pcr primers Search Results


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    PCR Primer mix to confirm AAVS1 integration amplifying the left integration junction 2 nmols
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    99
    Thermo Fisher pcr primers
    Detection of aberrant splicing products in HMeso01A harboring the BAP1 c . 2054 A > T (p.E685V) mutation. <t>PCR</t> fragments generated from amplification of BAP1 <t>exons</t> 14–17 on cDNAs from HMeso01A and 4 controls were separated by agarose gel electrophoresis. Marker: DNA marker PhiX 174-HaeIII digest ladder.
    Pcr Primers, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 19014 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sangon Biotech pcr primers
    Radiation elevates <t>IL-1β</t> levels in MSCs after radiation. ( A ) Dose-dependent increase in IL-1β secretion and ( B , C ) dose-dependent increases in IL-1β mRNA and protein expression. Cells were exposed to various doses of radiation (0, 2, 4, and 8 Gy), and after 24 h, extracellular and intracellular protein and mRNA levels of IL-1β were determined by ELISA, Western blot, and quantitative real-time <t>PCR</t> analyses, respectively. The values are presented as the mean ± SD ( n = 3). * p
    Pcr Primers, supplied by Sangon Biotech, used in various techniques. Bioz Stars score: 92/100, based on 2020 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rt pcr
    Radiation elevates <t>IL-1β</t> levels in MSCs after radiation. ( A ) Dose-dependent increase in IL-1β secretion and ( B , C ) dose-dependent increases in IL-1β mRNA and protein expression. Cells were exposed to various doses of radiation (0, 2, 4, and 8 Gy), and after 24 h, extracellular and intracellular protein and mRNA levels of IL-1β were determined by ELISA, Western blot, and quantitative real-time <t>PCR</t> analyses, respectively. The values are presented as the mean ± SD ( n = 3). * p
    Rt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 48164 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Eurofins pcr primers
    Inhibition of Mcl-1 or A1 leads to melanoma-specific cell death. (A) Primary human fibroblasts and 1205Lu melanoma cells were treated with the indicated <t>siRNAs</t> and expression of Bcl-2, Bcl-xL, Bcl-w, and Mcl-1 was analyzed after 48 hours by immunoblotting. (B) A1 mRNA was measured by quantitative <t>RT-PCR</t> in cells treated with A1-specific- or control siRNA for 48 hours (left and middle panel). Non-targeted Bcl-2 proteins were analyzed at the same time point by immunoblotting (right panel). (C) Cell death of fibroblasts was determined by quantification of Annexin V (AN)- and propidium iodide (PI)-positive cells 72 hours after transfection of the indicated siRNAs. (D) Cell death analysis of 1205Lu melanoma cells treated as described in C. Asterisks indicate a significant increase in cell death versus control siRNA-treated cells. (E) Fluorescence-activated cell sorting (FACS) of apoptotic and dead fibroblasts (upper panel) or 1205Lu melanoma cells (lower panel) treated as described in C. Numbers indicate the portion of cells in each quadrant that defines Annexin V- or propidium iodide-positive or negative cells. Mean +/− SD of at least 3 independent experiments is shown in B, C, and D. α-tubulin served as loading control in immunoblots. Blots are representative for 3 independent experiments.
    Pcr Primers, supplied by Eurofins, used in various techniques. Bioz Stars score: 92/100, based on 971 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bioneer Corporation pcr primers
    Transcriptional regulation of <t>XAF1</t> by BRD7 A. XAF1 was upregulated with BRD7 lentivirus or downregulated with BRD7 siRNAs in HMVECs. BRD7, CMPK2, ASPM, STIL, SQLE, PLAC8, RIOK2, XAF1, CFB, CBF4 and DTX3L expression levels were analyzed by real <t>time-PCR.</t> Each of the expression levels were normalized to GAPDH expression levels. B. Cells were transduced with BRD7 lentivirus or treated with 0, 20 or 100 ng/ml interferon-gamma (IFN-γ) for 24 h. XAF1, BRD7, IRF1, phospho-p53 and Ac-H3K9 protein levels were detected by Western blot analysis. C. Cells were transfected with BRD7 siRNAs and treated with IFN-γ for 24 h. XAF1, BRD7, IRF1 and phsopho-p53 protein levels were detected by Western blot analysis. D. Cells were transduced with BRD7 lentivirus or transfected with BRD7 siRNAs. Each of cells was treated with 4 Gy IR for 24 h. Chromatin immunoprecipitation (ChIP) assay was performed using the anti-BRD7 antibody and the immunoprecipitated DNA was amplified using primers for p53, IRF1 or XAF1. E. The cells were transfected with the luciferase reporter constructs containing the XAF1 promoter. XAF1 promoter activity in BRD7-expressed cells was determined by luciferase analysis. * p
    Pcr Primers, supplied by Bioneer Corporation, used in various techniques. Bioz Stars score: 92/100, based on 847 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad pcr primers
    Mechanism of CGE silencing and effect of <t>Pif1</t> helicases on telomere length. (a) URA3 and (b) CAN1 mRNA levels in pif1-m2 rrm3 Δ+G4 CGE clones and controls (pre-GCR parental strains and ura3 Δ and can1 Δ cells). <t>qRT-PCR</t> was used to determine the ACT1, URA3, or CAN1 mRNA levels in the indicated strains. URA3 and CAN1 values were normalized to ACT1 levels in each strain; the 2 −ΔΔCt method 38 was used to determine URA3 and CAN1 levels relative to parental pre-GCR cells. *, **, and ***: p
    Pcr Primers, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 1118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sequenom pcr primers
    Correlation between <t>DNA</t> methylation and mRNA expression levels for PTPRH. DNA methylation (average values) (A) and mRNA expression levels (B) for PTPRH in samples of N and of T in LC-C2 were determined by the Infinium assay and <t>qRT-PCR</t> analysis, respectively. DNA methylation levels for PTPRH were significantly lower in T than in N samples and levels of PTPRH mRNA expression were significantly higher in T than in N samples. (C) Correlation of DNA methylation (average values) and mRNA expression levels for PTPRH in LC-C2 samples. PTPRH mRNA expression levels were inversely correlated with DNA methylation of the single CpG site. These results suggested that PTPRH DNA hypomethylation may result in increased mRNA expression in tissue samples from the same cohort. N, non-cancerous lung tissue; T, corresponding tumorous tissue; qRT-PCR, quantitative real-time reverse transcription-polymerase chain reaction.
    Pcr Primers, supplied by Sequenom, used in various techniques. Bioz Stars score: 92/100, based on 572 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PrimerDesign Inc pcr primer design
    Melt curve of SYBR green <t>PCR</t> products . The Y -axis represents the derivative reporter (−Rn) while x -axis represents the temperature (°C). The figure shows a melting temperature ( 31 ) of human <t>ompW</t> PCR products as 78.46°C.
    Pcr Primer Design, supplied by PrimerDesign Inc, used in various techniques. Bioz Stars score: 90/100, based on 329 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sigma-Genosys pcr primers
    Multiplex <t>PCR-RFLP.</t> a : Diagram demonstrating the introduction of <t>MaeIII</t> restriction sites by the mutation (11778A) and the combination of primer alterations and mutation (3460A, 14484C). Mutations are shown in red with the primer alterations in lower case. b : 2.5 % ethidium bromide stained agarose gel showing the results of the PCR-RFLP on patient DNA (black labels) and synthesised DNA controls (red labels). DNA was PCR amplified in a multiplex reaction using 3460 F/R, 11778 F/R and 14484 F/R and restricted using 1 unit of MaeIII as described. M: size marker; Uncut: non-restricted PCR products; All other lanes: patient (Black) or synthesised DNA controls (Red) containing the indicated mutation PCR amplified and restricted with MaeIII. The red arrow between the uncut and normal lanes demonstrates the internal control of restriction. Yellow arrows demonstrate mutation detection
    Pcr Primers, supplied by Sigma-Genosys, used in various techniques. Bioz Stars score: 92/100, based on 412 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Macrogen pcr primers
    Multiplex <t>PCR-RFLP.</t> a : Diagram demonstrating the introduction of <t>MaeIII</t> restriction sites by the mutation (11778A) and the combination of primer alterations and mutation (3460A, 14484C). Mutations are shown in red with the primer alterations in lower case. b : 2.5 % ethidium bromide stained agarose gel showing the results of the PCR-RFLP on patient DNA (black labels) and synthesised DNA controls (red labels). DNA was PCR amplified in a multiplex reaction using 3460 F/R, 11778 F/R and 14484 F/R and restricted using 1 unit of MaeIII as described. M: size marker; Uncut: non-restricted PCR products; All other lanes: patient (Black) or synthesised DNA controls (Red) containing the indicated mutation PCR amplified and restricted with MaeIII. The red arrow between the uncut and normal lanes demonstrates the internal control of restriction. Yellow arrows demonstrate mutation detection
    Pcr Primers, supplied by Macrogen, used in various techniques. Bioz Stars score: 92/100, based on 333 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Ribobio bulge loop mirna qrt pcr primer
    Subcellular localization of HAGLROS and its “sponge” function as a ceRNA competing with miR-100-5p. a RNA was extracted from the nuclear and the cytoplasmic fractions of SGC-7901 and BGC-823 cells and HAGLROS expression of the nuclear and the cytoplasmic fraction was measured by <t>qRT-PCR.</t> GAPDH was used as a cytosolic marker, and U6 was used as a nuclear marker. b FISH was used to confirm HAGLROS location in SGC-7901 and BGC-823 cells, using Cy3 probes for HAGLROS, DAPI for nuclear staining. c miR-100-5p expression was examined in SGC-7901 and BGC-823 cells with HAGLROS knockdown by siRNAs, and HAGLROS expression was tested to determine the transfection efficiencies. d HAGLROS levels were examined in SGC-7901 and BGC-823 cells transfected with miR-100-5p, and miR-100-5p levels were tested for transfection efficiencies. e The expression of miR-100-5p in tumor samples of GC compared to adjacent non-cancerous tissues. f Wild-type or mutant HAGLROS plasmid was co-transfected with miR-NC or miR-100-5p mimics into 293T cells, and relative luciferase activities were measured to determine the level of interaction between miR-100-5p and HAGLROS. g RNA levels in immunoprecipitates are presented as fold enrichment relative to IgG in AGO 2 cells by RIP experiment. Error bars indicate the means ± S.E.M. * P
    Bulge Loop Mirna Qrt Pcr Primer, supplied by Ribobio, used in various techniques. Bioz Stars score: 89/100, based on 412 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Kaneka Corp pcr primers
    IL-1ß downregulates the expression of <t>MITF-M</t> and upregulates the expression of miR-155 in 2 melanoma cell lines. ( A ) Expression of MITF-M was analyzed by quantitative <t>RT-PCR</t> in melanoma cell lines LB2201-MEL and LB2259-MEL incubated with IL-1ß (10 ng/ml) for 4h or 24h. The result was normalized to ß-actin expression (means ± SD for 3 or 4 independent experiments, respectively). ( B ) Expression of miRNAs potentially targeting MITF-M (miR-96, miR-137, miR-148a, miR-155, miR-182 and miR-340) was analyzed by quantitative RT-PCR in the same samples and normalized to RNU44 expression. The table indicates the miRNA fold change when cells were treated with IL-1ß for 4h or 24h.
    Pcr Primers, supplied by Kaneka Corp, used in various techniques. Bioz Stars score: 92/100, based on 479 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MWG-Biotech pcr primers
    RIG-I and MDA-5 induce expression of the proapoptotic BH3-only members of the Bcl-2 family Noxa, Puma, Bim, and Bik. ( A ) 1205Lu cells were treated with pppRNA or poly(I:C) (3 ng/ml) for 24 hours, and levels of Noxa, Puma, Bim, Bad, Bik, Bid, and Hrk mRNA were measured by quantitative <t>RT-PCR.</t> Relative mRNA levels compared with mock-transfected cells are depicted. Mean ± SD of 3 independent experiments is shown. * P ≤ 0.05 and NS compared with mock-transfected cells. ( B ) The melanoma cell lines 1205Lu, WM239A, WM278, and WM793 were treated with pppRNA or poly(I:C) (5 ng/ml), and Noxa and Puma proteins were quantified by immunoblotting. β-Actin served as loading control. Blots are representative of 2 independent experiments. ( C ) Left: Expression of the BH3-only proteins Noxa, Puma, Bim, Bad, Bik, and Bid was inhibited by treatment with respective <t>siRNAs</t> for 48 hours. Thereafter, cells were treated with pppRNA (left), poly(I:C) (5 ng/ml; right), or transfection reagent alone and analyzed for apoptotic cells by FACS. Annexin V–positive and propidium iodide–negative cells are represented. Mean ± SD of 3 independent experiments is shown. * P ≤ 0.05 compared with control siRNA–transfected cells treated with pppRNA.
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    Biotechnology Information pcr primers
    Distribution of injected cells in major organs. (A) Fluorescently labeled iPSCs in the hearts, livers, lungs, and kidneys of healthy (left panels) and lung-injured (right panels) mice 8 h after receiving bolus injections of 2.5×10 8 cells/kg body weight via tail vein. (B) <t>PCR</t> analysis of <t>Tet-On</t> and 18S (18S rRNA gene) DNA levels in the organs. The Tet-On gene was carried into iPSCs by lentiviruses used for the induction of iPSCs from mouse fibroblasts.
    Pcr Primers, supplied by Biotechnology Information, used in various techniques. Bioz Stars score: 92/100, based on 349 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene pcr primers
    Position and frequency of exoY <t>DNA</t> and amino acid sequence variations ( n = 9) and position of <t>PCR</t> primers and conserved regions between P. aeruginosa , B. pertussis , and B. anthracis adenylate cyclases. The x axis represents the nucleotide or amino acid position; the y axis represents the percentage of P. aeruginosa isolates that differed in sequence from PAO1 at each position. Two lysine residues (Lys-81 and Lys-88) in conserved region I and two arginine residues (Arg-212 and Arg-214) in conserved region II are essential for the adenylate cyclase activity of ExoY.
    Pcr Primers, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 552 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Ribobio pcr primers
    SMAD5 was the predominant target of miR-K12-11. (A) miR-K12-11 downregulates SMAD1/2/3/5 3′ UTR reporter activity in HEK293T cells. One hundred nanograms pGL3-SMAD1- 3′ UTR, pGL3-SMAD2- 3′ UTR, pGL3-SMAD3- 3′ UTR, or pGL3-SMAD5- 3′ UTR was cotransfected with either pCDH-miR-K12-11 or pCDH-copGFP (1 μg) into HEK293T cells. The pGL3-BACH1 3′ UTR was used as a positive control. (B) Western blotting of SMAD1/2/3/5 in Ramos-teton-miR-K12-11 cells after induction with or without doxycycline for the indicated times. As a loading control, β-actin was also detected in the same blotting. Values represent percentages of SMAD1/2/3/5 normalized against β-actin and compared with an untreated control. (C) Total RNA of the same samples was reverse-transcribed and then used as a template to determine the SMAD5 mRNA levels by standard <t>qRT-PCR.</t> Values were normalized against β-actin. (D) Ramos-teton-miR-K12-11 cells were cultured with or without doxycycline for 48 h and then starved for 12 h before the addition of TGF-β1 (10 ng/ml). Lysates of these cells were taken 1 or 2 h after TGF-β1 stimulation to quantify the levels of phospho-SMAD1/5 and SMAD5 proteins by Western blotting. As a normalizing control, β-actin was also detected in the same blotting. Densitometry values represent the percentage of p-SMAD1/5 normalized against β-actin and compared with an uninduced control.
    Pcr Primers, supplied by Ribobio, used in various techniques. Bioz Stars score: 92/100, based on 248 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher real time pcr primers
    SMAD5 was the predominant target of miR-K12-11. (A) miR-K12-11 downregulates SMAD1/2/3/5 3′ UTR reporter activity in HEK293T cells. One hundred nanograms pGL3-SMAD1- 3′ UTR, pGL3-SMAD2- 3′ UTR, pGL3-SMAD3- 3′ UTR, or pGL3-SMAD5- 3′ UTR was cotransfected with either pCDH-miR-K12-11 or pCDH-copGFP (1 μg) into HEK293T cells. The pGL3-BACH1 3′ UTR was used as a positive control. (B) Western blotting of SMAD1/2/3/5 in Ramos-teton-miR-K12-11 cells after induction with or without doxycycline for the indicated times. As a loading control, β-actin was also detected in the same blotting. Values represent percentages of SMAD1/2/3/5 normalized against β-actin and compared with an untreated control. (C) Total RNA of the same samples was reverse-transcribed and then used as a template to determine the SMAD5 mRNA levels by standard <t>qRT-PCR.</t> Values were normalized against β-actin. (D) Ramos-teton-miR-K12-11 cells were cultured with or without doxycycline for 48 h and then starved for 12 h before the addition of TGF-β1 (10 ng/ml). Lysates of these cells were taken 1 or 2 h after TGF-β1 stimulation to quantify the levels of phospho-SMAD1/5 and SMAD5 proteins by Western blotting. As a normalizing control, β-actin was also detected in the same blotting. Densitometry values represent the percentage of p-SMAD1/5 normalized against β-actin and compared with an uninduced control.
    Real Time Pcr Primers, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1540 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SABiosciences pcr primers
    The minimal connectivity of genes increased in the odontoblast layer (ODL) and pulp of carious teeth . This network shows known direct or one off interactions between genes measured to be significantly upregulated in this study. For each gene, the expression level (inverse of <t>PCR</t> threshold cycle) in ODL (A) and pulp (B) of carious teeth is rendered as node size. Differential expression in ODL (A) and pulp (B) of carious teeth versus normal teeth (fold increase) is depicted as a color heat map with white showing no change and saturated blue meaning greater up-regulation in carious ODL (A) and pulp (B). Network bottlenecks are highlighted in red, signifying the most important candidate inflammatory signal mediators: PIK3R1, IL1R1, TLR4, ARRβ1, CCL5, CCR5, IL8, JAK1, JAK2, RELA, and TYK2. The key receptors for inflammatory signals induced by caries in ODL appear to converge through IL1R1, CCR5, and IL8Rα/β. The gene expression data used for building this map were derived from PCR arrays and <t>qPCR</t> verification data of cDNA arrays.
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    MacVector pcr primers
    <t>CBD103</t> expression in AD. Quantitative <t>RT-PCR</t> analysis of absolute copy-number of mRNA transcripts encoding RPS5, CBD103 and S100A8 in RNA isolated from lesional and nonlesional skin biopsy specimens from dogs (n = 18) diagnosed with AD. Error bars represent standard error.
    Pcr Primers, supplied by MacVector, used in various techniques. Bioz Stars score: 92/100, based on 231 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The Jackson Laboratory pcr primers
    Involvement of NLRC4 and RICK in NTHi-induced human β-defensin 2 up-regulation. (A) Luciferase assays show that NTHi lysate-induced human β-defensin 2 up-regulation is enhanced by silencing of NLRC4 (a <t>NOD2</t> inhibitor) but is inhibited by silencing of RICK that is downstream to NOD2 in the HMEEC cells. NC: a control group transfected with a nonspecific siRNA, KD: a group transfected with a gene-specific siRNA. (B) <t>RT-PCR</t> analysis showing siRNA-mediated inhibition of NLRC4 and RICK expression in the HMEEC cells. (C) Luciferase assays demonstrate that NTHi lysate-induced NF-κB activation is inhibited by the siRNAs specific to NOD2 and RICK in the HMEEC cells. pTAL-luc: a control vector containing the firefly luciferase gene with a TATA-like promoter region from the Herpes simplex virus thymidine kinase promoter, pNFκB-luc: a vector containing multiple copies of the NF-κB consensus sequence fused to pTAL-luc, Tx: treatment. Results were expressed as fold-induction, taking the value of the non-treated group as 1. The experiments were performed in triplicate and repeated twice. Values are given as the mean ± standard deviation (n = 3). *: p
    Pcr Primers, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 92/100, based on 147 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies mycoplasma plus pcr primer set
    TNFα affects hfNBM cell phenotype. ( a , b ) Representative images showing ( a ) ChAT and ( b ) VAchT expression in hfNBMs as evaluated by immunofluorescence analysis (DAPI counterstained nuclei, scale bar 100 µm). ( c ) Relative mRNA expression by <t>qRT-PCR</t> analysis of TNFR1 and TNFR2 receptors normalized over 18S ribosomal subunit, taken as reference gene, and reported as mean ± SEM ( n = 6). ( d ) Immunofluorescent analysis of NF-κB p65 nuclear translocation after TNFα stimulus (10 ng/mL, 3 h) in comparison to untreated cells (CTL; scale bar 100 µm). ( e ) COX2 mRNA expression in hfNBMs by qRT-PCR after TNFα stimulation (10 ng/mL, 24 h). Data are normalized over 18S ribosomal RNA subunit and reported as percentage of untreated cells (CTL) and displayed as mean ± SEM of three separate experiments performed in triplicate (unpaired Student’s t -test; *** p
    Mycoplasma Plus Pcr Primer Set, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 89/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Takara Bio s PrimerArray Series offers primer sets for real time RT PCR qPCR that can be used for gene expression analysis of biological pathways Each PrimerArray Series primer set
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    Human CXCL1 Primer Pair for RT PCR reverse transcription followed by polymerase chain reaction analysis of mRNA expression Human CXCL1 Primer Pair
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    Detection of aberrant splicing products in HMeso01A harboring the BAP1 c . 2054 A > T (p.E685V) mutation. PCR fragments generated from amplification of BAP1 exons 14–17 on cDNAs from HMeso01A and 4 controls were separated by agarose gel electrophoresis. Marker: DNA marker PhiX 174-HaeIII digest ladder.

    Journal: PLoS ONE

    Article Title: BAP1 Missense Mutation c.2054 A > T (p.E685V) Completely Disrupts Normal Splicing through Creation of a Novel 5’ Splice Site in a Human Mesothelioma Cell Line

    doi: 10.1371/journal.pone.0119224

    Figure Lengend Snippet: Detection of aberrant splicing products in HMeso01A harboring the BAP1 c . 2054 A > T (p.E685V) mutation. PCR fragments generated from amplification of BAP1 exons 14–17 on cDNAs from HMeso01A and 4 controls were separated by agarose gel electrophoresis. Marker: DNA marker PhiX 174-HaeIII digest ladder.

    Article Snippet: DNA from colonies was amplified using the BAP1 primers covering cDNA regions of exons 14–17 and subjected to direct DNA sequencing analysis using the forward PCR primer (BigDye Terminator v3.1 Cycle Sequencing kit and 3730 DNA analyzer, Applied Biosystems, Foster City, CA).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Generated, Amplification, Agarose Gel Electrophoresis, Marker

    Summary of splicing variants cloned from RT-PCR products of HMeso01A harboring the c.2054 A > T (p.Glu685Val) mutation. A. Retention of introns 14, 15, 16; B. Retention of intron 15, 16; C. Retention of intron 14 and 4 bp deletion (GAAG) at the end of exon 16; D. Retention of intron 16; E. Partial retention of 3’ 69 bp in intron 14 and 4 bp deletion (GAAG) at the end of exon 16; F. Partial retention of 3’ 18 bp in intron 15 and 4 bp deletion (GAAG) at the end of exon 16; G. 4 bp deletion (GAAG) at then end of exon 16 (Δ4). //: partial exon.

    Journal: PLoS ONE

    Article Title: BAP1 Missense Mutation c.2054 A > T (p.E685V) Completely Disrupts Normal Splicing through Creation of a Novel 5’ Splice Site in a Human Mesothelioma Cell Line

    doi: 10.1371/journal.pone.0119224

    Figure Lengend Snippet: Summary of splicing variants cloned from RT-PCR products of HMeso01A harboring the c.2054 A > T (p.Glu685Val) mutation. A. Retention of introns 14, 15, 16; B. Retention of intron 15, 16; C. Retention of intron 14 and 4 bp deletion (GAAG) at the end of exon 16; D. Retention of intron 16; E. Partial retention of 3’ 69 bp in intron 14 and 4 bp deletion (GAAG) at the end of exon 16; F. Partial retention of 3’ 18 bp in intron 15 and 4 bp deletion (GAAG) at the end of exon 16; G. 4 bp deletion (GAAG) at then end of exon 16 (Δ4). //: partial exon.

    Article Snippet: DNA from colonies was amplified using the BAP1 primers covering cDNA regions of exons 14–17 and subjected to direct DNA sequencing analysis using the forward PCR primer (BigDye Terminator v3.1 Cycle Sequencing kit and 3730 DNA analyzer, Applied Biosystems, Foster City, CA).

    Techniques: Clone Assay, Reverse Transcription Polymerase Chain Reaction, Mutagenesis

    Radiation elevates IL-1β levels in MSCs after radiation. ( A ) Dose-dependent increase in IL-1β secretion and ( B , C ) dose-dependent increases in IL-1β mRNA and protein expression. Cells were exposed to various doses of radiation (0, 2, 4, and 8 Gy), and after 24 h, extracellular and intracellular protein and mRNA levels of IL-1β were determined by ELISA, Western blot, and quantitative real-time PCR analyses, respectively. The values are presented as the mean ± SD ( n = 3). * p

    Journal: International Journal of Molecular Sciences

    Article Title: Resveratrol Inhibits Ionising Irradiation-Induced Inflammation in MSCs by Activating SIRT1 and Limiting NLRP-3 Inflammasome Activation

    doi: 10.3390/ijms140714105

    Figure Lengend Snippet: Radiation elevates IL-1β levels in MSCs after radiation. ( A ) Dose-dependent increase in IL-1β secretion and ( B , C ) dose-dependent increases in IL-1β mRNA and protein expression. Cells were exposed to various doses of radiation (0, 2, 4, and 8 Gy), and after 24 h, extracellular and intracellular protein and mRNA levels of IL-1β were determined by ELISA, Western blot, and quantitative real-time PCR analyses, respectively. The values are presented as the mean ± SD ( n = 3). * p

    Article Snippet: PCR primers for the NLRP3, IL-1β, caspase-1, Sirt1, and GAPDH genes were obtained from Sangon Biotech (Shanghai, China).

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Real-time Polymerase Chain Reaction

    Resveratrol reduces IL-1β expression in MSCs. ( A ) Concentration-dependent decrease in IL-1β secretion and ( B , C ) concentration-dependent decrease in IL-1β protein and mRNA expression. Cells were treated with different concentrations of resveratrol for 1 h before radiation. Then, extracellular and intracellular protein and mRNA levels of IL-1β were determined by ELISA, Western blot, and quantitative real-time PCR analyses, respectively. Values are presented as the mean ± SD ( n = 3). * p

    Journal: International Journal of Molecular Sciences

    Article Title: Resveratrol Inhibits Ionising Irradiation-Induced Inflammation in MSCs by Activating SIRT1 and Limiting NLRP-3 Inflammasome Activation

    doi: 10.3390/ijms140714105

    Figure Lengend Snippet: Resveratrol reduces IL-1β expression in MSCs. ( A ) Concentration-dependent decrease in IL-1β secretion and ( B , C ) concentration-dependent decrease in IL-1β protein and mRNA expression. Cells were treated with different concentrations of resveratrol for 1 h before radiation. Then, extracellular and intracellular protein and mRNA levels of IL-1β were determined by ELISA, Western blot, and quantitative real-time PCR analyses, respectively. Values are presented as the mean ± SD ( n = 3). * p

    Article Snippet: PCR primers for the NLRP3, IL-1β, caspase-1, Sirt1, and GAPDH genes were obtained from Sangon Biotech (Shanghai, China).

    Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Real-time Polymerase Chain Reaction

    The knockdown of Sirt1 by shRNA suppresses resveratrol-mediated anti-inflammatory activity. ( A ) Cells were transfected with Sirt1 shRNA for 48, 72, or 96 h; ( B ) Sirt1 expression was down-regulated by Sirt1 shRNA. The cells were transfected with Sirt1 shRNA for 48 h, and mRNA expression of Sirt1 was then determined by quantitative real-time PCR; ( C – E ) The knockdown of Sirt1 obviously suppresses resveratrol-mediated anti-inflammatory activity, and radiation-induced IL-1β expression was significantly increased by Sirt1 RNAi. The cells were stimulated with radiation for 24 h after transfection with Sirt1 shRNA for 48 h; protein and mRNA expressions of IL-1β and NLRP3 were determined by Western blot and quantitative real-time PCR analyses, respectively, and IL-1β secretion was detected by ELISAs. Values are presented as the mean ± SD ( n = 3). * p

    Journal: International Journal of Molecular Sciences

    Article Title: Resveratrol Inhibits Ionising Irradiation-Induced Inflammation in MSCs by Activating SIRT1 and Limiting NLRP-3 Inflammasome Activation

    doi: 10.3390/ijms140714105

    Figure Lengend Snippet: The knockdown of Sirt1 by shRNA suppresses resveratrol-mediated anti-inflammatory activity. ( A ) Cells were transfected with Sirt1 shRNA for 48, 72, or 96 h; ( B ) Sirt1 expression was down-regulated by Sirt1 shRNA. The cells were transfected with Sirt1 shRNA for 48 h, and mRNA expression of Sirt1 was then determined by quantitative real-time PCR; ( C – E ) The knockdown of Sirt1 obviously suppresses resveratrol-mediated anti-inflammatory activity, and radiation-induced IL-1β expression was significantly increased by Sirt1 RNAi. The cells were stimulated with radiation for 24 h after transfection with Sirt1 shRNA for 48 h; protein and mRNA expressions of IL-1β and NLRP3 were determined by Western blot and quantitative real-time PCR analyses, respectively, and IL-1β secretion was detected by ELISAs. Values are presented as the mean ± SD ( n = 3). * p

    Article Snippet: PCR primers for the NLRP3, IL-1β, caspase-1, Sirt1, and GAPDH genes were obtained from Sangon Biotech (Shanghai, China).

    Techniques: shRNA, Activity Assay, Transfection, Expressing, Real-time Polymerase Chain Reaction, Western Blot

    Sirt1 inhibits IL-1β expression via the NF-κb pathway in MSCs. ( A – C ) Cells were pre-treated with BAY (5 μM) and either resveratrol (200 μM) or NAM (20 mM) for 1 h prior to stimulation with radiation (4 Gy). Extracellular and intracellular protein and mRNA levels of IL-1β were determined by ELISA, Western blot, and quantitative real-time PCR analyses, respectively. Values are presented as the mean ± SD ( n = 3). * p

    Journal: International Journal of Molecular Sciences

    Article Title: Resveratrol Inhibits Ionising Irradiation-Induced Inflammation in MSCs by Activating SIRT1 and Limiting NLRP-3 Inflammasome Activation

    doi: 10.3390/ijms140714105

    Figure Lengend Snippet: Sirt1 inhibits IL-1β expression via the NF-κb pathway in MSCs. ( A – C ) Cells were pre-treated with BAY (5 μM) and either resveratrol (200 μM) or NAM (20 mM) for 1 h prior to stimulation with radiation (4 Gy). Extracellular and intracellular protein and mRNA levels of IL-1β were determined by ELISA, Western blot, and quantitative real-time PCR analyses, respectively. Values are presented as the mean ± SD ( n = 3). * p

    Article Snippet: PCR primers for the NLRP3, IL-1β, caspase-1, Sirt1, and GAPDH genes were obtained from Sangon Biotech (Shanghai, China).

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Real-time Polymerase Chain Reaction

    Pharmacological modulation of Sirt1-regulated radiation-induced NLRP3 and IL-1β expression in MSCs. ( A – D ) Cells were pre-treated with resveratrol (200 μM) or NAM (20 mM) and subsequently stimulated with radiation for 24 h. Then, extracellular and intracellular protein and mRNA expression of IL-1β were determined by ELISA, Western blot, and quantitative real-time PCR analyses, respectively. The values are presented as the mean ± SD ( n = 3). * p

    Journal: International Journal of Molecular Sciences

    Article Title: Resveratrol Inhibits Ionising Irradiation-Induced Inflammation in MSCs by Activating SIRT1 and Limiting NLRP-3 Inflammasome Activation

    doi: 10.3390/ijms140714105

    Figure Lengend Snippet: Pharmacological modulation of Sirt1-regulated radiation-induced NLRP3 and IL-1β expression in MSCs. ( A – D ) Cells were pre-treated with resveratrol (200 μM) or NAM (20 mM) and subsequently stimulated with radiation for 24 h. Then, extracellular and intracellular protein and mRNA expression of IL-1β were determined by ELISA, Western blot, and quantitative real-time PCR analyses, respectively. The values are presented as the mean ± SD ( n = 3). * p

    Article Snippet: PCR primers for the NLRP3, IL-1β, caspase-1, Sirt1, and GAPDH genes were obtained from Sangon Biotech (Shanghai, China).

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Real-time Polymerase Chain Reaction

    Inhibition of Mcl-1 or A1 leads to melanoma-specific cell death. (A) Primary human fibroblasts and 1205Lu melanoma cells were treated with the indicated siRNAs and expression of Bcl-2, Bcl-xL, Bcl-w, and Mcl-1 was analyzed after 48 hours by immunoblotting. (B) A1 mRNA was measured by quantitative RT-PCR in cells treated with A1-specific- or control siRNA for 48 hours (left and middle panel). Non-targeted Bcl-2 proteins were analyzed at the same time point by immunoblotting (right panel). (C) Cell death of fibroblasts was determined by quantification of Annexin V (AN)- and propidium iodide (PI)-positive cells 72 hours after transfection of the indicated siRNAs. (D) Cell death analysis of 1205Lu melanoma cells treated as described in C. Asterisks indicate a significant increase in cell death versus control siRNA-treated cells. (E) Fluorescence-activated cell sorting (FACS) of apoptotic and dead fibroblasts (upper panel) or 1205Lu melanoma cells (lower panel) treated as described in C. Numbers indicate the portion of cells in each quadrant that defines Annexin V- or propidium iodide-positive or negative cells. Mean +/− SD of at least 3 independent experiments is shown in B, C, and D. α-tubulin served as loading control in immunoblots. Blots are representative for 3 independent experiments.

    Journal: PLoS ONE

    Article Title: Selective Induction of Cell Death in Melanoma Cell Lines through Targeting of Mcl-1 and A1

    doi: 10.1371/journal.pone.0030821

    Figure Lengend Snippet: Inhibition of Mcl-1 or A1 leads to melanoma-specific cell death. (A) Primary human fibroblasts and 1205Lu melanoma cells were treated with the indicated siRNAs and expression of Bcl-2, Bcl-xL, Bcl-w, and Mcl-1 was analyzed after 48 hours by immunoblotting. (B) A1 mRNA was measured by quantitative RT-PCR in cells treated with A1-specific- or control siRNA for 48 hours (left and middle panel). Non-targeted Bcl-2 proteins were analyzed at the same time point by immunoblotting (right panel). (C) Cell death of fibroblasts was determined by quantification of Annexin V (AN)- and propidium iodide (PI)-positive cells 72 hours after transfection of the indicated siRNAs. (D) Cell death analysis of 1205Lu melanoma cells treated as described in C. Asterisks indicate a significant increase in cell death versus control siRNA-treated cells. (E) Fluorescence-activated cell sorting (FACS) of apoptotic and dead fibroblasts (upper panel) or 1205Lu melanoma cells (lower panel) treated as described in C. Numbers indicate the portion of cells in each quadrant that defines Annexin V- or propidium iodide-positive or negative cells. Mean +/− SD of at least 3 independent experiments is shown in B, C, and D. α-tubulin served as loading control in immunoblots. Blots are representative for 3 independent experiments.

    Article Snippet: PCR primers and siRNAs were purchased from Eurofins MWG Operon (Ebersberg, Germany).

    Techniques: Inhibition, Expressing, Quantitative RT-PCR, Transfection, Fluorescence, FACS, Western Blot

    Combined inhibition of Mcl-1 and A1 results in efficient induction of cell death in a panel of melanoma cell lines, but not in keratinocytes. (A) Primary human keratinocytes (upper left panel), non-invasive melanoma cells (WM3211), invasive (WM793), and metastatic melanoma cells (WM1232, WM239A, WM1158) were transfected with the siRNAs as indicated. Cell death was determined 72 hours after transfection. Mean +/− SD of 3 (keratinocytes, WM1232, WM239A, WM1158) or 5 (WM3211 and WM793) independent experiments is shown. (B) A1 mRNA was measured by quantitative RT-PCR in indicated cells treated with A1-specific- or control siRNA for 48 hours. Mean +/− SD of 3 independent experiments is shown. Asterisks represent significant increase in cell death compared to control siRNA-treated cells. AN, Annexin V; PI, propidium iodide.

    Journal: PLoS ONE

    Article Title: Selective Induction of Cell Death in Melanoma Cell Lines through Targeting of Mcl-1 and A1

    doi: 10.1371/journal.pone.0030821

    Figure Lengend Snippet: Combined inhibition of Mcl-1 and A1 results in efficient induction of cell death in a panel of melanoma cell lines, but not in keratinocytes. (A) Primary human keratinocytes (upper left panel), non-invasive melanoma cells (WM3211), invasive (WM793), and metastatic melanoma cells (WM1232, WM239A, WM1158) were transfected with the siRNAs as indicated. Cell death was determined 72 hours after transfection. Mean +/− SD of 3 (keratinocytes, WM1232, WM239A, WM1158) or 5 (WM3211 and WM793) independent experiments is shown. (B) A1 mRNA was measured by quantitative RT-PCR in indicated cells treated with A1-specific- or control siRNA for 48 hours. Mean +/− SD of 3 independent experiments is shown. Asterisks represent significant increase in cell death compared to control siRNA-treated cells. AN, Annexin V; PI, propidium iodide.

    Article Snippet: PCR primers and siRNAs were purchased from Eurofins MWG Operon (Ebersberg, Germany).

    Techniques: Inhibition, Transfection, Quantitative RT-PCR

    Melanoma-specific cell death can be enhanced by combined inhibition of Mcl-1 and A1. (A) Fibroblasts (left panel) and 1205Lu melanoma cells (right panel) were transfected with the indicated siRNAs for 48 hours. Thereafter, 1 µM ABT-737 was added and cell death was assessed 24 hours after ABT-737 treatment. (B) Fibroblasts were simultaneously transfected with two siRNAs as indicated. 48 hours after transfection the respective protein was analyzed by immunoblotting (upper panels) or RT-PCR (lower panel). Blots are representative for 3 (upper left panel) or 2 (upper right panel) independent experiments. Cell death analysis of (C) fibroblasts or (D) 1205Lu melanoma cells treated with the indicated siRNAs for 72 hours. Mean +/− SD of 3 independent experiments is shown in A, B, C and D. α-tubulin or β-actin served as loading control in immunoblots. White lines indicate lanes that were run at the same blot but are not contiguous. Asterisks represent significant increase in cell death compared to control siRNA-treated cells. AN, Annexin V; PI, propidium iodide.

    Journal: PLoS ONE

    Article Title: Selective Induction of Cell Death in Melanoma Cell Lines through Targeting of Mcl-1 and A1

    doi: 10.1371/journal.pone.0030821

    Figure Lengend Snippet: Melanoma-specific cell death can be enhanced by combined inhibition of Mcl-1 and A1. (A) Fibroblasts (left panel) and 1205Lu melanoma cells (right panel) were transfected with the indicated siRNAs for 48 hours. Thereafter, 1 µM ABT-737 was added and cell death was assessed 24 hours after ABT-737 treatment. (B) Fibroblasts were simultaneously transfected with two siRNAs as indicated. 48 hours after transfection the respective protein was analyzed by immunoblotting (upper panels) or RT-PCR (lower panel). Blots are representative for 3 (upper left panel) or 2 (upper right panel) independent experiments. Cell death analysis of (C) fibroblasts or (D) 1205Lu melanoma cells treated with the indicated siRNAs for 72 hours. Mean +/− SD of 3 independent experiments is shown in A, B, C and D. α-tubulin or β-actin served as loading control in immunoblots. White lines indicate lanes that were run at the same blot but are not contiguous. Asterisks represent significant increase in cell death compared to control siRNA-treated cells. AN, Annexin V; PI, propidium iodide.

    Article Snippet: PCR primers and siRNAs were purchased from Eurofins MWG Operon (Ebersberg, Germany).

    Techniques: Inhibition, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Transcriptional regulation of XAF1 by BRD7 A. XAF1 was upregulated with BRD7 lentivirus or downregulated with BRD7 siRNAs in HMVECs. BRD7, CMPK2, ASPM, STIL, SQLE, PLAC8, RIOK2, XAF1, CFB, CBF4 and DTX3L expression levels were analyzed by real time-PCR. Each of the expression levels were normalized to GAPDH expression levels. B. Cells were transduced with BRD7 lentivirus or treated with 0, 20 or 100 ng/ml interferon-gamma (IFN-γ) for 24 h. XAF1, BRD7, IRF1, phospho-p53 and Ac-H3K9 protein levels were detected by Western blot analysis. C. Cells were transfected with BRD7 siRNAs and treated with IFN-γ for 24 h. XAF1, BRD7, IRF1 and phsopho-p53 protein levels were detected by Western blot analysis. D. Cells were transduced with BRD7 lentivirus or transfected with BRD7 siRNAs. Each of cells was treated with 4 Gy IR for 24 h. Chromatin immunoprecipitation (ChIP) assay was performed using the anti-BRD7 antibody and the immunoprecipitated DNA was amplified using primers for p53, IRF1 or XAF1. E. The cells were transfected with the luciferase reporter constructs containing the XAF1 promoter. XAF1 promoter activity in BRD7-expressed cells was determined by luciferase analysis. * p

    Journal: Oncotarget

    Article Title: XIAP-associating factor 1, a transcriptional target of BRD7, contributes to endothelial cell senescence

    doi: 10.18632/oncotarget.6962

    Figure Lengend Snippet: Transcriptional regulation of XAF1 by BRD7 A. XAF1 was upregulated with BRD7 lentivirus or downregulated with BRD7 siRNAs in HMVECs. BRD7, CMPK2, ASPM, STIL, SQLE, PLAC8, RIOK2, XAF1, CFB, CBF4 and DTX3L expression levels were analyzed by real time-PCR. Each of the expression levels were normalized to GAPDH expression levels. B. Cells were transduced with BRD7 lentivirus or treated with 0, 20 or 100 ng/ml interferon-gamma (IFN-γ) for 24 h. XAF1, BRD7, IRF1, phospho-p53 and Ac-H3K9 protein levels were detected by Western blot analysis. C. Cells were transfected with BRD7 siRNAs and treated with IFN-γ for 24 h. XAF1, BRD7, IRF1 and phsopho-p53 protein levels were detected by Western blot analysis. D. Cells were transduced with BRD7 lentivirus or transfected with BRD7 siRNAs. Each of cells was treated with 4 Gy IR for 24 h. Chromatin immunoprecipitation (ChIP) assay was performed using the anti-BRD7 antibody and the immunoprecipitated DNA was amplified using primers for p53, IRF1 or XAF1. E. The cells were transfected with the luciferase reporter constructs containing the XAF1 promoter. XAF1 promoter activity in BRD7-expressed cells was determined by luciferase analysis. * p

    Article Snippet: The PCR primers for XAF1, p53, p16 and GAPDH were obtained from Bioneer (Daejeon, Korea).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Transduction, Western Blot, Transfection, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Luciferase, Construct, Activity Assay

    Effects of XAF1 on the upregulation of cellular senescence in young HMVECs A. Young cells were transduced with XAF1 or negative control lentiviruses and incubated for 3 days. XAF1 mRNA expression levels were measured by semi-quantitative PCR and XAF1, cyclin A, p53 and p21 protein levels were detected by Western blot analysis. B. Cell proliferation was measured by cell counting and C. the percentages of SA-β-gal positive cells were analyzed. * p

    Journal: Oncotarget

    Article Title: XIAP-associating factor 1, a transcriptional target of BRD7, contributes to endothelial cell senescence

    doi: 10.18632/oncotarget.6962

    Figure Lengend Snippet: Effects of XAF1 on the upregulation of cellular senescence in young HMVECs A. Young cells were transduced with XAF1 or negative control lentiviruses and incubated for 3 days. XAF1 mRNA expression levels were measured by semi-quantitative PCR and XAF1, cyclin A, p53 and p21 protein levels were detected by Western blot analysis. B. Cell proliferation was measured by cell counting and C. the percentages of SA-β-gal positive cells were analyzed. * p

    Article Snippet: The PCR primers for XAF1, p53, p16 and GAPDH were obtained from Bioneer (Daejeon, Korea).

    Techniques: Transduction, Negative Control, Incubation, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Cell Counting

    Partial reverse of premature senescence by XAF1 downregulation in HMVECs A. HMVECs treated with doxorubicin (Doxo, 1 μM) or ionizing radiation (IR, 4 Gy). The cells were incubated for 48 h before transfection with XAF1 siRNAs (siXAF1) or negative control siRNAs. XAF1 knockdown in Doxo- or IR-treated cells was confirmed by RT-PCR (upper panel) and Western blot analysis (lower panel). B. Effects of XAF1 knockdown on premature senescence by Doxo or IR in XAF1 siRNAs-transfected cells were examined by cell proliferation and C. SA-β-gal staining (100x). D. Cell cycle profiles were analyzed by PI staining and flow cytometry. The figure shows representative data from three independent experiments. Values are expressed as the mean ± SD of three independent experiments. * p

    Journal: Oncotarget

    Article Title: XIAP-associating factor 1, a transcriptional target of BRD7, contributes to endothelial cell senescence

    doi: 10.18632/oncotarget.6962

    Figure Lengend Snippet: Partial reverse of premature senescence by XAF1 downregulation in HMVECs A. HMVECs treated with doxorubicin (Doxo, 1 μM) or ionizing radiation (IR, 4 Gy). The cells were incubated for 48 h before transfection with XAF1 siRNAs (siXAF1) or negative control siRNAs. XAF1 knockdown in Doxo- or IR-treated cells was confirmed by RT-PCR (upper panel) and Western blot analysis (lower panel). B. Effects of XAF1 knockdown on premature senescence by Doxo or IR in XAF1 siRNAs-transfected cells were examined by cell proliferation and C. SA-β-gal staining (100x). D. Cell cycle profiles were analyzed by PI staining and flow cytometry. The figure shows representative data from three independent experiments. Values are expressed as the mean ± SD of three independent experiments. * p

    Article Snippet: The PCR primers for XAF1, p53, p16 and GAPDH were obtained from Bioneer (Daejeon, Korea).

    Techniques: Incubation, Transfection, Negative Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Flow Cytometry, Cytometry

    Effects of p53 or p16 knockdown on cell growth arrest induced by XAF1 A. Cells were transduced with p53- or p16-shRNA retroviruses and incubated for 2 days. P53 and p16 knockdown was confirmed by RT-PCR analysis. The p53- or p16-downregulated cells were transduced with XAF1 or negative control lentiviruses and incubated for 2 days. Cell proliferation was measured by B. cell counting for 2 days and C. SA-β-gal staining (100x). Values are expressed as the mean ± SD of three independent experiments. Representative data from three independent experiments are shown. * p

    Journal: Oncotarget

    Article Title: XIAP-associating factor 1, a transcriptional target of BRD7, contributes to endothelial cell senescence

    doi: 10.18632/oncotarget.6962

    Figure Lengend Snippet: Effects of p53 or p16 knockdown on cell growth arrest induced by XAF1 A. Cells were transduced with p53- or p16-shRNA retroviruses and incubated for 2 days. P53 and p16 knockdown was confirmed by RT-PCR analysis. The p53- or p16-downregulated cells were transduced with XAF1 or negative control lentiviruses and incubated for 2 days. Cell proliferation was measured by B. cell counting for 2 days and C. SA-β-gal staining (100x). Values are expressed as the mean ± SD of three independent experiments. Representative data from three independent experiments are shown. * p

    Article Snippet: The PCR primers for XAF1, p53, p16 and GAPDH were obtained from Bioneer (Daejeon, Korea).

    Techniques: Transduction, shRNA, Incubation, Reverse Transcription Polymerase Chain Reaction, Negative Control, Cell Counting, Staining

    Effects of XAF1 expression in lung cancer cells A. XAF1 was upregulated in A549 lung cancer cells and its expression levels were confirmed by semi-quantitative RT-PCR and western blot analysis. Cell proliferation was measured by B. cell counting for 2 days and C. SA-β-gal staining (100x) and the percentage of SA-β-gal positive cells were analyzed in XAF1 upregulated cells C. . * p

    Journal: Oncotarget

    Article Title: XIAP-associating factor 1, a transcriptional target of BRD7, contributes to endothelial cell senescence

    doi: 10.18632/oncotarget.6962

    Figure Lengend Snippet: Effects of XAF1 expression in lung cancer cells A. XAF1 was upregulated in A549 lung cancer cells and its expression levels were confirmed by semi-quantitative RT-PCR and western blot analysis. Cell proliferation was measured by B. cell counting for 2 days and C. SA-β-gal staining (100x) and the percentage of SA-β-gal positive cells were analyzed in XAF1 upregulated cells C. . * p

    Article Snippet: The PCR primers for XAF1, p53, p16 and GAPDH were obtained from Bioneer (Daejeon, Korea).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Cell Counting, Staining

    Mechanism of CGE silencing and effect of Pif1 helicases on telomere length. (a) URA3 and (b) CAN1 mRNA levels in pif1-m2 rrm3 Δ+G4 CGE clones and controls (pre-GCR parental strains and ura3 Δ and can1 Δ cells). qRT-PCR was used to determine the ACT1, URA3, or CAN1 mRNA levels in the indicated strains. URA3 and CAN1 values were normalized to ACT1 levels in each strain; the 2 −ΔΔCt method 38 was used to determine URA3 and CAN1 levels relative to parental pre-GCR cells. *, **, and ***: p

    Journal: Nature

    Article Title: Pif1 family helicases suppress genome instability at G-quadruplex motifs

    doi: 10.1038/nature12149

    Figure Lengend Snippet: Mechanism of CGE silencing and effect of Pif1 helicases on telomere length. (a) URA3 and (b) CAN1 mRNA levels in pif1-m2 rrm3 Δ+G4 CGE clones and controls (pre-GCR parental strains and ura3 Δ and can1 Δ cells). qRT-PCR was used to determine the ACT1, URA3, or CAN1 mRNA levels in the indicated strains. URA3 and CAN1 values were normalized to ACT1 levels in each strain; the 2 −ΔΔCt method 38 was used to determine URA3 and CAN1 levels relative to parental pre-GCR cells. *, **, and ***: p

    Article Snippet: PCR primers were designed to amplify the Pif1-like helicase genes from the above mentioned organisms (see ) with iProof HF Master Mix (BioRad).

    Techniques: Clone Assay, Quantitative RT-PCR

    Pif1 family helicases suppress G4-induced GCR events in pif1-m2 rrm3 Δ+G4 cells. (a, b) The G4-insert region was PCR-amplified and sequenced from 19 ( pif1-m2 rrm3 Δ) or 17 (others) GCR clones. (b) Examples of G4 mutations. G-tracts involved in G4 formation are denoted with dashed boxes. Mutated Gs, red; dashes, deletions. (c) GCR events in pif1-m2 rrm3Δ +G4 cells expressing the indicated helicase. Six of 150 spots/strain are shown. GCR events are white colonies on a gray background of non-growing cells. Average±SD colonies/spot is indicated; *, p

    Journal: Nature

    Article Title: Pif1 family helicases suppress genome instability at G-quadruplex motifs

    doi: 10.1038/nature12149

    Figure Lengend Snippet: Pif1 family helicases suppress G4-induced GCR events in pif1-m2 rrm3 Δ+G4 cells. (a, b) The G4-insert region was PCR-amplified and sequenced from 19 ( pif1-m2 rrm3 Δ) or 17 (others) GCR clones. (b) Examples of G4 mutations. G-tracts involved in G4 formation are denoted with dashed boxes. Mutated Gs, red; dashes, deletions. (c) GCR events in pif1-m2 rrm3Δ +G4 cells expressing the indicated helicase. Six of 150 spots/strain are shown. GCR events are white colonies on a gray background of non-growing cells. Average±SD colonies/spot is indicated; *, p

    Article Snippet: PCR primers were designed to amplify the Pif1-like helicase genes from the above mentioned organisms (see ) with iProof HF Master Mix (BioRad).

    Techniques: Polymerase Chain Reaction, Amplification, Expressing

    Correlation between DNA methylation and mRNA expression levels for PTPRH. DNA methylation (average values) (A) and mRNA expression levels (B) for PTPRH in samples of N and of T in LC-C2 were determined by the Infinium assay and qRT-PCR analysis, respectively. DNA methylation levels for PTPRH were significantly lower in T than in N samples and levels of PTPRH mRNA expression were significantly higher in T than in N samples. (C) Correlation of DNA methylation (average values) and mRNA expression levels for PTPRH in LC-C2 samples. PTPRH mRNA expression levels were inversely correlated with DNA methylation of the single CpG site. These results suggested that PTPRH DNA hypomethylation may result in increased mRNA expression in tissue samples from the same cohort. N, non-cancerous lung tissue; T, corresponding tumorous tissue; qRT-PCR, quantitative real-time reverse transcription-polymerase chain reaction.

    Journal: Oncology Reports

    Article Title: Prognostic implication of PTPRH hypomethylation in non-small cell lung cancer

    doi: 10.3892/or.2015.4082

    Figure Lengend Snippet: Correlation between DNA methylation and mRNA expression levels for PTPRH. DNA methylation (average values) (A) and mRNA expression levels (B) for PTPRH in samples of N and of T in LC-C2 were determined by the Infinium assay and qRT-PCR analysis, respectively. DNA methylation levels for PTPRH were significantly lower in T than in N samples and levels of PTPRH mRNA expression were significantly higher in T than in N samples. (C) Correlation of DNA methylation (average values) and mRNA expression levels for PTPRH in LC-C2 samples. PTPRH mRNA expression levels were inversely correlated with DNA methylation of the single CpG site. These results suggested that PTPRH DNA hypomethylation may result in increased mRNA expression in tissue samples from the same cohort. N, non-cancerous lung tissue; T, corresponding tumorous tissue; qRT-PCR, quantitative real-time reverse transcription-polymerase chain reaction.

    Article Snippet: Specific PCR primers for bisulfite-converted DNA were designed using the EpiDesigner software package ( http://www.epidesigner.com ; Sequenom).

    Techniques: DNA Methylation Assay, Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

    DNA methylation and mRNA expression levels after 5-aza-2′-deoxycytidine (5-aza-dC) treatment. (A) DNA methylation (average values) and (B) mRNA expression levels for PTPRH were determined by the Infinium assay and qRT-PCR analysis, respectively. The error bars represent the standard deviation for triplicate qRT-PCR analyses. DNA methylation and mRNA expression levels on days 3 and 6 were compared with those of untreated cells. After 5-aza-dC treatment, reduction of DNA methylation levels and restoration of the PTPRH mRNA expression levels were observed in both of the cell lines used. qRT-PCR, quantitative real-time reverse transcription-polymerase chain reaction.

    Journal: Oncology Reports

    Article Title: Prognostic implication of PTPRH hypomethylation in non-small cell lung cancer

    doi: 10.3892/or.2015.4082

    Figure Lengend Snippet: DNA methylation and mRNA expression levels after 5-aza-2′-deoxycytidine (5-aza-dC) treatment. (A) DNA methylation (average values) and (B) mRNA expression levels for PTPRH were determined by the Infinium assay and qRT-PCR analysis, respectively. The error bars represent the standard deviation for triplicate qRT-PCR analyses. DNA methylation and mRNA expression levels on days 3 and 6 were compared with those of untreated cells. After 5-aza-dC treatment, reduction of DNA methylation levels and restoration of the PTPRH mRNA expression levels were observed in both of the cell lines used. qRT-PCR, quantitative real-time reverse transcription-polymerase chain reaction.

    Article Snippet: Specific PCR primers for bisulfite-converted DNA were designed using the EpiDesigner software package ( http://www.epidesigner.com ; Sequenom).

    Techniques: DNA Methylation Assay, Expressing, Quantitative RT-PCR, Standard Deviation, Reverse Transcription Polymerase Chain Reaction

    Melt curve of SYBR green PCR products . The Y -axis represents the derivative reporter (−Rn) while x -axis represents the temperature (°C). The figure shows a melting temperature ( 31 ) of human ompW PCR products as 78.46°C.

    Journal: Frontiers in Public Health

    Article Title: Development and Validation of a Novel Real-time Assay for the Detection and Quantification of Vibrio cholerae

    doi: 10.3389/fpubh.2017.00109

    Figure Lengend Snippet: Melt curve of SYBR green PCR products . The Y -axis represents the derivative reporter (−Rn) while x -axis represents the temperature (°C). The figure shows a melting temperature ( 31 ) of human ompW PCR products as 78.46°C.

    Article Snippet: PCR Primer Design The Outer Membrane Protein W-OMPW Sequence of eight reference strains (Table ) was downloaded from the NCBI database.

    Techniques: SYBR Green Assay, Polymerase Chain Reaction

    Maternal HFD‐induced Pgc-1α hypermethylation is maintained with reduced gene expression and abnormal metabolic function in aging mice. Pgc-1α promoter methylation and mRNA expression were assessed by pyrosequencing and real-time PCR, respectively, in offspring skeletal muscle at 12 months of age. Graphs show Pgc-1α promoter methylation at CpG site −260 ( A ), Pgc-1α mRNA expression ( B ), correlation between Pgc-1α methylation and gene expression ( C ), and mRNA expression of Glut4 , Cox4 , Cyt c , Myh2a , and Sod1 ( D ) in skeletal muscle at 12 months of age. Body weight and composition are presented as a growth profile from birth to 12 months ( E ); percentages of lean body mass ( F ) and fat mass ( G ) as measured by dual-energy X-ray absorptiometry at 12 months of age in female offspring are also shown. * P

    Journal: Diabetes

    Article Title: Exercise Prevents Maternal High-Fat Diet–Induced Hypermethylation of the Pgc-1α Gene and Age-Dependent Metabolic Dysfunction in the Offspring

    doi: 10.2337/db13-1614

    Figure Lengend Snippet: Maternal HFD‐induced Pgc-1α hypermethylation is maintained with reduced gene expression and abnormal metabolic function in aging mice. Pgc-1α promoter methylation and mRNA expression were assessed by pyrosequencing and real-time PCR, respectively, in offspring skeletal muscle at 12 months of age. Graphs show Pgc-1α promoter methylation at CpG site −260 ( A ), Pgc-1α mRNA expression ( B ), correlation between Pgc-1α methylation and gene expression ( C ), and mRNA expression of Glut4 , Cox4 , Cyt c , Myh2a , and Sod1 ( D ) in skeletal muscle at 12 months of age. Body weight and composition are presented as a growth profile from birth to 12 months ( E ); percentages of lean body mass ( F ) and fat mass ( G ) as measured by dual-energy X-ray absorptiometry at 12 months of age in female offspring are also shown. * P

    Article Snippet: PCR primers spanning the CpG site −260 of the Pgc-1α promoter were designed using PyroMark primer design software (Qiagen, Valencia, CA).

    Techniques: Pyrolysis Gas Chromatography, Expressing, Mouse Assay, Methylation, Real-time Polymerase Chain Reaction

    Maternal exercise prevents maternal HFD-induced hypermethylation of the Pgc-1α promoter in skeletal muscle in offspring. Pgc-1α promoter methylation and mRNA expression were assessed by pyrosequencing and real-time PCR, respectively, in offspring skeletal muscle and liver at birth. A : Schematic presentation of the structural feature of the Pgc-1α promoter. Circles represent CpG islands, labeled by the base pair number relative to the transcription start site, with site −260 highlighted in red. Open rectangles represent transcription factor binding sites. The arrow marks the transcription start site. Graphs show Pgc-1α promoter methylation at CpG site −260 in muscle ( B ) and liver ( C ). Graphs also show Pgc-1α mRNA in offspring skeletal muscle ( D ) and its correlation with Pgc-1α methylation status ( E ). * P

    Journal: Diabetes

    Article Title: Exercise Prevents Maternal High-Fat Diet–Induced Hypermethylation of the Pgc-1α Gene and Age-Dependent Metabolic Dysfunction in the Offspring

    doi: 10.2337/db13-1614

    Figure Lengend Snippet: Maternal exercise prevents maternal HFD-induced hypermethylation of the Pgc-1α promoter in skeletal muscle in offspring. Pgc-1α promoter methylation and mRNA expression were assessed by pyrosequencing and real-time PCR, respectively, in offspring skeletal muscle and liver at birth. A : Schematic presentation of the structural feature of the Pgc-1α promoter. Circles represent CpG islands, labeled by the base pair number relative to the transcription start site, with site −260 highlighted in red. Open rectangles represent transcription factor binding sites. The arrow marks the transcription start site. Graphs show Pgc-1α promoter methylation at CpG site −260 in muscle ( B ) and liver ( C ). Graphs also show Pgc-1α mRNA in offspring skeletal muscle ( D ) and its correlation with Pgc-1α methylation status ( E ). * P

    Article Snippet: PCR primers spanning the CpG site −260 of the Pgc-1α promoter were designed using PyroMark primer design software (Qiagen, Valencia, CA).

    Techniques: Pyrolysis Gas Chromatography, Methylation, Expressing, Real-time Polymerase Chain Reaction, Labeling, Binding Assay

    Multiplex PCR-RFLP. a : Diagram demonstrating the introduction of MaeIII restriction sites by the mutation (11778A) and the combination of primer alterations and mutation (3460A, 14484C). Mutations are shown in red with the primer alterations in lower case. b : 2.5 % ethidium bromide stained agarose gel showing the results of the PCR-RFLP on patient DNA (black labels) and synthesised DNA controls (red labels). DNA was PCR amplified in a multiplex reaction using 3460 F/R, 11778 F/R and 14484 F/R and restricted using 1 unit of MaeIII as described. M: size marker; Uncut: non-restricted PCR products; All other lanes: patient (Black) or synthesised DNA controls (Red) containing the indicated mutation PCR amplified and restricted with MaeIII. The red arrow between the uncut and normal lanes demonstrates the internal control of restriction. Yellow arrows demonstrate mutation detection

    Journal: Eye and Vision

    Article Title: Development and validation of a novel PCR-RFLP based method for the detection of 3 primary mitochondrial mutations in Leber's hereditary optic neuropathy patients

    doi: 10.1186/s40662-015-0028-0

    Figure Lengend Snippet: Multiplex PCR-RFLP. a : Diagram demonstrating the introduction of MaeIII restriction sites by the mutation (11778A) and the combination of primer alterations and mutation (3460A, 14484C). Mutations are shown in red with the primer alterations in lower case. b : 2.5 % ethidium bromide stained agarose gel showing the results of the PCR-RFLP on patient DNA (black labels) and synthesised DNA controls (red labels). DNA was PCR amplified in a multiplex reaction using 3460 F/R, 11778 F/R and 14484 F/R and restricted using 1 unit of MaeIII as described. M: size marker; Uncut: non-restricted PCR products; All other lanes: patient (Black) or synthesised DNA controls (Red) containing the indicated mutation PCR amplified and restricted with MaeIII. The red arrow between the uncut and normal lanes demonstrates the internal control of restriction. Yellow arrows demonstrate mutation detection

    Article Snippet: MaeIII was chosen because the 11778 mutation naturally introduces a MaeIII restriction site and minor alterations of the PCR primers as shown in Fig. and Table , the 3460A and 14484C mutations also introduce a MaeIII site to allow the development of a multiplex PCR and RFLP strategy based on the modified primers and MaeIII restriction enzyme.

    Techniques: Multiplex Assay, Polymerase Chain Reaction, Mutagenesis, Staining, Agarose Gel Electrophoresis, Amplification, Marker

    Workflow and expected results for the Multiplex PCR-RFLP. a : Sizes of PCR products generated in the multiplex PCR with products of 333 bp, 164 bp, and 236 bp. b : Size of restriction products generated by MaeIII restriction of the multiplex PCR products. In non-mutated samples, only the internal control of restriction in the 3460 product is restricted resulting in the removal of 26 bp from the 333 bp PCR product. c : Schematic representation of the band pattern expected from the diagnostic test. DNA samples with the 3460A, 11778A and 14484C mutations restricted with MaeIII yield the restriction products indicated. The red arrow indicates the control of restriction and the yellow arrows indicate mutation detection

    Journal: Eye and Vision

    Article Title: Development and validation of a novel PCR-RFLP based method for the detection of 3 primary mitochondrial mutations in Leber's hereditary optic neuropathy patients

    doi: 10.1186/s40662-015-0028-0

    Figure Lengend Snippet: Workflow and expected results for the Multiplex PCR-RFLP. a : Sizes of PCR products generated in the multiplex PCR with products of 333 bp, 164 bp, and 236 bp. b : Size of restriction products generated by MaeIII restriction of the multiplex PCR products. In non-mutated samples, only the internal control of restriction in the 3460 product is restricted resulting in the removal of 26 bp from the 333 bp PCR product. c : Schematic representation of the band pattern expected from the diagnostic test. DNA samples with the 3460A, 11778A and 14484C mutations restricted with MaeIII yield the restriction products indicated. The red arrow indicates the control of restriction and the yellow arrows indicate mutation detection

    Article Snippet: MaeIII was chosen because the 11778 mutation naturally introduces a MaeIII restriction site and minor alterations of the PCR primers as shown in Fig. and Table , the 3460A and 14484C mutations also introduce a MaeIII site to allow the development of a multiplex PCR and RFLP strategy based on the modified primers and MaeIII restriction enzyme.

    Techniques: Multiplex Assay, Polymerase Chain Reaction, Generated, Diagnostic Assay, Mutagenesis

    Subcellular localization of HAGLROS and its “sponge” function as a ceRNA competing with miR-100-5p. a RNA was extracted from the nuclear and the cytoplasmic fractions of SGC-7901 and BGC-823 cells and HAGLROS expression of the nuclear and the cytoplasmic fraction was measured by qRT-PCR. GAPDH was used as a cytosolic marker, and U6 was used as a nuclear marker. b FISH was used to confirm HAGLROS location in SGC-7901 and BGC-823 cells, using Cy3 probes for HAGLROS, DAPI for nuclear staining. c miR-100-5p expression was examined in SGC-7901 and BGC-823 cells with HAGLROS knockdown by siRNAs, and HAGLROS expression was tested to determine the transfection efficiencies. d HAGLROS levels were examined in SGC-7901 and BGC-823 cells transfected with miR-100-5p, and miR-100-5p levels were tested for transfection efficiencies. e The expression of miR-100-5p in tumor samples of GC compared to adjacent non-cancerous tissues. f Wild-type or mutant HAGLROS plasmid was co-transfected with miR-NC or miR-100-5p mimics into 293T cells, and relative luciferase activities were measured to determine the level of interaction between miR-100-5p and HAGLROS. g RNA levels in immunoprecipitates are presented as fold enrichment relative to IgG in AGO 2 cells by RIP experiment. Error bars indicate the means ± S.E.M. * P

    Journal: Molecular Cancer

    Article Title: STAT3-induced lncRNA HAGLROS overexpression contributes to the malignant progression of gastric cancer cells via mTOR signal-mediated inhibition of autophagy

    doi: 10.1186/s12943-017-0756-y

    Figure Lengend Snippet: Subcellular localization of HAGLROS and its “sponge” function as a ceRNA competing with miR-100-5p. a RNA was extracted from the nuclear and the cytoplasmic fractions of SGC-7901 and BGC-823 cells and HAGLROS expression of the nuclear and the cytoplasmic fraction was measured by qRT-PCR. GAPDH was used as a cytosolic marker, and U6 was used as a nuclear marker. b FISH was used to confirm HAGLROS location in SGC-7901 and BGC-823 cells, using Cy3 probes for HAGLROS, DAPI for nuclear staining. c miR-100-5p expression was examined in SGC-7901 and BGC-823 cells with HAGLROS knockdown by siRNAs, and HAGLROS expression was tested to determine the transfection efficiencies. d HAGLROS levels were examined in SGC-7901 and BGC-823 cells transfected with miR-100-5p, and miR-100-5p levels were tested for transfection efficiencies. e The expression of miR-100-5p in tumor samples of GC compared to adjacent non-cancerous tissues. f Wild-type or mutant HAGLROS plasmid was co-transfected with miR-NC or miR-100-5p mimics into 293T cells, and relative luciferase activities were measured to determine the level of interaction between miR-100-5p and HAGLROS. g RNA levels in immunoprecipitates are presented as fold enrichment relative to IgG in AGO 2 cells by RIP experiment. Error bars indicate the means ± S.E.M. * P

    Article Snippet: For miRNA quantification, the Bulge-loop™ miRNA qRT-PCR Primer Sets (one RT primer and a pair of qRT-PCR primers for each set) specific for miR-100-5p is designed by RiboBio (Guangzhou, China). cDNA was synthesized using PrimeScript™ RT reagent Kit (Takara Bio USA, No. RR037A).

    Techniques: Expressing, Quantitative RT-PCR, Marker, Fluorescence In Situ Hybridization, Staining, Transfection, Mutagenesis, Plasmid Preparation, Luciferase

    HAGLROS antagonized miR-100-5p-mediated mTOR mRNA degradation. a The relative expression levels of autophagy-related signals mTOR and ATG9A/9B were validated by qRT-PCR upon HAGLROS knockdown in accordance with RNA high-throughput sequencing guidelines. b Bioinformatic analysis of the interactions of HAGLROS with miR-100-5p and mTOR mRNA. c The effect of miR-100-5p overexpression by transfection with miR-100-5p mimics on mTOR mRNA level in GC cells. d Relative luciferase activity of mTOR mRNA 3’-UTR was determined after transfection with miR-100-5p mimics, miR-100-5p inhibitor or HAGLROS plasmid. Error bars indicate the means ± S.E.M. * P

    Journal: Molecular Cancer

    Article Title: STAT3-induced lncRNA HAGLROS overexpression contributes to the malignant progression of gastric cancer cells via mTOR signal-mediated inhibition of autophagy

    doi: 10.1186/s12943-017-0756-y

    Figure Lengend Snippet: HAGLROS antagonized miR-100-5p-mediated mTOR mRNA degradation. a The relative expression levels of autophagy-related signals mTOR and ATG9A/9B were validated by qRT-PCR upon HAGLROS knockdown in accordance with RNA high-throughput sequencing guidelines. b Bioinformatic analysis of the interactions of HAGLROS with miR-100-5p and mTOR mRNA. c The effect of miR-100-5p overexpression by transfection with miR-100-5p mimics on mTOR mRNA level in GC cells. d Relative luciferase activity of mTOR mRNA 3’-UTR was determined after transfection with miR-100-5p mimics, miR-100-5p inhibitor or HAGLROS plasmid. Error bars indicate the means ± S.E.M. * P

    Article Snippet: For miRNA quantification, the Bulge-loop™ miRNA qRT-PCR Primer Sets (one RT primer and a pair of qRT-PCR primers for each set) specific for miR-100-5p is designed by RiboBio (Guangzhou, China). cDNA was synthesized using PrimeScript™ RT reagent Kit (Takara Bio USA, No. RR037A).

    Techniques: Expressing, Quantitative RT-PCR, Next-Generation Sequencing, Over Expression, Transfection, Luciferase, Activity Assay, Plasmid Preparation

    HAGLROS regulates GC cell proliferation and invasion in vitro. a Analysis of HAGLROS expression levels in GC cell lines compared with GES-1 cells by qRT-PCR. b Cell proliferation was determined by MTT assay after SGC-7901 and BGC-823 cells were transfected with siRNAs against HAGLROS and AGS cells were transfected with HAGLROS plasmid. c The representative results of colony formation assays using SGC-7901 and BGC-823 cells transfected with siRNAs against HAGLROS and AGS cells transfected with HAGLROS plasmid. d Cell migration was monitored by wound scratch assay; cell lines were treated the same as in ( b ) and ( c ). e Cell invasion was measured by Transwell assay; cell lines were treated the same as in ( b ) and ( c )

    Journal: Molecular Cancer

    Article Title: STAT3-induced lncRNA HAGLROS overexpression contributes to the malignant progression of gastric cancer cells via mTOR signal-mediated inhibition of autophagy

    doi: 10.1186/s12943-017-0756-y

    Figure Lengend Snippet: HAGLROS regulates GC cell proliferation and invasion in vitro. a Analysis of HAGLROS expression levels in GC cell lines compared with GES-1 cells by qRT-PCR. b Cell proliferation was determined by MTT assay after SGC-7901 and BGC-823 cells were transfected with siRNAs against HAGLROS and AGS cells were transfected with HAGLROS plasmid. c The representative results of colony formation assays using SGC-7901 and BGC-823 cells transfected with siRNAs against HAGLROS and AGS cells transfected with HAGLROS plasmid. d Cell migration was monitored by wound scratch assay; cell lines were treated the same as in ( b ) and ( c ). e Cell invasion was measured by Transwell assay; cell lines were treated the same as in ( b ) and ( c )

    Article Snippet: For miRNA quantification, the Bulge-loop™ miRNA qRT-PCR Primer Sets (one RT primer and a pair of qRT-PCR primers for each set) specific for miR-100-5p is designed by RiboBio (Guangzhou, China). cDNA was synthesized using PrimeScript™ RT reagent Kit (Takara Bio USA, No. RR037A).

    Techniques: In Vitro, Expressing, Quantitative RT-PCR, MTT Assay, Transfection, Plasmid Preparation, Migration, Wound Healing Assay, Transwell Assay

    HAGLROS promotes GC cell tumorigenesis in vivo. a BGC-823 cells transfected with Ctrl shRNA and HAGLROS shRNA were injected respectively into nude mice ( n = 7), which were killed by carbon dioxide euthanasia 20 days after injection. b Tumor volumes were calculated every 3 days beginning 5 days after injection. Bars indicate SD. c Tumor weights were represented as the means of tumor weights ± SD. d Transfection efficiency was tested by qRT-PCR. e The tumor sections underwent IHC staining using antibodies against Ki-67 and HE staining. Error bars indicate means ± S.E.M. * P

    Journal: Molecular Cancer

    Article Title: STAT3-induced lncRNA HAGLROS overexpression contributes to the malignant progression of gastric cancer cells via mTOR signal-mediated inhibition of autophagy

    doi: 10.1186/s12943-017-0756-y

    Figure Lengend Snippet: HAGLROS promotes GC cell tumorigenesis in vivo. a BGC-823 cells transfected with Ctrl shRNA and HAGLROS shRNA were injected respectively into nude mice ( n = 7), which were killed by carbon dioxide euthanasia 20 days after injection. b Tumor volumes were calculated every 3 days beginning 5 days after injection. Bars indicate SD. c Tumor weights were represented as the means of tumor weights ± SD. d Transfection efficiency was tested by qRT-PCR. e The tumor sections underwent IHC staining using antibodies against Ki-67 and HE staining. Error bars indicate means ± S.E.M. * P

    Article Snippet: For miRNA quantification, the Bulge-loop™ miRNA qRT-PCR Primer Sets (one RT primer and a pair of qRT-PCR primers for each set) specific for miR-100-5p is designed by RiboBio (Guangzhou, China). cDNA was synthesized using PrimeScript™ RT reagent Kit (Takara Bio USA, No. RR037A).

    Techniques: In Vivo, Transfection, shRNA, Injection, Mouse Assay, Quantitative RT-PCR, Immunohistochemistry, Staining

    miR-23a is positively and direct regulated by Runx2. ( a ) Runx2 expression level in Hca-P and Hepa1–6 cells, as measured by Western blotting (upper) and qRT-PCR (lower). The numbers upside WB figure present relative intensity of the bands normalized by corresponding GAPDH bands. ( b ) The putative Runx2-binding sequence (E-BOX) in the miR-23a gene promoter is shown. A mutation was generated in the miR-23a promoter at the complementary site for the E-BOX of Runx2 (red). ( c ) Luciferase assays of reporter activity after co-transfection of either the partial miR-23a promoter or the mutant promoter with the pCMV-Runx2 plasmid into Hepa1–6 cells. ( d ) ChIP assays were performed according to the EZ-Magna ChIP TM protocol using chromatin from Hepa1–6 cells, and both anti-Runx2 antibody and normal rabbit IgG were used as the immunoprecipitating antibodies in an overnight incubation. Purified DNA was then analyzed by qRT-PCR (upper) and RT-PCR (lower) using primers specific for the miR-23a promoter. For the ChIP assay positive controls, see also Supplementary Fig. S4 .

    Journal: Scientific Reports

    Article Title: MiR-23a transcriptional activated by Runx2 increases metastatic potential of mouse hepatoma cell via directly targeting Mgat3

    doi: 10.1038/s41598-018-25768-z

    Figure Lengend Snippet: miR-23a is positively and direct regulated by Runx2. ( a ) Runx2 expression level in Hca-P and Hepa1–6 cells, as measured by Western blotting (upper) and qRT-PCR (lower). The numbers upside WB figure present relative intensity of the bands normalized by corresponding GAPDH bands. ( b ) The putative Runx2-binding sequence (E-BOX) in the miR-23a gene promoter is shown. A mutation was generated in the miR-23a promoter at the complementary site for the E-BOX of Runx2 (red). ( c ) Luciferase assays of reporter activity after co-transfection of either the partial miR-23a promoter or the mutant promoter with the pCMV-Runx2 plasmid into Hepa1–6 cells. ( d ) ChIP assays were performed according to the EZ-Magna ChIP TM protocol using chromatin from Hepa1–6 cells, and both anti-Runx2 antibody and normal rabbit IgG were used as the immunoprecipitating antibodies in an overnight incubation. Purified DNA was then analyzed by qRT-PCR (upper) and RT-PCR (lower) using primers specific for the miR-23a promoter. For the ChIP assay positive controls, see also Supplementary Fig. S4 .

    Article Snippet: RT-specific forward and reverse primers for qPCR analysis of miR-23a were designed as part of the Bulge-Loop™ miRNA qRT-PCR primer set (Ribo-bio, China).

    Techniques: Expressing, High Content Screening, Western Blot, Quantitative RT-PCR, Binding Assay, Sequencing, Mutagenesis, Generated, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation, Chromatin Immunoprecipitation, Incubation, Purification, Reverse Transcription Polymerase Chain Reaction

    Mgat3 is a direct target of miR-23a. ( a ) The putative miR-23a-binding sequences in the 3′UTR of Mgat3 mRNA from several species are shown 19 . A mutation was generated in the Mgat3 3′UTR sequence at the complementary site for the seed region of miR-23a (red). ( b ) The luciferase reporter assays show reporter activity after co-transfection of either psiCHECK-2-Mgat3-3′UTR (2180–2751 bp) or psiCHECK-2-mutant Mgat3 3′UTR with miR-23a mimic in Hepa1–6 cells. ( c , d ) miR-23a and Mgat3 expression levels in mouse HCC cells transfected with miR-23a mimic or miR-23a inhibitor relative to those transfected with CP transfection reagent only (mock) or scrambled miR-23a (nc) as measured by qRT-PCR and Western blotting. The numbers upside WB figure present relative intensity of the bands normalized by corresponding GAPDH bands. Data are presented as the median with error bars (*p

    Journal: Scientific Reports

    Article Title: MiR-23a transcriptional activated by Runx2 increases metastatic potential of mouse hepatoma cell via directly targeting Mgat3

    doi: 10.1038/s41598-018-25768-z

    Figure Lengend Snippet: Mgat3 is a direct target of miR-23a. ( a ) The putative miR-23a-binding sequences in the 3′UTR of Mgat3 mRNA from several species are shown 19 . A mutation was generated in the Mgat3 3′UTR sequence at the complementary site for the seed region of miR-23a (red). ( b ) The luciferase reporter assays show reporter activity after co-transfection of either psiCHECK-2-Mgat3-3′UTR (2180–2751 bp) or psiCHECK-2-mutant Mgat3 3′UTR with miR-23a mimic in Hepa1–6 cells. ( c , d ) miR-23a and Mgat3 expression levels in mouse HCC cells transfected with miR-23a mimic or miR-23a inhibitor relative to those transfected with CP transfection reagent only (mock) or scrambled miR-23a (nc) as measured by qRT-PCR and Western blotting. The numbers upside WB figure present relative intensity of the bands normalized by corresponding GAPDH bands. Data are presented as the median with error bars (*p

    Article Snippet: RT-specific forward and reverse primers for qPCR analysis of miR-23a were designed as part of the Bulge-Loop™ miRNA qRT-PCR primer set (Ribo-bio, China).

    Techniques: Binding Assay, Mutagenesis, Generated, Sequencing, Luciferase, Activity Assay, Cotransfection, Expressing, Transfection, Quantitative RT-PCR, Western Blot

    Runx2 regulates Mgat3 expression via miR-23a. ( a ) Runx2, miR-23a and Mgat3 expression levels were measured by qRT-PCR (upper) and Western blot (lower) after transfection of 3.6 µg of pCMV-Runx2 plasmid or 150 nM miR-23a inhibitor and 3 µg of pCMV-Runx2 plasmid into Hepa1–6 cells relative to transfection of CP transfection reagent only (mock). ( b ) Runx2, miR-23a and Mgat3 mRNA levels were determined in Hca-P cells transfected with 100 nM Runx2 siRNA, 100 nM Runx2 siRNA and 3 µg of pCMV-Runx2 plasmid (Runx2 rescued after 24 h), or 150 nM miR-23a mimic and 100 nM Runx2 siRNA relative to the mock control. The numbers upside WB figure present relative intensity of the bands normalized by corresponding GAPDH bands. ( c ) FCM analysis of the levels of bisecting structures on the cell surface recognized by FITC-PHA-E after transfection of Hca-P cells with 100 nM Runx2 siRNA, 100 nM Runx2 siRNA and 3 µg of pCMV-Runx2 plasmid (Runx2 rescued after 24 h), or 150 nM miR-23a mimic and 100 nM Runx2 siRNA relative to the mock control. ( d ) FCM analysis of the levels of bisecting structures on the cell surface recognized by FITC-PHA-E after transfection of Hepa1–6 cells with 3.6 µg of pCMV-Runx2 plasmid or 150 nM miR-23a inhibitor and 3 µg pCMV-Runx2 plasmid relative to the mock control. See also Supplementary Fig. S2 for additional FCM analysis of the levels of β-1,6 branching of N-glycans. Data are presented as median with error bars (*p

    Journal: Scientific Reports

    Article Title: MiR-23a transcriptional activated by Runx2 increases metastatic potential of mouse hepatoma cell via directly targeting Mgat3

    doi: 10.1038/s41598-018-25768-z

    Figure Lengend Snippet: Runx2 regulates Mgat3 expression via miR-23a. ( a ) Runx2, miR-23a and Mgat3 expression levels were measured by qRT-PCR (upper) and Western blot (lower) after transfection of 3.6 µg of pCMV-Runx2 plasmid or 150 nM miR-23a inhibitor and 3 µg of pCMV-Runx2 plasmid into Hepa1–6 cells relative to transfection of CP transfection reagent only (mock). ( b ) Runx2, miR-23a and Mgat3 mRNA levels were determined in Hca-P cells transfected with 100 nM Runx2 siRNA, 100 nM Runx2 siRNA and 3 µg of pCMV-Runx2 plasmid (Runx2 rescued after 24 h), or 150 nM miR-23a mimic and 100 nM Runx2 siRNA relative to the mock control. The numbers upside WB figure present relative intensity of the bands normalized by corresponding GAPDH bands. ( c ) FCM analysis of the levels of bisecting structures on the cell surface recognized by FITC-PHA-E after transfection of Hca-P cells with 100 nM Runx2 siRNA, 100 nM Runx2 siRNA and 3 µg of pCMV-Runx2 plasmid (Runx2 rescued after 24 h), or 150 nM miR-23a mimic and 100 nM Runx2 siRNA relative to the mock control. ( d ) FCM analysis of the levels of bisecting structures on the cell surface recognized by FITC-PHA-E after transfection of Hepa1–6 cells with 3.6 µg of pCMV-Runx2 plasmid or 150 nM miR-23a inhibitor and 3 µg pCMV-Runx2 plasmid relative to the mock control. See also Supplementary Fig. S2 for additional FCM analysis of the levels of β-1,6 branching of N-glycans. Data are presented as median with error bars (*p

    Article Snippet: RT-specific forward and reverse primers for qPCR analysis of miR-23a were designed as part of the Bulge-Loop™ miRNA qRT-PCR primer set (Ribo-bio, China).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, High Content Screening

    Constitutive expression of miR-23a and Mgat3 in mouse HCC cell lines. ( a ) miRNA microarray was performed to compare the miRNA expression profiles of Hca-P and Hepa1–6 cells (left) 27 . The relative expression of miR-23a and several miRNAs, which are related to tumor malignancy 9 , 10 , 16 , 20 , 21 , 28 , 29 , measured by microarray is displayed as a histogram (right). ( b ) The relative expression of miR-23a in Hca-P and Hepa1–6 cells as measured by qRT-PCR. ( c ) Mgat3 expression level in Hca-P and Hepa1–6 cells, as measured by Western blotting (upper) and qRT-PCR (below). The numbers upside WB figure present relative intensity of the bands normalized by corresponding GAPDH bands. Data are presented as the median with error bars (*p

    Journal: Scientific Reports

    Article Title: MiR-23a transcriptional activated by Runx2 increases metastatic potential of mouse hepatoma cell via directly targeting Mgat3

    doi: 10.1038/s41598-018-25768-z

    Figure Lengend Snippet: Constitutive expression of miR-23a and Mgat3 in mouse HCC cell lines. ( a ) miRNA microarray was performed to compare the miRNA expression profiles of Hca-P and Hepa1–6 cells (left) 27 . The relative expression of miR-23a and several miRNAs, which are related to tumor malignancy 9 , 10 , 16 , 20 , 21 , 28 , 29 , measured by microarray is displayed as a histogram (right). ( b ) The relative expression of miR-23a in Hca-P and Hepa1–6 cells as measured by qRT-PCR. ( c ) Mgat3 expression level in Hca-P and Hepa1–6 cells, as measured by Western blotting (upper) and qRT-PCR (below). The numbers upside WB figure present relative intensity of the bands normalized by corresponding GAPDH bands. Data are presented as the median with error bars (*p

    Article Snippet: RT-specific forward and reverse primers for qPCR analysis of miR-23a were designed as part of the Bulge-Loop™ miRNA qRT-PCR primer set (Ribo-bio, China).

    Techniques: Expressing, Microarray, High Content Screening, Quantitative RT-PCR, Western Blot

    IL-1ß downregulates the expression of MITF-M and upregulates the expression of miR-155 in 2 melanoma cell lines. ( A ) Expression of MITF-M was analyzed by quantitative RT-PCR in melanoma cell lines LB2201-MEL and LB2259-MEL incubated with IL-1ß (10 ng/ml) for 4h or 24h. The result was normalized to ß-actin expression (means ± SD for 3 or 4 independent experiments, respectively). ( B ) Expression of miRNAs potentially targeting MITF-M (miR-96, miR-137, miR-148a, miR-155, miR-182 and miR-340) was analyzed by quantitative RT-PCR in the same samples and normalized to RNU44 expression. The table indicates the miRNA fold change when cells were treated with IL-1ß for 4h or 24h.

    Journal: PLoS ONE

    Article Title: microRNA-155, Induced by Interleukin-1ß, Represses the Expression of Microphthalmia-Associated Transcription Factor (MITF-M) in Melanoma Cells

    doi: 10.1371/journal.pone.0122517

    Figure Lengend Snippet: IL-1ß downregulates the expression of MITF-M and upregulates the expression of miR-155 in 2 melanoma cell lines. ( A ) Expression of MITF-M was analyzed by quantitative RT-PCR in melanoma cell lines LB2201-MEL and LB2259-MEL incubated with IL-1ß (10 ng/ml) for 4h or 24h. The result was normalized to ß-actin expression (means ± SD for 3 or 4 independent experiments, respectively). ( B ) Expression of miRNAs potentially targeting MITF-M (miR-96, miR-137, miR-148a, miR-155, miR-182 and miR-340) was analyzed by quantitative RT-PCR in the same samples and normalized to RNU44 expression. The table indicates the miRNA fold change when cells were treated with IL-1ß for 4h or 24h.

    Article Snippet: The PCR primers (Eurogentec) used were: human MITF-M sense 5’-GGAATTATAGAAAGTAGAGGGA-3’ and human MITF-M antisense 5’-ACATGGCAAGCTCAGGAC-3’; human tyrosinase sense 5’- CCAGCATCATTCTTCTCCTCTTG-3’ and human tyrosinase antisense 5’-GTGGACTAGCAAATCCTTCCAG-3’; human ß-actin sense 5’-GGCATCGTGATGGACTCCG-3’ and human ß-actin antisense 5’-GCTGGAAGGTGGACAGCGA-3’; murine MITF-M sense 5’-AGGAGGACTAAGTGGTCTGCG-3’ and murine MITF-M antisense 5’-CCCTGGTTGCTGTAGAGGTCG-3’; murine tyrosinase sense 5’-CTAACTTACTCAGCCCAGCATC-3’ and murine tyrosinase antisense 5’-GGGTTTTGGCTTTGTCATGG-3’; murine IL-1ß sense 5’-ACAGGCTCCGAGATGAACAA-3’ and murine IL-1ß antisense 5’-TTGCTTGGGATCCACACTCTC-3’; murine ß-actin sense 5’-CTCTGGCTCCTAGCACCATGAAG-3’ and murine ß-actin antisense 5’- GCTGGAAGGTGGACAGTGAG-3’.

    Techniques: Expressing, Quantitative RT-PCR, Incubation

    miR-155 downregulates MITF-M in LB2201-MEL melanoma cell line. ( A ) MITF-M and ( B ) Tyrosinase expression was analyzed by quantitative RT-PCR in melanoma cell line LB2201-MEL 24h and 48h after the transfection with a mimic of miR-155 (30 nM). ß-actin expression was used to normalize MITF-M expression (means ± SD for 3 independent experiments). ( C ) MITF-M and Tyrosinase protein expression was analyzed by Western Blot in the same cell line 24h and 48h after the transfection with the mimic of miR-155 (30 nM) (1 of 3 independent experiments).

    Journal: PLoS ONE

    Article Title: microRNA-155, Induced by Interleukin-1ß, Represses the Expression of Microphthalmia-Associated Transcription Factor (MITF-M) in Melanoma Cells

    doi: 10.1371/journal.pone.0122517

    Figure Lengend Snippet: miR-155 downregulates MITF-M in LB2201-MEL melanoma cell line. ( A ) MITF-M and ( B ) Tyrosinase expression was analyzed by quantitative RT-PCR in melanoma cell line LB2201-MEL 24h and 48h after the transfection with a mimic of miR-155 (30 nM). ß-actin expression was used to normalize MITF-M expression (means ± SD for 3 independent experiments). ( C ) MITF-M and Tyrosinase protein expression was analyzed by Western Blot in the same cell line 24h and 48h after the transfection with the mimic of miR-155 (30 nM) (1 of 3 independent experiments).

    Article Snippet: The PCR primers (Eurogentec) used were: human MITF-M sense 5’-GGAATTATAGAAAGTAGAGGGA-3’ and human MITF-M antisense 5’-ACATGGCAAGCTCAGGAC-3’; human tyrosinase sense 5’- CCAGCATCATTCTTCTCCTCTTG-3’ and human tyrosinase antisense 5’-GTGGACTAGCAAATCCTTCCAG-3’; human ß-actin sense 5’-GGCATCGTGATGGACTCCG-3’ and human ß-actin antisense 5’-GCTGGAAGGTGGACAGCGA-3’; murine MITF-M sense 5’-AGGAGGACTAAGTGGTCTGCG-3’ and murine MITF-M antisense 5’-CCCTGGTTGCTGTAGAGGTCG-3’; murine tyrosinase sense 5’-CTAACTTACTCAGCCCAGCATC-3’ and murine tyrosinase antisense 5’-GGGTTTTGGCTTTGTCATGG-3’; murine IL-1ß sense 5’-ACAGGCTCCGAGATGAACAA-3’ and murine IL-1ß antisense 5’-TTGCTTGGGATCCACACTCTC-3’; murine ß-actin sense 5’-CTCTGGCTCCTAGCACCATGAAG-3’ and murine ß-actin antisense 5’- GCTGGAAGGTGGACAGTGAG-3’.

    Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot

    In mouse melanoma samples, there is an anti-correlation between the expression of MITF-M or Tyrosinase and that of IL-1ß or miR-155. 24 tumor samples were collected from 23 Tirp10B mice [ 21 ] and the expression of MITF-M, Tyrosinase, IL-1ß and miR-155 was analyzed by quantitative RT-PCR. ß-actin was used as an internal control for MITF-M, Tyrosinase and IL-1ß whereas U6 was used as an internal control for miR-155. Taken together, our results suggest that the inverse correlation between the expression levels of miR-155 on the one side, and MITF-M and tyrosinase on the other side is also observed in vivo .

    Journal: PLoS ONE

    Article Title: microRNA-155, Induced by Interleukin-1ß, Represses the Expression of Microphthalmia-Associated Transcription Factor (MITF-M) in Melanoma Cells

    doi: 10.1371/journal.pone.0122517

    Figure Lengend Snippet: In mouse melanoma samples, there is an anti-correlation between the expression of MITF-M or Tyrosinase and that of IL-1ß or miR-155. 24 tumor samples were collected from 23 Tirp10B mice [ 21 ] and the expression of MITF-M, Tyrosinase, IL-1ß and miR-155 was analyzed by quantitative RT-PCR. ß-actin was used as an internal control for MITF-M, Tyrosinase and IL-1ß whereas U6 was used as an internal control for miR-155. Taken together, our results suggest that the inverse correlation between the expression levels of miR-155 on the one side, and MITF-M and tyrosinase on the other side is also observed in vivo .

    Article Snippet: The PCR primers (Eurogentec) used were: human MITF-M sense 5’-GGAATTATAGAAAGTAGAGGGA-3’ and human MITF-M antisense 5’-ACATGGCAAGCTCAGGAC-3’; human tyrosinase sense 5’- CCAGCATCATTCTTCTCCTCTTG-3’ and human tyrosinase antisense 5’-GTGGACTAGCAAATCCTTCCAG-3’; human ß-actin sense 5’-GGCATCGTGATGGACTCCG-3’ and human ß-actin antisense 5’-GCTGGAAGGTGGACAGCGA-3’; murine MITF-M sense 5’-AGGAGGACTAAGTGGTCTGCG-3’ and murine MITF-M antisense 5’-CCCTGGTTGCTGTAGAGGTCG-3’; murine tyrosinase sense 5’-CTAACTTACTCAGCCCAGCATC-3’ and murine tyrosinase antisense 5’-GGGTTTTGGCTTTGTCATGG-3’; murine IL-1ß sense 5’-ACAGGCTCCGAGATGAACAA-3’ and murine IL-1ß antisense 5’-TTGCTTGGGATCCACACTCTC-3’; murine ß-actin sense 5’-CTCTGGCTCCTAGCACCATGAAG-3’ and murine ß-actin antisense 5’- GCTGGAAGGTGGACAGTGAG-3’.

    Techniques: Expressing, Mouse Assay, Quantitative RT-PCR, In Vivo

    Inverse correlation between the expression of MITF-M and that of miR-155 in melanoma cell lines incubated with IL-1ß. ( A ) The expression of MITF-M, Tyrosinase and miR-155 was analyzed by quantitative RT-PCR in 8 melanoma cell lines incubated with IL-1ß (10 ng/ml) for 4h or 24h. The results were normalized to ß-actin expression for MITF-M and Tyrosinase and to RNU44 for miR-155 (means ± SD for 3 independent experiments). ( B ) The expression of MITF-M and Tyrosinase proteins was analyzed by Western Blot in the same samples (1 of 3 independent experiments).

    Journal: PLoS ONE

    Article Title: microRNA-155, Induced by Interleukin-1ß, Represses the Expression of Microphthalmia-Associated Transcription Factor (MITF-M) in Melanoma Cells

    doi: 10.1371/journal.pone.0122517

    Figure Lengend Snippet: Inverse correlation between the expression of MITF-M and that of miR-155 in melanoma cell lines incubated with IL-1ß. ( A ) The expression of MITF-M, Tyrosinase and miR-155 was analyzed by quantitative RT-PCR in 8 melanoma cell lines incubated with IL-1ß (10 ng/ml) for 4h or 24h. The results were normalized to ß-actin expression for MITF-M and Tyrosinase and to RNU44 for miR-155 (means ± SD for 3 independent experiments). ( B ) The expression of MITF-M and Tyrosinase proteins was analyzed by Western Blot in the same samples (1 of 3 independent experiments).

    Article Snippet: The PCR primers (Eurogentec) used were: human MITF-M sense 5’-GGAATTATAGAAAGTAGAGGGA-3’ and human MITF-M antisense 5’-ACATGGCAAGCTCAGGAC-3’; human tyrosinase sense 5’- CCAGCATCATTCTTCTCCTCTTG-3’ and human tyrosinase antisense 5’-GTGGACTAGCAAATCCTTCCAG-3’; human ß-actin sense 5’-GGCATCGTGATGGACTCCG-3’ and human ß-actin antisense 5’-GCTGGAAGGTGGACAGCGA-3’; murine MITF-M sense 5’-AGGAGGACTAAGTGGTCTGCG-3’ and murine MITF-M antisense 5’-CCCTGGTTGCTGTAGAGGTCG-3’; murine tyrosinase sense 5’-CTAACTTACTCAGCCCAGCATC-3’ and murine tyrosinase antisense 5’-GGGTTTTGGCTTTGTCATGG-3’; murine IL-1ß sense 5’-ACAGGCTCCGAGATGAACAA-3’ and murine IL-1ß antisense 5’-TTGCTTGGGATCCACACTCTC-3’; murine ß-actin sense 5’-CTCTGGCTCCTAGCACCATGAAG-3’ and murine ß-actin antisense 5’- GCTGGAAGGTGGACAGTGAG-3’.

    Techniques: Expressing, Incubation, Quantitative RT-PCR, Western Blot

    RIG-I and MDA-5 induce expression of the proapoptotic BH3-only members of the Bcl-2 family Noxa, Puma, Bim, and Bik. ( A ) 1205Lu cells were treated with pppRNA or poly(I:C) (3 ng/ml) for 24 hours, and levels of Noxa, Puma, Bim, Bad, Bik, Bid, and Hrk mRNA were measured by quantitative RT-PCR. Relative mRNA levels compared with mock-transfected cells are depicted. Mean ± SD of 3 independent experiments is shown. * P ≤ 0.05 and NS compared with mock-transfected cells. ( B ) The melanoma cell lines 1205Lu, WM239A, WM278, and WM793 were treated with pppRNA or poly(I:C) (5 ng/ml), and Noxa and Puma proteins were quantified by immunoblotting. β-Actin served as loading control. Blots are representative of 2 independent experiments. ( C ) Left: Expression of the BH3-only proteins Noxa, Puma, Bim, Bad, Bik, and Bid was inhibited by treatment with respective siRNAs for 48 hours. Thereafter, cells were treated with pppRNA (left), poly(I:C) (5 ng/ml; right), or transfection reagent alone and analyzed for apoptotic cells by FACS. Annexin V–positive and propidium iodide–negative cells are represented. Mean ± SD of 3 independent experiments is shown. * P ≤ 0.05 compared with control siRNA–transfected cells treated with pppRNA.

    Journal: The Journal of Clinical Investigation

    Article Title: Proapoptotic signaling induced by RIG-I and MDA-5 results in type I interferon-independent apoptosis in human melanoma cells

    doi: 10.1172/JCI37155

    Figure Lengend Snippet: RIG-I and MDA-5 induce expression of the proapoptotic BH3-only members of the Bcl-2 family Noxa, Puma, Bim, and Bik. ( A ) 1205Lu cells were treated with pppRNA or poly(I:C) (3 ng/ml) for 24 hours, and levels of Noxa, Puma, Bim, Bad, Bik, Bid, and Hrk mRNA were measured by quantitative RT-PCR. Relative mRNA levels compared with mock-transfected cells are depicted. Mean ± SD of 3 independent experiments is shown. * P ≤ 0.05 and NS compared with mock-transfected cells. ( B ) The melanoma cell lines 1205Lu, WM239A, WM278, and WM793 were treated with pppRNA or poly(I:C) (5 ng/ml), and Noxa and Puma proteins were quantified by immunoblotting. β-Actin served as loading control. Blots are representative of 2 independent experiments. ( C ) Left: Expression of the BH3-only proteins Noxa, Puma, Bim, Bad, Bik, and Bid was inhibited by treatment with respective siRNAs for 48 hours. Thereafter, cells were treated with pppRNA (left), poly(I:C) (5 ng/ml; right), or transfection reagent alone and analyzed for apoptotic cells by FACS. Annexin V–positive and propidium iodide–negative cells are represented. Mean ± SD of 3 independent experiments is shown. * P ≤ 0.05 compared with control siRNA–transfected cells treated with pppRNA.

    Article Snippet: PCR primers and siRNAs were purchased from MWG Biotech.

    Techniques: Multiple Displacement Amplification, Expressing, Quantitative RT-PCR, Transfection, FACS

    Increased apoptotic sensitivity of melanoma cells to RNA ligands of RIG-I and MDA-5. ( A ) Cell viability of 1205Lu melanoma cells was compared with that of human melanocytes, primary human fibroblasts, or primary human keratinocytes. Cells were transfected with pppRNA or poly(I:C) (20 ng/ml) for 24 hours. The viability of mock-transfected cells was set to 100% for each cell type. Mean of 3 transfections of 1205Lu is indicated; the mean ± SEM of 2 or 3 donors measured in triplicate for primary cells is shown. * P ≤ 0.05 compared with melanocytes, fibroblasts, or keratinocytes. ( B ) Primary cells were treated with pppRNA, poly(I:C) (3 ng/ml) with or without transfection reagent, or with transfection reagent alone. IFN-β expression was analyzed by quantitative RT-PCR 17 hours after transfection. Mean ± SD of triplicate measurements of RNAs pooled from 3 (melanocytes) or 2 donors (fibroblasts and keratinocytes) is shown. ( C ) Primary cells were transfected with pppRNA for 17 hours or poly(I:C) (10 ng/ml) for 24 hours, and expression of Puma and Noxa protein was quantified by immunoblotting. Blots are representative of 3 independent experiments. ( D ) Primary fibroblasts, primary keratinocytes, or 1205Lu melanoma cells were treated with pppRNA, poly(I:C) (3 ng/ml), or transfection reagent alone 48 hours after transfection of siRNAs specific for antiapoptotic Bcl-2, Bcl-x L , Bcl-w, or control siRNA. Cell death was determined by FACS 17 hours after treatment with pppRNA or poly(I:C). Annexin V– and propidium iodide–positive cells are represented. Mean ± SD of 3 experiments with different donors for primary cells or different passages of 1205Lu cells is shown. * P ≤ 0.05, primary cells compared with cells transfected with control siRNA and the respective stimulus, pppRNA or poly(I:C). ( E ) Primary fibroblasts were treated with the indicated siRNAs and analyzed 48 hours after transfection by immunoblotting. Blots are representative of 3 independent experiments. ( F ) Expression of Bcl-2, Bcl-x L , and Bcl-w upon transfection with RIG-I and MDA-5 ligands. Melanocytes of 2 donors or 1205Lu melanoma cells were treated with pppRNA for 17 hours or poly(I:C) (10 ng/ml) for 24 hours. Expression was quantified by immunoblotting. Blots are representative of 3 independent experiments for 1205Lu melanoma cells. In C , E , and F , β-actin served as loading control.

    Journal: The Journal of Clinical Investigation

    Article Title: Proapoptotic signaling induced by RIG-I and MDA-5 results in type I interferon-independent apoptosis in human melanoma cells

    doi: 10.1172/JCI37155

    Figure Lengend Snippet: Increased apoptotic sensitivity of melanoma cells to RNA ligands of RIG-I and MDA-5. ( A ) Cell viability of 1205Lu melanoma cells was compared with that of human melanocytes, primary human fibroblasts, or primary human keratinocytes. Cells were transfected with pppRNA or poly(I:C) (20 ng/ml) for 24 hours. The viability of mock-transfected cells was set to 100% for each cell type. Mean of 3 transfections of 1205Lu is indicated; the mean ± SEM of 2 or 3 donors measured in triplicate for primary cells is shown. * P ≤ 0.05 compared with melanocytes, fibroblasts, or keratinocytes. ( B ) Primary cells were treated with pppRNA, poly(I:C) (3 ng/ml) with or without transfection reagent, or with transfection reagent alone. IFN-β expression was analyzed by quantitative RT-PCR 17 hours after transfection. Mean ± SD of triplicate measurements of RNAs pooled from 3 (melanocytes) or 2 donors (fibroblasts and keratinocytes) is shown. ( C ) Primary cells were transfected with pppRNA for 17 hours or poly(I:C) (10 ng/ml) for 24 hours, and expression of Puma and Noxa protein was quantified by immunoblotting. Blots are representative of 3 independent experiments. ( D ) Primary fibroblasts, primary keratinocytes, or 1205Lu melanoma cells were treated with pppRNA, poly(I:C) (3 ng/ml), or transfection reagent alone 48 hours after transfection of siRNAs specific for antiapoptotic Bcl-2, Bcl-x L , Bcl-w, or control siRNA. Cell death was determined by FACS 17 hours after treatment with pppRNA or poly(I:C). Annexin V– and propidium iodide–positive cells are represented. Mean ± SD of 3 experiments with different donors for primary cells or different passages of 1205Lu cells is shown. * P ≤ 0.05, primary cells compared with cells transfected with control siRNA and the respective stimulus, pppRNA or poly(I:C). ( E ) Primary fibroblasts were treated with the indicated siRNAs and analyzed 48 hours after transfection by immunoblotting. Blots are representative of 3 independent experiments. ( F ) Expression of Bcl-2, Bcl-x L , and Bcl-w upon transfection with RIG-I and MDA-5 ligands. Melanocytes of 2 donors or 1205Lu melanoma cells were treated with pppRNA for 17 hours or poly(I:C) (10 ng/ml) for 24 hours. Expression was quantified by immunoblotting. Blots are representative of 3 independent experiments for 1205Lu melanoma cells. In C , E , and F , β-actin served as loading control.

    Article Snippet: PCR primers and siRNAs were purchased from MWG Biotech.

    Techniques: Multiple Displacement Amplification, Transfection, Expressing, Quantitative RT-PCR, FACS

    Apoptosis induction in melanoma cells requires RIG-I and MDA-5. ( A ) 1205Lu cells were treated with siRNAs specific for RIG-I or MDA-5 or with control siRNA (Ctrl) for 48 hours. Then cells were treated (+) with pppRNA or poly(I:C) (3 ng/ml) or with transfection reagent alone (–). Expression of RIG-I and MDA-5 mRNA was analyzed by quantitative RT-PCR. Mean ± SD of 3 independent experiments is shown. rel. U, relative units. ( B ) 1205Lu cells were treated with siRNAs and pppRNA or poly(I:C) (5 ng/ml) as described for A and analyzed for apoptosis by FACS. Annexin V–positive and propidium iodide–negative cells are represented. Mean ± SD of 3 independent experiments is shown. * P ≤ 0.05 compared with control siRNA–transfected cells treated with pppRNA. ( C ) 1205Lu cells were treated with siRNA specific for TLR3 or control siRNA and pppRNA, poly(I:C), or transfection reagent alone as described for A . Left: Quantification of TLR3 mRNA by quantitative RT-PCR. Right: Analysis of apoptotic cells. Mean ± SD of 3 independent experiments is shown. ( D ) 1205Lu cells were treated with siRNA specific for PKR or control siRNA and pppRNA, poly(I:C), or transfection reagent alone as described for A . Left: Quantification of PKR mRNA by quantitative RT-PCR. Right: Analysis of apoptotic cells. Mean ± SD of 3 independent experiments is shown.

    Journal: The Journal of Clinical Investigation

    Article Title: Proapoptotic signaling induced by RIG-I and MDA-5 results in type I interferon-independent apoptosis in human melanoma cells

    doi: 10.1172/JCI37155

    Figure Lengend Snippet: Apoptosis induction in melanoma cells requires RIG-I and MDA-5. ( A ) 1205Lu cells were treated with siRNAs specific for RIG-I or MDA-5 or with control siRNA (Ctrl) for 48 hours. Then cells were treated (+) with pppRNA or poly(I:C) (3 ng/ml) or with transfection reagent alone (–). Expression of RIG-I and MDA-5 mRNA was analyzed by quantitative RT-PCR. Mean ± SD of 3 independent experiments is shown. rel. U, relative units. ( B ) 1205Lu cells were treated with siRNAs and pppRNA or poly(I:C) (5 ng/ml) as described for A and analyzed for apoptosis by FACS. Annexin V–positive and propidium iodide–negative cells are represented. Mean ± SD of 3 independent experiments is shown. * P ≤ 0.05 compared with control siRNA–transfected cells treated with pppRNA. ( C ) 1205Lu cells were treated with siRNA specific for TLR3 or control siRNA and pppRNA, poly(I:C), or transfection reagent alone as described for A . Left: Quantification of TLR3 mRNA by quantitative RT-PCR. Right: Analysis of apoptotic cells. Mean ± SD of 3 independent experiments is shown. ( D ) 1205Lu cells were treated with siRNA specific for PKR or control siRNA and pppRNA, poly(I:C), or transfection reagent alone as described for A . Left: Quantification of PKR mRNA by quantitative RT-PCR. Right: Analysis of apoptotic cells. Mean ± SD of 3 independent experiments is shown.

    Article Snippet: PCR primers and siRNAs were purchased from MWG Biotech.

    Techniques: Multiple Displacement Amplification, Transfection, Expressing, Quantitative RT-PCR, FACS

    Apoptosis as well as IFN-β induction by pppRNA and poly(I:C) are mediated via IPS-1 in melanoma, but apoptosis is independent of IFN-β secretion. ( A ) 1205Lu cells were treated with pppRNA or poly(I:C) (3 ng/ml) 48 hours after transfection of siRNAs specific for RIG-I, MDA-5, or IPS-1 or control siRNA. IFN-β expression was analyzed by quantitative RT-PCR. ( B ) 1205Lu cells were treated with pppRNA or poly(I:C) (5 ng/ml) 48 hours after transfection of siRNAs specific for IPS-1 or control siRNA, and rates of apoptosis were determined by FACS. Annexin V–positive and propidium iodide–negative cells are represented. * P ≤ 0.05 compared with control siRNA–transfected cells treated with pppRNA1ds. ( C ). Twenty-four hours after vector transfection, cells were treated with pppRNA. Left: Apoptotic and dead cells (AN + /PI + ) were measured. * P ≤ 0.05 compared with mNS3/4- or mock-transfected cells treated with pppRNA. Right: Analysis of IFN-β expression by quantitative RT-PCR. ( D ) 1205Lu cells were treated with siRNA specific for the type I IFN receptor (IFNAR) or control siRNA and pppRNA, poly(I:C), or transfection reagent alone as described for A . Left: quantification of IFNAR mRNA. Middle: Quantification of IFN-β expression. Right: FACS analysis of apoptotic cells (AN + /PI – ) treated with pppRNA, poly(I:C), or transfection reagent alone. ( E ) 1205Lu cells were treated with an IRF-3–specific siRNA or control siRNA and pppRNA as described for A . Left: Analysis of IRF-3 mRNA expression. Right: Analysis of apoptotic cells (AN + /PI – ). For all panels, mean ± SD of 3 independent experiments is shown.

    Journal: The Journal of Clinical Investigation

    Article Title: Proapoptotic signaling induced by RIG-I and MDA-5 results in type I interferon-independent apoptosis in human melanoma cells

    doi: 10.1172/JCI37155

    Figure Lengend Snippet: Apoptosis as well as IFN-β induction by pppRNA and poly(I:C) are mediated via IPS-1 in melanoma, but apoptosis is independent of IFN-β secretion. ( A ) 1205Lu cells were treated with pppRNA or poly(I:C) (3 ng/ml) 48 hours after transfection of siRNAs specific for RIG-I, MDA-5, or IPS-1 or control siRNA. IFN-β expression was analyzed by quantitative RT-PCR. ( B ) 1205Lu cells were treated with pppRNA or poly(I:C) (5 ng/ml) 48 hours after transfection of siRNAs specific for IPS-1 or control siRNA, and rates of apoptosis were determined by FACS. Annexin V–positive and propidium iodide–negative cells are represented. * P ≤ 0.05 compared with control siRNA–transfected cells treated with pppRNA1ds. ( C ). Twenty-four hours after vector transfection, cells were treated with pppRNA. Left: Apoptotic and dead cells (AN + /PI + ) were measured. * P ≤ 0.05 compared with mNS3/4- or mock-transfected cells treated with pppRNA. Right: Analysis of IFN-β expression by quantitative RT-PCR. ( D ) 1205Lu cells were treated with siRNA specific for the type I IFN receptor (IFNAR) or control siRNA and pppRNA, poly(I:C), or transfection reagent alone as described for A . Left: quantification of IFNAR mRNA. Middle: Quantification of IFN-β expression. Right: FACS analysis of apoptotic cells (AN + /PI – ) treated with pppRNA, poly(I:C), or transfection reagent alone. ( E ) 1205Lu cells were treated with an IRF-3–specific siRNA or control siRNA and pppRNA as described for A . Left: Analysis of IRF-3 mRNA expression. Right: Analysis of apoptotic cells (AN + /PI – ). For all panels, mean ± SD of 3 independent experiments is shown.

    Article Snippet: PCR primers and siRNAs were purchased from MWG Biotech.

    Techniques: Transfection, Multiple Displacement Amplification, Expressing, Quantitative RT-PCR, FACS, Plasmid Preparation

    Distribution of injected cells in major organs. (A) Fluorescently labeled iPSCs in the hearts, livers, lungs, and kidneys of healthy (left panels) and lung-injured (right panels) mice 8 h after receiving bolus injections of 2.5×10 8 cells/kg body weight via tail vein. (B) PCR analysis of Tet-On and 18S (18S rRNA gene) DNA levels in the organs. The Tet-On gene was carried into iPSCs by lentiviruses used for the induction of iPSCs from mouse fibroblasts.

    Journal: Theranostics

    Article Title: Direct in vivo application of induced pluripotent stem cells is feasible and can be safe

    doi: 10.7150/thno.28671

    Figure Lengend Snippet: Distribution of injected cells in major organs. (A) Fluorescently labeled iPSCs in the hearts, livers, lungs, and kidneys of healthy (left panels) and lung-injured (right panels) mice 8 h after receiving bolus injections of 2.5×10 8 cells/kg body weight via tail vein. (B) PCR analysis of Tet-On and 18S (18S rRNA gene) DNA levels in the organs. The Tet-On gene was carried into iPSCs by lentiviruses used for the induction of iPSCs from mouse fibroblasts.

    Article Snippet: The following Tet-on -specific PCR primers were designed and confirmed using BLASTN searches (U.S. National Center for Biotechnology Information): sense 5′-AGCACAACTACGCCGCACCC-3′; antisense 5′-ATGCACCAGAGTTTCGAAGC-3′.

    Techniques: Injection, Labeling, Mouse Assay, Polymerase Chain Reaction

    Homing, survival, differentiation and therapeutic effect of intravenously injected iPSC in paraquat-injured lungs. (A) Representative fluorescence images of lungs harvested 1, 3, 7, or 28 days after paraquat injection along with PBS or iPSC administration. iPSCs were labeled with the red fluorescent dye PKH26 before injection. The slides were counterstained with DAPI. (B) The relationship between iPSC number and abundance of PCR product from the Tet-on gene, which was embedded in the mouse iPSCs (left half), and abundance of PCR product from the Tet-on gene isolated from paraquat-injured lungs of syngeneic iPSC-treated mice 1, 3, 7, 28, or 90 days after treatment (right half). The image was captured with the best exposure for showing the relationship between iPSC number and abundance of PCR product. (C) Statistical results of iPSC number in paraquat-induced lungs based on abundance of PCR product from the Tet-on gene. n =3 per time point. Data are represented as the mean ± SEM. (D) Abundance of PCR product from the Tet-on gene isolated from heart, liver, spleen, lung and kidney of paraquat-injected mice receiving syngeneic iPSCs 3 and 28 days before sample harvesting. (E) Detectable rate of Tet-on gene in major organs shown in paraquat-injected mice receiving syngeneic iPSCs. (F) Co-localization of injected cells and type 2 pneumocytes. Red: injected cells. Green: type 2 pneumocytes. Yellow: red and green fluorescence signals co-localized. (G) Intravenously injected syngeneic iPSCs improved pulmonary function of paraquat-injured lungs as shown by inspiratory and expiratory resistances and dynamic pulmonary compliance.

    Journal: Theranostics

    Article Title: Direct in vivo application of induced pluripotent stem cells is feasible and can be safe

    doi: 10.7150/thno.28671

    Figure Lengend Snippet: Homing, survival, differentiation and therapeutic effect of intravenously injected iPSC in paraquat-injured lungs. (A) Representative fluorescence images of lungs harvested 1, 3, 7, or 28 days after paraquat injection along with PBS or iPSC administration. iPSCs were labeled with the red fluorescent dye PKH26 before injection. The slides were counterstained with DAPI. (B) The relationship between iPSC number and abundance of PCR product from the Tet-on gene, which was embedded in the mouse iPSCs (left half), and abundance of PCR product from the Tet-on gene isolated from paraquat-injured lungs of syngeneic iPSC-treated mice 1, 3, 7, 28, or 90 days after treatment (right half). The image was captured with the best exposure for showing the relationship between iPSC number and abundance of PCR product. (C) Statistical results of iPSC number in paraquat-induced lungs based on abundance of PCR product from the Tet-on gene. n =3 per time point. Data are represented as the mean ± SEM. (D) Abundance of PCR product from the Tet-on gene isolated from heart, liver, spleen, lung and kidney of paraquat-injected mice receiving syngeneic iPSCs 3 and 28 days before sample harvesting. (E) Detectable rate of Tet-on gene in major organs shown in paraquat-injected mice receiving syngeneic iPSCs. (F) Co-localization of injected cells and type 2 pneumocytes. Red: injected cells. Green: type 2 pneumocytes. Yellow: red and green fluorescence signals co-localized. (G) Intravenously injected syngeneic iPSCs improved pulmonary function of paraquat-injured lungs as shown by inspiratory and expiratory resistances and dynamic pulmonary compliance.

    Article Snippet: The following Tet-on -specific PCR primers were designed and confirmed using BLASTN searches (U.S. National Center for Biotechnology Information): sense 5′-AGCACAACTACGCCGCACCC-3′; antisense 5′-ATGCACCAGAGTTTCGAAGC-3′.

    Techniques: Injection, Fluorescence, Labeling, Polymerase Chain Reaction, Isolation, Mouse Assay

    Position and frequency of exoY DNA and amino acid sequence variations ( n = 9) and position of PCR primers and conserved regions between P. aeruginosa , B. pertussis , and B. anthracis adenylate cyclases. The x axis represents the nucleotide or amino acid position; the y axis represents the percentage of P. aeruginosa isolates that differed in sequence from PAO1 at each position. Two lysine residues (Lys-81 and Lys-88) in conserved region I and two arginine residues (Arg-212 and Arg-214) in conserved region II are essential for the adenylate cyclase activity of ExoY.

    Journal: Journal of Clinical Microbiology

    Article Title: Single-Nucleotide-Polymorphism Mapping of the Pseudomonas aeruginosa Type III Secretion Toxins for Development of a Diagnostic Multiplex PCR System

    doi: 10.1128/JCM.41.8.3526-3531.2003

    Figure Lengend Snippet: Position and frequency of exoY DNA and amino acid sequence variations ( n = 9) and position of PCR primers and conserved regions between P. aeruginosa , B. pertussis , and B. anthracis adenylate cyclases. The x axis represents the nucleotide or amino acid position; the y axis represents the percentage of P. aeruginosa isolates that differed in sequence from PAO1 at each position. Two lysine residues (Lys-81 and Lys-88) in conserved region I and two arginine residues (Arg-212 and Arg-214) in conserved region II are essential for the adenylate cyclase activity of ExoY.

    Article Snippet: Each gene was amplified in total by using a single set of PCR primers (Table ), the proofreading DNA polymerase PFU (Stratagene, San Diego, Calif.), and an optimal protocol.

    Techniques: Sequencing, Polymerase Chain Reaction, Activity Assay

    Position and frequency of exoS DNA and amino acid sequence variations ( n = 13) and position of PCR primers, GAP, and ADP-ribosyltransferase functional domains. The x axis represents the nucleotide or amino acid position; the y axis represents the percentage of P. aeruginosa isolates that differed in sequence from PAO1 at each position. Arg-146 is essential for Rho GAP activity, whereas Glu-381 is essential for ADP-ribosyltransferase activity. E-son in the ADP-ribosyltransferase domain is the binding site of a 14-3-3 protein (factor for activating ExoS [FAS]). The exoS ) was used in this analysis.

    Journal: Journal of Clinical Microbiology

    Article Title: Single-Nucleotide-Polymorphism Mapping of the Pseudomonas aeruginosa Type III Secretion Toxins for Development of a Diagnostic Multiplex PCR System

    doi: 10.1128/JCM.41.8.3526-3531.2003

    Figure Lengend Snippet: Position and frequency of exoS DNA and amino acid sequence variations ( n = 13) and position of PCR primers, GAP, and ADP-ribosyltransferase functional domains. The x axis represents the nucleotide or amino acid position; the y axis represents the percentage of P. aeruginosa isolates that differed in sequence from PAO1 at each position. Arg-146 is essential for Rho GAP activity, whereas Glu-381 is essential for ADP-ribosyltransferase activity. E-son in the ADP-ribosyltransferase domain is the binding site of a 14-3-3 protein (factor for activating ExoS [FAS]). The exoS ) was used in this analysis.

    Article Snippet: Each gene was amplified in total by using a single set of PCR primers (Table ), the proofreading DNA polymerase PFU (Stratagene, San Diego, Calif.), and an optimal protocol.

    Techniques: Sequencing, Polymerase Chain Reaction, Functional Assay, Activity Assay, Binding Assay

    Genotyping of exoS , exoT , exoU , and exoY in clinical and laboratory P. aeruginosa isolates by multiplex PCR. Agarose gel electrophoresis shows bands representing amplified DNA fragments of each gene. All isolates were genotype positive for exoT , but only strain 19660 was genotype negative for exoY . Strains positive for exoU were genotype negative for exoS . M.W.M., molecular weight marker; standard, standard DNA fragments representing each gene; bps, base pairs.

    Journal: Journal of Clinical Microbiology

    Article Title: Single-Nucleotide-Polymorphism Mapping of the Pseudomonas aeruginosa Type III Secretion Toxins for Development of a Diagnostic Multiplex PCR System

    doi: 10.1128/JCM.41.8.3526-3531.2003

    Figure Lengend Snippet: Genotyping of exoS , exoT , exoU , and exoY in clinical and laboratory P. aeruginosa isolates by multiplex PCR. Agarose gel electrophoresis shows bands representing amplified DNA fragments of each gene. All isolates were genotype positive for exoT , but only strain 19660 was genotype negative for exoY . Strains positive for exoU were genotype negative for exoS . M.W.M., molecular weight marker; standard, standard DNA fragments representing each gene; bps, base pairs.

    Article Snippet: Each gene was amplified in total by using a single set of PCR primers (Table ), the proofreading DNA polymerase PFU (Stratagene, San Diego, Calif.), and an optimal protocol.

    Techniques: Multiplex Assay, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Molecular Weight, Marker

    Position and frequency of exoT DNA and amino acid sequence variations ( n = 8) and the position of PCR primers and signal sequence, GAP, and ADP-ribosyltransferase domains. The x axis represents the nucleotide or amino acid position; the y axis represents the percentage of P. aeruginosa isolates that differed in sequence from PAO1 at each position.

    Journal: Journal of Clinical Microbiology

    Article Title: Single-Nucleotide-Polymorphism Mapping of the Pseudomonas aeruginosa Type III Secretion Toxins for Development of a Diagnostic Multiplex PCR System

    doi: 10.1128/JCM.41.8.3526-3531.2003

    Figure Lengend Snippet: Position and frequency of exoT DNA and amino acid sequence variations ( n = 8) and the position of PCR primers and signal sequence, GAP, and ADP-ribosyltransferase domains. The x axis represents the nucleotide or amino acid position; the y axis represents the percentage of P. aeruginosa isolates that differed in sequence from PAO1 at each position.

    Article Snippet: Each gene was amplified in total by using a single set of PCR primers (Table ), the proofreading DNA polymerase PFU (Stratagene, San Diego, Calif.), and an optimal protocol.

    Techniques: Sequencing, Polymerase Chain Reaction

    SMAD5 was the predominant target of miR-K12-11. (A) miR-K12-11 downregulates SMAD1/2/3/5 3′ UTR reporter activity in HEK293T cells. One hundred nanograms pGL3-SMAD1- 3′ UTR, pGL3-SMAD2- 3′ UTR, pGL3-SMAD3- 3′ UTR, or pGL3-SMAD5- 3′ UTR was cotransfected with either pCDH-miR-K12-11 or pCDH-copGFP (1 μg) into HEK293T cells. The pGL3-BACH1 3′ UTR was used as a positive control. (B) Western blotting of SMAD1/2/3/5 in Ramos-teton-miR-K12-11 cells after induction with or without doxycycline for the indicated times. As a loading control, β-actin was also detected in the same blotting. Values represent percentages of SMAD1/2/3/5 normalized against β-actin and compared with an untreated control. (C) Total RNA of the same samples was reverse-transcribed and then used as a template to determine the SMAD5 mRNA levels by standard qRT-PCR. Values were normalized against β-actin. (D) Ramos-teton-miR-K12-11 cells were cultured with or without doxycycline for 48 h and then starved for 12 h before the addition of TGF-β1 (10 ng/ml). Lysates of these cells were taken 1 or 2 h after TGF-β1 stimulation to quantify the levels of phospho-SMAD1/5 and SMAD5 proteins by Western blotting. As a normalizing control, β-actin was also detected in the same blotting. Densitometry values represent the percentage of p-SMAD1/5 normalized against β-actin and compared with an uninduced control.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus-Encoded MicroRNA miR-K12-11 Attenuates Transforming Growth Factor Beta Signaling through Suppression of SMAD5

    doi: 10.1128/JVI.06245-11

    Figure Lengend Snippet: SMAD5 was the predominant target of miR-K12-11. (A) miR-K12-11 downregulates SMAD1/2/3/5 3′ UTR reporter activity in HEK293T cells. One hundred nanograms pGL3-SMAD1- 3′ UTR, pGL3-SMAD2- 3′ UTR, pGL3-SMAD3- 3′ UTR, or pGL3-SMAD5- 3′ UTR was cotransfected with either pCDH-miR-K12-11 or pCDH-copGFP (1 μg) into HEK293T cells. The pGL3-BACH1 3′ UTR was used as a positive control. (B) Western blotting of SMAD1/2/3/5 in Ramos-teton-miR-K12-11 cells after induction with or without doxycycline for the indicated times. As a loading control, β-actin was also detected in the same blotting. Values represent percentages of SMAD1/2/3/5 normalized against β-actin and compared with an untreated control. (C) Total RNA of the same samples was reverse-transcribed and then used as a template to determine the SMAD5 mRNA levels by standard qRT-PCR. Values were normalized against β-actin. (D) Ramos-teton-miR-K12-11 cells were cultured with or without doxycycline for 48 h and then starved for 12 h before the addition of TGF-β1 (10 ng/ml). Lysates of these cells were taken 1 or 2 h after TGF-β1 stimulation to quantify the levels of phospho-SMAD1/5 and SMAD5 proteins by Western blotting. As a normalizing control, β-actin was also detected in the same blotting. Densitometry values represent the percentage of p-SMAD1/5 normalized against β-actin and compared with an uninduced control.

    Article Snippet: The primers used to detect SMAD5 are listed in , and bulge-loop miRNA quantitative real-time (qRT)-PCR primer sets (one reverse transcription primer and a pair of quantitative PCR primers for each set) specific for miR-K12-11 and miR-155 were designed by RiboBio (Guangzhou, China).

    Techniques: Activity Assay, Positive Control, Western Blot, Quantitative RT-PCR, Cell Culture

    SMAD5 was significantly underexpressed in KSHV-positive PEL cells. (A) miR-K155 or miR-K12-11 expression levels were determined in the indicated cells by bulge-loop qRT-PCR. The percentage of SMAD5 normalized against β-actin and compared with the untreated control is shown. (B) The mRNA levels of SMAD5 in the indicated cells were detected by qRT-PCR. (C) Western blotting was used to monitor the expression levels of SMAD1/2/3/5 in KSHV-positive cells in contrast to KSHV-negative cells. Densitometry values denote the percentage of SMAD5 normalized against β-actin and compared with Ramos cells. (D) Sponge/K12-11 rescued SMAD5 expression in BCBL1 or BC3 cells.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus-Encoded MicroRNA miR-K12-11 Attenuates Transforming Growth Factor Beta Signaling through Suppression of SMAD5

    doi: 10.1128/JVI.06245-11

    Figure Lengend Snippet: SMAD5 was significantly underexpressed in KSHV-positive PEL cells. (A) miR-K155 or miR-K12-11 expression levels were determined in the indicated cells by bulge-loop qRT-PCR. The percentage of SMAD5 normalized against β-actin and compared with the untreated control is shown. (B) The mRNA levels of SMAD5 in the indicated cells were detected by qRT-PCR. (C) Western blotting was used to monitor the expression levels of SMAD1/2/3/5 in KSHV-positive cells in contrast to KSHV-negative cells. Densitometry values denote the percentage of SMAD5 normalized against β-actin and compared with Ramos cells. (D) Sponge/K12-11 rescued SMAD5 expression in BCBL1 or BC3 cells.

    Article Snippet: The primers used to detect SMAD5 are listed in , and bulge-loop miRNA quantitative real-time (qRT)-PCR primer sets (one reverse transcription primer and a pair of quantitative PCR primers for each set) specific for miR-K12-11 and miR-155 were designed by RiboBio (Guangzhou, China).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    Ectopic expression of miR-K12-11 attenuated the cytostatic effect of TGF-β1 in Ramos cells. (A) Ramos-teton-miR-K12-11 cells were treated with doxycycline to upregulate the expression of miR-K12-11. RNA from these samples was reverse-transcribed and then used to quantify the mature miR-K12-11 and housekeeping U6 levels with a bulge-loop qRT-PCR. KSHV-positive cell lines, BC3 or JSC, were used as a positive control. All values are expressed as fold induction of the untreated Ramos cells. (B) Growth of Ramos cells overexpressing miR-K12-11 or control miRNA treated with either TGF-β1 or vehicle over 3 days. Error bars represent SDs. (C) Cell cycle analyses show G 0 /G 1 arrest after TGF-β1 exposure in Ramos cells overexpressing control miRNA, miR-K12-11, or miR-K12-11 in combination with SMAD5. The corresponding P value was calculated for each group, and P values are shown at the top (Student's t test).

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus-Encoded MicroRNA miR-K12-11 Attenuates Transforming Growth Factor Beta Signaling through Suppression of SMAD5

    doi: 10.1128/JVI.06245-11

    Figure Lengend Snippet: Ectopic expression of miR-K12-11 attenuated the cytostatic effect of TGF-β1 in Ramos cells. (A) Ramos-teton-miR-K12-11 cells were treated with doxycycline to upregulate the expression of miR-K12-11. RNA from these samples was reverse-transcribed and then used to quantify the mature miR-K12-11 and housekeeping U6 levels with a bulge-loop qRT-PCR. KSHV-positive cell lines, BC3 or JSC, were used as a positive control. All values are expressed as fold induction of the untreated Ramos cells. (B) Growth of Ramos cells overexpressing miR-K12-11 or control miRNA treated with either TGF-β1 or vehicle over 3 days. Error bars represent SDs. (C) Cell cycle analyses show G 0 /G 1 arrest after TGF-β1 exposure in Ramos cells overexpressing control miRNA, miR-K12-11, or miR-K12-11 in combination with SMAD5. The corresponding P value was calculated for each group, and P values are shown at the top (Student's t test).

    Article Snippet: The primers used to detect SMAD5 are listed in , and bulge-loop miRNA quantitative real-time (qRT)-PCR primer sets (one reverse transcription primer and a pair of quantitative PCR primers for each set) specific for miR-K12-11 and miR-155 were designed by RiboBio (Guangzhou, China).

    Techniques: Expressing, Quantitative RT-PCR, Positive Control

    SMAD5 was downregulated by miR-K12-11 in de novo KSHV-infected HEK293T cells. (A) The expression levels of miR-K155 or miR-K12-11 were detected 72 h after rKSHV.219 infection (MOI = 10) in HEK293T cells and control cells. (B and C) SMAD5 expression was downregulated in response to rKSHV.219 de novo infection. Western blotting and qRT-PCR were used to detect SMAD5 expression at the protein and mRNA levels, respectively. Densitometry values represent the percentage of SMAD5 normalized against β-actin compared with control cells. (D) Sponge/K12-11 can rescue SMAD5 expression in HEK293T/219 cells. HEK293T/219 cells were transduced with a lentiviral miR-K12-11-specific sponge inhibitor or a control vector, and then the expression levels of SMAD5 and β-actin were determined by Western blotting.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus-Encoded MicroRNA miR-K12-11 Attenuates Transforming Growth Factor Beta Signaling through Suppression of SMAD5

    doi: 10.1128/JVI.06245-11

    Figure Lengend Snippet: SMAD5 was downregulated by miR-K12-11 in de novo KSHV-infected HEK293T cells. (A) The expression levels of miR-K155 or miR-K12-11 were detected 72 h after rKSHV.219 infection (MOI = 10) in HEK293T cells and control cells. (B and C) SMAD5 expression was downregulated in response to rKSHV.219 de novo infection. Western blotting and qRT-PCR were used to detect SMAD5 expression at the protein and mRNA levels, respectively. Densitometry values represent the percentage of SMAD5 normalized against β-actin compared with control cells. (D) Sponge/K12-11 can rescue SMAD5 expression in HEK293T/219 cells. HEK293T/219 cells were transduced with a lentiviral miR-K12-11-specific sponge inhibitor or a control vector, and then the expression levels of SMAD5 and β-actin were determined by Western blotting.

    Article Snippet: The primers used to detect SMAD5 are listed in , and bulge-loop miRNA quantitative real-time (qRT)-PCR primer sets (one reverse transcription primer and a pair of quantitative PCR primers for each set) specific for miR-K12-11 and miR-155 were designed by RiboBio (Guangzhou, China).

    Techniques: Infection, Expressing, Western Blot, Quantitative RT-PCR, Transduction, Plasmid Preparation

    The minimal connectivity of genes increased in the odontoblast layer (ODL) and pulp of carious teeth . This network shows known direct or one off interactions between genes measured to be significantly upregulated in this study. For each gene, the expression level (inverse of PCR threshold cycle) in ODL (A) and pulp (B) of carious teeth is rendered as node size. Differential expression in ODL (A) and pulp (B) of carious teeth versus normal teeth (fold increase) is depicted as a color heat map with white showing no change and saturated blue meaning greater up-regulation in carious ODL (A) and pulp (B). Network bottlenecks are highlighted in red, signifying the most important candidate inflammatory signal mediators: PIK3R1, IL1R1, TLR4, ARRβ1, CCL5, CCR5, IL8, JAK1, JAK2, RELA, and TYK2. The key receptors for inflammatory signals induced by caries in ODL appear to converge through IL1R1, CCR5, and IL8Rα/β. The gene expression data used for building this map were derived from PCR arrays and qPCR verification data of cDNA arrays.

    Journal: BMC Immunology

    Article Title: Caries induced cytokine network in the odontoblast layer of human teeth

    doi: 10.1186/1471-2172-12-9

    Figure Lengend Snippet: The minimal connectivity of genes increased in the odontoblast layer (ODL) and pulp of carious teeth . This network shows known direct or one off interactions between genes measured to be significantly upregulated in this study. For each gene, the expression level (inverse of PCR threshold cycle) in ODL (A) and pulp (B) of carious teeth is rendered as node size. Differential expression in ODL (A) and pulp (B) of carious teeth versus normal teeth (fold increase) is depicted as a color heat map with white showing no change and saturated blue meaning greater up-regulation in carious ODL (A) and pulp (B). Network bottlenecks are highlighted in red, signifying the most important candidate inflammatory signal mediators: PIK3R1, IL1R1, TLR4, ARRβ1, CCL5, CCR5, IL8, JAK1, JAK2, RELA, and TYK2. The key receptors for inflammatory signals induced by caries in ODL appear to converge through IL1R1, CCR5, and IL8Rα/β. The gene expression data used for building this map were derived from PCR arrays and qPCR verification data of cDNA arrays.

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction (qPCR) PCR reagents and all PCR primers for cytokines and receptors except those for detecting β-defensin genes were purchased from SA Biosciences.

    Techniques: Expressing, Inverse PCR, Derivative Assay, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    Quantitative analysis for differential changes in the odontoblast layer (ODL) and pulp of carious teeth . Expression of inflammatory genes in ODL and pulp of normal versus carious teeth was determined by real-time quantitative PCR arrays (A-C). The fold change of gene expression detected by PCR arrays was more robust than that determined by cDNA arrays. However, the overall regulatory profile was similar: the increase of inflammatory gene expression in carious teeth was much more profound in ODL than in the pulp. Values are reported as relative fold change in mRNA transcription of carious versus normal samples. The data represent means and standard errors from triplicate sets of array analyses. Abbreviations: IFNA2, Interferon alpha 2; CEBPB, Enhancer binding protein beta; CRP, C-reactive protein; C4A, complement component 4A; BCL6, B-cell CLL/lymphoma 6; ABCF1, ATP-binding cassette, subfamily F member 1; CD40LG, CD40 ligand; TNF, Tumor necrosis factor alpha; SPP1, Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 1); LTB4R, Leukotriene B4 receptor; LTB, Lymphotoxin beta; LTA, Lymphotoxin alpha; TOLLIP, Toll-interacting protein; ICEBERG, ICEBERG caspase-1 inhibitor; IL, Interleukin; IL1R1, Interleukin 1 receptor, type I; IL1RN, Interleukin 1 receptor antagonist; IL10RA, interleukin 10 receptor alpha; IL10RB; interleukin 10 receptor beta; IL1F9, Interleukin 1 family member 9; XCR1, Chemokine (C motif) receptor 1; CX3CR1, Chemokine (C-X3-C motif) receptor 1; CCR, Chemokine (C-C motif) receptor; SCYE1, Small inducible cytokine subfamily E member1 (endothelial monocyte activating); MIF, Macrophage migration inhibitory factor; CXCL, Chemokine (C-X-C motif) ligand; CCL, Chemokine (C-C motif) ligand.

    Journal: BMC Immunology

    Article Title: Caries induced cytokine network in the odontoblast layer of human teeth

    doi: 10.1186/1471-2172-12-9

    Figure Lengend Snippet: Quantitative analysis for differential changes in the odontoblast layer (ODL) and pulp of carious teeth . Expression of inflammatory genes in ODL and pulp of normal versus carious teeth was determined by real-time quantitative PCR arrays (A-C). The fold change of gene expression detected by PCR arrays was more robust than that determined by cDNA arrays. However, the overall regulatory profile was similar: the increase of inflammatory gene expression in carious teeth was much more profound in ODL than in the pulp. Values are reported as relative fold change in mRNA transcription of carious versus normal samples. The data represent means and standard errors from triplicate sets of array analyses. Abbreviations: IFNA2, Interferon alpha 2; CEBPB, Enhancer binding protein beta; CRP, C-reactive protein; C4A, complement component 4A; BCL6, B-cell CLL/lymphoma 6; ABCF1, ATP-binding cassette, subfamily F member 1; CD40LG, CD40 ligand; TNF, Tumor necrosis factor alpha; SPP1, Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 1); LTB4R, Leukotriene B4 receptor; LTB, Lymphotoxin beta; LTA, Lymphotoxin alpha; TOLLIP, Toll-interacting protein; ICEBERG, ICEBERG caspase-1 inhibitor; IL, Interleukin; IL1R1, Interleukin 1 receptor, type I; IL1RN, Interleukin 1 receptor antagonist; IL10RA, interleukin 10 receptor alpha; IL10RB; interleukin 10 receptor beta; IL1F9, Interleukin 1 family member 9; XCR1, Chemokine (C motif) receptor 1; CX3CR1, Chemokine (C-X3-C motif) receptor 1; CCR, Chemokine (C-C motif) receptor; SCYE1, Small inducible cytokine subfamily E member1 (endothelial monocyte activating); MIF, Macrophage migration inhibitory factor; CXCL, Chemokine (C-X-C motif) ligand; CCL, Chemokine (C-C motif) ligand.

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction (qPCR) PCR reagents and all PCR primers for cytokines and receptors except those for detecting β-defensin genes were purchased from SA Biosciences.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Binding Assay, Activation Assay, Migration

    Innate immunity in the odontoblast layer (ODL) . The effect of IL-1β/IL1B, TNF-α/TNFA, IFNγ/IFNG, and LPS on antimicrobial peptide regulation determined by real-time quantitative PCR (A). The effect of caries on HBD2 mRNA expression was detected by semi-quantitative PCR (B). IL-1β, TNF-α, and LPS amplify HBD2 transcription in odontoblast-like cells in vitro . Consistently, caries increase expression of HBD2 mRNA in cells of ODL in vivo . Values are reported as relative fold change in mRNA transcription of unstimulated versus stimulated samples. The data represent means and standard errors from triplicate wells of one experiment and are representative of at least three independent experiments. Asterisks indicate statistically significant changes, with P

    Journal: BMC Immunology

    Article Title: Caries induced cytokine network in the odontoblast layer of human teeth

    doi: 10.1186/1471-2172-12-9

    Figure Lengend Snippet: Innate immunity in the odontoblast layer (ODL) . The effect of IL-1β/IL1B, TNF-α/TNFA, IFNγ/IFNG, and LPS on antimicrobial peptide regulation determined by real-time quantitative PCR (A). The effect of caries on HBD2 mRNA expression was detected by semi-quantitative PCR (B). IL-1β, TNF-α, and LPS amplify HBD2 transcription in odontoblast-like cells in vitro . Consistently, caries increase expression of HBD2 mRNA in cells of ODL in vivo . Values are reported as relative fold change in mRNA transcription of unstimulated versus stimulated samples. The data represent means and standard errors from triplicate wells of one experiment and are representative of at least three independent experiments. Asterisks indicate statistically significant changes, with P

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction (qPCR) PCR reagents and all PCR primers for cytokines and receptors except those for detecting β-defensin genes were purchased from SA Biosciences.

    Techniques: Real-time Polymerase Chain Reaction, Expressing, In Vitro, In Vivo

    cDNA array analysis for cytokines, chemokines, and receptors involved in the responses of teeth to caries . Expression of inflammatory genes in the odontoblast layer (ODL) and pulp of normal versus carious teeth was determined by cDNA arrays (A B) and verified by real-time quantitative PCR (qPCR) (C-K). C-C chemokine receptor 2 (CCR2), CCR4, CCR5, C-C chemokine ligand 3 (CCL3), CCL23, interleukin-1β (IL-1B), tumor necrosis factor α (TNFA), and lymphotoxin α (LTA) mRNA significantly increased in ODL and pulp of carious teeth. CCR9 was significantly upregulated only in the ODL of carious teeth. These genes may play important roles in the responses of teeth to caries. Black Hexagons indicate house keeping genes (glyceraldehyde 3-phosphate dehydrogense, GAPDH; and beta-actin, B-ACTIN). Black-boxed marks indicate no statistically significant changes in gene expression levels between normal and carious teeth. Red-boxed marks label genes with statistically significant increases in carious teeth while green-boxed marks label genes with statistically significant decreases in carious teeth. Genes labeled with a solid line box were verified by qPCR but those with a dashed line box were not verified by qPCR. Values are reported as relative fold change in mRNA transcription of normal versus carious samples. The data represent means and standard errors from triplicate wells of one experiment and are representative of three independent experiments. Asterisks indicate statistically significant changes, with P

    Journal: BMC Immunology

    Article Title: Caries induced cytokine network in the odontoblast layer of human teeth

    doi: 10.1186/1471-2172-12-9

    Figure Lengend Snippet: cDNA array analysis for cytokines, chemokines, and receptors involved in the responses of teeth to caries . Expression of inflammatory genes in the odontoblast layer (ODL) and pulp of normal versus carious teeth was determined by cDNA arrays (A B) and verified by real-time quantitative PCR (qPCR) (C-K). C-C chemokine receptor 2 (CCR2), CCR4, CCR5, C-C chemokine ligand 3 (CCL3), CCL23, interleukin-1β (IL-1B), tumor necrosis factor α (TNFA), and lymphotoxin α (LTA) mRNA significantly increased in ODL and pulp of carious teeth. CCR9 was significantly upregulated only in the ODL of carious teeth. These genes may play important roles in the responses of teeth to caries. Black Hexagons indicate house keeping genes (glyceraldehyde 3-phosphate dehydrogense, GAPDH; and beta-actin, B-ACTIN). Black-boxed marks indicate no statistically significant changes in gene expression levels between normal and carious teeth. Red-boxed marks label genes with statistically significant increases in carious teeth while green-boxed marks label genes with statistically significant decreases in carious teeth. Genes labeled with a solid line box were verified by qPCR but those with a dashed line box were not verified by qPCR. Values are reported as relative fold change in mRNA transcription of normal versus carious samples. The data represent means and standard errors from triplicate wells of one experiment and are representative of three independent experiments. Asterisks indicate statistically significant changes, with P

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction (qPCR) PCR reagents and all PCR primers for cytokines and receptors except those for detecting β-defensin genes were purchased from SA Biosciences.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Labeling

    CBD103 expression in AD. Quantitative RT-PCR analysis of absolute copy-number of mRNA transcripts encoding RPS5, CBD103 and S100A8 in RNA isolated from lesional and nonlesional skin biopsy specimens from dogs (n = 18) diagnosed with AD. Error bars represent standard error.

    Journal: Journal of Innate Immunity

    Article Title: Activity, Expression and Genetic Variation of Canine ?-Defensin 103: A Multifunctional Antimicrobial Peptide in the Skin of Domestic Dogs

    doi: 10.1159/000334566

    Figure Lengend Snippet: CBD103 expression in AD. Quantitative RT-PCR analysis of absolute copy-number of mRNA transcripts encoding RPS5, CBD103 and S100A8 in RNA isolated from lesional and nonlesional skin biopsy specimens from dogs (n = 18) diagnosed with AD. Error bars represent standard error.

    Article Snippet: To determine the prevalence of the ΔG23 genetic variation in CBD103 , PCR primers were designed (MacVector) to flank the CBD103 ) [ ].

    Techniques: Expressing, Quantitative RT-PCR, Isolation

    Expression of canine β-defensins. qRT-PCR analysis of absolute copy number of mRNA transcripts encoding CBD1 ( a ) and CBD103 ( b ) in RNA isolated from various tissues including the skin, gastrointestinal tract (tongue, stomach, duodenum, jejunum, ileum, colon, rectum), lungs, kidneys, pancreas, bone marrow, testes and epididymis. Each bar represents an average of replicate assays (standard deviation

    Journal: Journal of Innate Immunity

    Article Title: Activity, Expression and Genetic Variation of Canine ?-Defensin 103: A Multifunctional Antimicrobial Peptide in the Skin of Domestic Dogs

    doi: 10.1159/000334566

    Figure Lengend Snippet: Expression of canine β-defensins. qRT-PCR analysis of absolute copy number of mRNA transcripts encoding CBD1 ( a ) and CBD103 ( b ) in RNA isolated from various tissues including the skin, gastrointestinal tract (tongue, stomach, duodenum, jejunum, ileum, colon, rectum), lungs, kidneys, pancreas, bone marrow, testes and epididymis. Each bar represents an average of replicate assays (standard deviation

    Article Snippet: To determine the prevalence of the ΔG23 genetic variation in CBD103 , PCR primers were designed (MacVector) to flank the CBD103 ) [ ].

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

    Epidermal mRNA and peptide expression of CBD103. a qRT-PCR analysis of absolute copy number of mRNA transcripts encoding CBD103 in RNA isolated from the caudodorsal (dorsal) and inguinal (ventral) skin of dogs. Calculated mean of the 6 dogs, dorsal and ventral skin. Error bars represent standard error. b Immunostaining of CBD103 in canine skin (2×) using a polyclonal antibody (1:500) raised against hBD3. CBD103 localizes to keratinocytes and follicular epithelial cells. Inset is taken at 20×. c Negative control for staining with secondary antibody alone (2×). Inset is taken at 20×. Arrowheads indicate positive staining. Size bar in 2× view: 300 μm, and in 20× view: 30 μm.

    Journal: Journal of Innate Immunity

    Article Title: Activity, Expression and Genetic Variation of Canine ?-Defensin 103: A Multifunctional Antimicrobial Peptide in the Skin of Domestic Dogs

    doi: 10.1159/000334566

    Figure Lengend Snippet: Epidermal mRNA and peptide expression of CBD103. a qRT-PCR analysis of absolute copy number of mRNA transcripts encoding CBD103 in RNA isolated from the caudodorsal (dorsal) and inguinal (ventral) skin of dogs. Calculated mean of the 6 dogs, dorsal and ventral skin. Error bars represent standard error. b Immunostaining of CBD103 in canine skin (2×) using a polyclonal antibody (1:500) raised against hBD3. CBD103 localizes to keratinocytes and follicular epithelial cells. Inset is taken at 20×. c Negative control for staining with secondary antibody alone (2×). Inset is taken at 20×. Arrowheads indicate positive staining. Size bar in 2× view: 300 μm, and in 20× view: 30 μm.

    Article Snippet: To determine the prevalence of the ΔG23 genetic variation in CBD103 , PCR primers were designed (MacVector) to flank the CBD103 ) [ ].

    Techniques: Expressing, Quantitative RT-PCR, Isolation, Immunostaining, Negative Control, Staining

    Involvement of NLRC4 and RICK in NTHi-induced human β-defensin 2 up-regulation. (A) Luciferase assays show that NTHi lysate-induced human β-defensin 2 up-regulation is enhanced by silencing of NLRC4 (a NOD2 inhibitor) but is inhibited by silencing of RICK that is downstream to NOD2 in the HMEEC cells. NC: a control group transfected with a nonspecific siRNA, KD: a group transfected with a gene-specific siRNA. (B) RT-PCR analysis showing siRNA-mediated inhibition of NLRC4 and RICK expression in the HMEEC cells. (C) Luciferase assays demonstrate that NTHi lysate-induced NF-κB activation is inhibited by the siRNAs specific to NOD2 and RICK in the HMEEC cells. pTAL-luc: a control vector containing the firefly luciferase gene with a TATA-like promoter region from the Herpes simplex virus thymidine kinase promoter, pNFκB-luc: a vector containing multiple copies of the NF-κB consensus sequence fused to pTAL-luc, Tx: treatment. Results were expressed as fold-induction, taking the value of the non-treated group as 1. The experiments were performed in triplicate and repeated twice. Values are given as the mean ± standard deviation (n = 3). *: p

    Journal: PLoS ONE

    Article Title: NOD2/RICK-Dependent ?-Defensin 2 Regulation Is Protective for Nontypeable Haemophilus influenzae-Induced Middle Ear Infection

    doi: 10.1371/journal.pone.0090933

    Figure Lengend Snippet: Involvement of NLRC4 and RICK in NTHi-induced human β-defensin 2 up-regulation. (A) Luciferase assays show that NTHi lysate-induced human β-defensin 2 up-regulation is enhanced by silencing of NLRC4 (a NOD2 inhibitor) but is inhibited by silencing of RICK that is downstream to NOD2 in the HMEEC cells. NC: a control group transfected with a nonspecific siRNA, KD: a group transfected with a gene-specific siRNA. (B) RT-PCR analysis showing siRNA-mediated inhibition of NLRC4 and RICK expression in the HMEEC cells. (C) Luciferase assays demonstrate that NTHi lysate-induced NF-κB activation is inhibited by the siRNAs specific to NOD2 and RICK in the HMEEC cells. pTAL-luc: a control vector containing the firefly luciferase gene with a TATA-like promoter region from the Herpes simplex virus thymidine kinase promoter, pNFκB-luc: a vector containing multiple copies of the NF-κB consensus sequence fused to pTAL-luc, Tx: treatment. Results were expressed as fold-induction, taking the value of the non-treated group as 1. The experiments were performed in triplicate and repeated twice. Values are given as the mean ± standard deviation (n = 3). *: p

    Article Snippet: TLR2−/− NOD2−/− mice were generated by intercrossing of TLR2−/− and NOD2−/− , and genotypes were determined by PCR primers provided by Jackson Laboratory.

    Techniques: Luciferase, Transfection, Reverse Transcription Polymerase Chain Reaction, Inhibition, Expressing, Activation Assay, Plasmid Preparation, Sequencing, Standard Deviation

    NOD2 is involved in bacterial clearance from the mouse middle ear. (A) Quantitative RT-PCR analysis shows that NOD2-deficient middle ear epithelial cells less up-regulate mouse Defb2 in response to the NTHi molecules, compared to the wild type cells. Values are given as the mean ± standard deviation (n = 3). *: p

    Journal: PLoS ONE

    Article Title: NOD2/RICK-Dependent ?-Defensin 2 Regulation Is Protective for Nontypeable Haemophilus influenzae-Induced Middle Ear Infection

    doi: 10.1371/journal.pone.0090933

    Figure Lengend Snippet: NOD2 is involved in bacterial clearance from the mouse middle ear. (A) Quantitative RT-PCR analysis shows that NOD2-deficient middle ear epithelial cells less up-regulate mouse Defb2 in response to the NTHi molecules, compared to the wild type cells. Values are given as the mean ± standard deviation (n = 3). *: p

    Article Snippet: TLR2−/− NOD2−/− mice were generated by intercrossing of TLR2−/− and NOD2−/− , and genotypes were determined by PCR primers provided by Jackson Laboratory.

    Techniques: Quantitative RT-PCR, Standard Deviation

    Membrane pore formation enhances NOD2-dependent human β-defensin 2 up-regulation. (A) Fluorescent microscopic images showing that α-hemolysin, a pore-forming toxin, enhances internalization of live NTHi (green) in the HMEEC cells. (B) An influx of calcein AM into the HMEEC cells was increased by α-hemolysin. (C) Quantitative RT-PCR analysis shows that α-hemolysin markedly enhances human β-defensin 2 up-regulation induced by a suboptimal dose (1 µg/ml) of NTHi lysate in the HMEEC cells. DEFB4: human β-defensin 2. (D) Note that silencing of NOD2 inhibits α-hemolysin-mediated enhancement of NTHi lysate-induced human β-defensin 2 up-regulation in the HMEEC cells. NC: a control group transfected with a nonspecific siRNA, KD: a group transfected with a gene-specific siRNA. Results were expressed as fold-induction, taking the value of the non-treated group as 1. The experiments were performed in triplicate and repeated twice. Values are given as the mean ± standard deviation (n = 3). *: p

    Journal: PLoS ONE

    Article Title: NOD2/RICK-Dependent ?-Defensin 2 Regulation Is Protective for Nontypeable Haemophilus influenzae-Induced Middle Ear Infection

    doi: 10.1371/journal.pone.0090933

    Figure Lengend Snippet: Membrane pore formation enhances NOD2-dependent human β-defensin 2 up-regulation. (A) Fluorescent microscopic images showing that α-hemolysin, a pore-forming toxin, enhances internalization of live NTHi (green) in the HMEEC cells. (B) An influx of calcein AM into the HMEEC cells was increased by α-hemolysin. (C) Quantitative RT-PCR analysis shows that α-hemolysin markedly enhances human β-defensin 2 up-regulation induced by a suboptimal dose (1 µg/ml) of NTHi lysate in the HMEEC cells. DEFB4: human β-defensin 2. (D) Note that silencing of NOD2 inhibits α-hemolysin-mediated enhancement of NTHi lysate-induced human β-defensin 2 up-regulation in the HMEEC cells. NC: a control group transfected with a nonspecific siRNA, KD: a group transfected with a gene-specific siRNA. Results were expressed as fold-induction, taking the value of the non-treated group as 1. The experiments were performed in triplicate and repeated twice. Values are given as the mean ± standard deviation (n = 3). *: p

    Article Snippet: TLR2−/− NOD2−/− mice were generated by intercrossing of TLR2−/− and NOD2−/− , and genotypes were determined by PCR primers provided by Jackson Laboratory.

    Techniques: Quantitative RT-PCR, Transfection, Standard Deviation

    NOD2 contributes to NTHi-induced human β-defensin 2 up-regulation. (A) Quantitative RT-PCR analysis shows that NOD2-specific siRNA inhibits NTHi lysate-induced human β-defensin 2 up-regulation more than NOD1-specific siRNA in the HMEEC cells. ELISA analysis shows that NTHi lysate-induced human β-defensin 2 production is suppressed by the NOD2-specific siRNA (B) but is enhanced by NOD2 overexpression (C) in the HMEEC cells. DEFB4: human β-defensin 2, NC: a control group silenced with a nonspecific negative control siRNA, KD: a group silenced with a gene-specific siRNA, pcDNA: a mock transfection, hNOD2: a construct expressing human NOD2. Results were expressed as fold induction, taking the value of the non-treated group as 1. The experiments were performed in triplicate and repeated twice. Values are given as the mean ± standard deviation (n = 3). *: p

    Journal: PLoS ONE

    Article Title: NOD2/RICK-Dependent ?-Defensin 2 Regulation Is Protective for Nontypeable Haemophilus influenzae-Induced Middle Ear Infection

    doi: 10.1371/journal.pone.0090933

    Figure Lengend Snippet: NOD2 contributes to NTHi-induced human β-defensin 2 up-regulation. (A) Quantitative RT-PCR analysis shows that NOD2-specific siRNA inhibits NTHi lysate-induced human β-defensin 2 up-regulation more than NOD1-specific siRNA in the HMEEC cells. ELISA analysis shows that NTHi lysate-induced human β-defensin 2 production is suppressed by the NOD2-specific siRNA (B) but is enhanced by NOD2 overexpression (C) in the HMEEC cells. DEFB4: human β-defensin 2, NC: a control group silenced with a nonspecific negative control siRNA, KD: a group silenced with a gene-specific siRNA, pcDNA: a mock transfection, hNOD2: a construct expressing human NOD2. Results were expressed as fold induction, taking the value of the non-treated group as 1. The experiments were performed in triplicate and repeated twice. Values are given as the mean ± standard deviation (n = 3). *: p

    Article Snippet: TLR2−/− NOD2−/− mice were generated by intercrossing of TLR2−/− and NOD2−/− , and genotypes were determined by PCR primers provided by Jackson Laboratory.

    Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Over Expression, Negative Control, Transfection, Construct, Expressing, Standard Deviation

    TNFα affects hfNBM cell phenotype. ( a , b ) Representative images showing ( a ) ChAT and ( b ) VAchT expression in hfNBMs as evaluated by immunofluorescence analysis (DAPI counterstained nuclei, scale bar 100 µm). ( c ) Relative mRNA expression by qRT-PCR analysis of TNFR1 and TNFR2 receptors normalized over 18S ribosomal subunit, taken as reference gene, and reported as mean ± SEM ( n = 6). ( d ) Immunofluorescent analysis of NF-κB p65 nuclear translocation after TNFα stimulus (10 ng/mL, 3 h) in comparison to untreated cells (CTL; scale bar 100 µm). ( e ) COX2 mRNA expression in hfNBMs by qRT-PCR after TNFα stimulation (10 ng/mL, 24 h). Data are normalized over 18S ribosomal RNA subunit and reported as percentage of untreated cells (CTL) and displayed as mean ± SEM of three separate experiments performed in triplicate (unpaired Student’s t -test; *** p

    Journal: International Journal of Molecular Sciences

    Article Title: Tumor Necrosis Factor α Influences Phenotypic Plasticity and Promotes Epigenetic Changes in Human Basal Forebrain Cholinergic Neuroblasts

    doi: 10.3390/ijms21176128

    Figure Lengend Snippet: TNFα affects hfNBM cell phenotype. ( a , b ) Representative images showing ( a ) ChAT and ( b ) VAchT expression in hfNBMs as evaluated by immunofluorescence analysis (DAPI counterstained nuclei, scale bar 100 µm). ( c ) Relative mRNA expression by qRT-PCR analysis of TNFR1 and TNFR2 receptors normalized over 18S ribosomal subunit, taken as reference gene, and reported as mean ± SEM ( n = 6). ( d ) Immunofluorescent analysis of NF-κB p65 nuclear translocation after TNFα stimulus (10 ng/mL, 3 h) in comparison to untreated cells (CTL; scale bar 100 µm). ( e ) COX2 mRNA expression in hfNBMs by qRT-PCR after TNFα stimulation (10 ng/mL, 24 h). Data are normalized over 18S ribosomal RNA subunit and reported as percentage of untreated cells (CTL) and displayed as mean ± SEM of three separate experiments performed in triplicate (unpaired Student’s t -test; *** p

    Article Snippet: The cell suspension obtained was cultured in dishes at 37° C in 5 % CO2 atmosphere and routinely checked for mycoplasma contamination by PCR assay (mycoplasma plus™ PCR primer set, Agilent technologies, Santa Clara, CA, USA; #302008).

    Techniques: Expressing, Immunofluorescence, Quantitative RT-PCR, Translocation Assay