s adenosylmethionine  (New England Biolabs)


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    Structured Review

    New England Biolabs s adenosylmethionine
    ( 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 <t>S-adenosylmethionine</t> (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.
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    Images

    1) 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

    2) Product Images from "Two Distinctive POMC Promoters Modify Gene Expression in Cushing Disease"

    Article Title: Two Distinctive POMC Promoters Modify Gene Expression in Cushing Disease

    Journal: The Journal of Clinical Endocrinology and Metabolism

    doi: 10.1210/clinem/dgab387

    First and second proopiomelanocortin (POMC) promoter activity with DNA methylation. A, Structure of luciferase reporter plasmids using the dinucleotide 5′-CG-3′ (CpG) sequence free luciferase vector CpG-free luciferase reporter plasmid (pCpGL) and first (hPro1; –428 to +68) and second promoter (hPro2; +6657 to +7136) fragments. The relative position of the CpG island and potential STAT and CREB binding sites on these fragments are indicated. B, First (hPro1) and second (hPro2) promoter reporter plasmids treated with methyltransferase with (+) or without (–) S-adenosylmethionine (SAM), digested with no enzyme (–), Hpa II, or Msp I, and analyzed by agarose gel electrophoresis. C, Luciferase assays using first (hPro1) and second (hPro2) promoter reporter plasmids treated with (+) and without (–) SAM, with generated luciferase activities compared to negative control plasmid CpG-free luciferase reporter plasmid (pCpGLbasic) (Basic). D, Methylation levels in first (hPro1) and second (hPro2) promoters analyzed by bisulfite-conversion–based methylation-specific polymerase chain reaction using DNA isolated from 3 pituitary adrenocorticotropin (ACTH)-secreting tumors (pituitary #1, #2, and #3; see Fig. 1A ) and 2 ectopic ACTH-secreting tumors (thymus #1 and lung #2; see Fig. 1A ), with normal pituitary obtained at autopsy (normal) as control. Tumor characteristics are shown in Tables 1 and 2 .
    Figure Legend Snippet: First and second proopiomelanocortin (POMC) promoter activity with DNA methylation. A, Structure of luciferase reporter plasmids using the dinucleotide 5′-CG-3′ (CpG) sequence free luciferase vector CpG-free luciferase reporter plasmid (pCpGL) and first (hPro1; –428 to +68) and second promoter (hPro2; +6657 to +7136) fragments. The relative position of the CpG island and potential STAT and CREB binding sites on these fragments are indicated. B, First (hPro1) and second (hPro2) promoter reporter plasmids treated with methyltransferase with (+) or without (–) S-adenosylmethionine (SAM), digested with no enzyme (–), Hpa II, or Msp I, and analyzed by agarose gel electrophoresis. C, Luciferase assays using first (hPro1) and second (hPro2) promoter reporter plasmids treated with (+) and without (–) SAM, with generated luciferase activities compared to negative control plasmid CpG-free luciferase reporter plasmid (pCpGLbasic) (Basic). D, Methylation levels in first (hPro1) and second (hPro2) promoters analyzed by bisulfite-conversion–based methylation-specific polymerase chain reaction using DNA isolated from 3 pituitary adrenocorticotropin (ACTH)-secreting tumors (pituitary #1, #2, and #3; see Fig. 1A ) and 2 ectopic ACTH-secreting tumors (thymus #1 and lung #2; see Fig. 1A ), with normal pituitary obtained at autopsy (normal) as control. Tumor characteristics are shown in Tables 1 and 2 .

    Techniques Used: Activity Assay, DNA Methylation Assay, Luciferase, Sequencing, Plasmid Preparation, Binding Assay, Agarose Gel Electrophoresis, Generated, Negative Control, Methylation, Polymerase Chain Reaction, Isolation

    3) Product Images from "Methyl donor S-adenosylmethionine (SAM) supplementation attenuates breast cancer growth, invasion, and metastasis in vivo; therapeutic and chemopreventive applications"

    Article Title: Methyl donor S-adenosylmethionine (SAM) supplementation attenuates breast cancer growth, invasion, and metastasis in vivo; therapeutic and chemopreventive applications

    Journal: Oncotarget

    doi: 10.18632/oncotarget.23704

    Effect of S-adenosylmethionine (SAM) on breast cancer cell proliferation, migration, invasion, anchorage-independent growth, and apoptosis in vitro (A) Schematic diagram of the treatment strategy for all the in vitro experiments. Human breast cancer cells MDA-MB-231 and Hs578T were treated with SAM (100 and 200 μM) by directly adding it to regular growth medium every other day from day 2 until they were harvested. (B) Human breast cancer cells MDA-MB-231 and Hs578T were plated in 6-well plates and treated with vehicle alone as control or SAM (100 and 200 μM). Cell growth rate in each group was determined on day 1, 3, 5, and 7 by Coulter counter as described in Methods. Results are shown as bar graphs of data obtained from three different experiments. (C) Wound healing assay for determining the migration capacity of the cells was carried out by making a cross-like scratch on the plate when they reached 90% confluency. Control and SAM (100 and 200 μM) treated cells were grown in culture media containing 2% FBS and migrating cells were photographed and recorded at different time points, and percentage of wound healing with respect to initial scratch (T0) was calculated using the equation described in ‘Supplementary Materials’. The results are represented as bar graphs obtained from three experiments. (D) Boyden chamber Matrigel invasion assay was used to measure the invasiveness of control and SAM-treated (100 and 200 μM) MDA-MB-231 and Hs578T cells. The cells were placed in the upper chamber, and conditioned media used as ‘chemoattractant’ was added into the lower chamber. Following an incubation period of 18 hours, the invasion process was stopped and the invaded cells from control and 100 and 200 μM SAM-treated groups were fixed, stained and randomly selected fields were counted under the microscope and averaged. Representative image of one randomly selected field for each treatment for both cell lines along with the number of cells invaded per field are shown. (E) After the usual treatment regimen, 5 × 10 3 cell from control and SAM-treated (100 μM and 200 μM) groups were plated onto soft agar for anchorage-independent growth assay. The culture media was replenished every other day for two weeks, and the number of colonies was counted. (F) Apoptosis was determined by flow cytometry after staining the control and SAM-treated cells with Annexin V/propidium iodide. Representative contour plots of annexinV-FITC staining of apoptotic cells vs. PI staining for both control and SAM-treated (100 μM) cells are shown. The bar graphs on the right panels show the total percentages of apoptotic cells for different treatments. Results are presented as the mean ± SEM from control and SAM-treated experimental cells. Significant differences were determined using ANOVA followed by post hoc Bonferroni test and are represented by asterisks ( * P
    Figure Legend Snippet: Effect of S-adenosylmethionine (SAM) on breast cancer cell proliferation, migration, invasion, anchorage-independent growth, and apoptosis in vitro (A) Schematic diagram of the treatment strategy for all the in vitro experiments. Human breast cancer cells MDA-MB-231 and Hs578T were treated with SAM (100 and 200 μM) by directly adding it to regular growth medium every other day from day 2 until they were harvested. (B) Human breast cancer cells MDA-MB-231 and Hs578T were plated in 6-well plates and treated with vehicle alone as control or SAM (100 and 200 μM). Cell growth rate in each group was determined on day 1, 3, 5, and 7 by Coulter counter as described in Methods. Results are shown as bar graphs of data obtained from three different experiments. (C) Wound healing assay for determining the migration capacity of the cells was carried out by making a cross-like scratch on the plate when they reached 90% confluency. Control and SAM (100 and 200 μM) treated cells were grown in culture media containing 2% FBS and migrating cells were photographed and recorded at different time points, and percentage of wound healing with respect to initial scratch (T0) was calculated using the equation described in ‘Supplementary Materials’. The results are represented as bar graphs obtained from three experiments. (D) Boyden chamber Matrigel invasion assay was used to measure the invasiveness of control and SAM-treated (100 and 200 μM) MDA-MB-231 and Hs578T cells. The cells were placed in the upper chamber, and conditioned media used as ‘chemoattractant’ was added into the lower chamber. Following an incubation period of 18 hours, the invasion process was stopped and the invaded cells from control and 100 and 200 μM SAM-treated groups were fixed, stained and randomly selected fields were counted under the microscope and averaged. Representative image of one randomly selected field for each treatment for both cell lines along with the number of cells invaded per field are shown. (E) After the usual treatment regimen, 5 × 10 3 cell from control and SAM-treated (100 μM and 200 μM) groups were plated onto soft agar for anchorage-independent growth assay. The culture media was replenished every other day for two weeks, and the number of colonies was counted. (F) Apoptosis was determined by flow cytometry after staining the control and SAM-treated cells with Annexin V/propidium iodide. Representative contour plots of annexinV-FITC staining of apoptotic cells vs. PI staining for both control and SAM-treated (100 μM) cells are shown. The bar graphs on the right panels show the total percentages of apoptotic cells for different treatments. Results are presented as the mean ± SEM from control and SAM-treated experimental cells. Significant differences were determined using ANOVA followed by post hoc Bonferroni test and are represented by asterisks ( * P

    Techniques Used: Migration, In Vitro, Multiple Displacement Amplification, Wound Healing Assay, Invasion Assay, Incubation, Staining, Microscopy, Growth Assay, Flow Cytometry, Cytometry

    4) Product Images from "Transcriptional activation of Il36A by C/EBPβ via a half-CRE•C/EBP element in murine macrophages is independent of its CpG methylation level"

    Article Title: Transcriptional activation of Il36A by C/EBPβ via a half-CRE•C/EBP element in murine macrophages is independent of its CpG methylation level

    Journal: bioRxiv

    doi: 10.1101/2021.04.24.441265

    Inhibition of the Il36A promoter activity by methylation of the CpG sites in the half-CRE•C/EBP element. (A) Schematic representation of the pCpGL- Il36A -357/-45 -Luciferase (Luc) reporter construct. Transcription factor binding sites are indicated. (B) In vitro methylation pCpGL- Il36A -357/-45 of using SssI CpG) methylase and S-adenosylmethionine. Methylation was confirmed by enzymatic digestion of the plasmid followed by gel electrophoresis. The unmethylated plasmid (lanes 1-3) is completely linearized by Hpy CH4IV (asterisk) whereas Hpy CH4IV does not linearize the methylated plasmid (lanes 4-6). Digestion with Pst I and Kpn I releases the Il36A -357/-45 insert from the plasmid (C) RAW 264.7 cells were co-transfected with the methylated/non-methylated pCpGL- Il36A -357/-45 construct along with the pRL-TK vector. 24 h after transfection cells were stimulated with LPS (100 ng/ml) for 8 h or left untreated. Firefly luciferase activity was normalized to that of Renilla luciferase and is expressed as fold change in relative luciferase induction (ratio LPS vs. ctrl) from n = 4 experiments. Individual data points and summary measurements (mean ± SD) are plotted on the left-hand side of the panel; effect size (mean differences, black dot) with bootstrapped 95% confidence intervals and resampling distribution are shown on the right-hand side of the panel.
    Figure Legend Snippet: Inhibition of the Il36A promoter activity by methylation of the CpG sites in the half-CRE•C/EBP element. (A) Schematic representation of the pCpGL- Il36A -357/-45 -Luciferase (Luc) reporter construct. Transcription factor binding sites are indicated. (B) In vitro methylation pCpGL- Il36A -357/-45 of using SssI CpG) methylase and S-adenosylmethionine. Methylation was confirmed by enzymatic digestion of the plasmid followed by gel electrophoresis. The unmethylated plasmid (lanes 1-3) is completely linearized by Hpy CH4IV (asterisk) whereas Hpy CH4IV does not linearize the methylated plasmid (lanes 4-6). Digestion with Pst I and Kpn I releases the Il36A -357/-45 insert from the plasmid (C) RAW 264.7 cells were co-transfected with the methylated/non-methylated pCpGL- Il36A -357/-45 construct along with the pRL-TK vector. 24 h after transfection cells were stimulated with LPS (100 ng/ml) for 8 h or left untreated. Firefly luciferase activity was normalized to that of Renilla luciferase and is expressed as fold change in relative luciferase induction (ratio LPS vs. ctrl) from n = 4 experiments. Individual data points and summary measurements (mean ± SD) are plotted on the left-hand side of the panel; effect size (mean differences, black dot) with bootstrapped 95% confidence intervals and resampling distribution are shown on the right-hand side of the panel.

    Techniques Used: Inhibition, Activity Assay, Methylation, Luciferase, Construct, Binding Assay, In Vitro, Plasmid Preparation, Nucleic Acid Electrophoresis, Transfection

    5) Product Images from "Dynamic chromosomal interactions and control of heterochromatin positioning by Ki67"

    Article Title: Dynamic chromosomal interactions and control of heterochromatin positioning by Ki67

    Journal: bioRxiv

    doi: 10.1101/2021.10.20.465140

    Visualization and genome-wide profiling of DNA-Ki67 interactions using pA-DamID (A) Schematic overview of pA-DamID [ 24 ]. Permeabilized cells are incubated with a primary antibody (e.g. against Ki67), followed by a fusion of proteinA and Dam (pA-Dam). After removal of unbound pA-Dam, the Dam enzyme is activated by addition of S-adenosylmethionine (SAM), resulting in local deposition of m6A marks. m6A-marked DNA can be processed for high-throughput sequencing, or alternatively cells can be fixed and m6A marks visualized using the m6A-Tracer protein. (B) Representative confocal microscopy sections of HCT116 cells following pA-DamID with free Dam (top panel) or Ki67 antibody (bottom panel), labeled with m6A-Tracer protein and stained for Ki67. Scale bar: 5 μm. (C) Quantification of the enrichment of Ki67 antibody and m6A-Tracer signals relative to segmented Ki67 domains (that we interpret as nucleoli) in different cell lines. For every cell, the enrichment is calculated by pixel-distances relative to the mean signal of that cell, and represented as a log2-ratio. Negative distances are outside of Ki67 domains, a distance of zero marks the domain boundary and positive distances are inside of Ki67 domains. Every thin line corresponds to an individual cell and the thick line is the mean of all cells. Results are combined from three (hTERT-RPE) or one (HCT116 and K562) biological replicates. (D) Comparison of Ki67 pA-DamID profiles (log2-ratios over the free Dam control) across two chromosomes in hTERT-RPE, HCT116 and K562 cells. Sequenced reads are counted and normalized in 50 kb bins. Data are averages of n biological replicates and smoothed with a running mean across nine 50 kb bins for visualization purposes. Centromeres are highlighted by black bars. (E) Distributions of Ki67 interactions for all chromosomes, ordered by decreasing chromosome size. rDNA-containing chromosomes are highlighted by black borders. Boxplots: horizontal lines represent 25th, 50th, and 75th percentiles; whiskers extend to 5th and 95th percentiles. (F) Distributions of Ki67 interactions nearby centromeres. Boxplots are drawn for every 0.5 Mb, following the specification as in (E) with the 50th percentile highlighted in red. (G) The mean Ki67 interaction score near centromeres is plotted for each chromosome ordered by size (within 2Mb of centromeres, overlapping the enrichment in (F)). rDNA-containing chromosomes are highlighted in red.
    Figure Legend Snippet: Visualization and genome-wide profiling of DNA-Ki67 interactions using pA-DamID (A) Schematic overview of pA-DamID [ 24 ]. Permeabilized cells are incubated with a primary antibody (e.g. against Ki67), followed by a fusion of proteinA and Dam (pA-Dam). After removal of unbound pA-Dam, the Dam enzyme is activated by addition of S-adenosylmethionine (SAM), resulting in local deposition of m6A marks. m6A-marked DNA can be processed for high-throughput sequencing, or alternatively cells can be fixed and m6A marks visualized using the m6A-Tracer protein. (B) Representative confocal microscopy sections of HCT116 cells following pA-DamID with free Dam (top panel) or Ki67 antibody (bottom panel), labeled with m6A-Tracer protein and stained for Ki67. Scale bar: 5 μm. (C) Quantification of the enrichment of Ki67 antibody and m6A-Tracer signals relative to segmented Ki67 domains (that we interpret as nucleoli) in different cell lines. For every cell, the enrichment is calculated by pixel-distances relative to the mean signal of that cell, and represented as a log2-ratio. Negative distances are outside of Ki67 domains, a distance of zero marks the domain boundary and positive distances are inside of Ki67 domains. Every thin line corresponds to an individual cell and the thick line is the mean of all cells. Results are combined from three (hTERT-RPE) or one (HCT116 and K562) biological replicates. (D) Comparison of Ki67 pA-DamID profiles (log2-ratios over the free Dam control) across two chromosomes in hTERT-RPE, HCT116 and K562 cells. Sequenced reads are counted and normalized in 50 kb bins. Data are averages of n biological replicates and smoothed with a running mean across nine 50 kb bins for visualization purposes. Centromeres are highlighted by black bars. (E) Distributions of Ki67 interactions for all chromosomes, ordered by decreasing chromosome size. rDNA-containing chromosomes are highlighted by black borders. Boxplots: horizontal lines represent 25th, 50th, and 75th percentiles; whiskers extend to 5th and 95th percentiles. (F) Distributions of Ki67 interactions nearby centromeres. Boxplots are drawn for every 0.5 Mb, following the specification as in (E) with the 50th percentile highlighted in red. (G) The mean Ki67 interaction score near centromeres is plotted for each chromosome ordered by size (within 2Mb of centromeres, overlapping the enrichment in (F)). rDNA-containing chromosomes are highlighted in red.

    Techniques Used: Genome Wide, Incubation, Next-Generation Sequencing, Confocal Microscopy, Labeling, Staining

    6) Product Images from "Polycomb repressive complex 2 in an autoinhibited state"

    Article Title: Polycomb repressive complex 2 in an autoinhibited state

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.787572

    Schematic of the different stable conformational states of ct PRC2. Letter codes correspond to autoinhibited (A); SAM-bound, autoinhibited (A′), basal (B), and H3K27me3-stimulated (S) states based on the observed structural transitions of ct PRC2. A dotted black box indicates a destabilized conformation compared with that in a neighboring structural state. Note that although all the structural transitions are based on a minimally active PRC2 complex ( i.e. Ezh2–Eed–Suz12(VEFS)), Suz12(VEFS) is omitted from all the schematics, and Eed is only shown in the S state for clarity. SRM , stimulation-responsive motif.
    Figure Legend Snippet: Schematic of the different stable conformational states of ct PRC2. Letter codes correspond to autoinhibited (A); SAM-bound, autoinhibited (A′), basal (B), and H3K27me3-stimulated (S) states based on the observed structural transitions of ct PRC2. A dotted black box indicates a destabilized conformation compared with that in a neighboring structural state. Note that although all the structural transitions are based on a minimally active PRC2 complex ( i.e. Ezh2–Eed–Suz12(VEFS)), Suz12(VEFS) is omitted from all the schematics, and Eed is only shown in the S state for clarity. SRM , stimulation-responsive motif.

    Techniques Used:

    7) Product Images from "Integrating molecular dynamics simulations with chemical probing experiments using SHAPE-FIT"

    Article Title: Integrating molecular dynamics simulations with chemical probing experiments using SHAPE-FIT

    Journal: Methods in enzymology

    doi: 10.1016/bs.mie.2014.10.061

    The T. tengcongensis metF SAM-I riboswitch aptamer domain in the off-state. (a) Secondary structure of the aptamer domain with different colors representing secondary structure elements. (b) Tertiary structure of the sequence in the presence of S-adenosylmethionine
    Figure Legend Snippet: The T. tengcongensis metF SAM-I riboswitch aptamer domain in the off-state. (a) Secondary structure of the aptamer domain with different colors representing secondary structure elements. (b) Tertiary structure of the sequence in the presence of S-adenosylmethionine

    Techniques Used: Sequencing

    8) Product Images from "A post‐translational modification switch controls coactivator function of histone methyltransferases G9a and GLP"

    Article Title: A post‐translational modification switch controls coactivator function of histone methyltransferases G9a and GLP

    Journal: EMBO Reports

    doi: 10.15252/embr.201744060

    G9a and GLP are methylated on their N‐terminal domain in cells Schematic representation of the related proteins GLP (EHMT1) and G9a (EHMT2). N: N‐terminal coactivator domain, E: polyglutamate domain, Cys: cysteine‐rich region, ANK: six ankyrin repeats, SET: SET‐domain containing methyltransferase activity. Partial protein sequence of hG9a and hGLP homologs shows the hypothetical methylated lysine residues (K) in red. After protein methylation reactions, in vitro methylated proteins were detected by immunoblot with pan‐methyllysine antibody (pan met‐K). The corresponding Coomassie‐stained gels are shown as loading controls. SAM, S‐adenosylmethionine. Cos‐7 cells were transfected with plasmids encoding full‐length HA‐hG9a wild type or K185R mutant, or full‐length HA‐hGLP wild type or K205R mutant. Lysates were immunoprecipitated (IP) with pan met‐K antibody and immunoblotted with HA antibody (top), or the usage of the two antibodies was reversed (bottom). Expression of HA‐tagged proteins and β‐actin (loading control) in the unfractionated extracts is shown at the bottom (Input). Cos‐7 cells were transfected with a plasmid encoding full‐length HA‐hG9a and treated with 2 μM UNC0646 or vehicle DMSO for 24 h. Lysates were immunoprecipitated with pan met‐K antibody and immunoblotted with HA antibody (top), or the usage of the two antibodies was reversed (bottom). Methylation and phosphorylation of endogenous G9a and GLP in A549 cells treated with 100 nM dex for 4 h were analyzed by immunoprecipitation with control IgG antibody, anti‐G9a (top), or anti‐GLP (bottom), followed by immunoblot with antibodies listed. Expression of G9a, GLP, and β‐actin (loading control) in the unfractionated extracts is shown at the bottom (Input).
    Figure Legend Snippet: G9a and GLP are methylated on their N‐terminal domain in cells Schematic representation of the related proteins GLP (EHMT1) and G9a (EHMT2). N: N‐terminal coactivator domain, E: polyglutamate domain, Cys: cysteine‐rich region, ANK: six ankyrin repeats, SET: SET‐domain containing methyltransferase activity. Partial protein sequence of hG9a and hGLP homologs shows the hypothetical methylated lysine residues (K) in red. After protein methylation reactions, in vitro methylated proteins were detected by immunoblot with pan‐methyllysine antibody (pan met‐K). The corresponding Coomassie‐stained gels are shown as loading controls. SAM, S‐adenosylmethionine. Cos‐7 cells were transfected with plasmids encoding full‐length HA‐hG9a wild type or K185R mutant, or full‐length HA‐hGLP wild type or K205R mutant. Lysates were immunoprecipitated (IP) with pan met‐K antibody and immunoblotted with HA antibody (top), or the usage of the two antibodies was reversed (bottom). Expression of HA‐tagged proteins and β‐actin (loading control) in the unfractionated extracts is shown at the bottom (Input). Cos‐7 cells were transfected with a plasmid encoding full‐length HA‐hG9a and treated with 2 μM UNC0646 or vehicle DMSO for 24 h. Lysates were immunoprecipitated with pan met‐K antibody and immunoblotted with HA antibody (top), or the usage of the two antibodies was reversed (bottom). Methylation and phosphorylation of endogenous G9a and GLP in A549 cells treated with 100 nM dex for 4 h were analyzed by immunoprecipitation with control IgG antibody, anti‐G9a (top), or anti‐GLP (bottom), followed by immunoblot with antibodies listed. Expression of G9a, GLP, and β‐actin (loading control) in the unfractionated extracts is shown at the bottom (Input).

    Techniques Used: Methylation, Activity Assay, Sequencing, In Vitro, Staining, Transfection, Mutagenesis, Immunoprecipitation, Expressing, Plasmid Preparation

    9) Product Images from "Transcriptional activation of Il36A by C/EBPβ via a half-CRE•C/EBP element in murine macrophages is independent of its CpG methylation level"

    Article Title: Transcriptional activation of Il36A by C/EBPβ via a half-CRE•C/EBP element in murine macrophages is independent of its CpG methylation level

    Journal: bioRxiv

    doi: 10.1101/2021.04.24.441265

    Inhibition of the Il36A promoter activity by methylation of the CpG sites in the half-CRE•C/EBP element. (A) Schematic representation of the pCpGL-Il36A-357/-45-Luciferase reporter construct. Transcription factor binding sites are indicated. (B) In vitro methylation pCpGL-Il36A-357/-45 of using Sss I CpG) methylase and S-adenosylmethionine. Methylation was confirmed by enzymatic digestion of the plasmid followed by gel electrophoresis. The unmethylated plasmid (lanes 1-3) is completely linearized by Hpy CH4IV (asterisk) whereas Hpy CH4IV does not linearize the methylated plasmid (lanes 4-6). Digestion with Pst I and Kpn I releases the Il36A-357/-45 insert from the plasmid (C) RAW 264.7cells were co-transfected with the methylated/non-methylated pCpGL-Il36A-357/-45 construct along with the pRL-TK vector. 24 h after transfection cells were stimulated with LPS (100 ng/ml, red dots) for 8 h or left untreated (blue dots). Firefly luciferase activity was normalized to that of Renilla luciferase and is expressed as relative luciferase units (RLU, n = 4 experiments, *P
    Figure Legend Snippet: Inhibition of the Il36A promoter activity by methylation of the CpG sites in the half-CRE•C/EBP element. (A) Schematic representation of the pCpGL-Il36A-357/-45-Luciferase reporter construct. Transcription factor binding sites are indicated. (B) In vitro methylation pCpGL-Il36A-357/-45 of using Sss I CpG) methylase and S-adenosylmethionine. Methylation was confirmed by enzymatic digestion of the plasmid followed by gel electrophoresis. The unmethylated plasmid (lanes 1-3) is completely linearized by Hpy CH4IV (asterisk) whereas Hpy CH4IV does not linearize the methylated plasmid (lanes 4-6). Digestion with Pst I and Kpn I releases the Il36A-357/-45 insert from the plasmid (C) RAW 264.7cells were co-transfected with the methylated/non-methylated pCpGL-Il36A-357/-45 construct along with the pRL-TK vector. 24 h after transfection cells were stimulated with LPS (100 ng/ml, red dots) for 8 h or left untreated (blue dots). Firefly luciferase activity was normalized to that of Renilla luciferase and is expressed as relative luciferase units (RLU, n = 4 experiments, *P

    Techniques Used: Inhibition, Activity Assay, Methylation, Luciferase, Construct, Binding Assay, In Vitro, Plasmid Preparation, Nucleic Acid Electrophoresis, Transfection

    10) Product Images from "Ectopic Methylation of a Single Persistently Unmethylated CpG in the Promoter of the Vitellogenin Gene Abolishes Its Inducibility by Estrogen through Attenuation of Upstream Stimulating Factor Binding"

    Article Title: Ectopic Methylation of a Single Persistently Unmethylated CpG in the Promoter of the Vitellogenin Gene Abolishes Its Inducibility by Estrogen through Attenuation of Upstream Stimulating Factor Binding

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00436-19

    Estrogen inducibility of VTG-CpGL luciferase reporter vector transfected into LMH/2A cells. (A) Luciferase expression before and after in vitro methylation of the ERE C/C (wt) or ΔG vector with SssI. (B) Same as panel A, but the ERE sequence was replaced with oligonucleotides carrying the indicated cytosine modifications only in CpGs d and c. (C) Same as panel A, but with either wt VTG-CpGL or the mutated reporter in which all CpGs except for the ERE and CpG7 were substituted for TpGs, with or without SssI. (D) Luciferase expression from the reporter before and after methylation (M) with Eco72IM, HpaII, or SssI. Eco72IM without S -adenosylmethionine (−SAM) was used as the control, and the other methylation reactions were performed in the presence of SAM (+SAM). Significance was assessed using the Tukey test for multiple comparisons. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (E) Luciferase assay using VTG-CpGL linearized with the indicated enzymes, methylated with SssI, and religated. The purified circular DNA then was transfected into LMH/2A cells. In these substrates, the cleaved restriction site remained unmethylated after the circularization. The ratio between methylated and unmethylated is shown. (F) Luciferase assay using unmethylated VTG-CpGL in LMH/2A cells in which USF1 was depleted with siRNA; siLuc was used as a control (this siRNA does not recognize either of the luciferases expressed from our vectors). Relative luciferase units (RLU) are defined as the ratio between firefly and Renilla signal. The graphs show the means ± SD from three independent experiments. (G) RT-qPCR of VTG mRNA isolated from cells treated with siLuc or siUSF1 for 96 h and EtOH or E 2 for the last 24 h. The graph shows the means ± SD from three independent experiments. Significance was assessed using Sidak’s multiple-comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
    Figure Legend Snippet: Estrogen inducibility of VTG-CpGL luciferase reporter vector transfected into LMH/2A cells. (A) Luciferase expression before and after in vitro methylation of the ERE C/C (wt) or ΔG vector with SssI. (B) Same as panel A, but the ERE sequence was replaced with oligonucleotides carrying the indicated cytosine modifications only in CpGs d and c. (C) Same as panel A, but with either wt VTG-CpGL or the mutated reporter in which all CpGs except for the ERE and CpG7 were substituted for TpGs, with or without SssI. (D) Luciferase expression from the reporter before and after methylation (M) with Eco72IM, HpaII, or SssI. Eco72IM without S -adenosylmethionine (−SAM) was used as the control, and the other methylation reactions were performed in the presence of SAM (+SAM). Significance was assessed using the Tukey test for multiple comparisons. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (E) Luciferase assay using VTG-CpGL linearized with the indicated enzymes, methylated with SssI, and religated. The purified circular DNA then was transfected into LMH/2A cells. In these substrates, the cleaved restriction site remained unmethylated after the circularization. The ratio between methylated and unmethylated is shown. (F) Luciferase assay using unmethylated VTG-CpGL in LMH/2A cells in which USF1 was depleted with siRNA; siLuc was used as a control (this siRNA does not recognize either of the luciferases expressed from our vectors). Relative luciferase units (RLU) are defined as the ratio between firefly and Renilla signal. The graphs show the means ± SD from three independent experiments. (G) RT-qPCR of VTG mRNA isolated from cells treated with siLuc or siUSF1 for 96 h and EtOH or E 2 for the last 24 h. The graph shows the means ± SD from three independent experiments. Significance was assessed using Sidak’s multiple-comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

    Techniques Used: Luciferase, Plasmid Preparation, Transfection, Expressing, In Vitro, Methylation, Sequencing, Purification, Quantitative RT-PCR, Isolation

    11) Product Images from "Transcriptional activation of Il36A by C/EBPβ via a half-CRE•C/EBP element in murine macrophages is independent of its CpG methylation level"

    Article Title: Transcriptional activation of Il36A by C/EBPβ via a half-CRE•C/EBP element in murine macrophages is independent of its CpG methylation level

    Journal: bioRxiv

    doi: 10.1101/2021.04.24.441265

    Inhibition of the Il36A promoter activity by methylation of the CpG sites in the half-CRE•C/EBP element. (A) Schematic representation of the pCpGL- Il36A -357/-45 -Luciferase (Luc) reporter construct. Transcription factor binding sites are indicated. (B) In vitro methylation pCpGL- Il36A -357/-45 of using SssI CpG) methylase and S-adenosylmethionine. Methylation was confirmed by enzymatic digestion of the plasmid followed by gel electrophoresis. The unmethylated plasmid (lanes 1-3) is completely linearized by Hpy CH4IV (asterisk) whereas Hpy CH4IV does not linearize the methylated plasmid (lanes 4-6). Digestion with Pst I and Kpn I releases the Il36A -357/-45 insert from the plasmid (C) RAW 264.7 cells were co-transfected with the methylated/non-methylated pCpGL- Il36A -357/-45 construct along with the pRL-TK vector. 24 h after transfection cells were stimulated with LPS (100 ng/ml) for 8 h or left untreated. Firefly luciferase activity was normalized to that of Renilla luciferase and is expressed as fold change in relative luciferase induction (ratio LPS vs. ctrl) from n = 4 experiments. Individual data points and summary measurements (mean ± SD) are plotted on the left-hand side of the panel; effect size (mean differences, black dot) with bootstrapped 95% confidence intervals and resampling distribution are shown on the right-hand side of the panel.
    Figure Legend Snippet: Inhibition of the Il36A promoter activity by methylation of the CpG sites in the half-CRE•C/EBP element. (A) Schematic representation of the pCpGL- Il36A -357/-45 -Luciferase (Luc) reporter construct. Transcription factor binding sites are indicated. (B) In vitro methylation pCpGL- Il36A -357/-45 of using SssI CpG) methylase and S-adenosylmethionine. Methylation was confirmed by enzymatic digestion of the plasmid followed by gel electrophoresis. The unmethylated plasmid (lanes 1-3) is completely linearized by Hpy CH4IV (asterisk) whereas Hpy CH4IV does not linearize the methylated plasmid (lanes 4-6). Digestion with Pst I and Kpn I releases the Il36A -357/-45 insert from the plasmid (C) RAW 264.7 cells were co-transfected with the methylated/non-methylated pCpGL- Il36A -357/-45 construct along with the pRL-TK vector. 24 h after transfection cells were stimulated with LPS (100 ng/ml) for 8 h or left untreated. Firefly luciferase activity was normalized to that of Renilla luciferase and is expressed as fold change in relative luciferase induction (ratio LPS vs. ctrl) from n = 4 experiments. Individual data points and summary measurements (mean ± SD) are plotted on the left-hand side of the panel; effect size (mean differences, black dot) with bootstrapped 95% confidence intervals and resampling distribution are shown on the right-hand side of the panel.

    Techniques Used: Inhibition, Activity Assay, Methylation, Luciferase, Construct, Binding Assay, In Vitro, Plasmid Preparation, Nucleic Acid Electrophoresis, Transfection

    12) 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

    13) Product Images from "RNA matchmaking remodels lncRNA structure and promotes PRC2 activity"

    Article Title: RNA matchmaking remodels lncRNA structure and promotes PRC2 activity

    Journal: bioRxiv

    doi: 10.1101/2020.04.13.040071

    Duplex RNA promotes PRC2 activity. a , Native gel of di-nucleosomes reconstituted via salt dialysis using a DNA template containing two 601 sequences surrounding 40-bp of linker DNA. DNA and nucleosome samples were run on a 5% native polyacrylamide gel and stained with SYBR Gold. b , Recombinant human PRC2 complex includes SUZ12, EZH2, EED, RBBP4 and AEBP2, analyzed by SDS-PAGE and stained with Coomassie blue. c , Histone methyltransferase assay (HMTase assay) was performed with recombinant PRC2 complex, di-nucleosomes, S-Adenosylmethionine (SAM) with and without the co-factor JARID2 (amino acids 119-574). PRC2 activity was determined by SDS-PAGE followed by H3K27me3 and total H3 Western blot analysis. d , Native 0.5X TBE gel of RNA annealing titration with HOTAIR forward and reverse fragments to show formation of dsRNA. HMTase assay with annealed HOTAIR dsRNA titration.
    Figure Legend Snippet: Duplex RNA promotes PRC2 activity. a , Native gel of di-nucleosomes reconstituted via salt dialysis using a DNA template containing two 601 sequences surrounding 40-bp of linker DNA. DNA and nucleosome samples were run on a 5% native polyacrylamide gel and stained with SYBR Gold. b , Recombinant human PRC2 complex includes SUZ12, EZH2, EED, RBBP4 and AEBP2, analyzed by SDS-PAGE and stained with Coomassie blue. c , Histone methyltransferase assay (HMTase assay) was performed with recombinant PRC2 complex, di-nucleosomes, S-Adenosylmethionine (SAM) with and without the co-factor JARID2 (amino acids 119-574). PRC2 activity was determined by SDS-PAGE followed by H3K27me3 and total H3 Western blot analysis. d , Native 0.5X TBE gel of RNA annealing titration with HOTAIR forward and reverse fragments to show formation of dsRNA. HMTase assay with annealed HOTAIR dsRNA titration.

    Techniques Used: Activity Assay, Staining, Recombinant, SDS Page, HMT Assay, Western Blot, Titration

    14) Product Images from "Establishing RNA-RNA interactions remodels lncRNA structure and promotes PRC2 activity"

    Article Title: Establishing RNA-RNA interactions remodels lncRNA structure and promotes PRC2 activity

    Journal: Science Advances

    doi: 10.1126/sciadv.abc9191

    Duplex RNA promotes PRC2 activity. ( A ) Histograms comparing the predicted minimum free energy for RNA-RNA interactions between HOTAIR and 40,740 RNAs across the transcriptome (gray) or between HOTAIR and 885 transcripts from genes that gain PRC2 activity when HOTAIR is overexpressed in breast cancer cells (red). Data are from ( 23 , 25 , 38 ). ( B ) Native gel of dinucleosomes reconstituted via salt dialysis using a DNA template containing two 601 sequences surrounding 40 bp of linker DNA. DNA and nucleosome samples were run on a 5% native polyacrylamide gel and stained with SYBR Gold. ( C ) Recombinant human PRC2 complex includes SUZ12, EZH2, EED, RBBP4, and AEBP2, analyzed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE), and stained with Coomassie blue. ( D ) HMTase assay was performed with recombinant PRC2 complex, hexanucleosomes, S -adenosylmethionine with and without the cofactor JARID2 (amino acids 119 to 574). PRC2 activity was determined by SDS-PAGE followed by H3K27me3 and total H3 Western blot analysis. ( E ) Native 0.5× tris-borate-EDTA gel of RNA annealing titration with HOTAIR forward and reverse fragments to show formation of dsRNA. HMTase assay with annealed HOTAIR dsRNA titration analyzed by Western blot.
    Figure Legend Snippet: Duplex RNA promotes PRC2 activity. ( A ) Histograms comparing the predicted minimum free energy for RNA-RNA interactions between HOTAIR and 40,740 RNAs across the transcriptome (gray) or between HOTAIR and 885 transcripts from genes that gain PRC2 activity when HOTAIR is overexpressed in breast cancer cells (red). Data are from ( 23 , 25 , 38 ). ( B ) Native gel of dinucleosomes reconstituted via salt dialysis using a DNA template containing two 601 sequences surrounding 40 bp of linker DNA. DNA and nucleosome samples were run on a 5% native polyacrylamide gel and stained with SYBR Gold. ( C ) Recombinant human PRC2 complex includes SUZ12, EZH2, EED, RBBP4, and AEBP2, analyzed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE), and stained with Coomassie blue. ( D ) HMTase assay was performed with recombinant PRC2 complex, hexanucleosomes, S -adenosylmethionine with and without the cofactor JARID2 (amino acids 119 to 574). PRC2 activity was determined by SDS-PAGE followed by H3K27me3 and total H3 Western blot analysis. ( E ) Native 0.5× tris-borate-EDTA gel of RNA annealing titration with HOTAIR forward and reverse fragments to show formation of dsRNA. HMTase assay with annealed HOTAIR dsRNA titration analyzed by Western blot.

    Techniques Used: Activity Assay, Staining, Recombinant, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Titration

    15) Product Images from "RNA matchmaking remodels lncRNA structure and promotes PRC2 activity"

    Article Title: RNA matchmaking remodels lncRNA structure and promotes PRC2 activity

    Journal: bioRxiv

    doi: 10.1101/2020.04.13.040071

    Duplex RNA promotes PRC2 activity. (A) Histograms comparing the predicted minimum free energy for RNA-RNA interactions between HOTAIR and 40,740 RNAs across the transcriptome (grey) or between HOTAIR and 885 transcripts from genes that gain PRC2 activity when HOTAIR is overexpressed in breast cancer cells (red). Data from( 20 , 22 , 29 ). (B) Native gel of di-nucleosomes reconstituted via salt dialysis using a DNA template containing two 601 sequences surrounding 40-bp of linker DNA. DNA and nucleosome samples were run on a 5% native polyacrylamide gel and stained with SYBR Gold. (C) Recombinant human PRC2 complex includes SUZ12, EZH2, EED, RBBP4 and AEBP2, analyzed by SDS-PAGE and stained with Coomassie blue. (D) Histone methyltransferase assay (HMTase assay) was performed with recombinant PRC2 complex, di-nucleosomes, S-Adenosylmethionine (SAM) with and without the co-factor JARID2 (amino acids 119-574). PRC2 activity was determined by SDS-PAGE followed by H3K27me3 and total H3 Western blot analysis. (E) Native 0.5X TBE gel of RNA annealing titration with HOTAIR forward and reverse fragments to show formation of dsRNA. HMTase assay with annealed HOTAIR dsRNA titration.
    Figure Legend Snippet: Duplex RNA promotes PRC2 activity. (A) Histograms comparing the predicted minimum free energy for RNA-RNA interactions between HOTAIR and 40,740 RNAs across the transcriptome (grey) or between HOTAIR and 885 transcripts from genes that gain PRC2 activity when HOTAIR is overexpressed in breast cancer cells (red). Data from( 20 , 22 , 29 ). (B) Native gel of di-nucleosomes reconstituted via salt dialysis using a DNA template containing two 601 sequences surrounding 40-bp of linker DNA. DNA and nucleosome samples were run on a 5% native polyacrylamide gel and stained with SYBR Gold. (C) Recombinant human PRC2 complex includes SUZ12, EZH2, EED, RBBP4 and AEBP2, analyzed by SDS-PAGE and stained with Coomassie blue. (D) Histone methyltransferase assay (HMTase assay) was performed with recombinant PRC2 complex, di-nucleosomes, S-Adenosylmethionine (SAM) with and without the co-factor JARID2 (amino acids 119-574). PRC2 activity was determined by SDS-PAGE followed by H3K27me3 and total H3 Western blot analysis. (E) Native 0.5X TBE gel of RNA annealing titration with HOTAIR forward and reverse fragments to show formation of dsRNA. HMTase assay with annealed HOTAIR dsRNA titration.

    Techniques Used: Activity Assay, Staining, Recombinant, SDS Page, HMT Assay, Western Blot, Titration

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    ( 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 <t>S-adenosylmethionine</t> (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.
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    ( 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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkp524

    Figure Lengend 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.

    Article Snippet: Reaction were stopped by addition of 10 mM ADP and incubation on ice for 10 min. Methylation of the remodeled products was performed by incubation at 37°C for 15 min after addition of 5 U M.SssI and S-adenosylmethionine to a final concentration of 160 μM (New England BioLabs).

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

    First and second proopiomelanocortin (POMC) promoter activity with DNA methylation. A, Structure of luciferase reporter plasmids using the dinucleotide 5′-CG-3′ (CpG) sequence free luciferase vector CpG-free luciferase reporter plasmid (pCpGL) and first (hPro1; –428 to +68) and second promoter (hPro2; +6657 to +7136) fragments. The relative position of the CpG island and potential STAT and CREB binding sites on these fragments are indicated. B, First (hPro1) and second (hPro2) promoter reporter plasmids treated with methyltransferase with (+) or without (–) S-adenosylmethionine (SAM), digested with no enzyme (–), Hpa II, or Msp I, and analyzed by agarose gel electrophoresis. C, Luciferase assays using first (hPro1) and second (hPro2) promoter reporter plasmids treated with (+) and without (–) SAM, with generated luciferase activities compared to negative control plasmid CpG-free luciferase reporter plasmid (pCpGLbasic) (Basic). D, Methylation levels in first (hPro1) and second (hPro2) promoters analyzed by bisulfite-conversion–based methylation-specific polymerase chain reaction using DNA isolated from 3 pituitary adrenocorticotropin (ACTH)-secreting tumors (pituitary #1, #2, and #3; see Fig. 1A ) and 2 ectopic ACTH-secreting tumors (thymus #1 and lung #2; see Fig. 1A ), with normal pituitary obtained at autopsy (normal) as control. Tumor characteristics are shown in Tables 1 and 2 .

    Journal: The Journal of Clinical Endocrinology and Metabolism

    Article Title: Two Distinctive POMC Promoters Modify Gene Expression in Cushing Disease

    doi: 10.1210/clinem/dgab387

    Figure Lengend Snippet: First and second proopiomelanocortin (POMC) promoter activity with DNA methylation. A, Structure of luciferase reporter plasmids using the dinucleotide 5′-CG-3′ (CpG) sequence free luciferase vector CpG-free luciferase reporter plasmid (pCpGL) and first (hPro1; –428 to +68) and second promoter (hPro2; +6657 to +7136) fragments. The relative position of the CpG island and potential STAT and CREB binding sites on these fragments are indicated. B, First (hPro1) and second (hPro2) promoter reporter plasmids treated with methyltransferase with (+) or without (–) S-adenosylmethionine (SAM), digested with no enzyme (–), Hpa II, or Msp I, and analyzed by agarose gel electrophoresis. C, Luciferase assays using first (hPro1) and second (hPro2) promoter reporter plasmids treated with (+) and without (–) SAM, with generated luciferase activities compared to negative control plasmid CpG-free luciferase reporter plasmid (pCpGLbasic) (Basic). D, Methylation levels in first (hPro1) and second (hPro2) promoters analyzed by bisulfite-conversion–based methylation-specific polymerase chain reaction using DNA isolated from 3 pituitary adrenocorticotropin (ACTH)-secreting tumors (pituitary #1, #2, and #3; see Fig. 1A ) and 2 ectopic ACTH-secreting tumors (thymus #1 and lung #2; see Fig. 1A ), with normal pituitary obtained at autopsy (normal) as control. Tumor characteristics are shown in Tables 1 and 2 .

    Article Snippet: A total of 10 µg of CpG free plasmids were treated by methyltransferase (SssI) with or without 160 µM S-adenosylmethionine (SAM; catalog No. B9003S, New England Biolabs) for 4 hours at 37 °C.

    Techniques: Activity Assay, DNA Methylation Assay, Luciferase, Sequencing, Plasmid Preparation, Binding Assay, Agarose Gel Electrophoresis, Generated, Negative Control, Methylation, Polymerase Chain Reaction, Isolation

    Effect of S-adenosylmethionine (SAM) on breast cancer cell proliferation, migration, invasion, anchorage-independent growth, and apoptosis in vitro (A) Schematic diagram of the treatment strategy for all the in vitro experiments. Human breast cancer cells MDA-MB-231 and Hs578T were treated with SAM (100 and 200 μM) by directly adding it to regular growth medium every other day from day 2 until they were harvested. (B) Human breast cancer cells MDA-MB-231 and Hs578T were plated in 6-well plates and treated with vehicle alone as control or SAM (100 and 200 μM). Cell growth rate in each group was determined on day 1, 3, 5, and 7 by Coulter counter as described in Methods. Results are shown as bar graphs of data obtained from three different experiments. (C) Wound healing assay for determining the migration capacity of the cells was carried out by making a cross-like scratch on the plate when they reached 90% confluency. Control and SAM (100 and 200 μM) treated cells were grown in culture media containing 2% FBS and migrating cells were photographed and recorded at different time points, and percentage of wound healing with respect to initial scratch (T0) was calculated using the equation described in ‘Supplementary Materials’. The results are represented as bar graphs obtained from three experiments. (D) Boyden chamber Matrigel invasion assay was used to measure the invasiveness of control and SAM-treated (100 and 200 μM) MDA-MB-231 and Hs578T cells. The cells were placed in the upper chamber, and conditioned media used as ‘chemoattractant’ was added into the lower chamber. Following an incubation period of 18 hours, the invasion process was stopped and the invaded cells from control and 100 and 200 μM SAM-treated groups were fixed, stained and randomly selected fields were counted under the microscope and averaged. Representative image of one randomly selected field for each treatment for both cell lines along with the number of cells invaded per field are shown. (E) After the usual treatment regimen, 5 × 10 3 cell from control and SAM-treated (100 μM and 200 μM) groups were plated onto soft agar for anchorage-independent growth assay. The culture media was replenished every other day for two weeks, and the number of colonies was counted. (F) Apoptosis was determined by flow cytometry after staining the control and SAM-treated cells with Annexin V/propidium iodide. Representative contour plots of annexinV-FITC staining of apoptotic cells vs. PI staining for both control and SAM-treated (100 μM) cells are shown. The bar graphs on the right panels show the total percentages of apoptotic cells for different treatments. Results are presented as the mean ± SEM from control and SAM-treated experimental cells. Significant differences were determined using ANOVA followed by post hoc Bonferroni test and are represented by asterisks ( * P

    Journal: Oncotarget

    Article Title: Methyl donor S-adenosylmethionine (SAM) supplementation attenuates breast cancer growth, invasion, and metastasis in vivo; therapeutic and chemopreventive applications

    doi: 10.18632/oncotarget.23704

    Figure Lengend Snippet: Effect of S-adenosylmethionine (SAM) on breast cancer cell proliferation, migration, invasion, anchorage-independent growth, and apoptosis in vitro (A) Schematic diagram of the treatment strategy for all the in vitro experiments. Human breast cancer cells MDA-MB-231 and Hs578T were treated with SAM (100 and 200 μM) by directly adding it to regular growth medium every other day from day 2 until they were harvested. (B) Human breast cancer cells MDA-MB-231 and Hs578T were plated in 6-well plates and treated with vehicle alone as control or SAM (100 and 200 μM). Cell growth rate in each group was determined on day 1, 3, 5, and 7 by Coulter counter as described in Methods. Results are shown as bar graphs of data obtained from three different experiments. (C) Wound healing assay for determining the migration capacity of the cells was carried out by making a cross-like scratch on the plate when they reached 90% confluency. Control and SAM (100 and 200 μM) treated cells were grown in culture media containing 2% FBS and migrating cells were photographed and recorded at different time points, and percentage of wound healing with respect to initial scratch (T0) was calculated using the equation described in ‘Supplementary Materials’. The results are represented as bar graphs obtained from three experiments. (D) Boyden chamber Matrigel invasion assay was used to measure the invasiveness of control and SAM-treated (100 and 200 μM) MDA-MB-231 and Hs578T cells. The cells were placed in the upper chamber, and conditioned media used as ‘chemoattractant’ was added into the lower chamber. Following an incubation period of 18 hours, the invasion process was stopped and the invaded cells from control and 100 and 200 μM SAM-treated groups were fixed, stained and randomly selected fields were counted under the microscope and averaged. Representative image of one randomly selected field for each treatment for both cell lines along with the number of cells invaded per field are shown. (E) After the usual treatment regimen, 5 × 10 3 cell from control and SAM-treated (100 μM and 200 μM) groups were plated onto soft agar for anchorage-independent growth assay. The culture media was replenished every other day for two weeks, and the number of colonies was counted. (F) Apoptosis was determined by flow cytometry after staining the control and SAM-treated cells with Annexin V/propidium iodide. Representative contour plots of annexinV-FITC staining of apoptotic cells vs. PI staining for both control and SAM-treated (100 μM) cells are shown. The bar graphs on the right panels show the total percentages of apoptotic cells for different treatments. Results are presented as the mean ± SEM from control and SAM-treated experimental cells. Significant differences were determined using ANOVA followed by post hoc Bonferroni test and are represented by asterisks ( * P

    Article Snippet: Cells were treated with SAM (New England Biolabs, Mississauga, Ontario, Canada; Catalog # B9003S) by directly adding it to regular growth medium under sterile conditions following the treatment plan shown in Figure .

    Techniques: Migration, In Vitro, Multiple Displacement Amplification, Wound Healing Assay, Invasion Assay, Incubation, Staining, Microscopy, Growth Assay, Flow Cytometry, Cytometry

    Inhibition of the Il36A promoter activity by methylation of the CpG sites in the half-CRE•C/EBP element. (A) Schematic representation of the pCpGL- Il36A -357/-45 -Luciferase (Luc) reporter construct. Transcription factor binding sites are indicated. (B) In vitro methylation pCpGL- Il36A -357/-45 of using SssI CpG) methylase and S-adenosylmethionine. Methylation was confirmed by enzymatic digestion of the plasmid followed by gel electrophoresis. The unmethylated plasmid (lanes 1-3) is completely linearized by Hpy CH4IV (asterisk) whereas Hpy CH4IV does not linearize the methylated plasmid (lanes 4-6). Digestion with Pst I and Kpn I releases the Il36A -357/-45 insert from the plasmid (C) RAW 264.7 cells were co-transfected with the methylated/non-methylated pCpGL- Il36A -357/-45 construct along with the pRL-TK vector. 24 h after transfection cells were stimulated with LPS (100 ng/ml) for 8 h or left untreated. Firefly luciferase activity was normalized to that of Renilla luciferase and is expressed as fold change in relative luciferase induction (ratio LPS vs. ctrl) from n = 4 experiments. Individual data points and summary measurements (mean ± SD) are plotted on the left-hand side of the panel; effect size (mean differences, black dot) with bootstrapped 95% confidence intervals and resampling distribution are shown on the right-hand side of the panel.

    Journal: bioRxiv

    Article Title: Transcriptional activation of Il36A by C/EBPβ via a half-CRE•C/EBP element in murine macrophages is independent of its CpG methylation level

    doi: 10.1101/2021.04.24.441265

    Figure Lengend Snippet: Inhibition of the Il36A promoter activity by methylation of the CpG sites in the half-CRE•C/EBP element. (A) Schematic representation of the pCpGL- Il36A -357/-45 -Luciferase (Luc) reporter construct. Transcription factor binding sites are indicated. (B) In vitro methylation pCpGL- Il36A -357/-45 of using SssI CpG) methylase and S-adenosylmethionine. Methylation was confirmed by enzymatic digestion of the plasmid followed by gel electrophoresis. The unmethylated plasmid (lanes 1-3) is completely linearized by Hpy CH4IV (asterisk) whereas Hpy CH4IV does not linearize the methylated plasmid (lanes 4-6). Digestion with Pst I and Kpn I releases the Il36A -357/-45 insert from the plasmid (C) RAW 264.7 cells were co-transfected with the methylated/non-methylated pCpGL- Il36A -357/-45 construct along with the pRL-TK vector. 24 h after transfection cells were stimulated with LPS (100 ng/ml) for 8 h or left untreated. Firefly luciferase activity was normalized to that of Renilla luciferase and is expressed as fold change in relative luciferase induction (ratio LPS vs. ctrl) from n = 4 experiments. Individual data points and summary measurements (mean ± SD) are plotted on the left-hand side of the panel; effect size (mean differences, black dot) with bootstrapped 95% confidence intervals and resampling distribution are shown on the right-hand side of the panel.

    Article Snippet: In brief, 10-20 μg plasmid DNA was incubated with Sss I (2.5 U/μg plasmid DNA) in the presence of 160 μM S-adenosylmethionine (SAM; New England Biolabs) for four hours at 37°C.

    Techniques: Inhibition, Activity Assay, Methylation, Luciferase, Construct, Binding Assay, In Vitro, Plasmid Preparation, Nucleic Acid Electrophoresis, Transfection