected in strains A909 and BM110. In addition, coverage of the intergenic regions between sak1445 (or san1522) and orf under conditions of RNA-seq experiments reveals a transcriptional read-through originating from sak1445 , in A909 only, that could participate to the global level of PI-2b transcription in A909. (B) Primer extension analysis of the PI-2b mRNA. Primer elongation product obtained with oligonucleotide E3 and 15 μg of total RNA from A909 (lane1) or BM110 (lane 2). Lanes T, G, C, A, results of sequencing reactions performed with primer E3. Arrow indicated the transcriptional start site. (C) Schematic representation and genomic DNA sequence, from nucleotide positions -281 to +3 (numbering from the A of the ATG start codon of orf , negative in the -3'-to-5' direction and positive in the 5' to-3' direction) of the region upstream from the PI-2b locus. Transcription start site is indicated in boldface and arrow. Consensus -10 and -35 sequences are indicated by grey boxes. The 43-bp sequence, present in BM110 and absent in A909, is underlined. The ATG start codon of orf is indicated in boldface.

Techniques Used: Sequencing, Generated, RNA Sequencing Assay

31) Product Images from "Plasmid-Based Generation of Induced Neural Stem Cells from Adult Human Fibroblasts"

Article Title: Plasmid-Based Generation of Induced Neural Stem Cells from Adult Human Fibroblasts

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2016.00245

Evaluation of genomic stability and continuous expression of reprogramming factors: (a) Normal karyogram of an iNSC line. (b) Copy numbers of plasmid per genome as quantified by qPCR at day 5 after transfection and under proliferative conditions (two independent experiments with three cell lines, two technical replicates, error bars correspond to standard deviation). (c) Gel electrophoresis of a PCR with a primer pair specific for the endogenous SOX2 sequence (left): A reaction product is only present for the cDNA derived from iNSC lines, not from fibroblasts or when using the reprogramming plasmid carrying the SOX2 sequence. Equal levels of b-Actin for the cell lines are demonstrated on the right. Supercoiled plamid DNA is visible at the top (asterisk). (d) qPCR analysis of residual reprogramming factor expression in relation to the house keeping gene beta-actin (bAct) under proliferation of iNSCs (means of three independent iNSC lines, two technical replicates, error bars correspond to standard deviation).
Figure Legend Snippet: Evaluation of genomic stability and continuous expression of reprogramming factors: (a) Normal karyogram of an iNSC line. (b) Copy numbers of plasmid per genome as quantified by qPCR at day 5 after transfection and under proliferative conditions (two independent experiments with three cell lines, two technical replicates, error bars correspond to standard deviation). (c) Gel electrophoresis of a PCR with a primer pair specific for the endogenous SOX2 sequence (left): A reaction product is only present for the cDNA derived from iNSC lines, not from fibroblasts or when using the reprogramming plasmid carrying the SOX2 sequence. Equal levels of b-Actin for the cell lines are demonstrated on the right. Supercoiled plamid DNA is visible at the top (asterisk). (d) qPCR analysis of residual reprogramming factor expression in relation to the house keeping gene beta-actin (bAct) under proliferation of iNSCs (means of three independent iNSC lines, two technical replicates, error bars correspond to standard deviation).

Techniques Used: Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction, Transfection, Standard Deviation, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Sequencing, Derivative Assay

32) Product Images from "DNA methylation of the RUNX2 P1 promoter mediates MMP13 transcription in chondrocytes"

Article Title: DNA methylation of the RUNX2 P1 promoter mediates MMP13 transcription in chondrocytes

Journal: Scientific Reports

doi: 10.1038/s41598-017-08418-8

Long-term exposure to 5-aza-dC enhances RUNX2 gene expression associated with DNA demethylation at the −336-bp CpG site. ( A ) Relative mRNA levels of RUNX2 were analysed by qRT-PCR and normalized against GAPDH in untreated (control) and 5-aza-dC-treated cultures. ( B ) Percentage methylation of indicated CpG sites in the RUNX2 P1 promoter was analysed using bisulfite pyrosequencing in the same samples. ( C ) The relative mRNA levels of MMP13 were analyzed in the same samples. Values represent mean ± SD of 6 independent experiments. *P
Figure Legend Snippet: Long-term exposure to 5-aza-dC enhances RUNX2 gene expression associated with DNA demethylation at the −336-bp CpG site. ( A ) Relative mRNA levels of RUNX2 were analysed by qRT-PCR and normalized against GAPDH in untreated (control) and 5-aza-dC-treated cultures. ( B ) Percentage methylation of indicated CpG sites in the RUNX2 P1 promoter was analysed using bisulfite pyrosequencing in the same samples. ( C ) The relative mRNA levels of MMP13 were analyzed in the same samples. Values represent mean ± SD of 6 independent experiments. *P

Techniques Used: Expressing, Quantitative RT-PCR, Methylation

Higher levels of RUNX2 mRNA in deep zone NOF and OA chondrocytes than in superficial zone NOF chondrocytes are associated with the methylation status of specific CpG sites in the RUNX2 P1 promoter. ( A ) Relative mRNA levels of RUNX2 in the superficial (NOF-s) and deep zone (NOF-d) of NOF cartilage and OA cartilage were analysed by qRT-PCR and normalized against GAPDH. ( B ) Percentage methylation of the indicated CpG sites in the RUNX2 P1 promoter were analysed in genomic DNA simultaneously extracted from the same subjects by bisulfite pyrosequencing. The y-axis indicates non-adjusted percentage methylation. ( C ) RUNX2 mRNA levels in OA chondrocytes are plotted against the ages of subjects. Values are the mean ± SD. *P
Figure Legend Snippet: Higher levels of RUNX2 mRNA in deep zone NOF and OA chondrocytes than in superficial zone NOF chondrocytes are associated with the methylation status of specific CpG sites in the RUNX2 P1 promoter. ( A ) Relative mRNA levels of RUNX2 in the superficial (NOF-s) and deep zone (NOF-d) of NOF cartilage and OA cartilage were analysed by qRT-PCR and normalized against GAPDH. ( B ) Percentage methylation of the indicated CpG sites in the RUNX2 P1 promoter were analysed in genomic DNA simultaneously extracted from the same subjects by bisulfite pyrosequencing. The y-axis indicates non-adjusted percentage methylation. ( C ) RUNX2 mRNA levels in OA chondrocytes are plotted against the ages of subjects. Values are the mean ± SD. *P

Techniques Used: Methylation, Quantitative RT-PCR

MMP13 is highly expressed in OA chondrocytes with accompanying demethylation in the CpG sites of the MMP13 proximal promoter and is associated with RUNX2 but not OSX gene expression or MMP13 DNA methylation status. Non-cultured primary human chondrocytes were isolated from cartilage obtained from patients with femoral neck fracture (NOF) and OA patients. ( A ) Relative MMP13 mRNA levels were analysed separately in chondrocytes from the superficial (NOF-s) and deep zones (NOF-d) of NOF cartilage by qRT-PCR and normalized against GAPDH. ( B ) Percentage methylation of each indicated CpG site in the MMP13 proximal promoter was analysed in the same samples by bisulfite pyrosequencing. The y-axis shows non-adjusted percentage methylation. ( C – F ) MMP13 gene expression in OA chondrocytes was compared with the levels of RUNX2 mRNA ( C ) or OSX mRNA ( D ), and with the methylation status of the −14 bp ( E ) or −110 bp CpG site in the MMP13 proximal promoter ( E , F ). Values are the mean ± SD. *P
Figure Legend Snippet: MMP13 is highly expressed in OA chondrocytes with accompanying demethylation in the CpG sites of the MMP13 proximal promoter and is associated with RUNX2 but not OSX gene expression or MMP13 DNA methylation status. Non-cultured primary human chondrocytes were isolated from cartilage obtained from patients with femoral neck fracture (NOF) and OA patients. ( A ) Relative MMP13 mRNA levels were analysed separately in chondrocytes from the superficial (NOF-s) and deep zones (NOF-d) of NOF cartilage by qRT-PCR and normalized against GAPDH. ( B ) Percentage methylation of each indicated CpG site in the MMP13 proximal promoter was analysed in the same samples by bisulfite pyrosequencing. The y-axis shows non-adjusted percentage methylation. ( C – F ) MMP13 gene expression in OA chondrocytes was compared with the levels of RUNX2 mRNA ( C ) or OSX mRNA ( D ), and with the methylation status of the −14 bp ( E ) or −110 bp CpG site in the MMP13 proximal promoter ( E , F ). Values are the mean ± SD. *P

Techniques Used: Expressing, DNA Methylation Assay, Cell Culture, Isolation, Quantitative RT-PCR, Methylation

33) Product Images from "Presynaptic LRP4 promotes synapse number and function of excitatory CNS neurons"

Article Title: Presynaptic LRP4 promotes synapse number and function of excitatory CNS neurons

Journal: eLife

doi: 10.7554/eLife.27347

LRP4 reagents and patterns of LRP4 expression. ( A ) Genomic region of lrp4 . Top bar represents physical position on the X chromosome (in base pairs), and the blue arrow represents the lrp4 genomic region flanked by other genes (yellow). Primer sets are indicated by forward and reverse arrows (see B). The exon structure is displayed with 5’ and 3’ UTRs shaded in gray and coding exons numbered and shaded in beige. The region deleted by the lrp4 dalek mutation is indicated in pink. RNAi targets are shown below in orange. The position of the GAL4 in the GMR90B08-GAL4 line is shown below and region of the protein against which antibodies were raised are noted below. ( B ) PCR analysis of genomic DNA from control and lrp4 dalek adults. The presence of bands corresponding to Exon 2 and Exon 7–8 in control and heterozygous flies and their absence in lrp4 dalek demonstrate loss of the coding region. The presence of a 315 bp band in heterozygous and homozygous lrp4 dalek flies (Flank) but not in control is a result of non-homologous end joining of the 5’ and 3’ UTRs following deletion of the gene. ( C ) Representative confocal maximum intensity projections of the antennal lobe region of an lrp4-GAL4 animal expressing HA-tagged LRP4 and stained with antibodies to HA ( C , C’’ , green) and N-Cadherin ( C’ - C’’ , magenta). LRP4-HA localizes to regions of synaptic neuropil, similar to endogenous staining ( Figure 1 ). ( D–E ) Representative confocal maximum intensity projections of antennal lobes in animals expressing UAS-FRT-Stop-FRT-mCD8-GFP using lrp4-GAL4 but where FLP expression (removing the stop codon) is restricted to either ORNs using eyFLP ( D ) or PNs using GH146-FLP ( E ) and stained with antibodies to GFP (green) and N-Cadherin (magenta). Intersectional analysis reveals lrp4 expression in both ORNs as well as PNs. Scale bars = 10 µm ( C ), 5 μm ( D–E ). DOI: http://dx.doi.org/10.7554/eLife.27347.005
Figure Legend Snippet: LRP4 reagents and patterns of LRP4 expression. ( A ) Genomic region of lrp4 . Top bar represents physical position on the X chromosome (in base pairs), and the blue arrow represents the lrp4 genomic region flanked by other genes (yellow). Primer sets are indicated by forward and reverse arrows (see B). The exon structure is displayed with 5’ and 3’ UTRs shaded in gray and coding exons numbered and shaded in beige. The region deleted by the lrp4 dalek mutation is indicated in pink. RNAi targets are shown below in orange. The position of the GAL4 in the GMR90B08-GAL4 line is shown below and region of the protein against which antibodies were raised are noted below. ( B ) PCR analysis of genomic DNA from control and lrp4 dalek adults. The presence of bands corresponding to Exon 2 and Exon 7–8 in control and heterozygous flies and their absence in lrp4 dalek demonstrate loss of the coding region. The presence of a 315 bp band in heterozygous and homozygous lrp4 dalek flies (Flank) but not in control is a result of non-homologous end joining of the 5’ and 3’ UTRs following deletion of the gene. ( C ) Representative confocal maximum intensity projections of the antennal lobe region of an lrp4-GAL4 animal expressing HA-tagged LRP4 and stained with antibodies to HA ( C , C’’ , green) and N-Cadherin ( C’ - C’’ , magenta). LRP4-HA localizes to regions of synaptic neuropil, similar to endogenous staining ( Figure 1 ). ( D–E ) Representative confocal maximum intensity projections of antennal lobes in animals expressing UAS-FRT-Stop-FRT-mCD8-GFP using lrp4-GAL4 but where FLP expression (removing the stop codon) is restricted to either ORNs using eyFLP ( D ) or PNs using GH146-FLP ( E ) and stained with antibodies to GFP (green) and N-Cadherin (magenta). Intersectional analysis reveals lrp4 expression in both ORNs as well as PNs. Scale bars = 10 µm ( C ), 5 μm ( D–E ). DOI: http://dx.doi.org/10.7554/eLife.27347.005

Techniques Used: Expressing, Mutagenesis, Polymerase Chain Reaction, Non-Homologous End Joining, Staining

34) Product Images from "MeCP2 regulates Tet1-catalyzed demethylation, CTCF binding, and learning-dependent alternative splicing of the BDNF gene in Turtle"

Article Title: MeCP2 regulates Tet1-catalyzed demethylation, CTCF binding, and learning-dependent alternative splicing of the BDNF gene in Turtle

Journal: eLife

doi: 10.7554/eLife.25384

MeCP2 siRNA inhibits demethylation of the tBDNF coding sequence during conditioning. ( A ) BSP analysis of the coding sequence in naïve, conditioned preparations, and those treated with MeCP2 siRNA. Specific CG sites demethylated in normal conditioning show significantly greater levels of methylation after conditioning in MeCP2 siRNA. Data from normal naïve and conditioned are from Figure 2 and are shown here for comparison. MeCP2 siRNA data are from 3 × 10 clones/group. *Significant differences (p=0.01) between the C 15 min MeCP2 siRNA treated group compared to normal C 15 min . ( B ) Grouped data of CG sites 2–11 (left panel) and sites 2–4 (right panel) show significant attenuation of demethylation in conditioned preparations treated with MeCP2 siRNA compared to normal conditioning. ( C ) Schematic illustration of the putative stepwise oxidative demethylation pathway performed by Tet proteins and the thymine DNA glycosylase (TDG) and base excision repair system. Normal conditioning drives the process to the right toward unmethylated C, whereas treatment of naïve or conditioned preparations with MeCP2 siRNA that results in a reduction of Tet1 binding to tBDNF shifts the pathway to the left toward 5mC. 5fC, 5-formylcytosine; 5caC, 5-carboxylcytosine. ( D ) MeDIP assays show high levels of 5hmC content in the tBDNF coding sequence of naïve preparations. These levels are significantly reduced after normal conditioning and in naïve preparations treated with MeCP2 siRNA. DOI: http://dx.doi.org/10.7554/eLife.25384.007
Figure Legend Snippet: MeCP2 siRNA inhibits demethylation of the tBDNF coding sequence during conditioning. ( A ) BSP analysis of the coding sequence in naïve, conditioned preparations, and those treated with MeCP2 siRNA. Specific CG sites demethylated in normal conditioning show significantly greater levels of methylation after conditioning in MeCP2 siRNA. Data from normal naïve and conditioned are from Figure 2 and are shown here for comparison. MeCP2 siRNA data are from 3 × 10 clones/group. *Significant differences (p=0.01) between the C 15 min MeCP2 siRNA treated group compared to normal C 15 min . ( B ) Grouped data of CG sites 2–11 (left panel) and sites 2–4 (right panel) show significant attenuation of demethylation in conditioned preparations treated with MeCP2 siRNA compared to normal conditioning. ( C ) Schematic illustration of the putative stepwise oxidative demethylation pathway performed by Tet proteins and the thymine DNA glycosylase (TDG) and base excision repair system. Normal conditioning drives the process to the right toward unmethylated C, whereas treatment of naïve or conditioned preparations with MeCP2 siRNA that results in a reduction of Tet1 binding to tBDNF shifts the pathway to the left toward 5mC. 5fC, 5-formylcytosine; 5caC, 5-carboxylcytosine. ( D ) MeDIP assays show high levels of 5hmC content in the tBDNF coding sequence of naïve preparations. These levels are significantly reduced after normal conditioning and in naïve preparations treated with MeCP2 siRNA. DOI: http://dx.doi.org/10.7554/eLife.25384.007

Techniques Used: Sequencing, Methylation, Clone Assay, Binding Assay, Methylated DNA Immunoprecipitation

Conditioning-dependent alternative splicing of tBDNF is mediated by MeCP2. ( A ) Schematic diagram of tBDNF mRNA transcripts generated from non-coding exon II and the protein coding exon IV. Four transcripts designated tBDNF2a-d are produced in naïve preparations but only the tBDNF2a transcript undergoes an intraexonic splicing event in which 40 bp (13 amino acids) are removed from the distal region of the coding sequence (shown by the hatching at nt 607–646). ( B ) The amino acid sequence of the distal end of the protein coding sequence is shown for the full-length tBDNF transcripts. The deletion of the 13 amino acids in tBDNF2a (aa 183–195) is also shown which results in a frame shift and alternative C-terminal end with an early stop codon that generates the truncated tBDNF protein. Complete sequences of the tBDNF and tBDNF2a preproBDNF proteins are shown in Ambigapathy et al. (2013) . ( C ) The region of exon IV that undergoes the splicing event was further analyzed using primers flanking the splice site. The PCR products generated from genomic DNA produced a single band at 225 bp while cDNA produced two bands at 225 bp and 185 bp. Sequencing showed that the larger PCR band from cDNA was identical to tBDNF2b-d , while the smaller band was tBDNF2a (accession numbers: KC151267 – KC151270). ( D ) Four tBDNF exon II transcripts 2a-d are expressed in naïve ( N ) preparations. After 15 min of conditioning ( C 15 ), all transcripts are downregulated but only the 2a transcript is nearly completely suppressed. Application of a MeCP2 siRNA (200 nM, 24 hr) to naïve preparations inhibits tBDNF2a expression ( arrow ) while a control siRNA (Ctrl siRNA; 200 nM, 24 hr) does not. ( E ) Semi-quantitative data of tBDNF2a mRNA expression in the different experimental conditions is shown relative to naive. The tBDNF2a transcript is significantly reduced during conditioning and after treatment with MeCP2 siRNA. ( F ) Western blots confirm that the MeCP2 siRNA significantly inhibits total MeCP2 protein compared to normal naïve. ( G ) Expression of the remaining exon II transcripts, tBDNF2b-d , is not inhibited by application of MeCP2 siRNA. For this and all figures, p and n values are given in the text. DOI: http://dx.doi.org/10.7554/eLife.25384.002
Figure Legend Snippet: Conditioning-dependent alternative splicing of tBDNF is mediated by MeCP2. ( A ) Schematic diagram of tBDNF mRNA transcripts generated from non-coding exon II and the protein coding exon IV. Four transcripts designated tBDNF2a-d are produced in naïve preparations but only the tBDNF2a transcript undergoes an intraexonic splicing event in which 40 bp (13 amino acids) are removed from the distal region of the coding sequence (shown by the hatching at nt 607–646). ( B ) The amino acid sequence of the distal end of the protein coding sequence is shown for the full-length tBDNF transcripts. The deletion of the 13 amino acids in tBDNF2a (aa 183–195) is also shown which results in a frame shift and alternative C-terminal end with an early stop codon that generates the truncated tBDNF protein. Complete sequences of the tBDNF and tBDNF2a preproBDNF proteins are shown in Ambigapathy et al. (2013) . ( C ) The region of exon IV that undergoes the splicing event was further analyzed using primers flanking the splice site. The PCR products generated from genomic DNA produced a single band at 225 bp while cDNA produced two bands at 225 bp and 185 bp. Sequencing showed that the larger PCR band from cDNA was identical to tBDNF2b-d , while the smaller band was tBDNF2a (accession numbers: KC151267 – KC151270). ( D ) Four tBDNF exon II transcripts 2a-d are expressed in naïve ( N ) preparations. After 15 min of conditioning ( C 15 ), all transcripts are downregulated but only the 2a transcript is nearly completely suppressed. Application of a MeCP2 siRNA (200 nM, 24 hr) to naïve preparations inhibits tBDNF2a expression ( arrow ) while a control siRNA (Ctrl siRNA; 200 nM, 24 hr) does not. ( E ) Semi-quantitative data of tBDNF2a mRNA expression in the different experimental conditions is shown relative to naive. The tBDNF2a transcript is significantly reduced during conditioning and after treatment with MeCP2 siRNA. ( F ) Western blots confirm that the MeCP2 siRNA significantly inhibits total MeCP2 protein compared to normal naïve. ( G ) Expression of the remaining exon II transcripts, tBDNF2b-d , is not inhibited by application of MeCP2 siRNA. For this and all figures, p and n values are given in the text. DOI: http://dx.doi.org/10.7554/eLife.25384.002

Techniques Used: Generated, Produced, Sequencing, Polymerase Chain Reaction, Expressing, Western Blot

35) Product Images from "In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni"

Article Title: In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni

Journal: Nature Communications

doi: 10.1038/ncomms14500

Optimization of sgRNA length for CjCas9. ( a ) sgRNAs with variable lengths (19 to 23 nucleotide complementary with a target DNA sequence) were designed and transfected with CjCas9 plasmid into human HEK 293 cells. Genomic DNA was isolated 48 h after transfection. Indel frequencies were analysed by targeted deep sequencing. The first guanine nucleotide at the 5' end that does not match the target sequence is shown in lower case. PAM motifs are shown in red. Error bars indicate s.e.m. ( n =3). ( b ) Mutation frequencies at target sites in the mouse genome. Rosa26 and Tp53 -specific gX 22 guide RNAs were designed and transfected into mouse NIH 3T3 cells together with CjCas9 plasmid. Genome editing efficiencies were examined by deep sequencing using genome DNA isolated from cells after 48 h of transfection. PAM motifs are shown in red. Error bars indicate s.e.m. ( n =3). See also Supplementary Fig. 2 . ( c ) CjCas9-mediated genome editing at the human AAVS1 locus with different PAM sequences. sgRNAs targeting sites with a 5'-NNNNACAC-3' PAM (green; 12 sgRNAs), 5′-NNNNATAC-3′ PAM (light blue; 7 sgRNAs), 5′-NNNNGCAC-3′ PAM (dark blue; 10 sgRNAs), and 5′-NNNNGTAC-3′ PAM (yellow; 8 sgRNAs) were designed and their activities examined in HEK293 cells with deep sequencing. Error bars indicate s.e.m. ( n =3).
Figure Legend Snippet: Optimization of sgRNA length for CjCas9. ( a ) sgRNAs with variable lengths (19 to 23 nucleotide complementary with a target DNA sequence) were designed and transfected with CjCas9 plasmid into human HEK 293 cells. Genomic DNA was isolated 48 h after transfection. Indel frequencies were analysed by targeted deep sequencing. The first guanine nucleotide at the 5' end that does not match the target sequence is shown in lower case. PAM motifs are shown in red. Error bars indicate s.e.m. ( n =3). ( b ) Mutation frequencies at target sites in the mouse genome. Rosa26 and Tp53 -specific gX 22 guide RNAs were designed and transfected into mouse NIH 3T3 cells together with CjCas9 plasmid. Genome editing efficiencies were examined by deep sequencing using genome DNA isolated from cells after 48 h of transfection. PAM motifs are shown in red. Error bars indicate s.e.m. ( n =3). See also Supplementary Fig. 2 . ( c ) CjCas9-mediated genome editing at the human AAVS1 locus with different PAM sequences. sgRNAs targeting sites with a 5'-NNNNACAC-3' PAM (green; 12 sgRNAs), 5′-NNNNATAC-3′ PAM (light blue; 7 sgRNAs), 5′-NNNNGCAC-3′ PAM (dark blue; 10 sgRNAs), and 5′-NNNNGTAC-3′ PAM (yellow; 8 sgRNAs) were designed and their activities examined in HEK293 cells with deep sequencing. Error bars indicate s.e.m. ( n =3).

Techniques Used: Sequencing, Transfection, Plasmid Preparation, Isolation, Mutagenesis

36) Product Images from "Comprehensive evaluation of genome-wide 5-hydroxymethylcytosine profiling approaches in human DNA"

Article Title: Comprehensive evaluation of genome-wide 5-hydroxymethylcytosine profiling approaches in human DNA

Journal: Epigenetics & Chromatin

doi: 10.1186/s13072-017-0123-7

hMeDIP-seq hydroxymethylation profiling in cell line DNA. a Scatter plot showing the correlation between hMeDIP-seq replicates in LNCaP cells. For each genomic tile from one replicate the average enrichment score for the second replicate was calculated. Each dot represents one genomic tile. b Scatter plot showing the correlation between hMeDIP-seq in LNCaP cells and public hMeDIP-seq in MCF7 cells. c Scatter plot showing the correlation between hMeDIP-seq in LNCaP cells and brain hMeDIP-seq. d Genomic region showing the correspondence of hMeDIP-seq replicates in LNCaP cells as well as MCF7 cells. e Density plot showing the distribution of p Bis − p OxBis values in the Brain versus LNCaP WG Bis/OxBis data. f The percentages of significantly hydroxymethylated CpGs of all CpGs with at least 10× coverage on the WG Bis/OxBis in the Brain versus LNCaP. g Density plot showing the distribution of p Bis − p OxBis values in the Brain versus LNCaP HM450K Bis/OxBis data. h The percentages of significantly hydroxymethylated CpGs of all CpGs with at least 10× coverage on the HM450K Bis/OxBis in the Brain versus LNCaP
Figure Legend Snippet: hMeDIP-seq hydroxymethylation profiling in cell line DNA. a Scatter plot showing the correlation between hMeDIP-seq replicates in LNCaP cells. For each genomic tile from one replicate the average enrichment score for the second replicate was calculated. Each dot represents one genomic tile. b Scatter plot showing the correlation between hMeDIP-seq in LNCaP cells and public hMeDIP-seq in MCF7 cells. c Scatter plot showing the correlation between hMeDIP-seq in LNCaP cells and brain hMeDIP-seq. d Genomic region showing the correspondence of hMeDIP-seq replicates in LNCaP cells as well as MCF7 cells. e Density plot showing the distribution of p Bis − p OxBis values in the Brain versus LNCaP WG Bis/OxBis data. f The percentages of significantly hydroxymethylated CpGs of all CpGs with at least 10× coverage on the WG Bis/OxBis in the Brain versus LNCaP. g Density plot showing the distribution of p Bis − p OxBis values in the Brain versus LNCaP HM450K Bis/OxBis data. h The percentages of significantly hydroxymethylated CpGs of all CpGs with at least 10× coverage on the HM450K Bis/OxBis in the Brain versus LNCaP

Techniques Used:

37) Product Images from "The proline–arginine repeat protein linked to C9-ALS/FTD causes neuronal toxicity by inhibiting the DEAD-box RNA helicase-mediated ribosome biogenesis"

Article Title: The proline–arginine repeat protein linked to C9-ALS/FTD causes neuronal toxicity by inhibiting the DEAD-box RNA helicase-mediated ribosome biogenesis

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-1028-5

PR100 down-regulates the expression of ribosomal RNAs. a , b NSC-34 cells were transfected with the empty vector or the FLAG-PR100-encoding vector. At 48 h after the transfection, the cell lysates were immunoprecipitated (IP) with normal mouse IgG (Cont.) or FLAG antibody. Precipitates were then used for RNA immunoprecipitation (RIP) assay ( a ) and dot blotting analysis using FLAG antibody ( b ). RT (−) was used as negative control to monitor the PCR amplification from genomic DNA. Priming sites of primers are shown in Fig. S 1a . c NSC-34 cells were infected with adenovirus encoding LacZ or FLAG-PR100 at an MOI of 800 together with adenovirus encoding Cre-recombinase at an MOI of 40. At 48 h after the infection, quantitative real time PCR analysis of 45S pre-rRNA, 18S rRNA, and 28S rRNA was performed. Means ± SD, N = 3 biological replicates. Statistical analysis was determined by unpaired t test
Figure Legend Snippet: PR100 down-regulates the expression of ribosomal RNAs. a , b NSC-34 cells were transfected with the empty vector or the FLAG-PR100-encoding vector. At 48 h after the transfection, the cell lysates were immunoprecipitated (IP) with normal mouse IgG (Cont.) or FLAG antibody. Precipitates were then used for RNA immunoprecipitation (RIP) assay ( a ) and dot blotting analysis using FLAG antibody ( b ). RT (−) was used as negative control to monitor the PCR amplification from genomic DNA. Priming sites of primers are shown in Fig. S 1a . c NSC-34 cells were infected with adenovirus encoding LacZ or FLAG-PR100 at an MOI of 800 together with adenovirus encoding Cre-recombinase at an MOI of 40. At 48 h after the infection, quantitative real time PCR analysis of 45S pre-rRNA, 18S rRNA, and 28S rRNA was performed. Means ± SD, N = 3 biological replicates. Statistical analysis was determined by unpaired t test

Techniques Used: Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Negative Control, Polymerase Chain Reaction, Amplification, Infection, Real-time Polymerase Chain Reaction

38) Product Images from "Comparison of Conventional, Nested, and Real-Time PCR Assays for Rapid and Accurate Detection of Vibrio vulnificus ▿"

Article Title: Comparison of Conventional, Nested, and Real-Time PCR Assays for Rapid and Accurate Detection of Vibrio vulnificus ▿

Journal:

doi: 10.1128/JCM.00027-08

Sensitivities of C-PCR (A), first-round N-PCR with external primers Tox-100 and Tox-303 (B), and second-round N-PCR with internal primers Tox-130 and Tox-200 (C) to detect V. vulnificus in plasmid DNA. Lanes: 1, 100-bp ladder marker (Bioneer); 2, negative
Figure Legend Snippet: Sensitivities of C-PCR (A), first-round N-PCR with external primers Tox-100 and Tox-303 (B), and second-round N-PCR with internal primers Tox-130 and Tox-200 (C) to detect V. vulnificus in plasmid DNA. Lanes: 1, 100-bp ladder marker (Bioneer); 2, negative

Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Marker

Standard curves (5 × 10 8 to 5 × 10 0 copies/μl) from the Q-PCR assay. Plasmid DNA was used as the template. Circled numerals: 1, negative control (sterile distilled water); 2 to 10, plasmid DNA serially diluted from 5 ×
Figure Legend Snippet: Standard curves (5 × 10 8 to 5 × 10 0 copies/μl) from the Q-PCR assay. Plasmid DNA was used as the template. Circled numerals: 1, negative control (sterile distilled water); 2 to 10, plasmid DNA serially diluted from 5 ×

Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Negative Control

39) Product Images from "Allele-specific gene editing prevents deafness in a model of dominant progressive hearing loss"

Article Title: Allele-specific gene editing prevents deafness in a model of dominant progressive hearing loss

Journal: Nature medicine

doi: 10.1038/s41591-019-0500-9

Single nucleotide substitutions after Cas9+gRNA treatment. Cells were transfected on two different occasions (SpCas9 only and SpCas9 + gRNA 1.1 on four occasions) and genomic DNA from two independent biological samples on each transfection day were pooled for sequencing. Substitutions are given as percentages (i.e. normalized to total read counts) or non-normalized values (i.e. the number of reads with substitutions). Analysis was performed on non-segregated .fastq files in Tmc1 Bth/WT cells and in Tmc1 WT/WT cells (top) and segregated .fastq files in Tmc1 Bth/WT cells (bottom). Experimental conditions are the same as in . Substitutions were not frequent (0.1-0.5% of reads) and there was no difference between untreated and treated samples in the percentage or number of reads with single nucleotide substitutions. Fig. 1b
Figure Legend Snippet: Single nucleotide substitutions after Cas9+gRNA treatment. Cells were transfected on two different occasions (SpCas9 only and SpCas9 + gRNA 1.1 on four occasions) and genomic DNA from two independent biological samples on each transfection day were pooled for sequencing. Substitutions are given as percentages (i.e. normalized to total read counts) or non-normalized values (i.e. the number of reads with substitutions). Analysis was performed on non-segregated .fastq files in Tmc1 Bth/WT cells and in Tmc1 WT/WT cells (top) and segregated .fastq files in Tmc1 Bth/WT cells (bottom). Experimental conditions are the same as in . Substitutions were not frequent (0.1-0.5% of reads) and there was no difference between untreated and treated samples in the percentage or number of reads with single nucleotide substitutions. Fig. 1b

Techniques Used: Transfection, Sequencing

Number of indels based on targeted deep sequencing data from Tmc1 Bth/WT and Tmc1 WT/WT cell lines treated with different Cas9+gRNA combinations (from ). Note, that data points show non-normalized read counts. Cells were transfected on two different occasions (SpCas9 only and SpCas9 + gRNA 1.1 on four occasions) and genomic DNA from two independent biological samples on each transfection day were pooled for sequencing. Indels in the SaCas9-KKH treated Tmc1 WT/WT lines are not different from the background (i.e. untreated samples). This method revealed high sensitivity, as the indel rates in CRISPR treated samples were 40-160-fold higher than the background indel rates observed in untreated samples. Fig. 1b
Figure Legend Snippet: Number of indels based on targeted deep sequencing data from Tmc1 Bth/WT and Tmc1 WT/WT cell lines treated with different Cas9+gRNA combinations (from ). Note, that data points show non-normalized read counts. Cells were transfected on two different occasions (SpCas9 only and SpCas9 + gRNA 1.1 on four occasions) and genomic DNA from two independent biological samples on each transfection day were pooled for sequencing. Indels in the SaCas9-KKH treated Tmc1 WT/WT lines are not different from the background (i.e. untreated samples). This method revealed high sensitivity, as the indel rates in CRISPR treated samples were 40-160-fold higher than the background indel rates observed in untreated samples. Fig. 1b

Techniques Used: Sequencing, Transfection, CRISPR

Targeting Tmc1 Bth with SpCas9. ( a . Plasmids encoding SpCas9-2A-GFP, along with the different gRNAs, were transfected into fibroblasts. Four days after transfection, GFP-positive cells were sorted by FACS ( b ) Sanger sequencing traces from Tmc1 Bth/WT or Tmc1 WT/WT mouse fibroblasts transfected with SpCas9-2A-GFP with or without gRNA 1.1. GFP expressing cells were sorted by FACS 4 days after transfection. The mutation site is marked by red arrow. Additional peaks appearing downstream (marked by black arrowheads) of the mutation site demonstrate sequence heterogeneity and thus, indel formation. Similar results were obtained by all gRNAs from two technical replicates (forward and reverse sequencing). Genome editing is apparent both in Tmc1 Bth/WT or Tmc1 WT/WT cells with SpCas9 + gRNA 1.1. ( c ) Sanger sequencing data was analyzed by TIDE. The control sample (SpCas9-2A-GFP only, black) and the genome edited sample (SpCas9-2A-GFP + gRNA 1.1, green) are overlaid. Downstream of the expected cut site (blue dashed line) the percentage of aberrant sequences was quantified in the region for decomposition. ( d ) Indel percentages (mean ± standard deviation) in Tmc1 Bth/WT or Tmc1 WT/WT cells based on TIDE analysis. Cells were transfected in duplicates and two independent sequencing reactions (forward and reverse) were performed. No indel formation was observed in the case of 3.1, 3.2 and 3.3 gRNAs. gRNA 1.4 showed minimal, but specific genome editing on the Tmc1 Bth/WT cells. All the other gRNAs mediated efficient indel formation both in Tmc1 Bth/WT or Tmc1 WT/WT cells. ( e ) Targeted deep sequencing on control (SpCas9-2A-GFP only) cells, WT gRNA and the 3 most specific gRNAs (1.1, 2.1 and 2.4) in Tmc1 Bth/WT (top) Tmc1 WT/WT (bottom) cells. Indels were quantified after segregating Tmc1 Bth and Tmc1 WT reads by CRISPResso (only insertions and deletions were quantified, substitutions were ignored). None of the gRNAs are specific to the Tmc1 Bth allele, and mediate efficient indel formation on the Tmc1 WT allele as well (light blue). Sequencing was performed one time from pooled cells, transfected in triplicates. Numbers in pie charts represent the percentage of reads. Specificity was defined as the indel percentage towards the targeted allele among total indels. The gRNA with the highest selectivity towards the Tmc1 Bth allele was gRNA 2.4. ( f ) The most abundant reads in the SpCas9 + gRNA 2.4 treated cells, shown separately for Tmc1 Bth (top) and Tmc1 WT (bottom) reads. The CRISPR cut site is marked by a black dashed line. Dashes represent deleted nucleotides. Insertions are shown with nucleotides in red squares. Nucleotides in bold are substitutions, however these were not quantified as CRISPR actions. Sequences were aligned to Bth allele, thus in the bottom panel, WT reads appear as having a substitution (a T to A change). Mutation site is marked by red arrow. Indel formation is evident in both Tmc1 Bth and Tmc1 WT reads. ( g ) Indel profiles from SpCas9 + gRNA 2.4 transfected Tmc1 Bth/WT fibroblasts. Tmc1 Bth and Tmc1 WT reads are plotted separately. Minus numbers represent deletions, plus numbers represent insertions. Sequences without indels (value=0) are omitted from the chart. The most common indel event is a single base deletion. ( h ) Indels causing in-frame vs. frame shift mutations (percentages are shown) in the coding sequence after SpCas9 + gRNA 2.4 transfection. ( i ) GUIDE-Seq analysis on SpCas9 + gRNA 2.4 transfected Tmc1 Bth/WT fibroblasts. Genomic DNA was isolated from 3 biological replicates for sequencing on one occasion. Only one off-target site was identified. Numbers next to reads are read counts in the GUIDE-Seq assay.
Figure Legend Snippet: Targeting Tmc1 Bth with SpCas9. ( a . Plasmids encoding SpCas9-2A-GFP, along with the different gRNAs, were transfected into fibroblasts. Four days after transfection, GFP-positive cells were sorted by FACS ( b ) Sanger sequencing traces from Tmc1 Bth/WT or Tmc1 WT/WT mouse fibroblasts transfected with SpCas9-2A-GFP with or without gRNA 1.1. GFP expressing cells were sorted by FACS 4 days after transfection. The mutation site is marked by red arrow. Additional peaks appearing downstream (marked by black arrowheads) of the mutation site demonstrate sequence heterogeneity and thus, indel formation. Similar results were obtained by all gRNAs from two technical replicates (forward and reverse sequencing). Genome editing is apparent both in Tmc1 Bth/WT or Tmc1 WT/WT cells with SpCas9 + gRNA 1.1. ( c ) Sanger sequencing data was analyzed by TIDE. The control sample (SpCas9-2A-GFP only, black) and the genome edited sample (SpCas9-2A-GFP + gRNA 1.1, green) are overlaid. Downstream of the expected cut site (blue dashed line) the percentage of aberrant sequences was quantified in the region for decomposition. ( d ) Indel percentages (mean ± standard deviation) in Tmc1 Bth/WT or Tmc1 WT/WT cells based on TIDE analysis. Cells were transfected in duplicates and two independent sequencing reactions (forward and reverse) were performed. No indel formation was observed in the case of 3.1, 3.2 and 3.3 gRNAs. gRNA 1.4 showed minimal, but specific genome editing on the Tmc1 Bth/WT cells. All the other gRNAs mediated efficient indel formation both in Tmc1 Bth/WT or Tmc1 WT/WT cells. ( e ) Targeted deep sequencing on control (SpCas9-2A-GFP only) cells, WT gRNA and the 3 most specific gRNAs (1.1, 2.1 and 2.4) in Tmc1 Bth/WT (top) Tmc1 WT/WT (bottom) cells. Indels were quantified after segregating Tmc1 Bth and Tmc1 WT reads by CRISPResso (only insertions and deletions were quantified, substitutions were ignored). None of the gRNAs are specific to the Tmc1 Bth allele, and mediate efficient indel formation on the Tmc1 WT allele as well (light blue). Sequencing was performed one time from pooled cells, transfected in triplicates. Numbers in pie charts represent the percentage of reads. Specificity was defined as the indel percentage towards the targeted allele among total indels. The gRNA with the highest selectivity towards the Tmc1 Bth allele was gRNA 2.4. ( f ) The most abundant reads in the SpCas9 + gRNA 2.4 treated cells, shown separately for Tmc1 Bth (top) and Tmc1 WT (bottom) reads. The CRISPR cut site is marked by a black dashed line. Dashes represent deleted nucleotides. Insertions are shown with nucleotides in red squares. Nucleotides in bold are substitutions, however these were not quantified as CRISPR actions. Sequences were aligned to Bth allele, thus in the bottom panel, WT reads appear as having a substitution (a T to A change). Mutation site is marked by red arrow. Indel formation is evident in both Tmc1 Bth and Tmc1 WT reads. ( g ) Indel profiles from SpCas9 + gRNA 2.4 transfected Tmc1 Bth/WT fibroblasts. Tmc1 Bth and Tmc1 WT reads are plotted separately. Minus numbers represent deletions, plus numbers represent insertions. Sequences without indels (value=0) are omitted from the chart. The most common indel event is a single base deletion. ( h ) Indels causing in-frame vs. frame shift mutations (percentages are shown) in the coding sequence after SpCas9 + gRNA 2.4 transfection. ( i ) GUIDE-Seq analysis on SpCas9 + gRNA 2.4 transfected Tmc1 Bth/WT fibroblasts. Genomic DNA was isolated from 3 biological replicates for sequencing on one occasion. Only one off-target site was identified. Numbers next to reads are read counts in the GUIDE-Seq assay.

Techniques Used: Transfection, FACS, Sequencing, Expressing, Mutagenesis, Standard Deviation, CRISPR, Isolation

40) Product Images from "N6-methyladenine DNA Modification in Glioblastoma"

Article Title: N6-methyladenine DNA Modification in Glioblastoma

Journal: Cell

doi: 10.1016/j.cell.2018.10.006

Identification of N(6)-methyladenine ( N 6 . (A) Levels of the N 6 -mA DNA modification were assessed via DNA dot blot in (1) normal human astrocytes, (2) patient-derived GSC models (387, D456, GSC23, and 1919) and (3) primary human glioblastoma specimens (3028, CW2386) using an N 6 -mA-specific antibody. Methylene blue detected DNA loading. (B) Mass spectrometry analysis of N 6 -mA in two normal human astrocyte cell lines and two patient-derived GSC models (387 and D456). Data are presented as mean ± SD. Two replicates were used for each sample. Significance was determined by one-way ANOVA with Tukey multiple comparison test. P
Figure Legend Snippet: Identification of N(6)-methyladenine ( N 6 . (A) Levels of the N 6 -mA DNA modification were assessed via DNA dot blot in (1) normal human astrocytes, (2) patient-derived GSC models (387, D456, GSC23, and 1919) and (3) primary human glioblastoma specimens (3028, CW2386) using an N 6 -mA-specific antibody. Methylene blue detected DNA loading. (B) Mass spectrometry analysis of N 6 -mA in two normal human astrocyte cell lines and two patient-derived GSC models (387 and D456). Data are presented as mean ± SD. Two replicates were used for each sample. Significance was determined by one-way ANOVA with Tukey multiple comparison test. P

Techniques Used: Modification, Dot Blot, Derivative Assay, Mass Spectrometry

ALKBH1 is a N 6 -mA DNA demethylase in human glioblastoma and contributes to N 6 . (A) N 6 -mA labelled DNA oligonucleotides were treated in a cell-free in vitro demethylase reaction with recombinant human ALKBH1 proteins. Results are depicted by dot blot after treatment of two quantities of substrate DNA oligonucleotides. (B) In vitro demethylation reaction was quantified by LC-MS/MS mass spectrometry following addition of ALKBH1 protein to N 6 -mA labelled DNA oligonucleotides. Data are presented as mean ± standard deviation. (Student’s t-test. ***, P
Figure Legend Snippet: ALKBH1 is a N 6 -mA DNA demethylase in human glioblastoma and contributes to N 6 . (A) N 6 -mA labelled DNA oligonucleotides were treated in a cell-free in vitro demethylase reaction with recombinant human ALKBH1 proteins. Results are depicted by dot blot after treatment of two quantities of substrate DNA oligonucleotides. (B) In vitro demethylation reaction was quantified by LC-MS/MS mass spectrometry following addition of ALKBH1 protein to N 6 -mA labelled DNA oligonucleotides. Data are presented as mean ± standard deviation. (Student’s t-test. ***, P

Techniques Used: In Vitro, Recombinant, Dot Blot, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Standard Deviation

Related Articles

Nucleic Acid Electrophoresis:

Article Title: Mutational processes shape the landscape of TP53 mutations in human cancer
Article Snippet: .. All 12 PCR reactions for each gDNA sample were pooled, concentrated with a PCR cleanup kit (QIAGEN), loaded onto a 1% agarose gel, and separated by gel electrophoresis. .. Bands of the expected size were excised, and DNA was purified first using a QIAquick kit (QIAGEN) then an AMPure XP kit (Beckman Coulter).

Transfection:

Article Title: ASF1a inhibition induces p53-dependent growth arrest and senescence of cancer cells
Article Snippet: .. Southern blot HepG2 and LNcaP cells were transfected with negative control and ASF1a siRNAs and cultured for 72 h. Cells were harvested and genomic DNA was extracted with QIAamp DNA Blood Mini Kit (Cat no 51104; Qiagen, Hilden, Germany). .. Southern blot was performed by using Telo TAGGG Telomere Length Assay (Merck; Cat no. 12 209 136 001) according to the manufacturer’s instructions.

Agarose Gel Electrophoresis:

Article Title: Mutational processes shape the landscape of TP53 mutations in human cancer
Article Snippet: .. All 12 PCR reactions for each gDNA sample were pooled, concentrated with a PCR cleanup kit (QIAGEN), loaded onto a 1% agarose gel, and separated by gel electrophoresis. .. Bands of the expected size were excised, and DNA was purified first using a QIAquick kit (QIAGEN) then an AMPure XP kit (Beckman Coulter).

Southern Blot:

Article Title: ASF1a inhibition induces p53-dependent growth arrest and senescence of cancer cells
Article Snippet: .. Southern blot HepG2 and LNcaP cells were transfected with negative control and ASF1a siRNAs and cultured for 72 h. Cells were harvested and genomic DNA was extracted with QIAamp DNA Blood Mini Kit (Cat no 51104; Qiagen, Hilden, Germany). .. Southern blot was performed by using Telo TAGGG Telomere Length Assay (Merck; Cat no. 12 209 136 001) according to the manufacturer’s instructions.

Isolation:

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 *
Article Snippet: .. Genomic DNA was isolated from undenervated and denervated TA muscle of mice or C2C12 myoblasts using DNeasy® blood and tissue kit (Qiagen). .. Global DNA methylation was assayed using an ELISA-based commercial kit (MDQ1; Imprint methylated DNA quantification kit; Sigma-Aldrich).

Negative Control:

Article Title: ASF1a inhibition induces p53-dependent growth arrest and senescence of cancer cells
Article Snippet: .. Southern blot HepG2 and LNcaP cells were transfected with negative control and ASF1a siRNAs and cultured for 72 h. Cells were harvested and genomic DNA was extracted with QIAamp DNA Blood Mini Kit (Cat no 51104; Qiagen, Hilden, Germany). .. Southern blot was performed by using Telo TAGGG Telomere Length Assay (Merck; Cat no. 12 209 136 001) according to the manufacturer’s instructions.

Cell Culture:

Article Title: ASF1a inhibition induces p53-dependent growth arrest and senescence of cancer cells
Article Snippet: .. Southern blot HepG2 and LNcaP cells were transfected with negative control and ASF1a siRNAs and cultured for 72 h. Cells were harvested and genomic DNA was extracted with QIAamp DNA Blood Mini Kit (Cat no 51104; Qiagen, Hilden, Germany). .. Southern blot was performed by using Telo TAGGG Telomere Length Assay (Merck; Cat no. 12 209 136 001) according to the manufacturer’s instructions.

Mouse Assay:

Article Title: CD27 signaling on chronic myelogenous leukemia stem cells activates Wnt target genes and promotes disease progression
Article Snippet: .. 10 mg of spleens from primary and secondary BL/6 CML and Cd27–/– mice were collected, and genomic DNA was extracted and purified using the DNeasy blood and tissue kit (QIAGEN). .. For quantitative real-time PCR, we designed DNA primers and probes for human ABL (forward: 5′-AGGACAGCTCTTGATTTG-3′; reverse: 5′-GACAGATGGAAAGGACATG-3′; probe: 5′-AAACAGGGTGCTAAAGCCAAC-3′) and murine c-abl (forward: 5′-CTGCACTTGAAACTTCTC-3′; reverse: 5′-TACCGTCATTGAGCTATTC-3′; probe: 5′-CACAGCCAGTCTCAGTTCAGG-3′).

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 *
Article Snippet: .. Genomic DNA was isolated from undenervated and denervated TA muscle of mice or C2C12 myoblasts using DNeasy® blood and tissue kit (Qiagen). .. Global DNA methylation was assayed using an ELISA-based commercial kit (MDQ1; Imprint methylated DNA quantification kit; Sigma-Aldrich).

Polymerase Chain Reaction:

Article Title: Mutational processes shape the landscape of TP53 mutations in human cancer
Article Snippet: .. All 12 PCR reactions for each gDNA sample were pooled, concentrated with a PCR cleanup kit (QIAGEN), loaded onto a 1% agarose gel, and separated by gel electrophoresis. .. Bands of the expected size were excised, and DNA was purified first using a QIAquick kit (QIAGEN) then an AMPure XP kit (Beckman Coulter).

Generated:

Article Title: Performance evaluation of commercial library construction kits for PCR-based targeted sequencing using a unique molecular identifier
Article Snippet: .. More importantly, the targeted deep sequencing data generated from 6.25 ng of initial genomic DNA using the Qiagen kit resulted in sufficient depth of unique coverage for detecting variants present at an allele frequency as low as 1%. ..

Sequencing:

Article Title: Performance evaluation of commercial library construction kits for PCR-based targeted sequencing using a unique molecular identifier
Article Snippet: .. More importantly, the targeted deep sequencing data generated from 6.25 ng of initial genomic DNA using the Qiagen kit resulted in sufficient depth of unique coverage for detecting variants present at an allele frequency as low as 1%. ..

Purification:

Article Title: CD27 signaling on chronic myelogenous leukemia stem cells activates Wnt target genes and promotes disease progression
Article Snippet: .. 10 mg of spleens from primary and secondary BL/6 CML and Cd27–/– mice were collected, and genomic DNA was extracted and purified using the DNeasy blood and tissue kit (QIAGEN). .. For quantitative real-time PCR, we designed DNA primers and probes for human ABL (forward: 5′-AGGACAGCTCTTGATTTG-3′; reverse: 5′-GACAGATGGAAAGGACATG-3′; probe: 5′-AAACAGGGTGCTAAAGCCAAC-3′) and murine c-abl (forward: 5′-CTGCACTTGAAACTTCTC-3′; reverse: 5′-TACCGTCATTGAGCTATTC-3′; probe: 5′-CACAGCCAGTCTCAGTTCAGG-3′).

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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: 91/100, based on 7821 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen dna demethylation treatment genomic dna
    MiR-486-5p expression is regulated by <t>DNA</t> 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 <t>MSP</t> 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
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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

    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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-018-07201-1

    Figure Lengend 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

    Article Snippet: Genomic DNA of 3 hESCs and 7 hiPSCs were extracted using the DNeasy Blood & Tissue Kit (QIAGEN, 69506).

    Techniques: 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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-018-07201-1

    Figure Lengend 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

    Article Snippet: Genomic DNA of 3 hESCs and 7 hiPSCs were extracted using the DNeasy Blood & Tissue Kit (QIAGEN, 69506).

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

    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

    Journal: BMC Plant Biology

    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

    doi: 10.1186/s12870-018-1506-z

    Figure Lengend 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

    Article Snippet: PCR analysis Genomic DNA was isolated from young leaf tissue of Keumkang and Olgeuru parental lines, DH20 mutant line, Chinese Spring, and the nullisomic tetrasomic lines of Chinese Spring using the DNeasy Plant mini kit (Qiagen, Hilden, Germany) according to protocols provided by the manufacturer.

    Techniques: 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

    Journal: BMC Plant Biology

    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

    doi: 10.1186/s12870-018-1506-z

    Figure Lengend 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

    Article Snippet: PCR analysis Genomic DNA was isolated from young leaf tissue of Keumkang and Olgeuru parental lines, DH20 mutant line, Chinese Spring, and the nullisomic tetrasomic lines of Chinese Spring using the DNeasy Plant mini kit (Qiagen, Hilden, Germany) according to protocols provided by the manufacturer.

    Techniques: Polymerase Chain Reaction, Amplification, Molecular Weight

    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

    Journal: Cell Death & Disease

    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

    doi: 10.1038/s41419-018-1105-9

    Figure Lengend 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

    Article Snippet: MSP, BSP, and DNA demethylation treatment Genomic DNA was extracted from CRC and normal tissues by using the DNeasy Blood and Tissue Kit (Qiagen, Duesseldorf, Germany) and then exposed to bisulfite using an EZ DNA Methylation-GoldTM Kit (Zymo Research, CA, USA) according to the manufacturers’ instructions.

    Techniques: Expressing, DNA Methylation Assay, Methylation