poly-a-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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    99
    Illumina Inc truseq stranded mrna 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).
    Truseq Stranded Mrna Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 2507 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    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: 94/100, based on 4144 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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    94
    Illumina Inc mrna seq
    RNF20 represses ccRCC cell proliferation by inhibiting SREBP1. (A) ACHN ccRCC cells were infected with adenovirus containing Myc-RNF20 and/or <t>Flag-SREBP1c.</t> After infection, total cell lysates were subjected to SDS-PAGE followed by Western blotting. pSREBP1, precursor SREBP1; nSREBP1, nuclear SREBP1. (B) ACHN ccRCC cells were transduced with lentivirus for stable overexpression of RNF20 and/or SREBP1c. Relative <t>mRNA</t> levels were determined by qRT-PCR. (C) Intracellular triglyceride contents were measured in lentiviral RNF20- and/or SREBP1c-overexpressing ACHN cells. (D) RNF20- and/or SREBP1c-overexpressing ACHN ccRCC cells were subjected to qRT-PCR. (E) ACHN ccRCC cells were transduced with RNF20 and/or SREBP1c lentivirus, and their ability to form colonies was determined by crystal violet staining. (F) ACHN ccRCC cells were transduced with RNF20 and/or SREBP1c lentivirus, and cell proliferation was monitored by the Cell Counting Kit-8 (CCK-8) assay. (G) RNF20 and/or SREBP1 was suppressed in ACHN ccRCC cells using siRNAs, and cell lysates were subjected to Western blotting. (H) Intracellular triglyceride contents were measured in RNF20- and/or SREBP1-suppressing ACHN cells. (I) ACHN ccRCC cells were transfected with siRNF20 and/or siSREBP1, and relative cell growth rates were monitored using the CCK-8 assay. (J) ACHN ccRCC cells were transfected with siRNAs for suppression of RNF20 and/or SREBP1, and relative mRNA levels were determined by qRT-PCR. The data shown are representative results for at least three independent experiments. Data presented are the means ± SD. Significance versus negative control: #, P
    Mrna Seq, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 2649 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen oligotex mrna kit
    RNF20 represses ccRCC cell proliferation by inhibiting SREBP1. (A) ACHN ccRCC cells were infected with adenovirus containing Myc-RNF20 and/or <t>Flag-SREBP1c.</t> After infection, total cell lysates were subjected to SDS-PAGE followed by Western blotting. pSREBP1, precursor SREBP1; nSREBP1, nuclear SREBP1. (B) ACHN ccRCC cells were transduced with lentivirus for stable overexpression of RNF20 and/or SREBP1c. Relative <t>mRNA</t> levels were determined by qRT-PCR. (C) Intracellular triglyceride contents were measured in lentiviral RNF20- and/or SREBP1c-overexpressing ACHN cells. (D) RNF20- and/or SREBP1c-overexpressing ACHN ccRCC cells were subjected to qRT-PCR. (E) ACHN ccRCC cells were transduced with RNF20 and/or SREBP1c lentivirus, and their ability to form colonies was determined by crystal violet staining. (F) ACHN ccRCC cells were transduced with RNF20 and/or SREBP1c lentivirus, and cell proliferation was monitored by the Cell Counting Kit-8 (CCK-8) assay. (G) RNF20 and/or SREBP1 was suppressed in ACHN ccRCC cells using siRNAs, and cell lysates were subjected to Western blotting. (H) Intracellular triglyceride contents were measured in RNF20- and/or SREBP1-suppressing ACHN cells. (I) ACHN ccRCC cells were transfected with siRNF20 and/or siSREBP1, and relative cell growth rates were monitored using the CCK-8 assay. (J) ACHN ccRCC cells were transfected with siRNAs for suppression of RNF20 and/or SREBP1, and relative mRNA levels were determined by qRT-PCR. The data shown are representative results for at least three independent experiments. Data presented are the means ± SD. Significance versus negative control: #, P
    Oligotex Mrna Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1725 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Illumina Inc mrnas
    KEGG analysis of differentially expressed <t>lncRNAs</t> targets and <t>mRNAs</t> in primary wool follicle induction. (A,B) The top 20 KEGG enrichment pathways for differentially expressed lncRNA targets and mRNAs are presented. The longitudinal and horizontal axis represents the enrichment pathways and rich factor of these pathways, respectively. Spot size represents the number of differentially expressed genes enriched in each pathway, and the color of the spot represents the q -value of each pathway.
    Mrnas, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 1722 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher mrna expression
    KEGG analysis of differentially expressed <t>lncRNAs</t> targets and <t>mRNAs</t> in primary wool follicle induction. (A,B) The top 20 KEGG enrichment pathways for differentially expressed lncRNA targets and mRNAs are presented. The longitudinal and horizontal axis represents the enrichment pathways and rich factor of these pathways, respectively. Spot size represents the number of differentially expressed genes enriched in each pathway, and the color of the spot represents the q -value of each pathway.
    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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    Thermo Fisher microbexpress bacterial mrna enrichment kit
    KEGG analysis of differentially expressed <t>lncRNAs</t> targets and <t>mRNAs</t> in primary wool follicle induction. (A,B) The top 20 KEGG enrichment pathways for differentially expressed lncRNA targets and mRNAs are presented. The longitudinal and horizontal axis represents the enrichment pathways and rich factor of these pathways, respectively. Spot size represents the number of differentially expressed genes enriched in each pathway, and the color of the spot represents the q -value of each pathway.
    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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    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

    RNF20 represses ccRCC cell proliferation by inhibiting SREBP1. (A) ACHN ccRCC cells were infected with adenovirus containing Myc-RNF20 and/or Flag-SREBP1c. After infection, total cell lysates were subjected to SDS-PAGE followed by Western blotting. pSREBP1, precursor SREBP1; nSREBP1, nuclear SREBP1. (B) ACHN ccRCC cells were transduced with lentivirus for stable overexpression of RNF20 and/or SREBP1c. Relative mRNA levels were determined by qRT-PCR. (C) Intracellular triglyceride contents were measured in lentiviral RNF20- and/or SREBP1c-overexpressing ACHN cells. (D) RNF20- and/or SREBP1c-overexpressing ACHN ccRCC cells were subjected to qRT-PCR. (E) ACHN ccRCC cells were transduced with RNF20 and/or SREBP1c lentivirus, and their ability to form colonies was determined by crystal violet staining. (F) ACHN ccRCC cells were transduced with RNF20 and/or SREBP1c lentivirus, and cell proliferation was monitored by the Cell Counting Kit-8 (CCK-8) assay. (G) RNF20 and/or SREBP1 was suppressed in ACHN ccRCC cells using siRNAs, and cell lysates were subjected to Western blotting. (H) Intracellular triglyceride contents were measured in RNF20- and/or SREBP1-suppressing ACHN cells. (I) ACHN ccRCC cells were transfected with siRNF20 and/or siSREBP1, and relative cell growth rates were monitored using the CCK-8 assay. (J) ACHN ccRCC cells were transfected with siRNAs for suppression of RNF20 and/or SREBP1, and relative mRNA levels were determined by qRT-PCR. The data shown are representative results for at least three independent experiments. Data presented are the means ± SD. Significance versus negative control: #, P

    Journal: Molecular and Cellular Biology

    Article Title: RNF20 Suppresses Tumorigenesis by Inhibiting the SREBP1c-PTTG1 Axis in Kidney Cancer

    doi: 10.1128/MCB.00265-17

    Figure Lengend Snippet: RNF20 represses ccRCC cell proliferation by inhibiting SREBP1. (A) ACHN ccRCC cells were infected with adenovirus containing Myc-RNF20 and/or Flag-SREBP1c. After infection, total cell lysates were subjected to SDS-PAGE followed by Western blotting. pSREBP1, precursor SREBP1; nSREBP1, nuclear SREBP1. (B) ACHN ccRCC cells were transduced with lentivirus for stable overexpression of RNF20 and/or SREBP1c. Relative mRNA levels were determined by qRT-PCR. (C) Intracellular triglyceride contents were measured in lentiviral RNF20- and/or SREBP1c-overexpressing ACHN cells. (D) RNF20- and/or SREBP1c-overexpressing ACHN ccRCC cells were subjected to qRT-PCR. (E) ACHN ccRCC cells were transduced with RNF20 and/or SREBP1c lentivirus, and their ability to form colonies was determined by crystal violet staining. (F) ACHN ccRCC cells were transduced with RNF20 and/or SREBP1c lentivirus, and cell proliferation was monitored by the Cell Counting Kit-8 (CCK-8) assay. (G) RNF20 and/or SREBP1 was suppressed in ACHN ccRCC cells using siRNAs, and cell lysates were subjected to Western blotting. (H) Intracellular triglyceride contents were measured in RNF20- and/or SREBP1-suppressing ACHN cells. (I) ACHN ccRCC cells were transfected with siRNF20 and/or siSREBP1, and relative cell growth rates were monitored using the CCK-8 assay. (J) ACHN ccRCC cells were transfected with siRNAs for suppression of RNF20 and/or SREBP1, and relative mRNA levels were determined by qRT-PCR. The data shown are representative results for at least three independent experiments. Data presented are the means ± SD. Significance versus negative control: #, P

    Article Snippet: Liver transcriptome profiles of 12-week-old wild-type littermates and SREBP1c -deficient mice were generated by mRNA-seq, in duplicate, using Illumina HiSeq4000 by Macrogen, Inc. (South Korea).

    Techniques: Infection, SDS Page, Western Blot, Transduction, Over Expression, Quantitative RT-PCR, Staining, Cell Counting, CCK-8 Assay, Transfection, Negative Control

    In ccRCC cells, PTTG1 is involved in cell cycle regulation. (A) Lentivirus-mediated negative-control-infected (Mock) or RNF20-overexpressing ACHN cells were transfected with PTTG1 for 48 h. Relative mRNA levels were determined using qRT-PCR. Expression data are presented relative to the negative control. (B) ACHN ccRCC cells were transfected with RNF20 and/or PTTG1, and cell proliferation was monitored by the Cell Counting Kit-8 (CCK-8) assay. (C) Lentivirus-mediated negative-control-infected (Mock) or SREBP1c-overexpressing ACHN cells were transfected with PTTG1 siRNA for 48 h. Relative mRNA levels were determined using qRT-PCR. (D) ACHN ccRCC cells were transfected with SREBP1c and/or siPTTG1, and cell proliferation was monitored by the CCK-8 assay. Significance versus negative control: #, P

    Journal: Molecular and Cellular Biology

    Article Title: RNF20 Suppresses Tumorigenesis by Inhibiting the SREBP1c-PTTG1 Axis in Kidney Cancer

    doi: 10.1128/MCB.00265-17

    Figure Lengend Snippet: In ccRCC cells, PTTG1 is involved in cell cycle regulation. (A) Lentivirus-mediated negative-control-infected (Mock) or RNF20-overexpressing ACHN cells were transfected with PTTG1 for 48 h. Relative mRNA levels were determined using qRT-PCR. Expression data are presented relative to the negative control. (B) ACHN ccRCC cells were transfected with RNF20 and/or PTTG1, and cell proliferation was monitored by the Cell Counting Kit-8 (CCK-8) assay. (C) Lentivirus-mediated negative-control-infected (Mock) or SREBP1c-overexpressing ACHN cells were transfected with PTTG1 siRNA for 48 h. Relative mRNA levels were determined using qRT-PCR. (D) ACHN ccRCC cells were transfected with SREBP1c and/or siPTTG1, and cell proliferation was monitored by the CCK-8 assay. Significance versus negative control: #, P

    Article Snippet: Liver transcriptome profiles of 12-week-old wild-type littermates and SREBP1c -deficient mice were generated by mRNA-seq, in duplicate, using Illumina HiSeq4000 by Macrogen, Inc. (South Korea).

    Techniques: Negative Control, Infection, Transfection, Quantitative RT-PCR, Expressing, Cell Counting, CCK-8 Assay

    Inhibition of lipogenic activity does not affect PTTG1 expression but induces apoptosis in ccRCC cells. (A) ACHN ccRCC cells were treated with TOFA (10 μg/ml) or C75 (10 μg/ml) for 24 h, and intracellular triglyceride contents were measured. (B) ACHN ccRCC cells were treated with TOFA (10 μg/ml) or C75 (10 μg/ml) for 24 h. Relative mRNA levels are shown relative to those in the vehicle group. (C) ACHN ccRCC cells were treated with TOFA (10 μg/ml) or C75 (10 μg/ml), and cell proliferation was monitored by the Cell Counting Kit-8 (CCK-8) assay. (D) After treatment with TOFA (10 μg/ml) or C75 (10 μg/ml) for 24 h in ACHN ccRCC cells, PARP cleavage was detected by Western blotting. S.E., short exposure; L.E., long exposure. (E) ACHN ccRCC cells were treated with TOFA (10 μg/ml) or C75 (10 μg/ml) for 24 h, and relative mRNA levels were determined using qRT-PCR. (F) ACHN ccRCC cells were stained with annexin V after incubation with TOFA (10 μg/ml) or C75 (10 μg/ml) for 24 h, and apoptotic cells were measured by fluorescence-activated cell sorter (FACS) analysis. (G) Lentivirus-mediated negative-control-infected (Mock) or SREBP1c-overexpressing ACHN cells were transfected with FASN siRNA for 48 h, and relative mRNA levels were determined by qRT-PCR. mRNA levels are shown relative to those in the negative-control group. Significance versus negative control: #, P

    Journal: Molecular and Cellular Biology

    Article Title: RNF20 Suppresses Tumorigenesis by Inhibiting the SREBP1c-PTTG1 Axis in Kidney Cancer

    doi: 10.1128/MCB.00265-17

    Figure Lengend Snippet: Inhibition of lipogenic activity does not affect PTTG1 expression but induces apoptosis in ccRCC cells. (A) ACHN ccRCC cells were treated with TOFA (10 μg/ml) or C75 (10 μg/ml) for 24 h, and intracellular triglyceride contents were measured. (B) ACHN ccRCC cells were treated with TOFA (10 μg/ml) or C75 (10 μg/ml) for 24 h. Relative mRNA levels are shown relative to those in the vehicle group. (C) ACHN ccRCC cells were treated with TOFA (10 μg/ml) or C75 (10 μg/ml), and cell proliferation was monitored by the Cell Counting Kit-8 (CCK-8) assay. (D) After treatment with TOFA (10 μg/ml) or C75 (10 μg/ml) for 24 h in ACHN ccRCC cells, PARP cleavage was detected by Western blotting. S.E., short exposure; L.E., long exposure. (E) ACHN ccRCC cells were treated with TOFA (10 μg/ml) or C75 (10 μg/ml) for 24 h, and relative mRNA levels were determined using qRT-PCR. (F) ACHN ccRCC cells were stained with annexin V after incubation with TOFA (10 μg/ml) or C75 (10 μg/ml) for 24 h, and apoptotic cells were measured by fluorescence-activated cell sorter (FACS) analysis. (G) Lentivirus-mediated negative-control-infected (Mock) or SREBP1c-overexpressing ACHN cells were transfected with FASN siRNA for 48 h, and relative mRNA levels were determined by qRT-PCR. mRNA levels are shown relative to those in the negative-control group. Significance versus negative control: #, P

    Article Snippet: Liver transcriptome profiles of 12-week-old wild-type littermates and SREBP1c -deficient mice were generated by mRNA-seq, in duplicate, using Illumina HiSeq4000 by Macrogen, Inc. (South Korea).

    Techniques: Inhibition, Activity Assay, Expressing, Cell Counting, CCK-8 Assay, Western Blot, Quantitative RT-PCR, Staining, Incubation, Fluorescence, FACS, Negative Control, Infection, Transfection

    PTTG1 is a novel target gene of SREBP1c in ccRCC cells. (A) RNA-seq data for transcriptome profiling of liver tissues from wild-type and SREBP1c -deficient mice are presented as a scatter plot. (B) SRE motifs and E-box sequences in the PTTG1 promoters from several species. (C) ChIP assay results showing the human PTTG1 promoter occupancy by SREBP1 in ACHN ccRCC cells. (D) HEK293 cells were cotransfected with luciferase reporter plasmid containing the PTTG1 promoter and expression vectors for β-galactosidase, RNF20, and/or SREBP1c. Total cell lysates were subjected to luciferase and β-galactosidase assays. RLU, relative luminescence units. (E) ACHN ccRCC cells were transduced with RNF20 and/or SREBP1c lentivirus, and mRNA levels were determined by qRT-PCR. (F) ACHN ccRCC cells were transfected with siRNF20 and/or siSREBP1, and relative mRNA levels were determined using qRT-PCR. #, P

    Journal: Molecular and Cellular Biology

    Article Title: RNF20 Suppresses Tumorigenesis by Inhibiting the SREBP1c-PTTG1 Axis in Kidney Cancer

    doi: 10.1128/MCB.00265-17

    Figure Lengend Snippet: PTTG1 is a novel target gene of SREBP1c in ccRCC cells. (A) RNA-seq data for transcriptome profiling of liver tissues from wild-type and SREBP1c -deficient mice are presented as a scatter plot. (B) SRE motifs and E-box sequences in the PTTG1 promoters from several species. (C) ChIP assay results showing the human PTTG1 promoter occupancy by SREBP1 in ACHN ccRCC cells. (D) HEK293 cells were cotransfected with luciferase reporter plasmid containing the PTTG1 promoter and expression vectors for β-galactosidase, RNF20, and/or SREBP1c. Total cell lysates were subjected to luciferase and β-galactosidase assays. RLU, relative luminescence units. (E) ACHN ccRCC cells were transduced with RNF20 and/or SREBP1c lentivirus, and mRNA levels were determined by qRT-PCR. (F) ACHN ccRCC cells were transfected with siRNF20 and/or siSREBP1, and relative mRNA levels were determined using qRT-PCR. #, P

    Article Snippet: Liver transcriptome profiles of 12-week-old wild-type littermates and SREBP1c -deficient mice were generated by mRNA-seq, in duplicate, using Illumina HiSeq4000 by Macrogen, Inc. (South Korea).

    Techniques: RNA Sequencing Assay, Mouse Assay, Chromatin Immunoprecipitation, Luciferase, Plasmid Preparation, Expressing, Transduction, Quantitative RT-PCR, Transfection

    Pre-mRNA splicing defects in Qk conditional knockout cortex pre-mRNA splicing defects in Qk conditional knockout cortices. ( A ) Steady state of mRNA level between control and Qk cKO. Volcano plot diagrams shown. The significance ( p -value) versus fold change is plotted on the Y -axis and X -axis, respectively. ( B ) Scatter plot of the exon inclusion of control versus Qk cKO mRNA-SEQ significant hits. Each point represents the mean obtained from two biological replicates for an individual alternative splicing event. ( C ) Qki5 regulates many types of alternative splicing patterns. Asterisk indicates significant enrichment by Fisher’s exact test (* p

    Journal: International Journal of Molecular Sciences

    Article Title: An RNA Switch of a Large Exon of Ninein Is Regulated by the Neural Stem Cell Specific-RNA Binding Protein, Qki5

    doi: 10.3390/ijms20051010

    Figure Lengend Snippet: Pre-mRNA splicing defects in Qk conditional knockout cortex pre-mRNA splicing defects in Qk conditional knockout cortices. ( A ) Steady state of mRNA level between control and Qk cKO. Volcano plot diagrams shown. The significance ( p -value) versus fold change is plotted on the Y -axis and X -axis, respectively. ( B ) Scatter plot of the exon inclusion of control versus Qk cKO mRNA-SEQ significant hits. Each point represents the mean obtained from two biological replicates for an individual alternative splicing event. ( C ) Qki5 regulates many types of alternative splicing patterns. Asterisk indicates significant enrichment by Fisher’s exact test (* p

    Article Snippet: Embryonic day 14.5 mouse cortex RNA from two independent Qkfl /fl (control) and Nestin -cre:Qkfl /fl (Qk cKO ) littermates was used to prepare mRNA libraries through polyA selection and 150 base pair (bp) paired-end sequencing using the Illumina (San Diego, CA, USA) HiSeq system.

    Techniques: Knock-Out

    The mRNA-SEQ analysis from E14.5 cortices. ( A ) IGV (Integrative Genome Viewer) image showing mRNA expression of the Qk gene from control versus Qk cKO cortices. As expected, mRNA from cKO does not include exon 2 because of the loxP sites flanking exon 2 of the Qk gene for gene targeting to obtain conditional knockouts. ( B ) Bar graphs show the results of a qRT-PCR validation using control and Qk cKO cortices for Qk mRNA.

    Journal: International Journal of Molecular Sciences

    Article Title: An RNA Switch of a Large Exon of Ninein Is Regulated by the Neural Stem Cell Specific-RNA Binding Protein, Qki5

    doi: 10.3390/ijms20051010

    Figure Lengend Snippet: The mRNA-SEQ analysis from E14.5 cortices. ( A ) IGV (Integrative Genome Viewer) image showing mRNA expression of the Qk gene from control versus Qk cKO cortices. As expected, mRNA from cKO does not include exon 2 because of the loxP sites flanking exon 2 of the Qk gene for gene targeting to obtain conditional knockouts. ( B ) Bar graphs show the results of a qRT-PCR validation using control and Qk cKO cortices for Qk mRNA.

    Article Snippet: Embryonic day 14.5 mouse cortex RNA from two independent Qkfl /fl (control) and Nestin -cre:Qkfl /fl (Qk cKO ) littermates was used to prepare mRNA libraries through polyA selection and 150 base pair (bp) paired-end sequencing using the Illumina (San Diego, CA, USA) HiSeq system.

    Techniques: Expressing, Quantitative RT-PCR

    Ninein coding a large alternative exon is regulated by the Qki5 protein along with neuronal differentiation. ( A ) IGV image showing mRNA expression and Qki5 HITS-CLIP clusters along the Nin gene including the alternative large exon 18 and its flanking region from control ( Qk fl / fl ), Qk cKO cortices ( Nestin-cre:Qkfl/fl ), and cultured embryonic neural stem cells (NPC). Magnified view of Nin gene showing the Qki5-enhanced alternative exon and Qki5 CLIP clusters, including defined Qki5-binding site ACUAAC (highlighted in yellow). ( B ) Protein structure of NPC-Ninein, neuron-Ninein proteins, and NPC-neuron hybrid Ninein with centrosomal protein-binding sites. ( C – E ) The qRT-PCR analysis in control and Qk cKO cortices was performed to monitor the splicing of each alternative exon of Nin . ( F ) RT-PCR gel image of each pre-mRNA splicing of Nin exon 18 in control and Qk cKO, performed to monitor the splicing of alternative exons. ( Bottom panels ) Primers corresponding to each flanking exon were used to determine the expression of aberrantly processed mRNAs in Qk cKO. ( G ) Qki5-dependent pre-mRNA splicing model during NPC-to-neuron transition.

    Journal: International Journal of Molecular Sciences

    Article Title: An RNA Switch of a Large Exon of Ninein Is Regulated by the Neural Stem Cell Specific-RNA Binding Protein, Qki5

    doi: 10.3390/ijms20051010

    Figure Lengend Snippet: Ninein coding a large alternative exon is regulated by the Qki5 protein along with neuronal differentiation. ( A ) IGV image showing mRNA expression and Qki5 HITS-CLIP clusters along the Nin gene including the alternative large exon 18 and its flanking region from control ( Qk fl / fl ), Qk cKO cortices ( Nestin-cre:Qkfl/fl ), and cultured embryonic neural stem cells (NPC). Magnified view of Nin gene showing the Qki5-enhanced alternative exon and Qki5 CLIP clusters, including defined Qki5-binding site ACUAAC (highlighted in yellow). ( B ) Protein structure of NPC-Ninein, neuron-Ninein proteins, and NPC-neuron hybrid Ninein with centrosomal protein-binding sites. ( C – E ) The qRT-PCR analysis in control and Qk cKO cortices was performed to monitor the splicing of each alternative exon of Nin . ( F ) RT-PCR gel image of each pre-mRNA splicing of Nin exon 18 in control and Qk cKO, performed to monitor the splicing of alternative exons. ( Bottom panels ) Primers corresponding to each flanking exon were used to determine the expression of aberrantly processed mRNAs in Qk cKO. ( G ) Qki5-dependent pre-mRNA splicing model during NPC-to-neuron transition.

    Article Snippet: Embryonic day 14.5 mouse cortex RNA from two independent Qkfl /fl (control) and Nestin -cre:Qkfl /fl (Qk cKO ) littermates was used to prepare mRNA libraries through polyA selection and 150 base pair (bp) paired-end sequencing using the Illumina (San Diego, CA, USA) HiSeq system.

    Techniques: Expressing, Cross-linking Immunoprecipitation, Cell Culture, Binding Assay, Protein Binding, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

    sCLIP recapitulates previously reported RBP–RNA interactions data and identifies CSTF2tau binding to non-coding RNAs. ( A ) CSTF2tau preferentially binds to 3΄ and 5΄ untranslated regions (UTRs). The pie diagram (left) shows the distribution of binding sites of CSTF2tau on the genomic features in percent; the bar diagram (right) shows enrichment in coverage of sites over the total length of the genomic feature of the gene. ( B ) CSTF2tau binding sites are focused in the 3΄UTR and are located in close proximity to polyadenylation sites, illustrated for YY1AP1, FUBP1, GDI2 and BUB3 transcripts for two independent experiments; samples, which were generated by immunoprecipitation with an IgG antibody ‘IgG only’, serve as control (overall estimated proportion of CSTF2tau cCLIP Rep 1 and Rep 2 libraries, see also Figure 1 having the same reads as the IgG control sCLIP is 1.05% and 1.15%). A similar binding pattern can also be found for replication-dependent histones (see Supplementary Figure S2H ). ( C ) CSTF2tau CLIPs are predominantly located downstream of the endonucleolytic cleavage site (where 3΄end processing of polyadenylated mRNAs occurs) with a peak centred 50–70 nucleotides downstream of the poly(A) signal AAUAAA hexamer (further binding preferences are shown in Supplementary Figure S2E ). Of note, in replication dependent histones (in which processing at the mRNA 3΄end is executed by other mechanisms) the CTF2tau CLIPS are found to predominate at the ORF 5΄end ( Supplementary Figure S2H ) thereby confirming the specificity of the CSTF2tau CLIPs studied here. ( D ) Three most prevalent motifs found within a 100nt region around the center of the peak of CSTF2tau sCLIP tags. ( E ) In addition to protein-coding RNAs, CSTF2tau also binds non-coding RNAs (pie diagram; for further details on the distribution among the different classes of ncRNAs, see Supplementary Figure S2G ). The hypergeometric distribution analysis (bar diagram) shows that some classes are significantly overrepresented among the RNAs bound by CSTF2tau, while protein coding RNAs and pseudogenes are underrepresented (dashed line labels probability below 5%; red bars indicate over-representation; blue bars show under-representation; * indicates a statistically significant change, P -value

    Journal: Nucleic Acids Research

    Article Title: sCLIP—an integrated platform to study RNA–protein interactomes in biomedical research: identification of CSTF2tau in alternative processing of small nuclear RNAs

    doi: 10.1093/nar/gkx152

    Figure Lengend Snippet: sCLIP recapitulates previously reported RBP–RNA interactions data and identifies CSTF2tau binding to non-coding RNAs. ( A ) CSTF2tau preferentially binds to 3΄ and 5΄ untranslated regions (UTRs). The pie diagram (left) shows the distribution of binding sites of CSTF2tau on the genomic features in percent; the bar diagram (right) shows enrichment in coverage of sites over the total length of the genomic feature of the gene. ( B ) CSTF2tau binding sites are focused in the 3΄UTR and are located in close proximity to polyadenylation sites, illustrated for YY1AP1, FUBP1, GDI2 and BUB3 transcripts for two independent experiments; samples, which were generated by immunoprecipitation with an IgG antibody ‘IgG only’, serve as control (overall estimated proportion of CSTF2tau cCLIP Rep 1 and Rep 2 libraries, see also Figure 1 having the same reads as the IgG control sCLIP is 1.05% and 1.15%). A similar binding pattern can also be found for replication-dependent histones (see Supplementary Figure S2H ). ( C ) CSTF2tau CLIPs are predominantly located downstream of the endonucleolytic cleavage site (where 3΄end processing of polyadenylated mRNAs occurs) with a peak centred 50–70 nucleotides downstream of the poly(A) signal AAUAAA hexamer (further binding preferences are shown in Supplementary Figure S2E ). Of note, in replication dependent histones (in which processing at the mRNA 3΄end is executed by other mechanisms) the CTF2tau CLIPS are found to predominate at the ORF 5΄end ( Supplementary Figure S2H ) thereby confirming the specificity of the CSTF2tau CLIPs studied here. ( D ) Three most prevalent motifs found within a 100nt region around the center of the peak of CSTF2tau sCLIP tags. ( E ) In addition to protein-coding RNAs, CSTF2tau also binds non-coding RNAs (pie diagram; for further details on the distribution among the different classes of ncRNAs, see Supplementary Figure S2G ). The hypergeometric distribution analysis (bar diagram) shows that some classes are significantly overrepresented among the RNAs bound by CSTF2tau, while protein coding RNAs and pseudogenes are underrepresented (dashed line labels probability below 5%; red bars indicate over-representation; blue bars show under-representation; * indicates a statistically significant change, P -value

    Article Snippet: Next, the polyadenylated RNA was reversely transcribed using anchored oligo (dT) primers with Illumina adaptors followed by three different approaches to generate the sequencing library ( ).

    Techniques: Binding Assay, Generated, Immunoprecipitation

    sCLIP—a simplified platform for studying RNA–protein interactomes by using crosslinking immunoprecipitation (CLIP) sequencing with a highly sensitive and non-radioactive biochemistry for low input material. ( A ) Schematic overview of the sCLIP technique. Day 1: RNA–RBP interactions are preserved by in vivo UV-crosslinking. After crosslinking, the intact cells are lysed and the RNA not covered by crosslinked RBPs is partially digested. After immunoprecipitation (using antibodies against the RBP of interest; see necessary specificity controls in Supplementary Figure S1D and E ) an aliquot of the ribonucleoprotein (RNP) complexes is visualized by a non-radioactive labeling strategy (based on biotinylated ADP; see material and methods ; Day 2, Supplementary Figure S1F ). Following the IP the remaining material is digested with proteinase K and the bound RNA is released ( Supplementary Figure S1G ). Next, the RNA is polyadenylated and then reversely transcribed by using a modified oligo d(T) primer that harbors an in line and a random barcode along with a sequencing platform-compatible Illumina adaptor and a T7 promotor (Day 3). Following reverse transcription (RT) the cDNA is in vitro transcribed ( Supplementary Figure S1H ) and an Illumina adaptor is ligated to the mRNA 3΄end (Day 4). Finally, the amplified RNA is reversely transcribed and amplified with 10 cycles of PCR ( Supplementary Figure S1H ); afterward the libraries are subjected to high-throughput sequencing, and the sequencing data is analysed by an integrated sCLIP Data processing workflow (for further information see Supplementary Figures S1 and S2A and material and methods ; a detailed protocol of the procedure and the automated bioinformatics pipeline can be found in the supplementary informations ). ( B ) Reproducibility of binding sites between two replicates applying sCLIP against CSTF2tau (Pearson correlation between replicates was calculated to be 0.77; P -value = 2.2 × 10 −16 , for specificity controls see Supplementary Figure S1D–H ). ( C ) Out of all sequencing reads sCLIP delivers almost 60% of usable reads (green). In contrast the relative fraction of reads, which are either too short (cutoff

    Journal: Nucleic Acids Research

    Article Title: sCLIP—an integrated platform to study RNA–protein interactomes in biomedical research: identification of CSTF2tau in alternative processing of small nuclear RNAs

    doi: 10.1093/nar/gkx152

    Figure Lengend Snippet: sCLIP—a simplified platform for studying RNA–protein interactomes by using crosslinking immunoprecipitation (CLIP) sequencing with a highly sensitive and non-radioactive biochemistry for low input material. ( A ) Schematic overview of the sCLIP technique. Day 1: RNA–RBP interactions are preserved by in vivo UV-crosslinking. After crosslinking, the intact cells are lysed and the RNA not covered by crosslinked RBPs is partially digested. After immunoprecipitation (using antibodies against the RBP of interest; see necessary specificity controls in Supplementary Figure S1D and E ) an aliquot of the ribonucleoprotein (RNP) complexes is visualized by a non-radioactive labeling strategy (based on biotinylated ADP; see material and methods ; Day 2, Supplementary Figure S1F ). Following the IP the remaining material is digested with proteinase K and the bound RNA is released ( Supplementary Figure S1G ). Next, the RNA is polyadenylated and then reversely transcribed by using a modified oligo d(T) primer that harbors an in line and a random barcode along with a sequencing platform-compatible Illumina adaptor and a T7 promotor (Day 3). Following reverse transcription (RT) the cDNA is in vitro transcribed ( Supplementary Figure S1H ) and an Illumina adaptor is ligated to the mRNA 3΄end (Day 4). Finally, the amplified RNA is reversely transcribed and amplified with 10 cycles of PCR ( Supplementary Figure S1H ); afterward the libraries are subjected to high-throughput sequencing, and the sequencing data is analysed by an integrated sCLIP Data processing workflow (for further information see Supplementary Figures S1 and S2A and material and methods ; a detailed protocol of the procedure and the automated bioinformatics pipeline can be found in the supplementary informations ). ( B ) Reproducibility of binding sites between two replicates applying sCLIP against CSTF2tau (Pearson correlation between replicates was calculated to be 0.77; P -value = 2.2 × 10 −16 , for specificity controls see Supplementary Figure S1D–H ). ( C ) Out of all sequencing reads sCLIP delivers almost 60% of usable reads (green). In contrast the relative fraction of reads, which are either too short (cutoff

    Article Snippet: Next, the polyadenylated RNA was reversely transcribed using anchored oligo (dT) primers with Illumina adaptors followed by three different approaches to generate the sequencing library ( ).

    Techniques: Cross-linking Immunoprecipitation, Sequencing, In Vivo, Immunoprecipitation, Labeling, Modification, In Vitro, Amplification, Polymerase Chain Reaction, Next-Generation Sequencing, Binding Assay

    snRNAs are internally poly/oligoadenylated in a CSTF2tau dependent manner. ( A ) Illustration of the CSTF2tau binding on the snRNAs’ 3΄end (highlighted in red). The site of oligoadenylation is marked by a red arrow head and asterisk (see also Figure 4B ). ( B ) ePAT assay based identification of polyadenylated snRNA isoforms after cloning and conventional sanger sequencing. The canonical (matured) 3΄end of the respective snRNA is shown in bold, (internally) oligoadenylated snRNA isoforms are shown underneath (for further details see Supplementary Figure S4 ; some oligoadenylated isoforms are found more than once as indicated i.e. 4×, 2x etc.). ( C ) Quantitative RT-PCR-analysis to determine the fold change of the steady state RNA abundance of the total (blue) and poly/oligoadenylated (red) snRNA isoforms upon depletion of CSTF2tau (log 2 scale; for details on normalization controls see Supplementary Figure S4 , * indicates a statistically significant change, P -value

    Journal: Nucleic Acids Research

    Article Title: sCLIP—an integrated platform to study RNA–protein interactomes in biomedical research: identification of CSTF2tau in alternative processing of small nuclear RNAs

    doi: 10.1093/nar/gkx152

    Figure Lengend Snippet: snRNAs are internally poly/oligoadenylated in a CSTF2tau dependent manner. ( A ) Illustration of the CSTF2tau binding on the snRNAs’ 3΄end (highlighted in red). The site of oligoadenylation is marked by a red arrow head and asterisk (see also Figure 4B ). ( B ) ePAT assay based identification of polyadenylated snRNA isoforms after cloning and conventional sanger sequencing. The canonical (matured) 3΄end of the respective snRNA is shown in bold, (internally) oligoadenylated snRNA isoforms are shown underneath (for further details see Supplementary Figure S4 ; some oligoadenylated isoforms are found more than once as indicated i.e. 4×, 2x etc.). ( C ) Quantitative RT-PCR-analysis to determine the fold change of the steady state RNA abundance of the total (blue) and poly/oligoadenylated (red) snRNA isoforms upon depletion of CSTF2tau (log 2 scale; for details on normalization controls see Supplementary Figure S4 , * indicates a statistically significant change, P -value

    Article Snippet: Next, the polyadenylated RNA was reversely transcribed using anchored oligo (dT) primers with Illumina adaptors followed by three different approaches to generate the sequencing library ( ).

    Techniques: Binding Assay, Clone Assay, Sequencing, Quantitative RT-PCR

    Decreased efficiency of RNAP II termination and increased gene expression following the loss of histone H3.V. A region on chromosome 3 (617–670 kb) representing cSSR 3.3 and chromosome 7 (453–525 kb) representing cSSR 7.3 is shown where H3.V regulates transcription of a cluster of genes. (A-C) Base J and H3.V co-localize at sites of RNAP II termination within a PTU. H3.V ChIP-seq reads and base J IP-seq reads, ORFs, and mRNA-seq reads from wild type T . brucei are plotted for cSSR 3.3 (left) and cSSR 7.3 (right) as described in Fig 2 . (D) RT-qPCR analysis of genes numbered according to the ORF maps above in panel B. As described in Fig 2G , white bars: Wild type; grey bars: Wild type+DMOG; dark grey bars: H3 . V KO; black bars: H3 . V KO+DMOG. Transcripts were normalized against 40S ribosomal protein S11, and are plotted as the average and standard deviation of three replicates. P values were calculated using Student’s t test. *, p value ≤ 0.05; **, p value ≤ 0.01. The silent gene cluster at cSSR 7.3 consists of nine highly similar retrotransposon hot spot protein genes, therefore the primers used to analyze gene 2 also amplify the additional upstream genes. (E and F) Strand-specific RT-PCR analysis of read-through transcription of the two cSSRs analyzed in A-D. Above each panel is a schematic representation (not to scale) of primer location and direction at a transcription termination site (TTS). The vertical arrow indicates the proposed TTS as described in the text [ 35 ]. The long solid arrow indicates the direction of transcription and the dashed arrow indicates read-through transcription past the TTS. cDNA was synthesized using the reverse primer (relative to transcription). PCR was performed using the same reverse primer to make the cDNA plus the forward primer, as indicated. 40S ribosomal protein S11 provides a positive control and a minus RT (-RT) negative control is shown.

    Journal: PLoS Genetics

    Article Title: Histone H3 Variant Regulates RNA Polymerase II Transcription Termination and Dual Strand Transcription of siRNA Loci in Trypanosoma brucei

    doi: 10.1371/journal.pgen.1005758

    Figure Lengend Snippet: Decreased efficiency of RNAP II termination and increased gene expression following the loss of histone H3.V. A region on chromosome 3 (617–670 kb) representing cSSR 3.3 and chromosome 7 (453–525 kb) representing cSSR 7.3 is shown where H3.V regulates transcription of a cluster of genes. (A-C) Base J and H3.V co-localize at sites of RNAP II termination within a PTU. H3.V ChIP-seq reads and base J IP-seq reads, ORFs, and mRNA-seq reads from wild type T . brucei are plotted for cSSR 3.3 (left) and cSSR 7.3 (right) as described in Fig 2 . (D) RT-qPCR analysis of genes numbered according to the ORF maps above in panel B. As described in Fig 2G , white bars: Wild type; grey bars: Wild type+DMOG; dark grey bars: H3 . V KO; black bars: H3 . V KO+DMOG. Transcripts were normalized against 40S ribosomal protein S11, and are plotted as the average and standard deviation of three replicates. P values were calculated using Student’s t test. *, p value ≤ 0.05; **, p value ≤ 0.01. The silent gene cluster at cSSR 7.3 consists of nine highly similar retrotransposon hot spot protein genes, therefore the primers used to analyze gene 2 also amplify the additional upstream genes. (E and F) Strand-specific RT-PCR analysis of read-through transcription of the two cSSRs analyzed in A-D. Above each panel is a schematic representation (not to scale) of primer location and direction at a transcription termination site (TTS). The vertical arrow indicates the proposed TTS as described in the text [ 35 ]. The long solid arrow indicates the direction of transcription and the dashed arrow indicates read-through transcription past the TTS. cDNA was synthesized using the reverse primer (relative to transcription). PCR was performed using the same reverse primer to make the cDNA plus the forward primer, as indicated. 40S ribosomal protein S11 provides a positive control and a minus RT (-RT) negative control is shown.

    Article Snippet: 12 mRNA-seq libraries were constructed (triplicate WT, WT+DMOG, H3 .V KO, and H3 .V KO+DMOG) using Illumina TruSeq Stranded RNA LT Kit following the manufacturer’s instructions with limited modifications.

    Techniques: Expressing, Chromatin Immunoprecipitation, Quantitative RT-PCR, Standard Deviation, Reverse Transcription Polymerase Chain Reaction, Synthesized, Polymerase Chain Reaction, Positive Control, Negative Control

    Gene expression changes in the H3 . V KO. The average reads per kilobase per million reads mapped (RPKM) of triplicate mRNA-seq libraries is plotted on a log 2 scale. Genes differentially expressed by 2-fold or more in T . brucei following the loss of base J and/or H3.V fall above or below the dotted lines. Red dots indicate genes that are adjacent to H3.V ( S1 Table ). Only mRNAs with an RPKM≥1 are included.

    Journal: PLoS Genetics

    Article Title: Histone H3 Variant Regulates RNA Polymerase II Transcription Termination and Dual Strand Transcription of siRNA Loci in Trypanosoma brucei

    doi: 10.1371/journal.pgen.1005758

    Figure Lengend Snippet: Gene expression changes in the H3 . V KO. The average reads per kilobase per million reads mapped (RPKM) of triplicate mRNA-seq libraries is plotted on a log 2 scale. Genes differentially expressed by 2-fold or more in T . brucei following the loss of base J and/or H3.V fall above or below the dotted lines. Red dots indicate genes that are adjacent to H3.V ( S1 Table ). Only mRNAs with an RPKM≥1 are included.

    Article Snippet: 12 mRNA-seq libraries were constructed (triplicate WT, WT+DMOG, H3 .V KO, and H3 .V KO+DMOG) using Illumina TruSeq Stranded RNA LT Kit following the manufacturer’s instructions with limited modifications.

    Techniques: Expressing

    H3.V regulates VSG gene expression from silent telomeric bloodstream expression sites. (A) A schematic diagram of the silent ES15 (not to scale). The box with stripes represents the 70 bp repeats. Numbers indicate ESAG genes. Grey box represents the VSG pseudogene 11 (Tb427.BES126.13). (B-C) mRNA-seq and RT-qPCR analysis of the indicated VSG genes in silent expression sites. As described in Figs 3D and 5E , white bars: Wild type; grey bars: Wild type+DMOG; dark grey bars: H3 . V KO; black bars: H3 . V KO+DMOG. For mRNA-seq analysis, p values determined by Cuffdiff are indicated by asterisks: *, p value ≤ 0.05; **, p value ≤ 0.01. For RT-qPCR analysis, p values were calculated using Student’s t test. *, p value ≤ 0.05; **, p value ≤ 0.01.

    Journal: PLoS Genetics

    Article Title: Histone H3 Variant Regulates RNA Polymerase II Transcription Termination and Dual Strand Transcription of siRNA Loci in Trypanosoma brucei

    doi: 10.1371/journal.pgen.1005758

    Figure Lengend Snippet: H3.V regulates VSG gene expression from silent telomeric bloodstream expression sites. (A) A schematic diagram of the silent ES15 (not to scale). The box with stripes represents the 70 bp repeats. Numbers indicate ESAG genes. Grey box represents the VSG pseudogene 11 (Tb427.BES126.13). (B-C) mRNA-seq and RT-qPCR analysis of the indicated VSG genes in silent expression sites. As described in Figs 3D and 5E , white bars: Wild type; grey bars: Wild type+DMOG; dark grey bars: H3 . V KO; black bars: H3 . V KO+DMOG. For mRNA-seq analysis, p values determined by Cuffdiff are indicated by asterisks: *, p value ≤ 0.05; **, p value ≤ 0.01. For RT-qPCR analysis, p values were calculated using Student’s t test. *, p value ≤ 0.05; **, p value ≤ 0.01.

    Article Snippet: 12 mRNA-seq libraries were constructed (triplicate WT, WT+DMOG, H3 .V KO, and H3 .V KO+DMOG) using Illumina TruSeq Stranded RNA LT Kit following the manufacturer’s instructions with limited modifications.

    Techniques: Expressing, Quantitative RT-PCR

    Increased production of nascent RNA in cSSR following the loss of H3.V. A region on chromosome 2 (950–975 kb) representing cSSR 2.5 is shown where H3.V regulates transcription. (A) Base J and H3.V co-localize at sites of RNAP II termination within cSSRs [ 13 , 14 ]. H3.V ChIP-seq reads and base J IP-seq reads are plotted as reads per million reads (RPM), as previously described [ 14 , 35 ]. (B) ORFs are shown with the top strand in blue and the bottom strand in red. (C) mRNA-seq reads from wild type T . brucei are plotted as RPM. Reads that mapped to the top strand are shown in blue and reads that mapped to the bottom strand in red. (D) Small RNA-seq reads from WT and H3 . V KO are mapped as described in Fig 1A . (E-G) Strand-specific RT-PCR analysis of nascent RNA in cSSRs. (E) Schematic representation (not to scale) of primer location and direction at cSSR 2.5 (primers shown as green arrows in this and all subsequent figures). The arrowhead below the line indicates the poly(A) processing site for the final gene in the PTU. (F) Strand-specific RT-PCR analysis. cDNA was synthesized using the reverse primer. PCR was performed using the same reverse primer to make the cDNA plus the forward primer, as indicated. Data is also presented for an additional cSSR on chromosome 1 (cSSR 1.4, 635–637 kb). Wild type: WT; Wild type+DMOG: -J; H3 . V KO: KO; H3 . V KO+DMOG: KO-J. 40S ribosomal protein S11 provides a positive control and minus RT (-RT) negative control is shown. (G) Nested qPCR. Primers were designed within the PCR reaction in F to use in subsequent qPCR analysis. White bars: Wild type; grey bars: Wild type+DMOG; dark grey bars: H3 . V KO; black bars: H3 . V KO+DMOG. All products were normalized to 40S ribosomal protein S11. The average of three independent strand-specific RT-PCR nested qPCR experiments is plotted. Error bars represent the standard deviation. P values were calculated using Student’s t test. *, p value ≤ 0.05; **, p value ≤ 0.01.

    Journal: PLoS Genetics

    Article Title: Histone H3 Variant Regulates RNA Polymerase II Transcription Termination and Dual Strand Transcription of siRNA Loci in Trypanosoma brucei

    doi: 10.1371/journal.pgen.1005758

    Figure Lengend Snippet: Increased production of nascent RNA in cSSR following the loss of H3.V. A region on chromosome 2 (950–975 kb) representing cSSR 2.5 is shown where H3.V regulates transcription. (A) Base J and H3.V co-localize at sites of RNAP II termination within cSSRs [ 13 , 14 ]. H3.V ChIP-seq reads and base J IP-seq reads are plotted as reads per million reads (RPM), as previously described [ 14 , 35 ]. (B) ORFs are shown with the top strand in blue and the bottom strand in red. (C) mRNA-seq reads from wild type T . brucei are plotted as RPM. Reads that mapped to the top strand are shown in blue and reads that mapped to the bottom strand in red. (D) Small RNA-seq reads from WT and H3 . V KO are mapped as described in Fig 1A . (E-G) Strand-specific RT-PCR analysis of nascent RNA in cSSRs. (E) Schematic representation (not to scale) of primer location and direction at cSSR 2.5 (primers shown as green arrows in this and all subsequent figures). The arrowhead below the line indicates the poly(A) processing site for the final gene in the PTU. (F) Strand-specific RT-PCR analysis. cDNA was synthesized using the reverse primer. PCR was performed using the same reverse primer to make the cDNA plus the forward primer, as indicated. Data is also presented for an additional cSSR on chromosome 1 (cSSR 1.4, 635–637 kb). Wild type: WT; Wild type+DMOG: -J; H3 . V KO: KO; H3 . V KO+DMOG: KO-J. 40S ribosomal protein S11 provides a positive control and minus RT (-RT) negative control is shown. (G) Nested qPCR. Primers were designed within the PCR reaction in F to use in subsequent qPCR analysis. White bars: Wild type; grey bars: Wild type+DMOG; dark grey bars: H3 . V KO; black bars: H3 . V KO+DMOG. All products were normalized to 40S ribosomal protein S11. The average of three independent strand-specific RT-PCR nested qPCR experiments is plotted. Error bars represent the standard deviation. P values were calculated using Student’s t test. *, p value ≤ 0.05; **, p value ≤ 0.01.

    Article Snippet: 12 mRNA-seq libraries were constructed (triplicate WT, WT+DMOG, H3 .V KO, and H3 .V KO+DMOG) using Illumina TruSeq Stranded RNA LT Kit following the manufacturer’s instructions with limited modifications.

    Techniques: Chromatin Immunoprecipitation, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Synthesized, Polymerase Chain Reaction, Positive Control, Negative Control, Real-time Polymerase Chain Reaction, Standard Deviation

    H3.V and base J have independent yet additive roles in regulating termination and gene expression. (A) Heatmap of genes upregulated in the H3 . V KO. For the list of genes represented on the heatmap see S1 Table . Clustering of genes at the top indicate those that are further upregulated upon loss of base J in the H3 . V KO. J and H3.V columns indicate whether each gene is located within 10 kb of the modification (filled black box), as described in the Materials and Methods section. (B-D) H3.V/J localization, gene map, and mRNA-seq reads plotted for a gene cluster on chromosome 7 at cSSR 7.7 (position 1750–1800 kb shown) is illustrated as described in Fig 3 . (E) Plot of the mRNA-seq data for the genes indicated (numbered) in the ORF map. The average RPKM of triplicate mRNA-seq libraries was used to determine fold changes, with wild type set to 1. Error bars indicate the standard deviation between mRNA-seq replicates and p values, determined in Cuffdiff, are indicated by asterisks: *, p value ≤ 0.05; **, p value ≤ 0.01. (F) RT-qPCR analysis of gene expression for the indicated genes (according to the ORF map) as described in Fig 3D . White bars: Wild type; grey bars: Wild type+DMOG; dark grey bars: H3 . V KO; black bars: H3 . V KO+DMOG. P values were calculated using Student’s t test. *, p value ≤ 0.05; **, p value ≤ 0.01.

    Journal: PLoS Genetics

    Article Title: Histone H3 Variant Regulates RNA Polymerase II Transcription Termination and Dual Strand Transcription of siRNA Loci in Trypanosoma brucei

    doi: 10.1371/journal.pgen.1005758

    Figure Lengend Snippet: H3.V and base J have independent yet additive roles in regulating termination and gene expression. (A) Heatmap of genes upregulated in the H3 . V KO. For the list of genes represented on the heatmap see S1 Table . Clustering of genes at the top indicate those that are further upregulated upon loss of base J in the H3 . V KO. J and H3.V columns indicate whether each gene is located within 10 kb of the modification (filled black box), as described in the Materials and Methods section. (B-D) H3.V/J localization, gene map, and mRNA-seq reads plotted for a gene cluster on chromosome 7 at cSSR 7.7 (position 1750–1800 kb shown) is illustrated as described in Fig 3 . (E) Plot of the mRNA-seq data for the genes indicated (numbered) in the ORF map. The average RPKM of triplicate mRNA-seq libraries was used to determine fold changes, with wild type set to 1. Error bars indicate the standard deviation between mRNA-seq replicates and p values, determined in Cuffdiff, are indicated by asterisks: *, p value ≤ 0.05; **, p value ≤ 0.01. (F) RT-qPCR analysis of gene expression for the indicated genes (according to the ORF map) as described in Fig 3D . White bars: Wild type; grey bars: Wild type+DMOG; dark grey bars: H3 . V KO; black bars: H3 . V KO+DMOG. P values were calculated using Student’s t test. *, p value ≤ 0.05; **, p value ≤ 0.01.

    Article Snippet: 12 mRNA-seq libraries were constructed (triplicate WT, WT+DMOG, H3 .V KO, and H3 .V KO+DMOG) using Illumina TruSeq Stranded RNA LT Kit following the manufacturer’s instructions with limited modifications.

    Techniques: Expressing, Modification, Standard Deviation, Quantitative RT-PCR

    KEGG analysis of differentially expressed lncRNAs targets and mRNAs in primary wool follicle induction. (A,B) The top 20 KEGG enrichment pathways for differentially expressed lncRNA targets and mRNAs are presented. The longitudinal and horizontal axis represents the enrichment pathways and rich factor of these pathways, respectively. Spot size represents the number of differentially expressed genes enriched in each pathway, and the color of the spot represents the q -value of each pathway.

    Journal: Frontiers in Physiology

    Article Title: Transcriptome Reveals Long Non-coding RNAs and mRNAs Involved in Primary Wool Follicle Induction in Carpet Sheep Fetal Skin

    doi: 10.3389/fphys.2018.00446

    Figure Lengend Snippet: KEGG analysis of differentially expressed lncRNAs targets and mRNAs in primary wool follicle induction. (A,B) The top 20 KEGG enrichment pathways for differentially expressed lncRNA targets and mRNAs are presented. The longitudinal and horizontal axis represents the enrichment pathways and rich factor of these pathways, respectively. Spot size represents the number of differentially expressed genes enriched in each pathway, and the color of the spot represents the q -value of each pathway.

    Article Snippet: Identification of LncRNAs and mRNAs in Carpet Wool Sheep Fetal Skin Illumina sequencing of six cDNA libraries derived from carpet wool sheep whole skin yielded a total of 338,366,742 and 305,668,486 raw reads for stage 0 and stage 1 samples, respectively.

    Techniques:

    Gene Ontology (GO) analysis of differentially expressed lncRNAs targets and mRNAs in primary wool follicle induction. (A,B) The top 20 enrichment biological processes for differentially expressed lncRNA targets and mRNAs are listed. (C) One-to-one pairs of differentially expressed lncRNA targets and mRNAs involved in wool follicle and skin development. (D) One-to-one pairs of differentially expressed lncRNA targets and mRNAs involved in key signaling pathways in wool follicle development. The orange and blue circles represent the number of differentially expressed lncRNA targets and mRNAs, respectively. Their intersections represent the number of differentially expressed targets of differentially expressed lncRNAs.

    Journal: Frontiers in Physiology

    Article Title: Transcriptome Reveals Long Non-coding RNAs and mRNAs Involved in Primary Wool Follicle Induction in Carpet Sheep Fetal Skin

    doi: 10.3389/fphys.2018.00446

    Figure Lengend Snippet: Gene Ontology (GO) analysis of differentially expressed lncRNAs targets and mRNAs in primary wool follicle induction. (A,B) The top 20 enrichment biological processes for differentially expressed lncRNA targets and mRNAs are listed. (C) One-to-one pairs of differentially expressed lncRNA targets and mRNAs involved in wool follicle and skin development. (D) One-to-one pairs of differentially expressed lncRNA targets and mRNAs involved in key signaling pathways in wool follicle development. The orange and blue circles represent the number of differentially expressed lncRNA targets and mRNAs, respectively. Their intersections represent the number of differentially expressed targets of differentially expressed lncRNAs.

    Article Snippet: Identification of LncRNAs and mRNAs in Carpet Wool Sheep Fetal Skin Illumina sequencing of six cDNA libraries derived from carpet wool sheep whole skin yielded a total of 338,366,742 and 305,668,486 raw reads for stage 0 and stage 1 samples, respectively.

    Techniques:

    Expression levels of differently expressed mRNAs and lncRNAs involved in primary wool follicle induction were validated by qRT-PCR. Data are presented as mean ± SEM ( n = 6). ∗ p

    Journal: Frontiers in Physiology

    Article Title: Transcriptome Reveals Long Non-coding RNAs and mRNAs Involved in Primary Wool Follicle Induction in Carpet Sheep Fetal Skin

    doi: 10.3389/fphys.2018.00446

    Figure Lengend Snippet: Expression levels of differently expressed mRNAs and lncRNAs involved in primary wool follicle induction were validated by qRT-PCR. Data are presented as mean ± SEM ( n = 6). ∗ p

    Article Snippet: Identification of LncRNAs and mRNAs in Carpet Wool Sheep Fetal Skin Illumina sequencing of six cDNA libraries derived from carpet wool sheep whole skin yielded a total of 338,366,742 and 305,668,486 raw reads for stage 0 and stage 1 samples, respectively.

    Techniques: Expressing, Quantitative RT-PCR

    Interaction network in primary wool follicle induction. (A) mRNA–mRNA interaction network involved in hair follicle development was constructed and is presented. (B) lncRNA–mRNA interaction network related to hair follicle development is displayed. Red and green represent upregulated and downregulated, respectively. Circles and squares represent mRNAs and lncRNAs, respectively.

    Journal: Frontiers in Physiology

    Article Title: Transcriptome Reveals Long Non-coding RNAs and mRNAs Involved in Primary Wool Follicle Induction in Carpet Sheep Fetal Skin

    doi: 10.3389/fphys.2018.00446

    Figure Lengend Snippet: Interaction network in primary wool follicle induction. (A) mRNA–mRNA interaction network involved in hair follicle development was constructed and is presented. (B) lncRNA–mRNA interaction network related to hair follicle development is displayed. Red and green represent upregulated and downregulated, respectively. Circles and squares represent mRNAs and lncRNAs, respectively.

    Article Snippet: Identification of LncRNAs and mRNAs in Carpet Wool Sheep Fetal Skin Illumina sequencing of six cDNA libraries derived from carpet wool sheep whole skin yielded a total of 338,366,742 and 305,668,486 raw reads for stage 0 and stage 1 samples, respectively.

    Techniques: Construct

    Identification of long non-coding RNAs (lncRNAs) and mRNAs involved in primary wool follicle induction stages in carpet wool sheep fetal skins. (A,B) The morphology of skin and wool follicles at stages 0 and 1 of primary wool follicle development were observed by hematoxylin and eosin (H E) staining (Left) are 200×, scale bars represent 100 μm. (Right) are 400×, scale bars represent 50 μm, which is the magnification of the segment in the red frame). Epi, epidermis, Der, dermis, Pc, placode, Dc, dermal condensation. At stage 1 (B) , the hair placode and dermal condensation were clearly detected compared to the homogeneous epidermal layers at stage (A) . (C) Coding potentiality filter using Coding Potential Calculator, Pfam, Phylogenetic codon substitution frequency, and Coding-Non-Coding Index. (D) Exon number distribution of lncRNAs and mRNAs. (E) Transcript lengths distribution of lncRNAs and mRNAs. (F) Open reading frame length distribution of lncRNAs and mRNAs. (G) Conservation of the sequence in lncRNAs and mRNAs was analyzed using Phast (v1.3) software. (H) Differentially expressed transcripts in sheep skin between the two stages of primary wool follicle development. Red, green, and blue dots in the graph represent transcripts that were significantly upregulated, significantly downregulated, and not significantly changed between these two stages, respectively.

    Journal: Frontiers in Physiology

    Article Title: Transcriptome Reveals Long Non-coding RNAs and mRNAs Involved in Primary Wool Follicle Induction in Carpet Sheep Fetal Skin

    doi: 10.3389/fphys.2018.00446

    Figure Lengend Snippet: Identification of long non-coding RNAs (lncRNAs) and mRNAs involved in primary wool follicle induction stages in carpet wool sheep fetal skins. (A,B) The morphology of skin and wool follicles at stages 0 and 1 of primary wool follicle development were observed by hematoxylin and eosin (H E) staining (Left) are 200×, scale bars represent 100 μm. (Right) are 400×, scale bars represent 50 μm, which is the magnification of the segment in the red frame). Epi, epidermis, Der, dermis, Pc, placode, Dc, dermal condensation. At stage 1 (B) , the hair placode and dermal condensation were clearly detected compared to the homogeneous epidermal layers at stage (A) . (C) Coding potentiality filter using Coding Potential Calculator, Pfam, Phylogenetic codon substitution frequency, and Coding-Non-Coding Index. (D) Exon number distribution of lncRNAs and mRNAs. (E) Transcript lengths distribution of lncRNAs and mRNAs. (F) Open reading frame length distribution of lncRNAs and mRNAs. (G) Conservation of the sequence in lncRNAs and mRNAs was analyzed using Phast (v1.3) software. (H) Differentially expressed transcripts in sheep skin between the two stages of primary wool follicle development. Red, green, and blue dots in the graph represent transcripts that were significantly upregulated, significantly downregulated, and not significantly changed between these two stages, respectively.

    Article Snippet: Identification of LncRNAs and mRNAs in Carpet Wool Sheep Fetal Skin Illumina sequencing of six cDNA libraries derived from carpet wool sheep whole skin yielded a total of 338,366,742 and 305,668,486 raw reads for stage 0 and stage 1 samples, respectively.

    Techniques: Staining, Sequencing, Software