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Agilent technologies mouse microarrays
Mouse Microarrays, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 81/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Labeling:

Article Title: Fine mapping of regulatory loci for mammalian gene expression using radiation hybrids
Article Snippet: .. We labeled the samples and applied them to two mouse microarrays (Agilent) differing by a dye-swap ( ) . .. Labeled RNA from A23 cells served as the control channel.

Microarray:

Article Title: Transcriptional profiling of the acute pulmonary inflammatory response induced by LPS: role of neutrophils
Article Snippet: Paragraph title: RNA labelling and microarray hybridisation ... Dye incorporation rates were used to put appropriate amounts of Cy5 and Cy3 labelled samples (10 pmol each) together for hybridisation on Agilent 4× 44 K Mouse Microarrays (Agilent Technologies).

Generated:

Article Title: Transcriptional profiling of the acute pulmonary inflammatory response induced by LPS: role of neutrophils
Article Snippet: RNA labelling and microarray hybridisation Cyanine labelled cRNA was generated by using the Two-Colour Microarray based Gene Expression Analysis kit from Agilent Technologies. .. Dye incorporation rates were used to put appropriate amounts of Cy5 and Cy3 labelled samples (10 pmol each) together for hybridisation on Agilent 4× 44 K Mouse Microarrays (Agilent Technologies).

Expressing:

Article Title: Transcriptional profiling of the acute pulmonary inflammatory response induced by LPS: role of neutrophils
Article Snippet: RNA labelling and microarray hybridisation Cyanine labelled cRNA was generated by using the Two-Colour Microarray based Gene Expression Analysis kit from Agilent Technologies. .. Dye incorporation rates were used to put appropriate amounts of Cy5 and Cy3 labelled samples (10 pmol each) together for hybridisation on Agilent 4× 44 K Mouse Microarrays (Agilent Technologies).

Hybridization:

Article Title: Transcriptional profiling of the acute pulmonary inflammatory response induced by LPS: role of neutrophils
Article Snippet: .. Dye incorporation rates were used to put appropriate amounts of Cy5 and Cy3 labelled samples (10 pmol each) together for hybridisation on Agilent 4× 44 K Mouse Microarrays (Agilent Technologies). .. After hybridisation, slides were washed and dried with N2 gas before scanning.

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    Agilent technologies mecp2 ko
    High affinity of <t>MeCP2</t> for mCG, mCA and hmCA in electrophoretic mobility shift assays a, Binding of the recombinant methyl-binding domain (MBD) of MeCP2 (amino acids 81–170) to 32 P-end-labeled oligonucleotides containing a methylated cytosine in a CA (left) or a CG (right) context competed with unlabeled competitor substituted with unmethylated, methylated, or hydroxymethylated cytosine in a CG or CA context (indicated in bold). Representative full gels showing shifted and unshifted probe in the presence of 50-fold excess of unlabeled competitor (top); close-up of shifted bands over a range of unlabeled competitor (bottom). A mCA-containing oligonucleotide competes for MeCP2 binding with equal or higher efficacy to that of a symmetrically methylated CG oligonucleotide. While hmCG-containing probes compete with similar efficacy to an unmethylated probe, a hmCA-containing probe competes with high efficacy. This difference in affinity of MeCP2 for hmCA- and hmCG-containing probes may explain conflicting results reported for the affinity of MeCP2 for hydroxymethylated DNA 18 , 50 – 53 ( Supplementary Discussion ). b, Binding and competition of recombinant MeCP2 MBD (amino acids 78–162, left) or full-length MeCP2 (amino acids 1–486, right) incubated with 32 P-end-labeled oligonucleotides containing a methylated cytosine in a CA context and competed with oligonucleotides containing unmethylated, methylated, or hydroxymethylated cytosine in a CG, CA, CT, or CC context. Representative full gels showing 100-fold excess of unlabeled competitor (top); close-up of shifted bands over a range of unlabeled competitor (bottom). The results obtained from competitors containing mCG, mCA, hmCG and hmCA are similar to those shown in a . In addition, both (h)mCT- and (h)mCC-containing oligonucleotides compete for MeCP2 binding with similar efficacy to that of an unmethylated probe.
    Mecp2 Ko, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 83/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies tet mev 1 mice
    CC chemokine expression in liver tissues from mice fed normal chow or high-fat/high-sucrose diet. Total RNA was extracted from liver tissues of male wild type or <t>Tet-mev-1</t> mice ( +ROS ) that had been supplied with doxycycline-containing water and were subsequently fed a control, normal diet or high-fat/high-sucrose diet ( +HFHSD ) for 4 months at around the age of either 1 ( left ) or 2 years ( right ). The gene expression levels of CCL2 ( A ), CCL3 ( B ), CCL4 ( C ), CCL5 ( D ), CCL8 ( E ) and CCL12 ( F ) were quantified using specific primers. The values are expressed as means ± SD from four to five mice per group. The asterisks indicate that the differences between the groups are statistically significant (* p
    Tet Mev 1 Mice, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 87/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies gal 9 stimulation
    Real-time RT-PCR confirms downregulation of genes involved in the immune system in human NK cells treated with <t>Gal-9.</t> We used PCR to verify the 6 genes shown to be maximally differentially expressed (greater than 7-fold change) in the microarray analysis.
    Gal 9 Stimulation, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies hg treated podocytes
    Schematic depicting Sirt6 deficiency exacerbates <t>podocyte</t> injuries and proteinuria through targeting N otch signaling. Under normal conditions, SIRT6 inhibits the transcription of Notch1 and Notch4 genes by decreasing H3K9ac levels in the promoter region of Notch1 and Notch4 . Under pathological conditions, SIRT6 reduction leads to the increased levels of H3K9ac in the promoters of Notch1 and Notch4 , thereby enhancing the transcription of Notch1 and Notch4 gene. The activation of Notch signaling finally results in podocyte injury through inducing inflammation, apoptosis, actin cytoskeleton derangement, as well as the inhibition of autophagy
    Hg Treated Podocytes, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 82/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    High affinity of MeCP2 for mCG, mCA and hmCA in electrophoretic mobility shift assays a, Binding of the recombinant methyl-binding domain (MBD) of MeCP2 (amino acids 81–170) to 32 P-end-labeled oligonucleotides containing a methylated cytosine in a CA (left) or a CG (right) context competed with unlabeled competitor substituted with unmethylated, methylated, or hydroxymethylated cytosine in a CG or CA context (indicated in bold). Representative full gels showing shifted and unshifted probe in the presence of 50-fold excess of unlabeled competitor (top); close-up of shifted bands over a range of unlabeled competitor (bottom). A mCA-containing oligonucleotide competes for MeCP2 binding with equal or higher efficacy to that of a symmetrically methylated CG oligonucleotide. While hmCG-containing probes compete with similar efficacy to an unmethylated probe, a hmCA-containing probe competes with high efficacy. This difference in affinity of MeCP2 for hmCA- and hmCG-containing probes may explain conflicting results reported for the affinity of MeCP2 for hydroxymethylated DNA 18 , 50 – 53 ( Supplementary Discussion ). b, Binding and competition of recombinant MeCP2 MBD (amino acids 78–162, left) or full-length MeCP2 (amino acids 1–486, right) incubated with 32 P-end-labeled oligonucleotides containing a methylated cytosine in a CA context and competed with oligonucleotides containing unmethylated, methylated, or hydroxymethylated cytosine in a CG, CA, CT, or CC context. Representative full gels showing 100-fold excess of unlabeled competitor (top); close-up of shifted bands over a range of unlabeled competitor (bottom). The results obtained from competitors containing mCG, mCA, hmCG and hmCA are similar to those shown in a . In addition, both (h)mCT- and (h)mCC-containing oligonucleotides compete for MeCP2 binding with similar efficacy to that of an unmethylated probe.

    Journal: Nature

    Article Title: Disruption of DNA methylation-dependent long gene repression in Rett syndrome

    doi: 10.1038/nature14319

    Figure Lengend Snippet: High affinity of MeCP2 for mCG, mCA and hmCA in electrophoretic mobility shift assays a, Binding of the recombinant methyl-binding domain (MBD) of MeCP2 (amino acids 81–170) to 32 P-end-labeled oligonucleotides containing a methylated cytosine in a CA (left) or a CG (right) context competed with unlabeled competitor substituted with unmethylated, methylated, or hydroxymethylated cytosine in a CG or CA context (indicated in bold). Representative full gels showing shifted and unshifted probe in the presence of 50-fold excess of unlabeled competitor (top); close-up of shifted bands over a range of unlabeled competitor (bottom). A mCA-containing oligonucleotide competes for MeCP2 binding with equal or higher efficacy to that of a symmetrically methylated CG oligonucleotide. While hmCG-containing probes compete with similar efficacy to an unmethylated probe, a hmCA-containing probe competes with high efficacy. This difference in affinity of MeCP2 for hmCA- and hmCG-containing probes may explain conflicting results reported for the affinity of MeCP2 for hydroxymethylated DNA 18 , 50 – 53 ( Supplementary Discussion ). b, Binding and competition of recombinant MeCP2 MBD (amino acids 78–162, left) or full-length MeCP2 (amino acids 1–486, right) incubated with 32 P-end-labeled oligonucleotides containing a methylated cytosine in a CA context and competed with oligonucleotides containing unmethylated, methylated, or hydroxymethylated cytosine in a CG, CA, CT, or CC context. Representative full gels showing 100-fold excess of unlabeled competitor (top); close-up of shifted bands over a range of unlabeled competitor (bottom). The results obtained from competitors containing mCG, mCA, hmCG and hmCA are similar to those shown in a . In addition, both (h)mCT- and (h)mCC-containing oligonucleotides compete for MeCP2 binding with similar efficacy to that of an unmethylated probe.

    Article Snippet: To analyze gene expression genome-wide with respect to gene length, CEL files containing the raw hybridization data in from multiple MeCP2 KO and MeCP2 OE gene expression studies were downloaded from GEO ( http://www.ncbi.nlm.nih.gov/geo/ ; study details, sample numbers and genotypes are provided in ) and analyzed for expression at the gene level using using the GeneSpring software suite (Agilent Technologies) with RMA summarization of “Core” probesets.

    Techniques: Electrophoretic Mobility Shift Assay, Binding Assay, Recombinant, Labeling, Methylation, Incubation

    Disruption of Dnmt3a in the brain leads to length-dependent up-regulation of genes containing high levels of mCA a, Summary of genome-wide bisulfite-sequencing analysis of mCN (where N = G, A, T, or C) in control and Dnmt3a cKO cerebella ( n =2 per genotype). Dashed line represents mean background non-conversion rate of the bisulfite-seq assay (see Methods). b, Mean fold-change in gene expression versus gene-body mCA for MeCP2 KO (left) or Dnmt3a cKO (right) cerebella. Long (top 25%, > 60kb) and short (bottom 25%,

    Journal: Nature

    Article Title: Disruption of DNA methylation-dependent long gene repression in Rett syndrome

    doi: 10.1038/nature14319

    Figure Lengend Snippet: Disruption of Dnmt3a in the brain leads to length-dependent up-regulation of genes containing high levels of mCA a, Summary of genome-wide bisulfite-sequencing analysis of mCN (where N = G, A, T, or C) in control and Dnmt3a cKO cerebella ( n =2 per genotype). Dashed line represents mean background non-conversion rate of the bisulfite-seq assay (see Methods). b, Mean fold-change in gene expression versus gene-body mCA for MeCP2 KO (left) or Dnmt3a cKO (right) cerebella. Long (top 25%, > 60kb) and short (bottom 25%,

    Article Snippet: To analyze gene expression genome-wide with respect to gene length, CEL files containing the raw hybridization data in from multiple MeCP2 KO and MeCP2 OE gene expression studies were downloaded from GEO ( http://www.ncbi.nlm.nih.gov/geo/ ; study details, sample numbers and genotypes are provided in ) and analyzed for expression at the gene level using using the GeneSpring software suite (Agilent Technologies) with RMA summarization of “Core” probesets.

    Techniques: Genome Wide, Methylation Sequencing, Bisulfite Sequencing, Expressing

    Analysis of gene expression changes in Mecp2 mutant mice a, Heatmap of median gene lengths for genes identified as misregulated in Mecp2 mutant studies or sixteen different studies of neurological dysfunction and disease in mice. Mouse model and GEO accession number, or reference, are listed (for Strand et al. (1), 3NP treatment; (2), Human HD brain; (3), R2/6 Htt transgenic). b, Scatter plots of fold-change in gene expression in the MeCP2 KO for the amygdala (left), which shows robust length-dependent misregulation, and the liver (right), which does not. Fold-change values for genes (black points) and mean fold-change for 200 genes per bin with a 40 gene step are shown (mean, red line; ribbon, S.E.M.). c, The fraction of genes showing fold-change > 0 for datasets in b ; genes binned by length (100 gene bins, 50 gene step). d–f , Analysis of published microarray 5 – 9 ( d, e ) or RNA sequencing (RNA-seq) 18 ( f ) datasets from MeCP2 KO ( d, f ) or OE ( e ) mice. Mean fold-change in expression (200 gene bins, 40 gene step), red line; ribbon, S.E.M. For d–f , mean (black line) and two standard deviations (gray ribbon) are shown for 10,000 resamplings in which gene lengths were randomized with respect to fold-change. The spike in mean fold-change at ~1 kb in several plots corresponds to the olfactory receptor genes ( Supplementary Discussion ). g, Mean changes in expression of genes binned by length from RNA-seq analysis of MeCP2 KO cortex ( n = 3 per genotype). h, Mean changes in expression from microarray analysis of genes binned by length in MeCP2 R306C cerebellum ( n = 4 per genotype) i, Heatmap summary of fold-changes in gene expression from RNA-seq analysis of Mecp2 mutant mean in g compared to Nanostring nCounter (18 genes, top) or RT-qPCR (17 genes, bottom) analysis from cortex ( n =4 per genotype). Selected long genes ( > 100 kb) consistently up-regulated in the MeCP2 KO or down-regulated in MeCP2 OE mutant mice across brain tissues were tested ( Supplementary Table 2 ). A statistically significant up-regulation of these genes is observed in the cortex for both MeCP2 KO (nCounter, p = 0.00073; qPCR, p

    Journal: Nature

    Article Title: Disruption of DNA methylation-dependent long gene repression in Rett syndrome

    doi: 10.1038/nature14319

    Figure Lengend Snippet: Analysis of gene expression changes in Mecp2 mutant mice a, Heatmap of median gene lengths for genes identified as misregulated in Mecp2 mutant studies or sixteen different studies of neurological dysfunction and disease in mice. Mouse model and GEO accession number, or reference, are listed (for Strand et al. (1), 3NP treatment; (2), Human HD brain; (3), R2/6 Htt transgenic). b, Scatter plots of fold-change in gene expression in the MeCP2 KO for the amygdala (left), which shows robust length-dependent misregulation, and the liver (right), which does not. Fold-change values for genes (black points) and mean fold-change for 200 genes per bin with a 40 gene step are shown (mean, red line; ribbon, S.E.M.). c, The fraction of genes showing fold-change > 0 for datasets in b ; genes binned by length (100 gene bins, 50 gene step). d–f , Analysis of published microarray 5 – 9 ( d, e ) or RNA sequencing (RNA-seq) 18 ( f ) datasets from MeCP2 KO ( d, f ) or OE ( e ) mice. Mean fold-change in expression (200 gene bins, 40 gene step), red line; ribbon, S.E.M. For d–f , mean (black line) and two standard deviations (gray ribbon) are shown for 10,000 resamplings in which gene lengths were randomized with respect to fold-change. The spike in mean fold-change at ~1 kb in several plots corresponds to the olfactory receptor genes ( Supplementary Discussion ). g, Mean changes in expression of genes binned by length from RNA-seq analysis of MeCP2 KO cortex ( n = 3 per genotype). h, Mean changes in expression from microarray analysis of genes binned by length in MeCP2 R306C cerebellum ( n = 4 per genotype) i, Heatmap summary of fold-changes in gene expression from RNA-seq analysis of Mecp2 mutant mean in g compared to Nanostring nCounter (18 genes, top) or RT-qPCR (17 genes, bottom) analysis from cortex ( n =4 per genotype). Selected long genes ( > 100 kb) consistently up-regulated in the MeCP2 KO or down-regulated in MeCP2 OE mutant mice across brain tissues were tested ( Supplementary Table 2 ). A statistically significant up-regulation of these genes is observed in the cortex for both MeCP2 KO (nCounter, p = 0.00073; qPCR, p

    Article Snippet: To analyze gene expression genome-wide with respect to gene length, CEL files containing the raw hybridization data in from multiple MeCP2 KO and MeCP2 OE gene expression studies were downloaded from GEO ( http://www.ncbi.nlm.nih.gov/geo/ ; study details, sample numbers and genotypes are provided in ) and analyzed for expression at the gene level using using the GeneSpring software suite (Agilent Technologies) with RMA summarization of “Core” probesets.

    Techniques: Expressing, Mutagenesis, Mouse Assay, Transgenic Assay, Microarray, RNA Sequencing Assay, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    Timing and severity of gene expression changes in models of RTT a, Mean fold-change in gene expression versus gene length in the hippocampus of MeCP2 KO mice compared to wild type at four and nine weeks of age reveals increasing magnitude of length-dependent gene misregulation that parallels the onset of RTT-like symptoms in these animals 8 . b , Mean fold-change in gene expression versus gene length in hippocampus of mice expressing truncated forms of MeCP2 mimicking human disease-causing alleles at four weeks of age. Re-expression of a longer truncated form of MeCP2 (G273X) in the MeCP2 KO normalizes expression of long genes more effectively than expression of a shorter truncation of MeCP2 (R270X), and parallels the higher degree of phenotypic rescue observed in MeCP2 G273X-expressing mice compared to MeCP2 R270X-expressing mice 8 . c , Mean fold-change in gene expression versus gene length in hippocampus of mice expressing truncated MeCP2 at nine weeks of age. Consistent with the eventual onset of symptoms of these mouse strains, length-dependent gene misregulation is evident in both strains. d , Changes in gene expression for genes binned by length in human MECP2 null ES cells differentiated into neural progenitor cells, neurons cultured for 2 weeks, or neurons cultured for 4 weeks 15 . In all plots, lines represent mean fold-change in expression for each bin (200 gene bins, 40 gene step), and the ribbon is S.E.M. of genes within each bin.

    Journal: Nature

    Article Title: Disruption of DNA methylation-dependent long gene repression in Rett syndrome

    doi: 10.1038/nature14319

    Figure Lengend Snippet: Timing and severity of gene expression changes in models of RTT a, Mean fold-change in gene expression versus gene length in the hippocampus of MeCP2 KO mice compared to wild type at four and nine weeks of age reveals increasing magnitude of length-dependent gene misregulation that parallels the onset of RTT-like symptoms in these animals 8 . b , Mean fold-change in gene expression versus gene length in hippocampus of mice expressing truncated forms of MeCP2 mimicking human disease-causing alleles at four weeks of age. Re-expression of a longer truncated form of MeCP2 (G273X) in the MeCP2 KO normalizes expression of long genes more effectively than expression of a shorter truncation of MeCP2 (R270X), and parallels the higher degree of phenotypic rescue observed in MeCP2 G273X-expressing mice compared to MeCP2 R270X-expressing mice 8 . c , Mean fold-change in gene expression versus gene length in hippocampus of mice expressing truncated MeCP2 at nine weeks of age. Consistent with the eventual onset of symptoms of these mouse strains, length-dependent gene misregulation is evident in both strains. d , Changes in gene expression for genes binned by length in human MECP2 null ES cells differentiated into neural progenitor cells, neurons cultured for 2 weeks, or neurons cultured for 4 weeks 15 . In all plots, lines represent mean fold-change in expression for each bin (200 gene bins, 40 gene step), and the ribbon is S.E.M. of genes within each bin.

    Article Snippet: To analyze gene expression genome-wide with respect to gene length, CEL files containing the raw hybridization data in from multiple MeCP2 KO and MeCP2 OE gene expression studies were downloaded from GEO ( http://www.ncbi.nlm.nih.gov/geo/ ; study details, sample numbers and genotypes are provided in ) and analyzed for expression at the gene level using using the GeneSpring software suite (Agilent Technologies) with RMA summarization of “Core” probesets.

    Techniques: Expressing, Mouse Assay, Cell Culture

    Analysis of MeCP2-repressed genes and FMRP target genes a, Mean fold-change in mRNA expression for examples of MeCP2-repressed genes across three different Mecp2 mutant genotypes (KO, OE, and R306C) and six brain regions. p-values for each gene are derived from the mean z -scores for fold-change across all datasets (see Methods). b , Gene expression and CA methylation data from the cerebellum for selected MeCP2-repressed genes from a (right), as well as examples of extremely long genes ( > 100kb) that are not enriched for mCA and are not misregulated (left). Fold-changes in mRNA expression in Mecp2 mutants and the Dnmt3a cKO are shown (left axis), as well as mean mCA levels (gray; right axis). Red line indicates genomic median for gene body mCA/CA c, Boxplots of mCA levels in MeCP2-repressed genes compared to all genes. d, Mean fold-change for MeCP2-repressed genes in eight “training datasets” used to define these genes (see Methods), and nine “test datasets”: three Mecp2 mutant datasets not used to define MeCP2-repressed genes (CTX MeCP2 KO and CB MeCP2 R306C, generated in this study; HC MeCP2 KO 4wk, analyzed from Baker et al. 8 ), and six datasets from brains of mouse models of neurological dysfunction generated using the same microarray platforms as the MeCP2 datasets (Geo accession # in order: GSE22115, GSE27088, GSE43051, GSE47706, GSE44855, GSE52584). Error bars are SEM of MeCP2–repressed gene expression across samples (n=4–8 microarrays per genotype per dataset); ** p

    Journal: Nature

    Article Title: Disruption of DNA methylation-dependent long gene repression in Rett syndrome

    doi: 10.1038/nature14319

    Figure Lengend Snippet: Analysis of MeCP2-repressed genes and FMRP target genes a, Mean fold-change in mRNA expression for examples of MeCP2-repressed genes across three different Mecp2 mutant genotypes (KO, OE, and R306C) and six brain regions. p-values for each gene are derived from the mean z -scores for fold-change across all datasets (see Methods). b , Gene expression and CA methylation data from the cerebellum for selected MeCP2-repressed genes from a (right), as well as examples of extremely long genes ( > 100kb) that are not enriched for mCA and are not misregulated (left). Fold-changes in mRNA expression in Mecp2 mutants and the Dnmt3a cKO are shown (left axis), as well as mean mCA levels (gray; right axis). Red line indicates genomic median for gene body mCA/CA c, Boxplots of mCA levels in MeCP2-repressed genes compared to all genes. d, Mean fold-change for MeCP2-repressed genes in eight “training datasets” used to define these genes (see Methods), and nine “test datasets”: three Mecp2 mutant datasets not used to define MeCP2-repressed genes (CTX MeCP2 KO and CB MeCP2 R306C, generated in this study; HC MeCP2 KO 4wk, analyzed from Baker et al. 8 ), and six datasets from brains of mouse models of neurological dysfunction generated using the same microarray platforms as the MeCP2 datasets (Geo accession # in order: GSE22115, GSE27088, GSE43051, GSE47706, GSE44855, GSE52584). Error bars are SEM of MeCP2–repressed gene expression across samples (n=4–8 microarrays per genotype per dataset); ** p

    Article Snippet: To analyze gene expression genome-wide with respect to gene length, CEL files containing the raw hybridization data in from multiple MeCP2 KO and MeCP2 OE gene expression studies were downloaded from GEO ( http://www.ncbi.nlm.nih.gov/geo/ ; study details, sample numbers and genotypes are provided in ) and analyzed for expression at the gene level using using the GeneSpring software suite (Agilent Technologies) with RMA summarization of “Core” probesets.

    Techniques: Expressing, Mutagenesis, Derivative Assay, Methylation, Generated, Microarray

    MeCP2 represses long genes containing high levels of mCA a, EMSA analysis of the MeCP2 methyl-binding domain (amino acids 78–162) binding to 32 P-end-labeled mCA-containing DNA probe incubated with 100-fold excess of unlabeled competitor oligonucleotides containing unmodified, methylated, or hydroxymethylated cytosines at the dinucleotides indicated in bold; no competitor indicated by “−” (see Methods, Extended Data Fig. 3 ). b, Boxplots of MeCP2 ChIP-seq read density within genes > 100 kb plotted by quartile of mCA/CA in the cortex and cerebellum. c, Mean fold-change in gene expression binned according to gene length in MeCP2 KO cortical tissue for genes with high (mCA/CA > 0.034, top 25%) and low (mCA/CA

    Journal: Nature

    Article Title: Disruption of DNA methylation-dependent long gene repression in Rett syndrome

    doi: 10.1038/nature14319

    Figure Lengend Snippet: MeCP2 represses long genes containing high levels of mCA a, EMSA analysis of the MeCP2 methyl-binding domain (amino acids 78–162) binding to 32 P-end-labeled mCA-containing DNA probe incubated with 100-fold excess of unlabeled competitor oligonucleotides containing unmodified, methylated, or hydroxymethylated cytosines at the dinucleotides indicated in bold; no competitor indicated by “−” (see Methods, Extended Data Fig. 3 ). b, Boxplots of MeCP2 ChIP-seq read density within genes > 100 kb plotted by quartile of mCA/CA in the cortex and cerebellum. c, Mean fold-change in gene expression binned according to gene length in MeCP2 KO cortical tissue for genes with high (mCA/CA > 0.034, top 25%) and low (mCA/CA

    Article Snippet: To analyze gene expression genome-wide with respect to gene length, CEL files containing the raw hybridization data in from multiple MeCP2 KO and MeCP2 OE gene expression studies were downloaded from GEO ( http://www.ncbi.nlm.nih.gov/geo/ ; study details, sample numbers and genotypes are provided in ) and analyzed for expression at the gene level using using the GeneSpring software suite (Agilent Technologies) with RMA summarization of “Core” probesets.

    Techniques: Binding Assay, Labeling, Incubation, Methylation, Chromatin Immunoprecipitation, Expressing

    Consequences of long gene misregulation in neurons a , Mean expression of genes binned according to length in human neural and non-neural tissues. Mean expression for genes within each bin (200 gene bins, 40 gene step) is indicated by the line; ribbon represents the S.E.M. of genes within each bin. b , Western blot analysis of MeCP2 from primary cortical neurons after control or MeCP2 shRNA knockdown (KD) and treatment with DMSO vehicle (−) or topotecan (+). c, Heatmap summary of nCounter analysis for the expression of selected MeCP2-repressed (MR) genes from primary neurons treated with control or MeCP2 shRNA and topotecan (n = 3–4). Normalized log2 fold-change relative to the DMSO-treated, control KD is shown. MeCP2 KD conditions are significantly different from control, (p = 1e-4, repeated measures ANOVA across 8 genes). Newman-Keuls corrected, post-hoc comparisons: p

    Journal: Nature

    Article Title: Disruption of DNA methylation-dependent long gene repression in Rett syndrome

    doi: 10.1038/nature14319

    Figure Lengend Snippet: Consequences of long gene misregulation in neurons a , Mean expression of genes binned according to length in human neural and non-neural tissues. Mean expression for genes within each bin (200 gene bins, 40 gene step) is indicated by the line; ribbon represents the S.E.M. of genes within each bin. b , Western blot analysis of MeCP2 from primary cortical neurons after control or MeCP2 shRNA knockdown (KD) and treatment with DMSO vehicle (−) or topotecan (+). c, Heatmap summary of nCounter analysis for the expression of selected MeCP2-repressed (MR) genes from primary neurons treated with control or MeCP2 shRNA and topotecan (n = 3–4). Normalized log2 fold-change relative to the DMSO-treated, control KD is shown. MeCP2 KD conditions are significantly different from control, (p = 1e-4, repeated measures ANOVA across 8 genes). Newman-Keuls corrected, post-hoc comparisons: p

    Article Snippet: To analyze gene expression genome-wide with respect to gene length, CEL files containing the raw hybridization data in from multiple MeCP2 KO and MeCP2 OE gene expression studies were downloaded from GEO ( http://www.ncbi.nlm.nih.gov/geo/ ; study details, sample numbers and genotypes are provided in ) and analyzed for expression at the gene level using using the GeneSpring software suite (Agilent Technologies) with RMA summarization of “Core” probesets.

    Techniques: Expressing, Western Blot, shRNA

    Analysis of long gene expression and regulation in the brain a, Cumulative distribution function of gene lengths for all genes in the genome (black), MeCP2-repressed genes (red), and genes encoding putative FMRP target mRNAs 29 (blue); p

    Journal: Nature

    Article Title: Disruption of DNA methylation-dependent long gene repression in Rett syndrome

    doi: 10.1038/nature14319

    Figure Lengend Snippet: Analysis of long gene expression and regulation in the brain a, Cumulative distribution function of gene lengths for all genes in the genome (black), MeCP2-repressed genes (red), and genes encoding putative FMRP target mRNAs 29 (blue); p

    Article Snippet: To analyze gene expression genome-wide with respect to gene length, CEL files containing the raw hybridization data in from multiple MeCP2 KO and MeCP2 OE gene expression studies were downloaded from GEO ( http://www.ncbi.nlm.nih.gov/geo/ ; study details, sample numbers and genotypes are provided in ) and analyzed for expression at the gene level using using the GeneSpring software suite (Agilent Technologies) with RMA summarization of “Core” probesets.

    Techniques: Expressing

    Conditional knockout of Dnmt3a in vivo a , Diagram of the Dnmt3a locus and Cre-dependent conditional knockout strategy for Dnmt3a 26 . LoxP sites (green triangles) flank exon 17, which is removed following Cre -mediated recombination. Primers (purple arrows) were designed to flank exons 17 and 18. The wild-type (WT), floxed (FLX), and knockout (KO) allele are depicted. b , Representative PCR genotyping for tail DNA samples indicates presence or absence of the floxed (flx, ~800 bp), wild-type (WT, ~750 bp), and knockout (KO, ~500 bp) alleles. Separate genotyping reaction for the Nestin-cre transgene (~250 bp) is shown. c , Efficient excision of the floxed exon is detected in cerebellar DNA from conditional knockout ( Dnmt3a flx/flx ; Nestin-Cre +/− , Dnmt3a cKO) mice but not from and control animals ( Dnmt3a flx/flx , Control). d , Western blot analysis of Dnmt3a, MeCP2, and Gapdh (loading control) protein from the cerebellum of control and Dnmt3a cKO adult mice.

    Journal: Nature

    Article Title: Disruption of DNA methylation-dependent long gene repression in Rett syndrome

    doi: 10.1038/nature14319

    Figure Lengend Snippet: Conditional knockout of Dnmt3a in vivo a , Diagram of the Dnmt3a locus and Cre-dependent conditional knockout strategy for Dnmt3a 26 . LoxP sites (green triangles) flank exon 17, which is removed following Cre -mediated recombination. Primers (purple arrows) were designed to flank exons 17 and 18. The wild-type (WT), floxed (FLX), and knockout (KO) allele are depicted. b , Representative PCR genotyping for tail DNA samples indicates presence or absence of the floxed (flx, ~800 bp), wild-type (WT, ~750 bp), and knockout (KO, ~500 bp) alleles. Separate genotyping reaction for the Nestin-cre transgene (~250 bp) is shown. c , Efficient excision of the floxed exon is detected in cerebellar DNA from conditional knockout ( Dnmt3a flx/flx ; Nestin-Cre +/− , Dnmt3a cKO) mice but not from and control animals ( Dnmt3a flx/flx , Control). d , Western blot analysis of Dnmt3a, MeCP2, and Gapdh (loading control) protein from the cerebellum of control and Dnmt3a cKO adult mice.

    Article Snippet: To analyze gene expression genome-wide with respect to gene length, CEL files containing the raw hybridization data in from multiple MeCP2 KO and MeCP2 OE gene expression studies were downloaded from GEO ( http://www.ncbi.nlm.nih.gov/geo/ ; study details, sample numbers and genotypes are provided in ) and analyzed for expression at the gene level using using the GeneSpring software suite (Agilent Technologies) with RMA summarization of “Core” probesets.

    Techniques: Knock-Out, In Vivo, Polymerase Chain Reaction, Mouse Assay, Western Blot

    Genomic analysis of mCG, hmCG, and hmCA in length-dependent gene regulation by MeCP2 a–c, Mean methylation of CG dinucleotides (mCG/CG) within gene bodies (transcription start site +3 kb, up to transcription termination site) in the cortex ( a ), hippocampus ( b ) and cerebellum ( c ) for genes binned according to length. d–f , Mean fold-change in gene expression in MeCP2 KO compared to wild type in the cortex ( d ), hippocampus ( e ), and cerebellum ( f ) for genes binned according to mCG levels (mCG/CG) within gene bodies. g , Mean hmCG levels (hmCG/CG) within gene bodies in the frontal cortex 24 for genes binned according to length. h , Mean fold-change in gene expression in MeCP2 KO compared to wild type for genes binned according to hmCG levels (hmCG/CG) within gene bodies in the frontal cortex 24 i , Mean hmCA levels (hmCA/CA) within gene bodies in the frontal cortex 24 for genes binned according to length. j , Mean fold-change in gene expression in MeCP2 KO compared to wild type genes binned according to hmCA levels (hmCA/CA) within gene bodies in the frontal cortex 24 . In all panels, mean values for each bin are indicated as a line (200 gene bins, 40 gene step); ribbon depicts S.E.M. for genes within each bin.

    Journal: Nature

    Article Title: Disruption of DNA methylation-dependent long gene repression in Rett syndrome

    doi: 10.1038/nature14319

    Figure Lengend Snippet: Genomic analysis of mCG, hmCG, and hmCA in length-dependent gene regulation by MeCP2 a–c, Mean methylation of CG dinucleotides (mCG/CG) within gene bodies (transcription start site +3 kb, up to transcription termination site) in the cortex ( a ), hippocampus ( b ) and cerebellum ( c ) for genes binned according to length. d–f , Mean fold-change in gene expression in MeCP2 KO compared to wild type in the cortex ( d ), hippocampus ( e ), and cerebellum ( f ) for genes binned according to mCG levels (mCG/CG) within gene bodies. g , Mean hmCG levels (hmCG/CG) within gene bodies in the frontal cortex 24 for genes binned according to length. h , Mean fold-change in gene expression in MeCP2 KO compared to wild type for genes binned according to hmCG levels (hmCG/CG) within gene bodies in the frontal cortex 24 i , Mean hmCA levels (hmCA/CA) within gene bodies in the frontal cortex 24 for genes binned according to length. j , Mean fold-change in gene expression in MeCP2 KO compared to wild type genes binned according to hmCA levels (hmCA/CA) within gene bodies in the frontal cortex 24 . In all panels, mean values for each bin are indicated as a line (200 gene bins, 40 gene step); ribbon depicts S.E.M. for genes within each bin.

    Article Snippet: To analyze gene expression genome-wide with respect to gene length, CEL files containing the raw hybridization data in from multiple MeCP2 KO and MeCP2 OE gene expression studies were downloaded from GEO ( http://www.ncbi.nlm.nih.gov/geo/ ; study details, sample numbers and genotypes are provided in ) and analyzed for expression at the gene level using using the GeneSpring software suite (Agilent Technologies) with RMA summarization of “Core” probesets.

    Techniques: Methylation, Expressing

    Length-dependent gene misregulation in Mecp2 mutant mice and human RTT brain a, Boxplots summarizing lengths of genes (Refseq transcription start site to termination site) detected as misregulated in independent studies of Mecp2 mutant mice. HYP, hypothalamus 5 ; CB, cerebellum 6 ; AMG, amygdala 7 ; HC, hippocampus 8 ; STR, striatum 9 ; LVR, liver 9 . MeCP2-induced (blue), genes down-regulated in MeCP2 knockout (MeCP2 KO) and up-regulated in MeCP2 overexpression (MeCP2 OE) mice. MeCP2-repressed (red), genes up-regulated in MeCP2 KO and down-regulated in MeCP2 OE (see Methods). b , Mean expression changes across brain regions and liver of Mecp2 mutant mice for genes ≤100 kb (gray) and > 100 kb (red). c–d, Genome-wide changes in gene expression assessed by RNA-seq analysis of mouse cortical tissue from MeCP2 KO compared to wild type ( c ) or microarray analysis of human RTT brain samples compared to age-matched controls 16 ( d ). In c, d lines represent mean fold-change in expression for genes binned according to gene length (200 gene bins, 40 gene step; see Methods); the ribbon is S.E.M. of each bin. *, p

    Journal: Nature

    Article Title: Disruption of DNA methylation-dependent long gene repression in Rett syndrome

    doi: 10.1038/nature14319

    Figure Lengend Snippet: Length-dependent gene misregulation in Mecp2 mutant mice and human RTT brain a, Boxplots summarizing lengths of genes (Refseq transcription start site to termination site) detected as misregulated in independent studies of Mecp2 mutant mice. HYP, hypothalamus 5 ; CB, cerebellum 6 ; AMG, amygdala 7 ; HC, hippocampus 8 ; STR, striatum 9 ; LVR, liver 9 . MeCP2-induced (blue), genes down-regulated in MeCP2 knockout (MeCP2 KO) and up-regulated in MeCP2 overexpression (MeCP2 OE) mice. MeCP2-repressed (red), genes up-regulated in MeCP2 KO and down-regulated in MeCP2 OE (see Methods). b , Mean expression changes across brain regions and liver of Mecp2 mutant mice for genes ≤100 kb (gray) and > 100 kb (red). c–d, Genome-wide changes in gene expression assessed by RNA-seq analysis of mouse cortical tissue from MeCP2 KO compared to wild type ( c ) or microarray analysis of human RTT brain samples compared to age-matched controls 16 ( d ). In c, d lines represent mean fold-change in expression for genes binned according to gene length (200 gene bins, 40 gene step; see Methods); the ribbon is S.E.M. of each bin. *, p

    Article Snippet: To analyze gene expression genome-wide with respect to gene length, CEL files containing the raw hybridization data in from multiple MeCP2 KO and MeCP2 OE gene expression studies were downloaded from GEO ( http://www.ncbi.nlm.nih.gov/geo/ ; study details, sample numbers and genotypes are provided in ) and analyzed for expression at the gene level using using the GeneSpring software suite (Agilent Technologies) with RMA summarization of “Core” probesets.

    Techniques: Mutagenesis, Mouse Assay, Knock-Out, Over Expression, Expressing, Genome Wide, RNA Sequencing Assay, Microarray

    Genomic analysis supports a role for mCA in length-dependent gene regulation by MeCP2 a–c, Mean methylation at CA dinucleotides (mCA/CA) within gene bodies (TSS +3 kb to TTS) in cortex ( a ), hippocampus ( b ), and cerebellum ( c ) for genes binned by length. d–f, Mean changes in gene expression in cortex ( d ), hippocampus ( e ), and cerebellum ( f ) of MeCP2 KO for high mCA genes (top 25% mean gene body mCA/CA) and low mCA genes (bottom 66% mean gene body mCA/CA) binned by length. g–i , Mean changes in gene expression in cortex ( g ), hippocampus ( h ), and cerebellum ( i ) of MeCP2 KO for genes binned according to average gene body mCA/CA levels. j–l, Mean changes in gene expression in cortex ( j ), hippocampus ( k ), and cerebellum ( l ) of MeCP2 KO mice for long genes (top 25%) and short genes (bottom 25%) in each brain region binned by gene body mCA/CA level. A correlation between fold-change in the MeCP2 KO and mCA/CA for all genes is less prominent, or not observed, in the hippocampus and cerebellum for all genes together ( h , i ), but it is clear for the longest genes in the genome analyzed alone ( k , l ). Note that average levels of mCA appear lower in hippocampus and cerebellum compared to cortex (compare y-axis in a , b and c ), and may explain why a correlation across all genes in not detected in these brain regions. In long genes analyzed alone the cumulative effect of higher mCA levels and integration across the gene may be larger, resulting in a detectable effect. In all panels, the line indicates the mean for 200 gene bins, with a 40 gene step; ribbon depicts S.E.M. for genes within each bin. Note that, for completeness, data from analysis of the cortex presented in Figure 2 are re-presented here.

    Journal: Nature

    Article Title: Disruption of DNA methylation-dependent long gene repression in Rett syndrome

    doi: 10.1038/nature14319

    Figure Lengend Snippet: Genomic analysis supports a role for mCA in length-dependent gene regulation by MeCP2 a–c, Mean methylation at CA dinucleotides (mCA/CA) within gene bodies (TSS +3 kb to TTS) in cortex ( a ), hippocampus ( b ), and cerebellum ( c ) for genes binned by length. d–f, Mean changes in gene expression in cortex ( d ), hippocampus ( e ), and cerebellum ( f ) of MeCP2 KO for high mCA genes (top 25% mean gene body mCA/CA) and low mCA genes (bottom 66% mean gene body mCA/CA) binned by length. g–i , Mean changes in gene expression in cortex ( g ), hippocampus ( h ), and cerebellum ( i ) of MeCP2 KO for genes binned according to average gene body mCA/CA levels. j–l, Mean changes in gene expression in cortex ( j ), hippocampus ( k ), and cerebellum ( l ) of MeCP2 KO mice for long genes (top 25%) and short genes (bottom 25%) in each brain region binned by gene body mCA/CA level. A correlation between fold-change in the MeCP2 KO and mCA/CA for all genes is less prominent, or not observed, in the hippocampus and cerebellum for all genes together ( h , i ), but it is clear for the longest genes in the genome analyzed alone ( k , l ). Note that average levels of mCA appear lower in hippocampus and cerebellum compared to cortex (compare y-axis in a , b and c ), and may explain why a correlation across all genes in not detected in these brain regions. In long genes analyzed alone the cumulative effect of higher mCA levels and integration across the gene may be larger, resulting in a detectable effect. In all panels, the line indicates the mean for 200 gene bins, with a 40 gene step; ribbon depicts S.E.M. for genes within each bin. Note that, for completeness, data from analysis of the cortex presented in Figure 2 are re-presented here.

    Article Snippet: To analyze gene expression genome-wide with respect to gene length, CEL files containing the raw hybridization data in from multiple MeCP2 KO and MeCP2 OE gene expression studies were downloaded from GEO ( http://www.ncbi.nlm.nih.gov/geo/ ; study details, sample numbers and genotypes are provided in ) and analyzed for expression at the gene level using using the GeneSpring software suite (Agilent Technologies) with RMA summarization of “Core” probesets.

    Techniques: Methylation, Expressing, Mouse Assay

    ChIP-seq analysis of MeCP2 binding in vivo a, Boxplots of input-normalized read density within gene bodies (TSS +3 kb to TTS) for MeCP2 ChIP from the mouse frontal cortex plotted for genes according to quartile of mCA/CA, mCG/CG, hmCA/CA and hmCG/CG in the frontal cortex 24 for all genes and genes > 100 kb. b, Similar analysis of MeCP2 ChIP from the mouse cortex (left) or cerebellum (right) plotted for genes according to quartile of mCA/CA or mCG/CG for all genes and genes > 100 kb. MeCP2 ChIP-signal is correlated with mCA/CA levels from the frontal cortex, cortex, and cerebellum for all genes and this correlation is more prominent among genes > 100 kb. mCG does not show as prominent a correlation with MeCP2 ChIP signal, and hmCG trends toward anti-correlation with MeCP2 ChIP. These results suggest that MeCP2 has a lower affinity for hmCG than mCG, suggesting that, in vivo, hmCG is associated with reduced MeCP2 occupancy ( Supplementary Discussion ). c , High resolution analysis of high-coverage bisulfite sequencing data from the frontal cortex showing a correlation between MeCP2 ChIP signal and mCA. Input-normalized ChIP signal plotted for mCA levels for 500 bp bins tiled across all genes. d , Aggregate plots of MeCP2 input-normalized ChIP signal (top) and relative methylation (log2 enrichment in mC as compared to the flanking regions) for mCA, mCC, mCT, and mCG (bottom) are plotted around the 31,479 summits of MeCP2 ChIP enrichment identified using the MACS peak-calling algorithm 40 (red) or 31,479 randomly selected control sites (gray, see Methods).

    Journal: Nature

    Article Title: Disruption of DNA methylation-dependent long gene repression in Rett syndrome

    doi: 10.1038/nature14319

    Figure Lengend Snippet: ChIP-seq analysis of MeCP2 binding in vivo a, Boxplots of input-normalized read density within gene bodies (TSS +3 kb to TTS) for MeCP2 ChIP from the mouse frontal cortex plotted for genes according to quartile of mCA/CA, mCG/CG, hmCA/CA and hmCG/CG in the frontal cortex 24 for all genes and genes > 100 kb. b, Similar analysis of MeCP2 ChIP from the mouse cortex (left) or cerebellum (right) plotted for genes according to quartile of mCA/CA or mCG/CG for all genes and genes > 100 kb. MeCP2 ChIP-signal is correlated with mCA/CA levels from the frontal cortex, cortex, and cerebellum for all genes and this correlation is more prominent among genes > 100 kb. mCG does not show as prominent a correlation with MeCP2 ChIP signal, and hmCG trends toward anti-correlation with MeCP2 ChIP. These results suggest that MeCP2 has a lower affinity for hmCG than mCG, suggesting that, in vivo, hmCG is associated with reduced MeCP2 occupancy ( Supplementary Discussion ). c , High resolution analysis of high-coverage bisulfite sequencing data from the frontal cortex showing a correlation between MeCP2 ChIP signal and mCA. Input-normalized ChIP signal plotted for mCA levels for 500 bp bins tiled across all genes. d , Aggregate plots of MeCP2 input-normalized ChIP signal (top) and relative methylation (log2 enrichment in mC as compared to the flanking regions) for mCA, mCC, mCT, and mCG (bottom) are plotted around the 31,479 summits of MeCP2 ChIP enrichment identified using the MACS peak-calling algorithm 40 (red) or 31,479 randomly selected control sites (gray, see Methods).

    Article Snippet: To analyze gene expression genome-wide with respect to gene length, CEL files containing the raw hybridization data in from multiple MeCP2 KO and MeCP2 OE gene expression studies were downloaded from GEO ( http://www.ncbi.nlm.nih.gov/geo/ ; study details, sample numbers and genotypes are provided in ) and analyzed for expression at the gene level using using the GeneSpring software suite (Agilent Technologies) with RMA summarization of “Core” probesets.

    Techniques: Chromatin Immunoprecipitation, Binding Assay, In Vivo, Methylation Sequencing, Methylation, Magnetic Cell Separation

    CC chemokine expression in liver tissues from mice fed normal chow or high-fat/high-sucrose diet. Total RNA was extracted from liver tissues of male wild type or Tet-mev-1 mice ( +ROS ) that had been supplied with doxycycline-containing water and were subsequently fed a control, normal diet or high-fat/high-sucrose diet ( +HFHSD ) for 4 months at around the age of either 1 ( left ) or 2 years ( right ). The gene expression levels of CCL2 ( A ), CCL3 ( B ), CCL4 ( C ), CCL5 ( D ), CCL8 ( E ) and CCL12 ( F ) were quantified using specific primers. The values are expressed as means ± SD from four to five mice per group. The asterisks indicate that the differences between the groups are statistically significant (* p

    Journal: PLoS ONE

    Article Title: A Combination of Mitochondrial Oxidative Stress and Excess Fat/Calorie Intake Accelerates Steatohepatitis by Enhancing Hepatic CC Chemokine Production in Mice

    doi: 10.1371/journal.pone.0146592

    Figure Lengend Snippet: CC chemokine expression in liver tissues from mice fed normal chow or high-fat/high-sucrose diet. Total RNA was extracted from liver tissues of male wild type or Tet-mev-1 mice ( +ROS ) that had been supplied with doxycycline-containing water and were subsequently fed a control, normal diet or high-fat/high-sucrose diet ( +HFHSD ) for 4 months at around the age of either 1 ( left ) or 2 years ( right ). The gene expression levels of CCL2 ( A ), CCL3 ( B ), CCL4 ( C ), CCL5 ( D ), CCL8 ( E ) and CCL12 ( F ) were quantified using specific primers. The values are expressed as means ± SD from four to five mice per group. The asterisks indicate that the differences between the groups are statistically significant (* p

    Article Snippet: Extracted RNA obtained from wild type and Tet-mev-1 mice (n = 2 per group) was labeled and hybridized to the Whole Mouse Genome Microarray Kit (Agilent Technology, Santa Clara, CA).

    Techniques: Expressing, Mouse Assay

    Increased CCR5 expression in peripheral blood monocytes/macrophages of Tet-mev-1 mice. ( A ) Activated monocytes/macrophages were identified as an F4/80 + /CD11b + /Ly-6C high cell fraction ( P3 ) in male wild type ( WT ) and Tet-mev-1 mice that had been supplied with doxycycline-containing water and were subsequently fed a normal diet ( ND ) or high-fat/high-sucrose diet ( HFHSD ) for 4 months. ( B ) CCR5 expression in these cells was analyzed by measuring the intensities of APC fluorescence bound to anti-CCR5 antibodies using five to six mice per group. The asterisks indicate that the differences between the groups are statistically significant (* p

    Journal: PLoS ONE

    Article Title: A Combination of Mitochondrial Oxidative Stress and Excess Fat/Calorie Intake Accelerates Steatohepatitis by Enhancing Hepatic CC Chemokine Production in Mice

    doi: 10.1371/journal.pone.0146592

    Figure Lengend Snippet: Increased CCR5 expression in peripheral blood monocytes/macrophages of Tet-mev-1 mice. ( A ) Activated monocytes/macrophages were identified as an F4/80 + /CD11b + /Ly-6C high cell fraction ( P3 ) in male wild type ( WT ) and Tet-mev-1 mice that had been supplied with doxycycline-containing water and were subsequently fed a normal diet ( ND ) or high-fat/high-sucrose diet ( HFHSD ) for 4 months. ( B ) CCR5 expression in these cells was analyzed by measuring the intensities of APC fluorescence bound to anti-CCR5 antibodies using five to six mice per group. The asterisks indicate that the differences between the groups are statistically significant (* p

    Article Snippet: Extracted RNA obtained from wild type and Tet-mev-1 mice (n = 2 per group) was labeled and hybridized to the Whole Mouse Genome Microarray Kit (Agilent Technology, Santa Clara, CA).

    Techniques: Expressing, Mouse Assay, Fluorescence

    Experimental design. Male wild type ( WT ) and Tet-mev-1 mice had been continuously supplied with drinking water containing doxycycline (Dox ) at a concentration of either 0.1 mg/mL during the prenatal period (received through their mothers) or 0.4 mg/mL after weaning. They were divided into four groups (n = 8 in each group) which were subsequently fed a control, normal diet (ND ) or a high-fat/high-sucrose diet ( HFHSD ) at approximately either 1 ( upper ) or 2 years of age ( lower ). The mice were sacrificed under isoflurane anesthesia at the end of the 4-month feeding period, and the obtained serum and liver specimens were subjected to further analyses.

    Journal: PLoS ONE

    Article Title: A Combination of Mitochondrial Oxidative Stress and Excess Fat/Calorie Intake Accelerates Steatohepatitis by Enhancing Hepatic CC Chemokine Production in Mice

    doi: 10.1371/journal.pone.0146592

    Figure Lengend Snippet: Experimental design. Male wild type ( WT ) and Tet-mev-1 mice had been continuously supplied with drinking water containing doxycycline (Dox ) at a concentration of either 0.1 mg/mL during the prenatal period (received through their mothers) or 0.4 mg/mL after weaning. They were divided into four groups (n = 8 in each group) which were subsequently fed a control, normal diet (ND ) or a high-fat/high-sucrose diet ( HFHSD ) at approximately either 1 ( upper ) or 2 years of age ( lower ). The mice were sacrificed under isoflurane anesthesia at the end of the 4-month feeding period, and the obtained serum and liver specimens were subjected to further analyses.

    Article Snippet: Extracted RNA obtained from wild type and Tet-mev-1 mice (n = 2 per group) was labeled and hybridized to the Whole Mouse Genome Microarray Kit (Agilent Technology, Santa Clara, CA).

    Techniques: Mouse Assay, Concentration Assay

    Increased reactive oxygen species (ROS) production and decreased mitochondrial membrane potential in Tet-mev-1 hepatocytes. ( A ) Primary hepatocytes were isolated from 14-month-old male wild type ( WT ) or Tet-mev-1 mice that had been supplied with doxycycline-free water. After treatment with the indicated concentrations of doxycycline ( Dox ) for 72 hours, they were incubated with 100 μM dichlorofluorescein diacetate for 30 min. The fluorescence intensity was measured at 520 nm, and the values are expressed as means ± SD from six samples in each group. ( B ) Hepatocytes that had been isolated from the same mice used for the experiment shown in Fig 3A were incubated with 300 nM tetramethyl rhodamine methyl ester for 30 min. The fluorescence at 573 nm was viewed and analyzed using a confocal laser-scanning microscope with the same excitation strength and detection gain. Representative images are shown for hepatocytes obtained from wild type ( upper ) and Tet-mev-1 mice ( lower ). Scale bars , 20 μm. On the right side of the images, histograms indicating the values (means ± SD) of fluorescence intensities obtained from five randomly selected visual fields in each group are shown. The asterisks indicate that the differences between the groups are statistically significant (* p

    Journal: PLoS ONE

    Article Title: A Combination of Mitochondrial Oxidative Stress and Excess Fat/Calorie Intake Accelerates Steatohepatitis by Enhancing Hepatic CC Chemokine Production in Mice

    doi: 10.1371/journal.pone.0146592

    Figure Lengend Snippet: Increased reactive oxygen species (ROS) production and decreased mitochondrial membrane potential in Tet-mev-1 hepatocytes. ( A ) Primary hepatocytes were isolated from 14-month-old male wild type ( WT ) or Tet-mev-1 mice that had been supplied with doxycycline-free water. After treatment with the indicated concentrations of doxycycline ( Dox ) for 72 hours, they were incubated with 100 μM dichlorofluorescein diacetate for 30 min. The fluorescence intensity was measured at 520 nm, and the values are expressed as means ± SD from six samples in each group. ( B ) Hepatocytes that had been isolated from the same mice used for the experiment shown in Fig 3A were incubated with 300 nM tetramethyl rhodamine methyl ester for 30 min. The fluorescence at 573 nm was viewed and analyzed using a confocal laser-scanning microscope with the same excitation strength and detection gain. Representative images are shown for hepatocytes obtained from wild type ( upper ) and Tet-mev-1 mice ( lower ). Scale bars , 20 μm. On the right side of the images, histograms indicating the values (means ± SD) of fluorescence intensities obtained from five randomly selected visual fields in each group are shown. The asterisks indicate that the differences between the groups are statistically significant (* p

    Article Snippet: Extracted RNA obtained from wild type and Tet-mev-1 mice (n = 2 per group) was labeled and hybridized to the Whole Mouse Genome Microarray Kit (Agilent Technology, Santa Clara, CA).

    Techniques: Isolation, Mouse Assay, Incubation, Fluorescence, Laser-Scanning Microscopy

    Cytotoxicity of doxycycline to primary cultures of hepatocytes. Hepatocytes were isolated from 9-week-old female wild type ( A ) or Tet-mev-1 mice ( B ) that had been supplied with doxycycline-free water. The cells were subjected to primary culture, treated with the indicated concentrations of doxycycline ( Dox ) for 72 hours, and then examined using cell counting assays. The values are expressed as means ± SD from six samples in each group. The asterisk indicates that the difference is statistically significant between the groups (* p

    Journal: PLoS ONE

    Article Title: A Combination of Mitochondrial Oxidative Stress and Excess Fat/Calorie Intake Accelerates Steatohepatitis by Enhancing Hepatic CC Chemokine Production in Mice

    doi: 10.1371/journal.pone.0146592

    Figure Lengend Snippet: Cytotoxicity of doxycycline to primary cultures of hepatocytes. Hepatocytes were isolated from 9-week-old female wild type ( A ) or Tet-mev-1 mice ( B ) that had been supplied with doxycycline-free water. The cells were subjected to primary culture, treated with the indicated concentrations of doxycycline ( Dox ) for 72 hours, and then examined using cell counting assays. The values are expressed as means ± SD from six samples in each group. The asterisk indicates that the difference is statistically significant between the groups (* p

    Article Snippet: Extracted RNA obtained from wild type and Tet-mev-1 mice (n = 2 per group) was labeled and hybridized to the Whole Mouse Genome Microarray Kit (Agilent Technology, Santa Clara, CA).

    Techniques: Isolation, Mouse Assay, Cell Counting

    Increased infiltration of CCR5-positive cells and activation of hepatic stellate cells in liver tissues of Tet-mev-1 mice. Liver specimens were obtained from male wild type ( A , B , E and F ) or Tet-mev-1 mice ( C , D , G and H ) that had been supplied with doxycycline-containing water and were subsequently fed control, normal chow ( A to D ) or a high-fat/high-sucrose diet ( E to H ) for 4 months at around the age of 2 years. The tissue sections were subjected to immunohistochemical staining using antibodies against CCR5 ( A , C , E and G ) or α-smooth muscle actin ( B , D , F and H ). CCR5-positive cells are shown by arrows in panels E and G , while α-smooth actin-expressing myofibroblasts were indicated by arrows in panel H . Representative images are shown from five mice in each group. Scale bars , 100 μm. A part of panels E to H is presented under high magnification in the corresponding panels on the right.

    Journal: PLoS ONE

    Article Title: A Combination of Mitochondrial Oxidative Stress and Excess Fat/Calorie Intake Accelerates Steatohepatitis by Enhancing Hepatic CC Chemokine Production in Mice

    doi: 10.1371/journal.pone.0146592

    Figure Lengend Snippet: Increased infiltration of CCR5-positive cells and activation of hepatic stellate cells in liver tissues of Tet-mev-1 mice. Liver specimens were obtained from male wild type ( A , B , E and F ) or Tet-mev-1 mice ( C , D , G and H ) that had been supplied with doxycycline-containing water and were subsequently fed control, normal chow ( A to D ) or a high-fat/high-sucrose diet ( E to H ) for 4 months at around the age of 2 years. The tissue sections were subjected to immunohistochemical staining using antibodies against CCR5 ( A , C , E and G ) or α-smooth muscle actin ( B , D , F and H ). CCR5-positive cells are shown by arrows in panels E and G , while α-smooth actin-expressing myofibroblasts were indicated by arrows in panel H . Representative images are shown from five mice in each group. Scale bars , 100 μm. A part of panels E to H is presented under high magnification in the corresponding panels on the right.

    Article Snippet: Extracted RNA obtained from wild type and Tet-mev-1 mice (n = 2 per group) was labeled and hybridized to the Whole Mouse Genome Microarray Kit (Agilent Technology, Santa Clara, CA).

    Techniques: Activation Assay, Mouse Assay, Immunohistochemistry, Staining, Expressing

    Histopathological findings of liver tissues from aged mice fed normal chow or high-fat/high-sucrose diet. Liver specimens were obtained from male wild type ( A and C ) or Tet-mev-1 mice ( B and D ) that had been supplied with doxycycline-containing water throughout the prenatal and postnatal periods and, at around the age of 2 years, were subsequently fed a control, normal diet ( A and B ) or high-fat/high-sucrose diet ( C and D ) for 4 months. Serial sections were subjected to hematoxylin and eosin staining ( upper ) or Sirius red-Fast green FCF staining ( lower ). Representative images from eight mice in each group are shown. Scale bars , 100 μm.

    Journal: PLoS ONE

    Article Title: A Combination of Mitochondrial Oxidative Stress and Excess Fat/Calorie Intake Accelerates Steatohepatitis by Enhancing Hepatic CC Chemokine Production in Mice

    doi: 10.1371/journal.pone.0146592

    Figure Lengend Snippet: Histopathological findings of liver tissues from aged mice fed normal chow or high-fat/high-sucrose diet. Liver specimens were obtained from male wild type ( A and C ) or Tet-mev-1 mice ( B and D ) that had been supplied with doxycycline-containing water throughout the prenatal and postnatal periods and, at around the age of 2 years, were subsequently fed a control, normal diet ( A and B ) or high-fat/high-sucrose diet ( C and D ) for 4 months. Serial sections were subjected to hematoxylin and eosin staining ( upper ) or Sirius red-Fast green FCF staining ( lower ). Representative images from eight mice in each group are shown. Scale bars , 100 μm.

    Article Snippet: Extracted RNA obtained from wild type and Tet-mev-1 mice (n = 2 per group) was labeled and hybridized to the Whole Mouse Genome Microarray Kit (Agilent Technology, Santa Clara, CA).

    Techniques: Mouse Assay, Staining

    Histopathological findings of liver tissues from young mice fed normal chow or high-fat/high-sucrose diet. Liver specimens were obtained from male wild type ( A and C ) or Tet-mev-1 mice ( B and D ) that had been supplied with doxycycline-containing water throughout the prenatal and postnatal periods and, at around the age of 1 year, were subsequently fed a control, normal diet ( A and B ) or high-fat/high-sucrose diet ( C and D ) for 4 months. Serial sections were subjected to hematoxylin and eosin staining ( upper ) or Sirius red-Fast green FCF staining ( lower ). Representative images from eight mice in each group are shown. Scale bars , 100 μm.

    Journal: PLoS ONE

    Article Title: A Combination of Mitochondrial Oxidative Stress and Excess Fat/Calorie Intake Accelerates Steatohepatitis by Enhancing Hepatic CC Chemokine Production in Mice

    doi: 10.1371/journal.pone.0146592

    Figure Lengend Snippet: Histopathological findings of liver tissues from young mice fed normal chow or high-fat/high-sucrose diet. Liver specimens were obtained from male wild type ( A and C ) or Tet-mev-1 mice ( B and D ) that had been supplied with doxycycline-containing water throughout the prenatal and postnatal periods and, at around the age of 1 year, were subsequently fed a control, normal diet ( A and B ) or high-fat/high-sucrose diet ( C and D ) for 4 months. Serial sections were subjected to hematoxylin and eosin staining ( upper ) or Sirius red-Fast green FCF staining ( lower ). Representative images from eight mice in each group are shown. Scale bars , 100 μm.

    Article Snippet: Extracted RNA obtained from wild type and Tet-mev-1 mice (n = 2 per group) was labeled and hybridized to the Whole Mouse Genome Microarray Kit (Agilent Technology, Santa Clara, CA).

    Techniques: Mouse Assay, Staining

    Detection of lipid peroxidation and apoptosis in liver tissues. Liver specimens were obtained from male wild type ( A, C, E, and G ) or Tet-mev-1 mice ( B, D, F and H ) that had been supplied with doxycycline-containing water and were subsequently fed a control, normal diet ( A, B, E, and F ) or high-fat/high-sucrose diet ( C, D, G and H ) for 4 months at around the age of 2 years. They were subjected to 4-hydroxy-2-nonenal staining ( A to D ) or apoptosis detection using the TdT-mediated dUTP nick end labeling method ( E to H ). Hepatocytes and non-parenchymal cells positively stained for 4-hydroxy-2-nonenal are indicated by arrows and arrowheads, respectively, in panels C and D , while non-parenchymal cells showing apoptosis are shown by arrowheads in panels F and H . Representative images from five mice in each group are shown. Scale bars , 20 μm.

    Journal: PLoS ONE

    Article Title: A Combination of Mitochondrial Oxidative Stress and Excess Fat/Calorie Intake Accelerates Steatohepatitis by Enhancing Hepatic CC Chemokine Production in Mice

    doi: 10.1371/journal.pone.0146592

    Figure Lengend Snippet: Detection of lipid peroxidation and apoptosis in liver tissues. Liver specimens were obtained from male wild type ( A, C, E, and G ) or Tet-mev-1 mice ( B, D, F and H ) that had been supplied with doxycycline-containing water and were subsequently fed a control, normal diet ( A, B, E, and F ) or high-fat/high-sucrose diet ( C, D, G and H ) for 4 months at around the age of 2 years. They were subjected to 4-hydroxy-2-nonenal staining ( A to D ) or apoptosis detection using the TdT-mediated dUTP nick end labeling method ( E to H ). Hepatocytes and non-parenchymal cells positively stained for 4-hydroxy-2-nonenal are indicated by arrows and arrowheads, respectively, in panels C and D , while non-parenchymal cells showing apoptosis are shown by arrowheads in panels F and H . Representative images from five mice in each group are shown. Scale bars , 20 μm.

    Article Snippet: Extracted RNA obtained from wild type and Tet-mev-1 mice (n = 2 per group) was labeled and hybridized to the Whole Mouse Genome Microarray Kit (Agilent Technology, Santa Clara, CA).

    Techniques: Mouse Assay, Staining, End Labeling

    Degree of fibrosis and expression of profibrogenic genes in liver tissues from mice fed normal chow or high-fat/high-sucrose diet. Liver specimens were obtained and the total RNA was extracted from male wild type or Tet-mev-1 mice ( +ROS ) that had been supplied with doxycycline-containing water and were subsequently fed a control, normal diet or high-fat/high-sucrose diet (+ HFHSD ) for 4 months at around the age of either 1 ( left ) or 2 years ( right ). The degree of liver fibrosis was semi-quantified by measuring the mean relative areas stained positive for Sirius red ( A ), and the gene expression levels of proα1 type I collagen ( B ) and transforming growth factor-β1 ( C ) were quantified using specific primers. The values represent the mean ± SD obtained from eight mice per group. The asterisks indicate that the differences between the groups are statistically significant (* p

    Journal: PLoS ONE

    Article Title: A Combination of Mitochondrial Oxidative Stress and Excess Fat/Calorie Intake Accelerates Steatohepatitis by Enhancing Hepatic CC Chemokine Production in Mice

    doi: 10.1371/journal.pone.0146592

    Figure Lengend Snippet: Degree of fibrosis and expression of profibrogenic genes in liver tissues from mice fed normal chow or high-fat/high-sucrose diet. Liver specimens were obtained and the total RNA was extracted from male wild type or Tet-mev-1 mice ( +ROS ) that had been supplied with doxycycline-containing water and were subsequently fed a control, normal diet or high-fat/high-sucrose diet (+ HFHSD ) for 4 months at around the age of either 1 ( left ) or 2 years ( right ). The degree of liver fibrosis was semi-quantified by measuring the mean relative areas stained positive for Sirius red ( A ), and the gene expression levels of proα1 type I collagen ( B ) and transforming growth factor-β1 ( C ) were quantified using specific primers. The values represent the mean ± SD obtained from eight mice per group. The asterisks indicate that the differences between the groups are statistically significant (* p

    Article Snippet: Extracted RNA obtained from wild type and Tet-mev-1 mice (n = 2 per group) was labeled and hybridized to the Whole Mouse Genome Microarray Kit (Agilent Technology, Santa Clara, CA).

    Techniques: Expressing, Mouse Assay, Staining

    Real-time RT-PCR confirms downregulation of genes involved in the immune system in human NK cells treated with Gal-9. We used PCR to verify the 6 genes shown to be maximally differentially expressed (greater than 7-fold change) in the microarray analysis.

    Journal: Journal of Virology

    Article Title: Galectin-9 Functionally Impairs Natural Killer Cells in Humans and Mice

    doi: 10.1128/JVI.01085-12

    Figure Lengend Snippet: Real-time RT-PCR confirms downregulation of genes involved in the immune system in human NK cells treated with Gal-9. We used PCR to verify the 6 genes shown to be maximally differentially expressed (greater than 7-fold change) in the microarray analysis.

    Article Snippet: We investigated the cellular response of human NK cells from normal subjects to 24 h of Gal-9 stimulation by examining differential expression using Agilent 4×44K human microarrays.

    Techniques: Quantitative RT-PCR, Polymerase Chain Reaction, Microarray

    Microarray analysis of human NK cells stimulated by Gal-9.

    Journal: Journal of Virology

    Article Title: Galectin-9 Functionally Impairs Natural Killer Cells in Humans and Mice

    doi: 10.1128/JVI.01085-12

    Figure Lengend Snippet: Microarray analysis of human NK cells stimulated by Gal-9.

    Article Snippet: We investigated the cellular response of human NK cells from normal subjects to 24 h of Gal-9 stimulation by examining differential expression using Agilent 4×44K human microarrays.

    Techniques: Microarray

    Direct ex vivo characterization of NK cells from Gal-9 KO mice. (A) Flow-cytometric analysis of splenic NK cells (NK1.1 + CD3 − ) demonstrated that in the absence of exogenous stimulation, the percentage of NKs that degranulated (CD107a + ) was higher

    Journal: Journal of Virology

    Article Title: Galectin-9 Functionally Impairs Natural Killer Cells in Humans and Mice

    doi: 10.1128/JVI.01085-12

    Figure Lengend Snippet: Direct ex vivo characterization of NK cells from Gal-9 KO mice. (A) Flow-cytometric analysis of splenic NK cells (NK1.1 + CD3 − ) demonstrated that in the absence of exogenous stimulation, the percentage of NKs that degranulated (CD107a + ) was higher

    Article Snippet: We investigated the cellular response of human NK cells from normal subjects to 24 h of Gal-9 stimulation by examining differential expression using Agilent 4×44K human microarrays.

    Techniques: Ex Vivo, Mouse Assay, Flow Cytometry

    Gal-9 functionally impairs human NK cells in vitro . (A) The IL-2-induced lymphokine-activated killing (LAK; 48 h of culture with 25 ng/ml IL-2) activity of isolated NK cells ( n = 6 normal control subjects) against the NK cell-sensitive target cell line

    Journal: Journal of Virology

    Article Title: Galectin-9 Functionally Impairs Natural Killer Cells in Humans and Mice

    doi: 10.1128/JVI.01085-12

    Figure Lengend Snippet: Gal-9 functionally impairs human NK cells in vitro . (A) The IL-2-induced lymphokine-activated killing (LAK; 48 h of culture with 25 ng/ml IL-2) activity of isolated NK cells ( n = 6 normal control subjects) against the NK cell-sensitive target cell line

    Article Snippet: We investigated the cellular response of human NK cells from normal subjects to 24 h of Gal-9 stimulation by examining differential expression using Agilent 4×44K human microarrays.

    Techniques: In Vitro, Activity Assay, Isolation

    Downregulation of human NK cell-mediated cytotoxicity pathway genes in response to Gal-9. Among the genes downregulated in response to Gal-9 stimulation are genes of the NK cell-mediated cytotoxicity pathway, including the natural cytotoxicity triggering

    Journal: Journal of Virology

    Article Title: Galectin-9 Functionally Impairs Natural Killer Cells in Humans and Mice

    doi: 10.1128/JVI.01085-12

    Figure Lengend Snippet: Downregulation of human NK cell-mediated cytotoxicity pathway genes in response to Gal-9. Among the genes downregulated in response to Gal-9 stimulation are genes of the NK cell-mediated cytotoxicity pathway, including the natural cytotoxicity triggering

    Article Snippet: We investigated the cellular response of human NK cells from normal subjects to 24 h of Gal-9 stimulation by examining differential expression using Agilent 4×44K human microarrays.

    Techniques:

    Microarray analysis of human NK cells stimulated by Gal-9.

    Journal: Journal of Virology

    Article Title: Galectin-9 Functionally Impairs Natural Killer Cells in Humans and Mice

    doi: 10.1128/JVI.01085-12

    Figure Lengend Snippet: Microarray analysis of human NK cells stimulated by Gal-9.

    Article Snippet: We investigated the cellular response of human NK cells from normal subjects to 24 h of Gal-9 stimulation by examining differential expression using Agilent 4×44K human microarrays.

    Techniques: Microarray

    Real time RT-PCR analysis of human NK cells stimulated by Gal-9.

    Journal: Journal of Virology

    Article Title: Galectin-9 Functionally Impairs Natural Killer Cells in Humans and Mice

    doi: 10.1128/JVI.01085-12

    Figure Lengend Snippet: Real time RT-PCR analysis of human NK cells stimulated by Gal-9.

    Article Snippet: We investigated the cellular response of human NK cells from normal subjects to 24 h of Gal-9 stimulation by examining differential expression using Agilent 4×44K human microarrays.

    Techniques: Quantitative RT-PCR

    NK cells from Gal-9 KO mice respond to stimulation with enhanced cytokine production and degranulation. For the induction of IFN-γ, splenic cells were stimulated with cytokines IL-12 (1 ng/ml) and IL-18 (10 ng/ml) for 6 h in the presence of brefeldin

    Journal: Journal of Virology

    Article Title: Galectin-9 Functionally Impairs Natural Killer Cells in Humans and Mice

    doi: 10.1128/JVI.01085-12

    Figure Lengend Snippet: NK cells from Gal-9 KO mice respond to stimulation with enhanced cytokine production and degranulation. For the induction of IFN-γ, splenic cells were stimulated with cytokines IL-12 (1 ng/ml) and IL-18 (10 ng/ml) for 6 h in the presence of brefeldin

    Article Snippet: We investigated the cellular response of human NK cells from normal subjects to 24 h of Gal-9 stimulation by examining differential expression using Agilent 4×44K human microarrays.

    Techniques: Mouse Assay

    Gal-9 inhibition of human NK cell activity is Tim-3 independent. Blocking experiments were carried out as described in Materials and Methods. (A) PCR detection of the 6 genes shown to be maximally differentially expressed (greater than 7-fold change)

    Journal: Journal of Virology

    Article Title: Galectin-9 Functionally Impairs Natural Killer Cells in Humans and Mice

    doi: 10.1128/JVI.01085-12

    Figure Lengend Snippet: Gal-9 inhibition of human NK cell activity is Tim-3 independent. Blocking experiments were carried out as described in Materials and Methods. (A) PCR detection of the 6 genes shown to be maximally differentially expressed (greater than 7-fold change)

    Article Snippet: We investigated the cellular response of human NK cells from normal subjects to 24 h of Gal-9 stimulation by examining differential expression using Agilent 4×44K human microarrays.

    Techniques: Inhibition, Activity Assay, Blocking Assay, Polymerase Chain Reaction

    MCMV infection in Gal-9 KO mice. (A) Hepatic mononuclear cells were isolated from the livers of control ( n = 5) and Gal-9 KO ( n = 5) mice after 4 days of infection with MCMV (as described in Materials and Methods). Flow-cytometric analysis was used to

    Journal: Journal of Virology

    Article Title: Galectin-9 Functionally Impairs Natural Killer Cells in Humans and Mice

    doi: 10.1128/JVI.01085-12

    Figure Lengend Snippet: MCMV infection in Gal-9 KO mice. (A) Hepatic mononuclear cells were isolated from the livers of control ( n = 5) and Gal-9 KO ( n = 5) mice after 4 days of infection with MCMV (as described in Materials and Methods). Flow-cytometric analysis was used to

    Article Snippet: We investigated the cellular response of human NK cells from normal subjects to 24 h of Gal-9 stimulation by examining differential expression using Agilent 4×44K human microarrays.

    Techniques: Infection, Mouse Assay, Isolation, Flow Cytometry

    Gal-9 KO mice show proportionally greater frequency of NK cells demonstrating intermediate and mature phenotypes, particularly within the liver. Splenic and hepatic NK cells from wild-type ( n = 5) and Gal-9 KO ( n = 5) mice were assayed for maturation

    Journal: Journal of Virology

    Article Title: Galectin-9 Functionally Impairs Natural Killer Cells in Humans and Mice

    doi: 10.1128/JVI.01085-12

    Figure Lengend Snippet: Gal-9 KO mice show proportionally greater frequency of NK cells demonstrating intermediate and mature phenotypes, particularly within the liver. Splenic and hepatic NK cells from wild-type ( n = 5) and Gal-9 KO ( n = 5) mice were assayed for maturation

    Article Snippet: We investigated the cellular response of human NK cells from normal subjects to 24 h of Gal-9 stimulation by examining differential expression using Agilent 4×44K human microarrays.

    Techniques: Mouse Assay

    Schematic depicting Sirt6 deficiency exacerbates podocyte injuries and proteinuria through targeting N otch signaling. Under normal conditions, SIRT6 inhibits the transcription of Notch1 and Notch4 genes by decreasing H3K9ac levels in the promoter region of Notch1 and Notch4 . Under pathological conditions, SIRT6 reduction leads to the increased levels of H3K9ac in the promoters of Notch1 and Notch4 , thereby enhancing the transcription of Notch1 and Notch4 gene. The activation of Notch signaling finally results in podocyte injury through inducing inflammation, apoptosis, actin cytoskeleton derangement, as well as the inhibition of autophagy

    Journal: Nature Communications

    Article Title: Sirt6 deficiency exacerbates podocyte injury and proteinuria through targeting Notch signaling

    doi: 10.1038/s41467-017-00498-4

    Figure Lengend Snippet: Schematic depicting Sirt6 deficiency exacerbates podocyte injuries and proteinuria through targeting N otch signaling. Under normal conditions, SIRT6 inhibits the transcription of Notch1 and Notch4 genes by decreasing H3K9ac levels in the promoter region of Notch1 and Notch4 . Under pathological conditions, SIRT6 reduction leads to the increased levels of H3K9ac in the promoters of Notch1 and Notch4 , thereby enhancing the transcription of Notch1 and Notch4 gene. The activation of Notch signaling finally results in podocyte injury through inducing inflammation, apoptosis, actin cytoskeleton derangement, as well as the inhibition of autophagy

    Article Snippet: Moreover, we observed the changes of Notch signaling in HG-treated podocytes with or without Sirt6 overexpression by Agilent Whole Human Genome Oligo Microarray for global gene expression analysis (Fig. ).

    Techniques: Activation Assay, Inhibition

    In vivo overexpression of Sirt6 by intrarenal lentiviral gene delivery ameliorates renal injury in Cre + /Sirt6 fl/fl mice with ADR nephropathy. a The relative mRNA levels of Sirt6 in isolated glomeruli at 5-week after pGLV3- Sirt6 delivered into the mouse kidney by means of intraparenchymal injections. b UACR (urine albumin-to creatinine ratio) in different groups of mice. c Photomicrographs and quantifications showing typical changes in glomerular structure in different groups of mice. Scale bar : black 50 μm, red 25 μm. d Representative photomicrographs and quantifications of mean glomerular basement membrane (GBM) thickness, mean foot process width, and the number of foot processes in different groups of mice. Scale bar , 2 μm. e Representative confocal microscopic images showing the expressions of nephrin and podocin in podocytes from different groups of mice. Scale bar , 25 μm. f ChIP analysis showing the acetylation levels of H3K9 (H3K9ac) in the promoter of Notch1 using antibodies to H3K9ac in isolated glomeruli. g ChIP analysis showed that Sirt6 bound to promoters of Notch1 by using antibodies to Sirt6 in isolated glomeruli. * P

    Journal: Nature Communications

    Article Title: Sirt6 deficiency exacerbates podocyte injury and proteinuria through targeting Notch signaling

    doi: 10.1038/s41467-017-00498-4

    Figure Lengend Snippet: In vivo overexpression of Sirt6 by intrarenal lentiviral gene delivery ameliorates renal injury in Cre + /Sirt6 fl/fl mice with ADR nephropathy. a The relative mRNA levels of Sirt6 in isolated glomeruli at 5-week after pGLV3- Sirt6 delivered into the mouse kidney by means of intraparenchymal injections. b UACR (urine albumin-to creatinine ratio) in different groups of mice. c Photomicrographs and quantifications showing typical changes in glomerular structure in different groups of mice. Scale bar : black 50 μm, red 25 μm. d Representative photomicrographs and quantifications of mean glomerular basement membrane (GBM) thickness, mean foot process width, and the number of foot processes in different groups of mice. Scale bar , 2 μm. e Representative confocal microscopic images showing the expressions of nephrin and podocin in podocytes from different groups of mice. Scale bar , 25 μm. f ChIP analysis showing the acetylation levels of H3K9 (H3K9ac) in the promoter of Notch1 using antibodies to H3K9ac in isolated glomeruli. g ChIP analysis showed that Sirt6 bound to promoters of Notch1 by using antibodies to Sirt6 in isolated glomeruli. * P

    Article Snippet: Moreover, we observed the changes of Notch signaling in HG-treated podocytes with or without Sirt6 overexpression by Agilent Whole Human Genome Oligo Microarray for global gene expression analysis (Fig. ).

    Techniques: In Vivo, Over Expression, Mouse Assay, Isolation, Chromatin Immunoprecipitation

    Sirt6 reduces inflammatory responses, apoptosis and was essential for maintaining basal autophagy in podocytes with HG treatment: a Representative western blot gel documents and summarized data showing the relative protein level of Sirt6 by an Sirt6 -adenovirus transfection. b The levels of pro-inflammatory mediators in podocytes with different treatments. c Podocytes with different treatments were stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide (PI), and analyzed by flow cytometry to evaluate the role of Sirt6 in the prevention of apoptosis. Quantification of the apoptotic cells was showed at right panel . * P

    Journal: Nature Communications

    Article Title: Sirt6 deficiency exacerbates podocyte injury and proteinuria through targeting Notch signaling

    doi: 10.1038/s41467-017-00498-4

    Figure Lengend Snippet: Sirt6 reduces inflammatory responses, apoptosis and was essential for maintaining basal autophagy in podocytes with HG treatment: a Representative western blot gel documents and summarized data showing the relative protein level of Sirt6 by an Sirt6 -adenovirus transfection. b The levels of pro-inflammatory mediators in podocytes with different treatments. c Podocytes with different treatments were stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide (PI), and analyzed by flow cytometry to evaluate the role of Sirt6 in the prevention of apoptosis. Quantification of the apoptotic cells was showed at right panel . * P

    Article Snippet: Moreover, we observed the changes of Notch signaling in HG-treated podocytes with or without Sirt6 overexpression by Agilent Whole Human Genome Oligo Microarray for global gene expression analysis (Fig. ).

    Techniques: Western Blot, Transfection, Staining, Flow Cytometry, Cytometry

    ADR-treated Podocin- Cre Sirt6 fl/fl mice exhibites substantial podocyte injury and glomerulosclerosis. a UACR (urinary albumin-to-creatinine ratio) in different groups of mice. b Photomicrographs and quantifications showing typical changes in glomerular structure in different groups of mice. Scale bar : black 100 μm, red 25 μm. c Representative photomicrographs and quantifications of mean glomerular basement membrane (GBM) thickness, mean foot process width, and the number of foot processes in different groups of mice. Scale bar , 3 μm. d Representative confocal microscopic images showing the expressions of nephrin and podocin in podocytes of the kidney from different groups of mice. Scale bar , 25 μm. Podocyte-specific Sirt6 knockout mice: Cre + /Sirt6 fl/fl mice; Mice with two WT alleles and Cre expression ( Cre + /Sirt6 +/+ ) were used as controls. * P

    Journal: Nature Communications

    Article Title: Sirt6 deficiency exacerbates podocyte injury and proteinuria through targeting Notch signaling

    doi: 10.1038/s41467-017-00498-4

    Figure Lengend Snippet: ADR-treated Podocin- Cre Sirt6 fl/fl mice exhibites substantial podocyte injury and glomerulosclerosis. a UACR (urinary albumin-to-creatinine ratio) in different groups of mice. b Photomicrographs and quantifications showing typical changes in glomerular structure in different groups of mice. Scale bar : black 100 μm, red 25 μm. c Representative photomicrographs and quantifications of mean glomerular basement membrane (GBM) thickness, mean foot process width, and the number of foot processes in different groups of mice. Scale bar , 3 μm. d Representative confocal microscopic images showing the expressions of nephrin and podocin in podocytes of the kidney from different groups of mice. Scale bar , 25 μm. Podocyte-specific Sirt6 knockout mice: Cre + /Sirt6 fl/fl mice; Mice with two WT alleles and Cre expression ( Cre + /Sirt6 +/+ ) were used as controls. * P

    Article Snippet: Moreover, we observed the changes of Notch signaling in HG-treated podocytes with or without Sirt6 overexpression by Agilent Whole Human Genome Oligo Microarray for global gene expression analysis (Fig. ).

    Techniques: Mouse Assay, Knock-Out, Expressing

    Sirt6 modulates Notch signaling by deacetylating H3K9ac at promoters of Notch1 and Notch4. a Representative photomicrographs of H3K9ac immunohistochemical staining in human renal tissue from normal subjects and patients with focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy (DN). Scale bar : black 100 μm, red 25 μm. b Representative confocal microscopic images showing the levels of H3K9ac in podocytes of the kidney from different groups of mice, synaptopodin was used as a podocyte marker. Scale bar , 25 μm. c Representative western blot gel documents and summarized data showing the effect of overexpression Sirt6 on the levels of H3K9ac in podocytes treated with high glucose (HG, final concentration 40 mmol/l in medium) for 24 h. d Representative heatmap of gene expression levels by multiplex quantitative RT-PCR array. e Summarized data showing the effect of Sirt6 on the mRNA levels of Notch genes in podocytes treated with HG. f Chromatin immunoprecipitation (ChIP) analysis showing the acetylation levels of H3K9 (H3K9ac) in the promoters of Notch1 and Notch4 using antibodies to H3K9ac. g ChIP analysis showed that Sirt6 bound to promoters of Notch1 and Notch4 genes by using antibodies to Sirt6 in podocytes. Sirt6 was dissociated from the promoters of these genes in podocytes with HG treatment and recruited to them after overexpression of Sirt6. * P

    Journal: Nature Communications

    Article Title: Sirt6 deficiency exacerbates podocyte injury and proteinuria through targeting Notch signaling

    doi: 10.1038/s41467-017-00498-4

    Figure Lengend Snippet: Sirt6 modulates Notch signaling by deacetylating H3K9ac at promoters of Notch1 and Notch4. a Representative photomicrographs of H3K9ac immunohistochemical staining in human renal tissue from normal subjects and patients with focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy (DN). Scale bar : black 100 μm, red 25 μm. b Representative confocal microscopic images showing the levels of H3K9ac in podocytes of the kidney from different groups of mice, synaptopodin was used as a podocyte marker. Scale bar , 25 μm. c Representative western blot gel documents and summarized data showing the effect of overexpression Sirt6 on the levels of H3K9ac in podocytes treated with high glucose (HG, final concentration 40 mmol/l in medium) for 24 h. d Representative heatmap of gene expression levels by multiplex quantitative RT-PCR array. e Summarized data showing the effect of Sirt6 on the mRNA levels of Notch genes in podocytes treated with HG. f Chromatin immunoprecipitation (ChIP) analysis showing the acetylation levels of H3K9 (H3K9ac) in the promoters of Notch1 and Notch4 using antibodies to H3K9ac. g ChIP analysis showed that Sirt6 bound to promoters of Notch1 and Notch4 genes by using antibodies to Sirt6 in podocytes. Sirt6 was dissociated from the promoters of these genes in podocytes with HG treatment and recruited to them after overexpression of Sirt6. * P

    Article Snippet: Moreover, we observed the changes of Notch signaling in HG-treated podocytes with or without Sirt6 overexpression by Agilent Whole Human Genome Oligo Microarray for global gene expression analysis (Fig. ).

    Techniques: Immunohistochemistry, Staining, Mouse Assay, Marker, Western Blot, Over Expression, Concentration Assay, Expressing, Multiplex Assay, Quantitative RT-PCR, Chromatin Immunoprecipitation

    Inhibition of the Notch pathway rescues the functional defect in Sirt6 -deficient podocytes. a The relative levels of pro-inflammatory mediators in podocytes with different treatments. b Summarized data showing the level of podocyte apoptosis determined by flow cytometric analysis in podocytes with different treatments. Podocytes were stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide (PI), and analyzed by flow cytometry. * P

    Journal: Nature Communications

    Article Title: Sirt6 deficiency exacerbates podocyte injury and proteinuria through targeting Notch signaling

    doi: 10.1038/s41467-017-00498-4

    Figure Lengend Snippet: Inhibition of the Notch pathway rescues the functional defect in Sirt6 -deficient podocytes. a The relative levels of pro-inflammatory mediators in podocytes with different treatments. b Summarized data showing the level of podocyte apoptosis determined by flow cytometric analysis in podocytes with different treatments. Podocytes were stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide (PI), and analyzed by flow cytometry. * P

    Article Snippet: Moreover, we observed the changes of Notch signaling in HG-treated podocytes with or without Sirt6 overexpression by Agilent Whole Human Genome Oligo Microarray for global gene expression analysis (Fig. ).

    Techniques: Inhibition, Functional Assay, Flow Cytometry, Staining, Cytometry

    Sirt6 is significantly reduced in podocytes from mice with diabetic nephropathy or ADR nephropathy. a Representative western blot gel documents and summarized data showing the relative protein levels of SIRTs in the kidney from STZ-induced diabetic mice and ADR-treated mice. * P

    Journal: Nature Communications

    Article Title: Sirt6 deficiency exacerbates podocyte injury and proteinuria through targeting Notch signaling

    doi: 10.1038/s41467-017-00498-4

    Figure Lengend Snippet: Sirt6 is significantly reduced in podocytes from mice with diabetic nephropathy or ADR nephropathy. a Representative western blot gel documents and summarized data showing the relative protein levels of SIRTs in the kidney from STZ-induced diabetic mice and ADR-treated mice. * P

    Article Snippet: Moreover, we observed the changes of Notch signaling in HG-treated podocytes with or without Sirt6 overexpression by Agilent Whole Human Genome Oligo Microarray for global gene expression analysis (Fig. ).

    Techniques: Mouse Assay, Western Blot

    Podocyte-specific loss of Sirt6 exacerbates podocyte injury and proteinuria in DN. a UACR (urine albumin-to-creatinine ratio) in different groups of mice. b Photomicrographs and quantifications showing typical glomerular structure changes in different groups of mice. Scale bar : black 50 μm, red 25 μm. c Representative photomicrographs and quantifications of mean glomerular basement membrane (GBM) thickness, mean foot process width, and the number of foot processes in different groups of mice at different age by transmission electron microscopy (TEM) analyses. Scale bar , 3 μm. d Representative confocal microscopic images showing the expressions of nephrin and podocin in the kidney from different groups of mice. Scale bar , 25 μm. e Representative confocal microscopic images showing the expressions of uPAR in podocytes from different groups of mice. Scale bar , 25 μm. Podocyte-specific Sirt6 knockout mice: Cre + /Sirt6 fl/fl mice; Mice with two WT alleles and Cre expression ( Cre + /Sirt6 +/+ ) were used as controls. * P

    Journal: Nature Communications

    Article Title: Sirt6 deficiency exacerbates podocyte injury and proteinuria through targeting Notch signaling

    doi: 10.1038/s41467-017-00498-4

    Figure Lengend Snippet: Podocyte-specific loss of Sirt6 exacerbates podocyte injury and proteinuria in DN. a UACR (urine albumin-to-creatinine ratio) in different groups of mice. b Photomicrographs and quantifications showing typical glomerular structure changes in different groups of mice. Scale bar : black 50 μm, red 25 μm. c Representative photomicrographs and quantifications of mean glomerular basement membrane (GBM) thickness, mean foot process width, and the number of foot processes in different groups of mice at different age by transmission electron microscopy (TEM) analyses. Scale bar , 3 μm. d Representative confocal microscopic images showing the expressions of nephrin and podocin in the kidney from different groups of mice. Scale bar , 25 μm. e Representative confocal microscopic images showing the expressions of uPAR in podocytes from different groups of mice. Scale bar , 25 μm. Podocyte-specific Sirt6 knockout mice: Cre + /Sirt6 fl/fl mice; Mice with two WT alleles and Cre expression ( Cre + /Sirt6 +/+ ) were used as controls. * P

    Article Snippet: Moreover, we observed the changes of Notch signaling in HG-treated podocytes with or without Sirt6 overexpression by Agilent Whole Human Genome Oligo Microarray for global gene expression analysis (Fig. ).

    Techniques: Mouse Assay, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Knock-Out, Expressing

    Establishment of podocyte-specific Sirt6 knockout ( Cre + /Sirt6 fl/fl ) mice. a Generation of conditional knockout mice in which Sirt6 is specifically ablated in podocytes by using Cre–LoxP recombination system. Exon 2 and 3 are deleted upon NPHS2-Cre -mediated recombination. Genotyping was confirmed by tail preparation and PCR at 2 weeks of age. b Podocyte-specific loss of Sirt6 was confirmed by immunofluorescent staining of Sirt6 in podocytes, synaptopodin was used as a podocyte marker. The arrows indicate representative podocytes. Scale bar , 25 μm. c Representative western blot gel documents and summarized data showing the decreased expression of Sirt6 in isolated glomeruli from podocyte-specific Sirt6 knockout (Cre + /Sirt6 fl/fl ) mice. d UACR (urine albumin-to-creatinine ratio) in mice at different age. e Photomicrographs and quantifications showing typical glomerular structures in mice at different age. Scale bar : black 50 μm, red 25 μm. f Representative photomicrographs and quantifications of mean glomerular basement membrane (GBM) thickness, mean foot process width, and the number of foot processes in different groups of mice at different age by transmission electron microscopy (TEM) analyses. Scale bar , 2 μm. Podocyte-specific Sirt6 knockout mice: Cre + /Sirt6 fl/fl mice; Mice with two WT alleles and Cre expression ( Cre + /Sirt6 +/+ ) were used as controls. * P

    Journal: Nature Communications

    Article Title: Sirt6 deficiency exacerbates podocyte injury and proteinuria through targeting Notch signaling

    doi: 10.1038/s41467-017-00498-4

    Figure Lengend Snippet: Establishment of podocyte-specific Sirt6 knockout ( Cre + /Sirt6 fl/fl ) mice. a Generation of conditional knockout mice in which Sirt6 is specifically ablated in podocytes by using Cre–LoxP recombination system. Exon 2 and 3 are deleted upon NPHS2-Cre -mediated recombination. Genotyping was confirmed by tail preparation and PCR at 2 weeks of age. b Podocyte-specific loss of Sirt6 was confirmed by immunofluorescent staining of Sirt6 in podocytes, synaptopodin was used as a podocyte marker. The arrows indicate representative podocytes. Scale bar , 25 μm. c Representative western blot gel documents and summarized data showing the decreased expression of Sirt6 in isolated glomeruli from podocyte-specific Sirt6 knockout (Cre + /Sirt6 fl/fl ) mice. d UACR (urine albumin-to-creatinine ratio) in mice at different age. e Photomicrographs and quantifications showing typical glomerular structures in mice at different age. Scale bar : black 50 μm, red 25 μm. f Representative photomicrographs and quantifications of mean glomerular basement membrane (GBM) thickness, mean foot process width, and the number of foot processes in different groups of mice at different age by transmission electron microscopy (TEM) analyses. Scale bar , 2 μm. Podocyte-specific Sirt6 knockout mice: Cre + /Sirt6 fl/fl mice; Mice with two WT alleles and Cre expression ( Cre + /Sirt6 +/+ ) were used as controls. * P

    Article Snippet: Moreover, we observed the changes of Notch signaling in HG-treated podocytes with or without Sirt6 overexpression by Agilent Whole Human Genome Oligo Microarray for global gene expression analysis (Fig. ).

    Techniques: Knock-Out, Mouse Assay, Polymerase Chain Reaction, Staining, Marker, Western Blot, Expressing, Isolation, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy