Structured Review

Roche input dna
H1.5 knockdown increases DNase I sensitivity at target regions. (A) Venn diagram of the overlap between significant H1.5 peaks and DNase I hypersensitive sites. The p-value for exclusivity of these two sets of peaks is indicated ( Text S1 ). (B–E) Quantitative <t>PCR</t> of <t>DNA</t> fragments at indicated genes from genomic DNA treated with increasing amount of DNase I. Data points with t-test p-value
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Images

1) Product Images from "Dynamic Distribution of Linker Histone H1.5 in Cellular Differentiation"

Article Title: Dynamic Distribution of Linker Histone H1.5 in Cellular Differentiation

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1002879

H1.5 knockdown increases DNase I sensitivity at target regions. (A) Venn diagram of the overlap between significant H1.5 peaks and DNase I hypersensitive sites. The p-value for exclusivity of these two sets of peaks is indicated ( Text S1 ). (B–E) Quantitative PCR of DNA fragments at indicated genes from genomic DNA treated with increasing amount of DNase I. Data points with t-test p-value
Figure Legend Snippet: H1.5 knockdown increases DNase I sensitivity at target regions. (A) Venn diagram of the overlap between significant H1.5 peaks and DNase I hypersensitive sites. The p-value for exclusivity of these two sets of peaks is indicated ( Text S1 ). (B–E) Quantitative PCR of DNA fragments at indicated genes from genomic DNA treated with increasing amount of DNase I. Data points with t-test p-value

Techniques Used: Real-time Polymerase Chain Reaction

2) Product Images from "Activation of the Farnesoid X Receptor Provides Protection against Acetaminophen-Induced Hepatic Toxicity"

Article Title: Activation of the Farnesoid X Receptor Provides Protection against Acetaminophen-Induced Hepatic Toxicity

Journal: Molecular Endocrinology

doi: 10.1210/me.2010-0117

Representative snapshots of ChIP-Seq peaks for FXR-target genes. Shp ( upper panel ) and Gst α 4 ( lower panel ), mapped onto University of California at Santa Cruz genome browser. Shown is their chromosomal location (chr 4, chr 9) according to the July 2007 Mouse Genome Assembly (mm9). Blue and red tags represent positive and negative strands, respectively, identified after DNA sequencing of the chromatin after immunoprecipitation using antibody to FXR or a control IgG. Each bar represents a distinct hit/sequence.
Figure Legend Snippet: Representative snapshots of ChIP-Seq peaks for FXR-target genes. Shp ( upper panel ) and Gst α 4 ( lower panel ), mapped onto University of California at Santa Cruz genome browser. Shown is their chromosomal location (chr 4, chr 9) according to the July 2007 Mouse Genome Assembly (mm9). Blue and red tags represent positive and negative strands, respectively, identified after DNA sequencing of the chromatin after immunoprecipitation using antibody to FXR or a control IgG. Each bar represents a distinct hit/sequence.

Techniques Used: Chromatin Immunoprecipitation, DNA Sequencing, Immunoprecipitation, Sequencing

3) Product Images from "Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare"

Article Title: Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2018.00443

Mbo II digest results. Agarose gel showing Mbo II digests of GAA PCR products of FRDA samples. The expected 170bp (5′) and 120bp (3′) undigested GAA-flanking fragments from normal pure GAA repeat expansion FRDA samples are shown in lanes 2, 3, and 4. These band sizes can be seen in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder markers, which are loaded into lanes 1 and 11 of the gel. Lane 5 shows a large Mbo II band of approximately 600bp that was obtained from the positive interrupted GAA repeat sequence from the “NEP” BAC transgenic mouse that contains approximately 500 triplet repeats with the previously determined interrupted sequence of (GAA) 21 (GGAGAA) 5 (GGAGGAGAA) 70 (GAA) n ). In addition for this positive sample, we also identified the expected 5′ flanking band of 170bp, together with a smaller band of less than 100bp that we sequenced and we showed to contain a 27bp deletion in the 3′ flanking region. Lane 6 shows an abnormal band of 200bp representing the 80bp duplication in the 3′ GAA flanking region. Lane 7 shows an abnormal band of approximately 100bp representing the 19bp deletion in the 3′ GAA flanking region. Lanes 8, 9, and 10 contain abnormal bands of approximately 300, 100, and 180bp, respectively, that are likely to contain a region of interrupted GAA repeat sequence within the body of one or other of the large FRDA GAA repeat expansions.
Figure Legend Snippet: Mbo II digest results. Agarose gel showing Mbo II digests of GAA PCR products of FRDA samples. The expected 170bp (5′) and 120bp (3′) undigested GAA-flanking fragments from normal pure GAA repeat expansion FRDA samples are shown in lanes 2, 3, and 4. These band sizes can be seen in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder markers, which are loaded into lanes 1 and 11 of the gel. Lane 5 shows a large Mbo II band of approximately 600bp that was obtained from the positive interrupted GAA repeat sequence from the “NEP” BAC transgenic mouse that contains approximately 500 triplet repeats with the previously determined interrupted sequence of (GAA) 21 (GGAGAA) 5 (GGAGGAGAA) 70 (GAA) n ). In addition for this positive sample, we also identified the expected 5′ flanking band of 170bp, together with a smaller band of less than 100bp that we sequenced and we showed to contain a 27bp deletion in the 3′ flanking region. Lane 6 shows an abnormal band of 200bp representing the 80bp duplication in the 3′ GAA flanking region. Lane 7 shows an abnormal band of approximately 100bp representing the 19bp deletion in the 3′ GAA flanking region. Lanes 8, 9, and 10 contain abnormal bands of approximately 300, 100, and 180bp, respectively, that are likely to contain a region of interrupted GAA repeat sequence within the body of one or other of the large FRDA GAA repeat expansions.

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Sequencing, BAC Assay, Transgenic Assay

Mbo II digests of GAA repeat expansions from human FRDA somatic tissues and mouse FRDA intergenerational and somatic tissues. Agarose gels showing Mbo II digests of GAA PCR products of (A) FRDA patient cerebellum tissue samples, (B) YG8sR mouse ear biopsy samples and human FRDA blood samples, and (C) four tissues from one YG8sR mouse. In each case, the expected 170 and 120bp undigested GAA-flanking fragments can be identified in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder marker, which is loaded into the first lane of each gel. (A) Lanes 1–3 show the results from cerebellum tissue samples from three FRDA patients. (B) Lanes 1 and 2 are from FRDA patient blood samples; lanes 3–6 are from ear biopsy samples from 4 GAA repeat expansion-based YG8sR mice of four different generations, and lane 7 is from an ear biopsy sample from the Y47R mouse which has nine GAA repeats. (C) Lanes 1–4 are from brain, cerebellum, heart, and liver tissues of the YG8sR mouse, respectively.
Figure Legend Snippet: Mbo II digests of GAA repeat expansions from human FRDA somatic tissues and mouse FRDA intergenerational and somatic tissues. Agarose gels showing Mbo II digests of GAA PCR products of (A) FRDA patient cerebellum tissue samples, (B) YG8sR mouse ear biopsy samples and human FRDA blood samples, and (C) four tissues from one YG8sR mouse. In each case, the expected 170 and 120bp undigested GAA-flanking fragments can be identified in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder marker, which is loaded into the first lane of each gel. (A) Lanes 1–3 show the results from cerebellum tissue samples from three FRDA patients. (B) Lanes 1 and 2 are from FRDA patient blood samples; lanes 3–6 are from ear biopsy samples from 4 GAA repeat expansion-based YG8sR mice of four different generations, and lane 7 is from an ear biopsy sample from the Y47R mouse which has nine GAA repeats. (C) Lanes 1–4 are from brain, cerebellum, heart, and liver tissues of the YG8sR mouse, respectively.

Techniques Used: Polymerase Chain Reaction, Marker, Mouse Assay

4) Product Images from "Fumarates improve psoriasis and multiple sclerosis by inducing type II dendritic cells"

Article Title: Fumarates improve psoriasis and multiple sclerosis by inducing type II dendritic cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20100977

DMF-induced HO-1 selectively prevents IL-23 induction. (A) DCs were treated with DMSO or 70 µM DMF, and HO-1 mRNA expression was determined by quantitative RT-PCR. HO-1 data were normalized to β-actin, and HO-1 level in control siRNA–transfected DMF–treated DCs was set as 1.0. The results are representative of three independent experiments. Error bars represent SEM. (B) HO-1 was knocked down, and levels of IL-12/IL-23p40, IL-23p19, or IL-12p35 mRNA were determined by RT-PCR. Data (mean ± SEM) were normalized to β-actin, and message levels in control siRNA–transfected DMF-treated DCs were set as 1.0. (C) DCs were treated as in A and lysed, and nuclear or cytoplasmic cell extracts were analyzed by Western blotting using antibodies directed against C- or N-terminal HO-1 protein. (D and E) DCs treated as in A were activated with LPS, cross-linked, and immunoprecipitated with anti–HO-1 (D) or anti-H3Ac (E). Bound DNA was amplified by quantitative PCR for primer sites P1 (AP-1; position 412–422 bp), P2 (c-Rel; position 560–584 bp), and P3 (RelA/c-Rel; position 394–406 bp). Data were pooled from four separate experiments and represent mean ± SEM (*, P
Figure Legend Snippet: DMF-induced HO-1 selectively prevents IL-23 induction. (A) DCs were treated with DMSO or 70 µM DMF, and HO-1 mRNA expression was determined by quantitative RT-PCR. HO-1 data were normalized to β-actin, and HO-1 level in control siRNA–transfected DMF–treated DCs was set as 1.0. The results are representative of three independent experiments. Error bars represent SEM. (B) HO-1 was knocked down, and levels of IL-12/IL-23p40, IL-23p19, or IL-12p35 mRNA were determined by RT-PCR. Data (mean ± SEM) were normalized to β-actin, and message levels in control siRNA–transfected DMF-treated DCs were set as 1.0. (C) DCs were treated as in A and lysed, and nuclear or cytoplasmic cell extracts were analyzed by Western blotting using antibodies directed against C- or N-terminal HO-1 protein. (D and E) DCs treated as in A were activated with LPS, cross-linked, and immunoprecipitated with anti–HO-1 (D) or anti-H3Ac (E). Bound DNA was amplified by quantitative PCR for primer sites P1 (AP-1; position 412–422 bp), P2 (c-Rel; position 560–584 bp), and P3 (RelA/c-Rel; position 394–406 bp). Data were pooled from four separate experiments and represent mean ± SEM (*, P

Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction

5) Product Images from "A Novel CpG Island Set Identifies Tissue-Specific Methylation at Developmental Gene Loci"

Article Title: A Novel CpG Island Set Identifies Tissue-Specific Methylation at Developmental Gene Loci

Journal: PLoS Biology

doi: 10.1371/journal.pbio.0060022

The Immobilised CXXC Domain Specifically Retains DNA Containing Clusters of Nonmethylated CpGs (A) EMSA showing the CXXC complex with a DNA probe containing 27 nonmethylated CpG sites. Nonmethylated probe DNA (CG11) or methylated probe (MeCG11) was incubated with 0, 250, 500, 1,000, or 2,000 ng of recombinant CXXC protein. (B) A typical elution profile of bulk genomic DNA (blue line) from a CXXC affinity chromatography column. Genomic DNA (100 μg) was applied to the CXXC affinity matrix (see Methods ) in low salt (0.1 M NaCl) and eluted with a gradient of increasing NaCl (red line; see text). Eighteen fractions were interrogated by PCR (blue lines). The bracket above indicates fractions that were found to contain nonmethylated CGIs. (C) Elution of specific CGI sequences of known methylation status. Methylated CGIs ( NYESO and MAO in females) coelute with bulk genomic DNA (see bracket) whereas nonmethylated CGIs ( P48 and MAO ) elute at high NaCl concentration.
Figure Legend Snippet: The Immobilised CXXC Domain Specifically Retains DNA Containing Clusters of Nonmethylated CpGs (A) EMSA showing the CXXC complex with a DNA probe containing 27 nonmethylated CpG sites. Nonmethylated probe DNA (CG11) or methylated probe (MeCG11) was incubated with 0, 250, 500, 1,000, or 2,000 ng of recombinant CXXC protein. (B) A typical elution profile of bulk genomic DNA (blue line) from a CXXC affinity chromatography column. Genomic DNA (100 μg) was applied to the CXXC affinity matrix (see Methods ) in low salt (0.1 M NaCl) and eluted with a gradient of increasing NaCl (red line; see text). Eighteen fractions were interrogated by PCR (blue lines). The bracket above indicates fractions that were found to contain nonmethylated CGIs. (C) Elution of specific CGI sequences of known methylation status. Methylated CGIs ( NYESO and MAO in females) coelute with bulk genomic DNA (see bracket) whereas nonmethylated CGIs ( P48 and MAO ) elute at high NaCl concentration.

Techniques Used: Methylation, Incubation, Recombinant, Affinity Column, Polymerase Chain Reaction, Concentration Assay

Use of an Arrayed CGI Library to Detect CGI Methylation in Human Blood DNA (A) Schematic showing isolation of densely methylated CGIs using MBD affinity purification based on reference [ 20 ]. Open and filled circles represent nonmethylated and methylated CpG sites, respectively. (B) Examples of retention of known methylated CGIs by MBD affinity chromatography. Methylated XIST and NYESO CGIs elute at high salt concentration, whereas nonmethylated P48 and female XIST co-elute with bulk genomic DNA (blue line) at low salt concentration (red line). (C) M values (log 2 [MBD/Input]) > 1.5 (dashed vertical arrow) denote DNA fragments enriched by MAP. M values are plotted against the ratio of fragment abundance in the MAP probe versus input DNA as determined by quantitative PCR. Error bars represent ± standard deviation. (D–F) MAP CGI array hybridization identifies CGIs that are methylated on the inactive X chromosome. (D) Probes isolated by MAP from male and female whole blood DNA detected female-specific CGI methylation. (E) CGIs on the X chromosome (red dots) often showed female-specific methylation. (F) CGIs on Chromosome 16 (red dots) were indistinguishably methylated between male and female. (G and H) Confirmation of methylated CGIs by bisulfite genomic sequencing. CGI clones I1387 (G) and I9112 (H) are nonmethylated and methylated, respectively, as predicted by the microarray data. Open and filled circles represent nonmethylated and methylated CpG sites, respectively. The genomic locus including annotated transcripts and CpG maps (vertical strokes) are shown above each profile. Each column represents products of amplification by a single primer pair (brackets below CpG map). Each line corresponds to a sequenced DNA strand. Red bars indicate the location of the MseI fragment cloned in the CGI library. (I) The CGI array distinguishes genes inactivated on the X chromosome (inactive) from genes that escape inactivation (escaping). CGIs associated with inactivated genes ( n = 103) show significantly higher M values than CGIs at escaping genes ( n = 14; KS test: p = 1.2 ×10 −5 ).
Figure Legend Snippet: Use of an Arrayed CGI Library to Detect CGI Methylation in Human Blood DNA (A) Schematic showing isolation of densely methylated CGIs using MBD affinity purification based on reference [ 20 ]. Open and filled circles represent nonmethylated and methylated CpG sites, respectively. (B) Examples of retention of known methylated CGIs by MBD affinity chromatography. Methylated XIST and NYESO CGIs elute at high salt concentration, whereas nonmethylated P48 and female XIST co-elute with bulk genomic DNA (blue line) at low salt concentration (red line). (C) M values (log 2 [MBD/Input]) > 1.5 (dashed vertical arrow) denote DNA fragments enriched by MAP. M values are plotted against the ratio of fragment abundance in the MAP probe versus input DNA as determined by quantitative PCR. Error bars represent ± standard deviation. (D–F) MAP CGI array hybridization identifies CGIs that are methylated on the inactive X chromosome. (D) Probes isolated by MAP from male and female whole blood DNA detected female-specific CGI methylation. (E) CGIs on the X chromosome (red dots) often showed female-specific methylation. (F) CGIs on Chromosome 16 (red dots) were indistinguishably methylated between male and female. (G and H) Confirmation of methylated CGIs by bisulfite genomic sequencing. CGI clones I1387 (G) and I9112 (H) are nonmethylated and methylated, respectively, as predicted by the microarray data. Open and filled circles represent nonmethylated and methylated CpG sites, respectively. The genomic locus including annotated transcripts and CpG maps (vertical strokes) are shown above each profile. Each column represents products of amplification by a single primer pair (brackets below CpG map). Each line corresponds to a sequenced DNA strand. Red bars indicate the location of the MseI fragment cloned in the CGI library. (I) The CGI array distinguishes genes inactivated on the X chromosome (inactive) from genes that escape inactivation (escaping). CGIs associated with inactivated genes ( n = 103) show significantly higher M values than CGIs at escaping genes ( n = 14; KS test: p = 1.2 ×10 −5 ).

Techniques Used: Methylation, Isolation, Affinity Purification, Affinity Chromatography, Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation, Hybridization, Genomic Sequencing, Clone Assay, Microarray, Amplification

6) Product Images from "ZFR coordinates crosstalk between RNA decay and transcription in innate immunity"

Article Title: ZFR coordinates crosstalk between RNA decay and transcription in innate immunity

Journal: Nature Communications

doi: 10.1038/s41467-018-03326-5

mH2A1 is responsible for ZFR-mediated IFNβ repression. a qRT-PCR analysis of IFNB1 after transient transfection of a plasmid DNA-expressing 3xFLAG-tagged RIG-I CARD domain in HEK-293TO cells stably transduced with shZFR or shScr ( n = 3). b Schematic of experiment to analyze synthesis of IFNB mRNA after poly(I:C) stimulation (left). qRT-PCR of newly synthesized IFNB1 mRNA 24 h after poly(I:C) stimulation (right; n = 3). c Dual luciferase assay of cells transfected with plasmids expressing firefly luciferase under the control of the IFNB1 promoter, with and without poly(I:C) stimulation ( n = 3). A plasmid constitutively expressing Renilla luciferase was used as control. d qRT-PCR analysis of mH2A1 in HEK-293TO cells transfected with si-mH2A1 or si-NS with and without stimulation with poly(I:C) ( n = 3). e qRT-PCR analysis of IFNB1 as in D ( n = 3). f qRT-PCR analysis of mH2A1 in mouse BMDMs transfected with si-mH2A1 or si-NS ( n = 3). g qRT-PCR analysis of Ifnb1 in BMDMs transfected with the indicated siRNAs and stimulated with poly(I:C) ( n = 3). h qRT-PCR analysis of Ifit1 as in g ( n = 3). i Chromatin immunoprecipitation-qPCR (ChIP-qPCR) analysis of mH2A1 binding to the IFNB1 promoter region in HEK-293TO cells. Isotype-matched IgG was used as a control. Fold enrichment over input is shown for each location ( n = 3). j Immunoblot analysis of mH2A1 in BMDMs from WT or macroH2A double knockout mouse. Approximate MW based on size markers is shown. k qRT-PCR analysis of Ifnb1 in BMDMs stimulated with poly(I:C) or ( l ) LPS. Graphs indicate mean ± SD; * P
Figure Legend Snippet: mH2A1 is responsible for ZFR-mediated IFNβ repression. a qRT-PCR analysis of IFNB1 after transient transfection of a plasmid DNA-expressing 3xFLAG-tagged RIG-I CARD domain in HEK-293TO cells stably transduced with shZFR or shScr ( n = 3). b Schematic of experiment to analyze synthesis of IFNB mRNA after poly(I:C) stimulation (left). qRT-PCR of newly synthesized IFNB1 mRNA 24 h after poly(I:C) stimulation (right; n = 3). c Dual luciferase assay of cells transfected with plasmids expressing firefly luciferase under the control of the IFNB1 promoter, with and without poly(I:C) stimulation ( n = 3). A plasmid constitutively expressing Renilla luciferase was used as control. d qRT-PCR analysis of mH2A1 in HEK-293TO cells transfected with si-mH2A1 or si-NS with and without stimulation with poly(I:C) ( n = 3). e qRT-PCR analysis of IFNB1 as in D ( n = 3). f qRT-PCR analysis of mH2A1 in mouse BMDMs transfected with si-mH2A1 or si-NS ( n = 3). g qRT-PCR analysis of Ifnb1 in BMDMs transfected with the indicated siRNAs and stimulated with poly(I:C) ( n = 3). h qRT-PCR analysis of Ifit1 as in g ( n = 3). i Chromatin immunoprecipitation-qPCR (ChIP-qPCR) analysis of mH2A1 binding to the IFNB1 promoter region in HEK-293TO cells. Isotype-matched IgG was used as a control. Fold enrichment over input is shown for each location ( n = 3). j Immunoblot analysis of mH2A1 in BMDMs from WT or macroH2A double knockout mouse. Approximate MW based on size markers is shown. k qRT-PCR analysis of Ifnb1 in BMDMs stimulated with poly(I:C) or ( l ) LPS. Graphs indicate mean ± SD; * P

Techniques Used: Quantitative RT-PCR, Transfection, Plasmid Preparation, Expressing, Stable Transfection, Transduction, Synthesized, Luciferase, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Double Knockout

7) Product Images from "Molecular Interactions of GBF1 with HY5 and HYH Proteins during Light-mediated Seedling Development in Arabidopsis thaliana *"

Article Title: Molecular Interactions of GBF1 with HY5 and HYH Proteins during Light-mediated Seedling Development in Arabidopsis thaliana *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.333906

GBF1 and HY5 form a G-box-binding heterodimer. A , electrophoretic mobility shift assays (EMSAs) using recombinant GBF1 and HY5 proteins bind to the G-box of the RBCS-1A minimal promoter. Approximately 100 ng of each of the proteins was mixed and incubated at 50 °C for 5 min to dissociate pre-existing dimers prior to their addition to radioactively labeled probe. Approximately 200 ng of GST protein was added in lane 2 . The protein-DNA complexes were resolved on a 4% native 0.5× TBE polyacrylamide gel. Plus and minus signs show the presence and absence of the components in respective lanes . The star indicates the heterodimer complex. B , ChIP assays of RBCS-1A promoter of white light-grown WT, mutant, and overexpresser transgenic seedlings using antibodies to GBF1. The result of the real-time PCR is presented as the ratio of immunoprecipitated DNA to input DNA from various backgrounds. Error bars , S.D. n ≥ 3 independent experiments with similar results. C , in vivo interactions of GBF1 and HY5 proteins in ChIP assays. The cross-linked complex of GBF1-HY5 with the RBCS-1A promoter was pulled down by antibodies to HY5. The complex was reverse cross-linked and resolved onto SDS-PAGE. Both the input and immunoprecipitates were probed with antibodies to GBF1. D , ChIP assays of the RBCS-1A promoter of blue light-grown WT, mutant, and overexpresser transgenic seedlings using antibodies to GBF1. The result of the real-time PCR is presented as the ratio of immunoprecipitated DNA to input DNA from various backgrounds. Error bars , S.D. n ≥ 3 independent experiments with similar results. E , analysis of GBF1 protein levels in wild type, mutant, and overexpresser transgenic lines by Western blots using antibodies to GBF1.
Figure Legend Snippet: GBF1 and HY5 form a G-box-binding heterodimer. A , electrophoretic mobility shift assays (EMSAs) using recombinant GBF1 and HY5 proteins bind to the G-box of the RBCS-1A minimal promoter. Approximately 100 ng of each of the proteins was mixed and incubated at 50 °C for 5 min to dissociate pre-existing dimers prior to their addition to radioactively labeled probe. Approximately 200 ng of GST protein was added in lane 2 . The protein-DNA complexes were resolved on a 4% native 0.5× TBE polyacrylamide gel. Plus and minus signs show the presence and absence of the components in respective lanes . The star indicates the heterodimer complex. B , ChIP assays of RBCS-1A promoter of white light-grown WT, mutant, and overexpresser transgenic seedlings using antibodies to GBF1. The result of the real-time PCR is presented as the ratio of immunoprecipitated DNA to input DNA from various backgrounds. Error bars , S.D. n ≥ 3 independent experiments with similar results. C , in vivo interactions of GBF1 and HY5 proteins in ChIP assays. The cross-linked complex of GBF1-HY5 with the RBCS-1A promoter was pulled down by antibodies to HY5. The complex was reverse cross-linked and resolved onto SDS-PAGE. Both the input and immunoprecipitates were probed with antibodies to GBF1. D , ChIP assays of the RBCS-1A promoter of blue light-grown WT, mutant, and overexpresser transgenic seedlings using antibodies to GBF1. The result of the real-time PCR is presented as the ratio of immunoprecipitated DNA to input DNA from various backgrounds. Error bars , S.D. n ≥ 3 independent experiments with similar results. E , analysis of GBF1 protein levels in wild type, mutant, and overexpresser transgenic lines by Western blots using antibodies to GBF1.

Techniques Used: Binding Assay, Electrophoretic Mobility Shift Assay, Recombinant, Incubation, Labeling, Chromatin Immunoprecipitation, Mutagenesis, Transgenic Assay, Real-time Polymerase Chain Reaction, Immunoprecipitation, In Vivo, SDS Page, Western Blot

8) Product Images from "Dynamic Epigenetic Regulation of Gene Expression during the Life Cycle of Malaria Parasite Plasmodium falciparum"

Article Title: Dynamic Epigenetic Regulation of Gene Expression during the Life Cycle of Malaria Parasite Plasmodium falciparum

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003170

Histone modification patterns on ectopically integrated promoters. (A) Cloning strategy for ectopic integration of promoter regions . Four promoter regions (1.5–2 kb upstream of the ATG) corresponding to upstream regions of MAL13P1.122, PF14_0705, PFD0240c and PFC0210c were cloned upstream of the luciferase reporter gene pLN-luc (see Materials and Methods ). P. falciparum strain Dd2 attB was transfected with the above vectors to achieve integration at the cg6 locus and the transgenic cell lines were selected on blasticidin. Primer pair P2/P4 was used to confirm integration (data not shown). (B) H4K8ac occupancy at the four ectopically integrated promoter regions . The graphs represent real time PCR results carried out on H4K8ac-immunoprecipitated DNA from rings (R), trophozoites (T) and schizonts (S). In order to distinguish between the endogenous and luciferase tagged promoter, specific primers were designed to amplify regions spanning the 3′ end of the promoter and either the start of the endogenous gene or the start of the luciferase gene. The positions of forward (F) and reverse (R) primers are shown in panel A. Grey lines refer to the ChIP enrichment of the native cg6 locus in the untransfected parasites. Orange and green lines represent the ChIP enrichment of native promoters and integrated promoters, respectively, in the transfectants. The error bars give the standard deviation from triplicate experiments.
Figure Legend Snippet: Histone modification patterns on ectopically integrated promoters. (A) Cloning strategy for ectopic integration of promoter regions . Four promoter regions (1.5–2 kb upstream of the ATG) corresponding to upstream regions of MAL13P1.122, PF14_0705, PFD0240c and PFC0210c were cloned upstream of the luciferase reporter gene pLN-luc (see Materials and Methods ). P. falciparum strain Dd2 attB was transfected with the above vectors to achieve integration at the cg6 locus and the transgenic cell lines were selected on blasticidin. Primer pair P2/P4 was used to confirm integration (data not shown). (B) H4K8ac occupancy at the four ectopically integrated promoter regions . The graphs represent real time PCR results carried out on H4K8ac-immunoprecipitated DNA from rings (R), trophozoites (T) and schizonts (S). In order to distinguish between the endogenous and luciferase tagged promoter, specific primers were designed to amplify regions spanning the 3′ end of the promoter and either the start of the endogenous gene or the start of the luciferase gene. The positions of forward (F) and reverse (R) primers are shown in panel A. Grey lines refer to the ChIP enrichment of the native cg6 locus in the untransfected parasites. Orange and green lines represent the ChIP enrichment of native promoters and integrated promoters, respectively, in the transfectants. The error bars give the standard deviation from triplicate experiments.

Techniques Used: Modification, Clone Assay, Luciferase, Transfection, Transgenic Assay, Real-time Polymerase Chain Reaction, Immunoprecipitation, Chromatin Immunoprecipitation, Standard Deviation

9) Product Images from "ZFR coordinates crosstalk between RNA decay and transcription in innate immunity"

Article Title: ZFR coordinates crosstalk between RNA decay and transcription in innate immunity

Journal: Nature Communications

doi: 10.1038/s41467-018-03326-5

mH2A1 is responsible for ZFR-mediated IFNβ repression. a qRT-PCR analysis of IFNB1 after transient transfection of a plasmid DNA-expressing 3xFLAG-tagged RIG-I CARD domain in HEK-293TO cells stably transduced with shZFR or shScr ( n = 3). b Schematic of experiment to analyze synthesis of IFNB mRNA after poly(I:C) stimulation (left). qRT-PCR of newly synthesized IFNB1 mRNA 24 h after poly(I:C) stimulation (right; n = 3). c Dual luciferase assay of cells transfected with plasmids expressing firefly luciferase under the control of the IFNB1 promoter, with and without poly(I:C) stimulation ( n = 3). A plasmid constitutively expressing Renilla luciferase was used as control. d qRT-PCR analysis of mH2A1 in HEK-293TO cells transfected with si-mH2A1 or si-NS with and without stimulation with poly(I:C) ( n = 3). e qRT-PCR analysis of IFNB1 as in D ( n = 3). f qRT-PCR analysis of mH2A1 in mouse BMDMs transfected with si-mH2A1 or si-NS ( n = 3). g qRT-PCR analysis of Ifnb1 in BMDMs transfected with the indicated siRNAs and stimulated with poly(I:C) ( n = 3). h qRT-PCR analysis of Ifit1 as in g ( n = 3). i Chromatin immunoprecipitation-qPCR (ChIP-qPCR) analysis of mH2A1 binding to the IFNB1 promoter region in HEK-293TO cells. Isotype-matched IgG was used as a control. Fold enrichment over input is shown for each location ( n = 3). j Immunoblot analysis of mH2A1 in BMDMs from WT or macroH2A double knockout mouse. Approximate MW based on size markers is shown. k qRT-PCR analysis of Ifnb1 in BMDMs stimulated with poly(I:C) or ( l ) LPS. Graphs indicate mean ± SD; * P
Figure Legend Snippet: mH2A1 is responsible for ZFR-mediated IFNβ repression. a qRT-PCR analysis of IFNB1 after transient transfection of a plasmid DNA-expressing 3xFLAG-tagged RIG-I CARD domain in HEK-293TO cells stably transduced with shZFR or shScr ( n = 3). b Schematic of experiment to analyze synthesis of IFNB mRNA after poly(I:C) stimulation (left). qRT-PCR of newly synthesized IFNB1 mRNA 24 h after poly(I:C) stimulation (right; n = 3). c Dual luciferase assay of cells transfected with plasmids expressing firefly luciferase under the control of the IFNB1 promoter, with and without poly(I:C) stimulation ( n = 3). A plasmid constitutively expressing Renilla luciferase was used as control. d qRT-PCR analysis of mH2A1 in HEK-293TO cells transfected with si-mH2A1 or si-NS with and without stimulation with poly(I:C) ( n = 3). e qRT-PCR analysis of IFNB1 as in D ( n = 3). f qRT-PCR analysis of mH2A1 in mouse BMDMs transfected with si-mH2A1 or si-NS ( n = 3). g qRT-PCR analysis of Ifnb1 in BMDMs transfected with the indicated siRNAs and stimulated with poly(I:C) ( n = 3). h qRT-PCR analysis of Ifit1 as in g ( n = 3). i Chromatin immunoprecipitation-qPCR (ChIP-qPCR) analysis of mH2A1 binding to the IFNB1 promoter region in HEK-293TO cells. Isotype-matched IgG was used as a control. Fold enrichment over input is shown for each location ( n = 3). j Immunoblot analysis of mH2A1 in BMDMs from WT or macroH2A double knockout mouse. Approximate MW based on size markers is shown. k qRT-PCR analysis of Ifnb1 in BMDMs stimulated with poly(I:C) or ( l ) LPS. Graphs indicate mean ± SD; * P

Techniques Used: Quantitative RT-PCR, Transfection, Plasmid Preparation, Expressing, Stable Transfection, Transduction, Synthesized, Luciferase, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Double Knockout

10) Product Images from "Investigating the Epigenetic Effects of a Prototype Smoke-Derived Carcinogen in Human Cells"

Article Title: Investigating the Epigenetic Effects of a Prototype Smoke-Derived Carcinogen in Human Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0010594

Comparison of DNA methylation profiles between B[ a ]PDE-treated cells and control by MIRA-assisted microarray analysis. Genomic DNA of normal human fibroblasts chronically treated with B[ a ]PDE vs DMSO was subjected to MIRA-assisted microarray analysis, as described in the text. Representative methylation array profiles from different chromosomal regions are displayed with corresponding genomic coordinates (indicated on the top). MIRA-T/MIRA-UT' = MIRA-enriched B[ a ]PDE-treated DNA vs MIRA-enriched DMSO-treated DNA, ‘MIRA-T/Input’ = MIRA-enriched B[ a ]PDE-treated DNA vs Input non-enriched B[ a ]PDE-treated DNA, and ‘MIRA-UT/Input’ = MIRA-enriched DMSO-treated DNA vs Input non-enriched DMSO-treated DNA.
Figure Legend Snippet: Comparison of DNA methylation profiles between B[ a ]PDE-treated cells and control by MIRA-assisted microarray analysis. Genomic DNA of normal human fibroblasts chronically treated with B[ a ]PDE vs DMSO was subjected to MIRA-assisted microarray analysis, as described in the text. Representative methylation array profiles from different chromosomal regions are displayed with corresponding genomic coordinates (indicated on the top). MIRA-T/MIRA-UT' = MIRA-enriched B[ a ]PDE-treated DNA vs MIRA-enriched DMSO-treated DNA, ‘MIRA-T/Input’ = MIRA-enriched B[ a ]PDE-treated DNA vs Input non-enriched B[ a ]PDE-treated DNA, and ‘MIRA-UT/Input’ = MIRA-enriched DMSO-treated DNA vs Input non-enriched DMSO-treated DNA.

Techniques Used: DNA Methylation Assay, Microarray, Methylation

Locus/gene-specific verification of DNA methylation profiles in B[ a ]PDE-treated cells vs control by COBRA and bisulfite genomic sequencing. Differentially, yet marginally, methylated target loci/genes identified by MIRA-assisted microarray analysis in B[ a ]PDE-treated cells vs control, were selected randomly, and subjected to conventional COBRA [32] and bisulfite sequencing analyses [33] to establish their methylation status, individually. The lack of ‘hypermethylation’ in the specified targets was confirmed by the COBRA [32] and/or genomic sequencing [33] methods. For comparison, we have presented readily detectable hypermethylation of one of these targets ( RASSF1A ) in A549 lung cancer cell line. Data from independent B[ a ]PDE-treated samples, indicated by superscript numbers, e.g. , B[ a ]PDE 1 , are shown. UT = DMSO-treated DNA; T = B[ a ]PDE-treated DNA. (•) = Methylated CpG; (○) = Unmethylated CpG; m CG: Absolute number of methylated CpGs/total CpGs (% methylated CpGs); None of the differences in m CG% between B[ a ]PDE-treated DNA vs control was statistically significant (Fisher's exact test).
Figure Legend Snippet: Locus/gene-specific verification of DNA methylation profiles in B[ a ]PDE-treated cells vs control by COBRA and bisulfite genomic sequencing. Differentially, yet marginally, methylated target loci/genes identified by MIRA-assisted microarray analysis in B[ a ]PDE-treated cells vs control, were selected randomly, and subjected to conventional COBRA [32] and bisulfite sequencing analyses [33] to establish their methylation status, individually. The lack of ‘hypermethylation’ in the specified targets was confirmed by the COBRA [32] and/or genomic sequencing [33] methods. For comparison, we have presented readily detectable hypermethylation of one of these targets ( RASSF1A ) in A549 lung cancer cell line. Data from independent B[ a ]PDE-treated samples, indicated by superscript numbers, e.g. , B[ a ]PDE 1 , are shown. UT = DMSO-treated DNA; T = B[ a ]PDE-treated DNA. (•) = Methylated CpG; (○) = Unmethylated CpG; m CG: Absolute number of methylated CpGs/total CpGs (% methylated CpGs); None of the differences in m CG% between B[ a ]PDE-treated DNA vs control was statistically significant (Fisher's exact test).

Techniques Used: DNA Methylation Assay, Combined Bisulfite Restriction Analysis Assay, Genomic Sequencing, Methylation, Microarray, Methylation Sequencing

11) Product Images from "Modulation of Neuroblastoma Disease Pathogenesis By An Extensive Network of Epigenetically Regulated MicroRNAs"

Article Title: Modulation of Neuroblastoma Disease Pathogenesis By An Extensive Network of Epigenetically Regulated MicroRNAs

Journal: Oncogene

doi: 10.1038/onc.2012.311

Identification of methylation sensitive miRNA ( a ) Flowchart representing study design. ( b ) Integrated heatmap and two-dimensional hierarchical clustering for 67 methylation sensitive miRNAs. Tumors are clustered horizontally while miRNA are clustered vertically. Characteristics of the tumors are displayed at the top of the heat map. The heat map is based on both levels of miRNA expression and levels of DNA methylation at each locus, with a key to the color coding presented in the upper left corner. The colored vertical bar on the left designates miRNAs from the same cluster which display similarity in expression levels due to being under the control of a common upstream site of methylation. The table on the right designates miRNAs that are associated with poor OS/EFS when under-expressed in tumours (black, n = 28) or over-expressed in tumors (grey, n = 10). miRNAs that are up-regulated in response to the demethylating agent, 5-aza-cytidine, in four different cell lines are designated in the table. ( c ) The distribution of all methylation peaks within 10kb of CpG Islands. The majority of peaks occur within regions defined as CpG shores.
Figure Legend Snippet: Identification of methylation sensitive miRNA ( a ) Flowchart representing study design. ( b ) Integrated heatmap and two-dimensional hierarchical clustering for 67 methylation sensitive miRNAs. Tumors are clustered horizontally while miRNA are clustered vertically. Characteristics of the tumors are displayed at the top of the heat map. The heat map is based on both levels of miRNA expression and levels of DNA methylation at each locus, with a key to the color coding presented in the upper left corner. The colored vertical bar on the left designates miRNAs from the same cluster which display similarity in expression levels due to being under the control of a common upstream site of methylation. The table on the right designates miRNAs that are associated with poor OS/EFS when under-expressed in tumours (black, n = 28) or over-expressed in tumors (grey, n = 10). miRNAs that are up-regulated in response to the demethylating agent, 5-aza-cytidine, in four different cell lines are designated in the table. ( c ) The distribution of all methylation peaks within 10kb of CpG Islands. The majority of peaks occur within regions defined as CpG shores.

Techniques Used: Methylation, Expressing, DNA Methylation Assay

DNA methylation and expressional alterations of miR-340 ( a ) Relative expression of miR-340 in SK-N-BE and SHSY-5Y at 7 days post-ATRA. ( b,c ) Kaplan Meier survival plots for OS ( b ) and EFS ( c ) in 237 neuroblastoma tumors based on miR-340 expression. ( d ) SignalMap image from MeDIP analysis for the miR-340 upstream region. Only the methylation peak highlighted with a bracket (~6 Kb upstream) exhibited significant de-methylation in SK-N-BE cells 7 days post-ATRA. This peak overlaps the predicted TSS for miR-340. ( e ) Scatter plot of methylation vs. miR-340 expression in tumors showing a significant inverse correlation using Pearson’s correlation coefficient. ( f ) Expression of miR-340 following 5’-Aza-2 treatment of SK-N-BE and SHSY-5Y (*P
Figure Legend Snippet: DNA methylation and expressional alterations of miR-340 ( a ) Relative expression of miR-340 in SK-N-BE and SHSY-5Y at 7 days post-ATRA. ( b,c ) Kaplan Meier survival plots for OS ( b ) and EFS ( c ) in 237 neuroblastoma tumors based on miR-340 expression. ( d ) SignalMap image from MeDIP analysis for the miR-340 upstream region. Only the methylation peak highlighted with a bracket (~6 Kb upstream) exhibited significant de-methylation in SK-N-BE cells 7 days post-ATRA. This peak overlaps the predicted TSS for miR-340. ( e ) Scatter plot of methylation vs. miR-340 expression in tumors showing a significant inverse correlation using Pearson’s correlation coefficient. ( f ) Expression of miR-340 following 5’-Aza-2 treatment of SK-N-BE and SHSY-5Y (*P

Techniques Used: DNA Methylation Assay, Expressing, Methylated DNA Immunoprecipitation, Methylation

12) Product Images from "B-MYB Is Essential for Normal Cell Cycle Progression and Chromosomal Stability of Embryonic Stem Cells"

Article Title: B-MYB Is Essential for Normal Cell Cycle Progression and Chromosomal Stability of Embryonic Stem Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0002478

B-Myb binds to and regulates Oct4 promoter activity. A) Chromosomal immunoprecipitation (ChIP) assays were performed with extracts from R1 mES cells. In these experiments, PCR amplifications were performed with total input DNA (positive control without immunoprecipitation) and following immunoprecipitation (IP) with antibodies to either B-MYB or to rabbit IgG (as a negative control). Amplifications of immunoprecipitated DNA were analyzed by agarose gel electrophoresis, and Myc was employed as a positive control. The presence of an amplification product in the anti-B-Myb immunoprecipitated DNA indicates that B-Myb binds a proximal region of the mouse pou5f1 promoter. B) In the second set of experiments, which were evaluated by qRT-PCR, we find that B-MYB dynamically binds to the endogenous pou5f1 promoter region. In undifferentiated cells, an amplification signal 10–15-fold above that seen in the IgG control is observed; however, this signal is reduced to only 2–4 fold above the IgG control following withdrawal of LIF and FCS. All qRT-PCR amplifications were performed using SYBR Green with primers located 5′ and 3′ to the putative binding site. C) The dynamic decrease in chromatin binding of B-Myb to the pou5f1 gene promoter in mES cells cultivated either in the presence or absence of FCS and LIF is illustrated graphically (p
Figure Legend Snippet: B-Myb binds to and regulates Oct4 promoter activity. A) Chromosomal immunoprecipitation (ChIP) assays were performed with extracts from R1 mES cells. In these experiments, PCR amplifications were performed with total input DNA (positive control without immunoprecipitation) and following immunoprecipitation (IP) with antibodies to either B-MYB or to rabbit IgG (as a negative control). Amplifications of immunoprecipitated DNA were analyzed by agarose gel electrophoresis, and Myc was employed as a positive control. The presence of an amplification product in the anti-B-Myb immunoprecipitated DNA indicates that B-Myb binds a proximal region of the mouse pou5f1 promoter. B) In the second set of experiments, which were evaluated by qRT-PCR, we find that B-MYB dynamically binds to the endogenous pou5f1 promoter region. In undifferentiated cells, an amplification signal 10–15-fold above that seen in the IgG control is observed; however, this signal is reduced to only 2–4 fold above the IgG control following withdrawal of LIF and FCS. All qRT-PCR amplifications were performed using SYBR Green with primers located 5′ and 3′ to the putative binding site. C) The dynamic decrease in chromatin binding of B-Myb to the pou5f1 gene promoter in mES cells cultivated either in the presence or absence of FCS and LIF is illustrated graphically (p

Techniques Used: Activity Assay, Immunoprecipitation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Positive Control, Negative Control, Agarose Gel Electrophoresis, Amplification, Quantitative RT-PCR, SYBR Green Assay, Binding Assay

13) Product Images from "NELF and DSIF cause promoter proximal pausing on the hsp70 promoter in Drosophila"

Article Title: NELF and DSIF cause promoter proximal pausing on the hsp70 promoter in Drosophila

Journal: Genes & Development

doi: 10.1101/gad.1091403

Chromatin immunoprecipitation analysis of NELF, DSIF, and GAGA factor. Drosophila Schneider 2 cells were subjected to formaldehyde cross-linking. Sonicated detergent lysates from the cells were immunoprecipited with preimmune serum (Pre-D or Pre-E) or with antiserum against NELF-E, NELF-D, DSIF, or GAGA factor. DNA was purified from each immunoprecipitate, and the presence of DNA from the 5′ and 3′ portions of the hsp70 gene was detected by PCR. Various amounts of material representing the input were amplified to quantify the amount of DNA recovered from the immunoprecipitates. ( A ) A schematic of the hsp70 transcription unit and the regions that were amplified. ( B ) The results from one of two independent experiments that were done. The bands represent radiolabeled PCR products that were detected after the DNA was run on an acrylamide gel and the gel was subjected to analysis with a PhosphorImager. Because preliminary experiments showed that DSIF and GAGA factor cross-linked more efficiently than NELF-D and NELF-E, 5× less of the DNA recovered from the DSIF and GAGA factor immunoprecipitates were used in the PCR reactions. The graph shows a compilation of the results from two independent experiments. The height of each bar shows the average percent of input material detected in immunoprecipitates from two experiments, and each error bar shows the range of measurements.
Figure Legend Snippet: Chromatin immunoprecipitation analysis of NELF, DSIF, and GAGA factor. Drosophila Schneider 2 cells were subjected to formaldehyde cross-linking. Sonicated detergent lysates from the cells were immunoprecipited with preimmune serum (Pre-D or Pre-E) or with antiserum against NELF-E, NELF-D, DSIF, or GAGA factor. DNA was purified from each immunoprecipitate, and the presence of DNA from the 5′ and 3′ portions of the hsp70 gene was detected by PCR. Various amounts of material representing the input were amplified to quantify the amount of DNA recovered from the immunoprecipitates. ( A ) A schematic of the hsp70 transcription unit and the regions that were amplified. ( B ) The results from one of two independent experiments that were done. The bands represent radiolabeled PCR products that were detected after the DNA was run on an acrylamide gel and the gel was subjected to analysis with a PhosphorImager. Because preliminary experiments showed that DSIF and GAGA factor cross-linked more efficiently than NELF-D and NELF-E, 5× less of the DNA recovered from the DSIF and GAGA factor immunoprecipitates were used in the PCR reactions. The graph shows a compilation of the results from two independent experiments. The height of each bar shows the average percent of input material detected in immunoprecipitates from two experiments, and each error bar shows the range of measurements.

Techniques Used: Chromatin Immunoprecipitation, Sonication, Purification, Polymerase Chain Reaction, Amplification, Acrylamide Gel Assay

14) Product Images from "Regulation of Steroid 5-alpha reductase type 2 (Srd5a2) by Sterol Regulatory Element Binding Proteins and Statins"

Article Title: Regulation of Steroid 5-alpha reductase type 2 (Srd5a2) by Sterol Regulatory Element Binding Proteins and Statins

Journal: Experimental cell research

doi: 10.1016/j.yexcr.2009.05.025

Identification of mouse Srd5a2 as SREBP-2 target gene. SREBP-2 ChIP-on-chip analysis was performed using 1.5 kb mouse whole genome promoter arrays from NimbleGen/Roche. ( A ) Location of the hybridization signals on the ChIP-chip array at the Srd5a2 locus on mouse chromosome 17 (Chr 17). The top panel shows log2 ratios of hybridization signals for tiled probes for SREBP-2 antibody-enriched ChIP DNA relative to the input DNA. The middle panel shows a similar analysis when a control IgG fraction was used as antibody. The X axis of [Fig 1A] is Enrichment Score relative to Input (Log2) and the lower panel in [Fig 1A] shows the relative position of the Srd5a2 coding region on Chr 17. ( B ) Gene-specific ChIP analysis. Chromatin from chow (open bars) or L/E (filled bars) treated mouse liver was analyzed for SREBP-2 binding to the indicated promoter regions using promoter specific oligonucleotides as described in Experimental Procedures. ( C ) Total RNAs from mice fed a control chow (open) or L/E supplemented diet were analyzed for expression of Srd5a2, SREBP-2, HMGCR and L32, as described under “Experimental Procedures.” Expression of each mRNA indicated was normalized to the expression of ribosomal L32 protein RNA in each sample, and the ratio in the normal chow sample was set at 1.0. Data are mean ± SEM; n = 3 for 3 separate experiments.
Figure Legend Snippet: Identification of mouse Srd5a2 as SREBP-2 target gene. SREBP-2 ChIP-on-chip analysis was performed using 1.5 kb mouse whole genome promoter arrays from NimbleGen/Roche. ( A ) Location of the hybridization signals on the ChIP-chip array at the Srd5a2 locus on mouse chromosome 17 (Chr 17). The top panel shows log2 ratios of hybridization signals for tiled probes for SREBP-2 antibody-enriched ChIP DNA relative to the input DNA. The middle panel shows a similar analysis when a control IgG fraction was used as antibody. The X axis of [Fig 1A] is Enrichment Score relative to Input (Log2) and the lower panel in [Fig 1A] shows the relative position of the Srd5a2 coding region on Chr 17. ( B ) Gene-specific ChIP analysis. Chromatin from chow (open bars) or L/E (filled bars) treated mouse liver was analyzed for SREBP-2 binding to the indicated promoter regions using promoter specific oligonucleotides as described in Experimental Procedures. ( C ) Total RNAs from mice fed a control chow (open) or L/E supplemented diet were analyzed for expression of Srd5a2, SREBP-2, HMGCR and L32, as described under “Experimental Procedures.” Expression of each mRNA indicated was normalized to the expression of ribosomal L32 protein RNA in each sample, and the ratio in the normal chow sample was set at 1.0. Data are mean ± SEM; n = 3 for 3 separate experiments.

Techniques Used: Chromatin Immunoprecipitation, Hybridization, Binding Assay, Mouse Assay, Expressing

15) Product Images from "Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare"

Article Title: Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2018.00443

Mbo II digest results. Agarose gel showing Mbo II digests of GAA PCR products of FRDA samples. The expected 170bp (5′) and 120bp (3′) undigested GAA-flanking fragments from normal pure GAA repeat expansion FRDA samples are shown in lanes 2, 3, and 4. These band sizes can be seen in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder markers, which are loaded into lanes 1 and 11 of the gel. Lane 5 shows a large Mbo II band of approximately 600bp that was obtained from the positive interrupted GAA repeat sequence from the “NEP” BAC transgenic mouse that contains approximately 500 triplet repeats with the previously determined interrupted sequence of (GAA) 21 (GGAGAA) 5 (GGAGGAGAA) 70 (GAA) n ( Holloway et al., 2011 ). In addition for this positive sample, we also identified the expected 5′ flanking band of 170bp, together with a smaller band of less than 100bp that we sequenced and we showed to contain a 27bp deletion in the 3′ flanking region. Lane 6 shows an abnormal band of 200bp representing the 80bp duplication in the 3′ GAA flanking region. Lane 7 shows an abnormal band of approximately 100bp representing the 19bp deletion in the 3′ GAA flanking region. Lanes 8, 9, and 10 contain abnormal bands of approximately 300, 100, and 180bp, respectively, that are likely to contain a region of interrupted GAA repeat sequence within the body of one or other of the large FRDA GAA repeat expansions.
Figure Legend Snippet: Mbo II digest results. Agarose gel showing Mbo II digests of GAA PCR products of FRDA samples. The expected 170bp (5′) and 120bp (3′) undigested GAA-flanking fragments from normal pure GAA repeat expansion FRDA samples are shown in lanes 2, 3, and 4. These band sizes can be seen in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder markers, which are loaded into lanes 1 and 11 of the gel. Lane 5 shows a large Mbo II band of approximately 600bp that was obtained from the positive interrupted GAA repeat sequence from the “NEP” BAC transgenic mouse that contains approximately 500 triplet repeats with the previously determined interrupted sequence of (GAA) 21 (GGAGAA) 5 (GGAGGAGAA) 70 (GAA) n ( Holloway et al., 2011 ). In addition for this positive sample, we also identified the expected 5′ flanking band of 170bp, together with a smaller band of less than 100bp that we sequenced and we showed to contain a 27bp deletion in the 3′ flanking region. Lane 6 shows an abnormal band of 200bp representing the 80bp duplication in the 3′ GAA flanking region. Lane 7 shows an abnormal band of approximately 100bp representing the 19bp deletion in the 3′ GAA flanking region. Lanes 8, 9, and 10 contain abnormal bands of approximately 300, 100, and 180bp, respectively, that are likely to contain a region of interrupted GAA repeat sequence within the body of one or other of the large FRDA GAA repeat expansions.

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Sequencing, BAC Assay, Transgenic Assay

Mbo II digests of GAA repeat expansions from human FRDA somatic tissues and mouse FRDA intergenerational and somatic tissues. Agarose gels showing Mbo II digests of GAA PCR products of (A) FRDA patient cerebellum tissue samples, (B) YG8sR mouse ear biopsy samples and human FRDA blood samples, and (C) four tissues from one YG8sR mouse. In each case, the expected 170 and 120bp undigested GAA-flanking fragments can be identified in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder marker, which is loaded into the first lane of each gel. (A) Lanes 1–3 show the results from cerebellum tissue samples from three FRDA patients. (B) Lanes 1 and 2 are from FRDA patient blood samples; lanes 3–6 are from ear biopsy samples from 4 GAA repeat expansion-based YG8sR mice of four different generations, and lane 7 is from an ear biopsy sample from the Y47R mouse which has nine GAA repeats. (C) Lanes 1–4 are from brain, cerebellum, heart, and liver tissues of the YG8sR mouse, respectively.
Figure Legend Snippet: Mbo II digests of GAA repeat expansions from human FRDA somatic tissues and mouse FRDA intergenerational and somatic tissues. Agarose gels showing Mbo II digests of GAA PCR products of (A) FRDA patient cerebellum tissue samples, (B) YG8sR mouse ear biopsy samples and human FRDA blood samples, and (C) four tissues from one YG8sR mouse. In each case, the expected 170 and 120bp undigested GAA-flanking fragments can be identified in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder marker, which is loaded into the first lane of each gel. (A) Lanes 1–3 show the results from cerebellum tissue samples from three FRDA patients. (B) Lanes 1 and 2 are from FRDA patient blood samples; lanes 3–6 are from ear biopsy samples from 4 GAA repeat expansion-based YG8sR mice of four different generations, and lane 7 is from an ear biopsy sample from the Y47R mouse which has nine GAA repeats. (C) Lanes 1–4 are from brain, cerebellum, heart, and liver tissues of the YG8sR mouse, respectively.

Techniques Used: Polymerase Chain Reaction, Marker, Mouse Assay

16) Product Images from "A genetic network mediating the control of bud break in hybrid aspen"

Article Title: A genetic network mediating the control of bud break in hybrid aspen

Journal: Nature Communications

doi: 10.1038/s41467-018-06696-y

SVL binds to FT1 , NCED3 , and TCP promoters in vivo in chromatin immunoprecipitation (ChIP) assays. a Diagrammatic representation of FT1 , NCED3 , and TCP18 / BRC1 promoters showing the CArG motif and their positions within a 3 kb region. F1–R1 indicates positions of DNA fragments used to assess DNA–protein interactions in ChIP assays, and F2–R2 indicates positions of DNA fragments with no CArG motif used as negative controls in the assays. b Enrichment of the DNA fragments containing the CArG motif quantified by ChIP-q-PCR. Presented values were first normalized by their respective input values, then fold enrichments in WT and Myc-SVL plants relative to negative controls were calculated. Bars show an average values from three independent biological replicates ± SEM. Asterisks indicate significant (* P
Figure Legend Snippet: SVL binds to FT1 , NCED3 , and TCP promoters in vivo in chromatin immunoprecipitation (ChIP) assays. a Diagrammatic representation of FT1 , NCED3 , and TCP18 / BRC1 promoters showing the CArG motif and their positions within a 3 kb region. F1–R1 indicates positions of DNA fragments used to assess DNA–protein interactions in ChIP assays, and F2–R2 indicates positions of DNA fragments with no CArG motif used as negative controls in the assays. b Enrichment of the DNA fragments containing the CArG motif quantified by ChIP-q-PCR. Presented values were first normalized by their respective input values, then fold enrichments in WT and Myc-SVL plants relative to negative controls were calculated. Bars show an average values from three independent biological replicates ± SEM. Asterisks indicate significant (* P

Techniques Used: In Vivo, Chromatin Immunoprecipitation, Polymerase Chain Reaction

17) Product Images from "A genetic network mediating the control of bud break in hybrid aspen"

Article Title: A genetic network mediating the control of bud break in hybrid aspen

Journal: Nature Communications

doi: 10.1038/s41467-018-06696-y

SVL binds to FT1 , NCED3 , and TCP promoters in vivo in chromatin immunoprecipitation (ChIP) assays. a Diagrammatic representation of FT1 , NCED3 , and TCP18 / BRC1 promoters showing the CArG motif and their positions within a 3 kb region. F1–R1 indicates positions of DNA fragments used to assess DNA–protein interactions in ChIP assays, and F2–R2 indicates positions of DNA fragments with no CArG motif used as negative controls in the assays. b Enrichment of the DNA fragments containing the CArG motif quantified by ChIP-q-PCR. Presented values were first normalized by their respective input values, then fold enrichments in WT and Myc-SVL plants relative to negative controls were calculated. Bars show an average values from three independent biological replicates ± SEM. Asterisks indicate significant (* P
Figure Legend Snippet: SVL binds to FT1 , NCED3 , and TCP promoters in vivo in chromatin immunoprecipitation (ChIP) assays. a Diagrammatic representation of FT1 , NCED3 , and TCP18 / BRC1 promoters showing the CArG motif and their positions within a 3 kb region. F1–R1 indicates positions of DNA fragments used to assess DNA–protein interactions in ChIP assays, and F2–R2 indicates positions of DNA fragments with no CArG motif used as negative controls in the assays. b Enrichment of the DNA fragments containing the CArG motif quantified by ChIP-q-PCR. Presented values were first normalized by their respective input values, then fold enrichments in WT and Myc-SVL plants relative to negative controls were calculated. Bars show an average values from three independent biological replicates ± SEM. Asterisks indicate significant (* P

Techniques Used: In Vivo, Chromatin Immunoprecipitation, Polymerase Chain Reaction

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Hybridization:

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Article Snippet: .. For ChIP-on-chip array hybridizations, mice were administered either vehicle or 50 mg/kg GW4064 dissolved in vehicle by oral gavage for 5 d. After ChIP, the FXR, IgG control, and input DNA for both control and GW4064-treated groups were prepared for hybridization to the 2.5-kb mouse promoter array (NimbleGen/Roche) using a random PCR amplification method according to the company’s protocol. .. The hybridization data were analyzed by using NimbleScan 2.3 and SignalMap software from NimbleGen/Roche.

SYBR Green Assay:

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Amplification:

Article Title: A Novel CpG Island Set Identifies Tissue-Specific Methylation at Developmental Gene Loci
Article Snippet: .. Purified DNA was resuspended in 1×TE and amplified in parallel with input DNA, using the GC Rich PCR system (Roche; 2 min at 95 °C; followed by 18 cycles of 95 °C for 1 min, Tann °C for 1 min, 72 °C for 4 min; and a final extension of 72 °C for 7 min; universal primer_GGT CCA TCC AAC CGA TCT TA). .. MAP and Input DNAs (200 ng) were fluorescently labeled by random priming using the Bioprime labeling kit (Invitrogen), 1 × dNTS (10× dNTPS; 2 mM of each dATP, dGTP, dTTP, 1 mM dCTP) and 1.5 nmol of Cy3 or Cy5-dCTP (GE Healthcare).

Article Title: Activation of the Farnesoid X Receptor Provides Protection against Acetaminophen-Induced Hepatic Toxicity
Article Snippet: .. For ChIP-on-chip array hybridizations, mice were administered either vehicle or 50 mg/kg GW4064 dissolved in vehicle by oral gavage for 5 d. After ChIP, the FXR, IgG control, and input DNA for both control and GW4064-treated groups were prepared for hybridization to the 2.5-kb mouse promoter array (NimbleGen/Roche) using a random PCR amplification method according to the company’s protocol. .. The hybridization data were analyzed by using NimbleScan 2.3 and SignalMap software from NimbleGen/Roche.

Purification:

Article Title: A Novel CpG Island Set Identifies Tissue-Specific Methylation at Developmental Gene Loci
Article Snippet: .. Purified DNA was resuspended in 1×TE and amplified in parallel with input DNA, using the GC Rich PCR system (Roche; 2 min at 95 °C; followed by 18 cycles of 95 °C for 1 min, Tann °C for 1 min, 72 °C for 4 min; and a final extension of 72 °C for 7 min; universal primer_GGT CCA TCC AAC CGA TCT TA). .. MAP and Input DNAs (200 ng) were fluorescently labeled by random priming using the Bioprime labeling kit (Invitrogen), 1 × dNTS (10× dNTPS; 2 mM of each dATP, dGTP, dTTP, 1 mM dCTP) and 1.5 nmol of Cy3 or Cy5-dCTP (GE Healthcare).

Real-time Polymerase Chain Reaction:

Article Title: Molecular Interactions of GBF1 with HY5 and HYH Proteins during Light-mediated Seedling Development in Arabidopsis thaliana *
Article Snippet: .. Both immunoprecipitated DNA and input DNA were analyzed by real-time PCR (Light Cycler; Roche Applied Science). .. Otherwise, after reverse cross-linking by boiling for 25 min, it was resolved on SDS-PAGE.

Article Title: Dynamic Distribution of Linker Histone H1.5 in Cellular Differentiation
Article Snippet: .. ChIP–quantitative PCR Real-time PCR was performed on ChIP and input DNA using SYBR Green Real-time PCR Master Mix (Roche). .. For each primer pair, an amplification standard curve was established by gradient amount of input DNA.

Article Title: Dynamic Epigenetic Regulation of Gene Expression during the Life Cycle of Malaria Parasite Plasmodium falciparum
Article Snippet: .. Quantitative real time PCR (RTQ-PCR) RTQ-PCR was carried out on immunoprecipitated and input DNA using the SYBR Green PCR Master Mix (Roche) according to manufacturer's instructions. .. ChIP enrichment was calculated by using the ΔCt method (Ct of immunoprecipitated target gene - Ct of input target gene) where Ct is the threshold cycle.

Article Title: ZFR coordinates crosstalk between RNA decay and transcription in innate immunity
Article Snippet: .. An aliquot of 1 μL each of immunoprecipitated and input DNA was analyzed in duplicate by qPCR using SYBR Green qPCR master mix (FastStart Essential DNA Green Master Mix, Roche) on a LightCycler 96 thermocycler (Roche). .. The relative cycle threshold (Ct) method was used to determine enrichment of immunoprecipitated DNA over input using the indicated primers; primer sequences are listed in Supplementary Data .

Immunoprecipitation:

Article Title: Molecular Interactions of GBF1 with HY5 and HYH Proteins during Light-mediated Seedling Development in Arabidopsis thaliana *
Article Snippet: .. Both immunoprecipitated DNA and input DNA were analyzed by real-time PCR (Light Cycler; Roche Applied Science). .. Otherwise, after reverse cross-linking by boiling for 25 min, it was resolved on SDS-PAGE.

Article Title: Dynamic Epigenetic Regulation of Gene Expression during the Life Cycle of Malaria Parasite Plasmodium falciparum
Article Snippet: .. Quantitative real time PCR (RTQ-PCR) RTQ-PCR was carried out on immunoprecipitated and input DNA using the SYBR Green PCR Master Mix (Roche) according to manufacturer's instructions. .. ChIP enrichment was calculated by using the ΔCt method (Ct of immunoprecipitated target gene - Ct of input target gene) where Ct is the threshold cycle.

Article Title: ZFR coordinates crosstalk between RNA decay and transcription in innate immunity
Article Snippet: .. An aliquot of 1 μL each of immunoprecipitated and input DNA was analyzed in duplicate by qPCR using SYBR Green qPCR master mix (FastStart Essential DNA Green Master Mix, Roche) on a LightCycler 96 thermocycler (Roche). .. The relative cycle threshold (Ct) method was used to determine enrichment of immunoprecipitated DNA over input using the indicated primers; primer sequences are listed in Supplementary Data .

Polymerase Chain Reaction:

Article Title: Dynamic Distribution of Linker Histone H1.5 in Cellular Differentiation
Article Snippet: .. ChIP–quantitative PCR Real-time PCR was performed on ChIP and input DNA using SYBR Green Real-time PCR Master Mix (Roche). .. For each primer pair, an amplification standard curve was established by gradient amount of input DNA.

Article Title: Dynamic Epigenetic Regulation of Gene Expression during the Life Cycle of Malaria Parasite Plasmodium falciparum
Article Snippet: .. Quantitative real time PCR (RTQ-PCR) RTQ-PCR was carried out on immunoprecipitated and input DNA using the SYBR Green PCR Master Mix (Roche) according to manufacturer's instructions. .. ChIP enrichment was calculated by using the ΔCt method (Ct of immunoprecipitated target gene - Ct of input target gene) where Ct is the threshold cycle.

Article Title: A Novel CpG Island Set Identifies Tissue-Specific Methylation at Developmental Gene Loci
Article Snippet: .. Purified DNA was resuspended in 1×TE and amplified in parallel with input DNA, using the GC Rich PCR system (Roche; 2 min at 95 °C; followed by 18 cycles of 95 °C for 1 min, Tann °C for 1 min, 72 °C for 4 min; and a final extension of 72 °C for 7 min; universal primer_GGT CCA TCC AAC CGA TCT TA). .. MAP and Input DNAs (200 ng) were fluorescently labeled by random priming using the Bioprime labeling kit (Invitrogen), 1 × dNTS (10× dNTPS; 2 mM of each dATP, dGTP, dTTP, 1 mM dCTP) and 1.5 nmol of Cy3 or Cy5-dCTP (GE Healthcare).

Article Title: Activation of the Farnesoid X Receptor Provides Protection against Acetaminophen-Induced Hepatic Toxicity
Article Snippet: .. For ChIP-on-chip array hybridizations, mice were administered either vehicle or 50 mg/kg GW4064 dissolved in vehicle by oral gavage for 5 d. After ChIP, the FXR, IgG control, and input DNA for both control and GW4064-treated groups were prepared for hybridization to the 2.5-kb mouse promoter array (NimbleGen/Roche) using a random PCR amplification method according to the company’s protocol. .. The hybridization data were analyzed by using NimbleScan 2.3 and SignalMap software from NimbleGen/Roche.

Article Title: Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare
Article Snippet: .. We then performed long-range PCR of the samples (approximately 100 ng input DNA) using either the Expand High Fidelity PCR System, dNTPack (Roche), or the Long Range PCR Kit (Qiagen) together with GAA-B-F (5′-AATGGATTTCCTGGCAGGACGC-3′) and GAA-B-R (5′-GCATTGGGCGATCTTGGCTTAA-3′) primers as previously described ( ). .. The thermocycling conditions used were (i) Roche Kit: 94°C for 2 min; 10 cycles of 94°C for 10 s, 60°C for 30 s, 68°C for 45 s; 20 cycles of 94°C for 10 s, 60°C for 30 s, 68°C for 1 min with 20 s increments; and a final cycle of 68°C for 10 mins, or (ii) Qiagen Kit: 93°C for 3 min; 35 cycles of 93°C for 15 s, 62°C for 30 s, 68°C for 5 min, and a final cycle of 68°C for 10 min.

Mouse Assay:

Article Title: Activation of the Farnesoid X Receptor Provides Protection against Acetaminophen-Induced Hepatic Toxicity
Article Snippet: .. For ChIP-on-chip array hybridizations, mice were administered either vehicle or 50 mg/kg GW4064 dissolved in vehicle by oral gavage for 5 d. After ChIP, the FXR, IgG control, and input DNA for both control and GW4064-treated groups were prepared for hybridization to the 2.5-kb mouse promoter array (NimbleGen/Roche) using a random PCR amplification method according to the company’s protocol. .. The hybridization data were analyzed by using NimbleScan 2.3 and SignalMap software from NimbleGen/Roche.

Chromatin Immunoprecipitation:

Article Title: Dynamic Distribution of Linker Histone H1.5 in Cellular Differentiation
Article Snippet: .. ChIP–quantitative PCR Real-time PCR was performed on ChIP and input DNA using SYBR Green Real-time PCR Master Mix (Roche). .. For each primer pair, an amplification standard curve was established by gradient amount of input DNA.

Article Title: Activation of the Farnesoid X Receptor Provides Protection against Acetaminophen-Induced Hepatic Toxicity
Article Snippet: .. For ChIP-on-chip array hybridizations, mice were administered either vehicle or 50 mg/kg GW4064 dissolved in vehicle by oral gavage for 5 d. After ChIP, the FXR, IgG control, and input DNA for both control and GW4064-treated groups were prepared for hybridization to the 2.5-kb mouse promoter array (NimbleGen/Roche) using a random PCR amplification method according to the company’s protocol. .. The hybridization data were analyzed by using NimbleScan 2.3 and SignalMap software from NimbleGen/Roche.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Fumarates improve psoriasis and multiple sclerosis by inducing type II dendritic cells
Article Snippet: .. RT-PCR was performed in duplicate on each sample and input DNA using LightCycler 480 SYBR Green I Master mix (Roche) according to manufacturer’s instructions in a LightCycler 480 system (Roche). .. The product specificity was monitored by melting curve analysis, and product size was visualized on agarose gel by electrophoresis.

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    Roche input dna
    H1.5 knockdown increases DNase I sensitivity at target regions. (A) Venn diagram of the overlap between significant H1.5 peaks and DNase I hypersensitive sites. The p-value for exclusivity of these two sets of peaks is indicated ( Text S1 ). (B–E) Quantitative <t>PCR</t> of <t>DNA</t> fragments at indicated genes from genomic DNA treated with increasing amount of DNase I. Data points with t-test p-value
    Input Dna, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    H1.5 knockdown increases DNase I sensitivity at target regions. (A) Venn diagram of the overlap between significant H1.5 peaks and DNase I hypersensitive sites. The p-value for exclusivity of these two sets of peaks is indicated ( Text S1 ). (B–E) Quantitative PCR of DNA fragments at indicated genes from genomic DNA treated with increasing amount of DNase I. Data points with t-test p-value

    Journal: PLoS Genetics

    Article Title: Dynamic Distribution of Linker Histone H1.5 in Cellular Differentiation

    doi: 10.1371/journal.pgen.1002879

    Figure Lengend Snippet: H1.5 knockdown increases DNase I sensitivity at target regions. (A) Venn diagram of the overlap between significant H1.5 peaks and DNase I hypersensitive sites. The p-value for exclusivity of these two sets of peaks is indicated ( Text S1 ). (B–E) Quantitative PCR of DNA fragments at indicated genes from genomic DNA treated with increasing amount of DNase I. Data points with t-test p-value

    Article Snippet: ChIP–quantitative PCR Real-time PCR was performed on ChIP and input DNA using SYBR Green Real-time PCR Master Mix (Roche).

    Techniques: Real-time Polymerase Chain Reaction

    Representative snapshots of ChIP-Seq peaks for FXR-target genes. Shp ( upper panel ) and Gst α 4 ( lower panel ), mapped onto University of California at Santa Cruz genome browser. Shown is their chromosomal location (chr 4, chr 9) according to the July 2007 Mouse Genome Assembly (mm9). Blue and red tags represent positive and negative strands, respectively, identified after DNA sequencing of the chromatin after immunoprecipitation using antibody to FXR or a control IgG. Each bar represents a distinct hit/sequence.

    Journal: Molecular Endocrinology

    Article Title: Activation of the Farnesoid X Receptor Provides Protection against Acetaminophen-Induced Hepatic Toxicity

    doi: 10.1210/me.2010-0117

    Figure Lengend Snippet: Representative snapshots of ChIP-Seq peaks for FXR-target genes. Shp ( upper panel ) and Gst α 4 ( lower panel ), mapped onto University of California at Santa Cruz genome browser. Shown is their chromosomal location (chr 4, chr 9) according to the July 2007 Mouse Genome Assembly (mm9). Blue and red tags represent positive and negative strands, respectively, identified after DNA sequencing of the chromatin after immunoprecipitation using antibody to FXR or a control IgG. Each bar represents a distinct hit/sequence.

    Article Snippet: For ChIP-on-chip array hybridizations, mice were administered either vehicle or 50 mg/kg GW4064 dissolved in vehicle by oral gavage for 5 d. After ChIP, the FXR, IgG control, and input DNA for both control and GW4064-treated groups were prepared for hybridization to the 2.5-kb mouse promoter array (NimbleGen/Roche) using a random PCR amplification method according to the company’s protocol.

    Techniques: Chromatin Immunoprecipitation, DNA Sequencing, Immunoprecipitation, Sequencing

    Mbo II digest results. Agarose gel showing Mbo II digests of GAA PCR products of FRDA samples. The expected 170bp (5′) and 120bp (3′) undigested GAA-flanking fragments from normal pure GAA repeat expansion FRDA samples are shown in lanes 2, 3, and 4. These band sizes can be seen in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder markers, which are loaded into lanes 1 and 11 of the gel. Lane 5 shows a large Mbo II band of approximately 600bp that was obtained from the positive interrupted GAA repeat sequence from the “NEP” BAC transgenic mouse that contains approximately 500 triplet repeats with the previously determined interrupted sequence of (GAA) 21 (GGAGAA) 5 (GGAGGAGAA) 70 (GAA) n ). In addition for this positive sample, we also identified the expected 5′ flanking band of 170bp, together with a smaller band of less than 100bp that we sequenced and we showed to contain a 27bp deletion in the 3′ flanking region. Lane 6 shows an abnormal band of 200bp representing the 80bp duplication in the 3′ GAA flanking region. Lane 7 shows an abnormal band of approximately 100bp representing the 19bp deletion in the 3′ GAA flanking region. Lanes 8, 9, and 10 contain abnormal bands of approximately 300, 100, and 180bp, respectively, that are likely to contain a region of interrupted GAA repeat sequence within the body of one or other of the large FRDA GAA repeat expansions.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare

    doi: 10.3389/fncel.2018.00443

    Figure Lengend Snippet: Mbo II digest results. Agarose gel showing Mbo II digests of GAA PCR products of FRDA samples. The expected 170bp (5′) and 120bp (3′) undigested GAA-flanking fragments from normal pure GAA repeat expansion FRDA samples are shown in lanes 2, 3, and 4. These band sizes can be seen in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder markers, which are loaded into lanes 1 and 11 of the gel. Lane 5 shows a large Mbo II band of approximately 600bp that was obtained from the positive interrupted GAA repeat sequence from the “NEP” BAC transgenic mouse that contains approximately 500 triplet repeats with the previously determined interrupted sequence of (GAA) 21 (GGAGAA) 5 (GGAGGAGAA) 70 (GAA) n ). In addition for this positive sample, we also identified the expected 5′ flanking band of 170bp, together with a smaller band of less than 100bp that we sequenced and we showed to contain a 27bp deletion in the 3′ flanking region. Lane 6 shows an abnormal band of 200bp representing the 80bp duplication in the 3′ GAA flanking region. Lane 7 shows an abnormal band of approximately 100bp representing the 19bp deletion in the 3′ GAA flanking region. Lanes 8, 9, and 10 contain abnormal bands of approximately 300, 100, and 180bp, respectively, that are likely to contain a region of interrupted GAA repeat sequence within the body of one or other of the large FRDA GAA repeat expansions.

    Article Snippet: We then performed long-range PCR of the samples (approximately 100 ng input DNA) using either the Expand High Fidelity PCR System, dNTPack (Roche), or the Long Range PCR Kit (Qiagen) together with GAA-B-F (5′-AATGGATTTCCTGGCAGGACGC-3′) and GAA-B-R (5′-GCATTGGGCGATCTTGGCTTAA-3′) primers as previously described ( ).

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Sequencing, BAC Assay, Transgenic Assay

    Mbo II digests of GAA repeat expansions from human FRDA somatic tissues and mouse FRDA intergenerational and somatic tissues. Agarose gels showing Mbo II digests of GAA PCR products of (A) FRDA patient cerebellum tissue samples, (B) YG8sR mouse ear biopsy samples and human FRDA blood samples, and (C) four tissues from one YG8sR mouse. In each case, the expected 170 and 120bp undigested GAA-flanking fragments can be identified in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder marker, which is loaded into the first lane of each gel. (A) Lanes 1–3 show the results from cerebellum tissue samples from three FRDA patients. (B) Lanes 1 and 2 are from FRDA patient blood samples; lanes 3–6 are from ear biopsy samples from 4 GAA repeat expansion-based YG8sR mice of four different generations, and lane 7 is from an ear biopsy sample from the Y47R mouse which has nine GAA repeats. (C) Lanes 1–4 are from brain, cerebellum, heart, and liver tissues of the YG8sR mouse, respectively.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare

    doi: 10.3389/fncel.2018.00443

    Figure Lengend Snippet: Mbo II digests of GAA repeat expansions from human FRDA somatic tissues and mouse FRDA intergenerational and somatic tissues. Agarose gels showing Mbo II digests of GAA PCR products of (A) FRDA patient cerebellum tissue samples, (B) YG8sR mouse ear biopsy samples and human FRDA blood samples, and (C) four tissues from one YG8sR mouse. In each case, the expected 170 and 120bp undigested GAA-flanking fragments can be identified in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder marker, which is loaded into the first lane of each gel. (A) Lanes 1–3 show the results from cerebellum tissue samples from three FRDA patients. (B) Lanes 1 and 2 are from FRDA patient blood samples; lanes 3–6 are from ear biopsy samples from 4 GAA repeat expansion-based YG8sR mice of four different generations, and lane 7 is from an ear biopsy sample from the Y47R mouse which has nine GAA repeats. (C) Lanes 1–4 are from brain, cerebellum, heart, and liver tissues of the YG8sR mouse, respectively.

    Article Snippet: We then performed long-range PCR of the samples (approximately 100 ng input DNA) using either the Expand High Fidelity PCR System, dNTPack (Roche), or the Long Range PCR Kit (Qiagen) together with GAA-B-F (5′-AATGGATTTCCTGGCAGGACGC-3′) and GAA-B-R (5′-GCATTGGGCGATCTTGGCTTAA-3′) primers as previously described ( ).

    Techniques: Polymerase Chain Reaction, Marker, Mouse Assay

    Optimization of virial transduction conditions for human MDA-MB-231 cells ( A ) Schematic representation of the non-invasive apoptosis detection sensor (NIADS). The fusion proteins of pepA-Nluc and pepB-Cluc fragment were linked with 3Xcaspase-3 cleavage sequences (DEVD). Once the cell is undergoing the apoptosis event, activated caspase-3 recognizes the DEVD sequence and cuts the fusion protein into two fragments. The proteins of pepA and pepB are known to have strong association force and therefore enable reconstitution of full-length luciferase. The bioluminescence activity can be further detected with substrate (Luciferin) addition; ( B ) NIADS DNA sequence was cloned into lentivirus plasmid and generated virus particles in medium. The virus titration from 10–40 μL of both RFP (red fluorescent protein) or NIADS was added in the culture medium of luciferase stable expressing MDA-MB-231 cells for three days. The cells were harvested and immunoblotted to luciferase antibody, whereas the full length of firefly luciferase was determined as protein loading control; ( C ) The virus titration from 10 to 40 μL RFP virus-containing medium was measured on MDA-MB-231 cells for three days. The image of RFP expression was captured under florescence microscopy. Scale bar: 50 μM; ( D ) Absolute qPCR analysis was measured by serial copy number of GUS (β-glucuronidase)-containing TA vector from a range of 10 2 (purple line) to 108 (blue line)/μL as a standard curve. The gene amplification curves were also determined for standard error and gene amplification efficiency. The qPCR efficiency analysis for standard curve is calculated by the formula of Efficiency = 10 −1/slope , whereas qPCR unexplained variance of R 2 value was calculated by the formula of R 2 = 1 − Error; ( E ) The purified and concentrated RFP and NIADS lentivirus were determined as virus copy number/μL by absolute qPCR analysis. Data is presented as the mean and standard error; ( F ) The purified and concentrated RFP lentivirus were measured as virus copy number by qPCR analysis. MDA-MB-231 cells were seeded in a 6-cm dish and infected with 6-, 12-, 30-, 60-, 120-, 240-fold concentrations of virus to MDA-MB-231 cell number for three days. The RFP-positive (infected) cell population was assessed using flow cytometry; ( G ) Linear curve comparison of virus input and the RFP-positive cell population; ( H ) The MOI (240-fold as MOI = 1, multiplicity of infection) from 0.1 to 6 of NIADS contain lentivirus were used on MDA-MB-231 cell. The NIADS fusion protein was immunoblotted by luciferase antibody.

    Journal: International Journal of Molecular Sciences

    Article Title: The Application of Non-Invasive Apoptosis Detection Sensor (NIADS) on Histone Deacetylation Inhibitor (HDACi)-Induced Breast Cancer Cell Death

    doi: 10.3390/ijms19020452

    Figure Lengend Snippet: Optimization of virial transduction conditions for human MDA-MB-231 cells ( A ) Schematic representation of the non-invasive apoptosis detection sensor (NIADS). The fusion proteins of pepA-Nluc and pepB-Cluc fragment were linked with 3Xcaspase-3 cleavage sequences (DEVD). Once the cell is undergoing the apoptosis event, activated caspase-3 recognizes the DEVD sequence and cuts the fusion protein into two fragments. The proteins of pepA and pepB are known to have strong association force and therefore enable reconstitution of full-length luciferase. The bioluminescence activity can be further detected with substrate (Luciferin) addition; ( B ) NIADS DNA sequence was cloned into lentivirus plasmid and generated virus particles in medium. The virus titration from 10–40 μL of both RFP (red fluorescent protein) or NIADS was added in the culture medium of luciferase stable expressing MDA-MB-231 cells for three days. The cells were harvested and immunoblotted to luciferase antibody, whereas the full length of firefly luciferase was determined as protein loading control; ( C ) The virus titration from 10 to 40 μL RFP virus-containing medium was measured on MDA-MB-231 cells for three days. The image of RFP expression was captured under florescence microscopy. Scale bar: 50 μM; ( D ) Absolute qPCR analysis was measured by serial copy number of GUS (β-glucuronidase)-containing TA vector from a range of 10 2 (purple line) to 108 (blue line)/μL as a standard curve. The gene amplification curves were also determined for standard error and gene amplification efficiency. The qPCR efficiency analysis for standard curve is calculated by the formula of Efficiency = 10 −1/slope , whereas qPCR unexplained variance of R 2 value was calculated by the formula of R 2 = 1 − Error; ( E ) The purified and concentrated RFP and NIADS lentivirus were determined as virus copy number/μL by absolute qPCR analysis. Data is presented as the mean and standard error; ( F ) The purified and concentrated RFP lentivirus were measured as virus copy number by qPCR analysis. MDA-MB-231 cells were seeded in a 6-cm dish and infected with 6-, 12-, 30-, 60-, 120-, 240-fold concentrations of virus to MDA-MB-231 cell number for three days. The RFP-positive (infected) cell population was assessed using flow cytometry; ( G ) Linear curve comparison of virus input and the RFP-positive cell population; ( H ) The MOI (240-fold as MOI = 1, multiplicity of infection) from 0.1 to 6 of NIADS contain lentivirus were used on MDA-MB-231 cell. The NIADS fusion protein was immunoblotted by luciferase antibody.

    Article Snippet: Luciferase gene expression from the qPCR analysis was normalized to GUS (β-glucuronidase) expression as an indicator of DNA input using the built-in Roche LightCycler Software, version 4 (Roche Molecular Biochemicals, Mannheim, German).

    Techniques: Transduction, Multiple Displacement Amplification, Sequencing, Luciferase, Activity Assay, Clone Assay, Plasmid Preparation, Generated, Titration, Expressing, Microscopy, Real-time Polymerase Chain Reaction, Amplification, Purification, Infection, Flow Cytometry, Cytometry