mrna Search Results


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  • 99
    New England Biolabs nebnext poly a mrna magnetic isolation module
    Holo-Seq accurately profiles total <t>RNAs</t> with a complete strand of origin information from single cells. a RPKM scatterplots of expressed genes between the combined dataset (total RNA with a complete strand of origin information from 10 mESCs single cells) and a directional bulk <t>mRNA-Seq.</t> b Comparison of the detected gene number in HEK293T single cells at the maximum exome-mapped depth of MATQ-Seq (UMI labeled reads) and 1.2M unique exome-mapped depth of Holo-Seq, SUPeR-Seq, and Smart-Seq2. c Read coverage across transcripts of different lengths of three methods in HEK293T single cells. The read coverage over the transcripts is displayed along with the percentage of the distance from their 3′ end. Shaded regions indicate the standard deviation
    Nebnext Poly A Mrna Magnetic Isolation Module, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3210 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher mrna
    Holo-Seq accurately profiles total <t>RNAs</t> with a complete strand of origin information from single cells. a RPKM scatterplots of expressed genes between the combined dataset (total RNA with a complete strand of origin information from 10 mESCs single cells) and a directional bulk <t>mRNA-Seq.</t> b Comparison of the detected gene number in HEK293T single cells at the maximum exome-mapped depth of MATQ-Seq (UMI labeled reads) and 1.2M unique exome-mapped depth of Holo-Seq, SUPeR-Seq, and Smart-Seq2. c Read coverage across transcripts of different lengths of three methods in HEK293T single cells. The read coverage over the transcripts is displayed along with the percentage of the distance from their 3′ end. Shaded regions indicate the standard deviation
    Mrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 89710 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Exosome Diagnostics mrnas
    Cell-cell communication via exosomes and microvesicles Exosomes are incorporated into vesicles in MVBs by budding into their lumen. MVBs are fused with the plasma membrane releasing internal exosomes to outside the cell by exocytosis. Microvesicles are formed and released directly from the plasma membrane by outward blebbing. These EVs are taken up by the other cell by fusion with the plasma membrane or endocytosis and EV cargoes are transferred horizontally from the donor cell to the recipient cell. EVs can contain various molecules including <t>mRNAs,</t> miRNAs, pre-miRNAs and proteins. These EV cargoes regulate physiological cell events in the recipient cell. Some EVs are circulating through the body in extracellular fluid and their cargoes can be useful as a biomarker to diagnose liver diseases.
    Mrnas, supplied by Exosome Diagnostics, used in various techniques. Bioz Stars score: 92/100, based on 1093 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Illumina Inc truseq stranded mrna library prep kit
    Cell-cell communication via exosomes and microvesicles Exosomes are incorporated into vesicles in MVBs by budding into their lumen. MVBs are fused with the plasma membrane releasing internal exosomes to outside the cell by exocytosis. Microvesicles are formed and released directly from the plasma membrane by outward blebbing. These EVs are taken up by the other cell by fusion with the plasma membrane or endocytosis and EV cargoes are transferred horizontally from the donor cell to the recipient cell. EVs can contain various molecules including <t>mRNAs,</t> miRNAs, pre-miRNAs and proteins. These EV cargoes regulate physiological cell events in the recipient cell. Some EVs are circulating through the body in extracellular fluid and their cargoes can be useful as a biomarker to diagnose liver diseases.
    Truseq Stranded Mrna Library Prep Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 2678 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dynabeads mrna purification kit
    Cell-cell communication via exosomes and microvesicles Exosomes are incorporated into vesicles in MVBs by budding into their lumen. MVBs are fused with the plasma membrane releasing internal exosomes to outside the cell by exocytosis. Microvesicles are formed and released directly from the plasma membrane by outward blebbing. These EVs are taken up by the other cell by fusion with the plasma membrane or endocytosis and EV cargoes are transferred horizontally from the donor cell to the recipient cell. EVs can contain various molecules including <t>mRNAs,</t> miRNAs, pre-miRNAs and proteins. These EV cargoes regulate physiological cell events in the recipient cell. Some EVs are circulating through the body in extracellular fluid and their cargoes can be useful as a biomarker to diagnose liver diseases.
    Dynabeads Mrna Purification Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4204 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore polyadenylated rna
    m 6 A increases upon KSHV reactivation. (A) Schematic of the experimental setup. iSLK.219 cells were induced with doxycycline for 5 days to induce the lytic cycle, and total <t>RNA</t> was collected and subjected to oligo dT selection to purify poly(A) RNA. <t>Polyadenylated</t> RNA was spiked with 10 μM of 5-fluorouridine and digested with nuclease P1 and alkaline phosphatase, and subjected to LC-MS/MS analysis. (B) Relative m 6 A content in iSLK.219 cells. The induced sample was normalized with respect to the uninduced sample (set to 1).
    Polyadenylated Rna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dynabeads mrna direct kit
    m 6 A increases upon KSHV reactivation. (A) Schematic of the experimental setup. iSLK.219 cells were induced with doxycycline for 5 days to induce the lytic cycle, and total <t>RNA</t> was collected and subjected to oligo dT selection to purify poly(A) RNA. <t>Polyadenylated</t> RNA was spiked with 10 μM of 5-fluorouridine and digested with nuclease P1 and alkaline phosphatase, and subjected to LC-MS/MS analysis. (B) Relative m 6 A content in iSLK.219 cells. The induced sample was normalized with respect to the uninduced sample (set to 1).
    Dynabeads Mrna Direct Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3417 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bio-Rad mrna levels
    MIIP–RelA facilitates H3-K9 acetylation at promoter region. a HCT116 cells expressed with WT H3 or H3 K9R were treated with or without EGF (100 ng/ml) for 10 h. Relative <t>mRNA</t> levels were analyzed by <t>q-PCR.</t> b , d HCT116 cells expressed with WT RelA or RelA K310R were treated with or without EGF for 10 h (100 ng/ml). c , e HCT116 cells expressed with WT MIIP or MIIP S303A were overexpressed with or without HDAC6; cells were treated with or without EGF (100 ng/ml) for 10 h. f HCT116 cells transfected with or without plasmid for expressing p300 shRNA were treated with or without EGF (100 ng/ml) for 10 h. In b – f , ChIP analyses with indicated antibodies were performed. The primers covering RelA binding site of MMP2 gene promoter region were used for the q-PCR. The Y axis shows the value normalized to the input. In a – f , the values are presented as mean ± s.e.m. ( n = 3 independent experiments), ** represents P
    Mrna Levels, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 4343 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen oligotex mrna mini kit
    MIIP–RelA facilitates H3-K9 acetylation at promoter region. a HCT116 cells expressed with WT H3 or H3 K9R were treated with or without EGF (100 ng/ml) for 10 h. Relative <t>mRNA</t> levels were analyzed by <t>q-PCR.</t> b , d HCT116 cells expressed with WT RelA or RelA K310R were treated with or without EGF for 10 h (100 ng/ml). c , e HCT116 cells expressed with WT MIIP or MIIP S303A were overexpressed with or without HDAC6; cells were treated with or without EGF (100 ng/ml) for 10 h. f HCT116 cells transfected with or without plasmid for expressing p300 shRNA were treated with or without EGF (100 ng/ml) for 10 h. In b – f , ChIP analyses with indicated antibodies were performed. The primers covering RelA binding site of MMP2 gene promoter region were used for the q-PCR. The Y axis shows the value normalized to the input. In a – f , the values are presented as mean ± s.e.m. ( n = 3 independent experiments), ** represents P
    Oligotex Mrna Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 3294 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Illumina Inc truseq stranded mrna
    MIIP–RelA facilitates H3-K9 acetylation at promoter region. a HCT116 cells expressed with WT H3 or H3 K9R were treated with or without EGF (100 ng/ml) for 10 h. Relative <t>mRNA</t> levels were analyzed by <t>q-PCR.</t> b , d HCT116 cells expressed with WT RelA or RelA K310R were treated with or without EGF for 10 h (100 ng/ml). c , e HCT116 cells expressed with WT MIIP or MIIP S303A were overexpressed with or without HDAC6; cells were treated with or without EGF (100 ng/ml) for 10 h. f HCT116 cells transfected with or without plasmid for expressing p300 shRNA were treated with or without EGF (100 ng/ml) for 10 h. In b – f , ChIP analyses with indicated antibodies were performed. The primers covering RelA binding site of MMP2 gene promoter region were used for the q-PCR. The Y axis shows the value normalized to the input. In a – f , the values are presented as mean ± s.e.m. ( n = 3 independent experiments), ** represents P
    Truseq Stranded Mrna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 3113 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen oligotex mrna kit
    MIIP–RelA facilitates H3-K9 acetylation at promoter region. a HCT116 cells expressed with WT H3 or H3 K9R were treated with or without EGF (100 ng/ml) for 10 h. Relative <t>mRNA</t> levels were analyzed by <t>q-PCR.</t> b , d HCT116 cells expressed with WT RelA or RelA K310R were treated with or without EGF for 10 h (100 ng/ml). c , e HCT116 cells expressed with WT MIIP or MIIP S303A were overexpressed with or without HDAC6; cells were treated with or without EGF (100 ng/ml) for 10 h. f HCT116 cells transfected with or without plasmid for expressing p300 shRNA were treated with or without EGF (100 ng/ml) for 10 h. In b – f , ChIP analyses with indicated antibodies were performed. The primers covering RelA binding site of MMP2 gene promoter region were used for the q-PCR. The Y axis shows the value normalized to the input. In a – f , the values are presented as mean ± s.e.m. ( n = 3 independent experiments), ** represents P
    Oligotex Mrna Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1860 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 1860 article reviews
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    99
    Thermo Fisher mrna expression
    MIIP–RelA facilitates H3-K9 acetylation at promoter region. a HCT116 cells expressed with WT H3 or H3 K9R were treated with or without EGF (100 ng/ml) for 10 h. Relative <t>mRNA</t> levels were analyzed by <t>q-PCR.</t> b , d HCT116 cells expressed with WT RelA or RelA K310R were treated with or without EGF for 10 h (100 ng/ml). c , e HCT116 cells expressed with WT MIIP or MIIP S303A were overexpressed with or without HDAC6; cells were treated with or without EGF (100 ng/ml) for 10 h. f HCT116 cells transfected with or without plasmid for expressing p300 shRNA were treated with or without EGF (100 ng/ml) for 10 h. In b – f , ChIP analyses with indicated antibodies were performed. The primers covering RelA binding site of MMP2 gene promoter region were used for the q-PCR. The Y axis shows the value normalized to the input. In a – f , the values are presented as mean ± s.e.m. ( n = 3 independent experiments), ** represents P
    Mrna Expression, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 17193 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 17193 article reviews
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    99
    Thermo Fisher microbexpress bacterial mrna enrichment kit
    MIIP–RelA facilitates H3-K9 acetylation at promoter region. a HCT116 cells expressed with WT H3 or H3 K9R were treated with or without EGF (100 ng/ml) for 10 h. Relative <t>mRNA</t> levels were analyzed by <t>q-PCR.</t> b , d HCT116 cells expressed with WT RelA or RelA K310R were treated with or without EGF for 10 h (100 ng/ml). c , e HCT116 cells expressed with WT MIIP or MIIP S303A were overexpressed with or without HDAC6; cells were treated with or without EGF (100 ng/ml) for 10 h. f HCT116 cells transfected with or without plasmid for expressing p300 shRNA were treated with or without EGF (100 ng/ml) for 10 h. In b – f , ChIP analyses with indicated antibodies were performed. The primers covering RelA binding site of MMP2 gene promoter region were used for the q-PCR. The Y axis shows the value normalized to the input. In a – f , the values are presented as mean ± s.e.m. ( n = 3 independent experiments), ** represents P
    Microbexpress Bacterial Mrna Enrichment Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1072 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 1072 article reviews
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    99
    Thermo Fisher dynabeads mrna direct micro kit
    MIIP–RelA facilitates H3-K9 acetylation at promoter region. a HCT116 cells expressed with WT H3 or H3 K9R were treated with or without EGF (100 ng/ml) for 10 h. Relative <t>mRNA</t> levels were analyzed by <t>q-PCR.</t> b , d HCT116 cells expressed with WT RelA or RelA K310R were treated with or without EGF for 10 h (100 ng/ml). c , e HCT116 cells expressed with WT MIIP or MIIP S303A were overexpressed with or without HDAC6; cells were treated with or without EGF (100 ng/ml) for 10 h. f HCT116 cells transfected with or without plasmid for expressing p300 shRNA were treated with or without EGF (100 ng/ml) for 10 h. In b – f , ChIP analyses with indicated antibodies were performed. The primers covering RelA binding site of MMP2 gene promoter region were used for the q-PCR. The Y axis shows the value normalized to the input. In a – f , the values are presented as mean ± s.e.m. ( n = 3 independent experiments), ** represents P
    Dynabeads Mrna Direct Micro Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1134 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dynabeads mrna direct micro kit/product/Thermo Fisher
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    92
    Bio-Rad mrnas
    Identifying eIF4E-dependent and independent <t>mRNAs</t> associating with Caf20p. A. Proportional Venn style diagram showing the overlap between the mRNAs associating with Caf20p and Eap1p TAP IPs. B. Quantitative RT-PCR of selected mRNAs showing their association with Caf20p and <t>Caf20</t> m2 p represented as fold-change over control. Statistically significant changes are indicated with a key (T-test). C. and D. Venn style diagrams showing C. mRNAs associating with Caf20-TAP, Caf20-FLAG and Caf20 m2 -FLAG, highlighting the core mRNAs identified in both studies and their dependence on the eIF4E interaction motif for binding, and D. Overlap between the core 4E-DEP and 4E-IND Caf20p interacting mRNAs and the Eap1-TAP associated proteins.
    Mrnas, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 1617 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Ipsen Group syntaxin mrna
    The 3′ UTR of Aplysia <t>syntaxin</t> <t>mRNA.</t> The stop codon is boxed. The CPE (UUUUAU) and polyadenylation signal (AAUAAA) are in bold and underlined. A poly(A) tail would be added after the terminal cytosine residue.
    Syntaxin Mrna, supplied by Ipsen Group, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa poly a rna
    The 3′ UTR of Aplysia <t>syntaxin</t> <t>mRNA.</t> The stop codon is boxed. The CPE (UUUUAU) and polyadenylation signal (AAUAAA) are in bold and underlined. A poly(A) tail would be added after the terminal cytosine residue.
    Poly A Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 828 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    poly a rna - by Bioz Stars, 2021-01
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    N/A
    Our poly A RNA is enriched for mRNA transcripts with two rounds of oligo dT cellulose purification The mRNA is isolated from our total RNA collection which is meticulously prepared
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    N/A
    Our poly A RNA is enriched for mRNA transcripts with two rounds of oligo dT cellulose purification The mRNA is isolated from our total RNA collection which is meticulously prepared
      Buy from Supplier

    N/A
    Our poly A RNA is enriched for mRNA transcripts with two rounds of oligo dT cellulose purification The mRNA is isolated from our total RNA collection which is meticulously prepared
      Buy from Supplier

    N/A
    The protein encoded by this gene is a component of the eukaryotic translation initiation factor 4F complex which recognizes the 7 methylguanosine cap structure at the 5 end of messenger
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    Image Search Results


    Holo-Seq accurately profiles total RNAs with a complete strand of origin information from single cells. a RPKM scatterplots of expressed genes between the combined dataset (total RNA with a complete strand of origin information from 10 mESCs single cells) and a directional bulk mRNA-Seq. b Comparison of the detected gene number in HEK293T single cells at the maximum exome-mapped depth of MATQ-Seq (UMI labeled reads) and 1.2M unique exome-mapped depth of Holo-Seq, SUPeR-Seq, and Smart-Seq2. c Read coverage across transcripts of different lengths of three methods in HEK293T single cells. The read coverage over the transcripts is displayed along with the percentage of the distance from their 3′ end. Shaded regions indicate the standard deviation

    Journal: Genome Biology

    Article Title: Holo-Seq: single-cell sequencing of holo-transcriptome

    doi: 10.1186/s13059-018-1553-7

    Figure Lengend Snippet: Holo-Seq accurately profiles total RNAs with a complete strand of origin information from single cells. a RPKM scatterplots of expressed genes between the combined dataset (total RNA with a complete strand of origin information from 10 mESCs single cells) and a directional bulk mRNA-Seq. b Comparison of the detected gene number in HEK293T single cells at the maximum exome-mapped depth of MATQ-Seq (UMI labeled reads) and 1.2M unique exome-mapped depth of Holo-Seq, SUPeR-Seq, and Smart-Seq2. c Read coverage across transcripts of different lengths of three methods in HEK293T single cells. The read coverage over the transcripts is displayed along with the percentage of the distance from their 3′ end. Shaded regions indicate the standard deviation

    Article Snippet: The poly-A RNAs were selected using a NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490), and the small RNAs remained in the supernatant after poly-A selection.

    Techniques: Labeling, Standard Deviation

    Cell-cell communication via exosomes and microvesicles Exosomes are incorporated into vesicles in MVBs by budding into their lumen. MVBs are fused with the plasma membrane releasing internal exosomes to outside the cell by exocytosis. Microvesicles are formed and released directly from the plasma membrane by outward blebbing. These EVs are taken up by the other cell by fusion with the plasma membrane or endocytosis and EV cargoes are transferred horizontally from the donor cell to the recipient cell. EVs can contain various molecules including mRNAs, miRNAs, pre-miRNAs and proteins. These EV cargoes regulate physiological cell events in the recipient cell. Some EVs are circulating through the body in extracellular fluid and their cargoes can be useful as a biomarker to diagnose liver diseases.

    Journal: Journal of hepatology

    Article Title: Exosomes in liver pathology

    doi: 10.1016/j.jhep.2016.03.004

    Figure Lengend Snippet: Cell-cell communication via exosomes and microvesicles Exosomes are incorporated into vesicles in MVBs by budding into their lumen. MVBs are fused with the plasma membrane releasing internal exosomes to outside the cell by exocytosis. Microvesicles are formed and released directly from the plasma membrane by outward blebbing. These EVs are taken up by the other cell by fusion with the plasma membrane or endocytosis and EV cargoes are transferred horizontally from the donor cell to the recipient cell. EVs can contain various molecules including mRNAs, miRNAs, pre-miRNAs and proteins. These EV cargoes regulate physiological cell events in the recipient cell. Some EVs are circulating through the body in extracellular fluid and their cargoes can be useful as a biomarker to diagnose liver diseases.

    Article Snippet: Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.

    Techniques: Biomarker Assay

    miRNA synthesis and contents of exosomes The pathway of miRNA synthesis and its function (top). In the nucleus, miRNAs are transcribed from DNA forming a primary transcript, pri-miRNA. The RNase III enzyme DROSHA cleaves pri-miRNA to produce a precursor, pre-miRNA that is transported to the cytoplasm through a nuclear export protein Exportin-5. In the cytoplasm, pre-miRNAs are cleaved by DICER to form a miRNA duplex. In general, only one strand of the miRNA duplex is used as the mature miRNA while the other strand is degraded because of less stability. Mature miRNA binds to the 3′UTR of target mRNA and is loaded into RNA-induced silencing complex (RISC). As a result, this complex inhibits the process of mRNA translation or enhances mRNA degradation leading to translational suppression. Contents of exosomes (bottom). Typical exosomes contain various cargoes including pre-miRNAs, mature miRNAs, and mRNAs as well as membrane marker proteins, endosome-associated (MVB-derived) proteins, and heat shock proteins (chaperones).

    Journal: Journal of hepatology

    Article Title: Exosomes in liver pathology

    doi: 10.1016/j.jhep.2016.03.004

    Figure Lengend Snippet: miRNA synthesis and contents of exosomes The pathway of miRNA synthesis and its function (top). In the nucleus, miRNAs are transcribed from DNA forming a primary transcript, pri-miRNA. The RNase III enzyme DROSHA cleaves pri-miRNA to produce a precursor, pre-miRNA that is transported to the cytoplasm through a nuclear export protein Exportin-5. In the cytoplasm, pre-miRNAs are cleaved by DICER to form a miRNA duplex. In general, only one strand of the miRNA duplex is used as the mature miRNA while the other strand is degraded because of less stability. Mature miRNA binds to the 3′UTR of target mRNA and is loaded into RNA-induced silencing complex (RISC). As a result, this complex inhibits the process of mRNA translation or enhances mRNA degradation leading to translational suppression. Contents of exosomes (bottom). Typical exosomes contain various cargoes including pre-miRNAs, mature miRNAs, and mRNAs as well as membrane marker proteins, endosome-associated (MVB-derived) proteins, and heat shock proteins (chaperones).

    Article Snippet: Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.

    Techniques: Marker, Derivative Assay

    m 6 A increases upon KSHV reactivation. (A) Schematic of the experimental setup. iSLK.219 cells were induced with doxycycline for 5 days to induce the lytic cycle, and total RNA was collected and subjected to oligo dT selection to purify poly(A) RNA. Polyadenylated RNA was spiked with 10 μM of 5-fluorouridine and digested with nuclease P1 and alkaline phosphatase, and subjected to LC-MS/MS analysis. (B) Relative m 6 A content in iSLK.219 cells. The induced sample was normalized with respect to the uninduced sample (set to 1).

    Journal: PLoS Pathogens

    Article Title: N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection

    doi: 10.1371/journal.ppat.1006995

    Figure Lengend Snippet: m 6 A increases upon KSHV reactivation. (A) Schematic of the experimental setup. iSLK.219 cells were induced with doxycycline for 5 days to induce the lytic cycle, and total RNA was collected and subjected to oligo dT selection to purify poly(A) RNA. Polyadenylated RNA was spiked with 10 μM of 5-fluorouridine and digested with nuclease P1 and alkaline phosphatase, and subjected to LC-MS/MS analysis. (B) Relative m 6 A content in iSLK.219 cells. The induced sample was normalized with respect to the uninduced sample (set to 1).

    Article Snippet: 100–200 ng of polyadenylated RNA was spiked with 10 μM of 5-fluorouridine (Sigma) and digested by nuclease P1 (1 U) in 25 μL of buffer containing 25 mM NaCl and 2.5 mM ZnCl2 at 42°C for 2–4 hr, followed by addition of NH4 HCO3 (1 M, 3 μL) and bacterial alkaline phosphatase (1 U) and incubation at 37°C for 2 hr.

    Techniques: Selection, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    MIIP–RelA facilitates H3-K9 acetylation at promoter region. a HCT116 cells expressed with WT H3 or H3 K9R were treated with or without EGF (100 ng/ml) for 10 h. Relative mRNA levels were analyzed by q-PCR. b , d HCT116 cells expressed with WT RelA or RelA K310R were treated with or without EGF for 10 h (100 ng/ml). c , e HCT116 cells expressed with WT MIIP or MIIP S303A were overexpressed with or without HDAC6; cells were treated with or without EGF (100 ng/ml) for 10 h. f HCT116 cells transfected with or without plasmid for expressing p300 shRNA were treated with or without EGF (100 ng/ml) for 10 h. In b – f , ChIP analyses with indicated antibodies were performed. The primers covering RelA binding site of MMP2 gene promoter region were used for the q-PCR. The Y axis shows the value normalized to the input. In a – f , the values are presented as mean ± s.e.m. ( n = 3 independent experiments), ** represents P

    Journal: Nature Communications

    Article Title: PKCε phosphorylates MIIP and promotes colorectal cancer metastasis through inhibition of RelA deacetylation

    doi: 10.1038/s41467-017-01024-2

    Figure Lengend Snippet: MIIP–RelA facilitates H3-K9 acetylation at promoter region. a HCT116 cells expressed with WT H3 or H3 K9R were treated with or without EGF (100 ng/ml) for 10 h. Relative mRNA levels were analyzed by q-PCR. b , d HCT116 cells expressed with WT RelA or RelA K310R were treated with or without EGF for 10 h (100 ng/ml). c , e HCT116 cells expressed with WT MIIP or MIIP S303A were overexpressed with or without HDAC6; cells were treated with or without EGF (100 ng/ml) for 10 h. f HCT116 cells transfected with or without plasmid for expressing p300 shRNA were treated with or without EGF (100 ng/ml) for 10 h. In b – f , ChIP analyses with indicated antibodies were performed. The primers covering RelA binding site of MMP2 gene promoter region were used for the q-PCR. The Y axis shows the value normalized to the input. In a – f , the values are presented as mean ± s.e.m. ( n = 3 independent experiments), ** represents P

    Article Snippet: We synthesized cDNA from 1 μg total RNA using iScript cDNA synthesis kit (Bio-Rad) and quantified mRNA levels by real-time qRT-PCR using SYBR Green (Bio-Rad).

    Techniques: Polymerase Chain Reaction, Transfection, Plasmid Preparation, Expressing, shRNA, Chromatin Immunoprecipitation, Binding Assay

    EGF induced the interaction between MIIP and RelA. a HCT116 cells were treated with or without EGF. Cellular nucleus-extracts subjected to immunoprecipitation with an anti-MIIP antibody. b HCT116 cells transfected with or without plasmid for expressing the indicated MIIP shRNA were treated with or without EGF for indicated periods of time. c , d HCT116 cells transfected with or without plasmid for expressing MIIP shRNA ( c ), and HCT116 cells expressed with wild type (WT) RelA and RelA K310R ( d ) were treated with or without EGF (100 ng/ml). Cell invasion assays were performed. e HCT116 cells expressed with WT RelA and RelA K310R were treated with or without EGF (100 ng/ml) for 10 h. Relative mRNA levels were analyzed by q-PCR. f HCT116 cells transfected with or without plasmid for expressing MIIP shRNA were treated with or without EGF (100 ng/ml) for 10 h. ChIP analyses with an anti-RelA Ac-K310 antibody were performed. The primers covering RelA binding site of MMP2 gene promoter region were used for the q-PCR. The Y axis shows the value normalized to the input. g HCT116 cells were pretreated with Bis-I (2 μM), U0126 (20 μM) for 1 h, prior to EGF treatment (100 ng/ml) for 30 min. Cellular extracts subjected to immunoprecipitation with an anti-Flag antibody. h Purified GST-MIIP protein was mixed with mitotic extracts from HCT116 cells treated with EGF (100 ng/ml) for 30 min. GST pull down analyses were performed (left panel). Purified GST–RelA protein was mixed with mitotic extracts from HCT116 cells treated with EGF (100 ng/ml) for 30 min (right panel). In a , b , g , h , immunoblotting analyses were performed using the indicated antibodies and data represent one out of three experiments. In c – f , the values are presented as mean ± s.e.m. ( n = 3 independent experiments), * represents P

    Journal: Nature Communications

    Article Title: PKCε phosphorylates MIIP and promotes colorectal cancer metastasis through inhibition of RelA deacetylation

    doi: 10.1038/s41467-017-01024-2

    Figure Lengend Snippet: EGF induced the interaction between MIIP and RelA. a HCT116 cells were treated with or without EGF. Cellular nucleus-extracts subjected to immunoprecipitation with an anti-MIIP antibody. b HCT116 cells transfected with or without plasmid for expressing the indicated MIIP shRNA were treated with or without EGF for indicated periods of time. c , d HCT116 cells transfected with or without plasmid for expressing MIIP shRNA ( c ), and HCT116 cells expressed with wild type (WT) RelA and RelA K310R ( d ) were treated with or without EGF (100 ng/ml). Cell invasion assays were performed. e HCT116 cells expressed with WT RelA and RelA K310R were treated with or without EGF (100 ng/ml) for 10 h. Relative mRNA levels were analyzed by q-PCR. f HCT116 cells transfected with or without plasmid for expressing MIIP shRNA were treated with or without EGF (100 ng/ml) for 10 h. ChIP analyses with an anti-RelA Ac-K310 antibody were performed. The primers covering RelA binding site of MMP2 gene promoter region were used for the q-PCR. The Y axis shows the value normalized to the input. g HCT116 cells were pretreated with Bis-I (2 μM), U0126 (20 μM) for 1 h, prior to EGF treatment (100 ng/ml) for 30 min. Cellular extracts subjected to immunoprecipitation with an anti-Flag antibody. h Purified GST-MIIP protein was mixed with mitotic extracts from HCT116 cells treated with EGF (100 ng/ml) for 30 min. GST pull down analyses were performed (left panel). Purified GST–RelA protein was mixed with mitotic extracts from HCT116 cells treated with EGF (100 ng/ml) for 30 min (right panel). In a , b , g , h , immunoblotting analyses were performed using the indicated antibodies and data represent one out of three experiments. In c – f , the values are presented as mean ± s.e.m. ( n = 3 independent experiments), * represents P

    Article Snippet: We synthesized cDNA from 1 μg total RNA using iScript cDNA synthesis kit (Bio-Rad) and quantified mRNA levels by real-time qRT-PCR using SYBR Green (Bio-Rad).

    Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Expressing, shRNA, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Binding Assay, Purification

    PKCε phosphorylated MIIP and promoted MIIP–RelA interaction. a HCT116 cells were treated with or without EGF. Cellular extracts subjected to immunoprecipitation with an anti-Flag antibody. The immunoprecipitates were treated with CIP (10 units), followed by immunoblotting analysis. b , c In vitro phosphorylation analyses were performed by mixing the purified active PKCε with the indicated purified GST-MIIP proteins in the presence of [γ-32P]ATP. Ser303 of MIIP is evolutionarily conserved in the indicated species ( c , left panel). d HCT116 cells expressed with WT MIIP or MIIP S303A were treated with or without EGF for 30 min. e HCT116 cells expressed with WT MIIP or MIIP S303A were treated with or without EGF (100 ng/ml) for 10 h. f HCT116 cells with depletion of MIIP, and reconstituted expression of WT rMIIP or rMIIP S303A were treated with or without EGF (100 ng/ml) for 10 h. g HCT116 cells with depletion of MIIP, and reconstituted expression of WT rMIIP or rMIIP S303A were treated with or without EGF for 10 h. Relative mRNA levels were analyzed by q-PCR. h HCT116 cells with depletion of MIIP, and reconstituted expression of WT rMIIP or rMIIP S303A were treated with or without EGF (100 ng/ml). Cell invasion assays were performed. In e , f , ChIP analyses with indicated antibodies were performed. The primers covering RelA binding site of MMP2 gene promoter region were used for the q-PCR. The Y axis shows the value normalized to the input. In a – d , immunoblotting analyses were performed using the indicated antibodies. In e – h , the values are presented as mean ± s.e.m. ( n = 3 independent experiments), ** represents P

    Journal: Nature Communications

    Article Title: PKCε phosphorylates MIIP and promotes colorectal cancer metastasis through inhibition of RelA deacetylation

    doi: 10.1038/s41467-017-01024-2

    Figure Lengend Snippet: PKCε phosphorylated MIIP and promoted MIIP–RelA interaction. a HCT116 cells were treated with or without EGF. Cellular extracts subjected to immunoprecipitation with an anti-Flag antibody. The immunoprecipitates were treated with CIP (10 units), followed by immunoblotting analysis. b , c In vitro phosphorylation analyses were performed by mixing the purified active PKCε with the indicated purified GST-MIIP proteins in the presence of [γ-32P]ATP. Ser303 of MIIP is evolutionarily conserved in the indicated species ( c , left panel). d HCT116 cells expressed with WT MIIP or MIIP S303A were treated with or without EGF for 30 min. e HCT116 cells expressed with WT MIIP or MIIP S303A were treated with or without EGF (100 ng/ml) for 10 h. f HCT116 cells with depletion of MIIP, and reconstituted expression of WT rMIIP or rMIIP S303A were treated with or without EGF (100 ng/ml) for 10 h. g HCT116 cells with depletion of MIIP, and reconstituted expression of WT rMIIP or rMIIP S303A were treated with or without EGF for 10 h. Relative mRNA levels were analyzed by q-PCR. h HCT116 cells with depletion of MIIP, and reconstituted expression of WT rMIIP or rMIIP S303A were treated with or without EGF (100 ng/ml). Cell invasion assays were performed. In e , f , ChIP analyses with indicated antibodies were performed. The primers covering RelA binding site of MMP2 gene promoter region were used for the q-PCR. The Y axis shows the value normalized to the input. In a – d , immunoblotting analyses were performed using the indicated antibodies. In e – h , the values are presented as mean ± s.e.m. ( n = 3 independent experiments), ** represents P

    Article Snippet: We synthesized cDNA from 1 μg total RNA using iScript cDNA synthesis kit (Bio-Rad) and quantified mRNA levels by real-time qRT-PCR using SYBR Green (Bio-Rad).

    Techniques: Immunoprecipitation, In Vitro, Purification, Expressing, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Binding Assay

    MIIP prevent HDAC6-mediated RelA deacetylation. a HCT116 cells were pretreated with or without 4SC-202 (0.6 μM), FK228(20 nM) and Nexturastat A (5 nM) for 1 h prior to EGF (100 ng/ml) treatment for 30 min. Immunoblotting analyses were performed. b HCT116 cells were pretreated with or without Bis-l for 1 h prior to EGF (100 ng/ml) treatment for 30 min. Cellular extracts subjected to immunoprecipitation with an anti-Flag, followed by Flag-beads washing and a second immunoprecipitation with an anti-MIIP (lanes 1–4 from left). Cellular extracts subjected to immunoprecipitation with an anti-Flag (lanes 5–8 from left). c – e HCT116 cells expressed with WT MIIP or MIIP S303A were overexpressed with or without HDAC6 shRNA; cells were treated with or without EGF (100 ng/ml) for 10 h. Immunoblotting analyses were performed ( c ). ChIP analyses with an anti-RelA Ac-K310 antibody were performed. The primers covering RelA binding site of Twist or MMP2 gene promoter region were used for the q-PCR. The Y axis shows the value normalized to the input. d Relative mRNA levels were analyzed by q-PCR ( e ). In a – c , immunoblotting analyses were performed using the indicated antibodies and data represent one out of three experiments. In d , e , the values are presented as mean ± s.e.m. ( n = 3 independent experiments), ** represents P

    Journal: Nature Communications

    Article Title: PKCε phosphorylates MIIP and promotes colorectal cancer metastasis through inhibition of RelA deacetylation

    doi: 10.1038/s41467-017-01024-2

    Figure Lengend Snippet: MIIP prevent HDAC6-mediated RelA deacetylation. a HCT116 cells were pretreated with or without 4SC-202 (0.6 μM), FK228(20 nM) and Nexturastat A (5 nM) for 1 h prior to EGF (100 ng/ml) treatment for 30 min. Immunoblotting analyses were performed. b HCT116 cells were pretreated with or without Bis-l for 1 h prior to EGF (100 ng/ml) treatment for 30 min. Cellular extracts subjected to immunoprecipitation with an anti-Flag, followed by Flag-beads washing and a second immunoprecipitation with an anti-MIIP (lanes 1–4 from left). Cellular extracts subjected to immunoprecipitation with an anti-Flag (lanes 5–8 from left). c – e HCT116 cells expressed with WT MIIP or MIIP S303A were overexpressed with or without HDAC6 shRNA; cells were treated with or without EGF (100 ng/ml) for 10 h. Immunoblotting analyses were performed ( c ). ChIP analyses with an anti-RelA Ac-K310 antibody were performed. The primers covering RelA binding site of Twist or MMP2 gene promoter region were used for the q-PCR. The Y axis shows the value normalized to the input. d Relative mRNA levels were analyzed by q-PCR ( e ). In a – c , immunoblotting analyses were performed using the indicated antibodies and data represent one out of three experiments. In d , e , the values are presented as mean ± s.e.m. ( n = 3 independent experiments), ** represents P

    Article Snippet: We synthesized cDNA from 1 μg total RNA using iScript cDNA synthesis kit (Bio-Rad) and quantified mRNA levels by real-time qRT-PCR using SYBR Green (Bio-Rad).

    Techniques: Immunoprecipitation, shRNA, Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction

    PP1 mediates MIIP dephosphorylation. a SW480 cells were treated with or without EGF. Cellular extracts subjected to immunoprecipitation with an anti-MIIP. b SW480 cells pretreated with Okadaic acid (a PP1 and PP2A inhibitor) (30 nM) were stimulated with or without EGF (100 ng/ml) for 30 min. c HCT116 cells transfected with or without plasmid for expressing PP1 were treated with or without EGF (100 ng/ml) for 30 min. d In vitro dephosphorylation analyses were performed by mixing the purified active PP1 with PKCε-phosphorylated GST-MIIP proteins in absence or presence of Na3VO4/Okadaic acid. e – h HCT116 cells expressing Flag-MIIP were transfected with or without plasmid for expressing PP1. Cells were treated with or without EGF (100 ng/ml) for 10 h. Cellular extracts subjected to immunoprecipitation with an anti-MIIP ( e ). ChIP analyses were performed. The primers covering RelA binding site of Twist or MMP2 gene promoter region were used for the q-PCR ( f , g ). Relative mRNA levels were analyzed by q-PCR ( h ). ( i ) HCT116 cells transfected with or without plasmid for expressing PP1 was treated with or without EGF (100 ng/ml). Cell invasion assays were performed. In f , g , ChIP analyses with indicated antibodies were performed. The primers covering RelA binding site of MMP2 gene promoter region were used for the q-PCR. The Y axis shows the value normalized to the input. In a – e , immunoblotting analyses were performed using the indicated antibodies. In a – e , immunoblotting analyses were performed using the indicated antibodies and data represent one out of three experiments. In g , f – i , the values are presented as mean ± s.e.m. ( n = 3 independent experiments), * represents P

    Journal: Nature Communications

    Article Title: PKCε phosphorylates MIIP and promotes colorectal cancer metastasis through inhibition of RelA deacetylation

    doi: 10.1038/s41467-017-01024-2

    Figure Lengend Snippet: PP1 mediates MIIP dephosphorylation. a SW480 cells were treated with or without EGF. Cellular extracts subjected to immunoprecipitation with an anti-MIIP. b SW480 cells pretreated with Okadaic acid (a PP1 and PP2A inhibitor) (30 nM) were stimulated with or without EGF (100 ng/ml) for 30 min. c HCT116 cells transfected with or without plasmid for expressing PP1 were treated with or without EGF (100 ng/ml) for 30 min. d In vitro dephosphorylation analyses were performed by mixing the purified active PP1 with PKCε-phosphorylated GST-MIIP proteins in absence or presence of Na3VO4/Okadaic acid. e – h HCT116 cells expressing Flag-MIIP were transfected with or without plasmid for expressing PP1. Cells were treated with or without EGF (100 ng/ml) for 10 h. Cellular extracts subjected to immunoprecipitation with an anti-MIIP ( e ). ChIP analyses were performed. The primers covering RelA binding site of Twist or MMP2 gene promoter region were used for the q-PCR ( f , g ). Relative mRNA levels were analyzed by q-PCR ( h ). ( i ) HCT116 cells transfected with or without plasmid for expressing PP1 was treated with or without EGF (100 ng/ml). Cell invasion assays were performed. In f , g , ChIP analyses with indicated antibodies were performed. The primers covering RelA binding site of MMP2 gene promoter region were used for the q-PCR. The Y axis shows the value normalized to the input. In a – e , immunoblotting analyses were performed using the indicated antibodies. In a – e , immunoblotting analyses were performed using the indicated antibodies and data represent one out of three experiments. In g , f – i , the values are presented as mean ± s.e.m. ( n = 3 independent experiments), * represents P

    Article Snippet: We synthesized cDNA from 1 μg total RNA using iScript cDNA synthesis kit (Bio-Rad) and quantified mRNA levels by real-time qRT-PCR using SYBR Green (Bio-Rad).

    Techniques: De-Phosphorylation Assay, Immunoprecipitation, Transfection, Plasmid Preparation, Expressing, In Vitro, Purification, Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction

    Neuronal PPARδ knockdown and brain morphology. (A) PPARδ gene expression in mediobasal hypothalamus of control (f/f), heterozygous KO (het) and homozygous KO (KO) PPARδ mice. Target gene PPARδ mRNA expression was measured by RT PCR and normalized to endogenous levels of the housekeeping gene RPL13A. (B) Representative Western blot of PPARδ protein levels in total cellular protein extracts from mediobasal hypothalamus of f/f and KO mice. β-tubulin was used as a loading control. (C) Quantification of PPARδ mRNA expression in peripheral and CNS tissues of f/f and KO mice (muscle, liver, white adipose tissue (WAT), brown adipose tissue (BAT), cerebral cortex and hypothalamus). Gene expression was measured by RT PCR and normalized to endogenous levels of the housekeeping gene RPL13A (n = 4–8). (D) Photomicrographs of Nissl staining in brains from f/f, nestin cre+ control and KO mice. Representative sections shown at the level of the hippocampus (top) and hypothalamus (bottom). No obvious differences or malformations in the structure of these or any other forebrain nuclei were observed across genotypes. Scale bar = 500 µm. Values in panels A and C represent the genotype group mean ± SEM, expressed relative to the levels of the f/f control group. Statistical significance is designated as * ( p

    Journal: PLoS ONE

    Article Title: Neuron-Specific Deletion of Peroxisome Proliferator-Activated Receptor Delta (PPAR?) in Mice Leads to Increased Susceptibility to Diet-Induced Obesity

    doi: 10.1371/journal.pone.0042981

    Figure Lengend Snippet: Neuronal PPARδ knockdown and brain morphology. (A) PPARδ gene expression in mediobasal hypothalamus of control (f/f), heterozygous KO (het) and homozygous KO (KO) PPARδ mice. Target gene PPARδ mRNA expression was measured by RT PCR and normalized to endogenous levels of the housekeeping gene RPL13A. (B) Representative Western blot of PPARδ protein levels in total cellular protein extracts from mediobasal hypothalamus of f/f and KO mice. β-tubulin was used as a loading control. (C) Quantification of PPARδ mRNA expression in peripheral and CNS tissues of f/f and KO mice (muscle, liver, white adipose tissue (WAT), brown adipose tissue (BAT), cerebral cortex and hypothalamus). Gene expression was measured by RT PCR and normalized to endogenous levels of the housekeeping gene RPL13A (n = 4–8). (D) Photomicrographs of Nissl staining in brains from f/f, nestin cre+ control and KO mice. Representative sections shown at the level of the hippocampus (top) and hypothalamus (bottom). No obvious differences or malformations in the structure of these or any other forebrain nuclei were observed across genotypes. Scale bar = 500 µm. Values in panels A and C represent the genotype group mean ± SEM, expressed relative to the levels of the f/f control group. Statistical significance is designated as * ( p

    Article Snippet: The resulting cDNA template was used to quantify mRNA expression via quantitative real-time PCR on a Bio-Rad iCycler using iQ SYBR green Supermix reagent (Bio-Rad; Hercules, CA).

    Techniques: Expressing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining

    White adipose tissue hypertrophy and inflammation. ( A ) Light micrographs (×10 magnification) of H E stained slides of WAT from f/f and KO mice fed chow or HFD for 33 weeks. Arrows point to crown-like structures (CLS), of areas of macrophage infiltration and inflammation. (B) Quantification of CLS (CLS/10× field, n = 4) corresponding to inflammatory macrophage infiltration around adipocytes in f/f and KO mice fed either a chow diet or a HFD. Adipose gene expression of inflammatory cytokine (C) TNFα and adipogenesis markers (D) PPARγ and (E) LPL measured by RT PCR. Target gene mRNA levels were normalized to endogenous RPL13A levels. Values are represented as group mean ± SEM relative to the f/f LF group. Statistical significance is designated as b ( p

    Journal: PLoS ONE

    Article Title: Neuron-Specific Deletion of Peroxisome Proliferator-Activated Receptor Delta (PPAR?) in Mice Leads to Increased Susceptibility to Diet-Induced Obesity

    doi: 10.1371/journal.pone.0042981

    Figure Lengend Snippet: White adipose tissue hypertrophy and inflammation. ( A ) Light micrographs (×10 magnification) of H E stained slides of WAT from f/f and KO mice fed chow or HFD for 33 weeks. Arrows point to crown-like structures (CLS), of areas of macrophage infiltration and inflammation. (B) Quantification of CLS (CLS/10× field, n = 4) corresponding to inflammatory macrophage infiltration around adipocytes in f/f and KO mice fed either a chow diet or a HFD. Adipose gene expression of inflammatory cytokine (C) TNFα and adipogenesis markers (D) PPARγ and (E) LPL measured by RT PCR. Target gene mRNA levels were normalized to endogenous RPL13A levels. Values are represented as group mean ± SEM relative to the f/f LF group. Statistical significance is designated as b ( p

    Article Snippet: The resulting cDNA template was used to quantify mRNA expression via quantitative real-time PCR on a Bio-Rad iCycler using iQ SYBR green Supermix reagent (Bio-Rad; Hercules, CA).

    Techniques: Staining, Mouse Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

    Neuronal PPARδ deletion alters hypothalamic neuropeptide gene expression and compensatory hyperphagia after prolonged fasting. Hypothalamic mRNA levels of neuropeptides in f/f and KO mice fed LFD or HFD for 33 weeks (n = 6–7). Target gene mRNA levels of (A) NPY and (B) POMC were assessed by quantitative RT PCR. (C–D) Fasting induced changes in hypothalamic neuropeptide mRNA levels of f/f and KO mice maintained on a chow diet or fasted for 24 hours. Target gene mRNA levels of (C) NPY, (D) POMC and (E) UCP2 in fed and fasted mice were normalized to RPL13A and are expressed relative to the f/f, fed control. (F) Refeeding after fasting was measured for an additional 24 hours in a separate cohort of individually housed chow fed mice (n = 6–7). Graph shows food intake normalized to basal, pre-fast lean mass (kcal/g lean mass). (G) Percent changes in body weight after a 24 hour fast, after fasting and refeeding for 24 hours, or an additional 7 days. Values represent the mean ± SEM. Statistical significance in panel A–E is designated as a ( p

    Journal: PLoS ONE

    Article Title: Neuron-Specific Deletion of Peroxisome Proliferator-Activated Receptor Delta (PPAR?) in Mice Leads to Increased Susceptibility to Diet-Induced Obesity

    doi: 10.1371/journal.pone.0042981

    Figure Lengend Snippet: Neuronal PPARδ deletion alters hypothalamic neuropeptide gene expression and compensatory hyperphagia after prolonged fasting. Hypothalamic mRNA levels of neuropeptides in f/f and KO mice fed LFD or HFD for 33 weeks (n = 6–7). Target gene mRNA levels of (A) NPY and (B) POMC were assessed by quantitative RT PCR. (C–D) Fasting induced changes in hypothalamic neuropeptide mRNA levels of f/f and KO mice maintained on a chow diet or fasted for 24 hours. Target gene mRNA levels of (C) NPY, (D) POMC and (E) UCP2 in fed and fasted mice were normalized to RPL13A and are expressed relative to the f/f, fed control. (F) Refeeding after fasting was measured for an additional 24 hours in a separate cohort of individually housed chow fed mice (n = 6–7). Graph shows food intake normalized to basal, pre-fast lean mass (kcal/g lean mass). (G) Percent changes in body weight after a 24 hour fast, after fasting and refeeding for 24 hours, or an additional 7 days. Values represent the mean ± SEM. Statistical significance in panel A–E is designated as a ( p

    Article Snippet: The resulting cDNA template was used to quantify mRNA expression via quantitative real-time PCR on a Bio-Rad iCycler using iQ SYBR green Supermix reagent (Bio-Rad; Hercules, CA).

    Techniques: Expressing, Mouse Assay, Quantitative RT-PCR

    Gene-diet interactions determine hypothalamic inflammatory signaling and gene expression in neuronal PPARδ KO mice. Protein and mRNA were isolated from bisected sections of mediobasal hypothalamus of f/f and KO mice fed LFD or HFD for 33 weeks. (A) Total hypothalamic protein extracts were subjected to Western blot analysis using an antibody directed against IκBα. Levels of β-tubulin were determined and used as a loading control. Densitometery of blots yielded relative intensity of protein levels (n = 6). Insert of A shows a representative Western blot. Hypothalamic mRNA levels of (B) IκBα and inflammatory cytokines (C) IL-6 and (D) IL-1β in f/f and KO mice measured by RT-PCR after the study period. Target gene mRNA levels were normalized to endogenous RPL13A levels. Values are represented as group mean ± SEM relative to the LF f/f control group. Statistical significance is designated by a ( p

    Journal: PLoS ONE

    Article Title: Neuron-Specific Deletion of Peroxisome Proliferator-Activated Receptor Delta (PPAR?) in Mice Leads to Increased Susceptibility to Diet-Induced Obesity

    doi: 10.1371/journal.pone.0042981

    Figure Lengend Snippet: Gene-diet interactions determine hypothalamic inflammatory signaling and gene expression in neuronal PPARδ KO mice. Protein and mRNA were isolated from bisected sections of mediobasal hypothalamus of f/f and KO mice fed LFD or HFD for 33 weeks. (A) Total hypothalamic protein extracts were subjected to Western blot analysis using an antibody directed against IκBα. Levels of β-tubulin were determined and used as a loading control. Densitometery of blots yielded relative intensity of protein levels (n = 6). Insert of A shows a representative Western blot. Hypothalamic mRNA levels of (B) IκBα and inflammatory cytokines (C) IL-6 and (D) IL-1β in f/f and KO mice measured by RT-PCR after the study period. Target gene mRNA levels were normalized to endogenous RPL13A levels. Values are represented as group mean ± SEM relative to the LF f/f control group. Statistical significance is designated by a ( p

    Article Snippet: The resulting cDNA template was used to quantify mRNA expression via quantitative real-time PCR on a Bio-Rad iCycler using iQ SYBR green Supermix reagent (Bio-Rad; Hercules, CA).

    Techniques: Expressing, Mouse Assay, Isolation, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Effects of neuronal PPARδ deletion on hypothalamic PPARγ and PPARα expression. Hypothalamic mRNA expression of PPAR isoforms in f/f and KO mice fed LFD or HFD for 33 weeks was assessed by quantitative real-time PCR. Changes in PPARδ, PPARγ and PPARα were normalized to endogenous RPL13A levels and expressed relative to that of the f/f LFD group. Values represent group mean±SEM (n = 6–7). Statistical significance is designated a ( p

    Journal: PLoS ONE

    Article Title: Neuron-Specific Deletion of Peroxisome Proliferator-Activated Receptor Delta (PPAR?) in Mice Leads to Increased Susceptibility to Diet-Induced Obesity

    doi: 10.1371/journal.pone.0042981

    Figure Lengend Snippet: Effects of neuronal PPARδ deletion on hypothalamic PPARγ and PPARα expression. Hypothalamic mRNA expression of PPAR isoforms in f/f and KO mice fed LFD or HFD for 33 weeks was assessed by quantitative real-time PCR. Changes in PPARδ, PPARγ and PPARα were normalized to endogenous RPL13A levels and expressed relative to that of the f/f LFD group. Values represent group mean±SEM (n = 6–7). Statistical significance is designated a ( p

    Article Snippet: The resulting cDNA template was used to quantify mRNA expression via quantitative real-time PCR on a Bio-Rad iCycler using iQ SYBR green Supermix reagent (Bio-Rad; Hercules, CA).

    Techniques: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    Effects of dietary fat and PPARδ deletion on brain lipids, fatty acid composition and lipid metabolism genes. (A) Total levels of triglyceride (TG), diglyceride (DG) and free fatty acid (FFA) extracted from total lipids from brains of f/f and KO mice fed LFD or HFD for 33 weeks (n = 6–7). Composition of individual FFA species making up the (C) FFA and (E) TG fractions were determined by GC-MS analysis, normalized to brain tissue mass (ng/mg tissue) and shown as group mean±SEM. (B) Changes in hypothalamic mRNA levels of target genes involved in (B) lipid uptake and storage (LPL, CD36, GPAT and DGAT), (D) lipid synthesis (FAS, ACC, SCD) and (F) fatty acid oxidation (ACO, PDK4, CPT1A, UCP2) were assessed by quantitative real-time PCR. Gene expression levels were normalized to endogenous RPL13A levels and are expressed as group mean±SEM relative to the level of the f/f LF diet control group. Statistical significance is designated as a ( p

    Journal: PLoS ONE

    Article Title: Neuron-Specific Deletion of Peroxisome Proliferator-Activated Receptor Delta (PPAR?) in Mice Leads to Increased Susceptibility to Diet-Induced Obesity

    doi: 10.1371/journal.pone.0042981

    Figure Lengend Snippet: Effects of dietary fat and PPARδ deletion on brain lipids, fatty acid composition and lipid metabolism genes. (A) Total levels of triglyceride (TG), diglyceride (DG) and free fatty acid (FFA) extracted from total lipids from brains of f/f and KO mice fed LFD or HFD for 33 weeks (n = 6–7). Composition of individual FFA species making up the (C) FFA and (E) TG fractions were determined by GC-MS analysis, normalized to brain tissue mass (ng/mg tissue) and shown as group mean±SEM. (B) Changes in hypothalamic mRNA levels of target genes involved in (B) lipid uptake and storage (LPL, CD36, GPAT and DGAT), (D) lipid synthesis (FAS, ACC, SCD) and (F) fatty acid oxidation (ACO, PDK4, CPT1A, UCP2) were assessed by quantitative real-time PCR. Gene expression levels were normalized to endogenous RPL13A levels and are expressed as group mean±SEM relative to the level of the f/f LF diet control group. Statistical significance is designated as a ( p

    Article Snippet: The resulting cDNA template was used to quantify mRNA expression via quantitative real-time PCR on a Bio-Rad iCycler using iQ SYBR green Supermix reagent (Bio-Rad; Hercules, CA).

    Techniques: Mouse Assay, Gas Chromatography-Mass Spectrometry, Real-time Polymerase Chain Reaction, Expressing

    HDACIs increase FOXO1 expression at the mRNA and protein levels. ( A ) HCT116 cells were treated with TSA (0.125, 0.25 or 0.5 μM for 24 h; or 0.5 μM for 6, 12, or 24 h); HepG2 cells were treated with TSA (0.25, 0.5 or 1 μM for 24 h; or 1 μM for 6, 12, or 24 h). The cells were then harvested and subjected to western blotting analysis to evaluate FOXO1, CDKN1A and acetylated FOXO1 (Ac-FOXO1) expression. ( B ) HCT116 cells were treated with TSA (0.25 μM) in the absence or presence of actinomycin D (1 μg/ml) for 16 h. Cell lysates were analyzed for protein levels of FOXO1 and CDKN1A. ( C ) HCT116 cells were treated with TSA (0.125, 0.25 or 0.5 μM) for 24 h; HepG2 cells were treated with TSA (0.25, 0.5 or 1 μM) for 24 h. Total mRNA was extracted and real-time PCR was performed to evaluate changes in FOXO1 mRNA level. GAPDH was used as a loading control for real-time PCR.

    Journal: Autophagy

    Article Title: Histone deacetylase inhibitors induce autophagy through FOXO1-dependent pathways

    doi: 10.1080/15548627.2015.1023981

    Figure Lengend Snippet: HDACIs increase FOXO1 expression at the mRNA and protein levels. ( A ) HCT116 cells were treated with TSA (0.125, 0.25 or 0.5 μM for 24 h; or 0.5 μM for 6, 12, or 24 h); HepG2 cells were treated with TSA (0.25, 0.5 or 1 μM for 24 h; or 1 μM for 6, 12, or 24 h). The cells were then harvested and subjected to western blotting analysis to evaluate FOXO1, CDKN1A and acetylated FOXO1 (Ac-FOXO1) expression. ( B ) HCT116 cells were treated with TSA (0.25 μM) in the absence or presence of actinomycin D (1 μg/ml) for 16 h. Cell lysates were analyzed for protein levels of FOXO1 and CDKN1A. ( C ) HCT116 cells were treated with TSA (0.125, 0.25 or 0.5 μM) for 24 h; HepG2 cells were treated with TSA (0.25, 0.5 or 1 μM) for 24 h. Total mRNA was extracted and real-time PCR was performed to evaluate changes in FOXO1 mRNA level. GAPDH was used as a loading control for real-time PCR.

    Article Snippet: The mRNA expression levels were determined by real-time PCR using SsoFast™ EvaGreen® Supermix (Bio-Rad, 172-5201) and CFX96 Touch™ Real-Time PCR Detection System (Bio Rad).

    Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction

    FOXO1 inhibits MTOR activity by elevating SESN3 / Sesn3 expression. ( A ) HCT116 cells were transiently transfected with the FOXO1 -specific siRNA followed by TSA treatment (0.5 μM) for 12 h (left panel). HCT116 cells were treated with 0.25 μM TSA in the absence or presence of FOXO1 inhibitor AS1842856 (100 nM) for 24 h (right panel). ( B ) Tsc2 +/+ and tsc2 −/− MEFs were treated with 1 μM TSA for 12 h and total RNA was isolated from cells. Total RNA was isolated from HCT116 cells, Tsc2 +/+ and tsc2 −/− MEFs, and the SESN3 / Sesn3 mRNA level was quantified using real-time PCR. The Foxo1 mRNA level was also measured in Tsc2 +/+ and tsc2 −/− MEFs. Fold change in mRNA levels was calculated by normalizing to respective Gapdh groups. ( C ) and ( D ) HCT116 cells were transiently transfected with a nonspecific siRNA or the SESN3 -specific siRNA followed by TSA treatment (0.5 μM) for 12 h. Total protein was extracted and subjected to immunoblotting for FOXO1, phospho-RPS6 (Ser235/236), RPS6, phospho-EIF4EBP1 (T37/46), and EIF4EBP1 antibodies. Total RNA was also isolated from HCT116 cells and the mRNA level of SESN3 was quantified using real-time PCR. Fold change in mRNA levels was calculated by normalizing to GAPDH .

    Journal: Autophagy

    Article Title: Histone deacetylase inhibitors induce autophagy through FOXO1-dependent pathways

    doi: 10.1080/15548627.2015.1023981

    Figure Lengend Snippet: FOXO1 inhibits MTOR activity by elevating SESN3 / Sesn3 expression. ( A ) HCT116 cells were transiently transfected with the FOXO1 -specific siRNA followed by TSA treatment (0.5 μM) for 12 h (left panel). HCT116 cells were treated with 0.25 μM TSA in the absence or presence of FOXO1 inhibitor AS1842856 (100 nM) for 24 h (right panel). ( B ) Tsc2 +/+ and tsc2 −/− MEFs were treated with 1 μM TSA for 12 h and total RNA was isolated from cells. Total RNA was isolated from HCT116 cells, Tsc2 +/+ and tsc2 −/− MEFs, and the SESN3 / Sesn3 mRNA level was quantified using real-time PCR. The Foxo1 mRNA level was also measured in Tsc2 +/+ and tsc2 −/− MEFs. Fold change in mRNA levels was calculated by normalizing to respective Gapdh groups. ( C ) and ( D ) HCT116 cells were transiently transfected with a nonspecific siRNA or the SESN3 -specific siRNA followed by TSA treatment (0.5 μM) for 12 h. Total protein was extracted and subjected to immunoblotting for FOXO1, phospho-RPS6 (Ser235/236), RPS6, phospho-EIF4EBP1 (T37/46), and EIF4EBP1 antibodies. Total RNA was also isolated from HCT116 cells and the mRNA level of SESN3 was quantified using real-time PCR. Fold change in mRNA levels was calculated by normalizing to GAPDH .

    Article Snippet: The mRNA expression levels were determined by real-time PCR using SsoFast™ EvaGreen® Supermix (Bio-Rad, 172-5201) and CFX96 Touch™ Real-Time PCR Detection System (Bio Rad).

    Techniques: Activity Assay, Expressing, Transfection, Isolation, Real-time Polymerase Chain Reaction

    FOXO1 transcriptional activity is increased by HDACIs. ( A ) Effect of TSA on nuclear retention of FOXO1. HCT116 cells were treated with TSA (0.5 μM for 6, 12, or 24 h); HepG2 cells were treated with TSA (1 μM for 6, 12, or 24 h). To track the subcellular localization of FOXO1, nuclear and cytosolic proteins from control and TSA-treated cells were probed for FOXO1. The same membrane was then stripped and reprobed for TUBA4A or LMNA to ensure equal protein loading. ( B ) The luciferase reporter plasmid under the control of the FOXO1 promoter was transfected into HCT116 and HepG2 cells. After 24 h, the cells were treated with TSA (0.5 or 1 μM) for another 12 h and the relative luciferase activity was measured. RLU refers to relative luciferase units. Error bars represent the standard deviation. ( C ) HCT116 cells were treated with TSA (0.125, 0.25 or 0.5 μM) for 24 h; HepG2 cells were treated with TSA (0.25, 0.5 or 1 μM) for 24 h. Total mRNA was extracted and real-time PCR was performed to determine changes in CDKN1A mRNA. GAPDH was used as a loading control.

    Journal: Autophagy

    Article Title: Histone deacetylase inhibitors induce autophagy through FOXO1-dependent pathways

    doi: 10.1080/15548627.2015.1023981

    Figure Lengend Snippet: FOXO1 transcriptional activity is increased by HDACIs. ( A ) Effect of TSA on nuclear retention of FOXO1. HCT116 cells were treated with TSA (0.5 μM for 6, 12, or 24 h); HepG2 cells were treated with TSA (1 μM for 6, 12, or 24 h). To track the subcellular localization of FOXO1, nuclear and cytosolic proteins from control and TSA-treated cells were probed for FOXO1. The same membrane was then stripped and reprobed for TUBA4A or LMNA to ensure equal protein loading. ( B ) The luciferase reporter plasmid under the control of the FOXO1 promoter was transfected into HCT116 and HepG2 cells. After 24 h, the cells were treated with TSA (0.5 or 1 μM) for another 12 h and the relative luciferase activity was measured. RLU refers to relative luciferase units. Error bars represent the standard deviation. ( C ) HCT116 cells were treated with TSA (0.125, 0.25 or 0.5 μM) for 24 h; HepG2 cells were treated with TSA (0.25, 0.5 or 1 μM) for 24 h. Total mRNA was extracted and real-time PCR was performed to determine changes in CDKN1A mRNA. GAPDH was used as a loading control.

    Article Snippet: The mRNA expression levels were determined by real-time PCR using SsoFast™ EvaGreen® Supermix (Bio-Rad, 172-5201) and CFX96 Touch™ Real-Time PCR Detection System (Bio Rad).

    Techniques: Activity Assay, Luciferase, Plasmid Preparation, Transfection, Standard Deviation, Real-time Polymerase Chain Reaction

    For figure legend, see page 636. Figure 5 (See previous page) . Role of FOXO1 in HDACIs-induced MTOR suppression. ( A ) FOXO1 knockdown increased MTOR activity. HCT116 were transiently transfected with a nonspecific siRNA or the FOXO1 -specific siRNA followed by TSA treatment (0.5 μM) for 12 h. ( B ) HCT116 cells were treated with 0.25 μM TSA in the absence or presence of FOXO1 inhibitor AS1842856 (100 nM) for 24 h. Total protein was extracted and subjected to immunoblotting for FOXO1, phospho-RPS6KB (T389), RPS6KB, phospho-RPS6 (Ser235/236), RPS6, phospho-EIF4EBP1 (T37/46), and EIF4EBP1 antibodies. ( C ) Tsc2 +/+ and tsc2 −/− MEFs were treated with 1 μM TSA for 2 different time points (6 or 12 h). Cell lysates were lysed, collected, and immunoblotted for TSC2, FOXO1, Ac-FOXO1, phospho-RPS6 ribosomal protein (Ser235/236), RPS6, phospho-EIF4EBP1 (T37/46), EIF4EBP1, LC3, and SQSTM1 levels. ( D ) Tsc2 +/+ and tsc2 −/− MEFs were treated with 1 μM TSA for 12 h and total RNA was isolated from cells. The mRNA levels of Atg s were also determined by real-time PCR, including Atg4b, Atg12, Pik3c3, Becn1 , and Map1lc3b . Fold change in mRNA levels was calculated by normalizing to Gapdh .

    Journal: Autophagy

    Article Title: Histone deacetylase inhibitors induce autophagy through FOXO1-dependent pathways

    doi: 10.1080/15548627.2015.1023981

    Figure Lengend Snippet: For figure legend, see page 636. Figure 5 (See previous page) . Role of FOXO1 in HDACIs-induced MTOR suppression. ( A ) FOXO1 knockdown increased MTOR activity. HCT116 were transiently transfected with a nonspecific siRNA or the FOXO1 -specific siRNA followed by TSA treatment (0.5 μM) for 12 h. ( B ) HCT116 cells were treated with 0.25 μM TSA in the absence or presence of FOXO1 inhibitor AS1842856 (100 nM) for 24 h. Total protein was extracted and subjected to immunoblotting for FOXO1, phospho-RPS6KB (T389), RPS6KB, phospho-RPS6 (Ser235/236), RPS6, phospho-EIF4EBP1 (T37/46), and EIF4EBP1 antibodies. ( C ) Tsc2 +/+ and tsc2 −/− MEFs were treated with 1 μM TSA for 2 different time points (6 or 12 h). Cell lysates were lysed, collected, and immunoblotted for TSC2, FOXO1, Ac-FOXO1, phospho-RPS6 ribosomal protein (Ser235/236), RPS6, phospho-EIF4EBP1 (T37/46), EIF4EBP1, LC3, and SQSTM1 levels. ( D ) Tsc2 +/+ and tsc2 −/− MEFs were treated with 1 μM TSA for 12 h and total RNA was isolated from cells. The mRNA levels of Atg s were also determined by real-time PCR, including Atg4b, Atg12, Pik3c3, Becn1 , and Map1lc3b . Fold change in mRNA levels was calculated by normalizing to Gapdh .

    Article Snippet: The mRNA expression levels were determined by real-time PCR using SsoFast™ EvaGreen® Supermix (Bio-Rad, 172-5201) and CFX96 Touch™ Real-Time PCR Detection System (Bio Rad).

    Techniques: Polyacrylamide Gel Electrophoresis, Activity Assay, Transfection, Isolation, Real-time Polymerase Chain Reaction

    Identifying eIF4E-dependent and independent mRNAs associating with Caf20p. A. Proportional Venn style diagram showing the overlap between the mRNAs associating with Caf20p and Eap1p TAP IPs. B. Quantitative RT-PCR of selected mRNAs showing their association with Caf20p and Caf20 m2 p represented as fold-change over control. Statistically significant changes are indicated with a key (T-test). C. and D. Venn style diagrams showing C. mRNAs associating with Caf20-TAP, Caf20-FLAG and Caf20 m2 -FLAG, highlighting the core mRNAs identified in both studies and their dependence on the eIF4E interaction motif for binding, and D. Overlap between the core 4E-DEP and 4E-IND Caf20p interacting mRNAs and the Eap1-TAP associated proteins.

    Journal: PLoS Genetics

    Article Title: The 4E-BP Caf20p Mediates Both eIF4E-Dependent and Independent Repression of Translation

    doi: 10.1371/journal.pgen.1005233

    Figure Lengend Snippet: Identifying eIF4E-dependent and independent mRNAs associating with Caf20p. A. Proportional Venn style diagram showing the overlap between the mRNAs associating with Caf20p and Eap1p TAP IPs. B. Quantitative RT-PCR of selected mRNAs showing their association with Caf20p and Caf20 m2 p represented as fold-change over control. Statistically significant changes are indicated with a key (T-test). C. and D. Venn style diagrams showing C. mRNAs associating with Caf20-TAP, Caf20-FLAG and Caf20 m2 -FLAG, highlighting the core mRNAs identified in both studies and their dependence on the eIF4E interaction motif for binding, and D. Overlap between the core 4E-DEP and 4E-IND Caf20p interacting mRNAs and the Eap1-TAP associated proteins.

    Article Snippet: For quantitative Reverse Transcriptase (qRT) PCR experiments (Figs and ), Primer pairs were designed to CAF20 and to a selection of Caf20p associated mRNAs ( ). qRT-PCR was performed using the CFx Connect Real-Time system with iTaq Universal SYBR Green Supermix (BioRad Laboratories, Hercules, CA).

    Techniques: Quantitative RT-PCR, T-Test, Binding Assay

    Models for 4E-DEP and 4E-IND Caf20p-mediated translational repression. A. Active translation. Short mRNA (blue line with grey ORF) shown with eIF4E (purple) bound to 5’cap (blue), eIF4G (blue oval) and Pab1p (green oval) facilitating mRNA circularisation and ribosome recruitment (cream ovals) that excludes Caf20p binding eIF4E. B. Caf20p mediated eIF4E-dependent translational repression. eIF4E/4G/Pab1p closed loop unstable on long mRNAs facilitating Caf20p binding (red). Caf20p binding to 80S ribosomes may contribute to its local recruitment. C. 4E-IND repression mechanisms. Caf20p 3’ UTR motif binding (may not be direct), either alone or in addition to 80S binding facilitates translational repression. eIF4E-Caf20p binding may contribute to repression on some of these mRNAs.

    Journal: PLoS Genetics

    Article Title: The 4E-BP Caf20p Mediates Both eIF4E-Dependent and Independent Repression of Translation

    doi: 10.1371/journal.pgen.1005233

    Figure Lengend Snippet: Models for 4E-DEP and 4E-IND Caf20p-mediated translational repression. A. Active translation. Short mRNA (blue line with grey ORF) shown with eIF4E (purple) bound to 5’cap (blue), eIF4G (blue oval) and Pab1p (green oval) facilitating mRNA circularisation and ribosome recruitment (cream ovals) that excludes Caf20p binding eIF4E. B. Caf20p mediated eIF4E-dependent translational repression. eIF4E/4G/Pab1p closed loop unstable on long mRNAs facilitating Caf20p binding (red). Caf20p binding to 80S ribosomes may contribute to its local recruitment. C. 4E-IND repression mechanisms. Caf20p 3’ UTR motif binding (may not be direct), either alone or in addition to 80S binding facilitates translational repression. eIF4E-Caf20p binding may contribute to repression on some of these mRNAs.

    Article Snippet: For quantitative Reverse Transcriptase (qRT) PCR experiments (Figs and ), Primer pairs were designed to CAF20 and to a selection of Caf20p associated mRNAs ( ). qRT-PCR was performed using the CFx Connect Real-Time system with iTaq Universal SYBR Green Supermix (BioRad Laboratories, Hercules, CA).

    Techniques: Binding Assay

    Features of Caf20p 4E-DEP and 4E-IND core bound mRNAs. A. Box and whisker plot showing impact of caf20 Δ on polysome association (blue) and transcript abundance (red) across sets of Caf20p-associated mRNAs [ 19 ]. A 95% confidence interval around the median is represented by a notch. Where notches do not overlap the medians are different. Inset: P values represent FDR (Mann-Whitney U tests) versus non-target RNAs. B-D: Box and whisker plots comparing mRNA features of Caf20 core mRNA targets and the 4E-DEP and 4E-IND subgroups. Histograms above each plot show binned total data with the vertical dashed line indicating the median of the total. P values represent FDR (Mann-Whitney U tests corrected for multiple hypothesis testing). Gene sets were statistically tested versus the set of mRNAs not bound to TAP or FLAG tagged Caf20p. B. Translation efficiency (TE) data was taken from ribosome footprinting experiments, median poly A tail length [ 37 ] and mRNA half-life [ 38 ]. C. 5’ UTR and ORF lengths and D. secondary structure information [ 40 ] are similarly shown. E. Sequence motif identified in 3’UTRs of 4E-IND mRNAs using the MEME Suite [ 43 ]. See S1 Dataset , sheet 11 for individual motifs identified. F. Caf20p interactions with 4E-IND mRNA 3’UTRs and with ORFs differ in sensitivity to glucose starvation. Fraction of each indicated RNA (left) isolated in complex with Caf20-FLAG (red) or Caf20 m2 -FLAG (blue bars) or an empty vector control (black bars) from caf20 Δ cells following formaldehyde cross-linking and RNase III digestion. qRT-PCR detection with primer pairs hybridizing along each RNA as indicated by colour coding in each cartoon (right). Samples were prepared from cells grown in SCD (+ glucose) or following 10 min of glucose starvation (- glucose). Statistically significant changes ± glucose indicated by ▲ (T-test).

    Journal: PLoS Genetics

    Article Title: The 4E-BP Caf20p Mediates Both eIF4E-Dependent and Independent Repression of Translation

    doi: 10.1371/journal.pgen.1005233

    Figure Lengend Snippet: Features of Caf20p 4E-DEP and 4E-IND core bound mRNAs. A. Box and whisker plot showing impact of caf20 Δ on polysome association (blue) and transcript abundance (red) across sets of Caf20p-associated mRNAs [ 19 ]. A 95% confidence interval around the median is represented by a notch. Where notches do not overlap the medians are different. Inset: P values represent FDR (Mann-Whitney U tests) versus non-target RNAs. B-D: Box and whisker plots comparing mRNA features of Caf20 core mRNA targets and the 4E-DEP and 4E-IND subgroups. Histograms above each plot show binned total data with the vertical dashed line indicating the median of the total. P values represent FDR (Mann-Whitney U tests corrected for multiple hypothesis testing). Gene sets were statistically tested versus the set of mRNAs not bound to TAP or FLAG tagged Caf20p. B. Translation efficiency (TE) data was taken from ribosome footprinting experiments, median poly A tail length [ 37 ] and mRNA half-life [ 38 ]. C. 5’ UTR and ORF lengths and D. secondary structure information [ 40 ] are similarly shown. E. Sequence motif identified in 3’UTRs of 4E-IND mRNAs using the MEME Suite [ 43 ]. See S1 Dataset , sheet 11 for individual motifs identified. F. Caf20p interactions with 4E-IND mRNA 3’UTRs and with ORFs differ in sensitivity to glucose starvation. Fraction of each indicated RNA (left) isolated in complex with Caf20-FLAG (red) or Caf20 m2 -FLAG (blue bars) or an empty vector control (black bars) from caf20 Δ cells following formaldehyde cross-linking and RNase III digestion. qRT-PCR detection with primer pairs hybridizing along each RNA as indicated by colour coding in each cartoon (right). Samples were prepared from cells grown in SCD (+ glucose) or following 10 min of glucose starvation (- glucose). Statistically significant changes ± glucose indicated by ▲ (T-test).

    Article Snippet: For quantitative Reverse Transcriptase (qRT) PCR experiments (Figs and ), Primer pairs were designed to CAF20 and to a selection of Caf20p associated mRNAs ( ). qRT-PCR was performed using the CFx Connect Real-Time system with iTaq Universal SYBR Green Supermix (BioRad Laboratories, Hercules, CA).

    Techniques: Whisker Assay, MANN-WHITNEY, Footprinting, Sequencing, Isolation, Plasmid Preparation, Quantitative RT-PCR, T-Test

    4E-BP-bound mRNAs enrich cell cycle and transcription factors. A. Functional GO class analysis of mRNAs associated with the 4E-BPs by RNA-seq. Over/underrepresentation colours depict FDR significance as per the key. Only a representative selection of the significant functional categories is shown. B. Summary of morphology changes noted within the Saccharomyces cerevisiae Morphological database (SCMD; http://yeast.gi.k.u-tokyo.ac.jp/datamine/ ) for 4E-BP deletion strains. Red/blue shading indicate over/under enrichment compared to wild type cells. C. D. Cell cycle progression defects in caf20 mutants. Flow cytometry analysis of DNA content using Sytox Green staining is shown at the indicated times (min) following release from alpha factor arrest for C. wild type and mutated cells, and D. plasmid complemented mutant cells.

    Journal: PLoS Genetics

    Article Title: The 4E-BP Caf20p Mediates Both eIF4E-Dependent and Independent Repression of Translation

    doi: 10.1371/journal.pgen.1005233

    Figure Lengend Snippet: 4E-BP-bound mRNAs enrich cell cycle and transcription factors. A. Functional GO class analysis of mRNAs associated with the 4E-BPs by RNA-seq. Over/underrepresentation colours depict FDR significance as per the key. Only a representative selection of the significant functional categories is shown. B. Summary of morphology changes noted within the Saccharomyces cerevisiae Morphological database (SCMD; http://yeast.gi.k.u-tokyo.ac.jp/datamine/ ) for 4E-BP deletion strains. Red/blue shading indicate over/under enrichment compared to wild type cells. C. D. Cell cycle progression defects in caf20 mutants. Flow cytometry analysis of DNA content using Sytox Green staining is shown at the indicated times (min) following release from alpha factor arrest for C. wild type and mutated cells, and D. plasmid complemented mutant cells.

    Article Snippet: For quantitative Reverse Transcriptase (qRT) PCR experiments (Figs and ), Primer pairs were designed to CAF20 and to a selection of Caf20p associated mRNAs ( ). qRT-PCR was performed using the CFx Connect Real-Time system with iTaq Universal SYBR Green Supermix (BioRad Laboratories, Hercules, CA).

    Techniques: Functional Assay, RNA Sequencing Assay, Selection, Flow Cytometry, Cytometry, Staining, Plasmid Preparation, Mutagenesis

    Caf20p and ERS1 3’UTR contribute to the translational repression of ERS1 . A. Interactions between Caf20 m2 -FLAG and ERS1 is diminished by loss of ERS1 3’UTR. Fraction of ERS1 or MET31 RNA (left) isolated in complex with Caf20-FLAG (red) or Caf20 m2 -FLAG (blue bars) or an empty vector control (black bars) from caf20 Δ cells following formaldehyde cross-linking and RNase III digestion. qRT-PCR detection with primer pairs hybridizing along each RNA as indicated by colour coding in each cartoon (right). Statistically significant changes ± ERS1 3’UTR indicated by ▲ (T-test). B. Polysome engagement of ERS1 is affected by Caf20p and the ERS1 3’UTR. Polysome profiles from sucrose gradient analyses of strains are shown (top) with free (F), monosomes (M) and polysome (P) regions indicated beneath the caf20 Δ profile. RNA collected from sucrose gradient fractions was analysed by qRT-PCR and quantified relative to a luciferase RNA ‘spike-in’ control. Proportions of RNA in ribosome free (F), monosomes (M) or polysome (P) are shown for ERS1 and ENO1 . Bar chart colors are as in A. P/M ratios for both mRNAs calculated from the data are indicated in the table below. C. ERS1 3’UTR confers Caf20p-dependent repression to firefly luciferase. Cartoon of plasmid dual luciferase construct and cloned 3’UTR fragments (left). Ratio of firefly to Renilla luciferase activities for transformants of caf20 Δ cells bearing indicated Caf20 plasmid. Mean ±SE for triplicate assays (right).

    Journal: PLoS Genetics

    Article Title: The 4E-BP Caf20p Mediates Both eIF4E-Dependent and Independent Repression of Translation

    doi: 10.1371/journal.pgen.1005233

    Figure Lengend Snippet: Caf20p and ERS1 3’UTR contribute to the translational repression of ERS1 . A. Interactions between Caf20 m2 -FLAG and ERS1 is diminished by loss of ERS1 3’UTR. Fraction of ERS1 or MET31 RNA (left) isolated in complex with Caf20-FLAG (red) or Caf20 m2 -FLAG (blue bars) or an empty vector control (black bars) from caf20 Δ cells following formaldehyde cross-linking and RNase III digestion. qRT-PCR detection with primer pairs hybridizing along each RNA as indicated by colour coding in each cartoon (right). Statistically significant changes ± ERS1 3’UTR indicated by ▲ (T-test). B. Polysome engagement of ERS1 is affected by Caf20p and the ERS1 3’UTR. Polysome profiles from sucrose gradient analyses of strains are shown (top) with free (F), monosomes (M) and polysome (P) regions indicated beneath the caf20 Δ profile. RNA collected from sucrose gradient fractions was analysed by qRT-PCR and quantified relative to a luciferase RNA ‘spike-in’ control. Proportions of RNA in ribosome free (F), monosomes (M) or polysome (P) are shown for ERS1 and ENO1 . Bar chart colors are as in A. P/M ratios for both mRNAs calculated from the data are indicated in the table below. C. ERS1 3’UTR confers Caf20p-dependent repression to firefly luciferase. Cartoon of plasmid dual luciferase construct and cloned 3’UTR fragments (left). Ratio of firefly to Renilla luciferase activities for transformants of caf20 Δ cells bearing indicated Caf20 plasmid. Mean ±SE for triplicate assays (right).

    Article Snippet: For quantitative Reverse Transcriptase (qRT) PCR experiments (Figs and ), Primer pairs were designed to CAF20 and to a selection of Caf20p associated mRNAs ( ). qRT-PCR was performed using the CFx Connect Real-Time system with iTaq Universal SYBR Green Supermix (BioRad Laboratories, Hercules, CA).

    Techniques: Isolation, Plasmid Preparation, Quantitative RT-PCR, T-Test, Luciferase, Construct, Clone Assay

    The 3′ UTR of Aplysia syntaxin mRNA. The stop codon is boxed. The CPE (UUUUAU) and polyadenylation signal (AAUAAA) are in bold and underlined. A poly(A) tail would be added after the terminal cytosine residue.

    Journal: The Journal of Neuroscience

    Article Title: Two mRNA-Binding Proteins Regulate the Distribution of Syntaxin mRNA in Aplysia Sensory Neurons

    doi: 10.1523/JNEUROSCI.4917-05.2006

    Figure Lengend Snippet: The 3′ UTR of Aplysia syntaxin mRNA. The stop codon is boxed. The CPE (UUUUAU) and polyadenylation signal (AAUAAA) are in bold and underlined. A poly(A) tail would be added after the terminal cytosine residue.

    Article Snippet: The distribution of Staufen protein is congruent with that of syntaxin mRNA: in untreated stable sensory neurons, both Staufen and syntaxin mRNA are located together in the region of the cell body opposite the axon hillock ( A ); after the treatment with 5-HT, both Staufen protein and syntaxin mRNA are expressed at their highest levels at the axon hillock ( A ).

    Techniques:

    The CPE regulates the movement of syntaxin mRNA to the axon hillock. A , Injecting the CPE blocked the 5-HT-induced accumulation of syntaxin mRNA at the axon hillock but did not affect the accumulation opposite the axon hillock in untreated sensory neurons. Sensory neurons were treated with 5-HT 6 h after the injection of oligonucleotides with control (reverse sequence of the CPE RNA) or the CPE RNA and fixed for in situ hybridization immediately after retesting the EPSPs 24 h after the treatment. Injecting the CPE did not affect the accumulation of syntaxin mRNA opposite the axon hillock or the accumulation of mRNA encoding the neuropeptide sensorin at the axon hillock in untreated sensory neurons (left micrographs). Injecting control oligonucleotides did not interfere with the 5-HT-induced accumulation of syntaxin mRNA or sensorin mRNA at the axon hillock (right micrographs). Injecting the CPE blocked the accumulation of syntaxin mRNA at the axon hillock after 5-HT but had no effect on the accumulation of sensorin mRNA at the axon hillock (middle micrographs). Scale bar, 25 μm. The histogram summarizes the effects of CPE or control injections on syntaxin mRNA distribution in the sensory neuron cell body. After CPE injections, staining intensity at the axon hillock (6:00) remains low (∼75%) relative to the accumulation opposite the axon hillock (12:00; normalized to 100%). Treatment with 5-HT after CPE produces a uniform distribution, whereas treatment with 5-HT after control injection produces a significant accumulation ( F = 5.269; p

    Journal: The Journal of Neuroscience

    Article Title: Two mRNA-Binding Proteins Regulate the Distribution of Syntaxin mRNA in Aplysia Sensory Neurons

    doi: 10.1523/JNEUROSCI.4917-05.2006

    Figure Lengend Snippet: The CPE regulates the movement of syntaxin mRNA to the axon hillock. A , Injecting the CPE blocked the 5-HT-induced accumulation of syntaxin mRNA at the axon hillock but did not affect the accumulation opposite the axon hillock in untreated sensory neurons. Sensory neurons were treated with 5-HT 6 h after the injection of oligonucleotides with control (reverse sequence of the CPE RNA) or the CPE RNA and fixed for in situ hybridization immediately after retesting the EPSPs 24 h after the treatment. Injecting the CPE did not affect the accumulation of syntaxin mRNA opposite the axon hillock or the accumulation of mRNA encoding the neuropeptide sensorin at the axon hillock in untreated sensory neurons (left micrographs). Injecting control oligonucleotides did not interfere with the 5-HT-induced accumulation of syntaxin mRNA or sensorin mRNA at the axon hillock (right micrographs). Injecting the CPE blocked the accumulation of syntaxin mRNA at the axon hillock after 5-HT but had no effect on the accumulation of sensorin mRNA at the axon hillock (middle micrographs). Scale bar, 25 μm. The histogram summarizes the effects of CPE or control injections on syntaxin mRNA distribution in the sensory neuron cell body. After CPE injections, staining intensity at the axon hillock (6:00) remains low (∼75%) relative to the accumulation opposite the axon hillock (12:00; normalized to 100%). Treatment with 5-HT after CPE produces a uniform distribution, whereas treatment with 5-HT after control injection produces a significant accumulation ( F = 5.269; p

    Article Snippet: The distribution of Staufen protein is congruent with that of syntaxin mRNA: in untreated stable sensory neurons, both Staufen and syntaxin mRNA are located together in the region of the cell body opposite the axon hillock ( A ); after the treatment with 5-HT, both Staufen protein and syntaxin mRNA are expressed at their highest levels at the axon hillock ( A ).

    Techniques: Injection, Sequencing, In Situ Hybridization, Staining

    CPEB moves syntaxin mRNA to the axon hillock. A , Injecting antisense oligonucleotides complementary to Aplysia CPEB blocked the 5-HT-induced accumulation of syntaxin mRNA at the axon hillock. Sensory neurons were treated with 5-HT 24 h after the injection and fixed for in situ hybridization immediately after retesting the EPSPs 24 h after the treatment. Injecting control (reverse of antisense) or antisense oligonucleotides did not affect the accumulation of syntaxin mRNA opposite the axon hillock in untreated sensory neurons (left micrographs). Injecting antisense blocked the accumulation at the axon hillock that is observed after control injections (right micrographs). Scale bar, 25 μm. B , Injecting antisense oligonucleotides complementary to Aplysia CPEB (AS-CPEB) blocked long-term facilitation. Representative EPSP traces were recorded before (0) and 24 h after the indicated treatments. The increase in EPSP amplitude produced by 5-HT was abolished when sensory neurons were injected with AS-CPEB but not after injection with the control oligonucleotides (control). Injection of AS-CPEB did not affect baseline synaptic efficacy. Calibration: vertical bar, 20 mV; horizontal bar, 25 ms. ANOVA indicates a significant effect of treatment (df = 3, 15; F = 8.665; ∗ p

    Journal: The Journal of Neuroscience

    Article Title: Two mRNA-Binding Proteins Regulate the Distribution of Syntaxin mRNA in Aplysia Sensory Neurons

    doi: 10.1523/JNEUROSCI.4917-05.2006

    Figure Lengend Snippet: CPEB moves syntaxin mRNA to the axon hillock. A , Injecting antisense oligonucleotides complementary to Aplysia CPEB blocked the 5-HT-induced accumulation of syntaxin mRNA at the axon hillock. Sensory neurons were treated with 5-HT 24 h after the injection and fixed for in situ hybridization immediately after retesting the EPSPs 24 h after the treatment. Injecting control (reverse of antisense) or antisense oligonucleotides did not affect the accumulation of syntaxin mRNA opposite the axon hillock in untreated sensory neurons (left micrographs). Injecting antisense blocked the accumulation at the axon hillock that is observed after control injections (right micrographs). Scale bar, 25 μm. B , Injecting antisense oligonucleotides complementary to Aplysia CPEB (AS-CPEB) blocked long-term facilitation. Representative EPSP traces were recorded before (0) and 24 h after the indicated treatments. The increase in EPSP amplitude produced by 5-HT was abolished when sensory neurons were injected with AS-CPEB but not after injection with the control oligonucleotides (control). Injection of AS-CPEB did not affect baseline synaptic efficacy. Calibration: vertical bar, 20 mV; horizontal bar, 25 ms. ANOVA indicates a significant effect of treatment (df = 3, 15; F = 8.665; ∗ p

    Article Snippet: The distribution of Staufen protein is congruent with that of syntaxin mRNA: in untreated stable sensory neurons, both Staufen and syntaxin mRNA are located together in the region of the cell body opposite the axon hillock ( A ); after the treatment with 5-HT, both Staufen protein and syntaxin mRNA are expressed at their highest levels at the axon hillock ( A ).

    Techniques: Injection, In Situ Hybridization, Produced, Mass Spectrometry

    Aplysia Staufen gene. A , The predicated amino acid sequence of Aplysia Staufen. The double-stranded RNA binding domains are in bold and underlined. These domains are 61 and 63% similar to those of the brain-specific forms of mouse and human Staufen2. Aplysia Staufen has several phosphorylation sites for PKA (Ser317, Ser836, and Ser876) and for p42/44 MAPK (Ser302, Thr435, and Thr651), two signaling pathways activated by the treatment with 5-HT. B , An antibody against Aplysia Staufen specifically recognizes a 125 kDa component on immunoblotting. The peptide antigen used to generate the antibody prevents the antibody from binding to Staufen (+). C , Aplysia Staufen is enriched in nervous tissue. Equal amounts of total protein (20 μg) from extracts of each tissue were fractionated by SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting. The gel shown is representative of three similar experiments. D , Staufen binds to syntaxin mRNA. Immunoprecipitation (IP) and RT-PCR was performed as described in Materials and Methods. The negative control experiment was performed without adding Staufen antibody (−Ab) during the immunoprecipitation step. For control of input, cDNA prepared from a total homogenate of pleural and pedal ganglia was used. Both 18S rRNA and syntaxin mRNA is present in the complex, whereas ApCAM mRNA is excluded.

    Journal: The Journal of Neuroscience

    Article Title: Two mRNA-Binding Proteins Regulate the Distribution of Syntaxin mRNA in Aplysia Sensory Neurons

    doi: 10.1523/JNEUROSCI.4917-05.2006

    Figure Lengend Snippet: Aplysia Staufen gene. A , The predicated amino acid sequence of Aplysia Staufen. The double-stranded RNA binding domains are in bold and underlined. These domains are 61 and 63% similar to those of the brain-specific forms of mouse and human Staufen2. Aplysia Staufen has several phosphorylation sites for PKA (Ser317, Ser836, and Ser876) and for p42/44 MAPK (Ser302, Thr435, and Thr651), two signaling pathways activated by the treatment with 5-HT. B , An antibody against Aplysia Staufen specifically recognizes a 125 kDa component on immunoblotting. The peptide antigen used to generate the antibody prevents the antibody from binding to Staufen (+). C , Aplysia Staufen is enriched in nervous tissue. Equal amounts of total protein (20 μg) from extracts of each tissue were fractionated by SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting. The gel shown is representative of three similar experiments. D , Staufen binds to syntaxin mRNA. Immunoprecipitation (IP) and RT-PCR was performed as described in Materials and Methods. The negative control experiment was performed without adding Staufen antibody (−Ab) during the immunoprecipitation step. For control of input, cDNA prepared from a total homogenate of pleural and pedal ganglia was used. Both 18S rRNA and syntaxin mRNA is present in the complex, whereas ApCAM mRNA is excluded.

    Article Snippet: The distribution of Staufen protein is congruent with that of syntaxin mRNA: in untreated stable sensory neurons, both Staufen and syntaxin mRNA are located together in the region of the cell body opposite the axon hillock ( A ); after the treatment with 5-HT, both Staufen protein and syntaxin mRNA are expressed at their highest levels at the axon hillock ( A ).

    Techniques: Sequencing, RNA Binding Assay, Binding Assay, SDS Page, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Negative Control

    Staufen is required for polar distribution of syntaxin mRNA. A , B , Injecting antisense oligonucleotides complimentary to Staufen reduces the expression of Staufen (top left micrograph compared with bottom left micrograph) and disperses syntaxin mRNA in the cell bodies of sensory neurons in both control untreated and 5-HT-treated neurons (right micrographs and histograms). Sensory neurons were injected with antisense oligonucleotides complementary to the Staufen gene (nucleotides 244–268) and control oligonucleotides (reverse sequence of the antisense). Neurons were treated with 5-HT 24 h later and fixed 24 h after the treatment to examine with double staining the distribution of Staufen protein (immunocytochemistry) and syntaxin mRNA ( in situ hybridization) in the same sensory neurons. Scale bar, 25 μm. In untreated sensory neurons injected with control oligonucleotides, Staufen protein staining and syntaxin mRNA staining were lowest at the 6:00 position. After injection with antisense, protein and mRNA staining were uniform (histogram in A ). After 5-HT and control injections, both Staufen protein and syntaxin mRNA staining were highest at the 6:00 position. Injection with antisense resulted in more uniform distribution for both protein and mRNA after treatment with 5-HT. C , Injection of Staufen antisense oligonucleotides did not disturb the distribution of ApCAM mRNA, which accumulates at the axon hillock under all conditions. D , Injecting antisense oligonucleotides complimentary to another RNA-binding protein, HnRNP L, does not affect the accumulation of syntaxin mRNA opposite the axon hillock in untreated sensory neurons (top) or the 5-HT-induced accumulation of syntaxin mRNA to the axon hillock (bottom). E , Injecting Staufen antisense oligonucleotides (AS-STF) into the cell bodies of sensory neurons blocked long-term facilitation. Representative EPSP traces were recorded before the injection and 24 h after the treatment with 5-HT. The increase in EPSP after treatment with 5-HT was abolished when sensory neurons were injected with AS-STF but not control oligonucleotides (control). Injection of AS-STF alone did not affect baseline synaptic transmission. ANOVA indicates a significant effect of treatment (df = 3, 24; F = 18.921; ∗ p

    Journal: The Journal of Neuroscience

    Article Title: Two mRNA-Binding Proteins Regulate the Distribution of Syntaxin mRNA in Aplysia Sensory Neurons

    doi: 10.1523/JNEUROSCI.4917-05.2006

    Figure Lengend Snippet: Staufen is required for polar distribution of syntaxin mRNA. A , B , Injecting antisense oligonucleotides complimentary to Staufen reduces the expression of Staufen (top left micrograph compared with bottom left micrograph) and disperses syntaxin mRNA in the cell bodies of sensory neurons in both control untreated and 5-HT-treated neurons (right micrographs and histograms). Sensory neurons were injected with antisense oligonucleotides complementary to the Staufen gene (nucleotides 244–268) and control oligonucleotides (reverse sequence of the antisense). Neurons were treated with 5-HT 24 h later and fixed 24 h after the treatment to examine with double staining the distribution of Staufen protein (immunocytochemistry) and syntaxin mRNA ( in situ hybridization) in the same sensory neurons. Scale bar, 25 μm. In untreated sensory neurons injected with control oligonucleotides, Staufen protein staining and syntaxin mRNA staining were lowest at the 6:00 position. After injection with antisense, protein and mRNA staining were uniform (histogram in A ). After 5-HT and control injections, both Staufen protein and syntaxin mRNA staining were highest at the 6:00 position. Injection with antisense resulted in more uniform distribution for both protein and mRNA after treatment with 5-HT. C , Injection of Staufen antisense oligonucleotides did not disturb the distribution of ApCAM mRNA, which accumulates at the axon hillock under all conditions. D , Injecting antisense oligonucleotides complimentary to another RNA-binding protein, HnRNP L, does not affect the accumulation of syntaxin mRNA opposite the axon hillock in untreated sensory neurons (top) or the 5-HT-induced accumulation of syntaxin mRNA to the axon hillock (bottom). E , Injecting Staufen antisense oligonucleotides (AS-STF) into the cell bodies of sensory neurons blocked long-term facilitation. Representative EPSP traces were recorded before the injection and 24 h after the treatment with 5-HT. The increase in EPSP after treatment with 5-HT was abolished when sensory neurons were injected with AS-STF but not control oligonucleotides (control). Injection of AS-STF alone did not affect baseline synaptic transmission. ANOVA indicates a significant effect of treatment (df = 3, 24; F = 18.921; ∗ p

    Article Snippet: The distribution of Staufen protein is congruent with that of syntaxin mRNA: in untreated stable sensory neurons, both Staufen and syntaxin mRNA are located together in the region of the cell body opposite the axon hillock ( A ); after the treatment with 5-HT, both Staufen protein and syntaxin mRNA are expressed at their highest levels at the axon hillock ( A ).

    Techniques: Expressing, Injection, Sequencing, Double Staining, Immunocytochemistry, In Situ Hybridization, Staining, RNA Binding Assay, Transmission Assay