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  • 99
    Thermo Fisher reverse transcription quantitative polymerase chain reaction rt pcr trizol
    Reverse Transcription Quantitative Polymerase Chain Reaction Rt Pcr Trizol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore quantitative real time polymerase chain reaction qrt pcr total rna
    Quantitative Real Time Polymerase Chain Reaction Qrt Pcr Total Rna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa quantitative polymerase chain reaction pcr total rna
    Effects of wogonin on IL-4-induced eotaxin-3 expression in human bronchial epithelial cells. (A) BEAS-2B cells transfected with eotaxin-3 promoter reporter were treated with the indicated concentrations of wogonin for 4 h. Luciferase activity was measured after 24 h of IL-4 stimulation. (B) BEAS-2B cells were pretreated with wogonin for 4 h and then stimulated with IL-4 for another 8 h. Levels of eotaxin-3 mRNA was analyzed by quantitative <t>RT-PCR.</t> (C) BEAS-2B cells were pretreated with wogonin for 4 h and then stimulated with IL-4 for 8 or 24 h. The protein levels of eotaxin-3 in culture supernatants were determined by ELISA. (D) Culture conditions were identical to those in (C). Total <t>RNA</t> was isolated and levels of eotaxin-3 mRNA was analyzed by RT-PCR. Data shown are the mean ± SD of three separate experiments. * p
    Quantitative Polymerase Chain Reaction Pcr Total Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche quantitative polymerase chain reaction pcr total rna
    MOV10 contributes to the regulation of INK4a in primary fibroblasts. ( a ) FDF cells were infected with lentiviruses encoding a control shRNA (Ctrl) and two independent shRNAs against MOV10 and CBX7 respectively (sh1 and sh2). The knockdown efficiency and the effect on p16 INK4a were assessed by immunoblotting with antibodies against MOV10, CBX7 and p16 INK4a . GAPDH was used as a loading control. ( b ) Effects of MOV10 and CBX7 shRNAs on INK4a and ARF <t>RNA</t> levels as determined by <t>qRT-PCR.</t> Error bars, s.d.; n =3. ( c ) Phase contrast photographs showing the enlarged and flattened appearance of cells expressing the MOV10 and CBX7 shRNAs. ( d ) Following knockdown of MOV10 (as in panel a ), cell lysates were immunoblotted for p53 and p21 CIP1 as indicated. GAPDH was used as a loading control.
    Quantitative Polymerase Chain Reaction Pcr Total Rna, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Molecular Research Center inc quantitative polymerase chain reaction pcr total rna
    Comparing the expression of glucocorticoid-regulated genes in inguinal fat of CRH-Tg and wild type mice. Total <t>RNA</t> of inguinal fat of wild type and CRH-Tg mice were isolated and converted to cDNA. Real-time <t>PCR</t> was then performed to monitor the expression of genes indicated. Data shows fold-induction of gene expression (CRH-Tg/wild type) from 6–8 mice (*, p
    Quantitative Polymerase Chain Reaction Pcr Total Rna, supplied by Molecular Research Center inc, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    OriGene quantitative polymerase chain reaction q pcr total rna
    Comparing the expression of glucocorticoid-regulated genes in inguinal fat of CRH-Tg and wild type mice. Total <t>RNA</t> of inguinal fat of wild type and CRH-Tg mice were isolated and converted to cDNA. Real-time <t>PCR</t> was then performed to monitor the expression of genes indicated. Data shows fold-induction of gene expression (CRH-Tg/wild type) from 6–8 mice (*, p
    Quantitative Polymerase Chain Reaction Q Pcr Total Rna, supplied by OriGene, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bioteke Corporation reverse transcription quantitative polymerase chain reaction pcr total rna
    Comparing the expression of glucocorticoid-regulated genes in inguinal fat of CRH-Tg and wild type mice. Total <t>RNA</t> of inguinal fat of wild type and CRH-Tg mice were isolated and converted to cDNA. Real-time <t>PCR</t> was then performed to monitor the expression of genes indicated. Data shows fold-induction of gene expression (CRH-Tg/wild type) from 6–8 mice (*, p
    Reverse Transcription Quantitative Polymerase Chain Reaction Pcr Total Rna, supplied by Bioteke Corporation, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher real time fluorescence quantitative polymerase chain reaction rt pcr total rna extraction kits
    Comparing the expression of glucocorticoid-regulated genes in inguinal fat of CRH-Tg and wild type mice. Total <t>RNA</t> of inguinal fat of wild type and CRH-Tg mice were isolated and converted to cDNA. Real-time <t>PCR</t> was then performed to monitor the expression of genes indicated. Data shows fold-induction of gene expression (CRH-Tg/wild type) from 6–8 mice (*, p
    Real Time Fluorescence Quantitative Polymerase Chain Reaction Rt Pcr Total Rna Extraction Kits, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MACHEREY NAGEL real time quantitative polymerase chain reaction pcr total rna
    Comparing the expression of glucocorticoid-regulated genes in inguinal fat of CRH-Tg and wild type mice. Total <t>RNA</t> of inguinal fat of wild type and CRH-Tg mice were isolated and converted to cDNA. Real-time <t>PCR</t> was then performed to monitor the expression of genes indicated. Data shows fold-induction of gene expression (CRH-Tg/wild type) from 6–8 mice (*, p
    Real Time Quantitative Polymerase Chain Reaction Pcr Total Rna, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sangon Biotech real time quantitative polymerase chain reaction rt pcr total rna
    Comparing the expression of glucocorticoid-regulated genes in inguinal fat of CRH-Tg and wild type mice. Total <t>RNA</t> of inguinal fat of wild type and CRH-Tg mice were isolated and converted to cDNA. Real-time <t>PCR</t> was then performed to monitor the expression of genes indicated. Data shows fold-induction of gene expression (CRH-Tg/wild type) from 6–8 mice (*, p
    Real Time Quantitative Polymerase Chain Reaction Rt Pcr Total Rna, supplied by Sangon Biotech, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa quantitative real time polymerase chain reaction pcr total rna
    RANKL enhances Snail and Twist expression and induces changes in the morphology of cells. (A) Analysis of cell morphology after cell treatment of with 100 ng/mL RANKL. RANKL induces changes in the epithelial morphology of 4T1, MCF-7, and NMuMG cells (×40 magnification). (B-D) Total <t>RNA</t> was extracted, and the mRNA expression levels of vimentin, E-cadherin, N-cadherin, Snail, Slug, and Twist were determined by real-time <t>PCR.</t> The results are expressed as treated over control ratio after correction to GAPDH mRNA levels. The results are representative of 5 independent experiments. *p
    Quantitative Real Time Polymerase Chain Reaction Pcr Total Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co reverse transcription quantitative polymerase chain reaction rt pcr total rna
    RANKL enhances Snail and Twist expression and induces changes in the morphology of cells. (A) Analysis of cell morphology after cell treatment of with 100 ng/mL RANKL. RANKL induces changes in the epithelial morphology of 4T1, MCF-7, and NMuMG cells (×40 magnification). (B-D) Total <t>RNA</t> was extracted, and the mRNA expression levels of vimentin, E-cadherin, N-cadherin, Snail, Slug, and Twist were determined by real-time <t>PCR.</t> The results are expressed as treated over control ratio after correction to GAPDH mRNA levels. The results are representative of 5 independent experiments. *p
    Reverse Transcription Quantitative Polymerase Chain Reaction Rt Pcr Total Rna, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 94/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa quantitative pcr qpcr total rna
    RANKL enhances Snail and Twist expression and induces changes in the morphology of cells. (A) Analysis of cell morphology after cell treatment of with 100 ng/mL RANKL. RANKL induces changes in the epithelial morphology of 4T1, MCF-7, and NMuMG cells (×40 magnification). (B-D) Total <t>RNA</t> was extracted, and the mRNA expression levels of vimentin, E-cadherin, N-cadherin, Snail, Slug, and Twist were determined by real-time <t>PCR.</t> The results are expressed as treated over control ratio after correction to GAPDH mRNA levels. The results are representative of 5 independent experiments. *p
    Quantitative Pcr Qpcr Total Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene quantitative pcr qpcr total rna
    Knockdown of endogenous nuclear IL-33 expression in primary human endothelial cells using two independent <t>RNA</t> silencing strategies. ( a,b ) Nuclear expression of endogenous IL-33 in confluent monolayers of primary human endothelial cells. IL-33 protein (red) was detected by indirect immunofluorescence staining with anti-IL-33 mAbs Nessy1 ( a ) and 305B ( b ). Nuclei were counterstained with DAPI (blue). ( c ) Knockdown of endogenous nuclear IL-33 expression. IL-33 expression was analyzed by indirect immunofluorescence with Nessy1 mAb 48 h after transfection of a pool of IL-33 siRNAs (siIL-33-Sm ) or control siRNA ( siCTL-Sm ). ( d–g ) Validation of the two independent RNA silencing strategies. Expression of IL-33 was analyzed at the protein level by western blot ( d,f ) and RNA level by <t>qPCR</t> ( e,g ), 72 h after the second siRNA transfection. Cells were treated with distinct pools of IL-33 siRNAs , siIL-33-Sm ( d,e ) or siIL-33-Mi ( f,g ), and corresponding controls, siCTL-Sm ( d,e ) and siCTL-Mi ( f,g ). RRL IL-33, human IL-33 protein produced by in vitro translation in rabbit reticulocyte lysates ( d,f ). For the qPCR experiments, relative mRNA levels were calculated by normalizing the signals to those of actin. Results are shown as means with s.d. from three separate datapoints. ( h,i ). Knockdown of endogenous nuclear IL-33 does not affect NFkB protein expression. Normalized protein quantities deduced from all peptides intensity values (LFQ intensities) are shown for NFkB p65 (RELA), NFkB p105 (NFKB1) and NFkB p100 (NFKB2). Endothelial cells were treated with siIL-33-Sm ( h ) or siIL-33-Mi ( i ), and corresponding controls. Results are shown as means with s.d. from three biological replicate experiments.
    Quantitative Pcr Qpcr Total Rna, supplied by Stratagene, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad quantitative pcr qpcr total rna
    Edited CCNI peptide level is correlated with its biological function. Correlation of CCNI peptide levels with CCNI and ADAR mRNA expression as well as Ted10 activation and Ted10 mediated tumour target killing. a Correlation between the number of edited CCNI peptide copies per cell determined by the AbsQuant ® method and mRNA editing levels determined by targeted <t>RNA-seq</t> for CCNI R75G . The scatterplot ( n = 8) includes the regression curve (red line) as well as the 95% confidence interval (grey band) and 95% prediction interval (dashed lines). b CCNI-R75G is edited by ADAR1 . HEK 293 stably expressing CCNI wildtype gene was transfected with empty vector or expression vectors of ADAR1 or ADAR2 . CCNI editing was measured by <t>RT-PCR</t> and followed by sequencing. The double peaks indicate nucleotide A to G conversion and the height of peaks reflect the level of editing. c ELISPOT assay showing IFNγ production by Ted10 incubated with peptide-pulsed or CCNI -transfected 293-A2 cells. d CTL killing assay showing that over-expression of edited CCNI gene increases the sensitivity of Ted10 mediated 293-A2 target killing ( n = 3), summarised as mean ± s.e.m. per titration. e IFNγ ELISPOT assay showing recognition of endogenous CCNI-ED antigen by Ted10. Mel-2391, mel-2400 and mel-2661 expressing both edited CCNI mRNA and HLA-A*02:01 are highly reactive to Ted10. Mel-2559, which was derived from the same patient as mel-2400 but does not have detectible edited CCNI mRNA, only reacted at background levels to Ted10. Mel-2357 and mel-2686, which express edited CCNI mRNA but do not express HLA-A*02:01, have no response to Ted10. f, Ted10 mediated target killing following incubation with mel-2400 and mel-2559 measured by caspase-3-based CTL killing assay (summarised as mean ± s.e.m. of the three triplicates)
    Quantitative Pcr Qpcr Total Rna, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies quantitative pcr qpcr total rna
    Edited CCNI peptide level is correlated with its biological function. Correlation of CCNI peptide levels with CCNI and ADAR mRNA expression as well as Ted10 activation and Ted10 mediated tumour target killing. a Correlation between the number of edited CCNI peptide copies per cell determined by the AbsQuant ® method and mRNA editing levels determined by targeted <t>RNA-seq</t> for CCNI R75G . The scatterplot ( n = 8) includes the regression curve (red line) as well as the 95% confidence interval (grey band) and 95% prediction interval (dashed lines). b CCNI-R75G is edited by ADAR1 . HEK 293 stably expressing CCNI wildtype gene was transfected with empty vector or expression vectors of ADAR1 or ADAR2 . CCNI editing was measured by <t>RT-PCR</t> and followed by sequencing. The double peaks indicate nucleotide A to G conversion and the height of peaks reflect the level of editing. c ELISPOT assay showing IFNγ production by Ted10 incubated with peptide-pulsed or CCNI -transfected 293-A2 cells. d CTL killing assay showing that over-expression of edited CCNI gene increases the sensitivity of Ted10 mediated 293-A2 target killing ( n = 3), summarised as mean ± s.e.m. per titration. e IFNγ ELISPOT assay showing recognition of endogenous CCNI-ED antigen by Ted10. Mel-2391, mel-2400 and mel-2661 expressing both edited CCNI mRNA and HLA-A*02:01 are highly reactive to Ted10. Mel-2559, which was derived from the same patient as mel-2400 but does not have detectible edited CCNI mRNA, only reacted at background levels to Ted10. Mel-2357 and mel-2686, which express edited CCNI mRNA but do not express HLA-A*02:01, have no response to Ted10. f, Ted10 mediated target killing following incubation with mel-2400 and mel-2559 measured by caspase-3-based CTL killing assay (summarised as mean ± s.e.m. of the three triplicates)
    Quantitative Pcr Qpcr Total Rna, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PEQLAB quantitative real time polymerase chain reaction pcr total rna
    Edited CCNI peptide level is correlated with its biological function. Correlation of CCNI peptide levels with CCNI and ADAR mRNA expression as well as Ted10 activation and Ted10 mediated tumour target killing. a Correlation between the number of edited CCNI peptide copies per cell determined by the AbsQuant ® method and mRNA editing levels determined by targeted <t>RNA-seq</t> for CCNI R75G . The scatterplot ( n = 8) includes the regression curve (red line) as well as the 95% confidence interval (grey band) and 95% prediction interval (dashed lines). b CCNI-R75G is edited by ADAR1 . HEK 293 stably expressing CCNI wildtype gene was transfected with empty vector or expression vectors of ADAR1 or ADAR2 . CCNI editing was measured by <t>RT-PCR</t> and followed by sequencing. The double peaks indicate nucleotide A to G conversion and the height of peaks reflect the level of editing. c ELISPOT assay showing IFNγ production by Ted10 incubated with peptide-pulsed or CCNI -transfected 293-A2 cells. d CTL killing assay showing that over-expression of edited CCNI gene increases the sensitivity of Ted10 mediated 293-A2 target killing ( n = 3), summarised as mean ± s.e.m. per titration. e IFNγ ELISPOT assay showing recognition of endogenous CCNI-ED antigen by Ted10. Mel-2391, mel-2400 and mel-2661 expressing both edited CCNI mRNA and HLA-A*02:01 are highly reactive to Ted10. Mel-2559, which was derived from the same patient as mel-2400 but does not have detectible edited CCNI mRNA, only reacted at background levels to Ted10. Mel-2357 and mel-2686, which express edited CCNI mRNA but do not express HLA-A*02:01, have no response to Ted10. f, Ted10 mediated target killing following incubation with mel-2400 and mel-2559 measured by caspase-3-based CTL killing assay (summarised as mean ± s.e.m. of the three triplicates)
    Quantitative Real Time Polymerase Chain Reaction Pcr Total Rna, supplied by PEQLAB, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa quantitative reverse transcription polymerase chain reaction rt pcr total rna
    Edited CCNI peptide level is correlated with its biological function. Correlation of CCNI peptide levels with CCNI and ADAR mRNA expression as well as Ted10 activation and Ted10 mediated tumour target killing. a Correlation between the number of edited CCNI peptide copies per cell determined by the AbsQuant ® method and mRNA editing levels determined by targeted <t>RNA-seq</t> for CCNI R75G . The scatterplot ( n = 8) includes the regression curve (red line) as well as the 95% confidence interval (grey band) and 95% prediction interval (dashed lines). b CCNI-R75G is edited by ADAR1 . HEK 293 stably expressing CCNI wildtype gene was transfected with empty vector or expression vectors of ADAR1 or ADAR2 . CCNI editing was measured by <t>RT-PCR</t> and followed by sequencing. The double peaks indicate nucleotide A to G conversion and the height of peaks reflect the level of editing. c ELISPOT assay showing IFNγ production by Ted10 incubated with peptide-pulsed or CCNI -transfected 293-A2 cells. d CTL killing assay showing that over-expression of edited CCNI gene increases the sensitivity of Ted10 mediated 293-A2 target killing ( n = 3), summarised as mean ± s.e.m. per titration. e IFNγ ELISPOT assay showing recognition of endogenous CCNI-ED antigen by Ted10. Mel-2391, mel-2400 and mel-2661 expressing both edited CCNI mRNA and HLA-A*02:01 are highly reactive to Ted10. Mel-2559, which was derived from the same patient as mel-2400 but does not have detectible edited CCNI mRNA, only reacted at background levels to Ted10. Mel-2357 and mel-2686, which express edited CCNI mRNA but do not express HLA-A*02:01, have no response to Ted10. f, Ted10 mediated target killing following incubation with mel-2400 and mel-2559 measured by caspase-3-based CTL killing assay (summarised as mean ± s.e.m. of the three triplicates)
    Quantitative Reverse Transcription Polymerase Chain Reaction Rt Pcr Total Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MACHEREY NAGEL quantitative polymerase chain reaction
    Edited CCNI peptide level is correlated with its biological function. Correlation of CCNI peptide levels with CCNI and ADAR mRNA expression as well as Ted10 activation and Ted10 mediated tumour target killing. a Correlation between the number of edited CCNI peptide copies per cell determined by the AbsQuant ® method and mRNA editing levels determined by targeted <t>RNA-seq</t> for CCNI R75G . The scatterplot ( n = 8) includes the regression curve (red line) as well as the 95% confidence interval (grey band) and 95% prediction interval (dashed lines). b CCNI-R75G is edited by ADAR1 . HEK 293 stably expressing CCNI wildtype gene was transfected with empty vector or expression vectors of ADAR1 or ADAR2 . CCNI editing was measured by <t>RT-PCR</t> and followed by sequencing. The double peaks indicate nucleotide A to G conversion and the height of peaks reflect the level of editing. c ELISPOT assay showing IFNγ production by Ted10 incubated with peptide-pulsed or CCNI -transfected 293-A2 cells. d CTL killing assay showing that over-expression of edited CCNI gene increases the sensitivity of Ted10 mediated 293-A2 target killing ( n = 3), summarised as mean ± s.e.m. per titration. e IFNγ ELISPOT assay showing recognition of endogenous CCNI-ED antigen by Ted10. Mel-2391, mel-2400 and mel-2661 expressing both edited CCNI mRNA and HLA-A*02:01 are highly reactive to Ted10. Mel-2559, which was derived from the same patient as mel-2400 but does not have detectible edited CCNI mRNA, only reacted at background levels to Ted10. Mel-2357 and mel-2686, which express edited CCNI mRNA but do not express HLA-A*02:01, have no response to Ted10. f, Ted10 mediated target killing following incubation with mel-2400 and mel-2559 measured by caspase-3-based CTL killing assay (summarised as mean ± s.e.m. of the three triplicates)
    Quantitative Polymerase Chain Reaction, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bioteke Corporation quantitative real time polymerase chain reaction pcr total rna
    Edited CCNI peptide level is correlated with its biological function. Correlation of CCNI peptide levels with CCNI and ADAR mRNA expression as well as Ted10 activation and Ted10 mediated tumour target killing. a Correlation between the number of edited CCNI peptide copies per cell determined by the AbsQuant ® method and mRNA editing levels determined by targeted <t>RNA-seq</t> for CCNI R75G . The scatterplot ( n = 8) includes the regression curve (red line) as well as the 95% confidence interval (grey band) and 95% prediction interval (dashed lines). b CCNI-R75G is edited by ADAR1 . HEK 293 stably expressing CCNI wildtype gene was transfected with empty vector or expression vectors of ADAR1 or ADAR2 . CCNI editing was measured by <t>RT-PCR</t> and followed by sequencing. The double peaks indicate nucleotide A to G conversion and the height of peaks reflect the level of editing. c ELISPOT assay showing IFNγ production by Ted10 incubated with peptide-pulsed or CCNI -transfected 293-A2 cells. d CTL killing assay showing that over-expression of edited CCNI gene increases the sensitivity of Ted10 mediated 293-A2 target killing ( n = 3), summarised as mean ± s.e.m. per titration. e IFNγ ELISPOT assay showing recognition of endogenous CCNI-ED antigen by Ted10. Mel-2391, mel-2400 and mel-2661 expressing both edited CCNI mRNA and HLA-A*02:01 are highly reactive to Ted10. Mel-2559, which was derived from the same patient as mel-2400 but does not have detectible edited CCNI mRNA, only reacted at background levels to Ted10. Mel-2357 and mel-2686, which express edited CCNI mRNA but do not express HLA-A*02:01, have no response to Ted10. f, Ted10 mediated target killing following incubation with mel-2400 and mel-2559 measured by caspase-3-based CTL killing assay (summarised as mean ± s.e.m. of the three triplicates)
    Quantitative Real Time Polymerase Chain Reaction Pcr Total Rna, supplied by Bioteke Corporation, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa quantitative pcr total rnas
    Edited CCNI peptide level is correlated with its biological function. Correlation of CCNI peptide levels with CCNI and ADAR mRNA expression as well as Ted10 activation and Ted10 mediated tumour target killing. a Correlation between the number of edited CCNI peptide copies per cell determined by the AbsQuant ® method and mRNA editing levels determined by targeted <t>RNA-seq</t> for CCNI R75G . The scatterplot ( n = 8) includes the regression curve (red line) as well as the 95% confidence interval (grey band) and 95% prediction interval (dashed lines). b CCNI-R75G is edited by ADAR1 . HEK 293 stably expressing CCNI wildtype gene was transfected with empty vector or expression vectors of ADAR1 or ADAR2 . CCNI editing was measured by <t>RT-PCR</t> and followed by sequencing. The double peaks indicate nucleotide A to G conversion and the height of peaks reflect the level of editing. c ELISPOT assay showing IFNγ production by Ted10 incubated with peptide-pulsed or CCNI -transfected 293-A2 cells. d CTL killing assay showing that over-expression of edited CCNI gene increases the sensitivity of Ted10 mediated 293-A2 target killing ( n = 3), summarised as mean ± s.e.m. per titration. e IFNγ ELISPOT assay showing recognition of endogenous CCNI-ED antigen by Ted10. Mel-2391, mel-2400 and mel-2661 expressing both edited CCNI mRNA and HLA-A*02:01 are highly reactive to Ted10. Mel-2559, which was derived from the same patient as mel-2400 but does not have detectible edited CCNI mRNA, only reacted at background levels to Ted10. Mel-2357 and mel-2686, which express edited CCNI mRNA but do not express HLA-A*02:01, have no response to Ted10. f, Ted10 mediated target killing following incubation with mel-2400 and mel-2559 measured by caspase-3-based CTL killing assay (summarised as mean ± s.e.m. of the three triplicates)
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    The protective effect of up-regulating ATP8A1 was related to apoptosis-related proteins. ( A ) The mRNA levels of Fas, Fasl, Bax, Bcl-2, and caspase-3 in each group were measured by <t>qRT-PCR.</t> ( B ) These apoptosis-related proteins expression levels were measured by Western blot. * P
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    The protective effect of up-regulating ATP8A1 was related to apoptosis-related proteins. ( A ) The mRNA levels of Fas, Fasl, Bax, Bcl-2, and caspase-3 in each group were measured by <t>qRT-PCR.</t> ( B ) These apoptosis-related proteins expression levels were measured by Western blot. * P
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    The protective effect of up-regulating ATP8A1 was related to apoptosis-related proteins. ( A ) The mRNA levels of Fas, Fasl, Bax, Bcl-2, and caspase-3 in each group were measured by <t>qRT-PCR.</t> ( B ) These apoptosis-related proteins expression levels were measured by Western blot. * P
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    Depletion of lincRNAs increases MAF mRNA levels in HEK293 cells. HEK293 cells were reverse-transfected with a control siRNA, 5 nM MAFTRR siRNA (A–C) or 5 nM LINC01229 siRNA (D–F) , and quantitative <t>PCR</t> was performed on <t>RNA</t> collected at 48 h post-transfection. (A) MAFTRR RNA transcript levels. (B) MAF transcript levels. (C) LINC01229 RNA transcript levels. (D) LINC01229 RNA transcript levels. (E) MAF RNA transcript levels. (F) MAFTRR transcript levels. Data represent four biological replicates; statistical significance was determined using unpaired t -test with one tail. Error bars denote standard error of the mean and asterisks indicate significance: ∗ p
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    tiangen biotech co quantitative pcr total rna
    Endogenous MAVS is degraded in the absence of its N-terminally truncated isoforms. ( a ) HEK293T WT, Mavs-(M2L) , Mavs-(M2 6L) , Mavs-(M2-5L) and Mavs-(M2-6L) cells were subjected to subcellular fractionation to obtain P5 fractions. Various MAVS isoforms were detected by immunoblotting with anti-MAVS-(C), anti-MAVS-(M) and anti-MAVS-(N) antibodies. See also Supplementary Fig. 4a . The original full blot can be found in Supplementary Fig. 13a . ( b ) Whole cell lysates of HEK293T WT, Mavs-(M2L) , Mavs-(M2 6L) , Mavs-(M2-5L) and Mavs-(M2-6L) cells was obtained to determine endogenous MAVS protein level by immunoblotting. Tubulin was immunoblotted as a loading control. <t>RT-PCR</t> was performed to measure mRNA level of various Mavs gene. GAPDH was analysed as an internal control. ( c – e ) HEK293T WT, Mavs-(M2L) , Mavs-(M2 6L) , Mavs-(M2-5L) and Mavs-(M2-6L) cells were infected with VSV (MOI=1). Twelve hours post infection, <t>RNA</t> was extracted and qPCR was performed to measure IFN induction ( c ). VSV titres were quantitated by plaque assay ( d ). Fluorescent images were taken to examine VSV proliferation eight hours after infection ( e ). Scale bar represents 10 micrometres. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student’s t -test. ** P
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    Endogenous MAVS is degraded in the absence of its N-terminally truncated isoforms. ( a ) HEK293T WT, Mavs-(M2L) , Mavs-(M2 6L) , Mavs-(M2-5L) and Mavs-(M2-6L) cells were subjected to subcellular fractionation to obtain P5 fractions. Various MAVS isoforms were detected by immunoblotting with anti-MAVS-(C), anti-MAVS-(M) and anti-MAVS-(N) antibodies. See also Supplementary Fig. 4a . The original full blot can be found in Supplementary Fig. 13a . ( b ) Whole cell lysates of HEK293T WT, Mavs-(M2L) , Mavs-(M2 6L) , Mavs-(M2-5L) and Mavs-(M2-6L) cells was obtained to determine endogenous MAVS protein level by immunoblotting. Tubulin was immunoblotted as a loading control. <t>RT-PCR</t> was performed to measure mRNA level of various Mavs gene. GAPDH was analysed as an internal control. ( c – e ) HEK293T WT, Mavs-(M2L) , Mavs-(M2 6L) , Mavs-(M2-5L) and Mavs-(M2-6L) cells were infected with VSV (MOI=1). Twelve hours post infection, <t>RNA</t> was extracted and qPCR was performed to measure IFN induction ( c ). VSV titres were quantitated by plaque assay ( d ). Fluorescent images were taken to examine VSV proliferation eight hours after infection ( e ). Scale bar represents 10 micrometres. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student’s t -test. ** P
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    Endogenous MAVS is degraded in the absence of its N-terminally truncated isoforms. ( a ) HEK293T WT, Mavs-(M2L) , Mavs-(M2 6L) , Mavs-(M2-5L) and Mavs-(M2-6L) cells were subjected to subcellular fractionation to obtain P5 fractions. Various MAVS isoforms were detected by immunoblotting with anti-MAVS-(C), anti-MAVS-(M) and anti-MAVS-(N) antibodies. See also Supplementary Fig. 4a . The original full blot can be found in Supplementary Fig. 13a . ( b ) Whole cell lysates of HEK293T WT, Mavs-(M2L) , Mavs-(M2 6L) , Mavs-(M2-5L) and Mavs-(M2-6L) cells was obtained to determine endogenous MAVS protein level by immunoblotting. Tubulin was immunoblotted as a loading control. <t>RT-PCR</t> was performed to measure mRNA level of various Mavs gene. GAPDH was analysed as an internal control. ( c – e ) HEK293T WT, Mavs-(M2L) , Mavs-(M2 6L) , Mavs-(M2-5L) and Mavs-(M2-6L) cells were infected with VSV (MOI=1). Twelve hours post infection, <t>RNA</t> was extracted and qPCR was performed to measure IFN induction ( c ). VSV titres were quantitated by plaque assay ( d ). Fluorescent images were taken to examine VSV proliferation eight hours after infection ( e ). Scale bar represents 10 micrometres. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student’s t -test. ** P
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    Endogenous MAVS is degraded in the absence of its N-terminally truncated isoforms. ( a ) HEK293T WT, Mavs-(M2L) , Mavs-(M2 6L) , Mavs-(M2-5L) and Mavs-(M2-6L) cells were subjected to subcellular fractionation to obtain P5 fractions. Various MAVS isoforms were detected by immunoblotting with anti-MAVS-(C), anti-MAVS-(M) and anti-MAVS-(N) antibodies. See also Supplementary Fig. 4a . The original full blot can be found in Supplementary Fig. 13a . ( b ) Whole cell lysates of HEK293T WT, Mavs-(M2L) , Mavs-(M2 6L) , Mavs-(M2-5L) and Mavs-(M2-6L) cells was obtained to determine endogenous MAVS protein level by immunoblotting. Tubulin was immunoblotted as a loading control. <t>RT-PCR</t> was performed to measure mRNA level of various Mavs gene. GAPDH was analysed as an internal control. ( c – e ) HEK293T WT, Mavs-(M2L) , Mavs-(M2 6L) , Mavs-(M2-5L) and Mavs-(M2-6L) cells were infected with VSV (MOI=1). Twelve hours post infection, <t>RNA</t> was extracted and qPCR was performed to measure IFN induction ( c ). VSV titres were quantitated by plaque assay ( d ). Fluorescent images were taken to examine VSV proliferation eight hours after infection ( e ). Scale bar represents 10 micrometres. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student’s t -test. ** P
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    Low-dose irradiation induces apoptosis of fibroblast-like synoviocytes (FLS) and slightly impacts on the inflammatory FLS phenotype. Inflammatory FLS obtained from h TNF- α tg mice were analyzed 96 h after irradiation with various doses of X-rays. Cell numbers (A) of living cells were counted using a Neubauer chamber and cell death was determined via flow cytometry analysis after staining with AxV-FITC/PI. Vital cells were defined as AxV − /PI − , apoptotic cells as AxV + /PI − (B) , and necrotic ones as AxV + /PI + (C) . Quantification of secreted IL-6 (D) , hTNF-α (E) , CXCL1 (F) , and TGF-β (G) was performed 96 h after irradiation in supernatants of FLS cultures via enzyme-linked immunosorbent assays. Total <t>RNA</t> levels were isolated using phenol–chloroform extraction. Gene expression was analyzed using SYBR Green quantitative <t>PCR</t> analysis (H–M) . Depicted is joint data obtained of five h TNF- α tg-FLS cell lines, examined in four independent experiments, each performed at least in triplicates. Data are presented as mean ± SEM, tested for normal distribution, and analyzed by students t test (A–G) or two-tailed Mann–Whitney U test (H–M) in comparison to mock-treated (w/o) controls at 96 h after irradiation (* p
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    Image Search Results


    Effects of wogonin on IL-4-induced eotaxin-3 expression in human bronchial epithelial cells. (A) BEAS-2B cells transfected with eotaxin-3 promoter reporter were treated with the indicated concentrations of wogonin for 4 h. Luciferase activity was measured after 24 h of IL-4 stimulation. (B) BEAS-2B cells were pretreated with wogonin for 4 h and then stimulated with IL-4 for another 8 h. Levels of eotaxin-3 mRNA was analyzed by quantitative RT-PCR. (C) BEAS-2B cells were pretreated with wogonin for 4 h and then stimulated with IL-4 for 8 or 24 h. The protein levels of eotaxin-3 in culture supernatants were determined by ELISA. (D) Culture conditions were identical to those in (C). Total RNA was isolated and levels of eotaxin-3 mRNA was analyzed by RT-PCR. Data shown are the mean ± SD of three separate experiments. * p

    Journal: Journal of Clinical Biochemistry and Nutrition

    Article Title: Wogonin, a plant flavone from Scutellariae radix, attenuated ovalbumin-induced airway inflammation in mouse model of asthma via the suppression of IL-4/STAT6 signaling

    doi: 10.3164/jcbn.15-45

    Figure Lengend Snippet: Effects of wogonin on IL-4-induced eotaxin-3 expression in human bronchial epithelial cells. (A) BEAS-2B cells transfected with eotaxin-3 promoter reporter were treated with the indicated concentrations of wogonin for 4 h. Luciferase activity was measured after 24 h of IL-4 stimulation. (B) BEAS-2B cells were pretreated with wogonin for 4 h and then stimulated with IL-4 for another 8 h. Levels of eotaxin-3 mRNA was analyzed by quantitative RT-PCR. (C) BEAS-2B cells were pretreated with wogonin for 4 h and then stimulated with IL-4 for 8 or 24 h. The protein levels of eotaxin-3 in culture supernatants were determined by ELISA. (D) Culture conditions were identical to those in (C). Total RNA was isolated and levels of eotaxin-3 mRNA was analyzed by RT-PCR. Data shown are the mean ± SD of three separate experiments. * p

    Article Snippet: Reverse Transcription and quantitative PCR Total RNA was isolated from cells using RNAiso Plus reagent (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s protocol.

    Techniques: Expressing, Transfection, Luciferase, Activity Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

    Isolation and cultivation of DPCs and BMSCs from GFP-transgenic rats, and expression of spinal cord injury repair-associated cytokines. (A-G) Isolation and cultivation of DPCs and BMSCs from GFP-transgenic rats. (A) Direct GFP-fluorescence and (B) phase-contrast images demonstrated the morphological characteristics of primary DPCs. (C and D) DPCs at passage 6. (E) Direct GFP-fluorescence and (F) phase-contrast images revealed the morphological characteristics of BMSCs. (G and H) BMSCs at passage 6. (I) Semi-quantitative RT-PCR analysis was performed to confirm the expression of NTFs, angiogenic factors and inflammation-associated cytokines in DPCs. (J) RT-qPCR analysis further revealed relative expression levels of NTFs, angiogenic factors and inflammation-associated cytokines in DPCs. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Rat vibrissa dermal papilla cells promote healing of spinal cord injury following transplantation

    doi: 10.3892/etm.2018.5916

    Figure Lengend Snippet: Isolation and cultivation of DPCs and BMSCs from GFP-transgenic rats, and expression of spinal cord injury repair-associated cytokines. (A-G) Isolation and cultivation of DPCs and BMSCs from GFP-transgenic rats. (A) Direct GFP-fluorescence and (B) phase-contrast images demonstrated the morphological characteristics of primary DPCs. (C and D) DPCs at passage 6. (E) Direct GFP-fluorescence and (F) phase-contrast images revealed the morphological characteristics of BMSCs. (G and H) BMSCs at passage 6. (I) Semi-quantitative RT-PCR analysis was performed to confirm the expression of NTFs, angiogenic factors and inflammation-associated cytokines in DPCs. (J) RT-qPCR analysis further revealed relative expression levels of NTFs, angiogenic factors and inflammation-associated cytokines in DPCs. *P

    Article Snippet: RT-quantitative PCR (RT-qPCR) Total RNA from DPCs and BMSCs was extracted using TRIzol reagent, according to the manufacturer's protocols, then reverse transcribed into cDNA using the Takara RNA PCR kit (AMV) Version 3.0 (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocols.

    Techniques: Isolation, Transgenic Assay, Expressing, Fluorescence, Quantitative RT-PCR

    The morphological and phenotypic characteristics of mouse spermatogonia, pachytene spermatocytes and round spermatids Phase-contrast microscope showed the morphological characteristics of the freshly isolated spermatogonia, pachytene spermatocytes and round spermatids a . Scale bars in a = 20 μm. RT-PCR revealed the transcripts of Gfra1, Thy1, Gpr125 , and Zbtb16 in the freshly isolated mouse spermatogonia and mouse testis b , Mlh1, Crest , and Scp3 in the freshly isolated pachytene spermatocytes and mouse testis c , Acr, Prm1 , and Tnp1 in the freshly isolated round spermatids and mouse testis d . Actb was employed as a loading control of total RNA, and RNA samples without RT (RT−) but with PCR by Actb primers were used as negative controls

    Journal: Cell Death & Disease

    Article Title: Transcriptional regulation of P63 on the apoptosis of male germ cells and three stages of spermatogenesis in mice

    doi: 10.1038/s41419-017-0046-z

    Figure Lengend Snippet: The morphological and phenotypic characteristics of mouse spermatogonia, pachytene spermatocytes and round spermatids Phase-contrast microscope showed the morphological characteristics of the freshly isolated spermatogonia, pachytene spermatocytes and round spermatids a . Scale bars in a = 20 μm. RT-PCR revealed the transcripts of Gfra1, Thy1, Gpr125 , and Zbtb16 in the freshly isolated mouse spermatogonia and mouse testis b , Mlh1, Crest , and Scp3 in the freshly isolated pachytene spermatocytes and mouse testis c , Acr, Prm1 , and Tnp1 in the freshly isolated round spermatids and mouse testis d . Actb was employed as a loading control of total RNA, and RNA samples without RT (RT−) but with PCR by Actb primers were used as negative controls

    Article Snippet: RNA extraction and reverse transcription (RT) and real-time quantitative PCR Total RNA was extracted from spermatogonia, pachytene spermatocytes, and round spermatids of P63(+/ − ) mice and wild-type mice using Trizol (Takara, Kusatsu, Japan).

    Techniques: Microscopy, Isolation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    MOV10 contributes to the regulation of INK4a in primary fibroblasts. ( a ) FDF cells were infected with lentiviruses encoding a control shRNA (Ctrl) and two independent shRNAs against MOV10 and CBX7 respectively (sh1 and sh2). The knockdown efficiency and the effect on p16 INK4a were assessed by immunoblotting with antibodies against MOV10, CBX7 and p16 INK4a . GAPDH was used as a loading control. ( b ) Effects of MOV10 and CBX7 shRNAs on INK4a and ARF RNA levels as determined by qRT-PCR. Error bars, s.d.; n =3. ( c ) Phase contrast photographs showing the enlarged and flattened appearance of cells expressing the MOV10 and CBX7 shRNAs. ( d ) Following knockdown of MOV10 (as in panel a ), cell lysates were immunoblotted for p53 and p21 CIP1 as indicated. GAPDH was used as a loading control.

    Journal: Nature structural & molecular biology

    Article Title: Role for the MOV10 RNA helicase in Polycomb-mediated repression of the INK4a tumor suppressor

    doi: 10.1038/nsmb.1824

    Figure Lengend Snippet: MOV10 contributes to the regulation of INK4a in primary fibroblasts. ( a ) FDF cells were infected with lentiviruses encoding a control shRNA (Ctrl) and two independent shRNAs against MOV10 and CBX7 respectively (sh1 and sh2). The knockdown efficiency and the effect on p16 INK4a were assessed by immunoblotting with antibodies against MOV10, CBX7 and p16 INK4a . GAPDH was used as a loading control. ( b ) Effects of MOV10 and CBX7 shRNAs on INK4a and ARF RNA levels as determined by qRT-PCR. Error bars, s.d.; n =3. ( c ) Phase contrast photographs showing the enlarged and flattened appearance of cells expressing the MOV10 and CBX7 shRNAs. ( d ) Following knockdown of MOV10 (as in panel a ), cell lysates were immunoblotted for p53 and p21 CIP1 as indicated. GAPDH was used as a loading control.

    Article Snippet: RNA extraction, quantitative reverse transcription and quantitative PCR Total RNA was prepared using the Ultra Pure RNA extraction Kit from Roche and the cDNA was generated using 0.25–1 μg of RNA using MultiScribe reverse transcriptase and random hexamer primers (Applied Biosystems).

    Techniques: Infection, shRNA, Quantitative RT-PCR, Expressing

    Detection of HA-mediated Nanog nuclear localization in MCF-7 and SK-OV-3.ipl cells. I, nuclear fraction isolated from MCF-7 ( I, panel A ) or SK-OV-3.ipl cells ( I, panel B ) (untreated ( lane 1 ) or treated with HA (50 μg/ml) for 30 min ( lane 2 ) or pretreated with anti-CD44 for 1 h followed by 30 min of HA (50 μg/ml) treatment ( lane 3 ) or pretreated with NanogsiRNA followed by 30 min of HA treatment ( lane 4 ) or pretreated with scrambled sequence siRNA followed by 30 min of HA treatment ( lane 5 ) or transfected with NanogcDNA ( lane 6 )) were immunoblotted with anti-Nanog antibody ( a ) or anti-lamin A/C antibody ( b ) (as a loading control). II, detection of Nanog target gene expression in both MCF-7 ( panel A ) and SK-OV-3.ipl cells ( panel B ). The expression of Nanog target genes ( e.g. Rex1 and Sox2 ) was measured using specific primers and Q-PCR in tumor cells according to the procedures described under “Materials and Methods.” Total RNA isolated from either MCF-7 ( panel A ) or SK-OV-3.ipl cells ( panel B ) (untreated ( lane 1 ) or treated with HA (50 μg/ml) for 24 h ( lane 2 ) or pretreated with anti-CD44 for 1 h followed by 24 h HA (50μg/ml) treatment ( lane 3 ) or pretreated with NanogsiRNA followed by 24 h HA treatment ( lane 4 ) or pretreated with scrambled sequence siRNA followed by 24 h HA treatment ( lane 5 ) or transfected with NanogcDNA ( lane 6 )) was reverse-transcribed and subjected to Q-PCR using Rex1 ( panel A, a and panel B, a )-specific or Sox2 ( panel A, b and panel B, b )-specific primer pairs as described under “Materials and Methods.” Relative mRNA expression levels of Rex1 or Sox2 in various treatments were calculated after normalization with 36B4 mRNA levels as determined by Q-PCR. The values expressed in this figure represent an average of triplicate determinations of three experiments with a standard deviation less than ±5%.

    Journal: The Journal of Biological Chemistry

    Article Title: Hyaluronan-CD44 Interaction Activates Stem Cell Marker Nanog, Stat-3-mediated MDR1 Gene Expression, and Ankyrin-regulated Multidrug Efflux in Breast and Ovarian Tumor Cells *

    doi: 10.1074/jbc.M800109200

    Figure Lengend Snippet: Detection of HA-mediated Nanog nuclear localization in MCF-7 and SK-OV-3.ipl cells. I, nuclear fraction isolated from MCF-7 ( I, panel A ) or SK-OV-3.ipl cells ( I, panel B ) (untreated ( lane 1 ) or treated with HA (50 μg/ml) for 30 min ( lane 2 ) or pretreated with anti-CD44 for 1 h followed by 30 min of HA (50 μg/ml) treatment ( lane 3 ) or pretreated with NanogsiRNA followed by 30 min of HA treatment ( lane 4 ) or pretreated with scrambled sequence siRNA followed by 30 min of HA treatment ( lane 5 ) or transfected with NanogcDNA ( lane 6 )) were immunoblotted with anti-Nanog antibody ( a ) or anti-lamin A/C antibody ( b ) (as a loading control). II, detection of Nanog target gene expression in both MCF-7 ( panel A ) and SK-OV-3.ipl cells ( panel B ). The expression of Nanog target genes ( e.g. Rex1 and Sox2 ) was measured using specific primers and Q-PCR in tumor cells according to the procedures described under “Materials and Methods.” Total RNA isolated from either MCF-7 ( panel A ) or SK-OV-3.ipl cells ( panel B ) (untreated ( lane 1 ) or treated with HA (50 μg/ml) for 24 h ( lane 2 ) or pretreated with anti-CD44 for 1 h followed by 24 h HA (50μg/ml) treatment ( lane 3 ) or pretreated with NanogsiRNA followed by 24 h HA treatment ( lane 4 ) or pretreated with scrambled sequence siRNA followed by 24 h HA treatment ( lane 5 ) or transfected with NanogcDNA ( lane 6 )) was reverse-transcribed and subjected to Q-PCR using Rex1 ( panel A, a and panel B, a )-specific or Sox2 ( panel A, b and panel B, b )-specific primer pairs as described under “Materials and Methods.” Relative mRNA expression levels of Rex1 or Sox2 in various treatments were calculated after normalization with 36B4 mRNA levels as determined by Q-PCR. The values expressed in this figure represent an average of triplicate determinations of three experiments with a standard deviation less than ±5%.

    Article Snippet: Quantitative PCR (Q-PCR) —Total RNA was isolated from MCF-7 or SK-OV-3.ipl cells (transfected with NanogcDNA or vector alone or treated with NanogsiRNA/Stat-3siRNA or siRNAs with scrambled sequences in the presence or absence of 24 h HA treatment) using Tripure isolation reagent kits (Roche Applied Science) as described above.

    Techniques: Isolation, Sequencing, Transfection, Expressing, Polymerase Chain Reaction, Standard Deviation

    Detection of Nanog expression in tumor cells. A, RT-PCR analysis of Nanog mRNA. Total RNA isolated from various cell types was reverse-transcribed and subjected to PCR using Nanog-specific primer pairs as described under “Materials and Methods.” DNA markers ( lane 1 ); RT-PCR product of Nanog detected in MCF-7 cells ( lane 2 ) or SK-OV-3.ipl cells ( lane 3 ) in the absence of reverse transcriptase; RT-PCR product of Nanog detected in normal skin fibroblasts ( lane 4 ) or MCF-7 cells ( lane 5 ) or SK-OV-3.ipl cells ( lane 6 ) in the presence of reverse transcriptase. RT-PCR product of 36B4 detected in samples used in lanes 4-6 in the presence of reverse transcriptase (as a loading control). B, detection of Nanog by anti-Nanog immunoblot analysis. Cell lysates isolated from MCF-7 or SK-OV-3.ipl cells were solubilized by 1% Non-idet P-40 buffer followed by immunoblotting with rabbit anti-Nanog antibody or anti-actin as described under “Materials and Methods.” Immunoblot of cell lysates isolated from MCF-7 cells ( lane 1 ) or SK-OV-3.ipl cells ( lane 2 ) with anti-Nanog antibody ( panel a ) and anti-actin ( panel b ) as a loading control.

    Journal: The Journal of Biological Chemistry

    Article Title: Hyaluronan-CD44 Interaction Activates Stem Cell Marker Nanog, Stat-3-mediated MDR1 Gene Expression, and Ankyrin-regulated Multidrug Efflux in Breast and Ovarian Tumor Cells *

    doi: 10.1074/jbc.M800109200

    Figure Lengend Snippet: Detection of Nanog expression in tumor cells. A, RT-PCR analysis of Nanog mRNA. Total RNA isolated from various cell types was reverse-transcribed and subjected to PCR using Nanog-specific primer pairs as described under “Materials and Methods.” DNA markers ( lane 1 ); RT-PCR product of Nanog detected in MCF-7 cells ( lane 2 ) or SK-OV-3.ipl cells ( lane 3 ) in the absence of reverse transcriptase; RT-PCR product of Nanog detected in normal skin fibroblasts ( lane 4 ) or MCF-7 cells ( lane 5 ) or SK-OV-3.ipl cells ( lane 6 ) in the presence of reverse transcriptase. RT-PCR product of 36B4 detected in samples used in lanes 4-6 in the presence of reverse transcriptase (as a loading control). B, detection of Nanog by anti-Nanog immunoblot analysis. Cell lysates isolated from MCF-7 or SK-OV-3.ipl cells were solubilized by 1% Non-idet P-40 buffer followed by immunoblotting with rabbit anti-Nanog antibody or anti-actin as described under “Materials and Methods.” Immunoblot of cell lysates isolated from MCF-7 cells ( lane 1 ) or SK-OV-3.ipl cells ( lane 2 ) with anti-Nanog antibody ( panel a ) and anti-actin ( panel b ) as a loading control.

    Article Snippet: Quantitative PCR (Q-PCR) —Total RNA was isolated from MCF-7 or SK-OV-3.ipl cells (transfected with NanogcDNA or vector alone or treated with NanogsiRNA/Stat-3siRNA or siRNAs with scrambled sequences in the presence or absence of 24 h HA treatment) using Tripure isolation reagent kits (Roche Applied Science) as described above.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction

    UPR to cisplatin-induced ER stress differed between cancer stem-like and cancer cells Monolayer (mono) or sphere-forming (sphere) cells were untreated (control: con) or treated with 20 μM cisplatin (CDDP) for 72 hours, and live cells were fractioned. A. Total RNA was extracted for RT-PCR and the ratio of spliced XBP1 mRNA to total XBP1 mRNA was calculated by the comparative Ct method. The values shown represent the means ± SEM (* p

    Journal: Oncotarget

    Article Title: Inhibition of endoplasmic reticulum (ER) stress sensors sensitizes cancer stem-like cells to ER stress-mediated apoptosis

    doi: 10.18632/oncotarget.10126

    Figure Lengend Snippet: UPR to cisplatin-induced ER stress differed between cancer stem-like and cancer cells Monolayer (mono) or sphere-forming (sphere) cells were untreated (control: con) or treated with 20 μM cisplatin (CDDP) for 72 hours, and live cells were fractioned. A. Total RNA was extracted for RT-PCR and the ratio of spliced XBP1 mRNA to total XBP1 mRNA was calculated by the comparative Ct method. The values shown represent the means ± SEM (* p

    Article Snippet: RT-quantitative PCR Total RNA was extracted from live cells using a Blood/Cultured Cell Total RNA Mini Kit (FAVORGEN), followed by reverse transcription. cDNA was amplified for 40 cycles in a Light Cycler 480 (Roche).

    Techniques: Reverse Transcription Polymerase Chain Reaction

    UPR to tunicamycin-induced ER stress differed between cancer stem-like and cancer cells Monolayer (mono) or sphere-forming (sphere) cells were untreated (control: con) or treated with 0.03 μM tunicamycin (TM) for 72 hours, and live cells were fractioned. A. Total RNA was extracted for RT-PCR and the ratio of spliced XBP1 mRNA to total XBP1 mRNA was calculated using the comparative Ct method. XBP1 splicing was increased by tunicamycin in monolayer cells. The values shown represent the means ± SEM (* p

    Journal: Oncotarget

    Article Title: Inhibition of endoplasmic reticulum (ER) stress sensors sensitizes cancer stem-like cells to ER stress-mediated apoptosis

    doi: 10.18632/oncotarget.10126

    Figure Lengend Snippet: UPR to tunicamycin-induced ER stress differed between cancer stem-like and cancer cells Monolayer (mono) or sphere-forming (sphere) cells were untreated (control: con) or treated with 0.03 μM tunicamycin (TM) for 72 hours, and live cells were fractioned. A. Total RNA was extracted for RT-PCR and the ratio of spliced XBP1 mRNA to total XBP1 mRNA was calculated using the comparative Ct method. XBP1 splicing was increased by tunicamycin in monolayer cells. The values shown represent the means ± SEM (* p

    Article Snippet: RT-quantitative PCR Total RNA was extracted from live cells using a Blood/Cultured Cell Total RNA Mini Kit (FAVORGEN), followed by reverse transcription. cDNA was amplified for 40 cycles in a Light Cycler 480 (Roche).

    Techniques: Reverse Transcription Polymerase Chain Reaction

    CSM regulates expression of TLR4 via ROS . (A) MDMs (5 × 10 6 cells) were stimulated by CSM (0.03, 0.06 and 0.12 OD) for 4 h with and without pretreatment with NAC (10 mM) for 30 min. RNA was extracted and TLR4 and GAPDH gene expression were quantified by real-time PCR. Results are expressed as copies of TLR4 vs. copies of GAPDH gene. (B) MDMs were preincubated with naturalizing anti-TLR4 or isotype control antibodies for 30 min and then stimulated with CSM (0.06 OD) for 4 h or LPS (100 ng/ml) and mRNA levels of TLR4 was determined by real-time PCR method. Values are expressed as mean +/- S.E.M (n = 3).*P

    Journal: Journal of Inflammation (London, England)

    Article Title: Cigarette smoke regulates the expression of TLR4 and IL-8 production by human macrophages

    doi: 10.1186/1476-9255-6-12

    Figure Lengend Snippet: CSM regulates expression of TLR4 via ROS . (A) MDMs (5 × 10 6 cells) were stimulated by CSM (0.03, 0.06 and 0.12 OD) for 4 h with and without pretreatment with NAC (10 mM) for 30 min. RNA was extracted and TLR4 and GAPDH gene expression were quantified by real-time PCR. Results are expressed as copies of TLR4 vs. copies of GAPDH gene. (B) MDMs were preincubated with naturalizing anti-TLR4 or isotype control antibodies for 30 min and then stimulated with CSM (0.06 OD) for 4 h or LPS (100 ng/ml) and mRNA levels of TLR4 was determined by real-time PCR method. Values are expressed as mean +/- S.E.M (n = 3).*P

    Article Snippet: Real-time quantitative PCR Total RNA was extracted using High Pure RNA Isolation Kit (Roche Applied Science) according to the manufacture's instruction.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    IL-8 expression is ROS dependent after CSM exposure . MDMs (5 × 10 6 cells/ml) were pretreated with NAC (10 mM) for 30 min and then stimulated by CSM (0.03, 0.06 and 0.12 OD) for 4 h. RNA was extracted and mRNA levels of IL-8 were quantified by real-time PCR (A). Results are expressed as copies of IL-8 vs. copies of GAPDH mRNA. (B) MDMs (1 × 10 6 cells/ml) were pretreated with NAC (10 mM) for 30 min and then stimulated by CSM (0.06 OD) for 16 h Supernatants were collected after 16 h incubation and IL-8 production was quantified using ELISA methods. *P

    Journal: Journal of Inflammation (London, England)

    Article Title: Cigarette smoke regulates the expression of TLR4 and IL-8 production by human macrophages

    doi: 10.1186/1476-9255-6-12

    Figure Lengend Snippet: IL-8 expression is ROS dependent after CSM exposure . MDMs (5 × 10 6 cells/ml) were pretreated with NAC (10 mM) for 30 min and then stimulated by CSM (0.03, 0.06 and 0.12 OD) for 4 h. RNA was extracted and mRNA levels of IL-8 were quantified by real-time PCR (A). Results are expressed as copies of IL-8 vs. copies of GAPDH mRNA. (B) MDMs (1 × 10 6 cells/ml) were pretreated with NAC (10 mM) for 30 min and then stimulated by CSM (0.06 OD) for 16 h Supernatants were collected after 16 h incubation and IL-8 production was quantified using ELISA methods. *P

    Article Snippet: Real-time quantitative PCR Total RNA was extracted using High Pure RNA Isolation Kit (Roche Applied Science) according to the manufacture's instruction.

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

    Comparing the expression of glucocorticoid-regulated genes in inguinal fat of CRH-Tg and wild type mice. Total RNA of inguinal fat of wild type and CRH-Tg mice were isolated and converted to cDNA. Real-time PCR was then performed to monitor the expression of genes indicated. Data shows fold-induction of gene expression (CRH-Tg/wild type) from 6–8 mice (*, p

    Journal: PLoS ONE

    Article Title: Genome-Wide Analysis of Glucocorticoid Receptor Binding Regions in Adipocytes Reveal Gene Network Involved in Triglyceride Homeostasis

    doi: 10.1371/journal.pone.0015188

    Figure Lengend Snippet: Comparing the expression of glucocorticoid-regulated genes in inguinal fat of CRH-Tg and wild type mice. Total RNA of inguinal fat of wild type and CRH-Tg mice were isolated and converted to cDNA. Real-time PCR was then performed to monitor the expression of genes indicated. Data shows fold-induction of gene expression (CRH-Tg/wild type) from 6–8 mice (*, p

    Article Snippet: RNA isolation and quantitative PCR Total RNA was isolated from mouse inguinal fat using TRI Reagent® RT (Molecular Research Center, Inc.).

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

    RANKL enhances Snail and Twist expression and induces changes in the morphology of cells. (A) Analysis of cell morphology after cell treatment of with 100 ng/mL RANKL. RANKL induces changes in the epithelial morphology of 4T1, MCF-7, and NMuMG cells (×40 magnification). (B-D) Total RNA was extracted, and the mRNA expression levels of vimentin, E-cadherin, N-cadherin, Snail, Slug, and Twist were determined by real-time PCR. The results are expressed as treated over control ratio after correction to GAPDH mRNA levels. The results are representative of 5 independent experiments. *p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Activation of NF-?B by the RANKL/RANK system up-regulates snail and twist expressions and induces epithelial-to-mesenchymal transition in mammary tumor cell lines

    doi: 10.1186/1756-9966-32-62

    Figure Lengend Snippet: RANKL enhances Snail and Twist expression and induces changes in the morphology of cells. (A) Analysis of cell morphology after cell treatment of with 100 ng/mL RANKL. RANKL induces changes in the epithelial morphology of 4T1, MCF-7, and NMuMG cells (×40 magnification). (B-D) Total RNA was extracted, and the mRNA expression levels of vimentin, E-cadherin, N-cadherin, Snail, Slug, and Twist were determined by real-time PCR. The results are expressed as treated over control ratio after correction to GAPDH mRNA levels. The results are representative of 5 independent experiments. *p

    Article Snippet: Quantitative real-time polymerase chain reaction (PCR) Total RNA was isolated using RNAiso (Takara Biomedical, Siga, Japan).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Effects of DMF on RANKL-induced EMT and EMT-related mRNA expression. (A) Analysis of 4T1 cell morphology after cell treatment of with 100 ng/mL RANKL or 100 μM DMF (× 40 magnification). (B) Total RNA was extracted, and the mRNA levels of vimentin, E-cadherin, N-cadherin, Snail, and Twist were determined by real-time PCR. The results are expressed as treated over control ratio after correction to GAPDH mRNA levels. The results are representative of 5 independent experiments. *p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Activation of NF-?B by the RANKL/RANK system up-regulates snail and twist expressions and induces epithelial-to-mesenchymal transition in mammary tumor cell lines

    doi: 10.1186/1756-9966-32-62

    Figure Lengend Snippet: Effects of DMF on RANKL-induced EMT and EMT-related mRNA expression. (A) Analysis of 4T1 cell morphology after cell treatment of with 100 ng/mL RANKL or 100 μM DMF (× 40 magnification). (B) Total RNA was extracted, and the mRNA levels of vimentin, E-cadherin, N-cadherin, Snail, and Twist were determined by real-time PCR. The results are expressed as treated over control ratio after correction to GAPDH mRNA levels. The results are representative of 5 independent experiments. *p

    Article Snippet: Quantitative real-time polymerase chain reaction (PCR) Total RNA was isolated using RNAiso (Takara Biomedical, Siga, Japan).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Knockdown of endogenous nuclear IL-33 expression in primary human endothelial cells using two independent RNA silencing strategies. ( a,b ) Nuclear expression of endogenous IL-33 in confluent monolayers of primary human endothelial cells. IL-33 protein (red) was detected by indirect immunofluorescence staining with anti-IL-33 mAbs Nessy1 ( a ) and 305B ( b ). Nuclei were counterstained with DAPI (blue). ( c ) Knockdown of endogenous nuclear IL-33 expression. IL-33 expression was analyzed by indirect immunofluorescence with Nessy1 mAb 48 h after transfection of a pool of IL-33 siRNAs (siIL-33-Sm ) or control siRNA ( siCTL-Sm ). ( d–g ) Validation of the two independent RNA silencing strategies. Expression of IL-33 was analyzed at the protein level by western blot ( d,f ) and RNA level by qPCR ( e,g ), 72 h after the second siRNA transfection. Cells were treated with distinct pools of IL-33 siRNAs , siIL-33-Sm ( d,e ) or siIL-33-Mi ( f,g ), and corresponding controls, siCTL-Sm ( d,e ) and siCTL-Mi ( f,g ). RRL IL-33, human IL-33 protein produced by in vitro translation in rabbit reticulocyte lysates ( d,f ). For the qPCR experiments, relative mRNA levels were calculated by normalizing the signals to those of actin. Results are shown as means with s.d. from three separate datapoints. ( h,i ). Knockdown of endogenous nuclear IL-33 does not affect NFkB protein expression. Normalized protein quantities deduced from all peptides intensity values (LFQ intensities) are shown for NFkB p65 (RELA), NFkB p105 (NFKB1) and NFkB p100 (NFKB2). Endothelial cells were treated with siIL-33-Sm ( h ) or siIL-33-Mi ( i ), and corresponding controls. Results are shown as means with s.d. from three biological replicate experiments.

    Journal: Scientific Reports

    Article Title: Extracellular IL-33 cytokine, but not endogenous nuclear IL-33, regulates protein expression in endothelial cells

    doi: 10.1038/srep34255

    Figure Lengend Snippet: Knockdown of endogenous nuclear IL-33 expression in primary human endothelial cells using two independent RNA silencing strategies. ( a,b ) Nuclear expression of endogenous IL-33 in confluent monolayers of primary human endothelial cells. IL-33 protein (red) was detected by indirect immunofluorescence staining with anti-IL-33 mAbs Nessy1 ( a ) and 305B ( b ). Nuclei were counterstained with DAPI (blue). ( c ) Knockdown of endogenous nuclear IL-33 expression. IL-33 expression was analyzed by indirect immunofluorescence with Nessy1 mAb 48 h after transfection of a pool of IL-33 siRNAs (siIL-33-Sm ) or control siRNA ( siCTL-Sm ). ( d–g ) Validation of the two independent RNA silencing strategies. Expression of IL-33 was analyzed at the protein level by western blot ( d,f ) and RNA level by qPCR ( e,g ), 72 h after the second siRNA transfection. Cells were treated with distinct pools of IL-33 siRNAs , siIL-33-Sm ( d,e ) or siIL-33-Mi ( f,g ), and corresponding controls, siCTL-Sm ( d,e ) and siCTL-Mi ( f,g ). RRL IL-33, human IL-33 protein produced by in vitro translation in rabbit reticulocyte lysates ( d,f ). For the qPCR experiments, relative mRNA levels were calculated by normalizing the signals to those of actin. Results are shown as means with s.d. from three separate datapoints. ( h,i ). Knockdown of endogenous nuclear IL-33 does not affect NFkB protein expression. Normalized protein quantities deduced from all peptides intensity values (LFQ intensities) are shown for NFkB p65 (RELA), NFkB p105 (NFKB1) and NFkB p100 (NFKB2). Endothelial cells were treated with siIL-33-Sm ( h ) or siIL-33-Mi ( i ), and corresponding controls. Results are shown as means with s.d. from three biological replicate experiments.

    Article Snippet: Quantitative PCR (qPCR) Total RNA was isolated using the Absolute RNA Kit from Stratagene (Agilent Technologies) and cDNAs were synthesized using SuperSript III First strand cDNA synthesis system for RT-PCR (Invitrogen) according to manufacturer’s instructions. qPCR was performed using the ABI7500 Prism SDS Real-Time PCR Detection System (Applied Biosystems) with a SYBR Green PCR Master Mix kit (Applied Biosystems) and a standard temperature protocol.

    Techniques: Expressing, Immunofluorescence, Staining, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Produced, In Vitro

    Edited CCNI peptide level is correlated with its biological function. Correlation of CCNI peptide levels with CCNI and ADAR mRNA expression as well as Ted10 activation and Ted10 mediated tumour target killing. a Correlation between the number of edited CCNI peptide copies per cell determined by the AbsQuant ® method and mRNA editing levels determined by targeted RNA-seq for CCNI R75G . The scatterplot ( n = 8) includes the regression curve (red line) as well as the 95% confidence interval (grey band) and 95% prediction interval (dashed lines). b CCNI-R75G is edited by ADAR1 . HEK 293 stably expressing CCNI wildtype gene was transfected with empty vector or expression vectors of ADAR1 or ADAR2 . CCNI editing was measured by RT-PCR and followed by sequencing. The double peaks indicate nucleotide A to G conversion and the height of peaks reflect the level of editing. c ELISPOT assay showing IFNγ production by Ted10 incubated with peptide-pulsed or CCNI -transfected 293-A2 cells. d CTL killing assay showing that over-expression of edited CCNI gene increases the sensitivity of Ted10 mediated 293-A2 target killing ( n = 3), summarised as mean ± s.e.m. per titration. e IFNγ ELISPOT assay showing recognition of endogenous CCNI-ED antigen by Ted10. Mel-2391, mel-2400 and mel-2661 expressing both edited CCNI mRNA and HLA-A*02:01 are highly reactive to Ted10. Mel-2559, which was derived from the same patient as mel-2400 but does not have detectible edited CCNI mRNA, only reacted at background levels to Ted10. Mel-2357 and mel-2686, which express edited CCNI mRNA but do not express HLA-A*02:01, have no response to Ted10. f, Ted10 mediated target killing following incubation with mel-2400 and mel-2559 measured by caspase-3-based CTL killing assay (summarised as mean ± s.e.m. of the three triplicates)

    Journal: Nature Communications

    Article Title: RNA editing derived epitopes function as cancer antigens to elicit immune responses

    doi: 10.1038/s41467-018-06405-9

    Figure Lengend Snippet: Edited CCNI peptide level is correlated with its biological function. Correlation of CCNI peptide levels with CCNI and ADAR mRNA expression as well as Ted10 activation and Ted10 mediated tumour target killing. a Correlation between the number of edited CCNI peptide copies per cell determined by the AbsQuant ® method and mRNA editing levels determined by targeted RNA-seq for CCNI R75G . The scatterplot ( n = 8) includes the regression curve (red line) as well as the 95% confidence interval (grey band) and 95% prediction interval (dashed lines). b CCNI-R75G is edited by ADAR1 . HEK 293 stably expressing CCNI wildtype gene was transfected with empty vector or expression vectors of ADAR1 or ADAR2 . CCNI editing was measured by RT-PCR and followed by sequencing. The double peaks indicate nucleotide A to G conversion and the height of peaks reflect the level of editing. c ELISPOT assay showing IFNγ production by Ted10 incubated with peptide-pulsed or CCNI -transfected 293-A2 cells. d CTL killing assay showing that over-expression of edited CCNI gene increases the sensitivity of Ted10 mediated 293-A2 target killing ( n = 3), summarised as mean ± s.e.m. per titration. e IFNγ ELISPOT assay showing recognition of endogenous CCNI-ED antigen by Ted10. Mel-2391, mel-2400 and mel-2661 expressing both edited CCNI mRNA and HLA-A*02:01 are highly reactive to Ted10. Mel-2559, which was derived from the same patient as mel-2400 but does not have detectible edited CCNI mRNA, only reacted at background levels to Ted10. Mel-2357 and mel-2686, which express edited CCNI mRNA but do not express HLA-A*02:01, have no response to Ted10. f, Ted10 mediated target killing following incubation with mel-2400 and mel-2559 measured by caspase-3-based CTL killing assay (summarised as mean ± s.e.m. of the three triplicates)

    Article Snippet: Quantitative PCR (qPCR) Total RNA was isolated and converted to cDNA as described above. qPCR was performed using iTaq™ Universal SYBR® Green Supermix reagent (Bio Red, 1725122) in a C1000TM Thermal Cycler CFX96 Real-Time System following manufacturer’s instructions (Bio-Rad).

    Techniques: Expressing, Activation Assay, RNA Sequencing Assay, Stable Transfection, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Sequencing, Enzyme-linked Immunospot, Incubation, CTL Assay, Over Expression, Titration, Derivative Assay

    The protective effect of up-regulating ATP8A1 was related to apoptosis-related proteins. ( A ) The mRNA levels of Fas, Fasl, Bax, Bcl-2, and caspase-3 in each group were measured by qRT-PCR. ( B ) These apoptosis-related proteins expression levels were measured by Western blot. * P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Over-Expression of ATPase II Alleviates Ethanol-Induced Hepatocyte Injury in HL-7702 Cells

    doi: 10.12659/MSM.910254

    Figure Lengend Snippet: The protective effect of up-regulating ATP8A1 was related to apoptosis-related proteins. ( A ) The mRNA levels of Fas, Fasl, Bax, Bcl-2, and caspase-3 in each group were measured by qRT-PCR. ( B ) These apoptosis-related proteins expression levels were measured by Western blot. * P

    Article Snippet: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) Total RNA was isolated according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA) a reverse transcription was performed using the protocol of for SuperScript III reverse transcriptase (Takara, Japan).

    Techniques: Quantitative RT-PCR, Expressing, Western Blot

    Depletion of lincRNAs increases MAF mRNA levels in HEK293 cells. HEK293 cells were reverse-transfected with a control siRNA, 5 nM MAFTRR siRNA (A–C) or 5 nM LINC01229 siRNA (D–F) , and quantitative PCR was performed on RNA collected at 48 h post-transfection. (A) MAFTRR RNA transcript levels. (B) MAF transcript levels. (C) LINC01229 RNA transcript levels. (D) LINC01229 RNA transcript levels. (E) MAF RNA transcript levels. (F) MAFTRR transcript levels. Data represent four biological replicates; statistical significance was determined using unpaired t -test with one tail. Error bars denote standard error of the mean and asterisks indicate significance: ∗ p

    Journal: Frontiers in Genetics

    Article Title: Functional Urate-Associated Genetic Variants Influence Expression of lincRNAs LINC01229 and MAFTRR

    doi: 10.3389/fgene.2018.00733

    Figure Lengend Snippet: Depletion of lincRNAs increases MAF mRNA levels in HEK293 cells. HEK293 cells were reverse-transfected with a control siRNA, 5 nM MAFTRR siRNA (A–C) or 5 nM LINC01229 siRNA (D–F) , and quantitative PCR was performed on RNA collected at 48 h post-transfection. (A) MAFTRR RNA transcript levels. (B) MAF transcript levels. (C) LINC01229 RNA transcript levels. (D) LINC01229 RNA transcript levels. (E) MAF RNA transcript levels. (F) MAFTRR transcript levels. Data represent four biological replicates; statistical significance was determined using unpaired t -test with one tail. Error bars denote standard error of the mean and asterisks indicate significance: ∗ p

    Article Snippet: Quantitative PCR Total RNA was isolated from control and siRNA-treated HEK293 cells at 48 h post-treatment using the NucleoSpin RNA kit (Macherey-Nagel). cDNA was synthesized with qScript cDNA SuperMix (Quanta Biosciences).

    Techniques: Transfection, Real-time Polymerase Chain Reaction

    Endogenous MAVS is degraded in the absence of its N-terminally truncated isoforms. ( a ) HEK293T WT, Mavs-(M2L) , Mavs-(M2 6L) , Mavs-(M2-5L) and Mavs-(M2-6L) cells were subjected to subcellular fractionation to obtain P5 fractions. Various MAVS isoforms were detected by immunoblotting with anti-MAVS-(C), anti-MAVS-(M) and anti-MAVS-(N) antibodies. See also Supplementary Fig. 4a . The original full blot can be found in Supplementary Fig. 13a . ( b ) Whole cell lysates of HEK293T WT, Mavs-(M2L) , Mavs-(M2 6L) , Mavs-(M2-5L) and Mavs-(M2-6L) cells was obtained to determine endogenous MAVS protein level by immunoblotting. Tubulin was immunoblotted as a loading control. RT-PCR was performed to measure mRNA level of various Mavs gene. GAPDH was analysed as an internal control. ( c – e ) HEK293T WT, Mavs-(M2L) , Mavs-(M2 6L) , Mavs-(M2-5L) and Mavs-(M2-6L) cells were infected with VSV (MOI=1). Twelve hours post infection, RNA was extracted and qPCR was performed to measure IFN induction ( c ). VSV titres were quantitated by plaque assay ( d ). Fluorescent images were taken to examine VSV proliferation eight hours after infection ( e ). Scale bar represents 10 micrometres. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student’s t -test. ** P

    Journal: Nature Communications

    Article Title: Multiple truncated isoforms of MAVS prevent its spontaneous aggregation in antiviral innate immune signalling

    doi: 10.1038/ncomms15676

    Figure Lengend Snippet: Endogenous MAVS is degraded in the absence of its N-terminally truncated isoforms. ( a ) HEK293T WT, Mavs-(M2L) , Mavs-(M2 6L) , Mavs-(M2-5L) and Mavs-(M2-6L) cells were subjected to subcellular fractionation to obtain P5 fractions. Various MAVS isoforms were detected by immunoblotting with anti-MAVS-(C), anti-MAVS-(M) and anti-MAVS-(N) antibodies. See also Supplementary Fig. 4a . The original full blot can be found in Supplementary Fig. 13a . ( b ) Whole cell lysates of HEK293T WT, Mavs-(M2L) , Mavs-(M2 6L) , Mavs-(M2-5L) and Mavs-(M2-6L) cells was obtained to determine endogenous MAVS protein level by immunoblotting. Tubulin was immunoblotted as a loading control. RT-PCR was performed to measure mRNA level of various Mavs gene. GAPDH was analysed as an internal control. ( c – e ) HEK293T WT, Mavs-(M2L) , Mavs-(M2 6L) , Mavs-(M2-5L) and Mavs-(M2-6L) cells were infected with VSV (MOI=1). Twelve hours post infection, RNA was extracted and qPCR was performed to measure IFN induction ( c ). VSV titres were quantitated by plaque assay ( d ). Fluorescent images were taken to examine VSV proliferation eight hours after infection ( e ). Scale bar represents 10 micrometres. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student’s t -test. ** P

    Article Snippet: Quantitative PCR Total RNA from cells was extracted with RNA simple total RNA kit (Catalogue number DP419, Tiangen, Shanghai, China) and reversed-transcribed using the GoScript Reverse Transcription system (Promega). cDNAs were then used as templates for qPCR assay using SuperReal Premix Plus (Tiangen).

    Techniques: Fractionation, Reverse Transcription Polymerase Chain Reaction, Infection, Real-time Polymerase Chain Reaction, Plaque Assay, Two Tailed Test

    Low-dose irradiation induces apoptosis of fibroblast-like synoviocytes (FLS) and slightly impacts on the inflammatory FLS phenotype. Inflammatory FLS obtained from h TNF- α tg mice were analyzed 96 h after irradiation with various doses of X-rays. Cell numbers (A) of living cells were counted using a Neubauer chamber and cell death was determined via flow cytometry analysis after staining with AxV-FITC/PI. Vital cells were defined as AxV − /PI − , apoptotic cells as AxV + /PI − (B) , and necrotic ones as AxV + /PI + (C) . Quantification of secreted IL-6 (D) , hTNF-α (E) , CXCL1 (F) , and TGF-β (G) was performed 96 h after irradiation in supernatants of FLS cultures via enzyme-linked immunosorbent assays. Total RNA levels were isolated using phenol–chloroform extraction. Gene expression was analyzed using SYBR Green quantitative PCR analysis (H–M) . Depicted is joint data obtained of five h TNF- α tg-FLS cell lines, examined in four independent experiments, each performed at least in triplicates. Data are presented as mean ± SEM, tested for normal distribution, and analyzed by students t test (A–G) or two-tailed Mann–Whitney U test (H–M) in comparison to mock-treated (w/o) controls at 96 h after irradiation (* p

    Journal: Frontiers in Immunology

    Article Title: Low-Dose Radiotherapy Ameliorates Advanced Arthritis in hTNF-α tg Mice by Particularly Positively Impacting on Bone Metabolism

    doi: 10.3389/fimmu.2018.01834

    Figure Lengend Snippet: Low-dose irradiation induces apoptosis of fibroblast-like synoviocytes (FLS) and slightly impacts on the inflammatory FLS phenotype. Inflammatory FLS obtained from h TNF- α tg mice were analyzed 96 h after irradiation with various doses of X-rays. Cell numbers (A) of living cells were counted using a Neubauer chamber and cell death was determined via flow cytometry analysis after staining with AxV-FITC/PI. Vital cells were defined as AxV − /PI − , apoptotic cells as AxV + /PI − (B) , and necrotic ones as AxV + /PI + (C) . Quantification of secreted IL-6 (D) , hTNF-α (E) , CXCL1 (F) , and TGF-β (G) was performed 96 h after irradiation in supernatants of FLS cultures via enzyme-linked immunosorbent assays. Total RNA levels were isolated using phenol–chloroform extraction. Gene expression was analyzed using SYBR Green quantitative PCR analysis (H–M) . Depicted is joint data obtained of five h TNF- α tg-FLS cell lines, examined in four independent experiments, each performed at least in triplicates. Data are presented as mean ± SEM, tested for normal distribution, and analyzed by students t test (A–G) or two-tailed Mann–Whitney U test (H–M) in comparison to mock-treated (w/o) controls at 96 h after irradiation (* p

    Article Snippet: RNA and Quantitative PCR Total RNA from cell culture was isolated using TriFast (peqlab, Darmstadt, Germany) and phenol–chloroform extraction.

    Techniques: Irradiation, Mouse Assay, Flow Cytometry, Cytometry, Staining, Isolation, Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction, Two Tailed Test, MANN-WHITNEY