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    Thermo Fisher pcr master mix
    Role of HIF-1α in the regulation of CXCR4 gene expression. (A) Expression of CXCR4 in HIF-1α KO mouse embryo fibroblast. Mouse embryo fibroblast from wild type (MEF +/+ ) or knockout for the α subunit of HIF-1 (MEF −/− ) were incubated under normoxic or hypoxic conditions for 6 h, and total RNA was tested for VEGF and CXCR4 mRNA levels by real-time <t>PCR.</t> (B) DFX was used as Hyp-inducing agent. Results are the average of three independent experiments. (C) Expression of CXCR4 in VHL WT and mutated renal carcinoma cells. Expression of CXCR4 and VEGF mRNAs was tested by real-time PCR in the renal cancer cell line 786.0 (VHL mutated) and WT2 (in which a WT VHL has been reintroduced). Results are the average of three independent experiments. (D) HIF-1–dependent transcriptional activation of CXCR4 promoter. MCF-7 breast carcinoma cells were transiently transfected with a plasmid containing a 2.6 kb fragment of the CXCR4 promoter linked to the luciferase reporter gene, with or without a HIF-1α expression vector. Cells were incubated under normoxic or hypoxic conditions for 24 h and evaluated for the luciferase activity. Results are the average of three independent experiments. (E) Hyp-induced HIF-1α recruitment to the CXCR4 promoter. <t>CAOV3</t> cells transfected with the p(HA)HIF-1α plasmid were cultured for 4 h in normoxic or hypoxic conditions. ChIP was performed to investigate the recruitment of HIF-1α on the CXCR4 promoter. (lane 1) Untransfected. (lane 2) Norm. (lane 3) Hyp.
    Pcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6891 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher universal pcr master mix
    Use of exo+ and exo− Taq polymerase. Eprobe mediated real-time <t>PCR</t> experiments were performed by using an exo+ <t>(Amplitaq)</t> and exo− (Genotyping Master) Taq polymerase and Eprobes with different melting temperatures. Amplification curves (Random fluorescent units (RFU) plotted against PCR cycle number) using a 7 times serial dilution of the DNA template are shown on the left. The R-squared values of the PCR efficiency plots are indicated in the graphs. Differential melting curve analysis by plotting –dF/dT against temperature is shown on the right. Main peaks are indicated in the graph to show the different T M values for both Eprobes. EGFR wild-type plasmid DNA concentrations are indicated by colors: Red: 1.5×10 8 copies, Dark blue: 1.5×10 7 copies, Yellow: 1.5×10 6 copies, Green: 1.5×10 5 copies, Pink: 1.5×10 4 copies, Sky blue: 1.5×10 3 copies, Brown: 150 copies, Orange: TE negative control. A: Amplitaq and Eprobe 215-21 wt TO. B: Genotyping Mastermix and Eprobe 215-21 TO. C: Amplitaq and Eprobe 205-13 wt TO. D: Genotyping Master and Eprobe 205-13 wt TO.
    Universal Pcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4926 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rt pcr master mix
    Differential effects of the unsaturated fatty acid DHA and palmitate on the expression levels and circadian expression profile of Bmal1. A : relative mRNA transcript levels of Bmal1 in the vehicle-treated group (DMSO-water, solid black circles), palmitate-treated group (DMSO-palmitate, open black circles), DHA-pretreated group (DHA-water, solid gray squares), and DHA-pretreated cells followed by DHA-palmitate cotreatment (DHA-palm; open gray squares). The mHypoE-37 neurons were pretreated for 1 h with either 25 μM DHA or DMSO vehicle, followed by synchronization. Next, medium was replaced into treatment media containing either cotreatment of 25 μM DHA and 25 μM palmitate, or vehicle (water). Total RNA was harvested every 3 h for 24 h and subsequently used for <t>qRT-PCR.</t> All values were normalized to Histone 3a . Values are plotted as means ± SE of four individual experiments. Symbols indicate significant differences with * P
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    Thermo Fisher taqman universal pcr master mix
    In silico and experimental evidence predict the 3’UTR of PDHX is targeted by MicroRNA-27b. a Diagram illustrating the stepwise approach used to select potential miR-27b targets for experimental validation. b Preliminary luciferase assay of MCF7 cells transiently cotransfected with a PDHX luciferase vector and miR-27b mimic or control. Luciferase expression is quantitated by measuring the degree of luminescence detected by a luminometer 24 h post-transfection for each assay. c and d <t>TaqMan</t> <t>qRT-PCR</t> assessing miR-27b levels in transfected MCF7 and 4175 cells to confirm adequate delivery of RNA oligonucleotides into the cell. e Nucleotide sequences indicating the location of miR-27b binding within the PDHX 3’UTR. The sequence of the original luciferase construct and the eight base pair substitutions present in the mutant construct are shown beneath. Luciferase assays in MCF7 ( f ) and 4175 ( g ) cells, showing miR-27b targeting of the PDHX-3’UTR sequence is lost upon mutation of the seed region. h Expression of PDHX in the Staunton Cell line dataset accessed online using Oncomine. PDHX Fold Change (vs average of all cancers): − 5.602; p -value: 0.023. Under-expression Gene Rank for PDHX: 335 (in top 7%)
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    Image Search Results


    Role of HIF-1α in the regulation of CXCR4 gene expression. (A) Expression of CXCR4 in HIF-1α KO mouse embryo fibroblast. Mouse embryo fibroblast from wild type (MEF +/+ ) or knockout for the α subunit of HIF-1 (MEF −/− ) were incubated under normoxic or hypoxic conditions for 6 h, and total RNA was tested for VEGF and CXCR4 mRNA levels by real-time PCR. (B) DFX was used as Hyp-inducing agent. Results are the average of three independent experiments. (C) Expression of CXCR4 in VHL WT and mutated renal carcinoma cells. Expression of CXCR4 and VEGF mRNAs was tested by real-time PCR in the renal cancer cell line 786.0 (VHL mutated) and WT2 (in which a WT VHL has been reintroduced). Results are the average of three independent experiments. (D) HIF-1–dependent transcriptional activation of CXCR4 promoter. MCF-7 breast carcinoma cells were transiently transfected with a plasmid containing a 2.6 kb fragment of the CXCR4 promoter linked to the luciferase reporter gene, with or without a HIF-1α expression vector. Cells were incubated under normoxic or hypoxic conditions for 24 h and evaluated for the luciferase activity. Results are the average of three independent experiments. (E) Hyp-induced HIF-1α recruitment to the CXCR4 promoter. CAOV3 cells transfected with the p(HA)HIF-1α plasmid were cultured for 4 h in normoxic or hypoxic conditions. ChIP was performed to investigate the recruitment of HIF-1α on the CXCR4 promoter. (lane 1) Untransfected. (lane 2) Norm. (lane 3) Hyp.

    Journal: The Journal of Experimental Medicine

    Article Title: Regulation of the Chemokine Receptor CXCR4 by Hypoxia

    doi: 10.1084/jem.20030267

    Figure Lengend Snippet: Role of HIF-1α in the regulation of CXCR4 gene expression. (A) Expression of CXCR4 in HIF-1α KO mouse embryo fibroblast. Mouse embryo fibroblast from wild type (MEF +/+ ) or knockout for the α subunit of HIF-1 (MEF −/− ) were incubated under normoxic or hypoxic conditions for 6 h, and total RNA was tested for VEGF and CXCR4 mRNA levels by real-time PCR. (B) DFX was used as Hyp-inducing agent. Results are the average of three independent experiments. (C) Expression of CXCR4 in VHL WT and mutated renal carcinoma cells. Expression of CXCR4 and VEGF mRNAs was tested by real-time PCR in the renal cancer cell line 786.0 (VHL mutated) and WT2 (in which a WT VHL has been reintroduced). Results are the average of three independent experiments. (D) HIF-1–dependent transcriptional activation of CXCR4 promoter. MCF-7 breast carcinoma cells were transiently transfected with a plasmid containing a 2.6 kb fragment of the CXCR4 promoter linked to the luciferase reporter gene, with or without a HIF-1α expression vector. Cells were incubated under normoxic or hypoxic conditions for 24 h and evaluated for the luciferase activity. Results are the average of three independent experiments. (E) Hyp-induced HIF-1α recruitment to the CXCR4 promoter. CAOV3 cells transfected with the p(HA)HIF-1α plasmid were cultured for 4 h in normoxic or hypoxic conditions. ChIP was performed to investigate the recruitment of HIF-1α on the CXCR4 promoter. (lane 1) Untransfected. (lane 2) Norm. (lane 3) Hyp.

    Article Snippet: Detection of SDF-1/CCL12 expression by the MCF7 and CAOV3 cell lines was performed by using a PCR master mix (SyBr Green; Applied Biosystem), and the following primers were used: human CXCL12, forward, 5′-ACACTCCAAACTGTGCCCTTCA-3′; and human CXCL12, reverse, 5′-CCACGTCTTTGCCCTTTCATC-3′.

    Techniques: Expressing, Knock-Out, Incubation, Real-time Polymerase Chain Reaction, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Cell Culture, Chromatin Immunoprecipitation

    Use of exo+ and exo− Taq polymerase. Eprobe mediated real-time PCR experiments were performed by using an exo+ (Amplitaq) and exo− (Genotyping Master) Taq polymerase and Eprobes with different melting temperatures. Amplification curves (Random fluorescent units (RFU) plotted against PCR cycle number) using a 7 times serial dilution of the DNA template are shown on the left. The R-squared values of the PCR efficiency plots are indicated in the graphs. Differential melting curve analysis by plotting –dF/dT against temperature is shown on the right. Main peaks are indicated in the graph to show the different T M values for both Eprobes. EGFR wild-type plasmid DNA concentrations are indicated by colors: Red: 1.5×10 8 copies, Dark blue: 1.5×10 7 copies, Yellow: 1.5×10 6 copies, Green: 1.5×10 5 copies, Pink: 1.5×10 4 copies, Sky blue: 1.5×10 3 copies, Brown: 150 copies, Orange: TE negative control. A: Amplitaq and Eprobe 215-21 wt TO. B: Genotyping Mastermix and Eprobe 215-21 TO. C: Amplitaq and Eprobe 205-13 wt TO. D: Genotyping Master and Eprobe 205-13 wt TO.

    Journal: PLoS ONE

    Article Title: Eprobe Mediated Real-Time PCR Monitoring and Melting Curve Analysis

    doi: 10.1371/journal.pone.0070942

    Figure Lengend Snippet: Use of exo+ and exo− Taq polymerase. Eprobe mediated real-time PCR experiments were performed by using an exo+ (Amplitaq) and exo− (Genotyping Master) Taq polymerase and Eprobes with different melting temperatures. Amplification curves (Random fluorescent units (RFU) plotted against PCR cycle number) using a 7 times serial dilution of the DNA template are shown on the left. The R-squared values of the PCR efficiency plots are indicated in the graphs. Differential melting curve analysis by plotting –dF/dT against temperature is shown on the right. Main peaks are indicated in the graph to show the different T M values for both Eprobes. EGFR wild-type plasmid DNA concentrations are indicated by colors: Red: 1.5×10 8 copies, Dark blue: 1.5×10 7 copies, Yellow: 1.5×10 6 copies, Green: 1.5×10 5 copies, Pink: 1.5×10 4 copies, Sky blue: 1.5×10 3 copies, Brown: 150 copies, Orange: TE negative control. A: Amplitaq and Eprobe 215-21 wt TO. B: Genotyping Mastermix and Eprobe 215-21 TO. C: Amplitaq and Eprobe 205-13 wt TO. D: Genotyping Master and Eprobe 205-13 wt TO.

    Article Snippet: We selected AmpliTaq Gold PCR Master Mix (Applied Biosystems, Carlsbad, USA) as a Taq DNA polymerase with an exonuclease activity (exo+) and LightCycler 480 Genotyping Master (Roche Diagnostics, Mannheim, Germany) as a Taq DNA polymerase having an N-terminal deletion lacking an exonuclease activity (exo−).

    Techniques: Real-time Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Serial Dilution, Plasmid Preparation, Negative Control

    Eprobe mediated real-time PCR monitoring. Real-time PCR experiments were performed using different Eprobes, a TaqMan probe, SYBR Green I, Amplitaq and different concentrations of an EGFR wild-type plasmid DNA template. Amplification curves (Random fluorescent units (RFU) plotted against PCR cycle number) using a 7 times serial dilution of the DNA template are shown on the left, and PCR efficiency plots (Ct values plotted against logarithm of plasmid DNA concentrations) are shown on the right. Plasmid DNA concentrations are indicated by colors: Red: 1.5×10 8 copies, Dark blue: 1.5×10 7 copies, Yellow: 1.5×10 6 copies, Green: 1.5×10 5 copies, Pink: 1.5×10 4 copies, Sky blue: 1.5×10 3 copies, Brown: 150 copies, Orange: TE negative control. A: Eprobe 203-10 wt TO, B: Eprobe 205-13 wt TO, C: Eprobe 215-21 wt TO, D: Eprobe 215-21 wt TP, E: SYBR Green I, F: TaqMan probe. Triplicate data are shown for each experiment. For Eprobe 203-10 wt early Ct values for the highest template concentration were not recorded by the PCR machine.

    Journal: PLoS ONE

    Article Title: Eprobe Mediated Real-Time PCR Monitoring and Melting Curve Analysis

    doi: 10.1371/journal.pone.0070942

    Figure Lengend Snippet: Eprobe mediated real-time PCR monitoring. Real-time PCR experiments were performed using different Eprobes, a TaqMan probe, SYBR Green I, Amplitaq and different concentrations of an EGFR wild-type plasmid DNA template. Amplification curves (Random fluorescent units (RFU) plotted against PCR cycle number) using a 7 times serial dilution of the DNA template are shown on the left, and PCR efficiency plots (Ct values plotted against logarithm of plasmid DNA concentrations) are shown on the right. Plasmid DNA concentrations are indicated by colors: Red: 1.5×10 8 copies, Dark blue: 1.5×10 7 copies, Yellow: 1.5×10 6 copies, Green: 1.5×10 5 copies, Pink: 1.5×10 4 copies, Sky blue: 1.5×10 3 copies, Brown: 150 copies, Orange: TE negative control. A: Eprobe 203-10 wt TO, B: Eprobe 205-13 wt TO, C: Eprobe 215-21 wt TO, D: Eprobe 215-21 wt TP, E: SYBR Green I, F: TaqMan probe. Triplicate data are shown for each experiment. For Eprobe 203-10 wt early Ct values for the highest template concentration were not recorded by the PCR machine.

    Article Snippet: We selected AmpliTaq Gold PCR Master Mix (Applied Biosystems, Carlsbad, USA) as a Taq DNA polymerase with an exonuclease activity (exo+) and LightCycler 480 Genotyping Master (Roche Diagnostics, Mannheim, Germany) as a Taq DNA polymerase having an N-terminal deletion lacking an exonuclease activity (exo−).

    Techniques: Real-time Polymerase Chain Reaction, SYBR Green Assay, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Serial Dilution, Negative Control, Concentration Assay

    Permissive and nonpermissive human cells support FeLV-B entry and early proviral DNA synthesis at similar levels. (A) Initial studies using standard PCR with primers based on FeLV LTR or envelope sequences showed marked increases in proviral DNA levels between 0.5 and 6 h after the exposure of cells to FeLV-B at an MOI of 1. Because no PIT-1-negative cell lines were identified, a receptor-negative control was generated for this assay by comparing the FeLV-A infection of permissive feline cells (3201 cells) and of Reh cells, which lack detectable expression of mRNA for the human homologue of FeLV-A receptor THTR1. Loading controls used human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or pan-Myc primers, which efficiently detect human and feline DNA. (B) A wider analysis of FeLV-B infection of human hematopoietic cell lines. Proviral DNA levels were analyzed by quantitative PCR. The line graphs show changes in FeLV DNA concentrations (detected by EnvB primers) relative to those of a housekeeping gene control (β2-microglobulin) between 0.5 and 6 h (top) or between 6 h and 14 days (bottom) after infection. The y axis shows the threshold cycle ( C T ) value relative to that of the β2-microglobulin control (taken as zero). Partially permissive cells were CEM, Jurkat, and Raji cells. Nonpermissive cells were PBMCs ( n = 2), Reh cells, and ALL/MIK cells. Lymphoblastoid cell lines were LCL30, LCL98, LCL113, and LCL114.

    Journal: Journal of Virology

    Article Title: Barriers to Infection of Human Cells by Feline Leukemia Virus: Insights into Resistance to Zoonosis

    doi: 10.1128/JVI.02119-16

    Figure Lengend Snippet: Permissive and nonpermissive human cells support FeLV-B entry and early proviral DNA synthesis at similar levels. (A) Initial studies using standard PCR with primers based on FeLV LTR or envelope sequences showed marked increases in proviral DNA levels between 0.5 and 6 h after the exposure of cells to FeLV-B at an MOI of 1. Because no PIT-1-negative cell lines were identified, a receptor-negative control was generated for this assay by comparing the FeLV-A infection of permissive feline cells (3201 cells) and of Reh cells, which lack detectable expression of mRNA for the human homologue of FeLV-A receptor THTR1. Loading controls used human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or pan-Myc primers, which efficiently detect human and feline DNA. (B) A wider analysis of FeLV-B infection of human hematopoietic cell lines. Proviral DNA levels were analyzed by quantitative PCR. The line graphs show changes in FeLV DNA concentrations (detected by EnvB primers) relative to those of a housekeeping gene control (β2-microglobulin) between 0.5 and 6 h (top) or between 6 h and 14 days (bottom) after infection. The y axis shows the threshold cycle ( C T ) value relative to that of the β2-microglobulin control (taken as zero). Partially permissive cells were CEM, Jurkat, and Raji cells. Nonpermissive cells were PBMCs ( n = 2), Reh cells, and ALL/MIK cells. Lymphoblastoid cell lines were LCL30, LCL98, LCL113, and LCL114.

    Article Snippet: FeLV gag or pol fragments were generated by amplification of 100-ng aliquots of infected-cell genomic DNA in Reddy Mix PCR master mix (Thermo Fisher) using primers FeLV p27gag F (5′-CCCAGTGGCCCTAACTAACC-3′) and R (5′-GCTGGCGTTTCCTCTTTCC-3′) or FeLV Pol F (5′-GCAACCGGTAAGGTGACTC-3′) and Pol R (5′-TTCAAAGGGTTTGGTGATATCTG-3′).

    Techniques: DNA Synthesis, Polymerase Chain Reaction, Negative Control, Generated, Infection, Expressing, Real-time Polymerase Chain Reaction

    Persistence of integrated FeLV-B DNA in human PBMCs. (A) Semiquantitative PCR reveals dose-dependent persistence of FeLV DNA in human PBMCs at 14 days postinfection. Fully permissive HEK293 control cells show stable levels by 3 days postinfection. (B) Results of a quantitative PCR time course analysis for FeLV sequences in PBMC DNA, with and without a preamplification step using primers to FeLV Gag and consensus human Alu sequences. The threshold cycle ( C T ) value is inversely proportional to the DNA content. (C) Results of semiquantitative PCR for LTR circles in FeLV-B-infected cells 3 days (PBMC, Reh, and KYO-1 cells) or 24 h (3201 and HEK293 cells) after infection. While single and double LTR circle forms are readily detected in productively infected HEK293 and 3201 cells, they are barely detectable in Reh cells and PBMCs relative to levels in permissive HEK293 control cells. The loading control was provided by PCR amplification with conserved Myc primers (pan-Myc).

    Journal: Journal of Virology

    Article Title: Barriers to Infection of Human Cells by Feline Leukemia Virus: Insights into Resistance to Zoonosis

    doi: 10.1128/JVI.02119-16

    Figure Lengend Snippet: Persistence of integrated FeLV-B DNA in human PBMCs. (A) Semiquantitative PCR reveals dose-dependent persistence of FeLV DNA in human PBMCs at 14 days postinfection. Fully permissive HEK293 control cells show stable levels by 3 days postinfection. (B) Results of a quantitative PCR time course analysis for FeLV sequences in PBMC DNA, with and without a preamplification step using primers to FeLV Gag and consensus human Alu sequences. The threshold cycle ( C T ) value is inversely proportional to the DNA content. (C) Results of semiquantitative PCR for LTR circles in FeLV-B-infected cells 3 days (PBMC, Reh, and KYO-1 cells) or 24 h (3201 and HEK293 cells) after infection. While single and double LTR circle forms are readily detected in productively infected HEK293 and 3201 cells, they are barely detectable in Reh cells and PBMCs relative to levels in permissive HEK293 control cells. The loading control was provided by PCR amplification with conserved Myc primers (pan-Myc).

    Article Snippet: FeLV gag or pol fragments were generated by amplification of 100-ng aliquots of infected-cell genomic DNA in Reddy Mix PCR master mix (Thermo Fisher) using primers FeLV p27gag F (5′-CCCAGTGGCCCTAACTAACC-3′) and R (5′-GCTGGCGTTTCCTCTTTCC-3′) or FeLV Pol F (5′-GCAACCGGTAAGGTGACTC-3′) and Pol R (5′-TTCAAAGGGTTTGGTGATATCTG-3′).

    Techniques: Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Infection, Amplification

    Hypermutation of the FeLV-B genome and APOBEC3G mRNA expression in human cells. FeLV genomic sequences (segments of Gag and Pol) were cloned by PCR from infected human cells, and individual templates were sequenced and compared to the reference input virus (pFGB clone). APOBEC3G mRNA expression was determined in the same cells (prior to infection) by quantitative real-time PCR (with SYBR green). (A) Representative plots of hypermutation visualized by the online HYPERMUT program ( www.hiv.lanl.gov/content/sequence/HYPERMUT/hypermut.html ), where sequence changes relative to the reference FeLV-B genome are color coded (red, GG→AG; cyan, GA→AA; green, GC→AC; magenta, GT→AT; black, non-G→A). (B) X/Y plots of G→A mutation (per kilobase) against APOBEC3G mRNA levels (where the level in HEK293 cells is taken as 1) with cell lines sorted according to FeLV restriction phenotype. Results for LCLs, which are discordant by virtue of their low levels of infectious virion release ( Table 2 ) but postinfection accumulation of proviral DNA ( Fig. 3 ), are enclosed by a dashed oval. (C) Percentage of G→A mutations that conform to the A3G signature ( 42 ) for all cell lines in which significant levels of mutations were detected. Blue-gray bars, hematopoietic cells; yellow bars, nonhematopoietic cells. (D) Relative levels of APOBEC3G mRNA (on a log 10 scale, with the level in HEK293 cells taken as 1) for all the cell lines tested, sorted into hematopoietic (blue circles) and nonhematopoietic (green circles) cell lines. Nonpermissive cells (nonspreading, with low virion release) are represented by black circles.

    Journal: Journal of Virology

    Article Title: Barriers to Infection of Human Cells by Feline Leukemia Virus: Insights into Resistance to Zoonosis

    doi: 10.1128/JVI.02119-16

    Figure Lengend Snippet: Hypermutation of the FeLV-B genome and APOBEC3G mRNA expression in human cells. FeLV genomic sequences (segments of Gag and Pol) were cloned by PCR from infected human cells, and individual templates were sequenced and compared to the reference input virus (pFGB clone). APOBEC3G mRNA expression was determined in the same cells (prior to infection) by quantitative real-time PCR (with SYBR green). (A) Representative plots of hypermutation visualized by the online HYPERMUT program ( www.hiv.lanl.gov/content/sequence/HYPERMUT/hypermut.html ), where sequence changes relative to the reference FeLV-B genome are color coded (red, GG→AG; cyan, GA→AA; green, GC→AC; magenta, GT→AT; black, non-G→A). (B) X/Y plots of G→A mutation (per kilobase) against APOBEC3G mRNA levels (where the level in HEK293 cells is taken as 1) with cell lines sorted according to FeLV restriction phenotype. Results for LCLs, which are discordant by virtue of their low levels of infectious virion release ( Table 2 ) but postinfection accumulation of proviral DNA ( Fig. 3 ), are enclosed by a dashed oval. (C) Percentage of G→A mutations that conform to the A3G signature ( 42 ) for all cell lines in which significant levels of mutations were detected. Blue-gray bars, hematopoietic cells; yellow bars, nonhematopoietic cells. (D) Relative levels of APOBEC3G mRNA (on a log 10 scale, with the level in HEK293 cells taken as 1) for all the cell lines tested, sorted into hematopoietic (blue circles) and nonhematopoietic (green circles) cell lines. Nonpermissive cells (nonspreading, with low virion release) are represented by black circles.

    Article Snippet: FeLV gag or pol fragments were generated by amplification of 100-ng aliquots of infected-cell genomic DNA in Reddy Mix PCR master mix (Thermo Fisher) using primers FeLV p27gag F (5′-CCCAGTGGCCCTAACTAACC-3′) and R (5′-GCTGGCGTTTCCTCTTTCC-3′) or FeLV Pol F (5′-GCAACCGGTAAGGTGACTC-3′) and Pol R (5′-TTCAAAGGGTTTGGTGATATCTG-3′).

    Techniques: Expressing, Genomic Sequencing, Clone Assay, Polymerase Chain Reaction, Infection, Real-time Polymerase Chain Reaction, SYBR Green Assay, Sequencing, Mutagenesis

    Analysis of BMP4 and CST6 mRNA levels following treatment with demethylating agent and histone deacetylase inhibitor. (A) A CpG island (506 bp) spans the transcription start site of the human BMP4 gene. (B) Relative BMP4 mRNA levels in vehicle (DMSO)-treated 231_ATCC and 231_HM.LNm5 cells by TaqMan qRT-PCR. Expression in 231_ATCC was set to 1. Mean±s.d. ( n =3). (C) TaqMan qRT-PCR analysis of BMP4 mRNA expression levels in 5-Aza-2′-deoxycytidine (5azadC)- and Trichostatin A (TSA)-treated 231_ATCC and 231_HM.LNm5 cells. Expression in vehicle-treated cells was set to 1. Mean±s.d. ( n =3). (D) A CpG island (370 bp) spans the transcription start site of the human CST6 gene. (E) Relative CST6 mRNA levels in vehicle (DMSO)-treated 231_ATCC and 231_HM.LNm5 cells by TaqMan qRT-PCR. Expression in 231_ATCC was set to 1. Mean±s.d. ( n =3). (F) TaqMan qRT-PCR analysis of CST6 mRNA expression levels in 5azadC- and TSA-treated 231_ATCC and 231_HM.LNm5 cells. Expression in vehicle-treated cells was set to 1. Mean±s.d. ( n =3).

    Journal: Disease Models & Mechanisms

    Article Title: Functional and genomic characterisation of a xenograft model system for the study of metastasis in triple-negative breast cancer

    doi: 10.1242/dmm.032250

    Figure Lengend Snippet: Analysis of BMP4 and CST6 mRNA levels following treatment with demethylating agent and histone deacetylase inhibitor. (A) A CpG island (506 bp) spans the transcription start site of the human BMP4 gene. (B) Relative BMP4 mRNA levels in vehicle (DMSO)-treated 231_ATCC and 231_HM.LNm5 cells by TaqMan qRT-PCR. Expression in 231_ATCC was set to 1. Mean±s.d. ( n =3). (C) TaqMan qRT-PCR analysis of BMP4 mRNA expression levels in 5-Aza-2′-deoxycytidine (5azadC)- and Trichostatin A (TSA)-treated 231_ATCC and 231_HM.LNm5 cells. Expression in vehicle-treated cells was set to 1. Mean±s.d. ( n =3). (D) A CpG island (370 bp) spans the transcription start site of the human CST6 gene. (E) Relative CST6 mRNA levels in vehicle (DMSO)-treated 231_ATCC and 231_HM.LNm5 cells by TaqMan qRT-PCR. Expression in 231_ATCC was set to 1. Mean±s.d. ( n =3). (F) TaqMan qRT-PCR analysis of CST6 mRNA expression levels in 5azadC- and TSA-treated 231_ATCC and 231_HM.LNm5 cells. Expression in vehicle-treated cells was set to 1. Mean±s.d. ( n =3).

    Article Snippet: Two-step qPCR was completed (50-250 ng cDNA template/reaction) using either TaqMan gene expression assays (CTSC, ENG, TIE1, BMP2, BMP4, RPL37A) with Fast Universal PCR Master Mix, no AmpErase™ UNG (Thermo Fisher Scientific) or Fast SYBR™ Green Master Mix (50-250 ng cDNA template/reaction) and the following oligonucleotide primers (Integrated DNA Technologies, Singapore).

    Techniques: Histone Deacetylase Assay, Quantitative RT-PCR, Expressing

    Differential effects of the unsaturated fatty acid DHA and palmitate on the expression levels and circadian expression profile of Bmal1. A : relative mRNA transcript levels of Bmal1 in the vehicle-treated group (DMSO-water, solid black circles), palmitate-treated group (DMSO-palmitate, open black circles), DHA-pretreated group (DHA-water, solid gray squares), and DHA-pretreated cells followed by DHA-palmitate cotreatment (DHA-palm; open gray squares). The mHypoE-37 neurons were pretreated for 1 h with either 25 μM DHA or DMSO vehicle, followed by synchronization. Next, medium was replaced into treatment media containing either cotreatment of 25 μM DHA and 25 μM palmitate, or vehicle (water). Total RNA was harvested every 3 h for 24 h and subsequently used for qRT-PCR. All values were normalized to Histone 3a . Values are plotted as means ± SE of four individual experiments. Symbols indicate significant differences with * P

    Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

    Article Title: Differential effects of omega-3 fatty acid docosahexaenoic acid and palmitate on the circadian transcriptional profile of clock genes in immortalized hypothalamic neurons

    doi: 10.1152/ajpregu.00100.2014

    Figure Lengend Snippet: Differential effects of the unsaturated fatty acid DHA and palmitate on the expression levels and circadian expression profile of Bmal1. A : relative mRNA transcript levels of Bmal1 in the vehicle-treated group (DMSO-water, solid black circles), palmitate-treated group (DMSO-palmitate, open black circles), DHA-pretreated group (DHA-water, solid gray squares), and DHA-pretreated cells followed by DHA-palmitate cotreatment (DHA-palm; open gray squares). The mHypoE-37 neurons were pretreated for 1 h with either 25 μM DHA or DMSO vehicle, followed by synchronization. Next, medium was replaced into treatment media containing either cotreatment of 25 μM DHA and 25 μM palmitate, or vehicle (water). Total RNA was harvested every 3 h for 24 h and subsequently used for qRT-PCR. All values were normalized to Histone 3a . Values are plotted as means ± SE of four individual experiments. Symbols indicate significant differences with * P

    Article Snippet: Single-stranded cDNA required for real-time quantitative RT-PCR (qRT-PCR) was synthesized using the high-capacity cDNA reverse transcription kit (Applied Biosystems) as per the manufacturer's instructions. cDNA was amplified using a qRT-PCR master mix (Platinum SYBR Green qPCR SuperMix-UDG with ROX; Invitrogen) and gene-specific primers.

    Techniques: Expressing, Quantitative RT-PCR

    Effects of the saturated fatty acid palmitate on inflammatory and metabolic gene transcription. The mHypoE-37 cells were treated with 25 μM of palmitate following serum shock. Total RNA was harvested every 3 h for 36 h and subsequently used for qRT-PCR. All values were normalized to Histone 3a . A–D : exposure to palmitate did not alter the expression of any examined inflammatory genes, IL-6 , TLR4 , or IκBα , or the orexigenic neuropeptide AgRP . Gray circles denote water, while black squares denote palmitate. Values are plotted as means ± SE of four individual experiments.

    Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

    Article Title: Differential effects of omega-3 fatty acid docosahexaenoic acid and palmitate on the circadian transcriptional profile of clock genes in immortalized hypothalamic neurons

    doi: 10.1152/ajpregu.00100.2014

    Figure Lengend Snippet: Effects of the saturated fatty acid palmitate on inflammatory and metabolic gene transcription. The mHypoE-37 cells were treated with 25 μM of palmitate following serum shock. Total RNA was harvested every 3 h for 36 h and subsequently used for qRT-PCR. All values were normalized to Histone 3a . A–D : exposure to palmitate did not alter the expression of any examined inflammatory genes, IL-6 , TLR4 , or IκBα , or the orexigenic neuropeptide AgRP . Gray circles denote water, while black squares denote palmitate. Values are plotted as means ± SE of four individual experiments.

    Article Snippet: Single-stranded cDNA required for real-time quantitative RT-PCR (qRT-PCR) was synthesized using the high-capacity cDNA reverse transcription kit (Applied Biosystems) as per the manufacturer's instructions. cDNA was amplified using a qRT-PCR master mix (Platinum SYBR Green qPCR SuperMix-UDG with ROX; Invitrogen) and gene-specific primers.

    Techniques: Quantitative RT-PCR, Expressing

    Effects of the unsaturated fatty acid DHA on inflammatory and metabolic gene transcription. The mHypoE-37 cell line was treated with 25 μM DHA following serum shock. Total RNA was harvested every 3 h for 36 h and subsequently used for qRT-PCR. All values were normalized to Histone 3a. A : exposure to 25 μM DHA significantly reduced the expression of IL-6 between the hours of 21 and 33, inclusively. However, DHA treatment did not alter the expression of TLR4, IκBα , or AgRP ( B–D ). Values are plotted as mean values ± SE of four individual experiments. * P

    Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

    Article Title: Differential effects of omega-3 fatty acid docosahexaenoic acid and palmitate on the circadian transcriptional profile of clock genes in immortalized hypothalamic neurons

    doi: 10.1152/ajpregu.00100.2014

    Figure Lengend Snippet: Effects of the unsaturated fatty acid DHA on inflammatory and metabolic gene transcription. The mHypoE-37 cell line was treated with 25 μM DHA following serum shock. Total RNA was harvested every 3 h for 36 h and subsequently used for qRT-PCR. All values were normalized to Histone 3a. A : exposure to 25 μM DHA significantly reduced the expression of IL-6 between the hours of 21 and 33, inclusively. However, DHA treatment did not alter the expression of TLR4, IκBα , or AgRP ( B–D ). Values are plotted as mean values ± SE of four individual experiments. * P

    Article Snippet: Single-stranded cDNA required for real-time quantitative RT-PCR (qRT-PCR) was synthesized using the high-capacity cDNA reverse transcription kit (Applied Biosystems) as per the manufacturer's instructions. cDNA was amplified using a qRT-PCR master mix (Platinum SYBR Green qPCR SuperMix-UDG with ROX; Invitrogen) and gene-specific primers.

    Techniques: Quantitative RT-PCR, Expressing

    In silico and experimental evidence predict the 3’UTR of PDHX is targeted by MicroRNA-27b. a Diagram illustrating the stepwise approach used to select potential miR-27b targets for experimental validation. b Preliminary luciferase assay of MCF7 cells transiently cotransfected with a PDHX luciferase vector and miR-27b mimic or control. Luciferase expression is quantitated by measuring the degree of luminescence detected by a luminometer 24 h post-transfection for each assay. c and d TaqMan qRT-PCR assessing miR-27b levels in transfected MCF7 and 4175 cells to confirm adequate delivery of RNA oligonucleotides into the cell. e Nucleotide sequences indicating the location of miR-27b binding within the PDHX 3’UTR. The sequence of the original luciferase construct and the eight base pair substitutions present in the mutant construct are shown beneath. Luciferase assays in MCF7 ( f ) and 4175 ( g ) cells, showing miR-27b targeting of the PDHX-3’UTR sequence is lost upon mutation of the seed region. h Expression of PDHX in the Staunton Cell line dataset accessed online using Oncomine. PDHX Fold Change (vs average of all cancers): − 5.602; p -value: 0.023. Under-expression Gene Rank for PDHX: 335 (in top 7%)

    Journal: Molecular Cancer

    Article Title: Suppression of PDHX by microRNA-27b deregulates cell metabolism and promotes growth in breast cancer

    doi: 10.1186/s12943-018-0851-8

    Figure Lengend Snippet: In silico and experimental evidence predict the 3’UTR of PDHX is targeted by MicroRNA-27b. a Diagram illustrating the stepwise approach used to select potential miR-27b targets for experimental validation. b Preliminary luciferase assay of MCF7 cells transiently cotransfected with a PDHX luciferase vector and miR-27b mimic or control. Luciferase expression is quantitated by measuring the degree of luminescence detected by a luminometer 24 h post-transfection for each assay. c and d TaqMan qRT-PCR assessing miR-27b levels in transfected MCF7 and 4175 cells to confirm adequate delivery of RNA oligonucleotides into the cell. e Nucleotide sequences indicating the location of miR-27b binding within the PDHX 3’UTR. The sequence of the original luciferase construct and the eight base pair substitutions present in the mutant construct are shown beneath. Luciferase assays in MCF7 ( f ) and 4175 ( g ) cells, showing miR-27b targeting of the PDHX-3’UTR sequence is lost upon mutation of the seed region. h Expression of PDHX in the Staunton Cell line dataset accessed online using Oncomine. PDHX Fold Change (vs average of all cancers): − 5.602; p -value: 0.023. Under-expression Gene Rank for PDHX: 335 (in top 7%)

    Article Snippet: The 20-μl PCR reactions included 1.33 μl of RT product, 10 μl of TaqMan Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems), and 1 μl of primer and probe mix (Applied Biosystems).

    Techniques: In Silico, Luciferase, Plasmid Preparation, Expressing, Transfection, Quantitative RT-PCR, Binding Assay, Sequencing, Construct, Mutagenesis

    PDHX mRNA and protein expression is suppressed by miR-27b in vitro. a and b Time course RT-PCR of cells transiently transfected with the indicated oligonucleotide. c and d RNA was collected from cells as above for evaluation of PDHX expression by quantitative RT-PCR using SYBR Green to probe for PDHX expression. e Cell lysates from transiently-transfected MCF7 and 4175 cells were collected and probed for PDHX protein and Vinculin loading control. ZR75 cells stably expressing elevated miR-27b or scr control, as well as 4175 cells expressing either the 27b-ZIP construct or scr control were used to replicate the experiments in transiently transfected cells. RNA was also collected from the stable cell and reverse transcribed for quantitation of PDHX expression by standard PCR followed by gel electrophoresis ( f ) as well as qPCR ( g and h ). Confirmation of the miR-27b expression status of the ZR75 ( i ) and 4175 ( j ) stable cell lines (previously generated in our lab) was completed by TaqMan qRT-PCR. The same RNA samples collected for PDHX qPCR were again used to measure the miR-27b level of these cells. k IB of PDHX protein using stable cell lysates as in panel e

    Journal: Molecular Cancer

    Article Title: Suppression of PDHX by microRNA-27b deregulates cell metabolism and promotes growth in breast cancer

    doi: 10.1186/s12943-018-0851-8

    Figure Lengend Snippet: PDHX mRNA and protein expression is suppressed by miR-27b in vitro. a and b Time course RT-PCR of cells transiently transfected with the indicated oligonucleotide. c and d RNA was collected from cells as above for evaluation of PDHX expression by quantitative RT-PCR using SYBR Green to probe for PDHX expression. e Cell lysates from transiently-transfected MCF7 and 4175 cells were collected and probed for PDHX protein and Vinculin loading control. ZR75 cells stably expressing elevated miR-27b or scr control, as well as 4175 cells expressing either the 27b-ZIP construct or scr control were used to replicate the experiments in transiently transfected cells. RNA was also collected from the stable cell and reverse transcribed for quantitation of PDHX expression by standard PCR followed by gel electrophoresis ( f ) as well as qPCR ( g and h ). Confirmation of the miR-27b expression status of the ZR75 ( i ) and 4175 ( j ) stable cell lines (previously generated in our lab) was completed by TaqMan qRT-PCR. The same RNA samples collected for PDHX qPCR were again used to measure the miR-27b level of these cells. k IB of PDHX protein using stable cell lysates as in panel e

    Article Snippet: The 20-μl PCR reactions included 1.33 μl of RT product, 10 μl of TaqMan Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems), and 1 μl of primer and probe mix (Applied Biosystems).

    Techniques: Expressing, In Vitro, Reverse Transcription Polymerase Chain Reaction, Transfection, Quantitative RT-PCR, SYBR Green Assay, Stable Transfection, Construct, Quantitation Assay, Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Real-time Polymerase Chain Reaction, Generated