qpcr reactions Search Results


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  • 99
    Thermo Fisher mirvana qrt pcr mirna detection kit
    Mirvana Qrt Pcr Mirna Detection Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 981 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore luminoct qpcr readymix
    Luminoct Qpcr Readymix, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 113 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Bio-Rad qpcr reactions
    Restriction of <t>HBV</t> by SAMHD1 is dependent upon dNTPase activity, is inhibited by phosphorylation of T592 and is rescued by addition of deoxynucleosides. ( a , b ) HepG2.2.15 cells were transfected with 1 μg of plasmids coding for wild-type or mutant FLAG-tagged SAMHD1 or with 1 μg of empty vector. Twenty-four hours after transfection, the cells were washed and cultured in serum-free medium without supplements for 48 hours. ( a ) Detection of overexpressed FLAG-SAMHD1 by Western blotting. Detection of actin was used as a loading control. ( b ) HBV DNA from the supernatant was quantified by <t>qPCR.</t> The mean ± SEM of technical triplicates from one representative experiment out of three independent experiments is represented. ( c ) HepG2 cells were transfected with 1 μg of plasmid coding for wild-type FLAG-tagged SAMHD1 or empty vector. Twenty-four hours after transfection, the cells were washed and cultured in serum-free medium without supplements to arrest the cell cycle. When indicated, 10 mM of hydroxyurea (HU) were added at the time of medium change. Quantification of dATP levels 3 days post medium change were determined by single-nucleotide incorporation assay. The mean ± SEM of technical duplicates is represented. ( d ) HepG2.2.15 cells were transfected with 1 μg of plasmid coding for wild-type FLAG-tagged SAMHD1 or with an empty vector. Twenty-four hours after transfection, the cells were washed and cultured in serum-free medium without supplements. When indicated, deoxynucleosides (dNs, 2 mM each) or solvent were added at the time of medium change. HBV DNA from the supernatant was quantified by qPCR 3 days post medium change. For each independent experiment, fold-changes to empty vector were calculated based on the median of three technical replicates. The mean ± SEM of the fold-changes of three independent experiments is represented. ( b , d ) Statistical significance was determined using a one-way ANOVA with multiple comparisons according to Dunnett (**p
    Qpcr Reactions, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 6786 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad qpcr reaction
    MethyLight duplex <t>dPCR.</t> (A) Duplex p14 dPCR assay showing data for p14_M and p14_M2 assays separately and with estimated targets for both assays combined. (B) dPCR heatmap showing distribution of p14_M (red) and p14_M2 (blue) positive chambers in a duplex reaction showing three example panels of a dPCR plate. (C) Correlation between MethyLight <t>qPCR</t> vs . duplex dPCR (estimated targets for both assays combined). All correlations were significant at p
    Qpcr Reaction, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 2028 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad real time quantitative pcr reactions
    IFN-γ treatment induced the deposition of active histone modifications throughout the HLA-DRA gene. (A) RNA levels for CIITA and HLA-DRA were measured by <t>qRT-PCR.</t> Transcript levels for A431 cells are shown before and after treatment with 500 U/ml of IFN-γ for 24 hrs. The results of three independent experiments were averaged and plotted with standard error. (B) A schematic of the HLA-DRA gene and open reading frame is illustrated with exons (black boxes) and introns (clear) indicated. The bars below the gene represent the relative position of the PCR amplicons used. (C) ChIP and <t>qPCR</t> were conducted on untreated control (clear) or 24 hrs IFN-γ treated (black) A431 cells using the indicated antisera and amplicons described in B. The data are presented as an average of the percent input value derived from three to four biological replicates and error bars represent standard error. For unmodified histone H3, the asterisk (*) indicates a Student’s t-test value of p
    Real Time Quantitative Pcr Reactions, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 675 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene qpcr reactions
    <t>qPCR</t> analysis of AtHEL (A) and <t>TaWRKY78</t> (B) transcript level in Arabidopsis leaves protoplasts transformed with empty vector (A, B) and 35S :: TaWRKY78 plasmid (A, B). AtHEL endogenous expression increases 18-fold when TaWRKY78 was provided in trans (A). High-level expression of TaWRKY78 in protoplasts transformed with 35S :: TaWRKY78 (B) indicates high transformation efficiency. Transcriptional activity of pHEL and Δ pHEL (C) and pPR4e and Δ pPR4e (D) with or without TaWRKY78 and AtWRKY20 transcription factor, respectively. Transient GUS expression driven by pHEL, pHEL plus TaWRKY78, Δ pHEL , and Δ pHEL plus TaWRKY78 (C); pPR4e, pPR4e plus AtWRKY20, Δ pPR4e , and Δ pPR4e plus AtWRKY20 (D). GUS expression increased 6-fold and 9-fold when TaWRKY78 was provided in trans to pHEL and Δ pHEL , respectively, and 11-fold and 8-fold when AtWRKY20 was provided in trans to pPR4e and Δ pPR4e , respectively. Bars represents the average of GUS-LUC activity ratios from five transformations. Error bars represent SDs.
    Qpcr Reactions, supplied by Stratagene, used in various techniques. Bioz Stars score: 93/100, based on 608 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher qpcr reactions
    <t>qPCR</t> analysis of AtHEL (A) and <t>TaWRKY78</t> (B) transcript level in Arabidopsis leaves protoplasts transformed with empty vector (A, B) and 35S :: TaWRKY78 plasmid (A, B). AtHEL endogenous expression increases 18-fold when TaWRKY78 was provided in trans (A). High-level expression of TaWRKY78 in protoplasts transformed with 35S :: TaWRKY78 (B) indicates high transformation efficiency. Transcriptional activity of pHEL and Δ pHEL (C) and pPR4e and Δ pPR4e (D) with or without TaWRKY78 and AtWRKY20 transcription factor, respectively. Transient GUS expression driven by pHEL, pHEL plus TaWRKY78, Δ pHEL , and Δ pHEL plus TaWRKY78 (C); pPR4e, pPR4e plus AtWRKY20, Δ pPR4e , and Δ pPR4e plus AtWRKY20 (D). GUS expression increased 6-fold and 9-fold when TaWRKY78 was provided in trans to pHEL and Δ pHEL , respectively, and 11-fold and 8-fold when AtWRKY20 was provided in trans to pPR4e and Δ pPR4e , respectively. Bars represents the average of GUS-LUC activity ratios from five transformations. Error bars represent SDs.
    Qpcr Reactions, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 15274 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad quantitative real time polymerase chain reaction qrt pcr
    Time course of CD200 expression in EAE. (A) Full-length Cd200 (Cd200full) and truncated Cd200 (Cd200tr) mRNAs were evaluated in spinal cords and brains by <t>qRT-PCR</t> during EAE, using Rn18s as the housekeeping gene. Bars are the means ± SEM of 4–6 animals. ∗ p
    Quantitative Real Time Polymerase Chain Reaction Qrt Pcr, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 678 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies qpcr reactions
    Mutation of a conserved 16‐mer sequence within the VSG 3′ UTR results in a drastic reduction in VSG transcript half‐life. A. Sequence of a relevant segment of the VSG117 3′ UTR with the conserved 9‐mer and 16‐mer sequences highlighted with boxes. The wild type (WT) 16‐mer sequence is shown, as well as the scrambled version present in Mutant 2 (mut 2). Relevant nucleotide numbers are shown in relation to the start of the 3′ UTR. B. Decrease in VSG <t>RNA</t> half‐life when the conserved 16‐mer in the VSG117 3′UTR is scrambled. RNA was isolated from cell lines expressing VSG117 with a wild type (WT) VSG 3′UTR ( T. brucei SM221/117 + WT 3′UTR) or a VSG 3′ UTR with a scrambled 16‐mer ( T. brucei SM221/117 + mut2 3′UTR). Cells were incubated with sinefungin to block trans‐splicing and actinomycin D to inhibit transcription, and total RNA was harvested at different time points. Transcript levels were determined by <t>qPCR.</t> Results are presented as a ratio of transcript levels obtained from single‐expresser cell lines expressing either only VSG117 or VSG221 . The mean of three independent experiments is shown with the standard deviation indicated with error bars. C. The half‐life in minutes (min) of VSG117 (red bars) or VSG221 (green bars) transcript in cell lines where ectopic VSG117 has either a wild type (WT) or mutant 2 (mut2) 3′UTR. Results are the mean of three independent experiments with the standard deviation indicated with error bars.
    Qpcr Reactions, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 532 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher vetmax plus qpcr master mix
    Mutation of a conserved 16‐mer sequence within the VSG 3′ UTR results in a drastic reduction in VSG transcript half‐life. A. Sequence of a relevant segment of the VSG117 3′ UTR with the conserved 9‐mer and 16‐mer sequences highlighted with boxes. The wild type (WT) 16‐mer sequence is shown, as well as the scrambled version present in Mutant 2 (mut 2). Relevant nucleotide numbers are shown in relation to the start of the 3′ UTR. B. Decrease in VSG <t>RNA</t> half‐life when the conserved 16‐mer in the VSG117 3′UTR is scrambled. RNA was isolated from cell lines expressing VSG117 with a wild type (WT) VSG 3′UTR ( T. brucei SM221/117 + WT 3′UTR) or a VSG 3′ UTR with a scrambled 16‐mer ( T. brucei SM221/117 + mut2 3′UTR). Cells were incubated with sinefungin to block trans‐splicing and actinomycin D to inhibit transcription, and total RNA was harvested at different time points. Transcript levels were determined by <t>qPCR.</t> Results are presented as a ratio of transcript levels obtained from single‐expresser cell lines expressing either only VSG117 or VSG221 . The mean of three independent experiments is shown with the standard deviation indicated with error bars. C. The half‐life in minutes (min) of VSG117 (red bars) or VSG221 (green bars) transcript in cell lines where ectopic VSG117 has either a wild type (WT) or mutant 2 (mut2) 3′UTR. Results are the mean of three independent experiments with the standard deviation indicated with error bars.
    Vetmax Plus Qpcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher platinum quantitative rt pcr thermoscript one step system
    Mutation of a conserved 16‐mer sequence within the VSG 3′ UTR results in a drastic reduction in VSG transcript half‐life. A. Sequence of a relevant segment of the VSG117 3′ UTR with the conserved 9‐mer and 16‐mer sequences highlighted with boxes. The wild type (WT) 16‐mer sequence is shown, as well as the scrambled version present in Mutant 2 (mut 2). Relevant nucleotide numbers are shown in relation to the start of the 3′ UTR. B. Decrease in VSG <t>RNA</t> half‐life when the conserved 16‐mer in the VSG117 3′UTR is scrambled. RNA was isolated from cell lines expressing VSG117 with a wild type (WT) VSG 3′UTR ( T. brucei SM221/117 + WT 3′UTR) or a VSG 3′ UTR with a scrambled 16‐mer ( T. brucei SM221/117 + mut2 3′UTR). Cells were incubated with sinefungin to block trans‐splicing and actinomycin D to inhibit transcription, and total RNA was harvested at different time points. Transcript levels were determined by <t>qPCR.</t> Results are presented as a ratio of transcript levels obtained from single‐expresser cell lines expressing either only VSG117 or VSG221 . The mean of three independent experiments is shown with the standard deviation indicated with error bars. C. The half‐life in minutes (min) of VSG117 (red bars) or VSG221 (green bars) transcript in cell lines where ectopic VSG117 has either a wild type (WT) or mutant 2 (mut2) 3′UTR. Results are the mean of three independent experiments with the standard deviation indicated with error bars.
    Platinum Quantitative Rt Pcr Thermoscript One Step System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 269 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad polymerase chain reaction qpcr
    ARID3B binds gene regulatory regions for genes in Wnt Signaling, Cell Death, Cell Division, and EGFR signaling. Chromatin Immunoprecipitation (ChIP) followed by <t>qPCR</t> was performed on parental and 6XHis-ARID3B Skov3IP and OVCA429 cells to detect binding of ARID3B to target genes in key pathways. All numbers are reported as relative binding compared to the corresponding input <t>DNA</t> sample. Brackets indicate that binding in ARID3B-ChIP samples is significantly higher than the corresponding background (IgG) sample (*—p-value
    Polymerase Chain Reaction Qpcr, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 298 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 7500 real time pcr system
    ARID3B binds gene regulatory regions for genes in Wnt Signaling, Cell Death, Cell Division, and EGFR signaling. Chromatin Immunoprecipitation (ChIP) followed by <t>qPCR</t> was performed on parental and 6XHis-ARID3B Skov3IP and OVCA429 cells to detect binding of ARID3B to target genes in key pathways. All numbers are reported as relative binding compared to the corresponding input <t>DNA</t> sample. Brackets indicate that binding in ARID3B-ChIP samples is significantly higher than the corresponding background (IgG) sample (*—p-value
    7500 Real Time Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 54115 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Quanta Biosciences qpcr reactions
    ARID3B binds gene regulatory regions for genes in Wnt Signaling, Cell Death, Cell Division, and EGFR signaling. Chromatin Immunoprecipitation (ChIP) followed by <t>qPCR</t> was performed on parental and 6XHis-ARID3B Skov3IP and OVCA429 cells to detect binding of ARID3B to target genes in key pathways. All numbers are reported as relative binding compared to the corresponding input <t>DNA</t> sample. Brackets indicate that binding in ARID3B-ChIP samples is significantly higher than the corresponding background (IgG) sample (*—p-value
    Qpcr Reactions, supplied by Quanta Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 291 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher qpcr reaction
    ARID3B binds gene regulatory regions for genes in Wnt Signaling, Cell Death, Cell Division, and EGFR signaling. Chromatin Immunoprecipitation (ChIP) followed by <t>qPCR</t> was performed on parental and 6XHis-ARID3B Skov3IP and OVCA429 cells to detect binding of ARID3B to target genes in key pathways. All numbers are reported as relative binding compared to the corresponding input <t>DNA</t> sample. Brackets indicate that binding in ARID3B-ChIP samples is significantly higher than the corresponding background (IgG) sample (*—p-value
    Qpcr Reaction, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5887 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher maxima probe qpcr master mix 2x
    ARID3B binds gene regulatory regions for genes in Wnt Signaling, Cell Death, Cell Division, and EGFR signaling. Chromatin Immunoprecipitation (ChIP) followed by <t>qPCR</t> was performed on parental and 6XHis-ARID3B Skov3IP and OVCA429 cells to detect binding of ARID3B to target genes in key pathways. All numbers are reported as relative binding compared to the corresponding input <t>DNA</t> sample. Brackets indicate that binding in ARID3B-ChIP samples is significantly higher than the corresponding background (IgG) sample (*—p-value
    Maxima Probe Qpcr Master Mix 2x, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 114 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quantitative real time polymerase chain reaction qrt pcr trizol reagent
    Circ-BIRC6 exerted its function by sponging miR-877-5p in HCC cells. ( A ) Predicted binding sites between circ-BIRC6 and miR-877-5p were shown. ( B and C ) Dual-luciferase reporter assay was conducted to determine luciferase activity in Huh-7 and Hep3B cells co-transfected with miR-877-5p or miR-NC and WT-circ-BIRC6 or MUT-circ-BIRC6. ( D and E ) The expression levels of circ-BIRC6 and miR-877-5p were detected through <t>qRT-PCR</t> analysis in control group or HCC cells (Huh-7 and Hep3B) transfected with si-NC, si-circ-BIRC6, pcDNA, or circ-BIRC6. ( F-I ) Huh-7 and Hep3B cells were divided into 5 groups: Control, si-NC, si-circ-BIRC6, si-circ-BIRC6 + anti-miR-NC, si-circ-BIRC6 + anti-miR-877-5p. ( F ) MiR-877-5p level was examined in transfected Huh-7 and Hep3B cells ( G ) CCK-8 assay was performed to examine cell viability. ( H ) The number of colonies was determined by colony formation assay. ( I ) Cell apoptosis was measured using the flow cytometry analysis.* P
    Quantitative Real Time Polymerase Chain Reaction Qrt Pcr Trizol Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Restriction of HBV by SAMHD1 is dependent upon dNTPase activity, is inhibited by phosphorylation of T592 and is rescued by addition of deoxynucleosides. ( a , b ) HepG2.2.15 cells were transfected with 1 μg of plasmids coding for wild-type or mutant FLAG-tagged SAMHD1 or with 1 μg of empty vector. Twenty-four hours after transfection, the cells were washed and cultured in serum-free medium without supplements for 48 hours. ( a ) Detection of overexpressed FLAG-SAMHD1 by Western blotting. Detection of actin was used as a loading control. ( b ) HBV DNA from the supernatant was quantified by qPCR. The mean ± SEM of technical triplicates from one representative experiment out of three independent experiments is represented. ( c ) HepG2 cells were transfected with 1 μg of plasmid coding for wild-type FLAG-tagged SAMHD1 or empty vector. Twenty-four hours after transfection, the cells were washed and cultured in serum-free medium without supplements to arrest the cell cycle. When indicated, 10 mM of hydroxyurea (HU) were added at the time of medium change. Quantification of dATP levels 3 days post medium change were determined by single-nucleotide incorporation assay. The mean ± SEM of technical duplicates is represented. ( d ) HepG2.2.15 cells were transfected with 1 μg of plasmid coding for wild-type FLAG-tagged SAMHD1 or with an empty vector. Twenty-four hours after transfection, the cells were washed and cultured in serum-free medium without supplements. When indicated, deoxynucleosides (dNs, 2 mM each) or solvent were added at the time of medium change. HBV DNA from the supernatant was quantified by qPCR 3 days post medium change. For each independent experiment, fold-changes to empty vector were calculated based on the median of three technical replicates. The mean ± SEM of the fold-changes of three independent experiments is represented. ( b , d ) Statistical significance was determined using a one-way ANOVA with multiple comparisons according to Dunnett (**p

    Journal: Scientific Reports

    Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle

    doi: 10.1038/srep26616

    Figure Lengend Snippet: Restriction of HBV by SAMHD1 is dependent upon dNTPase activity, is inhibited by phosphorylation of T592 and is rescued by addition of deoxynucleosides. ( a , b ) HepG2.2.15 cells were transfected with 1 μg of plasmids coding for wild-type or mutant FLAG-tagged SAMHD1 or with 1 μg of empty vector. Twenty-four hours after transfection, the cells were washed and cultured in serum-free medium without supplements for 48 hours. ( a ) Detection of overexpressed FLAG-SAMHD1 by Western blotting. Detection of actin was used as a loading control. ( b ) HBV DNA from the supernatant was quantified by qPCR. The mean ± SEM of technical triplicates from one representative experiment out of three independent experiments is represented. ( c ) HepG2 cells were transfected with 1 μg of plasmid coding for wild-type FLAG-tagged SAMHD1 or empty vector. Twenty-four hours after transfection, the cells were washed and cultured in serum-free medium without supplements to arrest the cell cycle. When indicated, 10 mM of hydroxyurea (HU) were added at the time of medium change. Quantification of dATP levels 3 days post medium change were determined by single-nucleotide incorporation assay. The mean ± SEM of technical duplicates is represented. ( d ) HepG2.2.15 cells were transfected with 1 μg of plasmid coding for wild-type FLAG-tagged SAMHD1 or with an empty vector. Twenty-four hours after transfection, the cells were washed and cultured in serum-free medium without supplements. When indicated, deoxynucleosides (dNs, 2 mM each) or solvent were added at the time of medium change. HBV DNA from the supernatant was quantified by qPCR 3 days post medium change. For each independent experiment, fold-changes to empty vector were calculated based on the median of three technical replicates. The mean ± SEM of the fold-changes of three independent experiments is represented. ( b , d ) Statistical significance was determined using a one-way ANOVA with multiple comparisons according to Dunnett (**p

    Article Snippet: For HBV total DNA, qPCR reactions were performed on undigested samples in SYBR green supermix (Bio-rad 172-5121) using HBV specific primers HBV-total-DNA-F and HBV-total-DNA-R (95 °C for 10 minutes, followed up by 95 °C for 10 seconds and 60 °C for 30 seconds for 40 cycles).

    Techniques: Activity Assay, Transfection, Mutagenesis, Plasmid Preparation, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction

    Overexpression of SAMHD1 reduces extracellular and intracellular levels of HBV DNA in HepG2.2.15 cells without affecting viral RNA. HepG2.2.15 cells were transfected with ( a ) increasing amounts or ( b – d ) 1 μg of a SAMHD1-coding plasmid. The total amount of plasmid DNA was adjusted to 1 μg by addition of empty vector. Twenty-four hours after transfection, the cells were washed with phosphate-buffered saline (PBS) and further cultured in serum-free medium without supplements. Forty-eight hours later, HBV DNA from the supernatant ( a , d ) and intracellular HBV DNA from cytoplasmic fractions ( c ) were quantified by qPCR. ( b ) The levels of intracellular HBV RNAs were determined by RT-qPCR using primer sets that amplify the indicated HBV RNAs (3.5 kb; 3.5 kb and 2.4 kb; 3.5 kb, 2.4 kb and 2.1 kb, or all four HBV RNAs). ( a ) The mean ± SEM of the technical triplicates is shown. Statistical significance was determined using a one-way ANOVA with multiple comparisons according to Dunnett (*p

    Journal: Scientific Reports

    Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle

    doi: 10.1038/srep26616

    Figure Lengend Snippet: Overexpression of SAMHD1 reduces extracellular and intracellular levels of HBV DNA in HepG2.2.15 cells without affecting viral RNA. HepG2.2.15 cells were transfected with ( a ) increasing amounts or ( b – d ) 1 μg of a SAMHD1-coding plasmid. The total amount of plasmid DNA was adjusted to 1 μg by addition of empty vector. Twenty-four hours after transfection, the cells were washed with phosphate-buffered saline (PBS) and further cultured in serum-free medium without supplements. Forty-eight hours later, HBV DNA from the supernatant ( a , d ) and intracellular HBV DNA from cytoplasmic fractions ( c ) were quantified by qPCR. ( b ) The levels of intracellular HBV RNAs were determined by RT-qPCR using primer sets that amplify the indicated HBV RNAs (3.5 kb; 3.5 kb and 2.4 kb; 3.5 kb, 2.4 kb and 2.1 kb, or all four HBV RNAs). ( a ) The mean ± SEM of the technical triplicates is shown. Statistical significance was determined using a one-way ANOVA with multiple comparisons according to Dunnett (*p

    Article Snippet: For HBV total DNA, qPCR reactions were performed on undigested samples in SYBR green supermix (Bio-rad 172-5121) using HBV specific primers HBV-total-DNA-F and HBV-total-DNA-R (95 °C for 10 minutes, followed up by 95 °C for 10 seconds and 60 °C for 30 seconds for 40 cycles).

    Techniques: Over Expression, Transfection, Plasmid Preparation, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    dATP levels are affected by HBV infection. SAMHD1 is de-phosphorylated at T592 in primary human hepatocytes, and is not affected by HBV infection. ( a ) HepG2-NTCP- control-shRNA cells were infected or not with HBV in presence of 2.5%DMSO and dATP content was measured 4 days post infection. The mean ± SEM of technical duplicates is represented. ( b , c ) Primary human hepatocytes (PHH) from one donnor were infected or not with HBV for 8 or 24 hours. ( b ) SAMHD1 phosphorylation on T592, total SAMHD1, cyclin B1 and GAPDH were detected by Western blotting. Cycling or serum starved HepG2.2.15-control-shRNA cells were used respectively as positive and negative controls for pT592-SAMHD1 and cyclin B1. A representative experiment out of two technical replicates is represented. ( c ) Levels of intracellular HBV RNAs from infected PHH used in ( b ) were determined by RT-qPCR using HBV primer set 3 (see Fig. 2b and Table 1 ; amplified HBV RNA species: 3.5 kb, 2.4 kb and 2.1 kb RNAs). B.d.: below detection. The mean ± SEM of technical triplicates is represented.

    Journal: Scientific Reports

    Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle

    doi: 10.1038/srep26616

    Figure Lengend Snippet: dATP levels are affected by HBV infection. SAMHD1 is de-phosphorylated at T592 in primary human hepatocytes, and is not affected by HBV infection. ( a ) HepG2-NTCP- control-shRNA cells were infected or not with HBV in presence of 2.5%DMSO and dATP content was measured 4 days post infection. The mean ± SEM of technical duplicates is represented. ( b , c ) Primary human hepatocytes (PHH) from one donnor were infected or not with HBV for 8 or 24 hours. ( b ) SAMHD1 phosphorylation on T592, total SAMHD1, cyclin B1 and GAPDH were detected by Western blotting. Cycling or serum starved HepG2.2.15-control-shRNA cells were used respectively as positive and negative controls for pT592-SAMHD1 and cyclin B1. A representative experiment out of two technical replicates is represented. ( c ) Levels of intracellular HBV RNAs from infected PHH used in ( b ) were determined by RT-qPCR using HBV primer set 3 (see Fig. 2b and Table 1 ; amplified HBV RNA species: 3.5 kb, 2.4 kb and 2.1 kb RNAs). B.d.: below detection. The mean ± SEM of technical triplicates is represented.

    Article Snippet: For HBV total DNA, qPCR reactions were performed on undigested samples in SYBR green supermix (Bio-rad 172-5121) using HBV specific primers HBV-total-DNA-F and HBV-total-DNA-R (95 °C for 10 minutes, followed up by 95 °C for 10 seconds and 60 °C for 30 seconds for 40 cycles).

    Techniques: Infection, shRNA, Western Blot, Quantitative RT-PCR, Amplification

    SAMHD1 restricts HBV replication in infected HepG2-NTCP cells. Detection of ( a ) SAMHD1 or ( b ) NTCP in SAMHD1 knockdown or control HepG2-NTCP cells by Western blotting. Actin or GAPDH were used as loading controls. ( c ) Equal cell growth and viability was assessed was assessed using ATPLite. Depicted are mean values (±SEM) of three biological replicates of one representative experiment out of three. ( d ) Control shRNA cells were cultured for 10 days in complete medium supplemented (+) or not (−) with 2.5% DMSO. When indicated cells were infected with HBV 48 hours after start of DMSO treatment. Phosphorylation at residue T592 in SAMHD1, as well as expression of total SAMHD1, cyclin B1 and actin, were detected by Western blotting. ( e ) Cells were cultured for 10 days in 2.5% DMSO-containing medium and infected with a single-round luciferase HIV-1 virus. Luciferase activity was detected 24 hours post infection. Depicted are mean values (±SEM) of three biological replicates of one representative experiment out of three. ( f-i ) Cells were infected with HBV inoculum, and where indicated, the entry inhibitor MyrcludexB (MyrB, 200 nM), or the reverse transcription inhibitor lamivudine (Lami, 0,5 μM) were added. Cells were cultured in medium containing 2.5% DMSO. Ten days post infection, HBV DNA from the supernatant ( f ), intracellular total HBV DNA ( g ) and cccDNA ( h ) were quantified by qPCR. ( i ) The intracellular HBV RNAs levels were determined 10 days post infection by RT-qPCR using primer sets that amplify the indicated HBV RNAs (3.5 kb; 3.5 kb and 2.4 kb; 3.5 kb, 2.4 kb and 2.1 kb, or all four HBV RNAs). ( f ) For each individual experiment, fold-changes to the untreated control shRNA were calculated based on the median of three biological replicates, each measured in three technical replicates. The mean ± SEM of the fold-changes of three independent experiments is depicted. ( g – i ) For each independent experiment, fold-changes to untreated control shRNA were calculated based on the median of three technical replicates. The mean ± SEM of the fold-changes of at least 3 independent experiments is represented. ( f – i ) Statistical significance was determined using a one-way ANOVA with multiple comparisons according to Dunnett (*p

    Journal: Scientific Reports

    Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle

    doi: 10.1038/srep26616

    Figure Lengend Snippet: SAMHD1 restricts HBV replication in infected HepG2-NTCP cells. Detection of ( a ) SAMHD1 or ( b ) NTCP in SAMHD1 knockdown or control HepG2-NTCP cells by Western blotting. Actin or GAPDH were used as loading controls. ( c ) Equal cell growth and viability was assessed was assessed using ATPLite. Depicted are mean values (±SEM) of three biological replicates of one representative experiment out of three. ( d ) Control shRNA cells were cultured for 10 days in complete medium supplemented (+) or not (−) with 2.5% DMSO. When indicated cells were infected with HBV 48 hours after start of DMSO treatment. Phosphorylation at residue T592 in SAMHD1, as well as expression of total SAMHD1, cyclin B1 and actin, were detected by Western blotting. ( e ) Cells were cultured for 10 days in 2.5% DMSO-containing medium and infected with a single-round luciferase HIV-1 virus. Luciferase activity was detected 24 hours post infection. Depicted are mean values (±SEM) of three biological replicates of one representative experiment out of three. ( f-i ) Cells were infected with HBV inoculum, and where indicated, the entry inhibitor MyrcludexB (MyrB, 200 nM), or the reverse transcription inhibitor lamivudine (Lami, 0,5 μM) were added. Cells were cultured in medium containing 2.5% DMSO. Ten days post infection, HBV DNA from the supernatant ( f ), intracellular total HBV DNA ( g ) and cccDNA ( h ) were quantified by qPCR. ( i ) The intracellular HBV RNAs levels were determined 10 days post infection by RT-qPCR using primer sets that amplify the indicated HBV RNAs (3.5 kb; 3.5 kb and 2.4 kb; 3.5 kb, 2.4 kb and 2.1 kb, or all four HBV RNAs). ( f ) For each individual experiment, fold-changes to the untreated control shRNA were calculated based on the median of three biological replicates, each measured in three technical replicates. The mean ± SEM of the fold-changes of three independent experiments is depicted. ( g – i ) For each independent experiment, fold-changes to untreated control shRNA were calculated based on the median of three technical replicates. The mean ± SEM of the fold-changes of at least 3 independent experiments is represented. ( f – i ) Statistical significance was determined using a one-way ANOVA with multiple comparisons according to Dunnett (*p

    Article Snippet: For HBV total DNA, qPCR reactions were performed on undigested samples in SYBR green supermix (Bio-rad 172-5121) using HBV specific primers HBV-total-DNA-F and HBV-total-DNA-R (95 °C for 10 minutes, followed up by 95 °C for 10 seconds and 60 °C for 30 seconds for 40 cycles).

    Techniques: Infection, Western Blot, shRNA, Cell Culture, Expressing, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Silencing of SAMHD1 increases HBV replication in resting HepG2.2.15 cells. ( a ) SAMHD1 or actin protein levels were detected in cell lysates from SAMHD1 knockdown or control HepG2.2.15 cells by Western blotting. ( b ) Phosphorylation on residue T592 of SAMHD1 and cyclin B1 expression were detected by Western blotting of lysates from control shRNA HepG2.2.15 cells cultured for 3 days in complete versus serum-free medium. ( c-e ) HepG2.2.15 cells stably expressing two different SAMHD1 shRNAs or a control shRNA were cultured in serum-free medium for 3 days. ( c ) Luciferase activity (relative luciferase units, RLU) was detected in cells 24 hours post infection with a single-round HIV-1-based lentiviral vector that encoded luciferase. ( d ) Equal cell growth and viability between different shRNA cell lines was assessed using ATPLite. ( e ) The amount of HBV DNA in the supernatant was determined by qPCR and normalized to the control shRNA. In ( c , d ), the mean ± standard error mean (SEM) of three biological replicates of one representative experiment is depicted. In ( e ), the fold-changes to negative control were calculated for each individual experiment based on the median of three biological replicates, each measured in three technical replicates. The mean ± SEM of the fold-changes of four independent experiments is depicted. Statistical significance was determined using a one-way ANOVA with multiple comparisons according to Dunnett (*p

    Journal: Scientific Reports

    Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle

    doi: 10.1038/srep26616

    Figure Lengend Snippet: Silencing of SAMHD1 increases HBV replication in resting HepG2.2.15 cells. ( a ) SAMHD1 or actin protein levels were detected in cell lysates from SAMHD1 knockdown or control HepG2.2.15 cells by Western blotting. ( b ) Phosphorylation on residue T592 of SAMHD1 and cyclin B1 expression were detected by Western blotting of lysates from control shRNA HepG2.2.15 cells cultured for 3 days in complete versus serum-free medium. ( c-e ) HepG2.2.15 cells stably expressing two different SAMHD1 shRNAs or a control shRNA were cultured in serum-free medium for 3 days. ( c ) Luciferase activity (relative luciferase units, RLU) was detected in cells 24 hours post infection with a single-round HIV-1-based lentiviral vector that encoded luciferase. ( d ) Equal cell growth and viability between different shRNA cell lines was assessed using ATPLite. ( e ) The amount of HBV DNA in the supernatant was determined by qPCR and normalized to the control shRNA. In ( c , d ), the mean ± standard error mean (SEM) of three biological replicates of one representative experiment is depicted. In ( e ), the fold-changes to negative control were calculated for each individual experiment based on the median of three biological replicates, each measured in three technical replicates. The mean ± SEM of the fold-changes of four independent experiments is depicted. Statistical significance was determined using a one-way ANOVA with multiple comparisons according to Dunnett (*p

    Article Snippet: For HBV total DNA, qPCR reactions were performed on undigested samples in SYBR green supermix (Bio-rad 172-5121) using HBV specific primers HBV-total-DNA-F and HBV-total-DNA-R (95 °C for 10 minutes, followed up by 95 °C for 10 seconds and 60 °C for 30 seconds for 40 cycles).

    Techniques: Western Blot, Expressing, shRNA, Cell Culture, Stable Transfection, Luciferase, Activity Assay, Infection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Negative Control

    Toll-like receptor 4 (TLR4) signal pathway play an important role in the production of inflammatory cytokines. The microarray result of TLR4 expression pattern was validated by real-time quantitative PCR (RT-qPCR) (A) . The promoter activity of the TLR4 gene was detected by reporter assays in NR8383 cells (B) . The inhibitory effect of TAK-242 on the TLR4 mRNA and protein expression was detected by RT-qPCR and Western Blot in NR8383 cells (C) . The transcription levels of inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), were detected by RT-qPCR in NR8383 cells (D) ; and their production levels in the culture medium were detected by enzyme-linked immune sorbent assay (E) . Data are presented as mean ± SD of three independent experimental repeats. * p

    Journal: Frontiers in Immunology

    Article Title: Hypoxia Exacerbates Inflammatory Acute Lung Injury via the Toll-Like Receptor 4 Signaling Pathway

    doi: 10.3389/fimmu.2018.01667

    Figure Lengend Snippet: Toll-like receptor 4 (TLR4) signal pathway play an important role in the production of inflammatory cytokines. The microarray result of TLR4 expression pattern was validated by real-time quantitative PCR (RT-qPCR) (A) . The promoter activity of the TLR4 gene was detected by reporter assays in NR8383 cells (B) . The inhibitory effect of TAK-242 on the TLR4 mRNA and protein expression was detected by RT-qPCR and Western Blot in NR8383 cells (C) . The transcription levels of inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), were detected by RT-qPCR in NR8383 cells (D) ; and their production levels in the culture medium were detected by enzyme-linked immune sorbent assay (E) . Data are presented as mean ± SD of three independent experimental repeats. * p

    Article Snippet: The RT-qPCR reactions were conducted using a Bio-Rad real-time PCR system with the following modified program: initial denaturation at 95°C for 2 min; followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and amplification at 72°C for 20 s. Products were verified by melting curve analysis and 1.5% agarose gel electrophoresis.

    Techniques: Microarray, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Activity Assay, Western Blot

    The production of inflammatory cytokines in the bronchi alveolar lavage fluid (BALF) of rats in four groups. The inflammatory cytokines, tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) in the BALF of rats were detected by enzyme-linked immune sorbent assay ( n = 10) (A–C) . The transcription level changes of TNF-α, IL-6, and IL-1β mRNA was determined by real-time quantitative PCR in NR8383 ( n = 3) and the total cells in BALF ( n = 5). The values of controls were normalized to 1 (D–F) . Data are presented as mean ± SD. * p

    Journal: Frontiers in Immunology

    Article Title: Hypoxia Exacerbates Inflammatory Acute Lung Injury via the Toll-Like Receptor 4 Signaling Pathway

    doi: 10.3389/fimmu.2018.01667

    Figure Lengend Snippet: The production of inflammatory cytokines in the bronchi alveolar lavage fluid (BALF) of rats in four groups. The inflammatory cytokines, tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) in the BALF of rats were detected by enzyme-linked immune sorbent assay ( n = 10) (A–C) . The transcription level changes of TNF-α, IL-6, and IL-1β mRNA was determined by real-time quantitative PCR in NR8383 ( n = 3) and the total cells in BALF ( n = 5). The values of controls were normalized to 1 (D–F) . Data are presented as mean ± SD. * p

    Article Snippet: The RT-qPCR reactions were conducted using a Bio-Rad real-time PCR system with the following modified program: initial denaturation at 95°C for 2 min; followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and amplification at 72°C for 20 s. Products were verified by melting curve analysis and 1.5% agarose gel electrophoresis.

    Techniques: Real-time Polymerase Chain Reaction

    The effect of manipulation of hypoxia-inducible factor 1 alpha (HIF-1α) on the production of inflammatory cytokine tumor necrosis factor alpha (TNF-α). We used 50 µM PX478 treatment for 20 h (A) or transfection of a small interfering RNA (siRNA) specific for HIF-1α for 48 h (C) to downregulate the HIF-1α protein level in NR8383 cells. We used 1 mM DMOG treatment for 8 h (B) or transfection of a plasmid specific for HIF-1α for 48 h (D) to upregulate the HIF-1α protein level in NR8383 cells. Then we detected TNF-α levels in the culture media by using enzyme-linked immune sorbent assay and its mRNA expression in NR8383 cells by using real-time quantitative PCR. Data are presented as mean ± SD of three independent experimental repeats. ** p

    Journal: Frontiers in Immunology

    Article Title: Hypoxia Exacerbates Inflammatory Acute Lung Injury via the Toll-Like Receptor 4 Signaling Pathway

    doi: 10.3389/fimmu.2018.01667

    Figure Lengend Snippet: The effect of manipulation of hypoxia-inducible factor 1 alpha (HIF-1α) on the production of inflammatory cytokine tumor necrosis factor alpha (TNF-α). We used 50 µM PX478 treatment for 20 h (A) or transfection of a small interfering RNA (siRNA) specific for HIF-1α for 48 h (C) to downregulate the HIF-1α protein level in NR8383 cells. We used 1 mM DMOG treatment for 8 h (B) or transfection of a plasmid specific for HIF-1α for 48 h (D) to upregulate the HIF-1α protein level in NR8383 cells. Then we detected TNF-α levels in the culture media by using enzyme-linked immune sorbent assay and its mRNA expression in NR8383 cells by using real-time quantitative PCR. Data are presented as mean ± SD of three independent experimental repeats. ** p

    Article Snippet: The RT-qPCR reactions were conducted using a Bio-Rad real-time PCR system with the following modified program: initial denaturation at 95°C for 2 min; followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and amplification at 72°C for 20 s. Products were verified by melting curve analysis and 1.5% agarose gel electrophoresis.

    Techniques: Transfection, Small Interfering RNA, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction

    Differential gene expression profiles were identified in the total cells of bronchi alveolar lavage fluid (BALF) by high-throughput microarray analysis. Before the gene chip detection, we mixed three animal samples into one pooled sample and total of three pooled samples were detected in each group. The clustering display was generated by Chip software with two-way data clustering. Each column represents an individual gene, and each row corresponds to an individual array. Gene expression values were standardized and color-coded relative to the mean (green, values less than the mean; red, values greater than the mean), the gene expression profiles of the COMB group are significant different from the other three groups (A) . We used the Scatter Plot to show the differential patterns of gene expression in the four groups (B) . We used a Venn diagram to show the distribution patterns of gene changes in lipopolysaccharides (LPS) group, HPO group, and COMB group (C) . Microarray findings of gene expression pattern were validated by using real-time quantitative PCR (RT-qPCR) (D) .

    Journal: Frontiers in Immunology

    Article Title: Hypoxia Exacerbates Inflammatory Acute Lung Injury via the Toll-Like Receptor 4 Signaling Pathway

    doi: 10.3389/fimmu.2018.01667

    Figure Lengend Snippet: Differential gene expression profiles were identified in the total cells of bronchi alveolar lavage fluid (BALF) by high-throughput microarray analysis. Before the gene chip detection, we mixed three animal samples into one pooled sample and total of three pooled samples were detected in each group. The clustering display was generated by Chip software with two-way data clustering. Each column represents an individual gene, and each row corresponds to an individual array. Gene expression values were standardized and color-coded relative to the mean (green, values less than the mean; red, values greater than the mean), the gene expression profiles of the COMB group are significant different from the other three groups (A) . We used the Scatter Plot to show the differential patterns of gene expression in the four groups (B) . We used a Venn diagram to show the distribution patterns of gene changes in lipopolysaccharides (LPS) group, HPO group, and COMB group (C) . Microarray findings of gene expression pattern were validated by using real-time quantitative PCR (RT-qPCR) (D) .

    Article Snippet: The RT-qPCR reactions were conducted using a Bio-Rad real-time PCR system with the following modified program: initial denaturation at 95°C for 2 min; followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and amplification at 72°C for 20 s. Products were verified by melting curve analysis and 1.5% agarose gel electrophoresis.

    Techniques: Expressing, High Throughput Screening Assay, Microarray, Chromatin Immunoprecipitation, Generated, Software, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    mosGCTL-3 facilitated DENV infections. ( A–B ) Silencing efficiency of mosGCTL-3 on both RNA level and protein level. 2 ug GFP or mosGCTL-3 dsRNA was inoculated into mosquitoes thorax respectively. At 3, 6 and 9 days after the treatment, the mosquitoes were sacrificed to determine silencing effect by qPCR and normalized by A. aegypti actin (A). The primers of dsRNA synthesis and qPCR detection were described in Table S3 . The mosGCTL-3 silenced or mock mosquitoes were grinded in lysis buffer and mosGCTL-3 was detected by immunoblotting (B). ( C–F ) mosGCTL-3 suppression impairs the infection of dengue serotypes. 10 M.I.D. 50 DENV-1 Hawaii strain (C), DENV-2 New Guinea C strain (D), DENV-3 Guangdong strain (E) and DENV-4 H241 strain (F) viruses were inoculated into mosGCTL-3 silenced mosquitoes by microinjection respectively. The viral load was determined at 6 days post-infection by qPCR and normalized by A. aegypti actin . The result was pooled from 3 independent experiments. ( G ) Diagram of mosGCTL-3 gene. An extra exon (Exon 1) of mosGCTL-3 , which encoded a peptide with 20 amino acid signal sequence, was identified by the 5′-RACE. ( H ) Expression and purification of mosGCTL-3 by a Drosophila expression system. mosGCTL-3 gene was cloned into pMT/BiP/V5-His-A DNA vector. The recombinant mosGCTL-3 was expressed and purified by a Cobalt-His column (Left panel). The expression was probed by anti-V5 mAb (Right panel). Mock was the concentrated supernatant of empty vector transfected Drosophila S2 cells. ( I ) Inoculation of mosGCTL-3 purified protein benefited DENV-2 infection in A. aegypti . 50 pg or 500 pg purified S2-expressed mosGCTL-3 protein was microinjected into each mosquito with 10 M.I.D. 50 DENV-2. The infected mosquitoes were sacrificed at 3 and 6 days post infection. The same amount of BSA was inoculated with DENV-2 as mocks. The viral load was determined by qPCR and normalized by A. aegypti actin . This experiment was repeated 3 times individually. One dot represented 1 mosquito and the horizontal line represented the mean value in all figures. We used the Mann-Whitney test for statistical analysis.

    Journal: PLoS Pathogens

    Article Title: Transmission-Blocking Antibodies against Mosquito C-Type Lectins for Dengue Prevention

    doi: 10.1371/journal.ppat.1003931

    Figure Lengend Snippet: mosGCTL-3 facilitated DENV infections. ( A–B ) Silencing efficiency of mosGCTL-3 on both RNA level and protein level. 2 ug GFP or mosGCTL-3 dsRNA was inoculated into mosquitoes thorax respectively. At 3, 6 and 9 days after the treatment, the mosquitoes were sacrificed to determine silencing effect by qPCR and normalized by A. aegypti actin (A). The primers of dsRNA synthesis and qPCR detection were described in Table S3 . The mosGCTL-3 silenced or mock mosquitoes were grinded in lysis buffer and mosGCTL-3 was detected by immunoblotting (B). ( C–F ) mosGCTL-3 suppression impairs the infection of dengue serotypes. 10 M.I.D. 50 DENV-1 Hawaii strain (C), DENV-2 New Guinea C strain (D), DENV-3 Guangdong strain (E) and DENV-4 H241 strain (F) viruses were inoculated into mosGCTL-3 silenced mosquitoes by microinjection respectively. The viral load was determined at 6 days post-infection by qPCR and normalized by A. aegypti actin . The result was pooled from 3 independent experiments. ( G ) Diagram of mosGCTL-3 gene. An extra exon (Exon 1) of mosGCTL-3 , which encoded a peptide with 20 amino acid signal sequence, was identified by the 5′-RACE. ( H ) Expression and purification of mosGCTL-3 by a Drosophila expression system. mosGCTL-3 gene was cloned into pMT/BiP/V5-His-A DNA vector. The recombinant mosGCTL-3 was expressed and purified by a Cobalt-His column (Left panel). The expression was probed by anti-V5 mAb (Right panel). Mock was the concentrated supernatant of empty vector transfected Drosophila S2 cells. ( I ) Inoculation of mosGCTL-3 purified protein benefited DENV-2 infection in A. aegypti . 50 pg or 500 pg purified S2-expressed mosGCTL-3 protein was microinjected into each mosquito with 10 M.I.D. 50 DENV-2. The infected mosquitoes were sacrificed at 3 and 6 days post infection. The same amount of BSA was inoculated with DENV-2 as mocks. The viral load was determined by qPCR and normalized by A. aegypti actin . This experiment was repeated 3 times individually. One dot represented 1 mosquito and the horizontal line represented the mean value in all figures. We used the Mann-Whitney test for statistical analysis.

    Article Snippet: Quantitative-Polymerase Chain Reaction (qPCR) The cDNAs of DENV genome were synthesized using a cDNA reverse transcription kit (170-8890; Bio-Rad) and quantified by qPCR with specific probes.

    Techniques: Real-time Polymerase Chain Reaction, Lysis, Infection, Sequencing, Expressing, Purification, Clone Assay, Plasmid Preparation, Recombinant, Transfection, MANN-WHITNEY

    Transmission-blocking effect of mosGCTLs antisera in the infection of low-passage DENV-2 strains. ( A–F ) Two low-passage DENV-2 strains, which were isolated from patients' sera, were employed to test transmission-blocking effect of mosGCTL antisera in A. aegypti . The pre-immune sera, mosGCTL-3 antisera and combined mosGCTLs antisera (1/9 mosGCTL antiserum each) with Vero cells-generatedDENV-2 AF2014178 strain (A to C)/JX470186 strain (D to F) and human blood were used for mosquitoes feeding. The antisera were serially diluted 100- (A and D), 1,000- (B and E) and 5,000- (C and F) fold for the investigation. The fed mosquitoes were sacrificed on 8 days post infection to determine the infectivity by qPCR. The numbers of infected mosquitoes/total mosquitoes are presented on the top of each column (Left panel of each sub-figure). The mosquito infective ratio (Right panel) indicates the ratio of DENV positive to total mosquitoes. One dot represents a mosquito. The results are pooled from 3 independent experiments.

    Journal: PLoS Pathogens

    Article Title: Transmission-Blocking Antibodies against Mosquito C-Type Lectins for Dengue Prevention

    doi: 10.1371/journal.ppat.1003931

    Figure Lengend Snippet: Transmission-blocking effect of mosGCTLs antisera in the infection of low-passage DENV-2 strains. ( A–F ) Two low-passage DENV-2 strains, which were isolated from patients' sera, were employed to test transmission-blocking effect of mosGCTL antisera in A. aegypti . The pre-immune sera, mosGCTL-3 antisera and combined mosGCTLs antisera (1/9 mosGCTL antiserum each) with Vero cells-generatedDENV-2 AF2014178 strain (A to C)/JX470186 strain (D to F) and human blood were used for mosquitoes feeding. The antisera were serially diluted 100- (A and D), 1,000- (B and E) and 5,000- (C and F) fold for the investigation. The fed mosquitoes were sacrificed on 8 days post infection to determine the infectivity by qPCR. The numbers of infected mosquitoes/total mosquitoes are presented on the top of each column (Left panel of each sub-figure). The mosquito infective ratio (Right panel) indicates the ratio of DENV positive to total mosquitoes. One dot represents a mosquito. The results are pooled from 3 independent experiments.

    Article Snippet: Quantitative-Polymerase Chain Reaction (qPCR) The cDNAs of DENV genome were synthesized using a cDNA reverse transcription kit (170-8890; Bio-Rad) and quantified by qPCR with specific probes.

    Techniques: Transmission Assay, Blocking Assay, Infection, Isolation, Real-time Polymerase Chain Reaction

    mosGCTL-3 antisera interrupted DENV-2 infection of A. aegypti . ( A ) Diagram of dengue life cycle and a transmission-blocking strategy for dengue prevention. ( B ) Inoculation of mosGCTL-3 antisera impaired DENV-2 infection of A. aegypti . The serial dilutions of mosGCTL-3 antisera or pre-immune sera with 10 M.I.D. 50 DENV-2 were inoculated into mosquitoes by microinjection. The infected mosquitoes were scarified at 3 days and 6 days post inoculation. DENV-2 was determined by qPCR and normalized by A. aegypti actin . One dot represented 1 mosquito and the horizontal line represented the mean value in all figures. The Mann-Whitney test was used for statistical analysis. The result was combined from 2 independent experiments. ( C–D ) mosGCTL-3 antisera interrupted DENV-2 infection by a blood meal. The mosGCTL-3 antisera or pre-immune sera were diluted 100-, 1,000- and 5,000-fold with fresh human blood and Vero cells-generated DENV-2. The mosquitoes were allowed to ingest the blood mixture by a membrane feeding. The fed mosquitoes were sacrificed 8 days later and DENV-2 infectivity was assessed by qPCR. The number of infected mosquitoes/total mosquitoes was presented on the top of each column (C). The mosquito infective ratio (D) was then interpreted from the Figure (C). One dot represented a mosquito. The result was pooled from 2 independent experiments.

    Journal: PLoS Pathogens

    Article Title: Transmission-Blocking Antibodies against Mosquito C-Type Lectins for Dengue Prevention

    doi: 10.1371/journal.ppat.1003931

    Figure Lengend Snippet: mosGCTL-3 antisera interrupted DENV-2 infection of A. aegypti . ( A ) Diagram of dengue life cycle and a transmission-blocking strategy for dengue prevention. ( B ) Inoculation of mosGCTL-3 antisera impaired DENV-2 infection of A. aegypti . The serial dilutions of mosGCTL-3 antisera or pre-immune sera with 10 M.I.D. 50 DENV-2 were inoculated into mosquitoes by microinjection. The infected mosquitoes were scarified at 3 days and 6 days post inoculation. DENV-2 was determined by qPCR and normalized by A. aegypti actin . One dot represented 1 mosquito and the horizontal line represented the mean value in all figures. The Mann-Whitney test was used for statistical analysis. The result was combined from 2 independent experiments. ( C–D ) mosGCTL-3 antisera interrupted DENV-2 infection by a blood meal. The mosGCTL-3 antisera or pre-immune sera were diluted 100-, 1,000- and 5,000-fold with fresh human blood and Vero cells-generated DENV-2. The mosquitoes were allowed to ingest the blood mixture by a membrane feeding. The fed mosquitoes were sacrificed 8 days later and DENV-2 infectivity was assessed by qPCR. The number of infected mosquitoes/total mosquitoes was presented on the top of each column (C). The mosquito infective ratio (D) was then interpreted from the Figure (C). One dot represented a mosquito. The result was pooled from 2 independent experiments.

    Article Snippet: Quantitative-Polymerase Chain Reaction (qPCR) The cDNAs of DENV genome were synthesized using a cDNA reverse transcription kit (170-8890; Bio-Rad) and quantified by qPCR with specific probes.

    Techniques: Infection, Transmission Assay, Blocking Assay, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Generated

    Wild-type tRNAs display strong Pol-III promoter activity and gRNA production. a Experimental strategy for testing functional gRNA production downstream of tRNAs. b Schematic diagram of molecular events occurring in cells transfected with tRNA-sgRNA constructs. c gRNA expression as measured by qPCR relative to Cas9 and U6 (ΔΔCt) ( n = 4 independent experiments, n = 3 for no promoter control; dashed line = gRNA levels for U6). Shaded area represents the 75% credible mass (Bayesian Estimation Supersedes the t test (BEST) test 17 , 18 ) for the no tRNA control. d 3′ processing ability of each wild-type tRNA tested. Efficiency represents the ratio of band intensity between the unprocessed and processed bands on a 2% agarose gel following RNA circularization and nested RT-PCR (thick lines = mean values). e Representative flow cytometry histograms of reporter levels downstream of U6 and various tRNA promoters. Values represent asinh(ECFP fluoresence /150) (thick lines = median fluorescent intensities). f Percent reporter ECFP + cells within transfected cells (thick lines = geometric mean values; n = 5 independent experiments; hollow downward triangles = points at or below the limit of detection). Source data are provided as a Source Data file

    Journal: Nature Communications

    Article Title: Decoupling tRNA promoter and processing activities enables specific Pol-II Cas9 guide RNA expression

    doi: 10.1038/s41467-019-09148-3

    Figure Lengend Snippet: Wild-type tRNAs display strong Pol-III promoter activity and gRNA production. a Experimental strategy for testing functional gRNA production downstream of tRNAs. b Schematic diagram of molecular events occurring in cells transfected with tRNA-sgRNA constructs. c gRNA expression as measured by qPCR relative to Cas9 and U6 (ΔΔCt) ( n = 4 independent experiments, n = 3 for no promoter control; dashed line = gRNA levels for U6). Shaded area represents the 75% credible mass (Bayesian Estimation Supersedes the t test (BEST) test 17 , 18 ) for the no tRNA control. d 3′ processing ability of each wild-type tRNA tested. Efficiency represents the ratio of band intensity between the unprocessed and processed bands on a 2% agarose gel following RNA circularization and nested RT-PCR (thick lines = mean values). e Representative flow cytometry histograms of reporter levels downstream of U6 and various tRNA promoters. Values represent asinh(ECFP fluoresence /150) (thick lines = median fluorescent intensities). f Percent reporter ECFP + cells within transfected cells (thick lines = geometric mean values; n = 5 independent experiments; hollow downward triangles = points at or below the limit of detection). Source data are provided as a Source Data file

    Article Snippet: Quantitative PCR (qPCR) reactions were performed on the resulting cDNA using SsoAdvanced™ Universal SYBR® Green Supermix (Biorad) on a CFX384 Touch™ Real-Time PCR Detection System (Biorad).

    Techniques: Activity Assay, Functional Assay, Transfection, Construct, Expressing, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Cytometry

    MethyLight duplex dPCR. (A) Duplex p14 dPCR assay showing data for p14_M and p14_M2 assays separately and with estimated targets for both assays combined. (B) dPCR heatmap showing distribution of p14_M (red) and p14_M2 (blue) positive chambers in a duplex reaction showing three example panels of a dPCR plate. (C) Correlation between MethyLight qPCR vs . duplex dPCR (estimated targets for both assays combined). All correlations were significant at p

    Journal: BMC Genomics

    Article Title: Quantification of epigenetic biomarkers: an evaluation of established and emerging methods for DNA methylation analysis

    doi: 10.1186/1471-2164-15-1174

    Figure Lengend Snippet: MethyLight duplex dPCR. (A) Duplex p14 dPCR assay showing data for p14_M and p14_M2 assays separately and with estimated targets for both assays combined. (B) dPCR heatmap showing distribution of p14_M (red) and p14_M2 (blue) positive chambers in a duplex reaction showing three example panels of a dPCR plate. (C) Correlation between MethyLight qPCR vs . duplex dPCR (estimated targets for both assays combined). All correlations were significant at p

    Article Snippet: [ ] report, the quantity of DNA analysed in our study by dPCR within the partitions of the IFC (0.65 ng) was lower than that analysed per qPCR reaction (5 ng). dPCR platforms with higher sample volume input and a greater number of partitions compared to the BioMark (such as the Bio-Rad Droplet Digital System and Life Technologies QuantStudio 3D Digital PCR System) may improve MethyLight- or RE-based dPCR precision.

    Techniques: Digital PCR, Real-time Polymerase Chain Reaction

    Restriction enzyme qPCR and dPCR. Correlation between expected and observed percent methylation for Methylation Dependent Restriction Enzyme (MDRE) (A,C) and Methylation Sensitive Restriction Enzyme (MSRE) (B,D) qPCR (A,B) and dPCR (C,D) analysis. Correlation performed with samples which comprise the optimal working range of the respective enzyme classes: 0-50% for MSRE (B,D) and 50-100% for MDRE (A,C) (dotted lines). Data points that were outside the viable range of the assay (

    Journal: BMC Genomics

    Article Title: Quantification of epigenetic biomarkers: an evaluation of established and emerging methods for DNA methylation analysis

    doi: 10.1186/1471-2164-15-1174

    Figure Lengend Snippet: Restriction enzyme qPCR and dPCR. Correlation between expected and observed percent methylation for Methylation Dependent Restriction Enzyme (MDRE) (A,C) and Methylation Sensitive Restriction Enzyme (MSRE) (B,D) qPCR (A,B) and dPCR (C,D) analysis. Correlation performed with samples which comprise the optimal working range of the respective enzyme classes: 0-50% for MSRE (B,D) and 50-100% for MDRE (A,C) (dotted lines). Data points that were outside the viable range of the assay (

    Article Snippet: [ ] report, the quantity of DNA analysed in our study by dPCR within the partitions of the IFC (0.65 ng) was lower than that analysed per qPCR reaction (5 ng). dPCR platforms with higher sample volume input and a greater number of partitions compared to the BioMark (such as the Bio-Rad Droplet Digital System and Life Technologies QuantStudio 3D Digital PCR System) may improve MethyLight- or RE-based dPCR precision.

    Techniques: Real-time Polymerase Chain Reaction, Digital PCR, Methylation

    MethyLight qPCR and singleplex dPCR. Correlation between expected and observed % methylation for MethyLight qPCR (A) and singleplex dPCR (B) using the p14_M assay. Error bars show ± Standard Deviation of three independent replicate measurements. (C) Correlation between MethyLight qPCR vs . singleplex dPCR. All correlations were significant at p

    Journal: BMC Genomics

    Article Title: Quantification of epigenetic biomarkers: an evaluation of established and emerging methods for DNA methylation analysis

    doi: 10.1186/1471-2164-15-1174

    Figure Lengend Snippet: MethyLight qPCR and singleplex dPCR. Correlation between expected and observed % methylation for MethyLight qPCR (A) and singleplex dPCR (B) using the p14_M assay. Error bars show ± Standard Deviation of three independent replicate measurements. (C) Correlation between MethyLight qPCR vs . singleplex dPCR. All correlations were significant at p

    Article Snippet: [ ] report, the quantity of DNA analysed in our study by dPCR within the partitions of the IFC (0.65 ng) was lower than that analysed per qPCR reaction (5 ng). dPCR platforms with higher sample volume input and a greater number of partitions compared to the BioMark (such as the Bio-Rad Droplet Digital System and Life Technologies QuantStudio 3D Digital PCR System) may improve MethyLight- or RE-based dPCR precision.

    Techniques: Real-time Polymerase Chain Reaction, Digital PCR, Methylation, Standard Deviation

    IFN-γ treatment induced the deposition of active histone modifications throughout the HLA-DRA gene. (A) RNA levels for CIITA and HLA-DRA were measured by qRT-PCR. Transcript levels for A431 cells are shown before and after treatment with 500 U/ml of IFN-γ for 24 hrs. The results of three independent experiments were averaged and plotted with standard error. (B) A schematic of the HLA-DRA gene and open reading frame is illustrated with exons (black boxes) and introns (clear) indicated. The bars below the gene represent the relative position of the PCR amplicons used. (C) ChIP and qPCR were conducted on untreated control (clear) or 24 hrs IFN-γ treated (black) A431 cells using the indicated antisera and amplicons described in B. The data are presented as an average of the percent input value derived from three to four biological replicates and error bars represent standard error. For unmodified histone H3, the asterisk (*) indicates a Student’s t-test value of p

    Journal: PLoS ONE

    Article Title: Multiple Histone Methyl and Acetyltransferase Complex Components Bind the HLA-DRA Gene

    doi: 10.1371/journal.pone.0037554

    Figure Lengend Snippet: IFN-γ treatment induced the deposition of active histone modifications throughout the HLA-DRA gene. (A) RNA levels for CIITA and HLA-DRA were measured by qRT-PCR. Transcript levels for A431 cells are shown before and after treatment with 500 U/ml of IFN-γ for 24 hrs. The results of three independent experiments were averaged and plotted with standard error. (B) A schematic of the HLA-DRA gene and open reading frame is illustrated with exons (black boxes) and introns (clear) indicated. The bars below the gene represent the relative position of the PCR amplicons used. (C) ChIP and qPCR were conducted on untreated control (clear) or 24 hrs IFN-γ treated (black) A431 cells using the indicated antisera and amplicons described in B. The data are presented as an average of the percent input value derived from three to four biological replicates and error bars represent standard error. For unmodified histone H3, the asterisk (*) indicates a Student’s t-test value of p

    Article Snippet: qPCR and Primers For all real-time quantitative PCR reactions, Bio-Rad iCycler instruments (Bio-Rad Laboratories, Inc., Hercules, CA) with an iQ optical module were used to measure the amount of SYBR incorporated amplicons.

    Techniques: Quantitative RT-PCR, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Derivative Assay

    MHC-II expressing B cells have active histone modifications distributed across the HLA-DRA gene. (A) The HLA-DRA gene region and additional PCR amplicons (−2000 and +5800) used in this and subsequent experiments are depicted. (B) Using the indicated antibodies, the distribution of unmodified H3, CIITA, and modified histones at the HLA-DRA gene was analyzed by ChIP-qPCR for the amplicons described in A in MHC-II expressing (Raji, black) and non-expressing (RJ2.2.5, CIITA-deficient, grey; SJO, RFX5-deficient, clear) B cell lines. The data are plotted as the average of the percent input values from three biological replicates and error bars represent standard error. In the unmodified histone H3 panel, an asterisk (*) indicates a Student’s t-test value of p

    Journal: PLoS ONE

    Article Title: Multiple Histone Methyl and Acetyltransferase Complex Components Bind the HLA-DRA Gene

    doi: 10.1371/journal.pone.0037554

    Figure Lengend Snippet: MHC-II expressing B cells have active histone modifications distributed across the HLA-DRA gene. (A) The HLA-DRA gene region and additional PCR amplicons (−2000 and +5800) used in this and subsequent experiments are depicted. (B) Using the indicated antibodies, the distribution of unmodified H3, CIITA, and modified histones at the HLA-DRA gene was analyzed by ChIP-qPCR for the amplicons described in A in MHC-II expressing (Raji, black) and non-expressing (RJ2.2.5, CIITA-deficient, grey; SJO, RFX5-deficient, clear) B cell lines. The data are plotted as the average of the percent input values from three biological replicates and error bars represent standard error. In the unmodified histone H3 panel, an asterisk (*) indicates a Student’s t-test value of p

    Article Snippet: qPCR and Primers For all real-time quantitative PCR reactions, Bio-Rad iCycler instruments (Bio-Rad Laboratories, Inc., Hercules, CA) with an iQ optical module were used to measure the amount of SYBR incorporated amplicons.

    Techniques: Expressing, Polymerase Chain Reaction, Modification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

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

    Journal: Cellular and Molecular Gastroenterology and Hepatology

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

    doi: 10.1016/j.jcmgh.2016.12.004

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

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

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

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

    Journal: Cellular and Molecular Gastroenterology and Hepatology

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

    doi: 10.1016/j.jcmgh.2016.12.004

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

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

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

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

    Journal: Cellular and Molecular Gastroenterology and Hepatology

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

    doi: 10.1016/j.jcmgh.2016.12.004

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

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

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

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

    Journal: Cellular and Molecular Gastroenterology and Hepatology

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

    doi: 10.1016/j.jcmgh.2016.12.004

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

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

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

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

    Journal: Cellular and Molecular Gastroenterology and Hepatology

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

    doi: 10.1016/j.jcmgh.2016.12.004

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

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

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

    NO donor (DETA NONOate) prevents NASH progression by inhibiting CYP2E1 activity and the resultant oxidative stress. (A) mRNA expression of liver α -SMA by quantitative real-time polymerase chain reaction (qRT-PCR) of DIO mice exposed to BDCM (DIO + BDCM) for 4 weeks, and DIO + BDCM mice treated with NO donor (DIO + BDCM + NO) for 4 weeks. The y -axis represents the fold of mRNA expression when normalized against the DIO-only group, n = 3. * P

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title:

    doi: 10.1124/jpet.114.218131

    Figure Lengend Snippet: NO donor (DETA NONOate) prevents NASH progression by inhibiting CYP2E1 activity and the resultant oxidative stress. (A) mRNA expression of liver α -SMA by quantitative real-time polymerase chain reaction (qRT-PCR) of DIO mice exposed to BDCM (DIO + BDCM) for 4 weeks, and DIO + BDCM mice treated with NO donor (DIO + BDCM + NO) for 4 weeks. The y -axis represents the fold of mRNA expression when normalized against the DIO-only group, n = 3. * P

    Article Snippet: We performed quantitative real-time polymerase chain reaction with gene-specific forward and reverse primers using SsoAdvanced universal SYBR Green supermix (Bio-Rad Laboratories, Hercules, CA) and a CFX96 thermal cycler (Bio-Rad Laboratories).

    Techniques: Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Mouse Assay

    NO donor (DETA NONOate) decreases oxidative stress by inhibiting CYP2E1 activity. (A) mRNA expression analysis of liver CYP2E1 by quantitative real-time polymerase chain reaction (qRT-PCR) of mice treated with DIO + BDCM (1 week) and DIO mice exposed to BDCM and treated with DETA NONOate for 1 week (DIO + BDCM + NO (1 week). The y -axis represents the fold of mRNA expression when normalized against the DIO-only group ( n = 3). * P

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title:

    doi: 10.1124/jpet.114.218131

    Figure Lengend Snippet: NO donor (DETA NONOate) decreases oxidative stress by inhibiting CYP2E1 activity. (A) mRNA expression analysis of liver CYP2E1 by quantitative real-time polymerase chain reaction (qRT-PCR) of mice treated with DIO + BDCM (1 week) and DIO mice exposed to BDCM and treated with DETA NONOate for 1 week (DIO + BDCM + NO (1 week). The y -axis represents the fold of mRNA expression when normalized against the DIO-only group ( n = 3). * P

    Article Snippet: We performed quantitative real-time polymerase chain reaction with gene-specific forward and reverse primers using SsoAdvanced universal SYBR Green supermix (Bio-Rad Laboratories, Hercules, CA) and a CFX96 thermal cycler (Bio-Rad Laboratories).

    Techniques: Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Mouse Assay

    M1 polarization depends on oxidative stress mediated by CYP2E1 activity. (A) Quantitative real-time polymerase chain reaction analysis of liver IL-1 β , IL-12, IL-23, and Dectin-1 mRNA expression of DIO, DIO + BDCM (1 week), CYP2E1 KO, and GdCl 3 -treated (macrophage-depleted) mice. The y -axis represents the mRNA fold expression when normalized against the DIO-only group ( n = 3). * P

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title:

    doi: 10.1124/jpet.114.218131

    Figure Lengend Snippet: M1 polarization depends on oxidative stress mediated by CYP2E1 activity. (A) Quantitative real-time polymerase chain reaction analysis of liver IL-1 β , IL-12, IL-23, and Dectin-1 mRNA expression of DIO, DIO + BDCM (1 week), CYP2E1 KO, and GdCl 3 -treated (macrophage-depleted) mice. The y -axis represents the mRNA fold expression when normalized against the DIO-only group ( n = 3). * P

    Article Snippet: We performed quantitative real-time polymerase chain reaction with gene-specific forward and reverse primers using SsoAdvanced universal SYBR Green supermix (Bio-Rad Laboratories, Hercules, CA) and a CFX96 thermal cycler (Bio-Rad Laboratories).

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

    Chronic exposure of BDCM leads to late M2 phenotypic shift. (A) quantitative real-time polymerase chain reaction (qRT-PCR) analysis of liver IL-4 and IL-13 mRNA expression in mice treated with DIO, DIO + BDCM (4 weeks), and GdCl 3 . The y -axis represents the fold of mRNA expression when normalized against the DIO-only group ( n = 3). * P

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title:

    doi: 10.1124/jpet.114.218131

    Figure Lengend Snippet: Chronic exposure of BDCM leads to late M2 phenotypic shift. (A) quantitative real-time polymerase chain reaction (qRT-PCR) analysis of liver IL-4 and IL-13 mRNA expression in mice treated with DIO, DIO + BDCM (4 weeks), and GdCl 3 . The y -axis represents the fold of mRNA expression when normalized against the DIO-only group ( n = 3). * P

    Article Snippet: We performed quantitative real-time polymerase chain reaction with gene-specific forward and reverse primers using SsoAdvanced universal SYBR Green supermix (Bio-Rad Laboratories, Hercules, CA) and a CFX96 thermal cycler (Bio-Rad Laboratories).

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

    NO donor DETA NONOate attenuates the CYP2E1-mediated M1 polarization bias. (A) mRNA expression analysis by quantitative real-time polymerase chain reaction (qRT-PCR) of liver IL-1 β , IL-12, IL-23, and Dectin-1 from DIO mice, DIO + BDCM (1 week) mice, and DIO mice exposed to BDCM and treated with DETA NONOate (DIO + BDCM + NO) for 1 week. The y -axis represents the fold of mRNA expression when normalized against the DIO-only group ( n = 3). * P

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title:

    doi: 10.1124/jpet.114.218131

    Figure Lengend Snippet: NO donor DETA NONOate attenuates the CYP2E1-mediated M1 polarization bias. (A) mRNA expression analysis by quantitative real-time polymerase chain reaction (qRT-PCR) of liver IL-1 β , IL-12, IL-23, and Dectin-1 from DIO mice, DIO + BDCM (1 week) mice, and DIO mice exposed to BDCM and treated with DETA NONOate (DIO + BDCM + NO) for 1 week. The y -axis represents the fold of mRNA expression when normalized against the DIO-only group ( n = 3). * P

    Article Snippet: We performed quantitative real-time polymerase chain reaction with gene-specific forward and reverse primers using SsoAdvanced universal SYBR Green supermix (Bio-Rad Laboratories, Hercules, CA) and a CFX96 thermal cycler (Bio-Rad Laboratories).

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

    qPCR analysis of AtHEL (A) and TaWRKY78 (B) transcript level in Arabidopsis leaves protoplasts transformed with empty vector (A, B) and 35S :: TaWRKY78 plasmid (A, B). AtHEL endogenous expression increases 18-fold when TaWRKY78 was provided in trans (A). High-level expression of TaWRKY78 in protoplasts transformed with 35S :: TaWRKY78 (B) indicates high transformation efficiency. Transcriptional activity of pHEL and Δ pHEL (C) and pPR4e and Δ pPR4e (D) with or without TaWRKY78 and AtWRKY20 transcription factor, respectively. Transient GUS expression driven by pHEL, pHEL plus TaWRKY78, Δ pHEL , and Δ pHEL plus TaWRKY78 (C); pPR4e, pPR4e plus AtWRKY20, Δ pPR4e , and Δ pPR4e plus AtWRKY20 (D). GUS expression increased 6-fold and 9-fold when TaWRKY78 was provided in trans to pHEL and Δ pHEL , respectively, and 11-fold and 8-fold when AtWRKY20 was provided in trans to pPR4e and Δ pPR4e , respectively. Bars represents the average of GUS-LUC activity ratios from five transformations. Error bars represent SDs.

    Journal: Journal of Experimental Botany

    Article Title: Cross activity of orthologous WRKY transcription factors in wheat and Arabidopsis

    doi: 10.1093/jxb/erq396

    Figure Lengend Snippet: qPCR analysis of AtHEL (A) and TaWRKY78 (B) transcript level in Arabidopsis leaves protoplasts transformed with empty vector (A, B) and 35S :: TaWRKY78 plasmid (A, B). AtHEL endogenous expression increases 18-fold when TaWRKY78 was provided in trans (A). High-level expression of TaWRKY78 in protoplasts transformed with 35S :: TaWRKY78 (B) indicates high transformation efficiency. Transcriptional activity of pHEL and Δ pHEL (C) and pPR4e and Δ pPR4e (D) with or without TaWRKY78 and AtWRKY20 transcription factor, respectively. Transient GUS expression driven by pHEL, pHEL plus TaWRKY78, Δ pHEL , and Δ pHEL plus TaWRKY78 (C); pPR4e, pPR4e plus AtWRKY20, Δ pPR4e , and Δ pPR4e plus AtWRKY20 (D). GUS expression increased 6-fold and 9-fold when TaWRKY78 was provided in trans to pHEL and Δ pHEL , respectively, and 11-fold and 8-fold when AtWRKY20 was provided in trans to pPR4e and Δ pPR4e , respectively. Bars represents the average of GUS-LUC activity ratios from five transformations. Error bars represent SDs.

    Article Snippet: Gene-specific primers for TaWRKY78 amplification are reported in . qPCR reactions were performed in a volume of 20 μl, containing 0.5 μl cDNA, 1 μl of gene-specific primers (10 pmol μl−1 ), 12.5 μl of SYBR Green Master Mix (Stratagene), and 0.375 μl of Rox, diluted 1:500.

    Techniques: Real-time Polymerase Chain Reaction, Transformation Assay, Plasmid Preparation, Expressing, Activity Assay

    qPCR analysis of TaWRKY78 transcript levels in wheat coleoptiles after SA (5 mM) and MeJA (5 mM) treatments, F. culmorum (Fc) infection, and wounding. Time-course expression was analysed in both control and treated coleoptiles harvested 4, 5, and 6 d after sowing. Bars represent means ±SD. Asterisks indicate statistically significant differences compare to control plants (Student's t test, P

    Journal: Journal of Experimental Botany

    Article Title: Cross activity of orthologous WRKY transcription factors in wheat and Arabidopsis

    doi: 10.1093/jxb/erq396

    Figure Lengend Snippet: qPCR analysis of TaWRKY78 transcript levels in wheat coleoptiles after SA (5 mM) and MeJA (5 mM) treatments, F. culmorum (Fc) infection, and wounding. Time-course expression was analysed in both control and treated coleoptiles harvested 4, 5, and 6 d after sowing. Bars represent means ±SD. Asterisks indicate statistically significant differences compare to control plants (Student's t test, P

    Article Snippet: Gene-specific primers for TaWRKY78 amplification are reported in . qPCR reactions were performed in a volume of 20 μl, containing 0.5 μl cDNA, 1 μl of gene-specific primers (10 pmol μl−1 ), 12.5 μl of SYBR Green Master Mix (Stratagene), and 0.375 μl of Rox, diluted 1:500.

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

    Time course of CD200 expression in EAE. (A) Full-length Cd200 (Cd200full) and truncated Cd200 (Cd200tr) mRNAs were evaluated in spinal cords and brains by qRT-PCR during EAE, using Rn18s as the housekeeping gene. Bars are the means ± SEM of 4–6 animals. ∗ p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Alterations in CD200-CD200R1 System during EAE Already Manifest at Presymptomatic Stages

    doi: 10.3389/fncel.2017.00129

    Figure Lengend Snippet: Time course of CD200 expression in EAE. (A) Full-length Cd200 (Cd200full) and truncated Cd200 (Cd200tr) mRNAs were evaluated in spinal cords and brains by qRT-PCR during EAE, using Rn18s as the housekeeping gene. Bars are the means ± SEM of 4–6 animals. ∗ p

    Article Snippet: Then, cDNA was diluted 1/30 to perform quantitative real-time polymerase chain reaction (qRT-PCR) with IQ SYBRGREEN SuperMix (Bio-Rad Laboratories, Hercules, CA, USA) as previously described ( ).

    Techniques: Expressing, Quantitative RT-PCR

    Glial activation in the CNS of MOG-EAE mice. (A) The mRNA expressions of Iba1 (microglial marker) and Gfap (astrocyte marker) were evaluated in spinal cord regions by qRT-PCR during EAE, using Rn18s as the housekeeping gene. (B) Iba1 and GFAP immunoreactivity (arrows) in CFA and MOG-EAE mice spinal cord at 14 and 21 DPI. Results from lumbar spinal cord, as representative of all spinal cord areas studied, are shown. (C) Iba1 and Gfap mRNA expressions in brain areas of CFA and MOG-EAE mice during EAE progression. (D) Immunocytochemistry showing Iba-1 and GFAP positive cells (red) in the CA1 field of the hippocampus (HP) and the temporal cortex (CX). Cells were counterstained with DAPI (blue). Bars represent the means ± SEM of 4–6 animals. ∗ p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Alterations in CD200-CD200R1 System during EAE Already Manifest at Presymptomatic Stages

    doi: 10.3389/fncel.2017.00129

    Figure Lengend Snippet: Glial activation in the CNS of MOG-EAE mice. (A) The mRNA expressions of Iba1 (microglial marker) and Gfap (astrocyte marker) were evaluated in spinal cord regions by qRT-PCR during EAE, using Rn18s as the housekeeping gene. (B) Iba1 and GFAP immunoreactivity (arrows) in CFA and MOG-EAE mice spinal cord at 14 and 21 DPI. Results from lumbar spinal cord, as representative of all spinal cord areas studied, are shown. (C) Iba1 and Gfap mRNA expressions in brain areas of CFA and MOG-EAE mice during EAE progression. (D) Immunocytochemistry showing Iba-1 and GFAP positive cells (red) in the CA1 field of the hippocampus (HP) and the temporal cortex (CX). Cells were counterstained with DAPI (blue). Bars represent the means ± SEM of 4–6 animals. ∗ p

    Article Snippet: Then, cDNA was diluted 1/30 to perform quantitative real-time polymerase chain reaction (qRT-PCR) with IQ SYBRGREEN SuperMix (Bio-Rad Laboratories, Hercules, CA, USA) as previously described ( ).

    Techniques: Activation Assay, Mouse Assay, Marker, Quantitative RT-PCR, Immunocytochemistry

    Time-course expression of pro-inflammatory genes in EAE. The mRNAs of the pro-inflammatory genes Nos2, Il1b, and Tnfa were evaluated in the spinal cord (A) and brain (B) by qRT-PCR during EAE. (C) The mRNA expression of COX2, pro- and anti-inflammatory gene, was evaluated in the spinal cord and brain by qRT-PCR. Rn18s was used as the housekeeping gene. Bars are the means ± SEM of 4–6 animals. ∗ p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Alterations in CD200-CD200R1 System during EAE Already Manifest at Presymptomatic Stages

    doi: 10.3389/fncel.2017.00129

    Figure Lengend Snippet: Time-course expression of pro-inflammatory genes in EAE. The mRNAs of the pro-inflammatory genes Nos2, Il1b, and Tnfa were evaluated in the spinal cord (A) and brain (B) by qRT-PCR during EAE. (C) The mRNA expression of COX2, pro- and anti-inflammatory gene, was evaluated in the spinal cord and brain by qRT-PCR. Rn18s was used as the housekeeping gene. Bars are the means ± SEM of 4–6 animals. ∗ p

    Article Snippet: Then, cDNA was diluted 1/30 to perform quantitative real-time polymerase chain reaction (qRT-PCR) with IQ SYBRGREEN SuperMix (Bio-Rad Laboratories, Hercules, CA, USA) as previously described ( ).

    Techniques: Expressing, Quantitative RT-PCR

    Time course of CD200R1 expression in EAE. (A) Cd200r1 mRNA was evaluated in the spinal cord and brain by qRT-PCR during EAE, using Rn18s as the housekeeping gene. Bars are the means ± SEM of 4–6 animals. ∗ p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Alterations in CD200-CD200R1 System during EAE Already Manifest at Presymptomatic Stages

    doi: 10.3389/fncel.2017.00129

    Figure Lengend Snippet: Time course of CD200R1 expression in EAE. (A) Cd200r1 mRNA was evaluated in the spinal cord and brain by qRT-PCR during EAE, using Rn18s as the housekeeping gene. Bars are the means ± SEM of 4–6 animals. ∗ p

    Article Snippet: Then, cDNA was diluted 1/30 to perform quantitative real-time polymerase chain reaction (qRT-PCR) with IQ SYBRGREEN SuperMix (Bio-Rad Laboratories, Hercules, CA, USA) as previously described ( ).

    Techniques: Expressing, Quantitative RT-PCR

    Time-course expression of anti-inflammatory genes in EAE. The mRNAs of anti-inflammatory genes Arg1, Il10, Mrc1, Socs3, and Tgfb were evaluated in the spinal cord (A) and brain (B) by qRT-PCR during EAE. Rn18s was used as the housekeeping gene. Bars are the means ± SEM of 4–6 animals. ∗ p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Alterations in CD200-CD200R1 System during EAE Already Manifest at Presymptomatic Stages

    doi: 10.3389/fncel.2017.00129

    Figure Lengend Snippet: Time-course expression of anti-inflammatory genes in EAE. The mRNAs of anti-inflammatory genes Arg1, Il10, Mrc1, Socs3, and Tgfb were evaluated in the spinal cord (A) and brain (B) by qRT-PCR during EAE. Rn18s was used as the housekeeping gene. Bars are the means ± SEM of 4–6 animals. ∗ p

    Article Snippet: Then, cDNA was diluted 1/30 to perform quantitative real-time polymerase chain reaction (qRT-PCR) with IQ SYBRGREEN SuperMix (Bio-Rad Laboratories, Hercules, CA, USA) as previously described ( ).

    Techniques: Expressing, Quantitative RT-PCR

    Mutation of a conserved 16‐mer sequence within the VSG 3′ UTR results in a drastic reduction in VSG transcript half‐life. A. Sequence of a relevant segment of the VSG117 3′ UTR with the conserved 9‐mer and 16‐mer sequences highlighted with boxes. The wild type (WT) 16‐mer sequence is shown, as well as the scrambled version present in Mutant 2 (mut 2). Relevant nucleotide numbers are shown in relation to the start of the 3′ UTR. B. Decrease in VSG RNA half‐life when the conserved 16‐mer in the VSG117 3′UTR is scrambled. RNA was isolated from cell lines expressing VSG117 with a wild type (WT) VSG 3′UTR ( T. brucei SM221/117 + WT 3′UTR) or a VSG 3′ UTR with a scrambled 16‐mer ( T. brucei SM221/117 + mut2 3′UTR). Cells were incubated with sinefungin to block trans‐splicing and actinomycin D to inhibit transcription, and total RNA was harvested at different time points. Transcript levels were determined by qPCR. Results are presented as a ratio of transcript levels obtained from single‐expresser cell lines expressing either only VSG117 or VSG221 . The mean of three independent experiments is shown with the standard deviation indicated with error bars. C. The half‐life in minutes (min) of VSG117 (red bars) or VSG221 (green bars) transcript in cell lines where ectopic VSG117 has either a wild type (WT) or mutant 2 (mut2) 3′UTR. Results are the mean of three independent experiments with the standard deviation indicated with error bars.

    Journal: Molecular Microbiology

    Article Title: The role of genomic location and flanking 3′UTR in the generation of functional levels of variant surface glycoprotein in Trypanosoma brucei

    doi: 10.1111/mmi.13838

    Figure Lengend Snippet: Mutation of a conserved 16‐mer sequence within the VSG 3′ UTR results in a drastic reduction in VSG transcript half‐life. A. Sequence of a relevant segment of the VSG117 3′ UTR with the conserved 9‐mer and 16‐mer sequences highlighted with boxes. The wild type (WT) 16‐mer sequence is shown, as well as the scrambled version present in Mutant 2 (mut 2). Relevant nucleotide numbers are shown in relation to the start of the 3′ UTR. B. Decrease in VSG RNA half‐life when the conserved 16‐mer in the VSG117 3′UTR is scrambled. RNA was isolated from cell lines expressing VSG117 with a wild type (WT) VSG 3′UTR ( T. brucei SM221/117 + WT 3′UTR) or a VSG 3′ UTR with a scrambled 16‐mer ( T. brucei SM221/117 + mut2 3′UTR). Cells were incubated with sinefungin to block trans‐splicing and actinomycin D to inhibit transcription, and total RNA was harvested at different time points. Transcript levels were determined by qPCR. Results are presented as a ratio of transcript levels obtained from single‐expresser cell lines expressing either only VSG117 or VSG221 . The mean of three independent experiments is shown with the standard deviation indicated with error bars. C. The half‐life in minutes (min) of VSG117 (red bars) or VSG221 (green bars) transcript in cell lines where ectopic VSG117 has either a wild type (WT) or mutant 2 (mut2) 3′UTR. Results are the mean of three independent experiments with the standard deviation indicated with error bars.

    Article Snippet: Synthesis of cDNA was carried out with 100 ng RNA per sample using an Omniscript RT kit (Qiagen). qPCR reactions were performed using Brilliant II SYBR low ROX master mix (Agilent) with relevant primers (Supporting Information Table S6) using an Applied Biosystems 7500 real time PCR machine. qPCR reactions were performed with 1 µl cDNA diluted ten‐fold with the exception of the mRNA decay assays where the cDNA was diluted 100‐fold.

    Techniques: Mutagenesis, Sequencing, Isolation, Expressing, Incubation, Blocking Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    Expression of ectopic VSG117 leads to significant reduction in levels of VSG221 mRNA but no significant attenuation of transcription at the active VSG221 telomere. A. Schematic of the VSG221 ES telomere indicating the relative positions of a single copy VSG pseudogene (ψ), 70 bp repeats, the VSG221 co‐transposed region (CTR) and VSG221 . The ES promoter is indicated with a flag and telomere repeats with horizontal arrows. The schematic is not to scale. B. Quantification of RNA transcript levels corresponding to the VSG pseudogene, CTR or VSG221 in cells where ectopic VSG117 with a VSG221 3′UTR was inserted into the active VSG221 ES or an rDNA spacer using qPCR. Transcript levels were normalised against actin, and data are presented as arbitrary units (2 –ΔCt ). The ‘single‐expresser’ SM221pur (221+) and SMΔ221/117 (117+) cell lines, as well as the HNI(VO2) cell line (V02+) with an active VSGV02 ES are included as controls. As expected, there was significant reduction in levels of VSG221 transcript on expression of ectopic VSG117 (* P

    Journal: Molecular Microbiology

    Article Title: The role of genomic location and flanking 3′UTR in the generation of functional levels of variant surface glycoprotein in Trypanosoma brucei

    doi: 10.1111/mmi.13838

    Figure Lengend Snippet: Expression of ectopic VSG117 leads to significant reduction in levels of VSG221 mRNA but no significant attenuation of transcription at the active VSG221 telomere. A. Schematic of the VSG221 ES telomere indicating the relative positions of a single copy VSG pseudogene (ψ), 70 bp repeats, the VSG221 co‐transposed region (CTR) and VSG221 . The ES promoter is indicated with a flag and telomere repeats with horizontal arrows. The schematic is not to scale. B. Quantification of RNA transcript levels corresponding to the VSG pseudogene, CTR or VSG221 in cells where ectopic VSG117 with a VSG221 3′UTR was inserted into the active VSG221 ES or an rDNA spacer using qPCR. Transcript levels were normalised against actin, and data are presented as arbitrary units (2 –ΔCt ). The ‘single‐expresser’ SM221pur (221+) and SMΔ221/117 (117+) cell lines, as well as the HNI(VO2) cell line (V02+) with an active VSGV02 ES are included as controls. As expected, there was significant reduction in levels of VSG221 transcript on expression of ectopic VSG117 (* P

    Article Snippet: Synthesis of cDNA was carried out with 100 ng RNA per sample using an Omniscript RT kit (Qiagen). qPCR reactions were performed using Brilliant II SYBR low ROX master mix (Agilent) with relevant primers (Supporting Information Table S6) using an Applied Biosystems 7500 real time PCR machine. qPCR reactions were performed with 1 µl cDNA diluted ten‐fold with the exception of the mRNA decay assays where the cDNA was diluted 100‐fold.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    The conserved 16‐mer sequence within the VSG 3′UTR is essential for high levels of VSG expression. A. Schematic of the cell line used for the VSG 3′UTR studies. Constructs containing VSG117 flanked downstream by either wild type (WT) or mutant VSG117 3′ UTRs were inserted into the active VSG221 expression site (ES) of the bloodstream form T. brucei 221VB1.1 cell line. The selectable marker was a puromycin (Pur) resistance gene. VSG221 RNAi can be induced in this cell line, which allows one to establish if ectopic VSG117 can compensate for lack of VSG221 transcript. Promoters are indicated with flags and transcription with arrows. B. Levels of VSG117 and VSG221 transcript as determined using qPCR. RNA was isolated from cell lines expressing ectopic VSG117 flanked downstream by either a wild type VSG117 UTR, or a VSG117 UTR with mutations in conserved features. The VSG 3′UTR mutant numbers (1–6) are described in Fig. 5 C. RNA from the SM221pur (221+) or SMΔ221/117 (117+) ‘single‐expresser’ strains is analysed as a control. Values normalised against actin are shown in arbitrary units (2 ‐ΔCt ), with the amount of VSG221 (red bars) or VSG117 (green bars) transcript shown as the mean of three independent experiments with the standard deviation indicated with error bars. The only significant reduction in VSG117 transcript compared with wild type (WT) was in mutants m2 and m3 containing scrambled 16‐mer sequences (** P

    Journal: Molecular Microbiology

    Article Title: The role of genomic location and flanking 3′UTR in the generation of functional levels of variant surface glycoprotein in Trypanosoma brucei

    doi: 10.1111/mmi.13838

    Figure Lengend Snippet: The conserved 16‐mer sequence within the VSG 3′UTR is essential for high levels of VSG expression. A. Schematic of the cell line used for the VSG 3′UTR studies. Constructs containing VSG117 flanked downstream by either wild type (WT) or mutant VSG117 3′ UTRs were inserted into the active VSG221 expression site (ES) of the bloodstream form T. brucei 221VB1.1 cell line. The selectable marker was a puromycin (Pur) resistance gene. VSG221 RNAi can be induced in this cell line, which allows one to establish if ectopic VSG117 can compensate for lack of VSG221 transcript. Promoters are indicated with flags and transcription with arrows. B. Levels of VSG117 and VSG221 transcript as determined using qPCR. RNA was isolated from cell lines expressing ectopic VSG117 flanked downstream by either a wild type VSG117 UTR, or a VSG117 UTR with mutations in conserved features. The VSG 3′UTR mutant numbers (1–6) are described in Fig. 5 C. RNA from the SM221pur (221+) or SMΔ221/117 (117+) ‘single‐expresser’ strains is analysed as a control. Values normalised against actin are shown in arbitrary units (2 ‐ΔCt ), with the amount of VSG221 (red bars) or VSG117 (green bars) transcript shown as the mean of three independent experiments with the standard deviation indicated with error bars. The only significant reduction in VSG117 transcript compared with wild type (WT) was in mutants m2 and m3 containing scrambled 16‐mer sequences (** P

    Article Snippet: Synthesis of cDNA was carried out with 100 ng RNA per sample using an Omniscript RT kit (Qiagen). qPCR reactions were performed using Brilliant II SYBR low ROX master mix (Agilent) with relevant primers (Supporting Information Table S6) using an Applied Biosystems 7500 real time PCR machine. qPCR reactions were performed with 1 µl cDNA diluted ten‐fold with the exception of the mRNA decay assays where the cDNA was diluted 100‐fold.

    Techniques: Sequencing, Expressing, Construct, Mutagenesis, Marker, Real-time Polymerase Chain Reaction, Isolation, Standard Deviation

    ARID3B binds gene regulatory regions for genes in Wnt Signaling, Cell Death, Cell Division, and EGFR signaling. Chromatin Immunoprecipitation (ChIP) followed by qPCR was performed on parental and 6XHis-ARID3B Skov3IP and OVCA429 cells to detect binding of ARID3B to target genes in key pathways. All numbers are reported as relative binding compared to the corresponding input DNA sample. Brackets indicate that binding in ARID3B-ChIP samples is significantly higher than the corresponding background (IgG) sample (*—p-value

    Journal: PLoS ONE

    Article Title: ARID3B Directly Regulates Ovarian Cancer Promoting Genes

    doi: 10.1371/journal.pone.0131961

    Figure Lengend Snippet: ARID3B binds gene regulatory regions for genes in Wnt Signaling, Cell Death, Cell Division, and EGFR signaling. Chromatin Immunoprecipitation (ChIP) followed by qPCR was performed on parental and 6XHis-ARID3B Skov3IP and OVCA429 cells to detect binding of ARID3B to target genes in key pathways. All numbers are reported as relative binding compared to the corresponding input DNA sample. Brackets indicate that binding in ARID3B-ChIP samples is significantly higher than the corresponding background (IgG) sample (*—p-value

    Article Snippet: ChIP DNA samples were analyzed with quantitative polymerase chain reaction (qPCR), using Sso Fast EvaGreen Supermix (Bio-Rad, Hercules, CA).

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay

    Circ-BIRC6 exerted its function by sponging miR-877-5p in HCC cells. ( A ) Predicted binding sites between circ-BIRC6 and miR-877-5p were shown. ( B and C ) Dual-luciferase reporter assay was conducted to determine luciferase activity in Huh-7 and Hep3B cells co-transfected with miR-877-5p or miR-NC and WT-circ-BIRC6 or MUT-circ-BIRC6. ( D and E ) The expression levels of circ-BIRC6 and miR-877-5p were detected through qRT-PCR analysis in control group or HCC cells (Huh-7 and Hep3B) transfected with si-NC, si-circ-BIRC6, pcDNA, or circ-BIRC6. ( F-I ) Huh-7 and Hep3B cells were divided into 5 groups: Control, si-NC, si-circ-BIRC6, si-circ-BIRC6 + anti-miR-NC, si-circ-BIRC6 + anti-miR-877-5p. ( F ) MiR-877-5p level was examined in transfected Huh-7 and Hep3B cells ( G ) CCK-8 assay was performed to examine cell viability. ( H ) The number of colonies was determined by colony formation assay. ( I ) Cell apoptosis was measured using the flow cytometry analysis.* P

    Journal: OncoTargets and therapy

    Article Title: Paclitaxel Suppresses Hepatocellular Carcinoma Tumorigenesis Through Regulating Circ-BIRC6/miR-877-5p/YWHAZ Axis

    doi: 10.2147/OTT.S261700

    Figure Lengend Snippet: Circ-BIRC6 exerted its function by sponging miR-877-5p in HCC cells. ( A ) Predicted binding sites between circ-BIRC6 and miR-877-5p were shown. ( B and C ) Dual-luciferase reporter assay was conducted to determine luciferase activity in Huh-7 and Hep3B cells co-transfected with miR-877-5p or miR-NC and WT-circ-BIRC6 or MUT-circ-BIRC6. ( D and E ) The expression levels of circ-BIRC6 and miR-877-5p were detected through qRT-PCR analysis in control group or HCC cells (Huh-7 and Hep3B) transfected with si-NC, si-circ-BIRC6, pcDNA, or circ-BIRC6. ( F-I ) Huh-7 and Hep3B cells were divided into 5 groups: Control, si-NC, si-circ-BIRC6, si-circ-BIRC6 + anti-miR-NC, si-circ-BIRC6 + anti-miR-877-5p. ( F ) MiR-877-5p level was examined in transfected Huh-7 and Hep3B cells ( G ) CCK-8 assay was performed to examine cell viability. ( H ) The number of colonies was determined by colony formation assay. ( I ) Cell apoptosis was measured using the flow cytometry analysis.* P

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Trizol reagent (Invitrogen) was utilized to gain total RNA from tissue samples and cells.

    Techniques: Binding Assay, Luciferase, Reporter Assay, Activity Assay, Transfection, Expressing, Quantitative RT-PCR, CCK-8 Assay, Colony Assay, Flow Cytometry

    Combination of circ-BIRC6 interference and paclitaxel treatment inhibited tumor growth in vivo. Xenograft model was established by injection Hep3B cells stably transfected with sh-NC or sh-circ-BIRC6. These mice were treated with PBS or paclitaxel. ( A and B ) Tumor volume and weight were detected. ( C ) Relative expression of circ-BIRC6 was analyzed using the qRT-PCR analysis in resected tumor tissues. * P

    Journal: OncoTargets and therapy

    Article Title: Paclitaxel Suppresses Hepatocellular Carcinoma Tumorigenesis Through Regulating Circ-BIRC6/miR-877-5p/YWHAZ Axis

    doi: 10.2147/OTT.S261700

    Figure Lengend Snippet: Combination of circ-BIRC6 interference and paclitaxel treatment inhibited tumor growth in vivo. Xenograft model was established by injection Hep3B cells stably transfected with sh-NC or sh-circ-BIRC6. These mice were treated with PBS or paclitaxel. ( A and B ) Tumor volume and weight were detected. ( C ) Relative expression of circ-BIRC6 was analyzed using the qRT-PCR analysis in resected tumor tissues. * P

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Trizol reagent (Invitrogen) was utilized to gain total RNA from tissue samples and cells.

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

    Paclitaxel exerted its function through regulating circ-BIRC6 in HCC cells. ( A ) The expression of circ-BIRC6 determined by qRT-PCR in Huh-7 and Hep3B cells treated with different concentrations of paclitaxel (2 nM, 1020 nM). ( B–F ) Huh-7 and Hep3B cells were divided into 4 groups: Control, paclitaxel, paclitaxel + pcDNA, paclitaxel + circ-BIRC6. ( B ) Circ-BIRC6 abundance was tested by qRT-PCR in treated Huh-7 and Hep3B cells. ( C and D ) CCK-8 assay was conducted to evaluate cell viability in treated Huh-7 and Hep3B cells. ( E ) The number of colonies was measured by colony formation assay in treated Huh-7 and Hep3B cells. ( F ) Cell apoptosis was determined using flow cytometry analysis in treated Huh-7 and Hep3B cells. * P

    Journal: OncoTargets and therapy

    Article Title: Paclitaxel Suppresses Hepatocellular Carcinoma Tumorigenesis Through Regulating Circ-BIRC6/miR-877-5p/YWHAZ Axis

    doi: 10.2147/OTT.S261700

    Figure Lengend Snippet: Paclitaxel exerted its function through regulating circ-BIRC6 in HCC cells. ( A ) The expression of circ-BIRC6 determined by qRT-PCR in Huh-7 and Hep3B cells treated with different concentrations of paclitaxel (2 nM, 1020 nM). ( B–F ) Huh-7 and Hep3B cells were divided into 4 groups: Control, paclitaxel, paclitaxel + pcDNA, paclitaxel + circ-BIRC6. ( B ) Circ-BIRC6 abundance was tested by qRT-PCR in treated Huh-7 and Hep3B cells. ( C and D ) CCK-8 assay was conducted to evaluate cell viability in treated Huh-7 and Hep3B cells. ( E ) The number of colonies was measured by colony formation assay in treated Huh-7 and Hep3B cells. ( F ) Cell apoptosis was determined using flow cytometry analysis in treated Huh-7 and Hep3B cells. * P

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Trizol reagent (Invitrogen) was utilized to gain total RNA from tissue samples and cells.

    Techniques: Expressing, Quantitative RT-PCR, CCK-8 Assay, Colony Assay, Flow Cytometry

    MiR-877-5p directly targeted YWHAZ in HCC cells. ( A ) The putative binding sites between miR-877-5p and YWHAZ were predicted by starBase v2.0. ( B and C ) Relative luciferase activity was assessed in Huh-7 and Hep3B cells co-transfected with YWHAZ 3ʹUTR-WT or YWHAZ 3ʹUTR-MUT and miR-877-5p or miR-NC. ( D and E ) The mRNA and protein expression of YWHAZ were determined in normal tissues and HCC tissues by qRT-PCR and Western blot analyses, respectively. ( F ) YWHAZ protein level was evaluated by Western blot assay in THLE-2 cells and HCC cells (Huh-7 and Hep3B). ( G ) The association between miR-877-5p abundance and YWHAZ mRNA level was measured in HCC tissues. ( H ) The expression of miR-877-5p was determined by qRT-PCR analyse in control group or HCC cells (Huh-7 and Hep3B) transfected with miR-NC, miR-877-5p, anti-miR-NC, or anti-miR-877-5p. ( I ) YWHAZ protein level was detected by Western blot analyses in control group or HCC cells (Huh-7 and Hep3B) transfected with miR-NC, miR-877-5p, anti-miR-NC, or anti-miR-877-5p. * P

    Journal: OncoTargets and therapy

    Article Title: Paclitaxel Suppresses Hepatocellular Carcinoma Tumorigenesis Through Regulating Circ-BIRC6/miR-877-5p/YWHAZ Axis

    doi: 10.2147/OTT.S261700

    Figure Lengend Snippet: MiR-877-5p directly targeted YWHAZ in HCC cells. ( A ) The putative binding sites between miR-877-5p and YWHAZ were predicted by starBase v2.0. ( B and C ) Relative luciferase activity was assessed in Huh-7 and Hep3B cells co-transfected with YWHAZ 3ʹUTR-WT or YWHAZ 3ʹUTR-MUT and miR-877-5p or miR-NC. ( D and E ) The mRNA and protein expression of YWHAZ were determined in normal tissues and HCC tissues by qRT-PCR and Western blot analyses, respectively. ( F ) YWHAZ protein level was evaluated by Western blot assay in THLE-2 cells and HCC cells (Huh-7 and Hep3B). ( G ) The association between miR-877-5p abundance and YWHAZ mRNA level was measured in HCC tissues. ( H ) The expression of miR-877-5p was determined by qRT-PCR analyse in control group or HCC cells (Huh-7 and Hep3B) transfected with miR-NC, miR-877-5p, anti-miR-NC, or anti-miR-877-5p. ( I ) YWHAZ protein level was detected by Western blot analyses in control group or HCC cells (Huh-7 and Hep3B) transfected with miR-NC, miR-877-5p, anti-miR-NC, or anti-miR-877-5p. * P

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Trizol reagent (Invitrogen) was utilized to gain total RNA from tissue samples and cells.

    Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Expressing, Quantitative RT-PCR, Western Blot

    Circ-BIRC6 was upregulated and miR-877-5p was downregulated in HCC. ( A ) The expression of circ-BIRC6 was detected by qRT-PCR in normal tissues, and HCC tissues. ( B ) Circ-BIRC6 level was detected by qRT-PCR human normal liver cells (THLE-2), and HCC cells (Huh-7 and Hep3B). ( C ) The level of miR-877-5p was measured by qRT-PCR in normal tissues and HCC tissues. ( D ) MiR-877-5p level was measured by qRT-PCR in THLE-2 cells, and HCC cells (Huh-7 and Hep3B). ( E ) The correlation between circ-BIRC6 and miR-877-5p was analyzed in HCC tissues. * P

    Journal: OncoTargets and therapy

    Article Title: Paclitaxel Suppresses Hepatocellular Carcinoma Tumorigenesis Through Regulating Circ-BIRC6/miR-877-5p/YWHAZ Axis

    doi: 10.2147/OTT.S261700

    Figure Lengend Snippet: Circ-BIRC6 was upregulated and miR-877-5p was downregulated in HCC. ( A ) The expression of circ-BIRC6 was detected by qRT-PCR in normal tissues, and HCC tissues. ( B ) Circ-BIRC6 level was detected by qRT-PCR human normal liver cells (THLE-2), and HCC cells (Huh-7 and Hep3B). ( C ) The level of miR-877-5p was measured by qRT-PCR in normal tissues and HCC tissues. ( D ) MiR-877-5p level was measured by qRT-PCR in THLE-2 cells, and HCC cells (Huh-7 and Hep3B). ( E ) The correlation between circ-BIRC6 and miR-877-5p was analyzed in HCC tissues. * P

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Trizol reagent (Invitrogen) was utilized to gain total RNA from tissue samples and cells.

    Techniques: Expressing, Quantitative RT-PCR

    Paclitaxel exerted its effect through modulating miR-877-5p in HCC cells. ( A ) The expression of miR-877-5p was evaluated by qRT-PCR in Huh-7 and Hep3B cells treated with various concentrations of paclitaxel (2 nM, 1020 nM). ( B–F ) Huh-7 and Hep3B cells were divided into 4 groups: Control, paclitaxel, paclitaxel + anti-miR-NC, paclitaxel + anti-miR-877-5p. ( B ) The abundance of miR-877-5p was calculated by qRT-PCR analysis. ( C and D ) Cell viability was assessed by CCK-8 assay. ( E ) Colony formation ability was determined using colony formation assay. ( F ) Cell apoptosis was examined using flow cytometry analysis. * P

    Journal: OncoTargets and therapy

    Article Title: Paclitaxel Suppresses Hepatocellular Carcinoma Tumorigenesis Through Regulating Circ-BIRC6/miR-877-5p/YWHAZ Axis

    doi: 10.2147/OTT.S261700

    Figure Lengend Snippet: Paclitaxel exerted its effect through modulating miR-877-5p in HCC cells. ( A ) The expression of miR-877-5p was evaluated by qRT-PCR in Huh-7 and Hep3B cells treated with various concentrations of paclitaxel (2 nM, 1020 nM). ( B–F ) Huh-7 and Hep3B cells were divided into 4 groups: Control, paclitaxel, paclitaxel + anti-miR-NC, paclitaxel + anti-miR-877-5p. ( B ) The abundance of miR-877-5p was calculated by qRT-PCR analysis. ( C and D ) Cell viability was assessed by CCK-8 assay. ( E ) Colony formation ability was determined using colony formation assay. ( F ) Cell apoptosis was examined using flow cytometry analysis. * P

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Trizol reagent (Invitrogen) was utilized to gain total RNA from tissue samples and cells.

    Techniques: Expressing, Quantitative RT-PCR, CCK-8 Assay, Colony Assay, Flow Cytometry