polymerase chain reaction pcr Search Results


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  • 99
    Thermo Fisher transcription polymerase chain reaction rt pcr
    Real-time <t>PCR</t> analysis of the 15 osteogenic genes at day 14 in vivo. ALP: alkaline phosphatase; TGF-β1: transforming growth factor-beta 1; VEGF-A: vascular endothelial growth factor A; bFGF: basic fibroblast growth factor; BMP-2: bone morphogenetic protein 2; BMP-4: bone morphogenetic protein 4; IGF-I: insulin-like growth factor I; RANK: receptor activator of nucleic factor kappa B; RANKL: receptor activator of nucleic factor kappa B ligand; Cbfa1: core binding factor alpha 1; OPG/OCIF: Osteoprotegerin/osteoclastogenesis inhibitory factor; PCR: polymerase chain reaction; mRNA: messenger <t>RNA.</t> Pooled samples ( n = 4) from each experimental group were assessed for mRNA amplification. The values are indicated as the mean relative to those for each gene expression in the control group on a logarithmic scale.
    Transcription Polymerase Chain Reaction Rt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1434 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Bio-Rad polymerase chain reaction pcr
    (A) Identification of new origins of replication, JunB and 5S rDNA in HT1080 cells. A real-time <t>PCR-based</t> nascent <t>DNA</t> enrichment assay ( A ) and ChIP assays with antibodies against components of pre-replication complex ( B , C ). cMyc origin was included as a positive control. Nascent DNA was normalized with JunB-nonORI (JunB-5). Precipitated chromatins for all antibodies were normalized with IgG. ( D, E ) Mapping of replication origin sites upstream the JunB gene in different human cell lines using nsDNA assay. Positions of primers used to map replication initiation sites within the JunB locus. A set of primers separated by 1.2 to 4.8 kb were chosen for mapping. Positions of primers sequences on the chromosome 19 in hg19 are shown in Supplementary Table S1. nsDNA enrichment in the JunB regions in different human cell lines. In each cell line, a profile of nascent DNA enrichment around JunB was characterized. In all analyzed cell lines, except for MCF-7 and NCI:OV:ADR-RES, the highest peak of nsDNA abundance corresponds to the JunB-2 sequence. In MCF-7 and NCI:OV:ADR-RES cells, origin of replication is mapped to the JunB-1 and JunB-3 sequences, respectively. Student's t -test was used: P
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    Stratagene polymerase chain reactions pcr
    The nucleotide and deduced amino acid sequences of Fus p 4.0101 (GenBank accession no. KF151224). Numbers to the right are the positions of the nucleotides and the deduced residues of the sequences. The stop codon TAA is denoted with an asterisk (*). The potential N-glycosylation site is indicated in bold letters and boxed. Eight conserved amino acid residues that are involved in the catalysis and the substrate binding of transaldolases are shaded and in bold letters. Nucleotide sequences in grey correspond to primer sequences synthesized for <t>PCR</t> experiments in the <t>cDNA</t> cloning of Fus p 4.0101 as described in the Materials and Methods. The sequences corresponding to primers Fu-TAase-f and Fu-TAase-r used in the preparation of rFus p 4.0101 are boxed.
    Polymerase Chain Reactions Pcr, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Eppendorf AG polymerase chain reactions pcr
    The nucleotide and deduced amino acid sequences of Fus p 4.0101 (GenBank accession no. KF151224). Numbers to the right are the positions of the nucleotides and the deduced residues of the sequences. The stop codon TAA is denoted with an asterisk (*). The potential N-glycosylation site is indicated in bold letters and boxed. Eight conserved amino acid residues that are involved in the catalysis and the substrate binding of transaldolases are shaded and in bold letters. Nucleotide sequences in grey correspond to primer sequences synthesized for <t>PCR</t> experiments in the <t>cDNA</t> cloning of Fus p 4.0101 as described in the Materials and Methods. The sequences corresponding to primers Fu-TAase-f and Fu-TAase-r used in the preparation of rFus p 4.0101 are boxed.
    Polymerase Chain Reactions Pcr, supplied by Eppendorf AG, used in various techniques. Bioz Stars score: 92/100, based on 173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher polymerase chain reaction
    Suppressed expression of ISGs in <t>NS5A-transgenic</t> mice. Expression levels of 2–5 OAS, PKR, and Mx1 genes were determined by quantitative real-time <t>PCR</t> in the livers of NS5A-transgenic (Tg) and non-transgenic (NT) mice injected with LPS (N = 5, each) or normal saline (N = 3, each). * p
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    93
    Bio-Rad quantitative polymerase chain reaction qpcr
    Fold decrements of template DNA of the <t>β-globin</t> and rhodopsin genes after MNase digestion of chromatin samples quantified by <t>qPCR.</t> Dark gray, phosphorylated samples. Light gray, unphosphorylated samples. ( A, C ) Results for the β-globin gene. ( B, D ) Results for the rhodopsin gene. Fold decrements were calculated as 2 ΔCt , using the Ct value of the initial undigested chromatin sample as reference for each gene. Error bars correspond to the interval 2 ΔCt±SD .
    Quantitative Polymerase Chain Reaction Qpcr, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 652 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad quantitative real time polymerase chain reaction qrt pcr
    The preventive effects of AMPK on the senescence associated with NAD + synthesis. (A) Cellular concentrations of NAD + on day 3 or day 5 after H 2 O 2 treatment. (B) Representative images of SA ‐β‐Gal staining of cells (left) and percentage of SA ‐β‐Gal‐positive cells (right). (C) Cellular concentrations of NAD + in the cells incubated with nicotinamide mononucleotide ( NMN ) for 3 days after H 2 O 2 treatment. (D) H 2 O 2 ‐treated cells were incubated with Met (10 mM ), BBR (10 μM), or CC (10 μM) for 3 days. The concentrations of NAD + are shown. (E) A schematic diagram of two pathways of NAD + synthesis. Red ones indicate rate‐limiting enzymes, and blue ones illustrate the enzyme inhibitors we used. (F) Relative fold‐changes in the mRNA levels of NAMPT , as monitored by <t>qRT</t> ‐ <t>PCR</t> (left), and representative images from immunoblot assays against NAMPT are shown (right). The ratio of NAMPT to actin was quantified by densitometry, based on the immunoblot images from three independent experiments. (G) Cells were incubated with Met, BBR , and CC for 3 days. Representative images from immunoblots against NAMPT are shown. The ratio of NAMPT to β‐actin was quantified by densitometry based on the immunoblot images from three independent experiments. (H) H 2 O 2 ‐treated NIH 3T3 cells were incubated with NAMPT inhibitor ( FK 866, 5 nM ), QPRT inhibitor ( PHTH , 1 mM ), Met (10 mM ), and BBR (10 μM) alone or in combination as indicated for 3 days. The percentages of SA ‐β‐Gal‐positive cells are shown. (I) The mRNA levels of NMNAT 1 , NMNAT 2 , and NMNAT 3 genes in the cells infected with corresponding lentivirus‐sh RNA or nontargeting sh RNA (sh CON ) as determined by qRT ‐ PCR . (J) Cellular concentrations of NAD + in sh RNA ‐infected cells. (K) Cells infected with sh RNA s were treated with H 2 O 2 and incubated with BBR for 3 days. Representative images of SA ‐β‐Gal staining of cells (left) and percentages of SA ‐β‐Gal‐positive cells (right). * P
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    Illumina Inc polymerase chain reaction pcr
    The preventive effects of AMPK on the senescence associated with NAD + synthesis. (A) Cellular concentrations of NAD + on day 3 or day 5 after H 2 O 2 treatment. (B) Representative images of SA ‐β‐Gal staining of cells (left) and percentage of SA ‐β‐Gal‐positive cells (right). (C) Cellular concentrations of NAD + in the cells incubated with nicotinamide mononucleotide ( NMN ) for 3 days after H 2 O 2 treatment. (D) H 2 O 2 ‐treated cells were incubated with Met (10 mM ), BBR (10 μM), or CC (10 μM) for 3 days. The concentrations of NAD + are shown. (E) A schematic diagram of two pathways of NAD + synthesis. Red ones indicate rate‐limiting enzymes, and blue ones illustrate the enzyme inhibitors we used. (F) Relative fold‐changes in the mRNA levels of NAMPT , as monitored by <t>qRT</t> ‐ <t>PCR</t> (left), and representative images from immunoblot assays against NAMPT are shown (right). The ratio of NAMPT to actin was quantified by densitometry, based on the immunoblot images from three independent experiments. (G) Cells were incubated with Met, BBR , and CC for 3 days. Representative images from immunoblots against NAMPT are shown. The ratio of NAMPT to β‐actin was quantified by densitometry based on the immunoblot images from three independent experiments. (H) H 2 O 2 ‐treated NIH 3T3 cells were incubated with NAMPT inhibitor ( FK 866, 5 nM ), QPRT inhibitor ( PHTH , 1 mM ), Met (10 mM ), and BBR (10 μM) alone or in combination as indicated for 3 days. The percentages of SA ‐β‐Gal‐positive cells are shown. (I) The mRNA levels of NMNAT 1 , NMNAT 2 , and NMNAT 3 genes in the cells infected with corresponding lentivirus‐sh RNA or nontargeting sh RNA (sh CON ) as determined by qRT ‐ PCR . (J) Cellular concentrations of NAD + in sh RNA ‐infected cells. (K) Cells infected with sh RNA s were treated with H 2 O 2 and incubated with BBR for 3 days. Representative images of SA ‐β‐Gal staining of cells (left) and percentages of SA ‐β‐Gal‐positive cells (right). * P
    Polymerase Chain Reaction Pcr, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 93/100, based on 149 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo polymerase chain reaction pcr
    The preventive effects of AMPK on the senescence associated with NAD + synthesis. (A) Cellular concentrations of NAD + on day 3 or day 5 after H 2 O 2 treatment. (B) Representative images of SA ‐β‐Gal staining of cells (left) and percentage of SA ‐β‐Gal‐positive cells (right). (C) Cellular concentrations of NAD + in the cells incubated with nicotinamide mononucleotide ( NMN ) for 3 days after H 2 O 2 treatment. (D) H 2 O 2 ‐treated cells were incubated with Met (10 mM ), BBR (10 μM), or CC (10 μM) for 3 days. The concentrations of NAD + are shown. (E) A schematic diagram of two pathways of NAD + synthesis. Red ones indicate rate‐limiting enzymes, and blue ones illustrate the enzyme inhibitors we used. (F) Relative fold‐changes in the mRNA levels of NAMPT , as monitored by <t>qRT</t> ‐ <t>PCR</t> (left), and representative images from immunoblot assays against NAMPT are shown (right). The ratio of NAMPT to actin was quantified by densitometry, based on the immunoblot images from three independent experiments. (G) Cells were incubated with Met, BBR , and CC for 3 days. Representative images from immunoblots against NAMPT are shown. The ratio of NAMPT to β‐actin was quantified by densitometry based on the immunoblot images from three independent experiments. (H) H 2 O 2 ‐treated NIH 3T3 cells were incubated with NAMPT inhibitor ( FK 866, 5 nM ), QPRT inhibitor ( PHTH , 1 mM ), Met (10 mM ), and BBR (10 μM) alone or in combination as indicated for 3 days. The percentages of SA ‐β‐Gal‐positive cells are shown. (I) The mRNA levels of NMNAT 1 , NMNAT 2 , and NMNAT 3 genes in the cells infected with corresponding lentivirus‐sh RNA or nontargeting sh RNA (sh CON ) as determined by qRT ‐ PCR . (J) Cellular concentrations of NAD + in sh RNA ‐infected cells. (K) Cells infected with sh RNA s were treated with H 2 O 2 and incubated with BBR for 3 days. Representative images of SA ‐β‐Gal staining of cells (left) and percentages of SA ‐β‐Gal‐positive cells (right). * P
    Polymerase Chain Reaction Pcr, supplied by Toyobo, used in various techniques. Bioz Stars score: 93/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad polymerase chain reactions qpcr
    Interaction networks of up-regulated genes and species-specific <t>qPCR</t> in xenogenic co-culture of bPCs and hMSCs. (A) The predicted interaction networks of the 180 up-regulated genes/proteins were shown. (B) 80% of human MSCs and 20% bovine chondrocytes were co-cultured for 2 days. <t>RNA</t> was then isolated. Expression of bovine genes was examined by real-time RT-PCR using bovine specific primers that did not cross-react with human genes. Data are expressed as fold change relative to the expression in chondrocyte monocultures and represent the mean of three independent experiments±S.D. (C) Expression of human genes was also demonstrated by real-time RT-PCR using human specific primers that did not cross-react with bovine genes. Data are expressed as fold change relative to the expression in MSC monocultures and represent the mean of three independent experiments±S.D. (D)
    Polymerase Chain Reactions Qpcr, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 90/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sequenom polymerase chain reactions pcr
    Interaction networks of up-regulated genes and species-specific <t>qPCR</t> in xenogenic co-culture of bPCs and hMSCs. (A) The predicted interaction networks of the 180 up-regulated genes/proteins were shown. (B) 80% of human MSCs and 20% bovine chondrocytes were co-cultured for 2 days. <t>RNA</t> was then isolated. Expression of bovine genes was examined by real-time RT-PCR using bovine specific primers that did not cross-react with human genes. Data are expressed as fold change relative to the expression in chondrocyte monocultures and represent the mean of three independent experiments±S.D. (C) Expression of human genes was also demonstrated by real-time RT-PCR using human specific primers that did not cross-react with bovine genes. Data are expressed as fold change relative to the expression in MSC monocultures and represent the mean of three independent experiments±S.D. (D)
    Polymerase Chain Reactions Pcr, supplied by Sequenom, used in various techniques. Bioz Stars score: 92/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ampflstr identifiler direct pcr amplification kit
    Interaction networks of up-regulated genes and species-specific <t>qPCR</t> in xenogenic co-culture of bPCs and hMSCs. (A) The predicted interaction networks of the 180 up-regulated genes/proteins were shown. (B) 80% of human MSCs and 20% bovine chondrocytes were co-cultured for 2 days. <t>RNA</t> was then isolated. Expression of bovine genes was examined by real-time RT-PCR using bovine specific primers that did not cross-react with human genes. Data are expressed as fold change relative to the expression in chondrocyte monocultures and represent the mean of three independent experiments±S.D. (C) Expression of human genes was also demonstrated by real-time RT-PCR using human specific primers that did not cross-react with bovine genes. Data are expressed as fold change relative to the expression in MSC monocultures and represent the mean of three independent experiments±S.D. (D)
    Ampflstr Identifiler Direct Pcr Amplification Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad quantitative real time polymerase chain reaction pcr
    CXCL1 and CCL3 are produced independently of TRIF in Kupffer cells, HSCs, and hepatocytes. ( A and B ) Primary Kupffer cells, ( C and D ) HSCs, and ( E and F ) hepatocytes were isolated from wild-type (WT) mice, ( A , C , and E ) TRIF -/- mice, and ( B , D , and F ) MyD88 -/- mice. Cells then were treated with LPS for 6 hours. Messenger <t>RNA</t> (mRNA) expression of CXCL1, CCL5, CCL3, IL10, Bambi, and Timp-1 was determined by quantitative real-time <t>PCR.</t> Similar results were obtained in 3 independent experiments. A representative result is shown. White square , wild-type mice; black square , TRIF -/- mice; gray square , MyD88 -/- mice. * P
    Quantitative Real Time Polymerase Chain Reaction Pcr, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 337 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher vetmax plus one step rt pcr kit
    CXCL1 and CCL3 are produced independently of TRIF in Kupffer cells, HSCs, and hepatocytes. ( A and B ) Primary Kupffer cells, ( C and D ) HSCs, and ( E and F ) hepatocytes were isolated from wild-type (WT) mice, ( A , C , and E ) TRIF -/- mice, and ( B , D , and F ) MyD88 -/- mice. Cells then were treated with LPS for 6 hours. Messenger <t>RNA</t> (mRNA) expression of CXCL1, CCL5, CCL3, IL10, Bambi, and Timp-1 was determined by quantitative real-time <t>PCR.</t> Similar results were obtained in 3 independent experiments. A representative result is shown. White square , wild-type mice; black square , TRIF -/- mice; gray square , MyD88 -/- mice. * P
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    Real-time PCR analysis of the 15 osteogenic genes at day 14 in vivo. ALP: alkaline phosphatase; TGF-β1: transforming growth factor-beta 1; VEGF-A: vascular endothelial growth factor A; bFGF: basic fibroblast growth factor; BMP-2: bone morphogenetic protein 2; BMP-4: bone morphogenetic protein 4; IGF-I: insulin-like growth factor I; RANK: receptor activator of nucleic factor kappa B; RANKL: receptor activator of nucleic factor kappa B ligand; Cbfa1: core binding factor alpha 1; OPG/OCIF: Osteoprotegerin/osteoclastogenesis inhibitory factor; PCR: polymerase chain reaction; mRNA: messenger RNA. Pooled samples ( n = 4) from each experimental group were assessed for mRNA amplification. The values are indicated as the mean relative to those for each gene expression in the control group on a logarithmic scale.

    Journal: Journal of Tissue Engineering

    Article Title: Effects of TGF-β1 and VEGF-A transgenes on the osteogenic potential of bone marrow stromal cells in vitro and in vivo

    doi: 10.1177/2041731412459745

    Figure Lengend Snippet: Real-time PCR analysis of the 15 osteogenic genes at day 14 in vivo. ALP: alkaline phosphatase; TGF-β1: transforming growth factor-beta 1; VEGF-A: vascular endothelial growth factor A; bFGF: basic fibroblast growth factor; BMP-2: bone morphogenetic protein 2; BMP-4: bone morphogenetic protein 4; IGF-I: insulin-like growth factor I; RANK: receptor activator of nucleic factor kappa B; RANKL: receptor activator of nucleic factor kappa B ligand; Cbfa1: core binding factor alpha 1; OPG/OCIF: Osteoprotegerin/osteoclastogenesis inhibitory factor; PCR: polymerase chain reaction; mRNA: messenger RNA. Pooled samples ( n = 4) from each experimental group were assessed for mRNA amplification. The values are indicated as the mean relative to those for each gene expression in the control group on a logarithmic scale.

    Article Snippet: Bone marrow cells from three wells per group were pooled and homogenized in TRIzol buffer (Invitrogen) at days 1, 2, 4, 7, and 14, followed by the extraction of total RNA using the SuperScript First-Strand Synthesis System for reverse transcription polymerase chain reaction (RT-PCR) (Invitrogen).

    Techniques: Real-time Polymerase Chain Reaction, In Vivo, ALP Assay, Binding Assay, Polymerase Chain Reaction, Amplification, Expressing

    (A) Identification of new origins of replication, JunB and 5S rDNA in HT1080 cells. A real-time PCR-based nascent DNA enrichment assay ( A ) and ChIP assays with antibodies against components of pre-replication complex ( B , C ). cMyc origin was included as a positive control. Nascent DNA was normalized with JunB-nonORI (JunB-5). Precipitated chromatins for all antibodies were normalized with IgG. ( D, E ) Mapping of replication origin sites upstream the JunB gene in different human cell lines using nsDNA assay. Positions of primers used to map replication initiation sites within the JunB locus. A set of primers separated by 1.2 to 4.8 kb were chosen for mapping. Positions of primers sequences on the chromosome 19 in hg19 are shown in Supplementary Table S1. nsDNA enrichment in the JunB regions in different human cell lines. In each cell line, a profile of nascent DNA enrichment around JunB was characterized. In all analyzed cell lines, except for MCF-7 and NCI:OV:ADR-RES, the highest peak of nsDNA abundance corresponds to the JunB-2 sequence. In MCF-7 and NCI:OV:ADR-RES cells, origin of replication is mapped to the JunB-1 and JunB-3 sequences, respectively. Student's t -test was used: P

    Journal: Nucleic Acids Research

    Article Title: Replication of alpha-satellite DNA arrays in endogenous human centromeric regions and in human artificial chromosome

    doi: 10.1093/nar/gku835

    Figure Lengend Snippet: (A) Identification of new origins of replication, JunB and 5S rDNA in HT1080 cells. A real-time PCR-based nascent DNA enrichment assay ( A ) and ChIP assays with antibodies against components of pre-replication complex ( B , C ). cMyc origin was included as a positive control. Nascent DNA was normalized with JunB-nonORI (JunB-5). Precipitated chromatins for all antibodies were normalized with IgG. ( D, E ) Mapping of replication origin sites upstream the JunB gene in different human cell lines using nsDNA assay. Positions of primers used to map replication initiation sites within the JunB locus. A set of primers separated by 1.2 to 4.8 kb were chosen for mapping. Positions of primers sequences on the chromosome 19 in hg19 are shown in Supplementary Table S1. nsDNA enrichment in the JunB regions in different human cell lines. In each cell line, a profile of nascent DNA enrichment around JunB was characterized. In all analyzed cell lines, except for MCF-7 and NCI:OV:ADR-RES, the highest peak of nsDNA abundance corresponds to the JunB-2 sequence. In MCF-7 and NCI:OV:ADR-RES cells, origin of replication is mapped to the JunB-1 and JunB-3 sequences, respectively. Student's t -test was used: P

    Article Snippet: The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time polymerase chain reaction (PCR) using iQ SYBR Green Supermix (Bio-Rad, USA).

    Techniques: Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Positive Control, Sequencing

    Replication timing of two different HORs, D5Z1 and D5Z2, on chromosome 5. Replication timing was measured by sorting BrdU-labeled cells into different S-phase fractions followed by BrdU-IPs. BrdU-labeled cells were stained with Hoechst 33342 and sorted to six fractions by FACS: G1, S1, S2, S3, S4 and G2/M. ( A, B ) Replication timing of β-globin and collagen origins. They replicate in early and late S phase, correspondingly. ( C, D ) Enrichment of BrdU-labeled alpha-satellite DNA in fractions identified by PCR using specific primers for D5Z1 and D5Z2 HORs. Statistically significant difference was observed between S2 and S3 phases for D5Z1 and D5Z2 in three experiments ( t -test, N = 3, P

    Journal: Nucleic Acids Research

    Article Title: Replication of alpha-satellite DNA arrays in endogenous human centromeric regions and in human artificial chromosome

    doi: 10.1093/nar/gku835

    Figure Lengend Snippet: Replication timing of two different HORs, D5Z1 and D5Z2, on chromosome 5. Replication timing was measured by sorting BrdU-labeled cells into different S-phase fractions followed by BrdU-IPs. BrdU-labeled cells were stained with Hoechst 33342 and sorted to six fractions by FACS: G1, S1, S2, S3, S4 and G2/M. ( A, B ) Replication timing of β-globin and collagen origins. They replicate in early and late S phase, correspondingly. ( C, D ) Enrichment of BrdU-labeled alpha-satellite DNA in fractions identified by PCR using specific primers for D5Z1 and D5Z2 HORs. Statistically significant difference was observed between S2 and S3 phases for D5Z1 and D5Z2 in three experiments ( t -test, N = 3, P

    Article Snippet: The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time polymerase chain reaction (PCR) using iQ SYBR Green Supermix (Bio-Rad, USA).

    Techniques: Labeling, Staining, FACS, Polymerase Chain Reaction

    Replication timing of alphoid arrays. ( A ) Replication timing was measured by sorting BrdU-labeled cells into different S-phase fractions followed by BrdU-IPs. BrdU-labeled cells were stained with Hoechst 33342 and sorted to six fractions by FACS: G1, S1, S2, S3, S4 and G2/M. ( B–D ) Replication timing of JunB , 5S rDNA and cMyc sites. They replicate early in S phase. ( E ) Enrichment of BrdU-labeled alpha-satellite DNA in fractions identified by PCR using primers for the alpha-satellite consensus sequence. This experiment suggests late replication of alphoid DNA.

    Journal: Nucleic Acids Research

    Article Title: Replication of alpha-satellite DNA arrays in endogenous human centromeric regions and in human artificial chromosome

    doi: 10.1093/nar/gku835

    Figure Lengend Snippet: Replication timing of alphoid arrays. ( A ) Replication timing was measured by sorting BrdU-labeled cells into different S-phase fractions followed by BrdU-IPs. BrdU-labeled cells were stained with Hoechst 33342 and sorted to six fractions by FACS: G1, S1, S2, S3, S4 and G2/M. ( B–D ) Replication timing of JunB , 5S rDNA and cMyc sites. They replicate early in S phase. ( E ) Enrichment of BrdU-labeled alpha-satellite DNA in fractions identified by PCR using primers for the alpha-satellite consensus sequence. This experiment suggests late replication of alphoid DNA.

    Article Snippet: The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time polymerase chain reaction (PCR) using iQ SYBR Green Supermix (Bio-Rad, USA).

    Techniques: Labeling, Staining, FACS, Polymerase Chain Reaction, Sequencing

    Mapping of replication initiation events within the alphoid tetO -HAC sequence. ( A ) Schematic of the HAC organization. The circular alphoid tetO -HAC was assembled from input DNA comprising ∼10 kb of RCA vector sequence (BAC/YAC) containing the Bsr gene and ∼40 kb of the synthetic tetO-alphoid array. The synthetic array is composed by tetO alphoid monomer (in green) arrange within every other monomer from chromosome 17 comprising CENP-B box (in gray). FISH demonstrates the presence of an autonomous form of alphoid tetO -HAC in HT1080 cells. The HAC was visualized using a vector probe (RCA) (in red) and tetO-alphoid probe (in green). ( B) DNA fiber-FISH analysis using a YAC/BAC vector probe demonstrating a regular structure of the HAC, i.e. a 10 kb YAC/BAC vector signals (in red) are flanked with a 40 kb alphoid tetO array (in green). ( C ) Real-time PCR-based nascent DNA enrichment assay to map origins of replication along the HAC sequence. ( D, E ) ChIP-qPCR with antibodies against Orc2 and Treslin shows enrichment of ORC on the Bsr sequences. JUNB-2 and cMyc were used as positive controls. ( F ) Chromatin profiling of the alphoid tetO -HAC shows H3K4me2 accumulation on the Bsr sequences. JUNB-2 and cMyc were used as positive controls. ( G ) ChIP analysis of H3K27me3 chromatin in the HAC. Student's t -test was used: P

    Journal: Nucleic Acids Research

    Article Title: Replication of alpha-satellite DNA arrays in endogenous human centromeric regions and in human artificial chromosome

    doi: 10.1093/nar/gku835

    Figure Lengend Snippet: Mapping of replication initiation events within the alphoid tetO -HAC sequence. ( A ) Schematic of the HAC organization. The circular alphoid tetO -HAC was assembled from input DNA comprising ∼10 kb of RCA vector sequence (BAC/YAC) containing the Bsr gene and ∼40 kb of the synthetic tetO-alphoid array. The synthetic array is composed by tetO alphoid monomer (in green) arrange within every other monomer from chromosome 17 comprising CENP-B box (in gray). FISH demonstrates the presence of an autonomous form of alphoid tetO -HAC in HT1080 cells. The HAC was visualized using a vector probe (RCA) (in red) and tetO-alphoid probe (in green). ( B) DNA fiber-FISH analysis using a YAC/BAC vector probe demonstrating a regular structure of the HAC, i.e. a 10 kb YAC/BAC vector signals (in red) are flanked with a 40 kb alphoid tetO array (in green). ( C ) Real-time PCR-based nascent DNA enrichment assay to map origins of replication along the HAC sequence. ( D, E ) ChIP-qPCR with antibodies against Orc2 and Treslin shows enrichment of ORC on the Bsr sequences. JUNB-2 and cMyc were used as positive controls. ( F ) Chromatin profiling of the alphoid tetO -HAC shows H3K4me2 accumulation on the Bsr sequences. JUNB-2 and cMyc were used as positive controls. ( G ) ChIP analysis of H3K27me3 chromatin in the HAC. Student's t -test was used: P

    Article Snippet: The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time polymerase chain reaction (PCR) using iQ SYBR Green Supermix (Bio-Rad, USA).

    Techniques: HAC Assay, Sequencing, Plasmid Preparation, BAC Assay, Fluorescence In Situ Hybridization, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

    CASP3, CASP7, CASP9, HSP27, HSP70, and HSP90 mRNA expression levels in satellite cells. The mRNA expressions of these genes were assayed by quantitative real-time PCR following 24 h transfection with shRNA expression constructs or control. ( A ) Showed the quantitative RT-PCR results of caspase-3, caspase-7, and caspase-9; ( B ) Showed the quantitative RT-PCR results of HSP27, HSP70, and HSP90. Values expressed as mean ± standard error of mean (SEM), calculated from three independent experiments. Gene expression was normalized to GAPDH expression and denoted as a gene/GAPDH ratio, * p

    Journal: In Vitro Cellular & Developmental Biology. Animal

    Article Title: Significant role of ?-calpain (CANP1) in proliferation/survival of bovine skeletal muscle satellite cells

    doi: 10.1007/s11626-013-9666-5

    Figure Lengend Snippet: CASP3, CASP7, CASP9, HSP27, HSP70, and HSP90 mRNA expression levels in satellite cells. The mRNA expressions of these genes were assayed by quantitative real-time PCR following 24 h transfection with shRNA expression constructs or control. ( A ) Showed the quantitative RT-PCR results of caspase-3, caspase-7, and caspase-9; ( B ) Showed the quantitative RT-PCR results of HSP27, HSP70, and HSP90. Values expressed as mean ± standard error of mean (SEM), calculated from three independent experiments. Gene expression was normalized to GAPDH expression and denoted as a gene/GAPDH ratio, * p

    Article Snippet: To assay the efficiency of CANP1 suppression by siRNA transfections and the possibility the siRNAs-induced cell death is regulated by particular genes, the analyses of CANP1, caspase-3 (CASP3), caspase-7 (CASP7), caspase-9 (CASP9), heat-shock protein 27 (HSP27), heat-shock protein 70 (HSP70), and heat-shock protein 90 (HSP90) mRNA expression in the shRNA expression constructs-transfected cells and control cells were carried out using real-time polymerase chain reaction (RT-PCR) (CFX96TM Real-Time PCR Detection Systems, Bio-Rad).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, shRNA, Construct, Quantitative RT-PCR

    The nucleotide and deduced amino acid sequences of Fus p 4.0101 (GenBank accession no. KF151224). Numbers to the right are the positions of the nucleotides and the deduced residues of the sequences. The stop codon TAA is denoted with an asterisk (*). The potential N-glycosylation site is indicated in bold letters and boxed. Eight conserved amino acid residues that are involved in the catalysis and the substrate binding of transaldolases are shaded and in bold letters. Nucleotide sequences in grey correspond to primer sequences synthesized for PCR experiments in the cDNA cloning of Fus p 4.0101 as described in the Materials and Methods. The sequences corresponding to primers Fu-TAase-f and Fu-TAase-r used in the preparation of rFus p 4.0101 are boxed.

    Journal: PLoS ONE

    Article Title: The Transaldolase, a Novel Allergen of Fusarium proliferatum, Demonstrates IgE Cross-Reactivity with Its Human Analogue

    doi: 10.1371/journal.pone.0103488

    Figure Lengend Snippet: The nucleotide and deduced amino acid sequences of Fus p 4.0101 (GenBank accession no. KF151224). Numbers to the right are the positions of the nucleotides and the deduced residues of the sequences. The stop codon TAA is denoted with an asterisk (*). The potential N-glycosylation site is indicated in bold letters and boxed. Eight conserved amino acid residues that are involved in the catalysis and the substrate binding of transaldolases are shaded and in bold letters. Nucleotide sequences in grey correspond to primer sequences synthesized for PCR experiments in the cDNA cloning of Fus p 4.0101 as described in the Materials and Methods. The sequences corresponding to primers Fu-TAase-f and Fu-TAase-r used in the preparation of rFus p 4.0101 are boxed.

    Article Snippet: cDNA Cloning The cDNA encoding the F. proliferatum transaldolase was isolated with polymerase chain reactions (PCR) using an AffinityScript Multiple Temperature cDNA Synthesis kit (Stratagene, La Jolla, Calif., USA) as previously described , .

    Techniques: Binding Assay, Synthesized, Polymerase Chain Reaction, Clone Assay

    Suppressed expression of ISGs in NS5A-transgenic mice. Expression levels of 2–5 OAS, PKR, and Mx1 genes were determined by quantitative real-time PCR in the livers of NS5A-transgenic (Tg) and non-transgenic (NT) mice injected with LPS (N = 5, each) or normal saline (N = 3, each). * p

    Journal: PLoS ONE

    Article Title: Nonstructural 5A Protein of Hepatitis C Virus Interferes with Toll-Like Receptor Signaling and Suppresses the Interferon Response in Mouse Liver

    doi: 10.1371/journal.pone.0170461

    Figure Lengend Snippet: Suppressed expression of ISGs in NS5A-transgenic mice. Expression levels of 2–5 OAS, PKR, and Mx1 genes were determined by quantitative real-time PCR in the livers of NS5A-transgenic (Tg) and non-transgenic (NT) mice injected with LPS (N = 5, each) or normal saline (N = 3, each). * p

    Article Snippet: Levels of NS5A mRNA were quantified by real-time polymerase chain reaction (PCR) with a specific probe and primers (Applied Biosystems, Foster City, CA).

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

    Expression of genes related to inflammatory response, and serum levels of transaminases in mice. (A) Expression levels of SAA1 and CRP genes were determined using quantitative real-time PCR in the livers of NS5A-transgenic (Tg) and non-transgenic (NT) mice 6 h after injection with LPS (N = 5, each) or normal saline (N = 3, each). (B) Serum levels of AST and ALT were determined in these mice using a dry chemistry technique. * statistically not significant.

    Journal: PLoS ONE

    Article Title: Nonstructural 5A Protein of Hepatitis C Virus Interferes with Toll-Like Receptor Signaling and Suppresses the Interferon Response in Mouse Liver

    doi: 10.1371/journal.pone.0170461

    Figure Lengend Snippet: Expression of genes related to inflammatory response, and serum levels of transaminases in mice. (A) Expression levels of SAA1 and CRP genes were determined using quantitative real-time PCR in the livers of NS5A-transgenic (Tg) and non-transgenic (NT) mice 6 h after injection with LPS (N = 5, each) or normal saline (N = 3, each). (B) Serum levels of AST and ALT were determined in these mice using a dry chemistry technique. * statistically not significant.

    Article Snippet: Levels of NS5A mRNA were quantified by real-time polymerase chain reaction (PCR) with a specific probe and primers (Applied Biosystems, Foster City, CA).

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

    Expression of intrahepatic cytokines and chemokines. (A) Expression levels of IL-6, TNF-α, and IFN-γ genes were determined using quantitative real-time PCR in the livers of NS5A-transgenic (Tg) and non-transgenic (NT) mice 6 h after injection with LPS (N = 5, each) or normal saline (N = 3, each). (B) Expression levels of CCL2, CCL4, and CCL5 were similarly determined using quantitative real-time PCR. * p

    Journal: PLoS ONE

    Article Title: Nonstructural 5A Protein of Hepatitis C Virus Interferes with Toll-Like Receptor Signaling and Suppresses the Interferon Response in Mouse Liver

    doi: 10.1371/journal.pone.0170461

    Figure Lengend Snippet: Expression of intrahepatic cytokines and chemokines. (A) Expression levels of IL-6, TNF-α, and IFN-γ genes were determined using quantitative real-time PCR in the livers of NS5A-transgenic (Tg) and non-transgenic (NT) mice 6 h after injection with LPS (N = 5, each) or normal saline (N = 3, each). (B) Expression levels of CCL2, CCL4, and CCL5 were similarly determined using quantitative real-time PCR. * p

    Article Snippet: Levels of NS5A mRNA were quantified by real-time polymerase chain reaction (PCR) with a specific probe and primers (Applied Biosystems, Foster City, CA).

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

    Expression of NS5A in transgenic mice. (A) Expression levels of NS5A protein in liver tissues of NS5A-transgenic (Tg) and non-transgenic (NT) mice were determined by immunoblotting with mouse anti-NS5A antibody. As a positive control (PC), lysates of the HCV subgenomic replicon cells were used. The arrow represents the NS5A protein. The arrowhead indicates a nonspecific band observed only in mouse tissues. (B) mRNA expression levels of NS5A in the liver of transgenic mice and HCV-infected patients were determined using quantitative real-time PCR. The levels were standardized to the β-actin gene. The median value of human samples was defined as 1 in Y-axis. (C) Liver tissue sections of the 6-month-old NS5A-transgenic (Tg) and non-transgenic (NT) mice were histologically examined with H E staining.

    Journal: PLoS ONE

    Article Title: Nonstructural 5A Protein of Hepatitis C Virus Interferes with Toll-Like Receptor Signaling and Suppresses the Interferon Response in Mouse Liver

    doi: 10.1371/journal.pone.0170461

    Figure Lengend Snippet: Expression of NS5A in transgenic mice. (A) Expression levels of NS5A protein in liver tissues of NS5A-transgenic (Tg) and non-transgenic (NT) mice were determined by immunoblotting with mouse anti-NS5A antibody. As a positive control (PC), lysates of the HCV subgenomic replicon cells were used. The arrow represents the NS5A protein. The arrowhead indicates a nonspecific band observed only in mouse tissues. (B) mRNA expression levels of NS5A in the liver of transgenic mice and HCV-infected patients were determined using quantitative real-time PCR. The levels were standardized to the β-actin gene. The median value of human samples was defined as 1 in Y-axis. (C) Liver tissue sections of the 6-month-old NS5A-transgenic (Tg) and non-transgenic (NT) mice were histologically examined with H E staining.

    Article Snippet: Levels of NS5A mRNA were quantified by real-time polymerase chain reaction (PCR) with a specific probe and primers (Applied Biosystems, Foster City, CA).

    Techniques: Expressing, Transgenic Assay, Mouse Assay, Positive Control, Infection, Real-time Polymerase Chain Reaction, Staining

    Fold decrements of template DNA of the β-globin and rhodopsin genes after MNase digestion of chromatin samples quantified by qPCR. Dark gray, phosphorylated samples. Light gray, unphosphorylated samples. ( A, C ) Results for the β-globin gene. ( B, D ) Results for the rhodopsin gene. Fold decrements were calculated as 2 ΔCt , using the Ct value of the initial undigested chromatin sample as reference for each gene. Error bars correspond to the interval 2 ΔCt±SD .

    Journal: Nucleic Acids Research

    Article Title: Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation

    doi: 10.1093/nar/gkv304

    Figure Lengend Snippet: Fold decrements of template DNA of the β-globin and rhodopsin genes after MNase digestion of chromatin samples quantified by qPCR. Dark gray, phosphorylated samples. Light gray, unphosphorylated samples. ( A, C ) Results for the β-globin gene. ( B, D ) Results for the rhodopsin gene. Fold decrements were calculated as 2 ΔCt , using the Ct value of the initial undigested chromatin sample as reference for each gene. Error bars correspond to the interval 2 ΔCt±SD .

    Article Snippet: Purified DNA was used as a template for the amplification of β-globin and rhodopsin genes by Quantitative Polymerase Chain Reaction (qPCR), following MIQE guidelines, in a C1000TM thermal cycler (BioRad).

    Techniques: Real-time Polymerase Chain Reaction

    The preventive effects of AMPK on the senescence associated with NAD + synthesis. (A) Cellular concentrations of NAD + on day 3 or day 5 after H 2 O 2 treatment. (B) Representative images of SA ‐β‐Gal staining of cells (left) and percentage of SA ‐β‐Gal‐positive cells (right). (C) Cellular concentrations of NAD + in the cells incubated with nicotinamide mononucleotide ( NMN ) for 3 days after H 2 O 2 treatment. (D) H 2 O 2 ‐treated cells were incubated with Met (10 mM ), BBR (10 μM), or CC (10 μM) for 3 days. The concentrations of NAD + are shown. (E) A schematic diagram of two pathways of NAD + synthesis. Red ones indicate rate‐limiting enzymes, and blue ones illustrate the enzyme inhibitors we used. (F) Relative fold‐changes in the mRNA levels of NAMPT , as monitored by qRT ‐ PCR (left), and representative images from immunoblot assays against NAMPT are shown (right). The ratio of NAMPT to actin was quantified by densitometry, based on the immunoblot images from three independent experiments. (G) Cells were incubated with Met, BBR , and CC for 3 days. Representative images from immunoblots against NAMPT are shown. The ratio of NAMPT to β‐actin was quantified by densitometry based on the immunoblot images from three independent experiments. (H) H 2 O 2 ‐treated NIH 3T3 cells were incubated with NAMPT inhibitor ( FK 866, 5 nM ), QPRT inhibitor ( PHTH , 1 mM ), Met (10 mM ), and BBR (10 μM) alone or in combination as indicated for 3 days. The percentages of SA ‐β‐Gal‐positive cells are shown. (I) The mRNA levels of NMNAT 1 , NMNAT 2 , and NMNAT 3 genes in the cells infected with corresponding lentivirus‐sh RNA or nontargeting sh RNA (sh CON ) as determined by qRT ‐ PCR . (J) Cellular concentrations of NAD + in sh RNA ‐infected cells. (K) Cells infected with sh RNA s were treated with H 2 O 2 and incubated with BBR for 3 days. Representative images of SA ‐β‐Gal staining of cells (left) and percentages of SA ‐β‐Gal‐positive cells (right). * P

    Journal: Aging Cell

    Article Title: AMPK activation protects cells from oxidative stress‐induced senescence via autophagic flux restoration and intracellular NAD+ elevation

    doi: 10.1111/acel.12446

    Figure Lengend Snippet: The preventive effects of AMPK on the senescence associated with NAD + synthesis. (A) Cellular concentrations of NAD + on day 3 or day 5 after H 2 O 2 treatment. (B) Representative images of SA ‐β‐Gal staining of cells (left) and percentage of SA ‐β‐Gal‐positive cells (right). (C) Cellular concentrations of NAD + in the cells incubated with nicotinamide mononucleotide ( NMN ) for 3 days after H 2 O 2 treatment. (D) H 2 O 2 ‐treated cells were incubated with Met (10 mM ), BBR (10 μM), or CC (10 μM) for 3 days. The concentrations of NAD + are shown. (E) A schematic diagram of two pathways of NAD + synthesis. Red ones indicate rate‐limiting enzymes, and blue ones illustrate the enzyme inhibitors we used. (F) Relative fold‐changes in the mRNA levels of NAMPT , as monitored by qRT ‐ PCR (left), and representative images from immunoblot assays against NAMPT are shown (right). The ratio of NAMPT to actin was quantified by densitometry, based on the immunoblot images from three independent experiments. (G) Cells were incubated with Met, BBR , and CC for 3 days. Representative images from immunoblots against NAMPT are shown. The ratio of NAMPT to β‐actin was quantified by densitometry based on the immunoblot images from three independent experiments. (H) H 2 O 2 ‐treated NIH 3T3 cells were incubated with NAMPT inhibitor ( FK 866, 5 nM ), QPRT inhibitor ( PHTH , 1 mM ), Met (10 mM ), and BBR (10 μM) alone or in combination as indicated for 3 days. The percentages of SA ‐β‐Gal‐positive cells are shown. (I) The mRNA levels of NMNAT 1 , NMNAT 2 , and NMNAT 3 genes in the cells infected with corresponding lentivirus‐sh RNA or nontargeting sh RNA (sh CON ) as determined by qRT ‐ PCR . (J) Cellular concentrations of NAD + in sh RNA ‐infected cells. (K) Cells infected with sh RNA s were treated with H 2 O 2 and incubated with BBR for 3 days. Representative images of SA ‐β‐Gal staining of cells (left) and percentages of SA ‐β‐Gal‐positive cells (right). * P

    Article Snippet: Quantitative real‐time polymerase chain reaction (qRT‐PCR) was performed using SYBR Green Supermix kit (Bio‐Rad, CA, USA) on a Bio‐Rad IQ5 system.

    Techniques: Staining, Incubation, Quantitative RT-PCR, Western Blot, Infection

    AMPK restored the autophagic flux associated with the amelioration of lysosomal function, mTOR inactivation, but not the nuclear translocation of TFEB . A‐C: H 2 O 2 ‐treated NIH 3T3 cells were incubated in complete medium with different reagents for 3 days as indicated. (A) Representative images from immunoblot assays against Cathepsin B protein. (B) The acid phosphatase activity in cells. (C) Representative images from immunoblot assays against phosphorylated mTOR (pm TOR , Ser2448), phosphorylated 70S6K (P‐p70S6K, Thr389), pAMPK (Thr172), and β‐actin. (D) Immunofluorescent images of TFEB after treatment with indicated reagents. The bar represents 20 μm. (E) Immunoblot assays against TFEB protein of total cytoplasmic and nuclear subcellular fractions obtained from NIH 3T3 cells with the indicated treatment. (F) Relative fold‐changes in mRNA levels of two TFEB target genes ( GNS and LAMP ‐1 ) were monitored by qRT ‐ PCR assays.* P

    Journal: Aging Cell

    Article Title: AMPK activation protects cells from oxidative stress‐induced senescence via autophagic flux restoration and intracellular NAD+ elevation

    doi: 10.1111/acel.12446

    Figure Lengend Snippet: AMPK restored the autophagic flux associated with the amelioration of lysosomal function, mTOR inactivation, but not the nuclear translocation of TFEB . A‐C: H 2 O 2 ‐treated NIH 3T3 cells were incubated in complete medium with different reagents for 3 days as indicated. (A) Representative images from immunoblot assays against Cathepsin B protein. (B) The acid phosphatase activity in cells. (C) Representative images from immunoblot assays against phosphorylated mTOR (pm TOR , Ser2448), phosphorylated 70S6K (P‐p70S6K, Thr389), pAMPK (Thr172), and β‐actin. (D) Immunofluorescent images of TFEB after treatment with indicated reagents. The bar represents 20 μm. (E) Immunoblot assays against TFEB protein of total cytoplasmic and nuclear subcellular fractions obtained from NIH 3T3 cells with the indicated treatment. (F) Relative fold‐changes in mRNA levels of two TFEB target genes ( GNS and LAMP ‐1 ) were monitored by qRT ‐ PCR assays.* P

    Article Snippet: Quantitative real‐time polymerase chain reaction (qRT‐PCR) was performed using SYBR Green Supermix kit (Bio‐Rad, CA, USA) on a Bio‐Rad IQ5 system.

    Techniques: Translocation Assay, Incubation, Activity Assay, Quantitative RT-PCR

    Activation of AMPK prevented H 2 O 2 ‐induced senescence. A to D: H 2 O 2 ‐treated NIH 3T3 cells were incubated in complete medium with metformin (Met, 5 to 10 mM ) or berberine ( BBR , 5 to 10 μM) for 3 days. (A) Representative images from immunoblot assays against pAMPK α (Thr172), AMPK α1, pACC (Ser79), and β‐actin. (B) Relative fold‐changes in mRNA levels of CPT ‐1 and FAS as determined by qRT ‐ PCR . (C) Representative images of SA ‐β‐Gal staining of cells (left), and percentages of SA ‐β‐Gal‐positive cells. D‐F: NIH 3T3 cells were treated with H 2 O 2 and incubated with Met (10 mM ), BBR (10 μM) and an AMPK inhibitor, Compound C ( CC , 10 μM), alone or in combination for 3 days. (D) Representative images from immunoblot assays. (E) Relative mRNA levels of CPT ‐1 and FAS as determined by qRT ‐ PCR . (F) Representative images of SA ‐β‐Gal staining of cells (left) and percentages of SA ‐β‐Gal‐positive cells (right). G‐I: NIH 3T3 cells without H 2 O 2 treatment were used. (G) The decrease in AMPK activity in DN ‐ AMPK ‐expressing NIH 3T3 cells was shown by the decrease in AMPK α phosphorylation. (H) Representative images of SA ‐β‐Gal staining of the nontransfected cells with or without CC (upper) and cells transfection with DN ‐ AMPK (lower). (I) The percentages of SA ‐β‐Gal‐positive cells were calculated based on the images represented in H. (J) H 2 O 2 ‐treated cells incubated with or without Met (10 mM ) or BBR (10 μM) for 3 days, representative images of SA ‐β‐Gal staining of cells transfected with empty vector (upper) or DN ‐ AMPK (lower). (K) The percentages of SA ‐β‐Gal‐positive cells were calculated based on the images presented in J. * P

    Journal: Aging Cell

    Article Title: AMPK activation protects cells from oxidative stress‐induced senescence via autophagic flux restoration and intracellular NAD+ elevation

    doi: 10.1111/acel.12446

    Figure Lengend Snippet: Activation of AMPK prevented H 2 O 2 ‐induced senescence. A to D: H 2 O 2 ‐treated NIH 3T3 cells were incubated in complete medium with metformin (Met, 5 to 10 mM ) or berberine ( BBR , 5 to 10 μM) for 3 days. (A) Representative images from immunoblot assays against pAMPK α (Thr172), AMPK α1, pACC (Ser79), and β‐actin. (B) Relative fold‐changes in mRNA levels of CPT ‐1 and FAS as determined by qRT ‐ PCR . (C) Representative images of SA ‐β‐Gal staining of cells (left), and percentages of SA ‐β‐Gal‐positive cells. D‐F: NIH 3T3 cells were treated with H 2 O 2 and incubated with Met (10 mM ), BBR (10 μM) and an AMPK inhibitor, Compound C ( CC , 10 μM), alone or in combination for 3 days. (D) Representative images from immunoblot assays. (E) Relative mRNA levels of CPT ‐1 and FAS as determined by qRT ‐ PCR . (F) Representative images of SA ‐β‐Gal staining of cells (left) and percentages of SA ‐β‐Gal‐positive cells (right). G‐I: NIH 3T3 cells without H 2 O 2 treatment were used. (G) The decrease in AMPK activity in DN ‐ AMPK ‐expressing NIH 3T3 cells was shown by the decrease in AMPK α phosphorylation. (H) Representative images of SA ‐β‐Gal staining of the nontransfected cells with or without CC (upper) and cells transfection with DN ‐ AMPK (lower). (I) The percentages of SA ‐β‐Gal‐positive cells were calculated based on the images represented in H. (J) H 2 O 2 ‐treated cells incubated with or without Met (10 mM ) or BBR (10 μM) for 3 days, representative images of SA ‐β‐Gal staining of cells transfected with empty vector (upper) or DN ‐ AMPK (lower). (K) The percentages of SA ‐β‐Gal‐positive cells were calculated based on the images presented in J. * P

    Article Snippet: Quantitative real‐time polymerase chain reaction (qRT‐PCR) was performed using SYBR Green Supermix kit (Bio‐Rad, CA, USA) on a Bio‐Rad IQ5 system.

    Techniques: Activation Assay, Incubation, Cycling Probe Technology, Quantitative RT-PCR, Staining, Activity Assay, Expressing, Transfection, Plasmid Preparation

    H 2 O 2 induced senescence and AMPK pathway inhibition in NIH 3T3 Cells. Cells were treated with H 2 O 2 and incubated in complete medium without H 2 O 2 for 3‐5 days. CTL means untreated cells. (A) Representative images of SA ‐β‐Gal staining of the cells (left) and percentages of SA ‐β‐Gal‐positive cells in a total of 1000 cells (right). (B) Representative images of SAHF s in cells (left) and percentages of SAHF s‐positive cells in 1000 cells (right). (C) Representative images from immunoblot assays against p53 and β‐actin. (D) Relative fold‐changes in the mRNA levels of the genes encoding p21, IL 6 and IL 8 , as determined by qRT ‐ PCR . (E) Representative images from immunoblot assays against phosphorylated AMPK α ( pAMPK , Thr172), AMPK α1, phosphorylated ACC ( pACC , Ser79), and β‐actin. (F) The ratio of pAMPK to total AMPK was quantified by densitometry based on immunoblot images from three independent experiments. (G) Relative fold‐changes in the mRNA levels of two AMPK target genes ( CPT ‐1 and FAS ) were monitored by qRT ‐ PCR assays. * P

    Journal: Aging Cell

    Article Title: AMPK activation protects cells from oxidative stress‐induced senescence via autophagic flux restoration and intracellular NAD+ elevation

    doi: 10.1111/acel.12446

    Figure Lengend Snippet: H 2 O 2 induced senescence and AMPK pathway inhibition in NIH 3T3 Cells. Cells were treated with H 2 O 2 and incubated in complete medium without H 2 O 2 for 3‐5 days. CTL means untreated cells. (A) Representative images of SA ‐β‐Gal staining of the cells (left) and percentages of SA ‐β‐Gal‐positive cells in a total of 1000 cells (right). (B) Representative images of SAHF s in cells (left) and percentages of SAHF s‐positive cells in 1000 cells (right). (C) Representative images from immunoblot assays against p53 and β‐actin. (D) Relative fold‐changes in the mRNA levels of the genes encoding p21, IL 6 and IL 8 , as determined by qRT ‐ PCR . (E) Representative images from immunoblot assays against phosphorylated AMPK α ( pAMPK , Thr172), AMPK α1, phosphorylated ACC ( pACC , Ser79), and β‐actin. (F) The ratio of pAMPK to total AMPK was quantified by densitometry based on immunoblot images from three independent experiments. (G) Relative fold‐changes in the mRNA levels of two AMPK target genes ( CPT ‐1 and FAS ) were monitored by qRT ‐ PCR assays. * P

    Article Snippet: Quantitative real‐time polymerase chain reaction (qRT‐PCR) was performed using SYBR Green Supermix kit (Bio‐Rad, CA, USA) on a Bio‐Rad IQ5 system.

    Techniques: Inhibition, Incubation, CTL Assay, Staining, Quantitative RT-PCR, Cycling Probe Technology

    Interaction networks of up-regulated genes and species-specific qPCR in xenogenic co-culture of bPCs and hMSCs. (A) The predicted interaction networks of the 180 up-regulated genes/proteins were shown. (B) 80% of human MSCs and 20% bovine chondrocytes were co-cultured for 2 days. RNA was then isolated. Expression of bovine genes was examined by real-time RT-PCR using bovine specific primers that did not cross-react with human genes. Data are expressed as fold change relative to the expression in chondrocyte monocultures and represent the mean of three independent experiments±S.D. (C) Expression of human genes was also demonstrated by real-time RT-PCR using human specific primers that did not cross-react with bovine genes. Data are expressed as fold change relative to the expression in MSC monocultures and represent the mean of three independent experiments±S.D. (D)

    Journal: Stem Cells and Development

    Article Title: Fibroblast Growth Factor-1 Is a Mesenchymal Stromal Cell-Secreted Factor Stimulating Proliferation of Osteoarthritic Chondrocytes in Co-Culture

    doi: 10.1089/scd.2013.0118

    Figure Lengend Snippet: Interaction networks of up-regulated genes and species-specific qPCR in xenogenic co-culture of bPCs and hMSCs. (A) The predicted interaction networks of the 180 up-regulated genes/proteins were shown. (B) 80% of human MSCs and 20% bovine chondrocytes were co-cultured for 2 days. RNA was then isolated. Expression of bovine genes was examined by real-time RT-PCR using bovine specific primers that did not cross-react with human genes. Data are expressed as fold change relative to the expression in chondrocyte monocultures and represent the mean of three independent experiments±S.D. (C) Expression of human genes was also demonstrated by real-time RT-PCR using human specific primers that did not cross-react with bovine genes. Data are expressed as fold change relative to the expression in MSC monocultures and represent the mean of three independent experiments±S.D. (D)

    Article Snippet: For quantitative polymerase chain reactions (qPCR), 1 μg of total RNA was reverse transcribed into cDNA using the iScript cDNA Synthesis kit (Bio-Rad). qPCR was performed on cDNA samples by using the iQ SYBR Green Supermix (Bio-Rad).

    Techniques: Real-time Polymerase Chain Reaction, Co-Culture Assay, Cell Culture, Isolation, Expressing, Quantitative RT-PCR

    CXCL1 and CCL3 are produced independently of TRIF in Kupffer cells, HSCs, and hepatocytes. ( A and B ) Primary Kupffer cells, ( C and D ) HSCs, and ( E and F ) hepatocytes were isolated from wild-type (WT) mice, ( A , C , and E ) TRIF -/- mice, and ( B , D , and F ) MyD88 -/- mice. Cells then were treated with LPS for 6 hours. Messenger RNA (mRNA) expression of CXCL1, CCL5, CCL3, IL10, Bambi, and Timp-1 was determined by quantitative real-time PCR. Similar results were obtained in 3 independent experiments. A representative result is shown. White square , wild-type mice; black square , TRIF -/- mice; gray square , MyD88 -/- mice. * P

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: TRIF Differentially Regulates Hepatic Steatosis and Inflammation/Fibrosis in Mice

    doi: 10.1016/j.jcmgh.2016.12.004

    Figure Lengend Snippet: CXCL1 and CCL3 are produced independently of TRIF in Kupffer cells, HSCs, and hepatocytes. ( A and B ) Primary Kupffer cells, ( C and D ) HSCs, and ( E and F ) hepatocytes were isolated from wild-type (WT) mice, ( A , C , and E ) TRIF -/- mice, and ( B , D , and F ) MyD88 -/- mice. Cells then were treated with LPS for 6 hours. Messenger RNA (mRNA) expression of CXCL1, CCL5, CCL3, IL10, Bambi, and Timp-1 was determined by quantitative real-time PCR. Similar results were obtained in 3 independent experiments. A representative result is shown. White square , wild-type mice; black square , TRIF -/- mice; gray square , MyD88 -/- mice. * P

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction Extracted RNA from livers and cells was subjected to reverse-transcription and subsequent quantitative real-time polymerase chain reaction (PCR) with the use of the CFX96 real-time PCR system (Bio-Rad, Hercules, CA).

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

    TRIF -/- mice showed severe inflammation and fibrosis but less steatosis in the murine NASH model induced by the CDAA diet. Wild-type (WT) and TRIF -/- mice were fed a choline-supplemented amino acid–defined diet (CS) or CDAA (CD) diet for 22 weeks (n = 5-9, each). ( A ) Liver sections were stained with H E and Oil Red O. Original magnification, ×200 for H E and Oil Red O staining. ( B ) Serum ALT levels. ( C ) NAFLD activity score. ( D ) Hepatic messenger RNA (mRNA) (TNF, IL10) expression was determined by quantitative real-time PCR. ( E ) Liver sections were stained with Sirius Red and used for immunohistochemistry for α-SMA. Original magnification, ×100 for Sirius Red staining, and ×200 for α-SMA staining. ( F ) Quantification of Sirius Red staining. ( G ) Hepatic fibrogenic genes (collagen α1[I], α-SMA) were determined by quantitative real-time PCR. White square , wild-type mice; black square , TRIF -/- mice. Similar results were obtained in 2 independent experiments. A representative result is shown. ** P

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: TRIF Differentially Regulates Hepatic Steatosis and Inflammation/Fibrosis in Mice

    doi: 10.1016/j.jcmgh.2016.12.004

    Figure Lengend Snippet: TRIF -/- mice showed severe inflammation and fibrosis but less steatosis in the murine NASH model induced by the CDAA diet. Wild-type (WT) and TRIF -/- mice were fed a choline-supplemented amino acid–defined diet (CS) or CDAA (CD) diet for 22 weeks (n = 5-9, each). ( A ) Liver sections were stained with H E and Oil Red O. Original magnification, ×200 for H E and Oil Red O staining. ( B ) Serum ALT levels. ( C ) NAFLD activity score. ( D ) Hepatic messenger RNA (mRNA) (TNF, IL10) expression was determined by quantitative real-time PCR. ( E ) Liver sections were stained with Sirius Red and used for immunohistochemistry for α-SMA. Original magnification, ×100 for Sirius Red staining, and ×200 for α-SMA staining. ( F ) Quantification of Sirius Red staining. ( G ) Hepatic fibrogenic genes (collagen α1[I], α-SMA) were determined by quantitative real-time PCR. White square , wild-type mice; black square , TRIF -/- mice. Similar results were obtained in 2 independent experiments. A representative result is shown. ** P

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction Extracted RNA from livers and cells was subjected to reverse-transcription and subsequent quantitative real-time polymerase chain reaction (PCR) with the use of the CFX96 real-time PCR system (Bio-Rad, Hercules, CA).

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

    The TLR4–TRIF signaling enhances palmitate-induced fat accumulation in hepatocytes. Wild-type (WT), TRIF -/- , and TLR4 -/- hepatocytes were treated with 200 μmol/L palmitate and/or 100 ng/mL LPS for 24 hours. ( A ) Representative pictures of Oil Red O staining. Original magnification, ×400. ( B ) Triglyceride (TG) concentrations in hepatocytes. ( C ) WT and TRIF -/- hepatocytes were treated with 200 μmol/L palmitate and/or 100 ng/mL LPS for 12 hours. Messenger RNA (mRNA) expression of DGAT2 was determined by quantitative real-time PCR. Similar results were obtained in 2 independent experiments. A representative result is shown. ( D ) WT and TRIF -/- mice were fed a choline-supplemented amino acid–defined diet (CS) or CDAA (CD) diet for 22 weeks (n = 5-9, each). Hepatic DGAT2 mRNA expression was determined by quantitative real-time PCR. White square , wild-type mice; black square , TRIF -/- mice; gray square , TLR4 -/- mice. ** P

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: TRIF Differentially Regulates Hepatic Steatosis and Inflammation/Fibrosis in Mice

    doi: 10.1016/j.jcmgh.2016.12.004

    Figure Lengend Snippet: The TLR4–TRIF signaling enhances palmitate-induced fat accumulation in hepatocytes. Wild-type (WT), TRIF -/- , and TLR4 -/- hepatocytes were treated with 200 μmol/L palmitate and/or 100 ng/mL LPS for 24 hours. ( A ) Representative pictures of Oil Red O staining. Original magnification, ×400. ( B ) Triglyceride (TG) concentrations in hepatocytes. ( C ) WT and TRIF -/- hepatocytes were treated with 200 μmol/L palmitate and/or 100 ng/mL LPS for 12 hours. Messenger RNA (mRNA) expression of DGAT2 was determined by quantitative real-time PCR. Similar results were obtained in 2 independent experiments. A representative result is shown. ( D ) WT and TRIF -/- mice were fed a choline-supplemented amino acid–defined diet (CS) or CDAA (CD) diet for 22 weeks (n = 5-9, each). Hepatic DGAT2 mRNA expression was determined by quantitative real-time PCR. White square , wild-type mice; black square , TRIF -/- mice; gray square , TLR4 -/- mice. ** P

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction Extracted RNA from livers and cells was subjected to reverse-transcription and subsequent quantitative real-time polymerase chain reaction (PCR) with the use of the CFX96 real-time PCR system (Bio-Rad, Hercules, CA).

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

    BM-derived and resident liver cells are required for TLR4-mediated hepatic steatosis, inflammation, and fibrosis in the murine NASH model induced by CDAA diet. ( A ) Wild-type mice were transplanted with BM from β-actin promoter-driven GFP-transgenic mice after whole-body irradiation. After 2 weeks of BMT, liposomal clodronate was injected. After 10 weeks of BMT, liver nonparenchymal cell fraction was separated and F4/80-positive GFP-expressing cells were examined by fluorescence-activated cell sorter analysis. ( B–I ) The TLR4 BM chimeric mice were fed the CDAA diet for 22 weeks (n = 5-9, each). ( B ) The successful engraftment of donor BM cells into TLR4 BM chimeric mice was determined by TLR4 messenger RNA (mRNA) expression in spleen cells. ( C ) Hepatic inflammation and steatosis were evaluated by H E staining and Oil Red O staining, respectively. Original magnification, ×100 for H E, and ×200 for Oil Red O. ( D ) ALT levels. ( E ) Hepatic TNF mRNA levels were measured by quantitative PCR. ( F ) NAFLD activity score. ( G ) Liver sections were stained with Sirius Red and used for immunohistochemistry for α-SMA. Original magnification, ×100 for Sirius Red staining, and ×200 for α-SMA staining. ( H ) Quantifications of Sirius red staining. ( I ) Hepatic fibrogenic genes (collagen α1[I], α-SMA) were determined by quantitative real-time PCR. Similar results were obtained in 2 independent experiments. A representative result is shown. * P

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: TRIF Differentially Regulates Hepatic Steatosis and Inflammation/Fibrosis in Mice

    doi: 10.1016/j.jcmgh.2016.12.004

    Figure Lengend Snippet: BM-derived and resident liver cells are required for TLR4-mediated hepatic steatosis, inflammation, and fibrosis in the murine NASH model induced by CDAA diet. ( A ) Wild-type mice were transplanted with BM from β-actin promoter-driven GFP-transgenic mice after whole-body irradiation. After 2 weeks of BMT, liposomal clodronate was injected. After 10 weeks of BMT, liver nonparenchymal cell fraction was separated and F4/80-positive GFP-expressing cells were examined by fluorescence-activated cell sorter analysis. ( B–I ) The TLR4 BM chimeric mice were fed the CDAA diet for 22 weeks (n = 5-9, each). ( B ) The successful engraftment of donor BM cells into TLR4 BM chimeric mice was determined by TLR4 messenger RNA (mRNA) expression in spleen cells. ( C ) Hepatic inflammation and steatosis were evaluated by H E staining and Oil Red O staining, respectively. Original magnification, ×100 for H E, and ×200 for Oil Red O. ( D ) ALT levels. ( E ) Hepatic TNF mRNA levels were measured by quantitative PCR. ( F ) NAFLD activity score. ( G ) Liver sections were stained with Sirius Red and used for immunohistochemistry for α-SMA. Original magnification, ×100 for Sirius Red staining, and ×200 for α-SMA staining. ( H ) Quantifications of Sirius red staining. ( I ) Hepatic fibrogenic genes (collagen α1[I], α-SMA) were determined by quantitative real-time PCR. Similar results were obtained in 2 independent experiments. A representative result is shown. * P

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction Extracted RNA from livers and cells was subjected to reverse-transcription and subsequent quantitative real-time polymerase chain reaction (PCR) with the use of the CFX96 real-time PCR system (Bio-Rad, Hercules, CA).

    Techniques: Derivative Assay, Mouse Assay, Transgenic Assay, Irradiation, Injection, Expressing, Fluorescence, Staining, Real-time Polymerase Chain Reaction, Activity Assay, Immunohistochemistry

    Increased CXCL1 and CCL3 levels accompanied by increased infiltration of neutrophils and macrophages in TRIF -/- mice. Wild-type (WT) and TRIF -/- mice were fed a choline-supplemented amino acid–defined diet (CS) or CDAA (CD) diet for 22 weeks (n = 4-10, each). ( A ) Hepatic CXCL1 messenger RNA (mRNA) expression was determined by quantitative real-time PCR. ( B ) Liver sections were used for immunofluorescence for Ly6G and their quantifications. Original magnification, ×200. ( C ) Hepatic CCL3 mRNA expression was determined by quantitative real-time PCR. ( D ) Liver sections were used for immunohistochemistry for F4/80 and their quantifications. Original magnification, ×200. White square , wild-type mice; black square , TRIF -/- mice. * P

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: TRIF Differentially Regulates Hepatic Steatosis and Inflammation/Fibrosis in Mice

    doi: 10.1016/j.jcmgh.2016.12.004

    Figure Lengend Snippet: Increased CXCL1 and CCL3 levels accompanied by increased infiltration of neutrophils and macrophages in TRIF -/- mice. Wild-type (WT) and TRIF -/- mice were fed a choline-supplemented amino acid–defined diet (CS) or CDAA (CD) diet for 22 weeks (n = 4-10, each). ( A ) Hepatic CXCL1 messenger RNA (mRNA) expression was determined by quantitative real-time PCR. ( B ) Liver sections were used for immunofluorescence for Ly6G and their quantifications. Original magnification, ×200. ( C ) Hepatic CCL3 mRNA expression was determined by quantitative real-time PCR. ( D ) Liver sections were used for immunohistochemistry for F4/80 and their quantifications. Original magnification, ×200. White square , wild-type mice; black square , TRIF -/- mice. * P

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction Extracted RNA from livers and cells was subjected to reverse-transcription and subsequent quantitative real-time polymerase chain reaction (PCR) with the use of the CFX96 real-time PCR system (Bio-Rad, Hercules, CA).

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