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    New England Biolabs cpg dna methylase m sssi
    Effect of global <t>CpG</t> methylation on HPV16 E2-dependent transcriptional activation. (A) Schematic of HPV16 LCR, BamC promoter, and luciferase reporter plasmid pKT267, illustrating the relative positions of the HPV16 LCR, the basic BamC promoter from EBV, and the firefly luciferase translational ORF. Asterisks, relative positions of E2BSs. (B) Bar graph summarizing the luciferase activities measured in lysates from cell populations transfected with methylated or unmethylated pKT267. Three micrograms of the plasmid pKT267 that had or had not been treated in vitro with CpG methylase was transfected into C33A cells by electroporation in the presence or absence of 9 μg of the HPV16 E2 expression vector pCMV 4 E2. Luciferase activities were measured 48 h posttransfection. All experiments were repeated at least three times. Bars: 1, no <t>DNA</t> transfection control; 2 methylated pKT267 alone; 3 methylated pKT267 plus pCMV 4 E2; 4 unmethylated pKT267 alone; 5 unmethylated pKT267 plus pCMV 4 E2 (error bar, standard deviation).
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    Effect of global CpG methylation on HPV16 E2-dependent transcriptional activation. (A) Schematic of HPV16 LCR, BamC promoter, and luciferase reporter plasmid pKT267, illustrating the relative positions of the HPV16 LCR, the basic BamC promoter from EBV, and the firefly luciferase translational ORF. Asterisks, relative positions of E2BSs. (B) Bar graph summarizing the luciferase activities measured in lysates from cell populations transfected with methylated or unmethylated pKT267. Three micrograms of the plasmid pKT267 that had or had not been treated in vitro with CpG methylase was transfected into C33A cells by electroporation in the presence or absence of 9 μg of the HPV16 E2 expression vector pCMV 4 E2. Luciferase activities were measured 48 h posttransfection. All experiments were repeated at least three times. Bars: 1, no DNA transfection control; 2 methylated pKT267 alone; 3 methylated pKT267 plus pCMV 4 E2; 4 unmethylated pKT267 alone; 5 unmethylated pKT267 plus pCMV 4 E2 (error bar, standard deviation).

    Journal: Journal of Virology

    Article Title: Methylation Patterns of Papillomavirus DNA, Its Influence on E2 Function, and Implications in Viral Infection

    doi: 10.1128/JVI.77.23.12450-12459.2003

    Figure Lengend Snippet: Effect of global CpG methylation on HPV16 E2-dependent transcriptional activation. (A) Schematic of HPV16 LCR, BamC promoter, and luciferase reporter plasmid pKT267, illustrating the relative positions of the HPV16 LCR, the basic BamC promoter from EBV, and the firefly luciferase translational ORF. Asterisks, relative positions of E2BSs. (B) Bar graph summarizing the luciferase activities measured in lysates from cell populations transfected with methylated or unmethylated pKT267. Three micrograms of the plasmid pKT267 that had or had not been treated in vitro with CpG methylase was transfected into C33A cells by electroporation in the presence or absence of 9 μg of the HPV16 E2 expression vector pCMV 4 E2. Luciferase activities were measured 48 h posttransfection. All experiments were repeated at least three times. Bars: 1, no DNA transfection control; 2 methylated pKT267 alone; 3 methylated pKT267 plus pCMV 4 E2; 4 unmethylated pKT267 alone; 5 unmethylated pKT267 plus pCMV 4 E2 (error bar, standard deviation).

    Article Snippet: In vitro DNA methylation was accomplished with CpG methylase ( Sss I methyltransferase), by following the procedure recommended by New England Biolabs, the commercial provider of Sss I.

    Techniques: CpG Methylation Assay, Activation Assay, Luciferase, Plasmid Preparation, Transfection, Methylation, In Vitro, Electroporation, Expressing, Standard Deviation

    Effect of the global CpG methylation on BPV1 E2-dependent transcriptional activation. (A) Schematic of BPV1 LCR, the BamC promoter, and luciferase reporter plasmid pKT260, illustrating the relative positions of the BPV1 sequences, the basic BamC promoter from EBV, and the firefly luciferase translational ORF. Asterisk, relative position of the E2BS. (B) Bar graph summarizing the luciferase activities measured in lysates from cell populations transfected with methylated or unmethylated pKT260. Eight micrograms of the plasmid pKT260 that had or had not been treated in vitro with CpG methylase was transfected into C33A cells by electroporation in the presence or absence of the BPV1 E2 expression vector pCGE2 (10 μg). Luciferase activities were measured 48 h posttransfection. All experiments were repeated at least three times. Bars (error bars, standard deviations): 1, no DNA transfection control; 2, methylated pKT260 alone; 3 methylated pKT260 plus pCGE2; 4, unmethylated pKT260 alone; 5, unmethylated pKT260 plus pCGE2.

    Journal: Journal of Virology

    Article Title: Methylation Patterns of Papillomavirus DNA, Its Influence on E2 Function, and Implications in Viral Infection

    doi: 10.1128/JVI.77.23.12450-12459.2003

    Figure Lengend Snippet: Effect of the global CpG methylation on BPV1 E2-dependent transcriptional activation. (A) Schematic of BPV1 LCR, the BamC promoter, and luciferase reporter plasmid pKT260, illustrating the relative positions of the BPV1 sequences, the basic BamC promoter from EBV, and the firefly luciferase translational ORF. Asterisk, relative position of the E2BS. (B) Bar graph summarizing the luciferase activities measured in lysates from cell populations transfected with methylated or unmethylated pKT260. Eight micrograms of the plasmid pKT260 that had or had not been treated in vitro with CpG methylase was transfected into C33A cells by electroporation in the presence or absence of the BPV1 E2 expression vector pCGE2 (10 μg). Luciferase activities were measured 48 h posttransfection. All experiments were repeated at least three times. Bars (error bars, standard deviations): 1, no DNA transfection control; 2, methylated pKT260 alone; 3 methylated pKT260 plus pCGE2; 4, unmethylated pKT260 alone; 5, unmethylated pKT260 plus pCGE2.

    Article Snippet: In vitro DNA methylation was accomplished with CpG methylase ( Sss I methyltransferase), by following the procedure recommended by New England Biolabs, the commercial provider of Sss I.

    Techniques: CpG Methylation Assay, Activation Assay, Luciferase, Plasmid Preparation, Transfection, Methylation, In Vitro, Electroporation, Expressing

    Effect of the global CpG methylation on HPV16 E2-dependent transcriptional activation. (A) Schematic of four E2BSs, the basic BamC promoter, and luciferase reporter plasmid pBS1073, illustrating the relative positions of the four E2BSs, the basic BamC promoter from EBV, and the firefly luciferase translational ORF. (B) Bar graph summarizing the luciferase activities measured in lysates from cell populations transfected with methylated or unmethylated pBS1073. Eight micrograms of the plasmid pBS1073 that had or had not been treated in vitro with CpG methylase was transfected into 293 cells by calcium phosphate precipitation in the presence or absence of the HPV16 E2 expression vector (5 μg) pCMV 4 E2. Luciferase activities were measured 48 h posttransfection. All experiments were repeated at least three times. Bars: 1, no DNA transfection control; 2, methylated pBS1073 alone; 3, methylated pBS1073 plus pCMV 4 E2; 4, unmethylated pBS1073 alone; 5, unmethylated pBS1073 plus pCMV 4 E2.

    Journal: Journal of Virology

    Article Title: Methylation Patterns of Papillomavirus DNA, Its Influence on E2 Function, and Implications in Viral Infection

    doi: 10.1128/JVI.77.23.12450-12459.2003

    Figure Lengend Snippet: Effect of the global CpG methylation on HPV16 E2-dependent transcriptional activation. (A) Schematic of four E2BSs, the basic BamC promoter, and luciferase reporter plasmid pBS1073, illustrating the relative positions of the four E2BSs, the basic BamC promoter from EBV, and the firefly luciferase translational ORF. (B) Bar graph summarizing the luciferase activities measured in lysates from cell populations transfected with methylated or unmethylated pBS1073. Eight micrograms of the plasmid pBS1073 that had or had not been treated in vitro with CpG methylase was transfected into 293 cells by calcium phosphate precipitation in the presence or absence of the HPV16 E2 expression vector (5 μg) pCMV 4 E2. Luciferase activities were measured 48 h posttransfection. All experiments were repeated at least three times. Bars: 1, no DNA transfection control; 2, methylated pBS1073 alone; 3, methylated pBS1073 plus pCMV 4 E2; 4, unmethylated pBS1073 alone; 5, unmethylated pBS1073 plus pCMV 4 E2.

    Article Snippet: In vitro DNA methylation was accomplished with CpG methylase ( Sss I methyltransferase), by following the procedure recommended by New England Biolabs, the commercial provider of Sss I.

    Techniques: CpG Methylation Assay, Activation Assay, Luciferase, Plasmid Preparation, Transfection, Methylation, In Vitro, Expressing

    Variance of the relative activity of Dam MTase in response to different concentrations of gentamycin (A) and 5-fluorouracil (B). The concentration of Dam MTase is 10 U mL –1 .

    Journal: Chemical Science

    Article Title: An integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity †Electronic supplementary information (ESI) available: Fig. S1 to S7 and Table S1. See DOI: 10.1039/c8sc05102j

    doi: 10.1039/c8sc05102j

    Figure Lengend Snippet: Variance of the relative activity of Dam MTase in response to different concentrations of gentamycin (A) and 5-fluorouracil (B). The concentration of Dam MTase is 10 U mL –1 .

    Article Snippet: Dam and M. SssI methyltransferase (MTase), endonuclease DpnI, Klenow Fragment Polymerase (3′–5′ exo-) (KFP), nicking enzyme Nb.BbvcI, S -adenyl methionine (SAM), EcoRI enzyme and the corresponding buffer solution were obtained from New England Biolabs (Beijing, China).

    Techniques: Activity Assay, Concentration Assay

    The Dam MTase activity assay by RT-qPCR. (A) Fluorescence–time plot and (B) logarithm of fluorescence–time plot obtained by RT-qPCR system under different concentrations of Dam MTase. The insert shows the linear relationship between the RT t value and the logarithm of the concentration of MTase at a threshold of 4.1.

    Journal: Chemical Science

    Article Title: An integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity †Electronic supplementary information (ESI) available: Fig. S1 to S7 and Table S1. See DOI: 10.1039/c8sc05102j

    doi: 10.1039/c8sc05102j

    Figure Lengend Snippet: The Dam MTase activity assay by RT-qPCR. (A) Fluorescence–time plot and (B) logarithm of fluorescence–time plot obtained by RT-qPCR system under different concentrations of Dam MTase. The insert shows the linear relationship between the RT t value and the logarithm of the concentration of MTase at a threshold of 4.1.

    Article Snippet: Dam and M. SssI methyltransferase (MTase), endonuclease DpnI, Klenow Fragment Polymerase (3′–5′ exo-) (KFP), nicking enzyme Nb.BbvcI, S -adenyl methionine (SAM), EcoRI enzyme and the corresponding buffer solution were obtained from New England Biolabs (Beijing, China).

    Techniques: Activity Assay, Quantitative RT-PCR, Fluorescence, Concentration Assay

    (A) PAGE result of the proposed sensing platform. Line M: DNA marker, line 1: SDA proceeding on shortened MB without Nb.BbvcI, line 2: SDA proceeding on shortened MB with primer 0, line 3: SDA proceeding on shortened MB without primers, line 4: complete SDA proceeding on shortened MB only, line 5: DpnI treated MB without Dam MTase, line 6: shortened MB cut by DpnI, line 7: MB only. (B) Fluorescent spectrum in response to the addition of different components.

    Journal: Chemical Science

    Article Title: An integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity †Electronic supplementary information (ESI) available: Fig. S1 to S7 and Table S1. See DOI: 10.1039/c8sc05102j

    doi: 10.1039/c8sc05102j

    Figure Lengend Snippet: (A) PAGE result of the proposed sensing platform. Line M: DNA marker, line 1: SDA proceeding on shortened MB without Nb.BbvcI, line 2: SDA proceeding on shortened MB with primer 0, line 3: SDA proceeding on shortened MB without primers, line 4: complete SDA proceeding on shortened MB only, line 5: DpnI treated MB without Dam MTase, line 6: shortened MB cut by DpnI, line 7: MB only. (B) Fluorescent spectrum in response to the addition of different components.

    Article Snippet: Dam and M. SssI methyltransferase (MTase), endonuclease DpnI, Klenow Fragment Polymerase (3′–5′ exo-) (KFP), nicking enzyme Nb.BbvcI, S -adenyl methionine (SAM), EcoRI enzyme and the corresponding buffer solution were obtained from New England Biolabs (Beijing, China).

    Techniques: Polyacrylamide Gel Electrophoresis, Marker

    Fluorescent spectrum in response to 0.01 U mL –1 Dam MTase and 0.1 U mL –1 M.SssI MTase. The insert shows the fluorescence intensity at 520 nm according to the spectrum.

    Journal: Chemical Science

    Article Title: An integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity †Electronic supplementary information (ESI) available: Fig. S1 to S7 and Table S1. See DOI: 10.1039/c8sc05102j

    doi: 10.1039/c8sc05102j

    Figure Lengend Snippet: Fluorescent spectrum in response to 0.01 U mL –1 Dam MTase and 0.1 U mL –1 M.SssI MTase. The insert shows the fluorescence intensity at 520 nm according to the spectrum.

    Article Snippet: Dam and M. SssI methyltransferase (MTase), endonuclease DpnI, Klenow Fragment Polymerase (3′–5′ exo-) (KFP), nicking enzyme Nb.BbvcI, S -adenyl methionine (SAM), EcoRI enzyme and the corresponding buffer solution were obtained from New England Biolabs (Beijing, China).

    Techniques: Fluorescence

    (A) The fluorescent spectrum in response to different concentrations of Dam MTase, from low to high; the curve revealed MTase concentrations of 0, 1 × 10 –5 , 5 × 10 –5 , 1 × 10 –4 , 5 × 10 –4 , 1 × 10 –3 , 5 × 10 –3 , 0.01, 0.1, 1 and 10 U mL –1 , respectively. (B) Variance of the fluorescence intensity with the concentration of Dam MTase in the range of 1 × 10 –5 to 1 U mL –1 . The insert shows the linear relationship between the fluorescence intensity and the logarithm of the concentration of Dam MTase.

    Journal: Chemical Science

    Article Title: An integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity †Electronic supplementary information (ESI) available: Fig. S1 to S7 and Table S1. See DOI: 10.1039/c8sc05102j

    doi: 10.1039/c8sc05102j

    Figure Lengend Snippet: (A) The fluorescent spectrum in response to different concentrations of Dam MTase, from low to high; the curve revealed MTase concentrations of 0, 1 × 10 –5 , 5 × 10 –5 , 1 × 10 –4 , 5 × 10 –4 , 1 × 10 –3 , 5 × 10 –3 , 0.01, 0.1, 1 and 10 U mL –1 , respectively. (B) Variance of the fluorescence intensity with the concentration of Dam MTase in the range of 1 × 10 –5 to 1 U mL –1 . The insert shows the linear relationship between the fluorescence intensity and the logarithm of the concentration of Dam MTase.

    Article Snippet: Dam and M. SssI methyltransferase (MTase), endonuclease DpnI, Klenow Fragment Polymerase (3′–5′ exo-) (KFP), nicking enzyme Nb.BbvcI, S -adenyl methionine (SAM), EcoRI enzyme and the corresponding buffer solution were obtained from New England Biolabs (Beijing, China).

    Techniques: Fluorescence, Concentration Assay

    In vitro oxidation of mC causes gene reactivation in ESCs. ( a ) Schematic representation of in vitro reporter DNA modification: Unmethylated pOct4-reporter DNA was methylated using the CpG methyltransferase M.SssI. Incubation with purified TET1CD results in oxidation of mC sites to hmC, fC and caC. ( b ) M.SssI treatment of pOct4-mCherry results in full methylation as shown after restriction with the methylation sensitive enzyme HpaII. MspI cuts irrespective of the methylation state. The hmC-specific restriction endonuclease PvuRts1I detects increasing hmC levels during incubation of methylated pOct4-mCherry with TET1CD. ( c ) Cytosine modification states of untreated, methylated and TET1CD oxidized pOct4-mCherry plasmid DNA were detected by slot blot. A 2-fold serial dilution of the plasmid DNA was loaded and detected using antibodies against mC, hmC, fC and caC. A gradual increase of hmC, fC and caC signals was obtained with longer incubation time with TET1CD while the mC signal decreases accordingly. ( d ) Quantification of the slot blot signals of pOct4-mCherry after treatment with TET1CD shows increasing oxidation of mC to hmC, fC and caC. The sum of all CpG modification signals was set to 100%. Error bars indicate standard deviation ( n = 3). ( e ) ESCs were transfected with pOct4-mCherry plasmids containing either unmodified (CpG), methylated (mCpG), TET1CD-oxidized ( ox CpG) or TET1CD mut -treated ( ox *CpG) cytosines. Confocal imaging and quantification show reporter gene silencing upon methylation and reactivation upon oxidation. Cells were fixed with formaldehyde and counterstained with DAPI. Scale bar: 5 μm. (Right: n = 200 000; error bars indicate standard deviation).

    Journal: Nucleic Acids Research

    Article Title: TET-mediated oxidation of methylcytosine causes TDG or NEIL glycosylase dependent gene reactivation

    doi: 10.1093/nar/gku552

    Figure Lengend Snippet: In vitro oxidation of mC causes gene reactivation in ESCs. ( a ) Schematic representation of in vitro reporter DNA modification: Unmethylated pOct4-reporter DNA was methylated using the CpG methyltransferase M.SssI. Incubation with purified TET1CD results in oxidation of mC sites to hmC, fC and caC. ( b ) M.SssI treatment of pOct4-mCherry results in full methylation as shown after restriction with the methylation sensitive enzyme HpaII. MspI cuts irrespective of the methylation state. The hmC-specific restriction endonuclease PvuRts1I detects increasing hmC levels during incubation of methylated pOct4-mCherry with TET1CD. ( c ) Cytosine modification states of untreated, methylated and TET1CD oxidized pOct4-mCherry plasmid DNA were detected by slot blot. A 2-fold serial dilution of the plasmid DNA was loaded and detected using antibodies against mC, hmC, fC and caC. A gradual increase of hmC, fC and caC signals was obtained with longer incubation time with TET1CD while the mC signal decreases accordingly. ( d ) Quantification of the slot blot signals of pOct4-mCherry after treatment with TET1CD shows increasing oxidation of mC to hmC, fC and caC. The sum of all CpG modification signals was set to 100%. Error bars indicate standard deviation ( n = 3). ( e ) ESCs were transfected with pOct4-mCherry plasmids containing either unmodified (CpG), methylated (mCpG), TET1CD-oxidized ( ox CpG) or TET1CD mut -treated ( ox *CpG) cytosines. Confocal imaging and quantification show reporter gene silencing upon methylation and reactivation upon oxidation. Cells were fixed with formaldehyde and counterstained with DAPI. Scale bar: 5 μm. (Right: n = 200 000; error bars indicate standard deviation).

    Article Snippet: In vitro methylation and oxidation of plasmid DNA In vitro methylation of pOct4-GFP plasmid DNA was performed using M.SssI methyltransferase (New England Biolabs) according to the manufacturer's instructions.

    Techniques: In Vitro, Modification, Methylation, Incubation, Purification, Plasmid Preparation, Dot Blot, Serial Dilution, Standard Deviation, Transfection, Imaging

    DNA methylation of Gpx1 decreases gene expression. In vitro methylation of Gpx1 target region inhibited transcriptional activity, as measured by a luciferase reporter assay. (A) Schematic representation of the plasmid construction containing the Gpx1 CpG island region. (B) Luciferase activity ratio of methylated (M.SssI treated) to unmethylated control (CTL) plasmids containing a CpG-free promoter or the Gpx1 CpG island region. The assay was repeated 4 times and data are mean ± SEM. *: p

    Journal: PLoS ONE

    Article Title: Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice

    doi: 10.1371/journal.pone.0151526

    Figure Lengend Snippet: DNA methylation of Gpx1 decreases gene expression. In vitro methylation of Gpx1 target region inhibited transcriptional activity, as measured by a luciferase reporter assay. (A) Schematic representation of the plasmid construction containing the Gpx1 CpG island region. (B) Luciferase activity ratio of methylated (M.SssI treated) to unmethylated control (CTL) plasmids containing a CpG-free promoter or the Gpx1 CpG island region. The assay was repeated 4 times and data are mean ± SEM. *: p

    Article Snippet: M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions.

    Techniques: DNA Methylation Assay, Expressing, In Vitro, Methylation, Activity Assay, Luciferase, Reporter Assay, Plasmid Preparation, CTL Assay

    Methylation status interfered the Sp1 binding to the CD147 gene promoter and inhibited CD147 transcriptional activity in vitro . (A) Methylation status interfered the Sp1 binding to the CD147 gene promoter in vitro . The binding of Sp1 to the CD147 gene promoter was determined by electrophoretic mobility shift analysis (EMSA). By using biotin-labelled 30-bp double-stranded oligonucleotides containing wild, mutated or methylated Sp1-binding sites as probes, EMSAs were performed with the same amount of nuclear extracts from HepG2 cells, and the products were separated on a 5% polyacrylamide gel (lanes 2–7). Lane 1, free probe; lane 2, biotin-labelled wild-type Sp1 consensus oligonucleotides were mixed with nuclear proteins; lane 3, the same reaction was performed as that in lane 2, except for the presence of a 100-fold excess of unlabelled wild-type Sp1 consensus oligonucleotides as a competitor; lanes 4, binding assays of biotin-labelled mutant-type Sp1 consensus oligonucleotides mixed with nuclear proteins; lanes 5–6, 1 μg each of IgG and anti-Sp1 antibody were added to the binding reaction mixtures with biotin-labelled wild-type probe; lane 7, binding assays of biotin-labelled methylated Sp1 consensus oligonucleotides mixed with nuclear proteins. (B) Analysis for the effect of in vitro DNA methylation on the CD147 promoter activity through the transfection of HEK-293 cells with methylated CpG reporter constructs. CD147P/pGL3 treated or untreated with SssI methylase was co-transfected with the pcDNA3.1 or Sp1/pcDNA3.1 into HEK-293 cells with pGL3-Basic as control. The relative luciferase activity was denoted as above-mentioned method and also expressed as the mean ± S.D. for three independent experiments. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma

    doi: 10.1111/j.1582-4934.2010.01124.x

    Figure Lengend Snippet: Methylation status interfered the Sp1 binding to the CD147 gene promoter and inhibited CD147 transcriptional activity in vitro . (A) Methylation status interfered the Sp1 binding to the CD147 gene promoter in vitro . The binding of Sp1 to the CD147 gene promoter was determined by electrophoretic mobility shift analysis (EMSA). By using biotin-labelled 30-bp double-stranded oligonucleotides containing wild, mutated or methylated Sp1-binding sites as probes, EMSAs were performed with the same amount of nuclear extracts from HepG2 cells, and the products were separated on a 5% polyacrylamide gel (lanes 2–7). Lane 1, free probe; lane 2, biotin-labelled wild-type Sp1 consensus oligonucleotides were mixed with nuclear proteins; lane 3, the same reaction was performed as that in lane 2, except for the presence of a 100-fold excess of unlabelled wild-type Sp1 consensus oligonucleotides as a competitor; lanes 4, binding assays of biotin-labelled mutant-type Sp1 consensus oligonucleotides mixed with nuclear proteins; lanes 5–6, 1 μg each of IgG and anti-Sp1 antibody were added to the binding reaction mixtures with biotin-labelled wild-type probe; lane 7, binding assays of biotin-labelled methylated Sp1 consensus oligonucleotides mixed with nuclear proteins. (B) Analysis for the effect of in vitro DNA methylation on the CD147 promoter activity through the transfection of HEK-293 cells with methylated CpG reporter constructs. CD147P/pGL3 treated or untreated with SssI methylase was co-transfected with the pcDNA3.1 or Sp1/pcDNA3.1 into HEK-293 cells with pGL3-Basic as control. The relative luciferase activity was denoted as above-mentioned method and also expressed as the mean ± S.D. for three independent experiments. * P

    Article Snippet: The methylation of the CD147 promoter probe was obtained through incubation with CpG methyltransferase M.SssI (New England BioLabs) as above-mentioned.

    Techniques: Methylation, Binding Assay, Activity Assay, In Vitro, Electrophoretic Mobility Shift Assay, Mutagenesis, DNA Methylation Assay, Transfection, Construct, Luciferase

    Classification and biological testing of CpG islands from chromosomes 21 and 22 A, Fifty-two of 1,358 chromosome 21/22 CpG islands were classified as MP by the PatMAn classifier (black ticks to right of each chromosome). A central moving average of CpG island density (CpGi/100kb) is indicated to the left of each chromosome. B, The methylation status of a subset of CpG islands from chromosome 21/22 were assessed by methylation-specific PCR (MSP) in normal fibroblasts (IMR90), 3 independent vector-only clones (Neo R ), and 3 independent DNMT1-overexpressing clones (DNMT1). DNA methylated in vitro with M.SssI is included as a positive control. U, unmethylated; M, methylated. C, Heatmap representation of MSP results. Each MSP was performed at least three times. The degree of methylation was estimated from the relative abundance of the methylated and unmethylated products and is scored on a 5 point scale ranging from completely unmethylated (white) to completely methylated (blue). Those CpG islands scored as truly MP are indicated by asterisks (*) and were defined as those that exhibited higher levels of methylation in at least 2 DNMT1-overexpressing clones compared to the average Neo R methylation.

    Journal: Cancer research

    Article Title: A Multi-Factorial Signature of DNA Sequence and Polycomb Binding Predicts Aberrant CpG Island Methylation

    doi: 10.1158/0008-5472.CAN-08-3274

    Figure Lengend Snippet: Classification and biological testing of CpG islands from chromosomes 21 and 22 A, Fifty-two of 1,358 chromosome 21/22 CpG islands were classified as MP by the PatMAn classifier (black ticks to right of each chromosome). A central moving average of CpG island density (CpGi/100kb) is indicated to the left of each chromosome. B, The methylation status of a subset of CpG islands from chromosome 21/22 were assessed by methylation-specific PCR (MSP) in normal fibroblasts (IMR90), 3 independent vector-only clones (Neo R ), and 3 independent DNMT1-overexpressing clones (DNMT1). DNA methylated in vitro with M.SssI is included as a positive control. U, unmethylated; M, methylated. C, Heatmap representation of MSP results. Each MSP was performed at least three times. The degree of methylation was estimated from the relative abundance of the methylated and unmethylated products and is scored on a 5 point scale ranging from completely unmethylated (white) to completely methylated (blue). Those CpG islands scored as truly MP are indicated by asterisks (*) and were defined as those that exhibited higher levels of methylation in at least 2 DNMT1-overexpressing clones compared to the average Neo R methylation.

    Article Snippet: As a methylation positive control, genomic DNA was in vitro methylated with the bacterial DNA methyltransferase M. SssI (New England Biolabs) according to manufacturer’s recommendations.

    Techniques: Methylation, Polymerase Chain Reaction, Plasmid Preparation, Clone Assay, In Vitro, Positive Control

    Reporter gene activities of human ERBB2 promoter constructs (A) Luciferase activities of progressive ERBB2 deletion constructs in A549 cells (see text for details). Normalized luciferase activities were expressed relative to the values from the −499 ERBB2 -pCpGL construct, which was assigned an arbitrary value of 100. (B) Effect of DNA methylation status on the luciferase activity of the −499 ERBB2 -pCpGL construct. CpG sites methylated by HpaII or SssI methyltransferase are indicated. Data represent the mean ± standard deviation of three independent experiments. *** p

    Journal: Gene

    Article Title: Investigation of the Role of DNA Methylation in the Expression of ERBB2 in Human Myocardium

    doi: 10.1016/j.gene.2017.07.058

    Figure Lengend Snippet: Reporter gene activities of human ERBB2 promoter constructs (A) Luciferase activities of progressive ERBB2 deletion constructs in A549 cells (see text for details). Normalized luciferase activities were expressed relative to the values from the −499 ERBB2 -pCpGL construct, which was assigned an arbitrary value of 100. (B) Effect of DNA methylation status on the luciferase activity of the −499 ERBB2 -pCpGL construct. CpG sites methylated by HpaII or SssI methyltransferase are indicated. Data represent the mean ± standard deviation of three independent experiments. *** p

    Article Snippet: The −499 ERBB2 -pCpGL reporter plasmid was methylated using the CpG methyltransferases M.SssI or HpaII (New England Biolabs, Massachusetts, USA).

    Techniques: Construct, Luciferase, DNA Methylation Assay, Activity Assay, Methylation, Standard Deviation

    Locus-specific modulation of CRMP4 expression by dTALEs (a) Illustration of the dTALEs. The synthetic TALE DNA-binding domain, the 23 bp targeting sequence from CRMP4 promoter region, nuclear localization signal (NLS), the truncated N-terminal domain (N95), the catalytic domain of Tet1 (Tet1c), the catalytic domain of DNMT3A (3Ac), and the other functional domain such as GFP are shown. The CRMP4 promoter structure (middle panel of Figure 2a ) is drawn on a non-proportional scale. TSS: translation start site. (b) Luciferase activities altered by dTALEs through locus-specific CpG modification. Co-transfections with dTALEs and the CpG-free CRMP4-pCpGL reporter pre-treated with and without M.SssI were performed in HEK293 and COS-1 cells. (c) Alteration of endogenous CRMP4 mRNA expression in prostate cancer cells detected using qRT-PCR. The PC3 and 22Rv1 cells were transfected with CRMP4-TAL-3Ac, CRMP4-TAL-Tet1c, and empty phCMV1 vector to induce locus-specific CpG modifications. (d) Alteration of endogenous CRMP4 protein expression in prostate cancer cells detected using Western blotting. The prostate cancer cells were treated as described for Figure 3c . (e) CpG methylation frequencies of CRMP4 promoter Region A and Region B detected in the PC3 cells using pyrosequencing. The PC3 cells were transfected with CRMP4-TAL-Tet1c or empty phCMV1 vector as control. (f) CpG methylation frequencies of CRMP4 promoter Region A and Region B detected in the 22Rv1 cells using pyrosequencing. The 22Rv1 cells were transfected with CRMP4-TAL-3Ac or empty phCMV1 vector as control. The P values in b–f were determined with the Student's t -test. The error bars in b–f are s.e.m.

    Journal: Oncotarget

    Article Title: Manipulation of prostate cancer metastasis by locus-specific modification of the CRMP4 promoter region using chimeric TALE DNA methyltransferase and demethylase

    doi:

    Figure Lengend Snippet: Locus-specific modulation of CRMP4 expression by dTALEs (a) Illustration of the dTALEs. The synthetic TALE DNA-binding domain, the 23 bp targeting sequence from CRMP4 promoter region, nuclear localization signal (NLS), the truncated N-terminal domain (N95), the catalytic domain of Tet1 (Tet1c), the catalytic domain of DNMT3A (3Ac), and the other functional domain such as GFP are shown. The CRMP4 promoter structure (middle panel of Figure 2a ) is drawn on a non-proportional scale. TSS: translation start site. (b) Luciferase activities altered by dTALEs through locus-specific CpG modification. Co-transfections with dTALEs and the CpG-free CRMP4-pCpGL reporter pre-treated with and without M.SssI were performed in HEK293 and COS-1 cells. (c) Alteration of endogenous CRMP4 mRNA expression in prostate cancer cells detected using qRT-PCR. The PC3 and 22Rv1 cells were transfected with CRMP4-TAL-3Ac, CRMP4-TAL-Tet1c, and empty phCMV1 vector to induce locus-specific CpG modifications. (d) Alteration of endogenous CRMP4 protein expression in prostate cancer cells detected using Western blotting. The prostate cancer cells were treated as described for Figure 3c . (e) CpG methylation frequencies of CRMP4 promoter Region A and Region B detected in the PC3 cells using pyrosequencing. The PC3 cells were transfected with CRMP4-TAL-Tet1c or empty phCMV1 vector as control. (f) CpG methylation frequencies of CRMP4 promoter Region A and Region B detected in the 22Rv1 cells using pyrosequencing. The 22Rv1 cells were transfected with CRMP4-TAL-3Ac or empty phCMV1 vector as control. The P values in b–f were determined with the Student's t -test. The error bars in b–f are s.e.m.

    Article Snippet: All reporters, except for CRMP4-Luc2p pGL4.27, were treated with non-specific CpG methyltransferase M.SssI ( http://NEB.com ) according to the manufacturer's instructions and the methylation was confirmed by Hae II digestion ( http://NEB.com ).

    Techniques: Expressing, Binding Assay, Sequencing, Functional Assay, Luciferase, Modification, Transfection, Quantitative RT-PCR, Plasmid Preparation, Western Blot, CpG Methylation Assay

    Regulation of CRMP4 promoter activity by CpG modification (a) Illustration of the four CRMP4 promoter-driven luciferase reporters designated as A+ (−867/+114), A− (−839/+114), B+ (−717/+114), and B− (−656/+114). (b) Luciferase activities of the four CRMP4 promoter reporters that were pre-treated with or without M.SssI. One-way ANOVA was used to analyze the difference among the four groups luciferase reporters designated, and the differences between groups determined by the Student's t -test were considered to be significant at a P value less than 0.05/3 after correction. The error bars in b are s.e.m.

    Journal: Oncotarget

    Article Title: Manipulation of prostate cancer metastasis by locus-specific modification of the CRMP4 promoter region using chimeric TALE DNA methyltransferase and demethylase

    doi:

    Figure Lengend Snippet: Regulation of CRMP4 promoter activity by CpG modification (a) Illustration of the four CRMP4 promoter-driven luciferase reporters designated as A+ (−867/+114), A− (−839/+114), B+ (−717/+114), and B− (−656/+114). (b) Luciferase activities of the four CRMP4 promoter reporters that were pre-treated with or without M.SssI. One-way ANOVA was used to analyze the difference among the four groups luciferase reporters designated, and the differences between groups determined by the Student's t -test were considered to be significant at a P value less than 0.05/3 after correction. The error bars in b are s.e.m.

    Article Snippet: All reporters, except for CRMP4-Luc2p pGL4.27, were treated with non-specific CpG methyltransferase M.SssI ( http://NEB.com ) according to the manufacturer's instructions and the methylation was confirmed by Hae II digestion ( http://NEB.com ).

    Techniques: Activity Assay, Modification, Luciferase