dna methylation treatment  (New England Biolabs)


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    Structured Review

    New England Biolabs dna methylation treatment
    mtDNA methylation identified by pyro-sequencing. Levels of <t>DNA</t> methylation at <t>CpG</t> sites in a HSP, b LSP, and c ND6 regions of 143B cells, 143B 143B early and 143B 143B late tumors were determined by pyro-sequencing. Statistical significance was determined by One-way ANOVA. Bars represent the mean of the percentage of DNA methylation (mean ± SEM; n = 3). * and ** indicate p values of
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    1) Product Images from "The degree of mitochondrial DNA methylation in tumor models of glioblastoma and osteosarcoma"

    Article Title: The degree of mitochondrial DNA methylation in tumor models of glioblastoma and osteosarcoma

    Journal: Clinical Epigenetics

    doi: 10.1186/s13148-018-0590-0

    mtDNA methylation identified by pyro-sequencing. Levels of DNA methylation at CpG sites in a HSP, b LSP, and c ND6 regions of 143B cells, 143B 143B early and 143B 143B late tumors were determined by pyro-sequencing. Statistical significance was determined by One-way ANOVA. Bars represent the mean of the percentage of DNA methylation (mean ± SEM; n = 3). * and ** indicate p values of
    Figure Legend Snippet: mtDNA methylation identified by pyro-sequencing. Levels of DNA methylation at CpG sites in a HSP, b LSP, and c ND6 regions of 143B cells, 143B 143B early and 143B 143B late tumors were determined by pyro-sequencing. Statistical significance was determined by One-way ANOVA. Bars represent the mean of the percentage of DNA methylation (mean ± SEM; n = 3). * and ** indicate p values of

    Techniques Used: Methylation, Sequencing, DNA Methylation Assay

    2) Product Images from "Dynamics of the Eukaryotic Replicative Helicase at Lagging-Strand Protein Barriers Support the Steric Exclusion Model"

    Article Title: Dynamics of the Eukaryotic Replicative Helicase at Lagging-Strand Protein Barriers Support the Steric Exclusion Model

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2019.01.086

    Single-Molecule Detection of CMG Pausing at a Lagging-Strand Methyltransferase Block (A) Schematic representation of experimental approach used in single-molecule DNA unwinding assays. (B) Images of a sample field of view showing accumulation of EGFP-RPA fluorescence signal at different time points from the addition of EGFP-RPA into the chamber. (C) Example unwinding traces of DNA substrates without a protein barrier. Traces exhibit a signal drop upon completion of unwinding due to dissociation of the leading-strand template (depicted in A). (D) Distribution of average fork rates measured in fully unwound substrates without MH (black) and after bypassing MH Lag (blue). Number of molecules are n(-MH) = 199, n(MH Lag after pause) = 20. (E) Sample unwinding traces of DNA substrates modified with MH Lag . Pausing observed at 800 bp is highlighted with gray rectangle. (F) Distribution of pause durations observed in molecules exhibiting a pausing event (n = 109). The solid line is a fit to a single exponential. See also Figure S6 .
    Figure Legend Snippet: Single-Molecule Detection of CMG Pausing at a Lagging-Strand Methyltransferase Block (A) Schematic representation of experimental approach used in single-molecule DNA unwinding assays. (B) Images of a sample field of view showing accumulation of EGFP-RPA fluorescence signal at different time points from the addition of EGFP-RPA into the chamber. (C) Example unwinding traces of DNA substrates without a protein barrier. Traces exhibit a signal drop upon completion of unwinding due to dissociation of the leading-strand template (depicted in A). (D) Distribution of average fork rates measured in fully unwound substrates without MH (black) and after bypassing MH Lag (blue). Number of molecules are n(-MH) = 199, n(MH Lag after pause) = 20. (E) Sample unwinding traces of DNA substrates modified with MH Lag . Pausing observed at 800 bp is highlighted with gray rectangle. (F) Distribution of pause durations observed in molecules exhibiting a pausing event (n = 109). The solid line is a fit to a single exponential. See also Figure S6 .

    Techniques Used: Blocking Assay, Recombinase Polymerase Amplification, Fluorescence, Modification

    3) Product Images from "Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori ▿"

    Article Title: Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00108-07

    Purified M.HpyAIV protects a GANTC-containing DNA fragment from HinfI digestion. Increasing concentrations of M.HpyAIV protein incubated with a 778-bp PCR fragment containing one GANTC site and S -adenosylmethionine. HinfI digestion of the GANTC-containing DNA fragment resulted in two fragments of 540 bp and 238 bp. The increased amount of undigested PCR products as a consequence of an increased M.HpyAIV concentration illustrates the in vitro capability of M.HpyAIV to protect GANTC sites from digestion in a concentration-dependent manner. L, ladder (samples in duplicate with increasing amounts of M.HpyAIV added [0, 200, 400, 800, and 1,200 nM]); UC, uncut control.
    Figure Legend Snippet: Purified M.HpyAIV protects a GANTC-containing DNA fragment from HinfI digestion. Increasing concentrations of M.HpyAIV protein incubated with a 778-bp PCR fragment containing one GANTC site and S -adenosylmethionine. HinfI digestion of the GANTC-containing DNA fragment resulted in two fragments of 540 bp and 238 bp. The increased amount of undigested PCR products as a consequence of an increased M.HpyAIV concentration illustrates the in vitro capability of M.HpyAIV to protect GANTC sites from digestion in a concentration-dependent manner. L, ladder (samples in duplicate with increasing amounts of M.HpyAIV added [0, 200, 400, 800, and 1,200 nM]); UC, uncut control.

    Techniques Used: Purification, Incubation, Polymerase Chain Reaction, Concentration Assay, In Vitro

    4) Product Images from "Epigenetic Regulation and Functional Characterization of MicroRNA-142 in Mesenchymal Cells"

    Article Title: Epigenetic Regulation and Functional Characterization of MicroRNA-142 in Mesenchymal Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0079231

    Assessment of the promoter activity of the upstream region of precursor mir-142 and identification of the putative core/proximal mir-142 promoter. ( A ) The 2,031 bp upstream region of pre-mir-142 was cloned into the promoter-less luciferase reporter construct pCpGL-basic to assess its promoter activity. The resulting pCpGL/2031 construct and empty pCpGL-basic were transfected into U-2 OS cells along with a Renilla reporter construct. The luciferase activity was measured after 48 hours and calculated relative to that of pCpGL-basic (set to 1). Each histogram shows the average relative luciferase activity, and the error bars show the standard deviation of biological experiments (n≥5). The Wilcoxon signed rank test was used to test for statistical differences. A P value ≤0.05 was considered as significant. ( B ) Schematic representation of the putative core/proximal mir-142 promoter region. The positions of the TATA-box, an enhancer box (E-box), 2 GC-boxes, a CACCC-box and a CGCCC-box (open boxes), and conserved binding sites for several transcription factors (filled boxes) are shown. Each vertical line represents an individual CpG. The numbers show the position relative to precursor mir-142 (+1 to +87). TSS, transcription start site.
    Figure Legend Snippet: Assessment of the promoter activity of the upstream region of precursor mir-142 and identification of the putative core/proximal mir-142 promoter. ( A ) The 2,031 bp upstream region of pre-mir-142 was cloned into the promoter-less luciferase reporter construct pCpGL-basic to assess its promoter activity. The resulting pCpGL/2031 construct and empty pCpGL-basic were transfected into U-2 OS cells along with a Renilla reporter construct. The luciferase activity was measured after 48 hours and calculated relative to that of pCpGL-basic (set to 1). Each histogram shows the average relative luciferase activity, and the error bars show the standard deviation of biological experiments (n≥5). The Wilcoxon signed rank test was used to test for statistical differences. A P value ≤0.05 was considered as significant. ( B ) Schematic representation of the putative core/proximal mir-142 promoter region. The positions of the TATA-box, an enhancer box (E-box), 2 GC-boxes, a CACCC-box and a CGCCC-box (open boxes), and conserved binding sites for several transcription factors (filled boxes) are shown. Each vertical line represents an individual CpG. The numbers show the position relative to precursor mir-142 (+1 to +87). TSS, transcription start site.

    Techniques Used: Activity Assay, Clone Assay, Luciferase, Construct, Transfection, Standard Deviation, Binding Assay

    5) Product Images from "Promoter DNA methylation regulates progranulin expression and is altered in FTLD"

    Article Title: Promoter DNA methylation regulates progranulin expression and is altered in FTLD

    Journal: Acta Neuropathologica Communications

    doi: 10.1186/2051-5960-1-16

    DNA methylation inhibits GRN promoter activity at distinct CpG units. In vitro methylated and unmethylated pCpGL plasmids containing the GRN core promoter region driving expression of firefly luciferase were transiently cotransfected into ( a ) HEK 293FT cells and ( b ) primary rat cortical neurons together with a Renilla luciferase expressing plasmid. The full length GRN promoter pCpGL plasmid, a GRN promoter construct with site specific mutations of the significant CpG units in amplicons A-1 and A-2, and a short GRN promoter construct lacking amplicons A-1 and A-2 were transiently cotransfected into ( c ) HEK 293FT cells and ( d ) primary rat cortical neurons together with a Renilla luciferase expressing plasmid. Luciferase reporter activity was measured 48 h (a + c, HEK 293FT) or 72 h (b + d, neurons) after transfection. Relative luciferase activity was determined by normalizing firefly luciferase against Renilla luciferase activity. The empty vector was used as negative control. Mean ± SEM, n ≥ 3. ***p
    Figure Legend Snippet: DNA methylation inhibits GRN promoter activity at distinct CpG units. In vitro methylated and unmethylated pCpGL plasmids containing the GRN core promoter region driving expression of firefly luciferase were transiently cotransfected into ( a ) HEK 293FT cells and ( b ) primary rat cortical neurons together with a Renilla luciferase expressing plasmid. The full length GRN promoter pCpGL plasmid, a GRN promoter construct with site specific mutations of the significant CpG units in amplicons A-1 and A-2, and a short GRN promoter construct lacking amplicons A-1 and A-2 were transiently cotransfected into ( c ) HEK 293FT cells and ( d ) primary rat cortical neurons together with a Renilla luciferase expressing plasmid. Luciferase reporter activity was measured 48 h (a + c, HEK 293FT) or 72 h (b + d, neurons) after transfection. Relative luciferase activity was determined by normalizing firefly luciferase against Renilla luciferase activity. The empty vector was used as negative control. Mean ± SEM, n ≥ 3. ***p

    Techniques Used: DNA Methylation Assay, Activity Assay, In Vitro, Methylation, Expressing, Luciferase, Plasmid Preparation, Construct, Transfection, Negative Control

    GRN expression in human lymphoblast cell lines is inversely correlated to its promoter methylation. ( a ) GRN net secretion was measured by ELISA in LCLs derived from neurologically healthy individuals (LCL #1-13), unaffected relatives of FTLD patients (LCL #14, 15, highlighted in blue) and FTLD-patients (LCL#16, 17, highlighted in red). n = 3, mean ± SEM. ( b ) Scheme of GRN promoter region. Red bars depict PCR-amplicons analyzed for DNA methylation levels by MassARRAY. Blue bars indicate full length and short promoter region that was cloned into the pCpGL vector for luciferase assays (compare Figure 3 ). White circles display CpG units in amplicons A-1 to A-5 and A-DAC quantified by MassARRAY. CpG units that were not analyzed are not shown. Asterisks indicate significant correlation between GRN mRNA expression or GRN secretion and GRN methylation at respective CpG unit (*p
    Figure Legend Snippet: GRN expression in human lymphoblast cell lines is inversely correlated to its promoter methylation. ( a ) GRN net secretion was measured by ELISA in LCLs derived from neurologically healthy individuals (LCL #1-13), unaffected relatives of FTLD patients (LCL #14, 15, highlighted in blue) and FTLD-patients (LCL#16, 17, highlighted in red). n = 3, mean ± SEM. ( b ) Scheme of GRN promoter region. Red bars depict PCR-amplicons analyzed for DNA methylation levels by MassARRAY. Blue bars indicate full length and short promoter region that was cloned into the pCpGL vector for luciferase assays (compare Figure 3 ). White circles display CpG units in amplicons A-1 to A-5 and A-DAC quantified by MassARRAY. CpG units that were not analyzed are not shown. Asterisks indicate significant correlation between GRN mRNA expression or GRN secretion and GRN methylation at respective CpG unit (*p

    Techniques Used: Expressing, Methylation, Enzyme-linked Immunosorbent Assay, Derivative Assay, Polymerase Chain Reaction, DNA Methylation Assay, Clone Assay, Plasmid Preparation, Luciferase

    6) Product Images from "DNA Methylation of the Gonadal Aromatase (cyp19a) Promoter Is Involved in Temperature-Dependent Sex Ratio Shifts in the European Sea Bass"

    Article Title: DNA Methylation of the Gonadal Aromatase (cyp19a) Promoter Is Involved in Temperature-Dependent Sex Ratio Shifts in the European Sea Bass

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002447

    Effects of methylation on sea bass cyp19a promoter activity in vitro . HEK293T cells were transfected with pGL3- cyp19a methylated and unmethylated promoter vectors. Transcription factors SF1 and Foxl2 were cotransfected with the sb cyp19a promoter to activate promoter luciferase activity. Transfected methylated and unmethylated groups were as follows (see Materials and Methods for amounts used): 1) plasmid β-galactosidase (Ctrl); 2) sb cyp19a promoter cloned into pGL3-basic luciferase reporter plasmid (cyp19a); 3) sb cyp19a and tilapia SF1 cloned into pCDNA3.1 expression plasmid (cyp19a+SF1); 4) sb cyp19a and tilapia Foxl2 cloned into pCDNA3.1 expression plasmid (cyp19a+Foxl2); 5) sb cyp19a , tilapia SF1 and Foxl2 cloned into pCDNA3.1 (cyp19a+SF1+Foxl2). The Student's t -test was used to compare methylated and unmethylated vectors. Significant differences are denoted by an asterisk and were as follows: cyp19a, P = 0.006; cyp19a+SF1, P = 0.006; cyp19a+Foxl2, P = 0.003; cyp19a+SF1+Foxl2, P = 0.013. Also, SF1 and Foxl2 exhibited an additive effect since the activation of sb cyp19a promoter was significantly higher when both were transfected together. Values are shown as mean ± S.E.M. (n = 2–4). Insert: Successful vector methylation verification by analysis of band patterns on electrophoresis gel after digestion of the purified plasmids with the McrBC enzyme. Lane 1, 0.5 µg sb cyp19a -pGL3; lane 2, 0.5 µg sb cyp19a -pGL3 treated with McrBC ; lane 3, 0.5 µg Sss I-methylated sb cyp19a -pGL3; lane 4, 0.5 µg Sss I-methylated sb cyp19a -pGL3 treated with McrBC ; lane 5, 1 Kb marker; lane 6, 100 bp marker. The electrophoresis gel shows that, as expected, only the methylated vector was digested.
    Figure Legend Snippet: Effects of methylation on sea bass cyp19a promoter activity in vitro . HEK293T cells were transfected with pGL3- cyp19a methylated and unmethylated promoter vectors. Transcription factors SF1 and Foxl2 were cotransfected with the sb cyp19a promoter to activate promoter luciferase activity. Transfected methylated and unmethylated groups were as follows (see Materials and Methods for amounts used): 1) plasmid β-galactosidase (Ctrl); 2) sb cyp19a promoter cloned into pGL3-basic luciferase reporter plasmid (cyp19a); 3) sb cyp19a and tilapia SF1 cloned into pCDNA3.1 expression plasmid (cyp19a+SF1); 4) sb cyp19a and tilapia Foxl2 cloned into pCDNA3.1 expression plasmid (cyp19a+Foxl2); 5) sb cyp19a , tilapia SF1 and Foxl2 cloned into pCDNA3.1 (cyp19a+SF1+Foxl2). The Student's t -test was used to compare methylated and unmethylated vectors. Significant differences are denoted by an asterisk and were as follows: cyp19a, P = 0.006; cyp19a+SF1, P = 0.006; cyp19a+Foxl2, P = 0.003; cyp19a+SF1+Foxl2, P = 0.013. Also, SF1 and Foxl2 exhibited an additive effect since the activation of sb cyp19a promoter was significantly higher when both were transfected together. Values are shown as mean ± S.E.M. (n = 2–4). Insert: Successful vector methylation verification by analysis of band patterns on electrophoresis gel after digestion of the purified plasmids with the McrBC enzyme. Lane 1, 0.5 µg sb cyp19a -pGL3; lane 2, 0.5 µg sb cyp19a -pGL3 treated with McrBC ; lane 3, 0.5 µg Sss I-methylated sb cyp19a -pGL3; lane 4, 0.5 µg Sss I-methylated sb cyp19a -pGL3 treated with McrBC ; lane 5, 1 Kb marker; lane 6, 100 bp marker. The electrophoresis gel shows that, as expected, only the methylated vector was digested.

    Techniques Used: Methylation, Activity Assay, In Vitro, Transfection, Luciferase, Plasmid Preparation, Clone Assay, Expressing, Activation Assay, Electrophoresis, Purification, Marker

    7) Product Images from "Analysis of individual remodeled nucleosomes reveals decreased histone-DNA contacts created by hSWI/SNF"

    Article Title: Analysis of individual remodeled nucleosomes reveals decreased histone-DNA contacts created by hSWI/SNF

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp524

    ( A ) Native electrophoresis of SNF2H remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of SNF2H (lane 2: 6 nM; lane 3: 19 nM; lanes 4 and 5: 57 nM) in the presence (lanes 2–4) or absence (lane 5) of ATP (1 mM) as indicated on top, for 1 h at 30°C. Reactions were stopped by addition of ADP (10 mM) and incubation on ice for 10 min. Methylation of the remodeled products were performed as follow. The reactions were incubated for 15 min at 37°C after addition of M.SssI (5 U) and S-adenosylmethionine (160 μM). The remodeling reactions were separated by native gel electrophoresis after addition of competitor plasmid DNA and visualized by ethidium bromide staining. Gel areas excised and used for analysis are delimited by a black frame. Back frames are connected by dashed lines when gel slices were combined. ( B–F ) Schematic representation of individual DNA molecules remodeled by SNF2H. Bisulfite-converted DNAs from gel slices (black frames, lanes 3–5) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 2 D. The number of remodeled molecules shown is proportional to the average intensity of the bands generated after remodeling at enzyme concentration allowing maximal remodeling in three independent experiments. ( G ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel F (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–E (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.
    Figure Legend Snippet: ( A ) Native electrophoresis of SNF2H remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of SNF2H (lane 2: 6 nM; lane 3: 19 nM; lanes 4 and 5: 57 nM) in the presence (lanes 2–4) or absence (lane 5) of ATP (1 mM) as indicated on top, for 1 h at 30°C. Reactions were stopped by addition of ADP (10 mM) and incubation on ice for 10 min. Methylation of the remodeled products were performed as follow. The reactions were incubated for 15 min at 37°C after addition of M.SssI (5 U) and S-adenosylmethionine (160 μM). The remodeling reactions were separated by native gel electrophoresis after addition of competitor plasmid DNA and visualized by ethidium bromide staining. Gel areas excised and used for analysis are delimited by a black frame. Back frames are connected by dashed lines when gel slices were combined. ( B–F ) Schematic representation of individual DNA molecules remodeled by SNF2H. Bisulfite-converted DNAs from gel slices (black frames, lanes 3–5) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 2 D. The number of remodeled molecules shown is proportional to the average intensity of the bands generated after remodeling at enzyme concentration allowing maximal remodeling in three independent experiments. ( G ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel F (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–E (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.

    Techniques Used: Electrophoresis, Incubation, Methylation, Nucleic Acid Electrophoresis, Plasmid Preparation, Staining, Amplification, Polymerase Chain Reaction, Clone Assay, Generated, Concentration Assay

    8) Product Images from "Early hypermethylation of hepatic Igfbp2 results in its reduced expression preceding fatty liver in mice"

    Article Title: Early hypermethylation of hepatic Igfbp2 results in its reduced expression preceding fatty liver in mice

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddw121

    pCpGL– Igfbp2 luciferase reporter assays. ( A ) Intragenic region of murine Igfbp2 gene spanning from 2320 to 2997 bp relative to TSS. Recognition sites for methyltransferases HpaII and M.SssI, CpG 2605 depicted in grey. ( B ) Luciferase activity upon selective methylation of pCpGL- Igfbp2 . Igfbp2 expression ( C ) and CpG 2605 methylation ( D ) in NZO mice. Correlation of CpG 2605 methylation and body weight ( E ), insulin concentrations ( F ) and LAR ( G ) of 6 weeks old HFD-fed C57BL/6J and NZO mice. All data represented as mean ± SEM of n = 3–4 independent experiments and n = 7 animals per group, respectively. * p
    Figure Legend Snippet: pCpGL– Igfbp2 luciferase reporter assays. ( A ) Intragenic region of murine Igfbp2 gene spanning from 2320 to 2997 bp relative to TSS. Recognition sites for methyltransferases HpaII and M.SssI, CpG 2605 depicted in grey. ( B ) Luciferase activity upon selective methylation of pCpGL- Igfbp2 . Igfbp2 expression ( C ) and CpG 2605 methylation ( D ) in NZO mice. Correlation of CpG 2605 methylation and body weight ( E ), insulin concentrations ( F ) and LAR ( G ) of 6 weeks old HFD-fed C57BL/6J and NZO mice. All data represented as mean ± SEM of n = 3–4 independent experiments and n = 7 animals per group, respectively. * p

    Techniques Used: Luciferase, Activity Assay, Methylation, Expressing, Mouse Assay

    9) Product Images from "Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule"

    Article Title: Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule

    Journal: Bioconjugate Chemistry

    doi: 10.1021/bc1000388

    Effect of TFO-141S treatment on plasmid conformation. (A) Agarose gel electrophoresis of plasmids p39E and p11-1 treated with the TFO, WT M.SssI, C141S, or the TFO−C141S conjugate: lane 1, untreated; lane 2, without TFO−C141S; lane 3, TFO; lane 4, M.SssI; lane 5, C141S; lane 6, TFO−C141S; lane 7, TFO- C141S without SAM; lane 8, 100-fold excess of TFO and TFO−C141S; lane 9, marker; lane 10, without TFO−C141S; lane 11, C141S; lane 12, TFO−C141S. (B) Agarose gel electrophoresis of plasmid p39E treated with active and heat-inactivated C141S and TFO−C141S. The supercoiled plasmid was incubated at 30 °C for different time points as indicated above the lanes (hours). Then the samples were deproteinized before electrophoresis as described in Experimental Procedures . (C) Agarose gel electrophoresis of plasmid p39C and p39E treated with the TFO−C141S conjugate only or in the presence of 100-fold excess of TFO. Plasmids were incubated as in part B. Lane a is purified plasmid.
    Figure Legend Snippet: Effect of TFO-141S treatment on plasmid conformation. (A) Agarose gel electrophoresis of plasmids p39E and p11-1 treated with the TFO, WT M.SssI, C141S, or the TFO−C141S conjugate: lane 1, untreated; lane 2, without TFO−C141S; lane 3, TFO; lane 4, M.SssI; lane 5, C141S; lane 6, TFO−C141S; lane 7, TFO- C141S without SAM; lane 8, 100-fold excess of TFO and TFO−C141S; lane 9, marker; lane 10, without TFO−C141S; lane 11, C141S; lane 12, TFO−C141S. (B) Agarose gel electrophoresis of plasmid p39E treated with active and heat-inactivated C141S and TFO−C141S. The supercoiled plasmid was incubated at 30 °C for different time points as indicated above the lanes (hours). Then the samples were deproteinized before electrophoresis as described in Experimental Procedures . (C) Agarose gel electrophoresis of plasmid p39C and p39E treated with the TFO−C141S conjugate only or in the presence of 100-fold excess of TFO. Plasmids were incubated as in part B. Lane a is purified plasmid.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Marker, Incubation, Electrophoresis, Purification

    Effect of TFO−C141S treatment on GFP expression in EpCAM positive SKOV3 cells. (A) Relative GFP expression measured 48 h after transfection of pretreated p39E. Plasmid p39E was treated as indicated: p39E = treatment without TFO−C141S, treated with TFO only, with untargeted M.SssI or C141S, with the TFO−C141S conjugate or with 100-fold excess of TFO and TFO−C141S (=competition). The value obtained with p39E without TFO−C141S was taken as 100%. Shown is the average GFP expression (±SD) of one representative transfection performed in triplicate. (B) Relative GFP expression measured 48 h after transfection of pretreated deletion derivatives p7-2 and p4-1. For each derivative, the values obtained with samples treated without TFO−C141S were taken as 100%. Shown is the average GFP expression (±SEM) of the mean of three independent transfections performed in triplicate. (C) Relative GFP expression was measured 48 h after transfection of pretreated p39E or p39C. Treatments were as indicated: (+) or (−) indicates the presence or absence of the methyl donor (SAM). Shown is the average GFP expression (±SEM) of the mean of three independent transfections performed in triplicate.
    Figure Legend Snippet: Effect of TFO−C141S treatment on GFP expression in EpCAM positive SKOV3 cells. (A) Relative GFP expression measured 48 h after transfection of pretreated p39E. Plasmid p39E was treated as indicated: p39E = treatment without TFO−C141S, treated with TFO only, with untargeted M.SssI or C141S, with the TFO−C141S conjugate or with 100-fold excess of TFO and TFO−C141S (=competition). The value obtained with p39E without TFO−C141S was taken as 100%. Shown is the average GFP expression (±SD) of one representative transfection performed in triplicate. (B) Relative GFP expression measured 48 h after transfection of pretreated deletion derivatives p7-2 and p4-1. For each derivative, the values obtained with samples treated without TFO−C141S were taken as 100%. Shown is the average GFP expression (±SEM) of the mean of three independent transfections performed in triplicate. (C) Relative GFP expression was measured 48 h after transfection of pretreated p39E or p39C. Treatments were as indicated: (+) or (−) indicates the presence or absence of the methyl donor (SAM). Shown is the average GFP expression (±SEM) of the mean of three independent transfections performed in triplicate.

    Techniques Used: Expressing, Transfection, Plasmid Preparation

    10) Product Images from "Effect of Regulatory Element DNA Methylation on Tissue-Type Plasminogen Activator Gene Expression"

    Article Title: Effect of Regulatory Element DNA Methylation on Tissue-Type Plasminogen Activator Gene Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0167588

    Effect of DNA methylation on PLAT promoter-driven reporter gene activity. Reporter gene activity of methylated (M) or unmethylated (NM) plasmids was compared after transfection of HeLa cells (top and center) or HT 1080 (bottom) with pCpGLprom401 and pCpGLprom151 plasmids, which contain the PLAT promoter region from -401 to +151 and -151 to +151, respectively, linked to a CpG-less firefly luciferase gene. Note that reporter gene activity of the methylated plasmids was the same as that of reporter gene plasmids lacking a promoter (empty) and markedly reduced compared to unmethylated plasmids. Results are presented as means ± SEM of 3 to 4 independent experiments.
    Figure Legend Snippet: Effect of DNA methylation on PLAT promoter-driven reporter gene activity. Reporter gene activity of methylated (M) or unmethylated (NM) plasmids was compared after transfection of HeLa cells (top and center) or HT 1080 (bottom) with pCpGLprom401 and pCpGLprom151 plasmids, which contain the PLAT promoter region from -401 to +151 and -151 to +151, respectively, linked to a CpG-less firefly luciferase gene. Note that reporter gene activity of the methylated plasmids was the same as that of reporter gene plasmids lacking a promoter (empty) and markedly reduced compared to unmethylated plasmids. Results are presented as means ± SEM of 3 to 4 independent experiments.

    Techniques Used: DNA Methylation Assay, Activity Assay, Methylation, Transfection, Luciferase

    11) Product Images from "Alterations of sorbin and SH3 domain containing 3 (SORBS3) in human skeletal muscle following Roux-en-Y gastric bypass surgery"

    Article Title: Alterations of sorbin and SH3 domain containing 3 (SORBS3) in human skeletal muscle following Roux-en-Y gastric bypass surgery

    Journal: Clinical Epigenetics

    doi: 10.1186/s13148-017-0396-5

    In vitro DNA methylation of the SORBS3 human promoter is associated with decreased gene expression. Data presented as mean ± SD. The mean represents four independent experiments with five replicates per experiment. * P
    Figure Legend Snippet: In vitro DNA methylation of the SORBS3 human promoter is associated with decreased gene expression. Data presented as mean ± SD. The mean represents four independent experiments with five replicates per experiment. * P

    Techniques Used: In Vitro, DNA Methylation Assay, Expressing

    Average methylation levels of SORBS3 DMCs from lean and obese participants in a previous study and the present study pre- and post-surgery levels. The average methylation was assessed with all DMCs, regardless of methylation direction ( a ) and of only the DMCs that were consistent in the direction of methylation ( b ). Data presented as mean ± SD
    Figure Legend Snippet: Average methylation levels of SORBS3 DMCs from lean and obese participants in a previous study and the present study pre- and post-surgery levels. The average methylation was assessed with all DMCs, regardless of methylation direction ( a ) and of only the DMCs that were consistent in the direction of methylation ( b ). Data presented as mean ± SD

    Techniques Used: Methylation

    Differentially methylated cytosine (DMC) distribution among the promoter and 5′ untranslated regions of sorbin and SH3 domain containing 3 ( SORBS3 ) variants 1 and 2. The DMCs are derived from a previous study in obesity (Ln = lean vs Ob = obese) and the present RYGB cohort (bariatric)
    Figure Legend Snippet: Differentially methylated cytosine (DMC) distribution among the promoter and 5′ untranslated regions of sorbin and SH3 domain containing 3 ( SORBS3 ) variants 1 and 2. The DMCs are derived from a previous study in obesity (Ln = lean vs Ob = obese) and the present RYGB cohort (bariatric)

    Techniques Used: Methylation, Derivative Assay

    Differentially methylated cytosines (DMCs) associated with SORBS3 detected using pyrosequencing on the sense strand ( a ) and antisense strand ( b ) pre- and post-surgery. Data presented as mean ± SD
    Figure Legend Snippet: Differentially methylated cytosines (DMCs) associated with SORBS3 detected using pyrosequencing on the sense strand ( a ) and antisense strand ( b ) pre- and post-surgery. Data presented as mean ± SD

    Techniques Used: Methylation

    12) Product Images from "Measuring quantitative effects of methylation on transcription factor–DNA binding affinity"

    Article Title: Measuring quantitative effects of methylation on transcription factor–DNA binding affinity

    Journal: Science Advances

    doi: 10.1126/sciadv.aao1799

    Overview of Methyl-Spec-seq. ( A ) Schematic representation of the general workflow of Methyl-Spec-seq (see Materials and Methods). Briefly, differentially barcoded DNA libraries with variable regions are mixed and used in protein-DNA binding reactions. The DNA libraries are either treated with M.SssI methyltransferase enzyme to incorporate methyl-CpGs or left untreated and can also have synthetic 5′-methyl cytidine (mC). The letters “M” and “W” in red represent mC and mC on the complementary strand opposing a G, respectively. The protein-DNA complex is separated from the unbound DNA, following the binding reaction, in 9% polyacrylamide gel. The bound and unbound fractions are then polymerase chain reaction (PCR)–amplified (eight cycles) using Illumina-specific primers (text S1), and the resulting indexed samples are sequenced to generate energy logos for the binding sites. ( B ) Randomized double-stranded DNA (dsDNA) library used to measure the binding specificity of ZFP57 and the effect of methylation on binding. The full-length DNA libraries are shown in text S1. The regions highlighted in blue are the unique barcodes to distinguish the libraries during sequencing, whereas “N” in bold represent variable regions within the libraries. ( C ) Relative binding energy for all 64 variants in R2 libraries with different types of methylation, ranked from low to high binding energies of the unmethylated DNA. The relative binding energies are represented in units of kT , where k is the Boltzmann constant and T is the temperature used in the binding experiments. The 64 sequences of R2 libraries are listed vertically, and the relative binding energies depending on the methylation status are plotted. ( D ) Meth-eLogo based on the regression of the ZFP57 reference site and all its single variants. The significant effect of methylation at positions 4 and 5, which is the binding site for finger 3 (F3), is also shown. The effect of CpG methylation (mCPG) on binding specificity was calculated from the ePWM listed in fig. S2.
    Figure Legend Snippet: Overview of Methyl-Spec-seq. ( A ) Schematic representation of the general workflow of Methyl-Spec-seq (see Materials and Methods). Briefly, differentially barcoded DNA libraries with variable regions are mixed and used in protein-DNA binding reactions. The DNA libraries are either treated with M.SssI methyltransferase enzyme to incorporate methyl-CpGs or left untreated and can also have synthetic 5′-methyl cytidine (mC). The letters “M” and “W” in red represent mC and mC on the complementary strand opposing a G, respectively. The protein-DNA complex is separated from the unbound DNA, following the binding reaction, in 9% polyacrylamide gel. The bound and unbound fractions are then polymerase chain reaction (PCR)–amplified (eight cycles) using Illumina-specific primers (text S1), and the resulting indexed samples are sequenced to generate energy logos for the binding sites. ( B ) Randomized double-stranded DNA (dsDNA) library used to measure the binding specificity of ZFP57 and the effect of methylation on binding. The full-length DNA libraries are shown in text S1. The regions highlighted in blue are the unique barcodes to distinguish the libraries during sequencing, whereas “N” in bold represent variable regions within the libraries. ( C ) Relative binding energy for all 64 variants in R2 libraries with different types of methylation, ranked from low to high binding energies of the unmethylated DNA. The relative binding energies are represented in units of kT , where k is the Boltzmann constant and T is the temperature used in the binding experiments. The 64 sequences of R2 libraries are listed vertically, and the relative binding energies depending on the methylation status are plotted. ( D ) Meth-eLogo based on the regression of the ZFP57 reference site and all its single variants. The significant effect of methylation at positions 4 and 5, which is the binding site for finger 3 (F3), is also shown. The effect of CpG methylation (mCPG) on binding specificity was calculated from the ePWM listed in fig. S2.

    Techniques Used: Binding Assay, Polymerase Chain Reaction, Amplification, Methylation, Sequencing, CpG Methylation Assay

    13) Product Images from "Twist1/Dnmt3a and miR186 establish a regulatory circuit that controls inflammation-associated prostate cancer progression"

    Article Title: Twist1/Dnmt3a and miR186 establish a regulatory circuit that controls inflammation-associated prostate cancer progression

    Journal: Oncogenesis

    doi: 10.1038/oncsis.2017.16

    Twist1 represses miR186 in a negative feedback loop through directly interacting with and recruiting Dnmt3a to the miR186 promoter. ( a ) Real-time PCR analysis for the relative expression of miR186 in BPH1-Vector and BPH1-Twist1, or P69-Vector and P69-Twist1 cells. *** P -values
    Figure Legend Snippet: Twist1 represses miR186 in a negative feedback loop through directly interacting with and recruiting Dnmt3a to the miR186 promoter. ( a ) Real-time PCR analysis for the relative expression of miR186 in BPH1-Vector and BPH1-Twist1, or P69-Vector and P69-Twist1 cells. *** P -values

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

    Twist1 recruiting Dnmt3a facilitates the site-specific CpG methylation of the miR186 promoter to decrease the binding and the transcriptional activity of NF-κB/P65. ( a ) ChIP analysis for Dnmt3a occupancy at miR186 promoter in BPH1 and LT-BPH1. ( b ) qChIP analysis for Dnmt3a occupancy at the miR186 promoter in BPH1 and BPH1-Twist1 cells. Data represent means±s.e.m. ( n =3), ** P -values
    Figure Legend Snippet: Twist1 recruiting Dnmt3a facilitates the site-specific CpG methylation of the miR186 promoter to decrease the binding and the transcriptional activity of NF-κB/P65. ( a ) ChIP analysis for Dnmt3a occupancy at miR186 promoter in BPH1 and LT-BPH1. ( b ) qChIP analysis for Dnmt3a occupancy at the miR186 promoter in BPH1 and BPH1-Twist1 cells. Data represent means±s.e.m. ( n =3), ** P -values

    Techniques Used: CpG Methylation Assay, Binding Assay, Activity Assay, Chromatin Immunoprecipitation

    MiR186 is directly activated by NF-κB under inflammation signals. ( a , b ) The miR186 levels in BPH1 ( a ) and P69 ( b ) treated with LPS (1 μg/ml) for different indicated time were measured by real-time PCR. U6 was used as internal control. The experiments were performed at least three independent times, and error bars indicate ±s.e.m., *** P -values
    Figure Legend Snippet: MiR186 is directly activated by NF-κB under inflammation signals. ( a , b ) The miR186 levels in BPH1 ( a ) and P69 ( b ) treated with LPS (1 μg/ml) for different indicated time were measured by real-time PCR. U6 was used as internal control. The experiments were performed at least three independent times, and error bars indicate ±s.e.m., *** P -values

    Techniques Used: Real-time Polymerase Chain Reaction

    High methylation of CpG islands in the miR186 promoter blocks its response to inflammation signals in transformed or malignant cells. ( a , b ) LT-BPH1 ( a ) and PC3 ( b ) were treated with 1 μg/ml LPS or 25 ng/ml TNFα for different time periods as indicated, and lysed for western blotting analysis of Twist1 and real-time PCR analysis of miR186. ( c , d ) MSP (methylation-specific PCR) analysis for the methylation levels of CpG islands in the miR186 promoter in BPH1 and LT-BPH1 ( c ), and in LT-BPH1 treated with 5 μ m 5-aza-CdR for 24 h ( d ). ‘U’ represents unmethylated DNA products amplified with non-methylation-specific primers, and ‘M’ refers to methylated DNA products amplified with methylation-specific primers. ( e ) Real-time PCR analysis for the miR186 levels in LT-BPH1 treated with or without 5 μ m 5-aza-CdR for 24 h. U6 was used as internal control. The experiments were performed at least three independent times, and error bars indicate ±s.e.m., ** P -values
    Figure Legend Snippet: High methylation of CpG islands in the miR186 promoter blocks its response to inflammation signals in transformed or malignant cells. ( a , b ) LT-BPH1 ( a ) and PC3 ( b ) were treated with 1 μg/ml LPS or 25 ng/ml TNFα for different time periods as indicated, and lysed for western blotting analysis of Twist1 and real-time PCR analysis of miR186. ( c , d ) MSP (methylation-specific PCR) analysis for the methylation levels of CpG islands in the miR186 promoter in BPH1 and LT-BPH1 ( c ), and in LT-BPH1 treated with 5 μ m 5-aza-CdR for 24 h ( d ). ‘U’ represents unmethylated DNA products amplified with non-methylation-specific primers, and ‘M’ refers to methylated DNA products amplified with methylation-specific primers. ( e ) Real-time PCR analysis for the miR186 levels in LT-BPH1 treated with or without 5 μ m 5-aza-CdR for 24 h. U6 was used as internal control. The experiments were performed at least three independent times, and error bars indicate ±s.e.m., ** P -values

    Techniques Used: Methylation, Transformation Assay, Western Blot, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification

    MiR186 is a key factor in inflammation-associated cell transformation. ( a ) Immunoblotting of epithelial and mesenchymal markers in BPH1 and LT-BPH1 cells. ( b ) Soft-agar colony-formation assays for BPH1 and LT-BPH1 cells. The culture medium containing 10% FBS with 0.35% agar was layered onto the base. Representative images of the colonies were shown, and the same scale bar (100 μm) was used in the images. ( c , i ) CCK8 cell-proliferation assays for BPH1 and LT-BPH1 cells ( c ), LT-BPH1-Vector and LT-BPH1-miR186 cells ( i ). ( d , j ) RTCA growth assays for BPH1 and LT-BPH1 ( d ), LT-BPH1-Vector and LT-BPH1-miR186 cells ( j ). The growth slopes were shown as histogram. Error bars indicate ±s.d., *** P -values
    Figure Legend Snippet: MiR186 is a key factor in inflammation-associated cell transformation. ( a ) Immunoblotting of epithelial and mesenchymal markers in BPH1 and LT-BPH1 cells. ( b ) Soft-agar colony-formation assays for BPH1 and LT-BPH1 cells. The culture medium containing 10% FBS with 0.35% agar was layered onto the base. Representative images of the colonies were shown, and the same scale bar (100 μm) was used in the images. ( c , i ) CCK8 cell-proliferation assays for BPH1 and LT-BPH1 cells ( c ), LT-BPH1-Vector and LT-BPH1-miR186 cells ( i ). ( d , j ) RTCA growth assays for BPH1 and LT-BPH1 ( d ), LT-BPH1-Vector and LT-BPH1-miR186 cells ( j ). The growth slopes were shown as histogram. Error bars indicate ±s.d., *** P -values

    Techniques Used: Transformation Assay, Plasmid Preparation

    The miR186 expression and its promoter methylation are related with inflammation-associated PCa. ( a ) Methylation scores of miR186 promoter were derived from the TCGA database, including prostate cancer samples ( n =270) and normal samples ( n =270). ( b ) Comparison of the methylation levels of miR186 promoter in the top 120 most significant difference paired clinical cases. The dots represent the relative methylation score of miR186 promoter and each pair of dots connected by a dash line denotes a set of paired specimens. ( c ) DNA isolated from normal, chronic inflammation or tumor samples was subjected to methylation-specific PCR using methylated-specific (‘M’) and unmethylated-specific (‘U’) primers from the miR186 promoter. ( d ) Assessment of miR186 levels by in situ hybridization in normal prostate tissues, chronic inflammation specimens, proliferative inflammatory atrophy tissues and adenocarcinoma specimens. ( e ) A schematic representation of the proposed Twist1-dependent site-specific CpG8 methylation through recruiting Dnmt3a to the miR186 promoter to block the transcriptional activity of NF-κB/p65.
    Figure Legend Snippet: The miR186 expression and its promoter methylation are related with inflammation-associated PCa. ( a ) Methylation scores of miR186 promoter were derived from the TCGA database, including prostate cancer samples ( n =270) and normal samples ( n =270). ( b ) Comparison of the methylation levels of miR186 promoter in the top 120 most significant difference paired clinical cases. The dots represent the relative methylation score of miR186 promoter and each pair of dots connected by a dash line denotes a set of paired specimens. ( c ) DNA isolated from normal, chronic inflammation or tumor samples was subjected to methylation-specific PCR using methylated-specific (‘M’) and unmethylated-specific (‘U’) primers from the miR186 promoter. ( d ) Assessment of miR186 levels by in situ hybridization in normal prostate tissues, chronic inflammation specimens, proliferative inflammatory atrophy tissues and adenocarcinoma specimens. ( e ) A schematic representation of the proposed Twist1-dependent site-specific CpG8 methylation through recruiting Dnmt3a to the miR186 promoter to block the transcriptional activity of NF-κB/p65.

    Techniques Used: Expressing, Methylation, Derivative Assay, Isolation, Polymerase Chain Reaction, In Situ Hybridization, Blocking Assay, Activity Assay

    14) Product Images from "Coordinated Changes in DNA Methylation in Antigen-Specific Memory CD4 T Cells"

    Article Title: Coordinated Changes in DNA Methylation in Antigen-Specific Memory CD4 T Cells

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1202267

    Transcriptional activity of a luciferase reporter gene in unmethylated and methylated DMR sequences from the introns of 15 genes. Transient transfections were performed with a control plasmid (pCpGL-EF1 promoter) or pCpGL-EF-DMR in P/I-treated EL-4 T
    Figure Legend Snippet: Transcriptional activity of a luciferase reporter gene in unmethylated and methylated DMR sequences from the introns of 15 genes. Transient transfections were performed with a control plasmid (pCpGL-EF1 promoter) or pCpGL-EF-DMR in P/I-treated EL-4 T

    Techniques Used: Activity Assay, Luciferase, Methylation, Transfection, Plasmid Preparation

    15) Product Images from "Epigenetic Regulation of ZBTB18 Promotes Glioblastoma Progression"

    Article Title: Epigenetic Regulation of ZBTB18 Promotes Glioblastoma Progression

    Journal: Molecular cancer research : MCR

    doi: 10.1158/1541-7786.MCR-16-0494

    The core promoter region of ZBTB18 is essential for promoter activity and is sensitive to DNA methylation. (A) Schematic representation of the ZBTB18 promoter regions cloned in the pGL3 luciferase reporter vector. (B) Analysis of ZBTB18 promoter constructs activity by dual luciferase assay. (C) In vitro methylation assay of ZBTB18 promoter 4. The plasmid DNA was methylated with SssI or HpaII methylase. (D) Control restriction digestion of the methylase reaction using the methylation sensitive (HpaII) and the methylation insensitive (McrBC) restriction enzymes.
    Figure Legend Snippet: The core promoter region of ZBTB18 is essential for promoter activity and is sensitive to DNA methylation. (A) Schematic representation of the ZBTB18 promoter regions cloned in the pGL3 luciferase reporter vector. (B) Analysis of ZBTB18 promoter constructs activity by dual luciferase assay. (C) In vitro methylation assay of ZBTB18 promoter 4. The plasmid DNA was methylated with SssI or HpaII methylase. (D) Control restriction digestion of the methylase reaction using the methylation sensitive (HpaII) and the methylation insensitive (McrBC) restriction enzymes.

    Techniques Used: Activity Assay, DNA Methylation Assay, Clone Assay, Luciferase, Plasmid Preparation, Construct, In Vitro, Methylation

    16) Product Images from "Synthesis of 5? cap-0 and cap-1 RNAs using solid-phase chemistry coupled with enzymatic methylation by human (guanine-N7)-methyl transferase"

    Article Title: Synthesis of 5? cap-0 and cap-1 RNAs using solid-phase chemistry coupled with enzymatic methylation by human (guanine-N7)-methyl transferase

    Journal: RNA

    doi: 10.1261/rna.030932.111

    Time course analysis of guanine- N 7 -methylation of GpppAGUUGU by N 7 -hMTase: 62, 125, 250, and 1000 nM of N 7 -hMTase was incubated with GpppAGUUGU (5) in the presence of methyl donor (AdoMet) containing a tritiated methyl group. The reaction products, collected
    Figure Legend Snippet: Time course analysis of guanine- N 7 -methylation of GpppAGUUGU by N 7 -hMTase: 62, 125, 250, and 1000 nM of N 7 -hMTase was incubated with GpppAGUUGU (5) in the presence of methyl donor (AdoMet) containing a tritiated methyl group. The reaction products, collected

    Techniques Used: Methylation, Incubation

    17) Product Images from "New Shuttle Vector-Based Expression System To Generate Polyhistidine-Tagged Fusion Proteins in Staphylococcus aureus and Escherichia coli"

    Article Title: New Shuttle Vector-Based Expression System To Generate Polyhistidine-Tagged Fusion Proteins in Staphylococcus aureus and Escherichia coli

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.03803-14

    One-step nickel affinity purification of Erm(44)-RGS-His 6 from S. aureus RN4220 (A) and E. coli AG100A or AG100 (B). Cells carried one of the erm (44)- rgs-his 6 expression vectors (pBJW13-RGS-His, pBJW13-HC-RGS-His, and pTJW13-RGS-His) or the empty pBUS1-P
    Figure Legend Snippet: One-step nickel affinity purification of Erm(44)-RGS-His 6 from S. aureus RN4220 (A) and E. coli AG100A or AG100 (B). Cells carried one of the erm (44)- rgs-his 6 expression vectors (pBJW13-RGS-His, pBJW13-HC-RGS-His, and pTJW13-RGS-His) or the empty pBUS1-P

    Techniques Used: Affinity Purification, Expressing

    18) Product Images from "DNA and a CpG Oligonucleotide Derived from Babesia bovis Are Mitogenic for Bovine B Cells"

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

    Journal: Infection and Immunity

    doi:

    Resistance of methylated B. bovis DNA to cleavage by Hpa II. DNAs prepared from E. coli or B. bovis were treated with Sss I methylase and compared with untreated DNA for sensitivity to Hpa II digestion. DNA was visualized after electrophoresis on ethidium bromide-stained agarose gels. Lanes: M, 1-kb markers; 1, untreated DNA; 2, untreated DNA incubated with Hpa II; 3, methylated DNA; 4, methylated DNA incubated with Hpa II.
    Figure Legend Snippet: Resistance of methylated B. bovis DNA to cleavage by Hpa II. DNAs prepared from E. coli or B. bovis were treated with Sss I methylase and compared with untreated DNA for sensitivity to Hpa II digestion. DNA was visualized after electrophoresis on ethidium bromide-stained agarose gels. Lanes: M, 1-kb markers; 1, untreated DNA; 2, untreated DNA incubated with Hpa II; 3, methylated DNA; 4, methylated DNA incubated with Hpa II.

    Techniques Used: Methylation, Electrophoresis, Staining, Incubation

    19) Product Images from "High-resolution functional mapping of a cloned gene by genetic footprinting"

    Article Title: High-resolution functional mapping of a cloned gene by genetic footprinting

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Genetic footprinting of supF . ( A ) Analysis of 36-bp insertional mutations in the plasmid πAN13. DNA from the unselected (U) and selected (S) libraries of mutant supF was used as template for a PCR containing a primer complementary to the insert oligonucleotide, paired with a radiolabeled primer complementary to a region 160 bp upstream of the transcription start site (π267L). A schematic diagram of the supF gene is shown to the right, aligned with the corresponding PCR products. Sequenced plasmid DNA and PCR products from selected sequenced mutations were run adjacent to the footprinted DNA as molecular weight markers and were used to draw the boxes representing various regions of the supF gene (boxes marked −10 and −35 correspond to promoter elements; P, regions of precursor tRNA that are removed upon processing; M, mature tRNA. Black box within the mature tRNA corresponds to the variable loop). ( B and C ) Analysis of libraries of 36-bp insertional (INS) or 12-bp substitution (SUB) mutations in the plasmid πchloro. In B , the labeled primer (CAM60supF) was complementary to a region 141 bp upstream of the transcription start site; in C , the labeled primer (π858U) was complementary to a region 183 bp downstream of the start site. In lanes 3, 4, 9, and 10, insertional mutants were analyzed by a PCR in which the inserted sequence served as a priming site, as in A . In lanes 5–8 and 11–14, PCR products generated using primers π858U and CAM60supF (one of which was labeled as indicated above) were digested with a restriction endonuclease, whose recognition sequence was unique to the inserted sequence. The ladder (LAD) in lane 15 was generated by pooling 12 unique sequenced clones of replacement mutants that were made in the plasmid πAN13. Ladder DNA was PCR amplified with primer π267L and labeled primer π858U, and digested with a restriction endonuclease as described. ( D ) The boxed region from lanes 13 and 14 is enlarged to show the PCR products corresponding to substitution mutants replacing all or part of the −10 element. The sequence corresponding to each mutant is indicated to the left with the substituted sequence indicated in boldface type. The wild-type sequence of this region is shown at the bottom, with the −10 element indicated by a box.
    Figure Legend Snippet: Genetic footprinting of supF . ( A ) Analysis of 36-bp insertional mutations in the plasmid πAN13. DNA from the unselected (U) and selected (S) libraries of mutant supF was used as template for a PCR containing a primer complementary to the insert oligonucleotide, paired with a radiolabeled primer complementary to a region 160 bp upstream of the transcription start site (π267L). A schematic diagram of the supF gene is shown to the right, aligned with the corresponding PCR products. Sequenced plasmid DNA and PCR products from selected sequenced mutations were run adjacent to the footprinted DNA as molecular weight markers and were used to draw the boxes representing various regions of the supF gene (boxes marked −10 and −35 correspond to promoter elements; P, regions of precursor tRNA that are removed upon processing; M, mature tRNA. Black box within the mature tRNA corresponds to the variable loop). ( B and C ) Analysis of libraries of 36-bp insertional (INS) or 12-bp substitution (SUB) mutations in the plasmid πchloro. In B , the labeled primer (CAM60supF) was complementary to a region 141 bp upstream of the transcription start site; in C , the labeled primer (π858U) was complementary to a region 183 bp downstream of the start site. In lanes 3, 4, 9, and 10, insertional mutants were analyzed by a PCR in which the inserted sequence served as a priming site, as in A . In lanes 5–8 and 11–14, PCR products generated using primers π858U and CAM60supF (one of which was labeled as indicated above) were digested with a restriction endonuclease, whose recognition sequence was unique to the inserted sequence. The ladder (LAD) in lane 15 was generated by pooling 12 unique sequenced clones of replacement mutants that were made in the plasmid πAN13. Ladder DNA was PCR amplified with primer π267L and labeled primer π858U, and digested with a restriction endonuclease as described. ( D ) The boxed region from lanes 13 and 14 is enlarged to show the PCR products corresponding to substitution mutants replacing all or part of the −10 element. The sequence corresponding to each mutant is indicated to the left with the substituted sequence indicated in boldface type. The wild-type sequence of this region is shown at the bottom, with the −10 element indicated by a box.

    Techniques Used: Footprinting, Plasmid Preparation, Mutagenesis, Polymerase Chain Reaction, Molecular Weight, Labeling, Sequencing, Generated, Clone Assay, Amplification

    20) Product Images from "DNA Methylation of the Gonadal Aromatase (cyp19a) Promoter Is Involved in Temperature-Dependent Sex Ratio Shifts in the European Sea Bass"

    Article Title: DNA Methylation of the Gonadal Aromatase (cyp19a) Promoter Is Involved in Temperature-Dependent Sex Ratio Shifts in the European Sea Bass

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002447

    Effects of methylation on sea bass cyp19a promoter activity in vitro . HEK293T cells were transfected with pGL3- cyp19a methylated and unmethylated promoter vectors. Transcription factors SF1 and Foxl2 were cotransfected with the sb cyp19a promoter to activate promoter luciferase activity. Transfected methylated and unmethylated groups were as follows (see Materials and Methods for amounts used): 1) plasmid β-galactosidase (Ctrl); 2) sb cyp19a promoter cloned into pGL3-basic luciferase reporter plasmid (cyp19a); 3) sb cyp19a and tilapia SF1 cloned into pCDNA3.1 expression plasmid (cyp19a+SF1); 4) sb cyp19a and tilapia Foxl2 cloned into pCDNA3.1 expression plasmid (cyp19a+Foxl2); 5) sb cyp19a , tilapia SF1 and Foxl2 cloned into pCDNA3.1 (cyp19a+SF1+Foxl2). The Student's t -test was used to compare methylated and unmethylated vectors. Significant differences are denoted by an asterisk and were as follows: cyp19a, P = 0.006; cyp19a+SF1, P = 0.006; cyp19a+Foxl2, P = 0.003; cyp19a+SF1+Foxl2, P = 0.013. Also, SF1 and Foxl2 exhibited an additive effect since the activation of sb cyp19a promoter was significantly higher when both were transfected together. Values are shown as mean ± S.E.M. (n = 2–4). Insert: Successful vector methylation verification by analysis of band patterns on electrophoresis gel after digestion of the purified plasmids with the McrBC enzyme. Lane 1, 0.5 µg sb cyp19a -pGL3; lane 2, 0.5 µg sb cyp19a -pGL3 treated with McrBC ; lane 3, 0.5 µg Sss I-methylated sb cyp19a -pGL3; lane 4, 0.5 µg Sss I-methylated sb cyp19a -pGL3 treated with McrBC ; lane 5, 1 Kb marker; lane 6, 100 bp marker. The electrophoresis gel shows that, as expected, only the methylated vector was digested.
    Figure Legend Snippet: Effects of methylation on sea bass cyp19a promoter activity in vitro . HEK293T cells were transfected with pGL3- cyp19a methylated and unmethylated promoter vectors. Transcription factors SF1 and Foxl2 were cotransfected with the sb cyp19a promoter to activate promoter luciferase activity. Transfected methylated and unmethylated groups were as follows (see Materials and Methods for amounts used): 1) plasmid β-galactosidase (Ctrl); 2) sb cyp19a promoter cloned into pGL3-basic luciferase reporter plasmid (cyp19a); 3) sb cyp19a and tilapia SF1 cloned into pCDNA3.1 expression plasmid (cyp19a+SF1); 4) sb cyp19a and tilapia Foxl2 cloned into pCDNA3.1 expression plasmid (cyp19a+Foxl2); 5) sb cyp19a , tilapia SF1 and Foxl2 cloned into pCDNA3.1 (cyp19a+SF1+Foxl2). The Student's t -test was used to compare methylated and unmethylated vectors. Significant differences are denoted by an asterisk and were as follows: cyp19a, P = 0.006; cyp19a+SF1, P = 0.006; cyp19a+Foxl2, P = 0.003; cyp19a+SF1+Foxl2, P = 0.013. Also, SF1 and Foxl2 exhibited an additive effect since the activation of sb cyp19a promoter was significantly higher when both were transfected together. Values are shown as mean ± S.E.M. (n = 2–4). Insert: Successful vector methylation verification by analysis of band patterns on electrophoresis gel after digestion of the purified plasmids with the McrBC enzyme. Lane 1, 0.5 µg sb cyp19a -pGL3; lane 2, 0.5 µg sb cyp19a -pGL3 treated with McrBC ; lane 3, 0.5 µg Sss I-methylated sb cyp19a -pGL3; lane 4, 0.5 µg Sss I-methylated sb cyp19a -pGL3 treated with McrBC ; lane 5, 1 Kb marker; lane 6, 100 bp marker. The electrophoresis gel shows that, as expected, only the methylated vector was digested.

    Techniques Used: Methylation, Activity Assay, In Vitro, Transfection, Luciferase, Plasmid Preparation, Clone Assay, Expressing, Activation Assay, Electrophoresis, Purification, Marker

    21) Product Images from "Uncovering human METTL12 as a mitochondrial methyltransferase that modulates citrate synthase activity through metabolite-sensitive lysine methylation"

    Article Title: Uncovering human METTL12 as a mitochondrial methyltransferase that modulates citrate synthase activity through metabolite-sensitive lysine methylation

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.808451

    Human METTL12 is a protein-specific MTase. A , targeting of METTL12 gene in human HAP1 WT cells by CRISPR/Cas9 generated METTL12 KO cells containing a 1 base pair insertion in the METTL12 gene, located upstream of motif Post I, resulting in generation of truncated METTL12 protein. The dashed lines interrupting the open reading frames correspond to 177 nucleotides, i.e. 59 amino acids. B , METTL12-dependent protein methylation in cell extracts. Mitochondrial extracts from HAP1 WT or METTL12 KO cells were incubated with [ 3 H]AdoMet and recombinant human METTL12. Methylation reactions were separated by SDS-PAGE and transferred to a membrane. Methylation was visualized by fluorography ( top ) of the Ponceau S-stained membrane ( bottom ). Arrows indicate the positions of the ∼48 kDa substrate and METTL12. C , D107A mutation abrogates enzymatic activity of METTL12. Mitochondrial extracts from METTL12 KO cells were incubated with [ 3 H]AdoMet and recombinant human METTL12, either WT or D107A-mutated. Methylation was analyzed as in B . Note: in panels B and C different levels of background (non-METTL12-dependent) methylation are observed; this is likely due to differences in the purity of the mitochondrial extracts.
    Figure Legend Snippet: Human METTL12 is a protein-specific MTase. A , targeting of METTL12 gene in human HAP1 WT cells by CRISPR/Cas9 generated METTL12 KO cells containing a 1 base pair insertion in the METTL12 gene, located upstream of motif Post I, resulting in generation of truncated METTL12 protein. The dashed lines interrupting the open reading frames correspond to 177 nucleotides, i.e. 59 amino acids. B , METTL12-dependent protein methylation in cell extracts. Mitochondrial extracts from HAP1 WT or METTL12 KO cells were incubated with [ 3 H]AdoMet and recombinant human METTL12. Methylation reactions were separated by SDS-PAGE and transferred to a membrane. Methylation was visualized by fluorography ( top ) of the Ponceau S-stained membrane ( bottom ). Arrows indicate the positions of the ∼48 kDa substrate and METTL12. C , D107A mutation abrogates enzymatic activity of METTL12. Mitochondrial extracts from METTL12 KO cells were incubated with [ 3 H]AdoMet and recombinant human METTL12, either WT or D107A-mutated. Methylation was analyzed as in B . Note: in panels B and C different levels of background (non-METTL12-dependent) methylation are observed; this is likely due to differences in the purity of the mitochondrial extracts.

    Techniques Used: CRISPR, Generated, Methylation, Incubation, Recombinant, SDS Page, Staining, Mutagenesis, Activity Assay

    22) Product Images from "Maintenance DNA methyltransferase activity in the presence of oxidized forms of 5-methylcytosine: structural basis for ten eleven translocation-mediated DNA demethylation"

    Article Title: Maintenance DNA methyltransferase activity in the presence of oxidized forms of 5-methylcytosine: structural basis for ten eleven translocation-mediated DNA demethylation

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.8b00683

    Molecular dynamics (MD) simulations demonstrate an incremental spatial displacement of oxo-mC from the TRD hydrophobic binding pocket. A . Residues Cys1501, Leu1502, and Met1535 make up the target recognition domain (TRD) and harbor the methyl group of mC, providing the specificity of DNMT1 for hemi-methylated DNA. The MD simulations quantify the displacement of the oxidized forms of mC from these residues in the TRD: B. Cys1501 C. Leu1502 D. Met1535
    Figure Legend Snippet: Molecular dynamics (MD) simulations demonstrate an incremental spatial displacement of oxo-mC from the TRD hydrophobic binding pocket. A . Residues Cys1501, Leu1502, and Met1535 make up the target recognition domain (TRD) and harbor the methyl group of mC, providing the specificity of DNMT1 for hemi-methylated DNA. The MD simulations quantify the displacement of the oxidized forms of mC from these residues in the TRD: B. Cys1501 C. Leu1502 D. Met1535

    Techniques Used: Binding Assay, Methylation

    23) Product Images from "Removing the needle from the haystack: Enrichment of Wolbachia endosymbiont transcripts from host nematode RNA by Cappable-seq™"

    Article Title: Removing the needle from the haystack: Enrichment of Wolbachia endosymbiont transcripts from host nematode RNA by Cappable-seq™

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0173186

    (A) Transcript abundance of all 940 annotated Wolbachia genes (listed by geneID number) reveals over 95% of Wolbachia transcripts from B . malayi adult male RNA were enriched using the Cappable-seq technique. Each transcript is indicated by in red (the FPKM value in the total RNA) and blue (the FPKM value in capped-RNA sample)(B) A closer view (note difference in y-axis scales between panel A and B) of transcript abundance reveals an enrichment in Wolbachia transcripts in the capped RNA sample.
    Figure Legend Snippet: (A) Transcript abundance of all 940 annotated Wolbachia genes (listed by geneID number) reveals over 95% of Wolbachia transcripts from B . malayi adult male RNA were enriched using the Cappable-seq technique. Each transcript is indicated by in red (the FPKM value in the total RNA) and blue (the FPKM value in capped-RNA sample)(B) A closer view (note difference in y-axis scales between panel A and B) of transcript abundance reveals an enrichment in Wolbachia transcripts in the capped RNA sample.

    Techniques Used:

    (A) Transcript abundance of all 940 annotated Wolbachia genes (listed by geneID number) reveals over 95% of Wolbachia transcripts from B . malayi MF RNA were enriched using the Cappable-seq technique. Each transcript is indicated by in red (the FPKM value in the total RNA) and blue (the FPKM value in capped-RNA sample)(B) A closer view (note difference in y-axis scales between panel A and B) of transcript abundance reveals an enrichment in Wolbachia transcripts in the capped RNA sample.
    Figure Legend Snippet: (A) Transcript abundance of all 940 annotated Wolbachia genes (listed by geneID number) reveals over 95% of Wolbachia transcripts from B . malayi MF RNA were enriched using the Cappable-seq technique. Each transcript is indicated by in red (the FPKM value in the total RNA) and blue (the FPKM value in capped-RNA sample)(B) A closer view (note difference in y-axis scales between panel A and B) of transcript abundance reveals an enrichment in Wolbachia transcripts in the capped RNA sample.

    Techniques Used:

    A. Transcript coverage (FPKM) of Wolbachia genes reveals over 88% of Wolbachia transcripts from B . malayi adult male RNA were enriched using the Cappable-seq technique. A closer view of transcript abundance (inset) reveals most Wolbachia transcripts in total RNA are present in very low abundance, whereas the Wolbachia transcripts are more abundant in the capped RNA sample. Points along the y-axis are indicative of Wolbachia transcripts that were undetectable in total RNA that were detected in the capped RNA sample. B. Transcript coverage (FPKM) of Wolbachia genes reveals over 95% of Wolbachia transcripts from B . malayi MF RNA were enriched using the Cappable-seq technique. A closer view of transcript abundance (inset) reveals most Wolbachia transcripts in total RNA are present in very low abundance, whereas the Wolbachia transcripts are more abundant in the capped RNA sample. Points along the y-axis are indicative of Wolbachia transcripts that were undetectable in total RNA that were detected in the capped RNA sample.
    Figure Legend Snippet: A. Transcript coverage (FPKM) of Wolbachia genes reveals over 88% of Wolbachia transcripts from B . malayi adult male RNA were enriched using the Cappable-seq technique. A closer view of transcript abundance (inset) reveals most Wolbachia transcripts in total RNA are present in very low abundance, whereas the Wolbachia transcripts are more abundant in the capped RNA sample. Points along the y-axis are indicative of Wolbachia transcripts that were undetectable in total RNA that were detected in the capped RNA sample. B. Transcript coverage (FPKM) of Wolbachia genes reveals over 95% of Wolbachia transcripts from B . malayi MF RNA were enriched using the Cappable-seq technique. A closer view of transcript abundance (inset) reveals most Wolbachia transcripts in total RNA are present in very low abundance, whereas the Wolbachia transcripts are more abundant in the capped RNA sample. Points along the y-axis are indicative of Wolbachia transcripts that were undetectable in total RNA that were detected in the capped RNA sample.

    Techniques Used:

    24) Product Images from "The use of Multiple Displacement Amplified DNA as a control for Methylation Specific PCR, Pyrosequencing, Bisulfite Sequencing and Methylation-Sensitive Restriction Enzyme PCR"

    Article Title: The use of Multiple Displacement Amplified DNA as a control for Methylation Specific PCR, Pyrosequencing, Bisulfite Sequencing and Methylation-Sensitive Restriction Enzyme PCR

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-8-91

    Bisulfite sequencing results for MMP-14 . a) When genomic DNA (lane 1) and bisulfite treated mDNA (lane 2) and uDNA (lane 3) were used as template for sequencing in combination with primers for MMP-14 a PCR product was generated for all samples but not the negative control (lane 4). Sequencing results for b) non-amplified genomic DNA, c) uDNA and d) mDNA demonstrate that MDA treatment generates DNA (uDNA) free of all methylation as when it is bisulfite treated all cytosine are converted to thymine [indicated by asterix (*)]. In addition, sequencing also demonstrates that M.SssI treatment (mDNA) methylates CpG motifs as cytosines are retained when present as part of a CpG dinucleotide (indicated by *).
    Figure Legend Snippet: Bisulfite sequencing results for MMP-14 . a) When genomic DNA (lane 1) and bisulfite treated mDNA (lane 2) and uDNA (lane 3) were used as template for sequencing in combination with primers for MMP-14 a PCR product was generated for all samples but not the negative control (lane 4). Sequencing results for b) non-amplified genomic DNA, c) uDNA and d) mDNA demonstrate that MDA treatment generates DNA (uDNA) free of all methylation as when it is bisulfite treated all cytosine are converted to thymine [indicated by asterix (*)]. In addition, sequencing also demonstrates that M.SssI treatment (mDNA) methylates CpG motifs as cytosines are retained when present as part of a CpG dinucleotide (indicated by *).

    Techniques Used: Methylation Sequencing, Sequencing, Polymerase Chain Reaction, Generated, Negative Control, Amplification, Multiple Displacement Amplification, Methylation

    Methylation-Sensitive Restriction Enzyme PCR for MMP-1 and MMP-3 . a) PCR using primers spanning the restriction site for MMP-1 and MMP-3 gave a PCR product with mDNA but not with uDNA. In contrast, undigested samples gave PCR products for both mDNA and uDNA. b) PCR using digested DNA from MDA-MB231 (231), MDA-MB468 (468) and HFFF2 identified that the CpG motif is methylated for all three cell lines in the MMP-3 amplicon, but only for MDA-MB468 (468) and HFFF2 for the MMP-1 amplicon, with the MDA-MB231 (231) being unmethylated. However, the undigested DNA gave a PCR product with all three cells lines.
    Figure Legend Snippet: Methylation-Sensitive Restriction Enzyme PCR for MMP-1 and MMP-3 . a) PCR using primers spanning the restriction site for MMP-1 and MMP-3 gave a PCR product with mDNA but not with uDNA. In contrast, undigested samples gave PCR products for both mDNA and uDNA. b) PCR using digested DNA from MDA-MB231 (231), MDA-MB468 (468) and HFFF2 identified that the CpG motif is methylated for all three cell lines in the MMP-3 amplicon, but only for MDA-MB468 (468) and HFFF2 for the MMP-1 amplicon, with the MDA-MB231 (231) being unmethylated. However, the undigested DNA gave a PCR product with all three cells lines.

    Techniques Used: Methylation, Polymerase Chain Reaction, Multiple Displacement Amplification, Amplification

    25) Product Images from "High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion"

    Article Title: High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0163184

    The workflow of the methyl-sensitive fluorescence polarization (MSFP) assay. Lambda (λ) and human genomic DNA are restricted by MspI (white) or by HpaII each alone or by a combination of HpaII and HpyCH4IV (grey) (Step 1). Subsequently, digested DNA with CpG overhangs at the 5' termini are terminally extended with fluorescence-labelled TAMRA-dCTP (C*, *C) (Step 2). TAMRA-dCTP incorporated into DNA is quantified by fluorescence polarization directly on the plate without additional purification procedures.
    Figure Legend Snippet: The workflow of the methyl-sensitive fluorescence polarization (MSFP) assay. Lambda (λ) and human genomic DNA are restricted by MspI (white) or by HpaII each alone or by a combination of HpaII and HpyCH4IV (grey) (Step 1). Subsequently, digested DNA with CpG overhangs at the 5' termini are terminally extended with fluorescence-labelled TAMRA-dCTP (C*, *C) (Step 2). TAMRA-dCTP incorporated into DNA is quantified by fluorescence polarization directly on the plate without additional purification procedures.

    Techniques Used: Fluorescence, Purification

    26) Product Images from "Epigenetic Inactivation of Heparan Sulfate (Glucosamine) 3-O-Sulfotransferase 2 in Lung Cancer and Its Role in Tumorigenesis"

    Article Title: Epigenetic Inactivation of Heparan Sulfate (Glucosamine) 3-O-Sulfotransferase 2 in Lung Cancer and Its Role in Tumorigenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0079634

    Reporter assay. The dual luciferase activities of four HS3ST2 promoter constructs were measured in H23 (A) and HEK-293T (B) cell lines. One kb promoter construct was serially deleted, and luciferase activity of each construct was compared between constructs treated with (gray bar) and without SssI methylase (black bar).
    Figure Legend Snippet: Reporter assay. The dual luciferase activities of four HS3ST2 promoter constructs were measured in H23 (A) and HEK-293T (B) cell lines. One kb promoter construct was serially deleted, and luciferase activity of each construct was compared between constructs treated with (gray bar) and without SssI methylase (black bar).

    Techniques Used: Reporter Assay, Luciferase, Construct, Activity Assay

    27) Product Images from "Target identification reveals protein arginine methyltransferase 1 is a potential target of phenyl vinyl sulfone and its derivatives"

    Article Title: Target identification reveals protein arginine methyltransferase 1 is a potential target of phenyl vinyl sulfone and its derivatives

    Journal: Bioscience Reports

    doi: 10.1042/BSR20171717

    Effects of PVS, PVSN, Bay 11-7082, and AMI-1 on the in vitro enzyme activity of PRMT1 ( A ) and anti-PVS detection of PVS, PVSN, and Bay 11-7082 tagging of PRMT1 ( B ) The reaction mixture contained 100 ng of recombinant PRMT1, 1 µg of full-length recombinant Histone H4, 1 µM S-adenosylmethionine, various concentrations of PVS, PVSN, Bay 11-7082, or AMI-1 (AMI) in a total volume of 100 µl in PBS, pH 7.4. DMSO was used in the control. The reaction was incubated at 37°C for 30 min and 10 µl of the reaction product was examined by SDS/PAGE and immunoblotting with anti-H4R3me2a for the recognition of Histone H4 asymmetric dimethylation at Arg3 ( A ) and anti-PVS ( B ). The 0 µM groups were treated with the same volume of DMSO.
    Figure Legend Snippet: Effects of PVS, PVSN, Bay 11-7082, and AMI-1 on the in vitro enzyme activity of PRMT1 ( A ) and anti-PVS detection of PVS, PVSN, and Bay 11-7082 tagging of PRMT1 ( B ) The reaction mixture contained 100 ng of recombinant PRMT1, 1 µg of full-length recombinant Histone H4, 1 µM S-adenosylmethionine, various concentrations of PVS, PVSN, Bay 11-7082, or AMI-1 (AMI) in a total volume of 100 µl in PBS, pH 7.4. DMSO was used in the control. The reaction was incubated at 37°C for 30 min and 10 µl of the reaction product was examined by SDS/PAGE and immunoblotting with anti-H4R3me2a for the recognition of Histone H4 asymmetric dimethylation at Arg3 ( A ) and anti-PVS ( B ). The 0 µM groups were treated with the same volume of DMSO.

    Techniques Used: In Vitro, Activity Assay, Recombinant, Incubation, SDS Page

    28) Product Images from "Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells"

    Article Title: Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0096512

    The short DNA regions containing the GC-rich region, the RARE half-site, and the TATA box in the 5′-flanking region of the Aldh1a2 genes are well conserved among various species. The sequence of the 5′-flanking region of the mouse ( Mus musculus ) Aldh1a2 gene was compared with that of the human ( Homo sapiens ) ALDH1A2 gene, that of rat ( Rattus norvegicus ) Aldh1a2 gene, that of cattle ( Bos taurus ) ALDH1A2 gene, that of chicken ( Gallus gallus ) ALDH1A2 gene, and that of zebrafish ( Danio rerio ) aldh1a2 gene. The sequence data were obtained using the NCBI MapViewer. The shaded regions indicate homology with the mouse sequence. The locations of the conserved RARE half-sites (RARE-h) and TATA boxes are indicated by boxes.
    Figure Legend Snippet: The short DNA regions containing the GC-rich region, the RARE half-site, and the TATA box in the 5′-flanking region of the Aldh1a2 genes are well conserved among various species. The sequence of the 5′-flanking region of the mouse ( Mus musculus ) Aldh1a2 gene was compared with that of the human ( Homo sapiens ) ALDH1A2 gene, that of rat ( Rattus norvegicus ) Aldh1a2 gene, that of cattle ( Bos taurus ) ALDH1A2 gene, that of chicken ( Gallus gallus ) ALDH1A2 gene, and that of zebrafish ( Danio rerio ) aldh1a2 gene. The sequence data were obtained using the NCBI MapViewer. The shaded regions indicate homology with the mouse sequence. The locations of the conserved RARE half-sites (RARE-h) and TATA boxes are indicated by boxes.

    Techniques Used: Sequencing

    RA enhances GM-CSF-induced Aldh1a2 expression via the RARα/RXRα heterodimer bound to the RARE half-site. ( A ) Localization of the putative RARE half-sites ( underlined ) in Probe C and their mutants, Probe C(RARE-h mt1) and Probe C(RARE-h mt2), are shown. COS-7 cells were transfected with the 0.5 µg of pSG5-RARα and/or pSG5-RXRα. One day after transfection, cell lysates were subjected to DNAP assay using the biotinylated DNA probes. The precipitates were analyzed by Western blotting using anti-RARα( upper panel ) and RXRα ( lower panel ) Abs. ( B ) Flt3L-generated BM-DCs were cultured with 10 ng/ml GM-CSF for 16 h in the presence or absence of 100 nM RA. LE540 (1 µM) was added to the indicated cultures. After the culture, Aldh1a2 mRNA expression was assessed by real-time PCR. The Aldh1a2 mRNA expression level in the cells incubated with medium alone for 16 h was set to 1. ( C ) BM-DCs were cultured with ( closed circle ) or without ( open circle ) 10 ng/ml GM-CSF for 16 h in the presence of graded concentrations of RA. After the culture, Aldh1a2 mRNA expression was assessed by real-time PCR. Relative expression levels were calculated by defining the Aldh1a2 mRNA expression level in the cells incubated with medium alone for 16 h was set to 1. Asterisks indicate a significant difference (*** p
    Figure Legend Snippet: RA enhances GM-CSF-induced Aldh1a2 expression via the RARα/RXRα heterodimer bound to the RARE half-site. ( A ) Localization of the putative RARE half-sites ( underlined ) in Probe C and their mutants, Probe C(RARE-h mt1) and Probe C(RARE-h mt2), are shown. COS-7 cells were transfected with the 0.5 µg of pSG5-RARα and/or pSG5-RXRα. One day after transfection, cell lysates were subjected to DNAP assay using the biotinylated DNA probes. The precipitates were analyzed by Western blotting using anti-RARα( upper panel ) and RXRα ( lower panel ) Abs. ( B ) Flt3L-generated BM-DCs were cultured with 10 ng/ml GM-CSF for 16 h in the presence or absence of 100 nM RA. LE540 (1 µM) was added to the indicated cultures. After the culture, Aldh1a2 mRNA expression was assessed by real-time PCR. The Aldh1a2 mRNA expression level in the cells incubated with medium alone for 16 h was set to 1. ( C ) BM-DCs were cultured with ( closed circle ) or without ( open circle ) 10 ng/ml GM-CSF for 16 h in the presence of graded concentrations of RA. After the culture, Aldh1a2 mRNA expression was assessed by real-time PCR. Relative expression levels were calculated by defining the Aldh1a2 mRNA expression level in the cells incubated with medium alone for 16 h was set to 1. Asterisks indicate a significant difference (*** p

    Techniques Used: Expressing, Transfection, Western Blot, Generated, Cell Culture, Real-time Polymerase Chain Reaction, Incubation

    Sp1 binds to the Aldh1a2 promoter region. ( A ) The locations and nucleotide sequences corresponding to Probe A, Probe B, and Probe C in the 5′-flanking region of the mouse Aldh1a2 gene are shown. COS-7 cells were transfected with the 0.5 µg of pCMV-Myc-Sp1 or control empty vector. One day after transfection, cell lysates were analyzed for DNA-binding activity by DNAP assay using the indicated biotinylated DNA probes and anti-Myc Ab. (B) Flt3L-generated BM-DCs were cultured in the presence or absence of 10 ng/ml GM-CSF. After 16 h, nuclear extracts were analyzed for the presence of lamin B1 and Sp1 by Western blotting using anti-lamin B1 and anti-Sp1 Abs ( left panel ), or assessed for DNA binding activity by DNAP assay using biotinylated DNA Probe C and anti-Sp1 Ab ( right panel ). Data are representative of at least three independent experiments.
    Figure Legend Snippet: Sp1 binds to the Aldh1a2 promoter region. ( A ) The locations and nucleotide sequences corresponding to Probe A, Probe B, and Probe C in the 5′-flanking region of the mouse Aldh1a2 gene are shown. COS-7 cells were transfected with the 0.5 µg of pCMV-Myc-Sp1 or control empty vector. One day after transfection, cell lysates were analyzed for DNA-binding activity by DNAP assay using the indicated biotinylated DNA probes and anti-Myc Ab. (B) Flt3L-generated BM-DCs were cultured in the presence or absence of 10 ng/ml GM-CSF. After 16 h, nuclear extracts were analyzed for the presence of lamin B1 and Sp1 by Western blotting using anti-lamin B1 and anti-Sp1 Abs ( left panel ), or assessed for DNA binding activity by DNAP assay using biotinylated DNA Probe C and anti-Sp1 Ab ( right panel ). Data are representative of at least three independent experiments.

    Techniques Used: Transfection, Plasmid Preparation, Binding Assay, Activity Assay, Generated, Cell Culture, Western Blot

    Methylation of the CpG island in the Aldh1a2 promoter prohibits Sp1 to activate the promoter, whereas the Aldh1a2 promoter is largely unmethylated in BM-pDCs as well as in BM-cDCs. ( A ) pCpGL-basic and pCpGL-RALDH2 (−873) reporter vectors were methylated with 1.25 µg of M.SssI. COS-7 cells were transfected with methylated or unmethylated pCpGL-basic or pCpGL-RALDH2 (−873) reporter vector in combination with or without the 0.5 µg of pCMV-Myc-Sp1 expression vector. One day after transfection, luciferase activity was measured. Relative promoter activities were calculated by arbitrarily defining the activity of pCpGL-basic alone as 1. ( B ) COS-7 cells were transfected with pCMV-Myc-Sp1. One day after transfection, cell lysates were analyzed for DNA binding activity by DNAP assay using DNA Probe B and Probe C methylated with M.SssI or left unmethylated. The bound proteins were analyzed by SDS-PAGE followed by Western blotting with anti-Myc Ab. ( C ) Flt3L-generated BM-DCs were stained with allophycocyanin-labeled anti-CD11c Ab and phycoerythrin-labeled anti-B220 Ab, and were sorted to cDC and pDC fractions with a FACSAria. ( D ) Sorted BM-pDCs and BM-cDCs were cultured for 16 h with or without 10 ng/ml GM-CSF. Expression of Aldh1a2 mRNA was analyzed by real-time PCR. Relative expression levels were calculated by defining the Aldh1a2 mRNA expression in the cells incubated with medium alone for 16 h was set to 1. Data in (A and D) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (*** p
    Figure Legend Snippet: Methylation of the CpG island in the Aldh1a2 promoter prohibits Sp1 to activate the promoter, whereas the Aldh1a2 promoter is largely unmethylated in BM-pDCs as well as in BM-cDCs. ( A ) pCpGL-basic and pCpGL-RALDH2 (−873) reporter vectors were methylated with 1.25 µg of M.SssI. COS-7 cells were transfected with methylated or unmethylated pCpGL-basic or pCpGL-RALDH2 (−873) reporter vector in combination with or without the 0.5 µg of pCMV-Myc-Sp1 expression vector. One day after transfection, luciferase activity was measured. Relative promoter activities were calculated by arbitrarily defining the activity of pCpGL-basic alone as 1. ( B ) COS-7 cells were transfected with pCMV-Myc-Sp1. One day after transfection, cell lysates were analyzed for DNA binding activity by DNAP assay using DNA Probe B and Probe C methylated with M.SssI or left unmethylated. The bound proteins were analyzed by SDS-PAGE followed by Western blotting with anti-Myc Ab. ( C ) Flt3L-generated BM-DCs were stained with allophycocyanin-labeled anti-CD11c Ab and phycoerythrin-labeled anti-B220 Ab, and were sorted to cDC and pDC fractions with a FACSAria. ( D ) Sorted BM-pDCs and BM-cDCs were cultured for 16 h with or without 10 ng/ml GM-CSF. Expression of Aldh1a2 mRNA was analyzed by real-time PCR. Relative expression levels were calculated by defining the Aldh1a2 mRNA expression in the cells incubated with medium alone for 16 h was set to 1. Data in (A and D) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (*** p

    Techniques Used: Methylation, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Binding Assay, SDS Page, Western Blot, Generated, Staining, Labeling, Cell Culture, Real-time Polymerase Chain Reaction, Incubation

    Sp1 participates in the Aldh1a2 expression. ( A ) The genomic organization of the mouse Aldh1a2 gene and its 5′-flanking region is shown. A fragment containing exon 1 and its 5′-flanking region from −2,600 to +182 was inserted into reporter vectors. DNA binding sites (STAT-binding sites, NF-κB binding sites, a SREBP binding site, and putative RARE half-sites (RARE-h)), the TATA box, and the GC-rich region in the fragment are indicated. ( B ) Flt3L-generated BM-DCs were cultured with 10 ng/ml GM-CSF for 16 h in the presence or absence of 1 µM mithramycin A. After the culture, Aldh1a2 mRNA expression was assessed by real-time PCR ( Left panel ), and protein expression of RALDH2 (ALDH1A2) and α-tubulin was analyzed by Western blotting ( Right panel ). Relative mRNA expression levels were calculated by defining the Aldh1a2 mRNA expression level in the cells incubated with medium alone for 16 h was set to 1 ( Left panel ). Data are representative of three ( Left panel ) or two ( Right panel ) independent experiments. ( C ) Serial-deletion fragments derived from the 5′-flanking region of the mouse Aldh1a2 gene were inserted in the reporter vector, pGL3-basic. COS-7 cells were transfected in triplicate with one of the deletion constructs (1.25 µg) or the pGL3-RALDH2 (−2,600) reporter vector (1.25 µg) in combination with or without the 0.5 µg of pCMV-Myc-Sp1 expression vector. One day after the transfection, luciferase activity was measured. Relative promoter activities were calculated by arbitrarily defining the activity of pGL3-basic alone as 1. Data are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (** p
    Figure Legend Snippet: Sp1 participates in the Aldh1a2 expression. ( A ) The genomic organization of the mouse Aldh1a2 gene and its 5′-flanking region is shown. A fragment containing exon 1 and its 5′-flanking region from −2,600 to +182 was inserted into reporter vectors. DNA binding sites (STAT-binding sites, NF-κB binding sites, a SREBP binding site, and putative RARE half-sites (RARE-h)), the TATA box, and the GC-rich region in the fragment are indicated. ( B ) Flt3L-generated BM-DCs were cultured with 10 ng/ml GM-CSF for 16 h in the presence or absence of 1 µM mithramycin A. After the culture, Aldh1a2 mRNA expression was assessed by real-time PCR ( Left panel ), and protein expression of RALDH2 (ALDH1A2) and α-tubulin was analyzed by Western blotting ( Right panel ). Relative mRNA expression levels were calculated by defining the Aldh1a2 mRNA expression level in the cells incubated with medium alone for 16 h was set to 1 ( Left panel ). Data are representative of three ( Left panel ) or two ( Right panel ) independent experiments. ( C ) Serial-deletion fragments derived from the 5′-flanking region of the mouse Aldh1a2 gene were inserted in the reporter vector, pGL3-basic. COS-7 cells were transfected in triplicate with one of the deletion constructs (1.25 µg) or the pGL3-RALDH2 (−2,600) reporter vector (1.25 µg) in combination with or without the 0.5 µg of pCMV-Myc-Sp1 expression vector. One day after the transfection, luciferase activity was measured. Relative promoter activities were calculated by arbitrarily defining the activity of pGL3-basic alone as 1. Data are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (** p

    Techniques Used: Expressing, Binding Assay, Generated, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Incubation, Derivative Assay, Plasmid Preparation, Transfection, Construct, Luciferase, Activity Assay

    The MEK1-ERK-signaling pathway and the p38 MAPK-signaling pathway are required for the GM-CSF-induced Aldh1a2 expression and nuclear translocation of Sp1 in BM-DCs. ( A ) Flt3L-generated BM-DCs were cultured with or without 10 ng/ml GM-CSF for 16 h in the presence or absence of 50 µM PD98059 (PD) or 25 µM SB203580 (SB). After the culture, Aldh1a2 gene expression was assessed by real-time PCR. The Aldh1a2 mRNA expression level in the cells incubated with medium alone for 16 h was set to 1. The results are shown as the mean + SD of triplicate cultures. Statistical significance was determined by the Student's t test (*** p
    Figure Legend Snippet: The MEK1-ERK-signaling pathway and the p38 MAPK-signaling pathway are required for the GM-CSF-induced Aldh1a2 expression and nuclear translocation of Sp1 in BM-DCs. ( A ) Flt3L-generated BM-DCs were cultured with or without 10 ng/ml GM-CSF for 16 h in the presence or absence of 50 µM PD98059 (PD) or 25 µM SB203580 (SB). After the culture, Aldh1a2 gene expression was assessed by real-time PCR. The Aldh1a2 mRNA expression level in the cells incubated with medium alone for 16 h was set to 1. The results are shown as the mean + SD of triplicate cultures. Statistical significance was determined by the Student's t test (*** p

    Techniques Used: Expressing, Translocation Assay, Generated, Cell Culture, Real-time Polymerase Chain Reaction, Incubation

    Sp1 and RARα/RXRα enhance each other's binding to the Aldh1a2 promoter and cooperatively enhance its activity. ( A ) COS-7 cells were transfected with the 0.5 µg of pCMV-Myc-Sp1, the combination of pSG5-RARα and pSG5-RXRα, or the three. One day after transfection, cell lysates were subjected to DNAP assay using anti-Myc Ab, anti-RARα Ab, or anti-RXRα Ab, and biotinylated DNA Probe C whose sequence is shown in Figure 3 . ( B ) COS-7 cells were transfected in triplicate with the 1.25 µg of pGL4-RALDH2 (−873) reporter vector and the 0.5 µg of expression vectors, pCMV-Myc-Sp1, pCMV-Myc-Sp1db, pSG5-RARα, and pSG5-RXRα, or control empty vectors. One day after transfection, cells were stimulated with or without 100 nM RA for 16 h. Then luciferase activities were measured. Relative promoter activities were calculated by arbitrarily defining the activity of pGL4-RALDH2 (−873) alone without RA as 1. ( C ) Flt3L-generated BM-DCs were cultured with or without 10 ng/ml GM-CSF or 10 nM RA. These cells were subjected to ChIP assay with anti-Sp1 or anti-RARα Ab or control IgG1. Binding of Sp1 and RARα proteins to the Aldh1a2 promoter site was estimated by real-time PCR. Data in (B and C) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (* p
    Figure Legend Snippet: Sp1 and RARα/RXRα enhance each other's binding to the Aldh1a2 promoter and cooperatively enhance its activity. ( A ) COS-7 cells were transfected with the 0.5 µg of pCMV-Myc-Sp1, the combination of pSG5-RARα and pSG5-RXRα, or the three. One day after transfection, cell lysates were subjected to DNAP assay using anti-Myc Ab, anti-RARα Ab, or anti-RXRα Ab, and biotinylated DNA Probe C whose sequence is shown in Figure 3 . ( B ) COS-7 cells were transfected in triplicate with the 1.25 µg of pGL4-RALDH2 (−873) reporter vector and the 0.5 µg of expression vectors, pCMV-Myc-Sp1, pCMV-Myc-Sp1db, pSG5-RARα, and pSG5-RXRα, or control empty vectors. One day after transfection, cells were stimulated with or without 100 nM RA for 16 h. Then luciferase activities were measured. Relative promoter activities were calculated by arbitrarily defining the activity of pGL4-RALDH2 (−873) alone without RA as 1. ( C ) Flt3L-generated BM-DCs were cultured with or without 10 ng/ml GM-CSF or 10 nM RA. These cells were subjected to ChIP assay with anti-Sp1 or anti-RARα Ab or control IgG1. Binding of Sp1 and RARα proteins to the Aldh1a2 promoter site was estimated by real-time PCR. Data in (B and C) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (* p

    Techniques Used: Binding Assay, Activity Assay, Transfection, Sequencing, Plasmid Preparation, Expressing, Luciferase, Generated, Cell Culture, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    29) Product Images from "Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells"

    Article Title: Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0096512

    RA enhances GM-CSF-induced Aldh1a2 expression via the RARα/RXRα heterodimer bound to the RARE half-site. ( A ) Localization of the putative RARE half-sites ( underlined ) in Probe C and their mutants, Probe C(RARE-h mt1) and Probe C(RARE-h mt2), are shown. COS-7 cells were transfected with the 0.5 µg of pSG5-RARα and/or pSG5-RXRα. One day after transfection, cell lysates were subjected to DNAP assay using the biotinylated DNA probes. The precipitates were analyzed by Western blotting using anti-RARα( upper panel ) and RXRα ( lower panel ) Abs. ( B ) Flt3L-generated BM-DCs were cultured with 10 ng/ml GM-CSF for 16 h in the presence or absence of 100 nM RA. LE540 (1 µM) was added to the indicated cultures. After the culture, Aldh1a2 mRNA expression was assessed by real-time PCR. The Aldh1a2 mRNA expression level in the cells incubated with medium alone for 16 h was set to 1. ( C ) BM-DCs were cultured with ( closed circle ) or without ( open circle ) 10 ng/ml GM-CSF for 16 h in the presence of graded concentrations of RA. After the culture, Aldh1a2 mRNA expression was assessed by real-time PCR. Relative expression levels were calculated by defining the Aldh1a2 mRNA expression level in the cells incubated with medium alone for 16 h was set to 1. Asterisks indicate a significant difference (*** p
    Figure Legend Snippet: RA enhances GM-CSF-induced Aldh1a2 expression via the RARα/RXRα heterodimer bound to the RARE half-site. ( A ) Localization of the putative RARE half-sites ( underlined ) in Probe C and their mutants, Probe C(RARE-h mt1) and Probe C(RARE-h mt2), are shown. COS-7 cells were transfected with the 0.5 µg of pSG5-RARα and/or pSG5-RXRα. One day after transfection, cell lysates were subjected to DNAP assay using the biotinylated DNA probes. The precipitates were analyzed by Western blotting using anti-RARα( upper panel ) and RXRα ( lower panel ) Abs. ( B ) Flt3L-generated BM-DCs were cultured with 10 ng/ml GM-CSF for 16 h in the presence or absence of 100 nM RA. LE540 (1 µM) was added to the indicated cultures. After the culture, Aldh1a2 mRNA expression was assessed by real-time PCR. The Aldh1a2 mRNA expression level in the cells incubated with medium alone for 16 h was set to 1. ( C ) BM-DCs were cultured with ( closed circle ) or without ( open circle ) 10 ng/ml GM-CSF for 16 h in the presence of graded concentrations of RA. After the culture, Aldh1a2 mRNA expression was assessed by real-time PCR. Relative expression levels were calculated by defining the Aldh1a2 mRNA expression level in the cells incubated with medium alone for 16 h was set to 1. Asterisks indicate a significant difference (*** p

    Techniques Used: Expressing, Transfection, Western Blot, Generated, Cell Culture, Real-time Polymerase Chain Reaction, Incubation

    Sp1 binds to the Aldh1a2 promoter region. ( A ) The locations and nucleotide sequences corresponding to Probe A, Probe B, and Probe C in the 5′-flanking region of the mouse Aldh1a2 gene are shown. COS-7 cells were transfected with the 0.5 µg of pCMV-Myc-Sp1 or control empty vector. One day after transfection, cell lysates were analyzed for DNA-binding activity by DNAP assay using the indicated biotinylated DNA probes and anti-Myc Ab. (B) Flt3L-generated BM-DCs were cultured in the presence or absence of 10 ng/ml GM-CSF. After 16 h, nuclear extracts were analyzed for the presence of lamin B1 and Sp1 by Western blotting using anti-lamin B1 and anti-Sp1 Abs ( left panel ), or assessed for DNA binding activity by DNAP assay using biotinylated DNA Probe C and anti-Sp1 Ab ( right panel ). Data are representative of at least three independent experiments.
    Figure Legend Snippet: Sp1 binds to the Aldh1a2 promoter region. ( A ) The locations and nucleotide sequences corresponding to Probe A, Probe B, and Probe C in the 5′-flanking region of the mouse Aldh1a2 gene are shown. COS-7 cells were transfected with the 0.5 µg of pCMV-Myc-Sp1 or control empty vector. One day after transfection, cell lysates were analyzed for DNA-binding activity by DNAP assay using the indicated biotinylated DNA probes and anti-Myc Ab. (B) Flt3L-generated BM-DCs were cultured in the presence or absence of 10 ng/ml GM-CSF. After 16 h, nuclear extracts were analyzed for the presence of lamin B1 and Sp1 by Western blotting using anti-lamin B1 and anti-Sp1 Abs ( left panel ), or assessed for DNA binding activity by DNAP assay using biotinylated DNA Probe C and anti-Sp1 Ab ( right panel ). Data are representative of at least three independent experiments.

    Techniques Used: Transfection, Plasmid Preparation, Binding Assay, Activity Assay, Generated, Cell Culture, Western Blot

    Methylation of the CpG island in the Aldh1a2 promoter prohibits Sp1 to activate the promoter, whereas the Aldh1a2 promoter is largely unmethylated in BM-pDCs as well as in BM-cDCs. ( A ) pCpGL-basic and pCpGL-RALDH2 (−873) reporter vectors were methylated with 1.25 µg of M.SssI. COS-7 cells were transfected with methylated or unmethylated pCpGL-basic or pCpGL-RALDH2 (−873) reporter vector in combination with or without the 0.5 µg of pCMV-Myc-Sp1 expression vector. One day after transfection, luciferase activity was measured. Relative promoter activities were calculated by arbitrarily defining the activity of pCpGL-basic alone as 1. ( B ) COS-7 cells were transfected with pCMV-Myc-Sp1. One day after transfection, cell lysates were analyzed for DNA binding activity by DNAP assay using DNA Probe B and Probe C methylated with M.SssI or left unmethylated. The bound proteins were analyzed by SDS-PAGE followed by Western blotting with anti-Myc Ab. ( C ) Flt3L-generated BM-DCs were stained with allophycocyanin-labeled anti-CD11c Ab and phycoerythrin-labeled anti-B220 Ab, and were sorted to cDC and pDC fractions with a FACSAria. ( D ) Sorted BM-pDCs and BM-cDCs were cultured for 16 h with or without 10 ng/ml GM-CSF. Expression of Aldh1a2 mRNA was analyzed by real-time PCR. Relative expression levels were calculated by defining the Aldh1a2 mRNA expression in the cells incubated with medium alone for 16 h was set to 1. Data in (A and D) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (*** p
    Figure Legend Snippet: Methylation of the CpG island in the Aldh1a2 promoter prohibits Sp1 to activate the promoter, whereas the Aldh1a2 promoter is largely unmethylated in BM-pDCs as well as in BM-cDCs. ( A ) pCpGL-basic and pCpGL-RALDH2 (−873) reporter vectors were methylated with 1.25 µg of M.SssI. COS-7 cells were transfected with methylated or unmethylated pCpGL-basic or pCpGL-RALDH2 (−873) reporter vector in combination with or without the 0.5 µg of pCMV-Myc-Sp1 expression vector. One day after transfection, luciferase activity was measured. Relative promoter activities were calculated by arbitrarily defining the activity of pCpGL-basic alone as 1. ( B ) COS-7 cells were transfected with pCMV-Myc-Sp1. One day after transfection, cell lysates were analyzed for DNA binding activity by DNAP assay using DNA Probe B and Probe C methylated with M.SssI or left unmethylated. The bound proteins were analyzed by SDS-PAGE followed by Western blotting with anti-Myc Ab. ( C ) Flt3L-generated BM-DCs were stained with allophycocyanin-labeled anti-CD11c Ab and phycoerythrin-labeled anti-B220 Ab, and were sorted to cDC and pDC fractions with a FACSAria. ( D ) Sorted BM-pDCs and BM-cDCs were cultured for 16 h with or without 10 ng/ml GM-CSF. Expression of Aldh1a2 mRNA was analyzed by real-time PCR. Relative expression levels were calculated by defining the Aldh1a2 mRNA expression in the cells incubated with medium alone for 16 h was set to 1. Data in (A and D) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (*** p

    Techniques Used: Methylation, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Binding Assay, SDS Page, Western Blot, Generated, Staining, Labeling, Cell Culture, Real-time Polymerase Chain Reaction, Incubation

    Sp1 and RARα/RXRα enhance each other's binding to the Aldh1a2 promoter and cooperatively enhance its activity. ( A ) COS-7 cells were transfected with the 0.5 µg of pCMV-Myc-Sp1, the combination of pSG5-RARα and pSG5-RXRα, or the three. One day after transfection, cell lysates were subjected to DNAP assay using anti-Myc Ab, anti-RARα Ab, or anti-RXRα Ab, and biotinylated DNA Probe C whose sequence is shown in Figure 3 . ( B ) COS-7 cells were transfected in triplicate with the 1.25 µg of pGL4-RALDH2 (−873) reporter vector and the 0.5 µg of expression vectors, pCMV-Myc-Sp1, pCMV-Myc-Sp1db, pSG5-RARα, and pSG5-RXRα, or control empty vectors. One day after transfection, cells were stimulated with or without 100 nM RA for 16 h. Then luciferase activities were measured. Relative promoter activities were calculated by arbitrarily defining the activity of pGL4-RALDH2 (−873) alone without RA as 1. ( C ) Flt3L-generated BM-DCs were cultured with or without 10 ng/ml GM-CSF or 10 nM RA. These cells were subjected to ChIP assay with anti-Sp1 or anti-RARα Ab or control IgG1. Binding of Sp1 and RARα proteins to the Aldh1a2 promoter site was estimated by real-time PCR. Data in (B and C) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (* p
    Figure Legend Snippet: Sp1 and RARα/RXRα enhance each other's binding to the Aldh1a2 promoter and cooperatively enhance its activity. ( A ) COS-7 cells were transfected with the 0.5 µg of pCMV-Myc-Sp1, the combination of pSG5-RARα and pSG5-RXRα, or the three. One day after transfection, cell lysates were subjected to DNAP assay using anti-Myc Ab, anti-RARα Ab, or anti-RXRα Ab, and biotinylated DNA Probe C whose sequence is shown in Figure 3 . ( B ) COS-7 cells were transfected in triplicate with the 1.25 µg of pGL4-RALDH2 (−873) reporter vector and the 0.5 µg of expression vectors, pCMV-Myc-Sp1, pCMV-Myc-Sp1db, pSG5-RARα, and pSG5-RXRα, or control empty vectors. One day after transfection, cells were stimulated with or without 100 nM RA for 16 h. Then luciferase activities were measured. Relative promoter activities were calculated by arbitrarily defining the activity of pGL4-RALDH2 (−873) alone without RA as 1. ( C ) Flt3L-generated BM-DCs were cultured with or without 10 ng/ml GM-CSF or 10 nM RA. These cells were subjected to ChIP assay with anti-Sp1 or anti-RARα Ab or control IgG1. Binding of Sp1 and RARα proteins to the Aldh1a2 promoter site was estimated by real-time PCR. Data in (B and C) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (* p

    Techniques Used: Binding Assay, Activity Assay, Transfection, Sequencing, Plasmid Preparation, Expressing, Luciferase, Generated, Cell Culture, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    30) Product Images from "The IGF1 P2 promoter is an epigenetic QTL for circulating IGF1 and human growth"

    Article Title: The IGF1 P2 promoter is an epigenetic QTL for circulating IGF1 and human growth

    Journal: Clinical Epigenetics

    doi: 10.1186/s13148-015-0062-8

    . The demethylation of the IGF1 -P2 region increases luciferase activity. Results of luciferase assays in transiently transfected HEK293 cells. Forty-eight hours after transfection, basal activity of unmethylated and methylated, Firefly luciferase reporter plasmids containing IGF1 promoter 2 CG fragment, and empty Firefly luciferase reporter plasmid, pCpGL-Basic, was measured, and normalized to the activity of co-transfected Renilla luciferase plasmid. Results were analyzed by paired t -test, ** P
    Figure Legend Snippet: . The demethylation of the IGF1 -P2 region increases luciferase activity. Results of luciferase assays in transiently transfected HEK293 cells. Forty-eight hours after transfection, basal activity of unmethylated and methylated, Firefly luciferase reporter plasmids containing IGF1 promoter 2 CG fragment, and empty Firefly luciferase reporter plasmid, pCpGL-Basic, was measured, and normalized to the activity of co-transfected Renilla luciferase plasmid. Results were analyzed by paired t -test, ** P

    Techniques Used: Luciferase, Activity Assay, Transfection, Methylation, Plasmid Preparation

    31) Product Images from "CTCF cis-Regulates Trinucleotide Repeat Instability in an Epigenetic Manner: A Novel Basis for Mutational Hot Spot Determination"

    Article Title: CTCF cis-Regulates Trinucleotide Repeat Instability in an Epigenetic Manner: A Novel Basis for Mutational Hot Spot Determination

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1000257

    Analysis and mutagenesis of the SCA7-CTCF-I binding site. (A) SCA7 genomic fragments used for transgenesis. Upper: SCA7-CTCF-I -wt; Middle: α-SCA7 3′ genomic deletion; Bottom: SCA7-CTCF-I -mut. Core CCCTC sequences are underlined, and sequence alterations in the SCA7-CTCF-I -mut transgenic construct are shown in gray. (B) Electrophoretic mobility shift assays with SCA7-CTCF-I -wt and -mut probe fragments were performed with probe only, empty lysate (no protein), full-length CTCF protein with pre-immune anti-CTCF sera (CTCF+pI), CTCF protein with anti-CTCF sera (CTCF+α-CTCF), or the 11 zinc-finger DNA binding domain region of CTCF. Arrows indicate shifted CTCF-DNA complexes. Addition of CTCF-DM1 probe as cold competitor prevented CTCF-DNA complex formation for SCA7-CTCF-I -wt fragment, while non-specific cold competitor did not (data not shown). (C) Methylation interference (Me I) and DNase I footprinting (DNase) on SCA7-CTCF-I fragment. Left and right panels correspond to the 5′-end labeled coding and anti-sense strands respectively. B, CTCF-bound DNA; F, free DNA; long bars, CTCF-protected from DNase I; arrows, DNase I hypersensitive sites created by CTCF binding; filled circles, contact guanine nucleotides essential for sequence recognition by CTCF. See panel ‘A’ for precise location of sites. (D) ChIP on cerebellar lysates from SCA7-CTCF-I -wt and -mut mice (n = 3/genotype). Significantly decreased occupancy at the CTCF-I site was detected with the 3′ amplicon (primer set B) in SCA7-CTCF-I -mut mice (p = 0.02, one-way ANOVA), as this amplicon is not in close proximity to the 5′ CTCF-II site. No differences in CTCF occupancy between SCA7-CTCF-I -wt and -mut mice were detected with primer set A (or other adjacent primer sets; data not shown) due to the close proximity of the two CTCF binding sites. Results are normalized to SCA7-CTCF-I -wt. Error bars are s.d.
    Figure Legend Snippet: Analysis and mutagenesis of the SCA7-CTCF-I binding site. (A) SCA7 genomic fragments used for transgenesis. Upper: SCA7-CTCF-I -wt; Middle: α-SCA7 3′ genomic deletion; Bottom: SCA7-CTCF-I -mut. Core CCCTC sequences are underlined, and sequence alterations in the SCA7-CTCF-I -mut transgenic construct are shown in gray. (B) Electrophoretic mobility shift assays with SCA7-CTCF-I -wt and -mut probe fragments were performed with probe only, empty lysate (no protein), full-length CTCF protein with pre-immune anti-CTCF sera (CTCF+pI), CTCF protein with anti-CTCF sera (CTCF+α-CTCF), or the 11 zinc-finger DNA binding domain region of CTCF. Arrows indicate shifted CTCF-DNA complexes. Addition of CTCF-DM1 probe as cold competitor prevented CTCF-DNA complex formation for SCA7-CTCF-I -wt fragment, while non-specific cold competitor did not (data not shown). (C) Methylation interference (Me I) and DNase I footprinting (DNase) on SCA7-CTCF-I fragment. Left and right panels correspond to the 5′-end labeled coding and anti-sense strands respectively. B, CTCF-bound DNA; F, free DNA; long bars, CTCF-protected from DNase I; arrows, DNase I hypersensitive sites created by CTCF binding; filled circles, contact guanine nucleotides essential for sequence recognition by CTCF. See panel ‘A’ for precise location of sites. (D) ChIP on cerebellar lysates from SCA7-CTCF-I -wt and -mut mice (n = 3/genotype). Significantly decreased occupancy at the CTCF-I site was detected with the 3′ amplicon (primer set B) in SCA7-CTCF-I -mut mice (p = 0.02, one-way ANOVA), as this amplicon is not in close proximity to the 5′ CTCF-II site. No differences in CTCF occupancy between SCA7-CTCF-I -wt and -mut mice were detected with primer set A (or other adjacent primer sets; data not shown) due to the close proximity of the two CTCF binding sites. Results are normalized to SCA7-CTCF-I -wt. Error bars are s.d.

    Techniques Used: Mutagenesis, Binding Assay, Sequencing, Transgenic Assay, Construct, Electrophoretic Mobility Shift Assay, Methylation, Footprinting, Labeling, Chromatin Immunoprecipitation, Mouse Assay, Amplification

    SCA7-CTCF-I -mut mice display increased somatic instability. (A) At 2 months of age, the SCA7 CAG repeat is stable in the SCA7-CTCF-I -wt line and in both SCA7-CTCF-I -mut lines. (B) With advancing age, tissue-specific instability is seen in SCA7-CTCF-I -wt mice; however, this tissue-specific instability is much more pronounced in SCA7-CTCF-I -mut mice. Results for individuals from the two different SCA7-CTCF-I -mut mice are shown here. (C) To permit quantification of somatic instability, we performed small-pool PCR on tissue DNA samples from SCA7-CTCF-I -wt and SCA7-CTCF-I -mut mice. As shown here for cortex, SCA7-CTCF-I -mut mice displayed significantly greater instability than SCA7-CTCF-I -wt mice (p = 8.6×10 −5 , Mann-Whitney two-tailed test). See Table 1 for a compiled list of repeat alleles. (D) Histogram of repeat length variation in the cortex of SCA7-CTCF-I -wt and SCA7-CTCF-I -mut mice. SCA7-CTCF-I -mut mice exhibit significantly greater instability than SCA7-CTCF-I -wt mice, and this expansion tendency exceeds that of SCA7-CTCF-I -wt mice, even when 2.5 months younger (p = 0.0003, Mann-Whitney two-tailed test). With advancing age, the expansion bias between the SCA7-CTCF-I -mut and -wt mice becomes more pronounced (p
    Figure Legend Snippet: SCA7-CTCF-I -mut mice display increased somatic instability. (A) At 2 months of age, the SCA7 CAG repeat is stable in the SCA7-CTCF-I -wt line and in both SCA7-CTCF-I -mut lines. (B) With advancing age, tissue-specific instability is seen in SCA7-CTCF-I -wt mice; however, this tissue-specific instability is much more pronounced in SCA7-CTCF-I -mut mice. Results for individuals from the two different SCA7-CTCF-I -mut mice are shown here. (C) To permit quantification of somatic instability, we performed small-pool PCR on tissue DNA samples from SCA7-CTCF-I -wt and SCA7-CTCF-I -mut mice. As shown here for cortex, SCA7-CTCF-I -mut mice displayed significantly greater instability than SCA7-CTCF-I -wt mice (p = 8.6×10 −5 , Mann-Whitney two-tailed test). See Table 1 for a compiled list of repeat alleles. (D) Histogram of repeat length variation in the cortex of SCA7-CTCF-I -wt and SCA7-CTCF-I -mut mice. SCA7-CTCF-I -mut mice exhibit significantly greater instability than SCA7-CTCF-I -wt mice, and this expansion tendency exceeds that of SCA7-CTCF-I -wt mice, even when 2.5 months younger (p = 0.0003, Mann-Whitney two-tailed test). With advancing age, the expansion bias between the SCA7-CTCF-I -mut and -wt mice becomes more pronounced (p

    Techniques Used: Mouse Assay, Polymerase Chain Reaction, MANN-WHITNEY, Two Tailed Test

    SCA7-CTCF-I -mut mice display increased germ line instability. (A) Comparison of CAG repeat instability in parent-offspring transmissions for SCA7-CTCF-I mice. Repeat lengths are plotted as % of total alleles scored for 53 SCA7-CTCF-I -wt and 95 SCA7-CTCF-I -mut mice. The repeat size range in the SCA7-CTCF-I -mut mice was significantly different from the distribution of repeat alleles in the SCA7-CTCF-I -wt mice (p = 0.002; Mann-Whitney two-tailed test). (B) Small-pool PCR of sperm DNAs in 16 month-old SCA7 transgenic mice. SCA7-CTCF-I -wt mice typically exhibited small repeat length changes, while SCA7-CTCF-I -mut mice displayed pronounced instability. (C) Compilation of small-pool PCR data. At 2 months of age, only modest instability was noted. At 16 months of age, SCA7-CTCF-I -wt mice displayed moderate instability, but SCA7-CTCF-I -mut mice exhibited significantly greater instability (p = 0.009; Mann-Whitney two-tailed test).
    Figure Legend Snippet: SCA7-CTCF-I -mut mice display increased germ line instability. (A) Comparison of CAG repeat instability in parent-offspring transmissions for SCA7-CTCF-I mice. Repeat lengths are plotted as % of total alleles scored for 53 SCA7-CTCF-I -wt and 95 SCA7-CTCF-I -mut mice. The repeat size range in the SCA7-CTCF-I -mut mice was significantly different from the distribution of repeat alleles in the SCA7-CTCF-I -wt mice (p = 0.002; Mann-Whitney two-tailed test). (B) Small-pool PCR of sperm DNAs in 16 month-old SCA7 transgenic mice. SCA7-CTCF-I -wt mice typically exhibited small repeat length changes, while SCA7-CTCF-I -mut mice displayed pronounced instability. (C) Compilation of small-pool PCR data. At 2 months of age, only modest instability was noted. At 16 months of age, SCA7-CTCF-I -wt mice displayed moderate instability, but SCA7-CTCF-I -mut mice exhibited significantly greater instability (p = 0.009; Mann-Whitney two-tailed test).

    Techniques Used: Mouse Assay, MANN-WHITNEY, Two Tailed Test, Polymerase Chain Reaction, Transgenic Assay

    Epigenetic regulation of CTCF binding modulates instability at the SCA7 locus. (A) CpG methylation prevents binding of CTCF to SCA7-CTCF-I site. Electrophoretic mobility shift assays with un-methylated (control) or methylated SCA7-CTCF-I fragments, using CTCF with no antisera (CTCF), CTCF with anti-CTCF antisera (CTCF+α-CTCF), or CTCF with pre-immune sera (CTCF+pI). Arrow indicates CTCF-bound probe. (B) Prominent somatic instability in kidney DNA (black arrowheads) from a SCA7-CTCF-I -wt mouse with CTCF-I site methylation ( SCA7-CTCF-I -wt*) contrasts with somatic stability in SCA7-CTCF-I -wt mice with un-methylated CTCF-I sites. Note that SCA7-CTCF-I -wt lines display bimodal CAG repeat alleles. Prominent somatic instability is apparent in kidney DNA (gray arrowhead) from a SCA7-CTCF-I -mut mouse. All mice were 6 months of age. (C) Kidney DNAs from the SCA7-CTCF-I -wt* mouse are highly methylated. Circles, CpG dyads; open circles, unmethylated; filled circles; methylated. Box highlights core CTCF binding site contact residue, based upon footprinting analysis. Diagrammed epigenotypes summarize results for five SCA7-CTCF-I -wt mice, eight SCA7-CTCF-I -mut mice, and the SCA7-CTCF-I -wt* mouse, and were consistent for at least 75% of all sequenced clones (n = 10−12/sample). (D) Liver DNAs from control SCA7-CTCF-I -wt mice are methylated. Bisulfite sequencing of the SCA7-CTCF-I region was performed upon liver DNAs from three SCA7-CTCF-I -wt mice at one year of age (n = 17 clones/mouse), and CpG methylation determined for the 13 CpG dyads in the SCA7-CTCF-I region. A number of CpG dyads, including the CpG-4 CTCF contact site, exhibit moderate to high levels of methylation.
    Figure Legend Snippet: Epigenetic regulation of CTCF binding modulates instability at the SCA7 locus. (A) CpG methylation prevents binding of CTCF to SCA7-CTCF-I site. Electrophoretic mobility shift assays with un-methylated (control) or methylated SCA7-CTCF-I fragments, using CTCF with no antisera (CTCF), CTCF with anti-CTCF antisera (CTCF+α-CTCF), or CTCF with pre-immune sera (CTCF+pI). Arrow indicates CTCF-bound probe. (B) Prominent somatic instability in kidney DNA (black arrowheads) from a SCA7-CTCF-I -wt mouse with CTCF-I site methylation ( SCA7-CTCF-I -wt*) contrasts with somatic stability in SCA7-CTCF-I -wt mice with un-methylated CTCF-I sites. Note that SCA7-CTCF-I -wt lines display bimodal CAG repeat alleles. Prominent somatic instability is apparent in kidney DNA (gray arrowhead) from a SCA7-CTCF-I -mut mouse. All mice were 6 months of age. (C) Kidney DNAs from the SCA7-CTCF-I -wt* mouse are highly methylated. Circles, CpG dyads; open circles, unmethylated; filled circles; methylated. Box highlights core CTCF binding site contact residue, based upon footprinting analysis. Diagrammed epigenotypes summarize results for five SCA7-CTCF-I -wt mice, eight SCA7-CTCF-I -mut mice, and the SCA7-CTCF-I -wt* mouse, and were consistent for at least 75% of all sequenced clones (n = 10−12/sample). (D) Liver DNAs from control SCA7-CTCF-I -wt mice are methylated. Bisulfite sequencing of the SCA7-CTCF-I region was performed upon liver DNAs from three SCA7-CTCF-I -wt mice at one year of age (n = 17 clones/mouse), and CpG methylation determined for the 13 CpG dyads in the SCA7-CTCF-I region. A number of CpG dyads, including the CpG-4 CTCF contact site, exhibit moderate to high levels of methylation.

    Techniques Used: Binding Assay, CpG Methylation Assay, Electrophoretic Mobility Shift Assay, Methylation, Mouse Assay, Footprinting, Clone Assay, Methylation Sequencing

    32) Product Images from "Regulation of the Bone-restricted IFITM-like (Bril) Gene Transcription by Sp and Gli Family Members and CpG Methylation *"

    Article Title: Regulation of the Bone-restricted IFITM-like (Bril) Gene Transcription by Sp and Gli Family Members and CpG Methylation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.457010

    Transcriptional activation of the rat, mouse, and human Bril promoter by Sp family members. The promoter Luc constructs (100 ng) were co-transfected with 300 ng of each expression plasmids encoding Sp1, Sp3-L1 (long form 1), Sp3-L2 (long form 2), Sp3-S (short form), and OSX. Luc activity was measured 48 h after transient transfection into MC3T3 osteoblasts ( A ) or HEK293 ( B and C ). C , the human −1434 bp promoter Luc construct (100 ng) was co-transfected with 150 ng of each plasmid encoding either GFP or Sp1, in combination with the other Sp members. The first two bars represent transfection of Sp1 (150 ng) with GFP (150 ng) or with itself (300 ng). Results are presented as -fold increase relative to the negative control plasmid encoding GFP. Results shown are mean ± S.E. ( error bars ) ( n = 3–5).
    Figure Legend Snippet: Transcriptional activation of the rat, mouse, and human Bril promoter by Sp family members. The promoter Luc constructs (100 ng) were co-transfected with 300 ng of each expression plasmids encoding Sp1, Sp3-L1 (long form 1), Sp3-L2 (long form 2), Sp3-S (short form), and OSX. Luc activity was measured 48 h after transient transfection into MC3T3 osteoblasts ( A ) or HEK293 ( B and C ). C , the human −1434 bp promoter Luc construct (100 ng) was co-transfected with 150 ng of each plasmid encoding either GFP or Sp1, in combination with the other Sp members. The first two bars represent transfection of Sp1 (150 ng) with GFP (150 ng) or with itself (300 ng). Results are presented as -fold increase relative to the negative control plasmid encoding GFP. Results shown are mean ± S.E. ( error bars ) ( n = 3–5).

    Techniques Used: Activation Assay, Construct, Transfection, Expressing, Activity Assay, Plasmid Preparation, Negative Control

    Transcriptional activity of the Bril promoter in UMR106 cells. The rat ( A ) and human ( B ) promoter regions were cloned into the pGL3-basic reporter plasmid, and Luc activity was measured 48 h after transient transfection in UMR106 cells. The longest constructs tested extended 1327 and 1434 bp upstream of the coding ATG for the rat and human, respectively. Progressive truncations from the 5′-end ( gray bars ) and internal deletions ( back bars ) indicated that maximal regulatory activity resides within −265 bp (rat) and −199 bp (human), with complete loss of activity down to −82 bp. Data represent relative Luc activity as compared with the −633 and −1434 bp for the rat and human constructs, respectively. Promoterless pGL3-basic was used as a negative control. Results shown are mean ± S.E. ( error bars ) ( n = 4).
    Figure Legend Snippet: Transcriptional activity of the Bril promoter in UMR106 cells. The rat ( A ) and human ( B ) promoter regions were cloned into the pGL3-basic reporter plasmid, and Luc activity was measured 48 h after transient transfection in UMR106 cells. The longest constructs tested extended 1327 and 1434 bp upstream of the coding ATG for the rat and human, respectively. Progressive truncations from the 5′-end ( gray bars ) and internal deletions ( back bars ) indicated that maximal regulatory activity resides within −265 bp (rat) and −199 bp (human), with complete loss of activity down to −82 bp. Data represent relative Luc activity as compared with the −633 and −1434 bp for the rat and human constructs, respectively. Promoterless pGL3-basic was used as a negative control. Results shown are mean ± S.E. ( error bars ) ( n = 4).

    Techniques Used: Activity Assay, Clone Assay, Plasmid Preparation, Transfection, Construct, Negative Control

    Activation of the human BRIL promoter and endogenous mouse Bril gene by GLI2 in MC3T3 cells. A , the human BRIL promoter contains an element between −191 and −204 matching the GLI consensus binding site ( top , core binding site highlighted ). The WT and mutant (having a 2-base substitution within the GLI core, lowercase at top ) human −1434 bp promoter Luc constructs (100 ng) were co-transfected in MC3T3 cells with 300 ng of plasmids encoding GFP, GLI1, GLI2, or GLI3. 42 h post-transfection, Luc activity was measured, and results are presented as -fold increase relative to GFP. Values above bars represent -fold increases. B , the human −1434 bp promoter Luc construct (100 ng) was co-transfected in MC3T3 cells with increasing amounts of plasmid encoding GLI2 ( open symbols ) or with 300 ng of Sp1 ( closed squares ). Luc activity was measured 15, 25, and 42 h post-transfection and expressed as -fold increase relative to a co-transfection with GFP. C , MC3T3 cells were co-transfected with the human −1434 bp promoter Luc construct (100 ng) with 150 ng each of GFP or GLI2, in combination with OSX, Sp1, or TCF1, and Luc activity was recorded after 25 h. D , real-time quantitative PCR analysis of endogenous Bril expression 25 h post-transfection with plasmids encoding GFP, GLI2, or Sp1. Values are normalized to β- actin and expressed as 2 −Δ C t relative to the GFP control. Results shown are mean ± S.E. ( error bars ) ( n = 3–5).
    Figure Legend Snippet: Activation of the human BRIL promoter and endogenous mouse Bril gene by GLI2 in MC3T3 cells. A , the human BRIL promoter contains an element between −191 and −204 matching the GLI consensus binding site ( top , core binding site highlighted ). The WT and mutant (having a 2-base substitution within the GLI core, lowercase at top ) human −1434 bp promoter Luc constructs (100 ng) were co-transfected in MC3T3 cells with 300 ng of plasmids encoding GFP, GLI1, GLI2, or GLI3. 42 h post-transfection, Luc activity was measured, and results are presented as -fold increase relative to GFP. Values above bars represent -fold increases. B , the human −1434 bp promoter Luc construct (100 ng) was co-transfected in MC3T3 cells with increasing amounts of plasmid encoding GLI2 ( open symbols ) or with 300 ng of Sp1 ( closed squares ). Luc activity was measured 15, 25, and 42 h post-transfection and expressed as -fold increase relative to a co-transfection with GFP. C , MC3T3 cells were co-transfected with the human −1434 bp promoter Luc construct (100 ng) with 150 ng each of GFP or GLI2, in combination with OSX, Sp1, or TCF1, and Luc activity was recorded after 25 h. D , real-time quantitative PCR analysis of endogenous Bril expression 25 h post-transfection with plasmids encoding GFP, GLI2, or Sp1. Values are normalized to β- actin and expressed as 2 −Δ C t relative to the GFP control. Results shown are mean ± S.E. ( error bars ) ( n = 3–5).

    Techniques Used: Activation Assay, Binding Assay, Mutagenesis, Construct, Transfection, Activity Assay, Plasmid Preparation, Cotransfection, Real-time Polymerase Chain Reaction, Expressing

    ZFP354C represses basal and Sp1-induced transcriptional activity of the Bril promoters. A , the rat, mouse, and human promoter Luc constructs (100 ng) were co-transfected in HEK293 cells with GFP or ZFP354C expression plasmids (300 ng). Luc activity was assayed 48 h later, and the raw values are presented. ZFP354C repressed the base-line Luc activity for the three species from 76 to 85%. B , the human −1434 bp promoter construct (100 ng) was co-transfected with a fixed amount of Sp1 (150 ng) and with increasing quantities (0, 25, 50, 100, and 150 ng) of ZFP354C. The amount of co-transfected plasmid was kept constant at 300 ng with GFP. The values represent the -fold increase relative to co-transfection with GFP alone (300 ng). Results shown are mean ± S.E. ( error bars ) ( n = 3).
    Figure Legend Snippet: ZFP354C represses basal and Sp1-induced transcriptional activity of the Bril promoters. A , the rat, mouse, and human promoter Luc constructs (100 ng) were co-transfected in HEK293 cells with GFP or ZFP354C expression plasmids (300 ng). Luc activity was assayed 48 h later, and the raw values are presented. ZFP354C repressed the base-line Luc activity for the three species from 76 to 85%. B , the human −1434 bp promoter construct (100 ng) was co-transfected with a fixed amount of Sp1 (150 ng) and with increasing quantities (0, 25, 50, 100, and 150 ng) of ZFP354C. The amount of co-transfected plasmid was kept constant at 300 ng with GFP. The values represent the -fold increase relative to co-transfection with GFP alone (300 ng). Results shown are mean ± S.E. ( error bars ) ( n = 3).

    Techniques Used: Activity Assay, Construct, Transfection, Expressing, Plasmid Preparation, Cotransfection

    33) Product Images from "Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule"

    Article Title: Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule

    Journal: Bioconjugate Chemistry

    doi: 10.1021/bc1000388

    Effect of TFO-141S treatment on plasmid conformation. (A) Agarose gel electrophoresis of plasmids p39E and p11-1 treated with the TFO, WT M.SssI, C141S, or the TFO−C141S conjugate: lane 1, untreated; lane 2, without TFO−C141S; lane 3, TFO; lane 4, M.SssI; lane 5, C141S; lane 6, TFO−C141S; lane 7, TFO- C141S without SAM; lane 8, 100-fold excess of TFO and TFO−C141S; lane 9, marker; lane 10, without TFO−C141S; lane 11, C141S; lane 12, TFO−C141S. (B) Agarose gel electrophoresis of plasmid p39E treated with active and heat-inactivated C141S and TFO−C141S. The supercoiled plasmid was incubated at 30 °C for different time points as indicated above the lanes (hours). Then the samples were deproteinized before electrophoresis as described in Experimental Procedures . (C) Agarose gel electrophoresis of plasmid p39C and p39E treated with the TFO−C141S conjugate only or in the presence of 100-fold excess of TFO. Plasmids were incubated as in part B. Lane a is purified plasmid.
    Figure Legend Snippet: Effect of TFO-141S treatment on plasmid conformation. (A) Agarose gel electrophoresis of plasmids p39E and p11-1 treated with the TFO, WT M.SssI, C141S, or the TFO−C141S conjugate: lane 1, untreated; lane 2, without TFO−C141S; lane 3, TFO; lane 4, M.SssI; lane 5, C141S; lane 6, TFO−C141S; lane 7, TFO- C141S without SAM; lane 8, 100-fold excess of TFO and TFO−C141S; lane 9, marker; lane 10, without TFO−C141S; lane 11, C141S; lane 12, TFO−C141S. (B) Agarose gel electrophoresis of plasmid p39E treated with active and heat-inactivated C141S and TFO−C141S. The supercoiled plasmid was incubated at 30 °C for different time points as indicated above the lanes (hours). Then the samples were deproteinized before electrophoresis as described in Experimental Procedures . (C) Agarose gel electrophoresis of plasmid p39C and p39E treated with the TFO−C141S conjugate only or in the presence of 100-fold excess of TFO. Plasmids were incubated as in part B. Lane a is purified plasmid.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Marker, Incubation, Electrophoresis, Purification

    Effect of TFO−C141S treatment on GFP expression in EpCAM positive SKOV3 cells. (A) Relative GFP expression measured 48 h after transfection of pretreated p39E. Plasmid p39E was treated as indicated: p39E = treatment without TFO−C141S, treated with TFO only, with untargeted M.SssI or C141S, with the TFO−C141S conjugate or with 100-fold excess of TFO and TFO−C141S (=competition). The value obtained with p39E without TFO−C141S was taken as 100%. Shown is the average GFP expression (±SD) of one representative transfection performed in triplicate. (B) Relative GFP expression measured 48 h after transfection of pretreated deletion derivatives p7-2 and p4-1. For each derivative, the values obtained with samples treated without TFO−C141S were taken as 100%. Shown is the average GFP expression (±SEM) of the mean of three independent transfections performed in triplicate. (C) Relative GFP expression was measured 48 h after transfection of pretreated p39E or p39C. Treatments were as indicated: (+) or (−) indicates the presence or absence of the methyl donor (SAM). Shown is the average GFP expression (±SEM) of the mean of three independent transfections performed in triplicate.
    Figure Legend Snippet: Effect of TFO−C141S treatment on GFP expression in EpCAM positive SKOV3 cells. (A) Relative GFP expression measured 48 h after transfection of pretreated p39E. Plasmid p39E was treated as indicated: p39E = treatment without TFO−C141S, treated with TFO only, with untargeted M.SssI or C141S, with the TFO−C141S conjugate or with 100-fold excess of TFO and TFO−C141S (=competition). The value obtained with p39E without TFO−C141S was taken as 100%. Shown is the average GFP expression (±SD) of one representative transfection performed in triplicate. (B) Relative GFP expression measured 48 h after transfection of pretreated deletion derivatives p7-2 and p4-1. For each derivative, the values obtained with samples treated without TFO−C141S were taken as 100%. Shown is the average GFP expression (±SEM) of the mean of three independent transfections performed in triplicate. (C) Relative GFP expression was measured 48 h after transfection of pretreated p39E or p39C. Treatments were as indicated: (+) or (−) indicates the presence or absence of the methyl donor (SAM). Shown is the average GFP expression (±SEM) of the mean of three independent transfections performed in triplicate.

    Techniques Used: Expressing, Transfection, Plasmid Preparation

    34) Product Images from "DNA METHYLATION INHIBITION INCREASES T CELL KIR EXPRESSION THROUGH EFFECTS ON BOTH PROMOTER METHYLATION AND TRANSCRIPTION FACTORS"

    Article Title: DNA METHYLATION INHIBITION INCREASES T CELL KIR EXPRESSION THROUGH EFFECTS ON BOTH PROMOTER METHYLATION AND TRANSCRIPTION FACTORS

    Journal: Clinical immunology (Orlando, Fla.)

    doi: 10.1016/j.clim.2008.08.009

    Effect of 5-azaC on KIR2DL2 and KIR2DL4 promoter methylation
    Figure Legend Snippet: Effect of 5-azaC on KIR2DL2 and KIR2DL4 promoter methylation

    Techniques Used: Methylation

    KIR2DL4 induced by 5-azaC is functional
    Figure Legend Snippet: KIR2DL4 induced by 5-azaC is functional

    Techniques Used: Functional Assay

    35) Product Images from "Regulation of Human RNA Polymerase III Transcription by DNMT1 and DNMT3a DNA Methyltransferases *"

    Article Title: Regulation of Human RNA Polymerase III Transcription by DNMT1 and DNMT3a DNA Methyltransferases *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.285601

    Multiple DNMTs impede human RNA polymerase III transcription. A , DNMT knockdown causes increased U6 transcription.  Top , human MCF7 cells were co-transfected with the U6 reporter plasmid ( lanes 2–9 ) along with different siRNA oligonucleotides against
    Figure Legend Snippet: Multiple DNMTs impede human RNA polymerase III transcription. A , DNMT knockdown causes increased U6 transcription. Top , human MCF7 cells were co-transfected with the U6 reporter plasmid ( lanes 2–9 ) along with different siRNA oligonucleotides against

    Techniques Used: Transfection, Plasmid Preparation

    DNA methylation inhibits U6 transcription by RNA polymerase III. A , schematic representation of endogenous U6 loci that harbor identical U6 coding regions. The positions of CpG dinucleotides are depicted by a red dot , and the relative positions of key
    Figure Legend Snippet: DNA methylation inhibits U6 transcription by RNA polymerase III. A , schematic representation of endogenous U6 loci that harbor identical U6 coding regions. The positions of CpG dinucleotides are depicted by a red dot , and the relative positions of key

    Techniques Used: DNA Methylation Assay

    RB can enable DNA methylation independently of nucleosomes. A , recombinant RB represses human U6 transcription by RNA polymerase III from naked DNA templates. In vitro U6 transcription assays were performed using HeLa nuclear extract and the U6 reporter
    Figure Legend Snippet: RB can enable DNA methylation independently of nucleosomes. A , recombinant RB represses human U6 transcription by RNA polymerase III from naked DNA templates. In vitro U6 transcription assays were performed using HeLa nuclear extract and the U6 reporter

    Techniques Used: DNA Methylation Assay, Recombinant, In Vitro

    36) Product Images from "5′-end NAD+ cap in human cells promotes RNA decay through DXO-mediated deNADding"

    Article Title: 5′-end NAD+ cap in human cells promotes RNA decay through DXO-mediated deNADding

    Journal: Cell

    doi: 10.1016/j.cell.2017.02.019

    DXO is a deNADding enzyme in cells and NAD-capped RNAs do not support translation (A) 32 P-NAD + cap-labeled RNA was transfected into HEK293T control knock-out (Con-KO), DXO knock-out (DXO-KO) or Dcp2 knock-out (Dcp2-KO) cell lines with Lipofectamine 3000. Untransfected RNAs were degraded with micrococcal nuclease (MN) and total RNA was isolated at the indicated time points following MN treatment and resolved on 8% Denaturing PAGE and exposed to a PhosphorImager. RNA remaining was quantitated and plotted from three independent experiments with ±SD denoted by the error bars. Stabilities: Con-KO t 1/2 =2.8 hr [95%CI: 2.0 – 4.5]; DXO-KO t 1/2 =6.3 hr [95%CI: 4.2 – 11.9] (ANOVA, p=6.10 × 10 −4 ); Dcp2-KO t 1/2 =3.2 hr, [95%CI: 2.3 – 5.8] (ANOVA, p=0.38). (B) Same as A except the NAD + -capped RNA was uniformly labeled 32 P. Stabilities: Con-KO t 1/2 =3.0 hr [95%CI: 2.4 – 4.3]; DXO-KO t 1/2 =5.7 hr [95%CI: 4.1 – 8.9] (ANOVA, p=7.88 × 10 −4 ); Dcp2-KO t 1/2 =3.3 hr, [95%CI: 2.7 – 4.4] (ANOVA, p=0.17). (C) Same as A except the RNA contained an m 7 G cap and was uniformly labeled within the RNA body with 32 P. Stabilities: Con-KO t 1/2 =4.1 hr [95%CI: 3.2 – 5.8]; DXO-KO t 1/2 =3.9 hr [95%CI: 3.0 – 5.4] (ANOVA, p=0.51); Dcp2-KO t 1/2 =4.1 hr, [95%CI: 3.1 – 5.9] (ANOVA, p=0.64). (D). Luciferase RNAs with a 5′-end triphosphate lacking a cap, 5′-end NAD + cap or m 7 G cap transfected into control or DXO-KO cells. Cell were treated with micrococcal nuclease to degrade untransfected RNA, harvested at the indicated times and remaining RNA levels quantitated from three independent experiments. The left panel shows RNAs transfections into control HEK293T (Con-KO) cells and the right panel presents RNAs introduced into either Con-KO or DXO-KO cells as indicated. Error bars denoted ±SD. Con-KO m 7 G-FLuc t 1/2 = 92.6 min (95% CI: 82.2 to 107.8 min, R2=0.96, p=2.10 × 10 −8 ); DXO-KO m 7 G-FLuc t 1/2 =125 min (95% CI: 92.2 to 174 min, R2=0.84, p=1.47 × 10 −5 ); Con-KO NAD-FLuc t 1/2 = 67.4 min (95% CI: 51.4 to 97.9 min, R2=0.82, p=3.05 × 10 −5 ); DXO-KO NAD-FLuc t 1/2 = 103.6 min (95% CI: 85.2 to 132.3 min, R2=0.91, p=1.22 × 10 −6 ); Con-KO pppA-RNA=110.7 min (95% CI: 88 to 149.2 min, R2=0.870, p=5.99 × 10 −5 ). (E) . Firefly luciferase mRNAs containing either a 5′-end NAD + cap, m 7 G cap or no cap and 3′ poly(A) 60 tail were co-transfected with m 7 G-capped Renilla Luciferase RNA into HEK293T cells. Cells were harvested and assayed at the indicated time points. Firefly Luciferase activity was plotted normalized to Renilla Luciferase and data from three independent experiments are presented with error bars representing +/−SD.
    Figure Legend Snippet: DXO is a deNADding enzyme in cells and NAD-capped RNAs do not support translation (A) 32 P-NAD + cap-labeled RNA was transfected into HEK293T control knock-out (Con-KO), DXO knock-out (DXO-KO) or Dcp2 knock-out (Dcp2-KO) cell lines with Lipofectamine 3000. Untransfected RNAs were degraded with micrococcal nuclease (MN) and total RNA was isolated at the indicated time points following MN treatment and resolved on 8% Denaturing PAGE and exposed to a PhosphorImager. RNA remaining was quantitated and plotted from three independent experiments with ±SD denoted by the error bars. Stabilities: Con-KO t 1/2 =2.8 hr [95%CI: 2.0 – 4.5]; DXO-KO t 1/2 =6.3 hr [95%CI: 4.2 – 11.9] (ANOVA, p=6.10 × 10 −4 ); Dcp2-KO t 1/2 =3.2 hr, [95%CI: 2.3 – 5.8] (ANOVA, p=0.38). (B) Same as A except the NAD + -capped RNA was uniformly labeled 32 P. Stabilities: Con-KO t 1/2 =3.0 hr [95%CI: 2.4 – 4.3]; DXO-KO t 1/2 =5.7 hr [95%CI: 4.1 – 8.9] (ANOVA, p=7.88 × 10 −4 ); Dcp2-KO t 1/2 =3.3 hr, [95%CI: 2.7 – 4.4] (ANOVA, p=0.17). (C) Same as A except the RNA contained an m 7 G cap and was uniformly labeled within the RNA body with 32 P. Stabilities: Con-KO t 1/2 =4.1 hr [95%CI: 3.2 – 5.8]; DXO-KO t 1/2 =3.9 hr [95%CI: 3.0 – 5.4] (ANOVA, p=0.51); Dcp2-KO t 1/2 =4.1 hr, [95%CI: 3.1 – 5.9] (ANOVA, p=0.64). (D). Luciferase RNAs with a 5′-end triphosphate lacking a cap, 5′-end NAD + cap or m 7 G cap transfected into control or DXO-KO cells. Cell were treated with micrococcal nuclease to degrade untransfected RNA, harvested at the indicated times and remaining RNA levels quantitated from three independent experiments. The left panel shows RNAs transfections into control HEK293T (Con-KO) cells and the right panel presents RNAs introduced into either Con-KO or DXO-KO cells as indicated. Error bars denoted ±SD. Con-KO m 7 G-FLuc t 1/2 = 92.6 min (95% CI: 82.2 to 107.8 min, R2=0.96, p=2.10 × 10 −8 ); DXO-KO m 7 G-FLuc t 1/2 =125 min (95% CI: 92.2 to 174 min, R2=0.84, p=1.47 × 10 −5 ); Con-KO NAD-FLuc t 1/2 = 67.4 min (95% CI: 51.4 to 97.9 min, R2=0.82, p=3.05 × 10 −5 ); DXO-KO NAD-FLuc t 1/2 = 103.6 min (95% CI: 85.2 to 132.3 min, R2=0.91, p=1.22 × 10 −6 ); Con-KO pppA-RNA=110.7 min (95% CI: 88 to 149.2 min, R2=0.870, p=5.99 × 10 −5 ). (E) . Firefly luciferase mRNAs containing either a 5′-end NAD + cap, m 7 G cap or no cap and 3′ poly(A) 60 tail were co-transfected with m 7 G-capped Renilla Luciferase RNA into HEK293T cells. Cells were harvested and assayed at the indicated time points. Firefly Luciferase activity was plotted normalized to Renilla Luciferase and data from three independent experiments are presented with error bars representing +/−SD.

    Techniques Used: Labeling, Transfection, Knock-Out, Isolation, Polyacrylamide Gel Electrophoresis, Luciferase, Activity Assay

    37) Product Images from "Structure of human IFIT1 with capped RNA reveals adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2′-O methylations"

    Article Title: Structure of human IFIT1 with capped RNA reveals adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2′-O methylations

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1612444114

    IFIT1 PPP- and m7Gppp- binding mechanism. ( A ) Simulated annealing 2 F o – F c omit map of the PPP moiety contoured at 1σ. ( B ) Cartoon/stick representation of residues making specific contacts with the triphosphate group from PPP-RNA–bound
    Figure Legend Snippet: IFIT1 PPP- and m7Gppp- binding mechanism. ( A ) Simulated annealing 2 F o – F c omit map of the PPP moiety contoured at 1σ. ( B ) Cartoon/stick representation of residues making specific contacts with the triphosphate group from PPP-RNA–bound

    Techniques Used: Binding Assay

    IFIT1 senses N1 and N2 ribose 2′-O methylation ( A and B ) EMSAs between 1 µM or 2.5 µM IFIT1 and differentially methylated m7Gppp-RNA ( C ) Comparison of IFIT1 binding to 35 nM Cap0-MHV, Cap1-MHV, or N2Me-MHV. Cap1 methylation reduces
    Figure Legend Snippet: IFIT1 senses N1 and N2 ribose 2′-O methylation ( A and B ) EMSAs between 1 µM or 2.5 µM IFIT1 and differentially methylated m7Gppp-RNA ( C ) Comparison of IFIT1 binding to 35 nM Cap0-MHV, Cap1-MHV, or N2Me-MHV. Cap1 methylation reduces

    Techniques Used: Methylation, Binding Assay

    38) Product Images from "Structure of human IFIT1 with capped RNA reveals adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2′-O methylations"

    Article Title: Structure of human IFIT1 with capped RNA reveals adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2′-O methylations

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1612444114

    IFIT1 PPP- and m7Gppp- binding mechanism. ( A ) Simulated annealing 2 F o – F c omit map of the PPP moiety contoured at 1σ. ( B ) Cartoon/stick representation of residues making specific contacts with the triphosphate group from PPP-RNA–bound
    Figure Legend Snippet: IFIT1 PPP- and m7Gppp- binding mechanism. ( A ) Simulated annealing 2 F o – F c omit map of the PPP moiety contoured at 1σ. ( B ) Cartoon/stick representation of residues making specific contacts with the triphosphate group from PPP-RNA–bound

    Techniques Used: Binding Assay

    IFIT1 mRNA cap-binding mechanism. ( A ) The IFIT1 PPP (blue) adopts an extended conformation compared with the “bent” IFIT5 PPP (pink). The γ-phosphate from PPP–RNA-bound IFIT1 points toward the nearby unoccupied cap-binding
    Figure Legend Snippet: IFIT1 mRNA cap-binding mechanism. ( A ) The IFIT1 PPP (blue) adopts an extended conformation compared with the “bent” IFIT5 PPP (pink). The γ-phosphate from PPP–RNA-bound IFIT1 points toward the nearby unoccupied cap-binding

    Techniques Used: Binding Assay

    39) Product Images from "scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells"

    Article Title: scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03149-4

    scNMT-seq overview and genome-wide coverage. a Protocol overview. Single-cells are lysed and accessible DNA is labelled using GpC methyltransferase. RNA is then separated and sequenced using Smart-seq2, whilst DNA undergoes scBS-seq library preparation and sequencing. Methylation and chromatin accessibility data are separated bioinformatically. b Theoretical maximum CpG coverage of genomic contexts with known regulatory roles. Shown is the proportion of loci in different contexts that contain at least 5 cytosines. ‘All CpG’ considers any C-G dinucleotides (e.g., as in scBS-seq), ‘NOMe-seq CpG’ considers A–C–G and T–C–G trinucleotides and ‘NOMe-seq GpC’ considers G–C–A, G–C–C and G–C–T trinucleotides. c Empirical coverage in 61 mouse ES cells considering the same contexts as in b . Shown is the coverage distribution across cells after QC; box plots show median coverage and the first and third quartile, whiskers show 1.5 × the interquartile range above and below the box. d . The profiles were computed as a running average in 50 bp windows. Shading denotes standard deviation across cells. e CpG methylation and GpC accessibility profiles at gene promoters. Promoters are stratified by average expression level of the corresponding gene (log normalised counts less than 2 (low), between 2 and 6 (medium) and higher than 6 (high). The profile is generated by computing a running average in 50 bp windows
    Figure Legend Snippet: scNMT-seq overview and genome-wide coverage. a Protocol overview. Single-cells are lysed and accessible DNA is labelled using GpC methyltransferase. RNA is then separated and sequenced using Smart-seq2, whilst DNA undergoes scBS-seq library preparation and sequencing. Methylation and chromatin accessibility data are separated bioinformatically. b Theoretical maximum CpG coverage of genomic contexts with known regulatory roles. Shown is the proportion of loci in different contexts that contain at least 5 cytosines. ‘All CpG’ considers any C-G dinucleotides (e.g., as in scBS-seq), ‘NOMe-seq CpG’ considers A–C–G and T–C–G trinucleotides and ‘NOMe-seq GpC’ considers G–C–A, G–C–C and G–C–T trinucleotides. c Empirical coverage in 61 mouse ES cells considering the same contexts as in b . Shown is the coverage distribution across cells after QC; box plots show median coverage and the first and third quartile, whiskers show 1.5 × the interquartile range above and below the box. d . The profiles were computed as a running average in 50 bp windows. Shading denotes standard deviation across cells. e CpG methylation and GpC accessibility profiles at gene promoters. Promoters are stratified by average expression level of the corresponding gene (log normalised counts less than 2 (low), between 2 and 6 (medium) and higher than 6 (high). The profile is generated by computing a running average in 50 bp windows

    Techniques Used: Genome Wide, Gel Permeation Chromatography, Sequencing, Methylation, Standard Deviation, CpG Methylation Assay, Expressing, Generated

    40) Product Images from "Methylation-mediated transcriptional repression of microRNAs during cervical carcinogenesis"

    Article Title: Methylation-mediated transcriptional repression of microRNAs during cervical carcinogenesis

    Journal: Epigenetics

    doi: 10.4161/epi.23605

    Figure 1. Methylation patterns of selected miRNAs in the cell line panel. Methylation as determined in human foreskin keratinocytes (HFKs), HPV-transformed keratinocyte cell lines FK16A, FK18A and FK18B (reminiscent of high-grade CIN), and cervical cancer cell lines SiHa, HeLa and CaSki is shown for ( A ) hsa-miR-149, ( B ) hsa-miR-203, ( C ) hsa-miR-375, ( D ) hsa-miR-572 and ( E ) hsa-miR-638. In ( F ) ACTB results are shown, indicating successful modification and comparable input for all samples. In vitro methylated DNA (IVD) and unmodified DNA (UD) were included as a positive and negative control, respectively.
    Figure Legend Snippet: Figure 1. Methylation patterns of selected miRNAs in the cell line panel. Methylation as determined in human foreskin keratinocytes (HFKs), HPV-transformed keratinocyte cell lines FK16A, FK18A and FK18B (reminiscent of high-grade CIN), and cervical cancer cell lines SiHa, HeLa and CaSki is shown for ( A ) hsa-miR-149, ( B ) hsa-miR-203, ( C ) hsa-miR-375, ( D ) hsa-miR-572 and ( E ) hsa-miR-638. In ( F ) ACTB results are shown, indicating successful modification and comparable input for all samples. In vitro methylated DNA (IVD) and unmodified DNA (UD) were included as a positive and negative control, respectively.

    Techniques Used: Methylation, Transformation Assay, Modification, In Vitro, Negative Control

    Related Articles

    In Vitro:

    Article Title: High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion
    Article Snippet: .. In vitro methylation of lambda DNA Fully methylated bacteriophage lambda (λ) DNA was acquired by incubating 1 μg of λ DNA in a 20-μl reaction containing 1 U of M.SssI methylase, 1X NEBuffer 2, and freshly-prepared 160 μM S-adenosylmethionine at 37°C for 1 h, followed by heat inactivation of the enzyme at 65°C for 20 min ( ). ..

    Methylation:

    Article Title: High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion
    Article Snippet: .. In vitro methylation of lambda DNA Fully methylated bacteriophage lambda (λ) DNA was acquired by incubating 1 μg of λ DNA in a 20-μl reaction containing 1 U of M.SssI methylase, 1X NEBuffer 2, and freshly-prepared 160 μM S-adenosylmethionine at 37°C for 1 h, followed by heat inactivation of the enzyme at 65°C for 20 min ( ). ..

    Lambda DNA Preparation:

    Article Title: Single Molecule Hydrodynamic Separation Allows Sensitive and Quantitative Analysis of DNA Conformation and Binding Interactions in Free Solution
    Article Snippet: .. Lambda DNA, HindIII digested Lambda DNA, 1 kb DNA Ladder, and Supercoiled DNA Ladder (all from New England Biolabs, Inc.) were used as double stranded DNA samples. .. Staining was performed at 5 or 10 ng/μL total dsDNA concentration and 1 μM TOTO-3 Iodide (Life Technologies) for at least 1 hour in the dark.

    Article Title: High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion
    Article Snippet: .. In vitro methylation of lambda DNA Fully methylated bacteriophage lambda (λ) DNA was acquired by incubating 1 μg of λ DNA in a 20-μl reaction containing 1 U of M.SssI methylase, 1X NEBuffer 2, and freshly-prepared 160 μM S-adenosylmethionine at 37°C for 1 h, followed by heat inactivation of the enzyme at 65°C for 20 min ( ). ..

    Purification:

    Article Title: Efficient modification of λ-DNA substrates for single-molecule studies
    Article Snippet: .. Inserting synthetic oligonucleotides into λ-DNA Recombinant λ-DNA was obtained from strain IF189, which was modified and purified as described above, and 25 μg of the DNA was incubated with 150 U of Nt.BspQI (NEB# R0644S) in a 250 µL reaction with 1X buffer 3.1 (NEB #B7203) at 55 °C for 1 hour. .. The reaction was halted with 1 U of proteinase K (NEB #P8107S) for 1 hour at 55 °C.

    Incubation:

    Article Title: Efficient modification of λ-DNA substrates for single-molecule studies
    Article Snippet: .. Inserting synthetic oligonucleotides into λ-DNA Recombinant λ-DNA was obtained from strain IF189, which was modified and purified as described above, and 25 μg of the DNA was incubated with 150 U of Nt.BspQI (NEB# R0644S) in a 250 µL reaction with 1X buffer 3.1 (NEB #B7203) at 55 °C for 1 hour. .. The reaction was halted with 1 U of proteinase K (NEB #P8107S) for 1 hour at 55 °C.

    Modification:

    Article Title: Efficient modification of λ-DNA substrates for single-molecule studies
    Article Snippet: .. Inserting synthetic oligonucleotides into λ-DNA Recombinant λ-DNA was obtained from strain IF189, which was modified and purified as described above, and 25 μg of the DNA was incubated with 150 U of Nt.BspQI (NEB# R0644S) in a 250 µL reaction with 1X buffer 3.1 (NEB #B7203) at 55 °C for 1 hour. .. The reaction was halted with 1 U of proteinase K (NEB #P8107S) for 1 hour at 55 °C.

    Recombinant:

    Article Title: Efficient modification of λ-DNA substrates for single-molecule studies
    Article Snippet: .. Inserting synthetic oligonucleotides into λ-DNA Recombinant λ-DNA was obtained from strain IF189, which was modified and purified as described above, and 25 μg of the DNA was incubated with 150 U of Nt.BspQI (NEB# R0644S) in a 250 µL reaction with 1X buffer 3.1 (NEB #B7203) at 55 °C for 1 hour. .. The reaction was halted with 1 U of proteinase K (NEB #P8107S) for 1 hour at 55 °C.

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    New England Biolabs adomet
    Human METTL12 is a protein-specific <t>MTase.</t> A , targeting of METTL12 gene in human HAP1 WT cells by CRISPR/Cas9 generated METTL12 KO cells containing a 1 base pair insertion in the METTL12 gene, located upstream of motif Post I, resulting in generation of truncated METTL12 protein. The dashed lines interrupting the open reading frames correspond to 177 nucleotides, i.e. 59 amino acids. B , METTL12-dependent protein methylation in cell extracts. Mitochondrial extracts from HAP1 WT or METTL12 KO cells were incubated with [ 3 <t>H]AdoMet</t> and recombinant human METTL12. Methylation reactions were separated by SDS-PAGE and transferred to a membrane. Methylation was visualized by fluorography ( top ) of the Ponceau S-stained membrane ( bottom ). Arrows indicate the positions of the ∼48 kDa substrate and METTL12. C , D107A mutation abrogates enzymatic activity of METTL12. Mitochondrial extracts from METTL12 KO cells were incubated with [ 3 H]AdoMet and recombinant human METTL12, either WT or D107A-mutated. Methylation was analyzed as in B . Note: in panels B and C different levels of background (non-METTL12-dependent) methylation are observed; this is likely due to differences in the purity of the mitochondrial extracts.
    Adomet, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    New England Biolabs non radioactive adomet
    Identification of METTL13 as an eEF1A-specific methyltransferase. a Workflow of mass spectrometry-based quantitative peptide pull-down screen. Synthetic peptides corresponding N-terminally trimethylated (Nt-Me3) and unmethylated (Nt-Me0) eEF1A were used as baits to enrich proteins from HAP-1 cell extracts. b . c Domain organization of METTL13. The boundaries for used constructs encompassing the N-terminal (MT13-N) and the C-terminal (MT13-C) methyltransferase domains are indicated. d , e Evaluation of METTL13 constructs for eEF1A-specific methyltransferase activity. MT13-N ( d ) and MT13-C ( e ) were incubated with [ 3 <t>H]-AdoMet</t> and eEF1A1 carrying an N-terminal or C-terminal His-tag in the absence of cofactors and in the presence of either GDP or GTP. Methylation was visualized by fluorography (top panels) and the membranes were stained with Ponceau S (bottom panels) to assess protein loading
    Non Radioactive Adomet, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/non radioactive adomet/product/New England Biolabs
    Average 91 stars, based on 2 article reviews
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    Human METTL12 is a protein-specific MTase. A , targeting of METTL12 gene in human HAP1 WT cells by CRISPR/Cas9 generated METTL12 KO cells containing a 1 base pair insertion in the METTL12 gene, located upstream of motif Post I, resulting in generation of truncated METTL12 protein. The dashed lines interrupting the open reading frames correspond to 177 nucleotides, i.e. 59 amino acids. B , METTL12-dependent protein methylation in cell extracts. Mitochondrial extracts from HAP1 WT or METTL12 KO cells were incubated with [ 3 H]AdoMet and recombinant human METTL12. Methylation reactions were separated by SDS-PAGE and transferred to a membrane. Methylation was visualized by fluorography ( top ) of the Ponceau S-stained membrane ( bottom ). Arrows indicate the positions of the ∼48 kDa substrate and METTL12. C , D107A mutation abrogates enzymatic activity of METTL12. Mitochondrial extracts from METTL12 KO cells were incubated with [ 3 H]AdoMet and recombinant human METTL12, either WT or D107A-mutated. Methylation was analyzed as in B . Note: in panels B and C different levels of background (non-METTL12-dependent) methylation are observed; this is likely due to differences in the purity of the mitochondrial extracts.

    Journal: The Journal of Biological Chemistry

    Article Title: Uncovering human METTL12 as a mitochondrial methyltransferase that modulates citrate synthase activity through metabolite-sensitive lysine methylation

    doi: 10.1074/jbc.M117.808451

    Figure Lengend Snippet: Human METTL12 is a protein-specific MTase. A , targeting of METTL12 gene in human HAP1 WT cells by CRISPR/Cas9 generated METTL12 KO cells containing a 1 base pair insertion in the METTL12 gene, located upstream of motif Post I, resulting in generation of truncated METTL12 protein. The dashed lines interrupting the open reading frames correspond to 177 nucleotides, i.e. 59 amino acids. B , METTL12-dependent protein methylation in cell extracts. Mitochondrial extracts from HAP1 WT or METTL12 KO cells were incubated with [ 3 H]AdoMet and recombinant human METTL12. Methylation reactions were separated by SDS-PAGE and transferred to a membrane. Methylation was visualized by fluorography ( top ) of the Ponceau S-stained membrane ( bottom ). Arrows indicate the positions of the ∼48 kDa substrate and METTL12. C , D107A mutation abrogates enzymatic activity of METTL12. Mitochondrial extracts from METTL12 KO cells were incubated with [ 3 H]AdoMet and recombinant human METTL12, either WT or D107A-mutated. Methylation was analyzed as in B . Note: in panels B and C different levels of background (non-METTL12-dependent) methylation are observed; this is likely due to differences in the purity of the mitochondrial extracts.

    Article Snippet: For scintillation counting and titration experiments, MTase reactions contained [3 H]AdoMet, which was diluted with nonradioactive AdoMet (New England BioLabs).

    Techniques: CRISPR, Generated, Methylation, Incubation, Recombinant, SDS Page, Staining, Mutagenesis, Activity Assay

    Mg18 2′O MTase activity. AdoMet-dependent MTase assays were performed on equimolar amounts of short capped RNAs substrates (GpppAN 13 ), N7- or 2′O-methylated capped RNA substrates ( 7Me GpppAN 13 or GpppA 2′OMe N 13 ), A. castellanii mRNAs or homopolymeric poly (U), (C), (G) and (A). Human N7 MTase and Vaccinia virus VP39 were use as controls.

    Journal: Nucleic Acids Research

    Article Title: mRNA maturation in giant viruses: variation on a theme

    doi: 10.1093/nar/gkv224

    Figure Lengend Snippet: Mg18 2′O MTase activity. AdoMet-dependent MTase assays were performed on equimolar amounts of short capped RNAs substrates (GpppAN 13 ), N7- or 2′O-methylated capped RNA substrates ( 7Me GpppAN 13 or GpppA 2′OMe N 13 ), A. castellanii mRNAs or homopolymeric poly (U), (C), (G) and (A). Human N7 MTase and Vaccinia virus VP39 were use as controls.

    Article Snippet: MTase activity assays were performed at 30°C in 40 mM Tris-HCl pH 7.5, 1 mM DTT, 1 mM MgCl2 , 0.7 μM RNA substrates, 10 μM AdoMet (NEB), and 0.03 mCi/ml 3 H-AdoMet (GE Healthcare).

    Techniques: Activity Assay, Methylation

    Purified M.HpyAIV protects a GANTC-containing DNA fragment from HinfI digestion. Increasing concentrations of M.HpyAIV protein incubated with a 778-bp PCR fragment containing one GANTC site and S -adenosylmethionine. HinfI digestion of the GANTC-containing DNA fragment resulted in two fragments of 540 bp and 238 bp. The increased amount of undigested PCR products as a consequence of an increased M.HpyAIV concentration illustrates the in vitro capability of M.HpyAIV to protect GANTC sites from digestion in a concentration-dependent manner. L, ladder (samples in duplicate with increasing amounts of M.HpyAIV added [0, 200, 400, 800, and 1,200 nM]); UC, uncut control.

    Journal: Journal of Bacteriology

    Article Title: Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori ▿

    doi: 10.1128/JB.00108-07

    Figure Lengend Snippet: Purified M.HpyAIV protects a GANTC-containing DNA fragment from HinfI digestion. Increasing concentrations of M.HpyAIV protein incubated with a 778-bp PCR fragment containing one GANTC site and S -adenosylmethionine. HinfI digestion of the GANTC-containing DNA fragment resulted in two fragments of 540 bp and 238 bp. The increased amount of undigested PCR products as a consequence of an increased M.HpyAIV concentration illustrates the in vitro capability of M.HpyAIV to protect GANTC sites from digestion in a concentration-dependent manner. L, ladder (samples in duplicate with increasing amounts of M.HpyAIV added [0, 200, 400, 800, and 1,200 nM]); UC, uncut control.

    Article Snippet: To investigate methylation and protection by the recombinant protein, we incubated 1 μg of a PCR fragment containing one GANTC site (778 bp, amplified with 1351GANTCF and 1351GANTCR) (Table ) with NEB2 buffer, S -adenosylmethionine (New England BioLabs), and different M.HpyAIV concentrations (0, 200, 400, 800, and 1,200 nM) and performed incubation for 1 h at room temperature followed by protein inactivation at 95°C for 10 min.

    Techniques: Purification, Incubation, Polymerase Chain Reaction, Concentration Assay, In Vitro

    Identification of METTL13 as an eEF1A-specific methyltransferase. a Workflow of mass spectrometry-based quantitative peptide pull-down screen. Synthetic peptides corresponding N-terminally trimethylated (Nt-Me3) and unmethylated (Nt-Me0) eEF1A were used as baits to enrich proteins from HAP-1 cell extracts. b . c Domain organization of METTL13. The boundaries for used constructs encompassing the N-terminal (MT13-N) and the C-terminal (MT13-C) methyltransferase domains are indicated. d , e Evaluation of METTL13 constructs for eEF1A-specific methyltransferase activity. MT13-N ( d ) and MT13-C ( e ) were incubated with [ 3 H]-AdoMet and eEF1A1 carrying an N-terminal or C-terminal His-tag in the absence of cofactors and in the presence of either GDP or GTP. Methylation was visualized by fluorography (top panels) and the membranes were stained with Ponceau S (bottom panels) to assess protein loading

    Journal: Nature Communications

    Article Title: The dual methyltransferase METTL13 targets N terminus and Lys55 of eEF1A and modulates codon-specific translation rates

    doi: 10.1038/s41467-018-05646-y

    Figure Lengend Snippet: Identification of METTL13 as an eEF1A-specific methyltransferase. a Workflow of mass spectrometry-based quantitative peptide pull-down screen. Synthetic peptides corresponding N-terminally trimethylated (Nt-Me3) and unmethylated (Nt-Me0) eEF1A were used as baits to enrich proteins from HAP-1 cell extracts. b . c Domain organization of METTL13. The boundaries for used constructs encompassing the N-terminal (MT13-N) and the C-terminal (MT13-C) methyltransferase domains are indicated. d , e Evaluation of METTL13 constructs for eEF1A-specific methyltransferase activity. MT13-N ( d ) and MT13-C ( e ) were incubated with [ 3 H]-AdoMet and eEF1A1 carrying an N-terminal or C-terminal His-tag in the absence of cofactors and in the presence of either GDP or GTP. Methylation was visualized by fluorography (top panels) and the membranes were stained with Ponceau S (bottom panels) to assess protein loading

    Article Snippet: For quantitative MTase assays, [3 H]-AdoMet was diluted with non-radioactive AdoMet (New England Biolabs) ([AdoMet]total = 32.6 μM) .

    Techniques: Mass Spectrometry, Construct, Activity Assay, Incubation, Methylation, Staining