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  • 79
    TaKaRa transcription polymerase chain reaction qrt pcr system
    HIF-1α regulated the expression of CCL2 in PDAC cells, PC, N and H indicate positive control, normoxia, and hypoxia, respectively. ( A ) HPDE6-C7,BxPC3 and MiaPaca-2 cells were transfected with siHIF-1α (50 nmol/L) and pcDNA3.1-HIF-1α plasmids (4 µg) for 48 h, respectively; the mRNA expression levels of HIF-1α and CCL2 were assessed by <t>qRT-PCR.</t> The experiments were performed thrice independently. * p
    Transcription Polymerase Chain Reaction Qrt Pcr System, supplied by TaKaRa, used in various techniques. Bioz Stars score: 79/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher transcript polymerase chain reaction qrt pcr kit
    HIF-1α regulated the expression of CCL2 in PDAC cells, PC, N and H indicate positive control, normoxia, and hypoxia, respectively. ( A ) HPDE6-C7,BxPC3 and MiaPaca-2 cells were transfected with siHIF-1α (50 nmol/L) and pcDNA3.1-HIF-1α plasmids (4 µg) for 48 h, respectively; the mRNA expression levels of HIF-1α and CCL2 were assessed by <t>qRT-PCR.</t> The experiments were performed thrice independently. * p
    Transcript Polymerase Chain Reaction Qrt Pcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 82/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad polymerase chain reaction qrt pcr
    CDKI expression in human terminal erythroid differentiation. (A) RNA-seq data showing CDKI members’ expression (fragments per kilobase of transcript per million) at each distinct stage of human terminal erythroid differentiation. Baso-E, basophilic erythroblast; ortho-E, orthochromatic erythroblast; poly-E, polychromatic erythroblast; pro-E, proerythroblast. (B) Representative image of western blotting of p19 INK4d , p18 INK4c , and p27 KIP1 expression in whole-cell lysates prepared from erythroblasts cultured for different times (left). Quantitative analysis of data from 3 independent experiments of protein expression levels (right). GAPDH was used as a loading control. (C) <t>qRT-PCR</t> results showing p19 INK4d expression in erythroblasts infected with lentivirus containing control (Lucif shRNA) or p19 INK4d shRNA on day (D) 7, D 11, and D 15 of culture. The results were normalized to GAPDH mRNA. (D) Representative images of western blotting showing p19 INK4d expression levels in erythroblasts infected with Lucif shRNA or p19 INK4d shRNA (left). Quantitative analysis of protein expression data from 3 independent experiments (right). The results were normalized to GAPDH protein. Statistical analysis of data of 3 independent experiments and bar plot represents mean ± SD of triplicate samples. *** P
    Polymerase Chain Reaction Qrt Pcr, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 80/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche real time polymerase chain reaction qrt pcr
    LPS / ATP activate NLRP 3 inflammasomes in VSMC s. Human primary VSMC s were treated with vehicle or the indicated concentrations of LPS for 16 hours and/or ATP for 1 hour. Some cells were transfected with control or NLRP 3‐specific si RNA or treated with vehicle DMSO or ZYVAD ‐ FMK , followed by treatment with LPS and/or ATP . The relative levels of NLRP 3 and cleaved caspase‐1 expression were determined by <t>qRT</t> ‐ <t>PCR</t> and Western blot. The distribution of HMGB 1 in the different groups of cells and their cultured supernatants were determined by Western blot and ELISA . Data are representative images or expressed as the mean± SD of each group from 4 separated experiments (Western blot) or 3 separated experiments with 4 duplicated wells ( qRT ‐ PCR and ELISA ). A, The levels of NLRP 3 mRNA transcripts. B, The levels of NLRP 3 proteins. C, The levels of cleaved caspase‐1. D, The levels of total HMGB 1. E, The levels of nuclear HMGB 1. F, The levels of cytoplasmic HMGB 1. G, The levels of HMGB 1 in the supernatants. * P
    Real Time Polymerase Chain Reaction Qrt Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 167 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher real time polymerase chain reaction qrt pcr
    Primer Design and <t>qRT-PCR</t>
    Real Time Polymerase Chain Reaction Qrt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1001 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche polymerase chain reaction qrt pcr
    POT1 mRNA expression as determined by <t>qRT-PCR</t> (a) and POT1 protein expression as determined by western blot analysis (b) in shRNA-infected human ovarian cancer SK-OV3 cells. POT1-KD significantly decreased cell proliferation (c) and colony formation (d) in the early POT1-KD SK-OV3 cells. “Negative control” indicates negative control SK-OV3 cells that express a nonspecific shRNA, and “POT1-KD” represents POT1-knockdown SK-OV3 cells that express POT1-specific shRNA. (c) shows the cPDs of the SK-OV3 cells. The cPDs of the negative control cells are depicted with black dots and lines, while the cPDs of the immediate effect POT1-KD cells are depicted with black squares and lines. (d) shows images from the soft agar colony formation assay that used SK-OV3 cells. The dark dots represent the colonies. Images of the negative control cells and the immediate effect POT1-KD cells were captured via digital phase-contrast microscopy at 4x magnification; scale bar: 1000 μ m. ∗∗ P
    Polymerase Chain Reaction Qrt Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 80/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher reverse transcriptase polymerase chain reaction qrt pcr
    POT1 mRNA expression as determined by <t>qRT-PCR</t> (a) and POT1 protein expression as determined by western blot analysis (b) in shRNA-infected human ovarian cancer SK-OV3 cells. POT1-KD significantly decreased cell proliferation (c) and colony formation (d) in the early POT1-KD SK-OV3 cells. “Negative control” indicates negative control SK-OV3 cells that express a nonspecific shRNA, and “POT1-KD” represents POT1-knockdown SK-OV3 cells that express POT1-specific shRNA. (c) shows the cPDs of the SK-OV3 cells. The cPDs of the negative control cells are depicted with black dots and lines, while the cPDs of the immediate effect POT1-KD cells are depicted with black squares and lines. (d) shows images from the soft agar colony formation assay that used SK-OV3 cells. The dark dots represent the colonies. Images of the negative control cells and the immediate effect POT1-KD cells were captured via digital phase-contrast microscopy at 4x magnification; scale bar: 1000 μ m. ∗∗ P
    Reverse Transcriptase Polymerase Chain Reaction Qrt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad real time polymerase chain reaction qrt pcr
    Elevated levels of Mg 2+ in APP/PS1 transgenic mice decrease the expression of IL-1β. The APP/PS1 transgenic mice at the age of 4 months were administered Mg 2+ (100 mg/kg/d) for 2 months before collecting the brain ( a ). In select experiments, the brains of APP/PS1 transgenic mice at the age of 3 months were harvested and sectioned (400 μm) using a cryostat ( b ). In separate experiments, the left cerebral ventricle was injected with Mg 2+ (2 μg/5 μl) or vehicle (PBS) and the right cerebral ventricle was injected (i.c.v.) with D1A cells, which was pre-transfected with IL-1β promoter in the right cerebral ventricle ( c ). In distinct experiments, the left cerebral ventricle was injected with Mg 2+ (2 μg/5 μl) or vehicle (PBS) before staining with IL-1β antibody and scanning under two-photon microscopy ( d ). The immunoreactivity of IL-1β was determined by immunohistochemistry using an anti-IL-1β antibody (a left panel, b). These images are representative of six independent experiments, all with similar results. IL-1β protein and mRNA levels were determined by <t>qRT-PCR</t> and IL-1β enzyme immunoassay kits, respectively (a right panel). The total amounts of GAPDH and protein served as an internal control. The experimental cartoon and real surgery images are shown (c, d upper panel). Luciferase activities from the different groups of mice were measured using a live animal imaging system (c lower panel). The immunofluorescence of IL-1β was scanned using a two-photon microscope (d lower panel). The data represent the means ± S.E. of three independent experiments. * p
    Real Time Polymerase Chain Reaction Qrt Pcr, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 244 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa real time polymerase chain reaction qrt pcr
    Relative expression of MabZIP genes in BX and FJ by <t>qRT-PCR.</t> ( A – D ) expression patterns of MabZIP61, MabZIP75, MabZIP25 and MabZIP11 in different organs. The mRNA fold difference was relative to that of BX-root samples used as calibrator. ( E – H ) expression patterns of MabZIP61, MabZIP75, MabZIP25 and MabZIP11 in different stages of fruit development and ripening. The mRNA fold difference was relative to that of BX-0DAF samples used as calibrator. ( I – L ) expression patterns of MabZIP3, MabZIP93, MabZIP40 and MabZIP101 in response to cold, salt and osmotic stresses. The mRNA fold difference was relative to that of untreated samples used as calibrator. Data are means ± SD of n = 3 biological replicates.
    Real Time Polymerase Chain Reaction Qrt Pcr, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 231 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher reverse transcription polymerase chain reaction qrt pcr
    Relative expression of MabZIP genes in BX and FJ by <t>qRT-PCR.</t> ( A – D ) expression patterns of MabZIP61, MabZIP75, MabZIP25 and MabZIP11 in different organs. The mRNA fold difference was relative to that of BX-root samples used as calibrator. ( E – H ) expression patterns of MabZIP61, MabZIP75, MabZIP25 and MabZIP11 in different stages of fruit development and ripening. The mRNA fold difference was relative to that of BX-0DAF samples used as calibrator. ( I – L ) expression patterns of MabZIP3, MabZIP93, MabZIP40 and MabZIP101 in response to cold, salt and osmotic stresses. The mRNA fold difference was relative to that of untreated samples used as calibrator. Data are means ± SD of n = 3 biological replicates.
    Reverse Transcription Polymerase Chain Reaction Qrt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 540 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche qrt pcr quantitative polymerase chain reaction qrt pcr
    <t>qRT-PCR</t> analysis of PTPC transcript levels in developing and germinating sorghum seeds. To normalize the values, 18S RNA was used as internal control in each sample. Data were normalized to stage VI for developing seeds (A, C, E), or 14h post-imbibition germinating seeds (B, D, F). Results are means ±SE of at least three independent experiments. Mean values that are not significantly different ( P
    Qrt Pcr Quantitative Polymerase Chain Reaction Qrt Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quantitative reverse transcription polymerase chain reaction qrt pcr
    ( A ) Hierarchal clustering of differentially expressed microRNAs. Forty-six differentially expressed microRNAs (for up- and down-regulation, respectively) compared to the negative control group in at least one sample were clustered hierarchically. Each rows represent individual microRNAs. The expression ratio representing colour ranges from green (low) to red (high), as indicated by the scale bar. The column bar chart shows the relative expression level of selected microRNAs by <t>qRT-PCR</t> (right column, white) as compared to the fold changes of the corresponding microRNA (left column, grey); ( B ) MicroRNAs expression of Group B versus Group A on the first day of treatment; ( C ) Group B versus Group A on the third day of treatment; ( D ) MicroRNAs expression of on the fifth day of treatment. U87 was used as housekeeping gene. Bars represent the mean ± standard error of three independent experiments; and ( E ) qRT-PCR product was confirmed by gel electrophoresis at 90 V for 40 min.
    Quantitative Reverse Transcription Polymerase Chain Reaction Qrt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1303 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genewiz reverse transcription polymerase chain reaction qrt pcr validation
    FA2H expression among breast cancer cell lines. (A) FA2H gene expression among different breast cancer cell lines by <t>qRT-PCR.</t> (B) FA2H protein expression among different breast cancer cell lines by Western-blotting. (C) FA2H protein expression quantified using the ImageJ software. (D) FA2H gene expression across 56 breast cancer cell lines ( 26 ).
    Reverse Transcription Polymerase Chain Reaction Qrt Pcr Validation, supplied by Genewiz, used in various techniques. Bioz Stars score: 83/100, based on 7 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
    <t>QRT-PCR</t> for 27-hydroxylase and ABCA1 message in NS398-treated THP-1 cells exposed to prostaglandins. (a) 27-Hydroxylase message is decreased by the COX-2 inhibitor NS398 and this decrease is reversed by prostaglandins E 1 , E 2 , and D 2 . THP-1 human monocytes were exposed to the following conditions represented by the six bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE 1 (0.1 μM) + NS398 (50 μM), (4) PGE 2 (0.1 μM) + NS398 (50 μM), (5) PGE 1 (0.1 μM) + PGE 2 (0.1 μM) + NS398 (50 μM), and (6) PGD 2 (14 μM) + NS398 (50 μM) (all 18-hour exposures). Cells were extracted for total RNA and were evaluated for 27-hydroxylase mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) ABCA1 message is decreased by the COX-2 inhibitor NS398 and this decrease is reversed by prostaglandins E 1 , E 2 , and D 2 . THP-1 human monocytes were exposed to the following conditions represented by the six bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE 1 (0.1 μM) + NS398 (50 μM), (4) PGE 2 (0.1 μM) + NS398 (50 μM), (5) PGE 1 (0.1 μM) + PGE 2 (0.1 μM) + NS398 (50 μM), and (6) PGD 2 (14 μM) + NS398 (50 μM) (all 18-hour exposures). Cells were extracted for total RNA and were evaluated for ABCA1 mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. ** p
    Quantitative Real Time Polymerase Chain Reaction Qrt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2201 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies quantitative real time polymerase chain reaction qrt pcr
    <t>QRT-PCR</t> for 27-hydroxylase and ABCA1 message in NS398-treated THP-1 cells exposed to prostaglandins. (a) 27-Hydroxylase message is decreased by the COX-2 inhibitor NS398 and this decrease is reversed by prostaglandins E 1 , E 2 , and D 2 . THP-1 human monocytes were exposed to the following conditions represented by the six bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE 1 (0.1 μM) + NS398 (50 μM), (4) PGE 2 (0.1 μM) + NS398 (50 μM), (5) PGE 1 (0.1 μM) + PGE 2 (0.1 μM) + NS398 (50 μM), and (6) PGD 2 (14 μM) + NS398 (50 μM) (all 18-hour exposures). Cells were extracted for total RNA and were evaluated for 27-hydroxylase mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) ABCA1 message is decreased by the COX-2 inhibitor NS398 and this decrease is reversed by prostaglandins E 1 , E 2 , and D 2 . THP-1 human monocytes were exposed to the following conditions represented by the six bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE 1 (0.1 μM) + NS398 (50 μM), (4) PGE 2 (0.1 μM) + NS398 (50 μM), (5) PGE 1 (0.1 μM) + PGE 2 (0.1 μM) + NS398 (50 μM), and (6) PGD 2 (14 μM) + NS398 (50 μM) (all 18-hour exposures). Cells were extracted for total RNA and were evaluated for ABCA1 mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. ** p
    Quantitative Real Time Polymerase Chain Reaction Qrt Pcr, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 87/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche polymerase chain reaction qrt pcr analysis
    <t>QRT-PCR</t> for 27-hydroxylase and ABCA1 message in NS398-treated THP-1 cells exposed to prostaglandins. (a) 27-Hydroxylase message is decreased by the COX-2 inhibitor NS398 and this decrease is reversed by prostaglandins E 1 , E 2 , and D 2 . THP-1 human monocytes were exposed to the following conditions represented by the six bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE 1 (0.1 μM) + NS398 (50 μM), (4) PGE 2 (0.1 μM) + NS398 (50 μM), (5) PGE 1 (0.1 μM) + PGE 2 (0.1 μM) + NS398 (50 μM), and (6) PGD 2 (14 μM) + NS398 (50 μM) (all 18-hour exposures). Cells were extracted for total RNA and were evaluated for 27-hydroxylase mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) ABCA1 message is decreased by the COX-2 inhibitor NS398 and this decrease is reversed by prostaglandins E 1 , E 2 , and D 2 . THP-1 human monocytes were exposed to the following conditions represented by the six bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE 1 (0.1 μM) + NS398 (50 μM), (4) PGE 2 (0.1 μM) + NS398 (50 μM), (5) PGE 1 (0.1 μM) + PGE 2 (0.1 μM) + NS398 (50 μM), and (6) PGD 2 (14 μM) + NS398 (50 μM) (all 18-hour exposures). Cells were extracted for total RNA and were evaluated for ABCA1 mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. ** p
    Polymerase Chain Reaction Qrt Pcr Analysis, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad quantitative real time polymerase chain reaction qrt pcr
    The preventive effects of AMPK on the senescence associated with NAD + synthesis. (A) Cellular concentrations of NAD + on day 3 or day 5 after H 2 O 2 treatment. (B) Representative images of SA ‐β‐Gal staining of cells (left) and percentage of SA ‐β‐Gal‐positive cells (right). (C) Cellular concentrations of NAD + in the cells incubated with nicotinamide mononucleotide ( NMN ) for 3 days after H 2 O 2 treatment. (D) H 2 O 2 ‐treated cells were incubated with Met (10 mM ), BBR (10 μM), or CC (10 μM) for 3 days. The concentrations of NAD + are shown. (E) A schematic diagram of two pathways of NAD + synthesis. Red ones indicate rate‐limiting enzymes, and blue ones illustrate the enzyme inhibitors we used. (F) Relative fold‐changes in the mRNA levels of NAMPT , as monitored by <t>qRT</t> ‐ <t>PCR</t> (left), and representative images from immunoblot assays against NAMPT are shown (right). The ratio of NAMPT to actin was quantified by densitometry, based on the immunoblot images from three independent experiments. (G) Cells were incubated with Met, BBR , and CC for 3 days. Representative images from immunoblots against NAMPT are shown. The ratio of NAMPT to β‐actin was quantified by densitometry based on the immunoblot images from three independent experiments. (H) H 2 O 2 ‐treated NIH 3T3 cells were incubated with NAMPT inhibitor ( FK 866, 5 nM ), QPRT inhibitor ( PHTH , 1 mM ), Met (10 mM ), and BBR (10 μM) alone or in combination as indicated for 3 days. The percentages of SA ‐β‐Gal‐positive cells are shown. (I) The mRNA levels of NMNAT 1 , NMNAT 2 , and NMNAT 3 genes in the cells infected with corresponding lentivirus‐sh RNA or nontargeting sh RNA (sh CON ) as determined by qRT ‐ PCR . (J) Cellular concentrations of NAD + in sh RNA ‐infected cells. (K) Cells infected with sh RNA s were treated with H 2 O 2 and incubated with BBR for 3 days. Representative images of SA ‐β‐Gal staining of cells (left) and percentages of SA ‐β‐Gal‐positive cells (right). * P
    Quantitative Real Time Polymerase Chain Reaction Qrt Pcr, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 560 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche transcription polymerase chain reaction qrt pcr
    The preventive effects of AMPK on the senescence associated with NAD + synthesis. (A) Cellular concentrations of NAD + on day 3 or day 5 after H 2 O 2 treatment. (B) Representative images of SA ‐β‐Gal staining of cells (left) and percentage of SA ‐β‐Gal‐positive cells (right). (C) Cellular concentrations of NAD + in the cells incubated with nicotinamide mononucleotide ( NMN ) for 3 days after H 2 O 2 treatment. (D) H 2 O 2 ‐treated cells were incubated with Met (10 mM ), BBR (10 μM), or CC (10 μM) for 3 days. The concentrations of NAD + are shown. (E) A schematic diagram of two pathways of NAD + synthesis. Red ones indicate rate‐limiting enzymes, and blue ones illustrate the enzyme inhibitors we used. (F) Relative fold‐changes in the mRNA levels of NAMPT , as monitored by <t>qRT</t> ‐ <t>PCR</t> (left), and representative images from immunoblot assays against NAMPT are shown (right). The ratio of NAMPT to actin was quantified by densitometry, based on the immunoblot images from three independent experiments. (G) Cells were incubated with Met, BBR , and CC for 3 days. Representative images from immunoblots against NAMPT are shown. The ratio of NAMPT to β‐actin was quantified by densitometry based on the immunoblot images from three independent experiments. (H) H 2 O 2 ‐treated NIH 3T3 cells were incubated with NAMPT inhibitor ( FK 866, 5 nM ), QPRT inhibitor ( PHTH , 1 mM ), Met (10 mM ), and BBR (10 μM) alone or in combination as indicated for 3 days. The percentages of SA ‐β‐Gal‐positive cells are shown. (I) The mRNA levels of NMNAT 1 , NMNAT 2 , and NMNAT 3 genes in the cells infected with corresponding lentivirus‐sh RNA or nontargeting sh RNA (sh CON ) as determined by qRT ‐ PCR . (J) Cellular concentrations of NAD + in sh RNA ‐infected cells. (K) Cells infected with sh RNA s were treated with H 2 O 2 and incubated with BBR for 3 days. Representative images of SA ‐β‐Gal staining of cells (left) and percentages of SA ‐β‐Gal‐positive cells (right). * P
    Transcription Polymerase Chain Reaction Qrt Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 84/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The preventive effects of AMPK on the senescence associated with NAD + synthesis. (A) Cellular concentrations of NAD + on day 3 or day 5 after H 2 O 2 treatment. (B) Representative images of SA ‐β‐Gal staining of cells (left) and percentage of SA ‐β‐Gal‐positive cells (right). (C) Cellular concentrations of NAD + in the cells incubated with nicotinamide mononucleotide ( NMN ) for 3 days after H 2 O 2 treatment. (D) H 2 O 2 ‐treated cells were incubated with Met (10 mM ), BBR (10 μM), or CC (10 μM) for 3 days. The concentrations of NAD + are shown. (E) A schematic diagram of two pathways of NAD + synthesis. Red ones indicate rate‐limiting enzymes, and blue ones illustrate the enzyme inhibitors we used. (F) Relative fold‐changes in the mRNA levels of NAMPT , as monitored by <t>qRT</t> ‐ <t>PCR</t> (left), and representative images from immunoblot assays against NAMPT are shown (right). The ratio of NAMPT to actin was quantified by densitometry, based on the immunoblot images from three independent experiments. (G) Cells were incubated with Met, BBR , and CC for 3 days. Representative images from immunoblots against NAMPT are shown. The ratio of NAMPT to β‐actin was quantified by densitometry based on the immunoblot images from three independent experiments. (H) H 2 O 2 ‐treated NIH 3T3 cells were incubated with NAMPT inhibitor ( FK 866, 5 nM ), QPRT inhibitor ( PHTH , 1 mM ), Met (10 mM ), and BBR (10 μM) alone or in combination as indicated for 3 days. The percentages of SA ‐β‐Gal‐positive cells are shown. (I) The mRNA levels of NMNAT 1 , NMNAT 2 , and NMNAT 3 genes in the cells infected with corresponding lentivirus‐sh RNA or nontargeting sh RNA (sh CON ) as determined by qRT ‐ PCR . (J) Cellular concentrations of NAD + in sh RNA ‐infected cells. (K) Cells infected with sh RNA s were treated with H 2 O 2 and incubated with BBR for 3 days. Representative images of SA ‐β‐Gal staining of cells (left) and percentages of SA ‐β‐Gal‐positive cells (right). * P
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    Inhibition of LINC01116 suppressed cell proliferation and migration in NPC cells. A, <t>qRT‐PCR</t> result of LINC01116 expression in NPC cell lines and the normal HNEpC cells. B, qRT‐PCR result of LINC01116 expression in CNE2 and 5‐8F cells under the transfection of shCtrl or two shRNAs against LINC01116. C, CCK‐8 assay was performed to evaluate cell viability in above cells. D, Cell proliferation was examined by EdU assay. E, Transwell assay for the assessment of cell migration in NPC cells with or without LINC01116 inhibition. * P
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    Inhibition of LINC01116 suppressed cell proliferation and migration in NPC cells. A, <t>qRT‐PCR</t> result of LINC01116 expression in NPC cell lines and the normal HNEpC cells. B, qRT‐PCR result of LINC01116 expression in CNE2 and 5‐8F cells under the transfection of shCtrl or two shRNAs against LINC01116. C, CCK‐8 assay was performed to evaluate cell viability in above cells. D, Cell proliferation was examined by EdU assay. E, Transwell assay for the assessment of cell migration in NPC cells with or without LINC01116 inhibition. * P
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    Inhibition of LINC01116 suppressed cell proliferation and migration in NPC cells. A, <t>qRT‐PCR</t> result of LINC01116 expression in NPC cell lines and the normal HNEpC cells. B, qRT‐PCR result of LINC01116 expression in CNE2 and 5‐8F cells under the transfection of shCtrl or two shRNAs against LINC01116. C, CCK‐8 assay was performed to evaluate cell viability in above cells. D, Cell proliferation was examined by EdU assay. E, Transwell assay for the assessment of cell migration in NPC cells with or without LINC01116 inhibition. * P
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    Inhibition of LINC01116 suppressed cell proliferation and migration in NPC cells. A, <t>qRT‐PCR</t> result of LINC01116 expression in NPC cell lines and the normal HNEpC cells. B, qRT‐PCR result of LINC01116 expression in CNE2 and 5‐8F cells under the transfection of shCtrl or two shRNAs against LINC01116. C, CCK‐8 assay was performed to evaluate cell viability in above cells. D, Cell proliferation was examined by EdU assay. E, Transwell assay for the assessment of cell migration in NPC cells with or without LINC01116 inhibition. * P
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    Graphic representation of NF-κB mRNA expression levels via <t>qRT-PCR</t> in the three groups. NF-κB mRNA expression levels in the model and propofol groups are significantly increased compared with those in the control group (**P
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    Graphic representation of NF-κB mRNA expression levels via <t>qRT-PCR</t> in the three groups. NF-κB mRNA expression levels in the model and propofol groups are significantly increased compared with those in the control group (**P
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    Site-specific contribution to differential gene expression in KD. (A) Comparison of gene expression between laser-captured dermal tissue ( in situ ) and fibroblasts for both keloid and normal skin. <t>qRT-PCR</t> graph for both TGFβ1 and CTGF (additional examples found in S2A Fig ). All data are mean ± SEM for at least three independent experiments. B) qRT-PCR for TGFβ1 and interleukin-8 ( IL-8 ) showing relative contributions of different keloid sites to overall expression and comparison with normal skin (additional genes available in S2B Fig ). Data are mean ± SEM where * p-value
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    MACC1 SNPs L31V, S515L, and R804T and MACC1 expression in colorectal cells and tumors A). MACC1 mRNA expression was determined in 60 colorectal tumors by <t>qRT-PCR.</t> Box plots show the mRNA expression compared to MACC1 genotypes. SNPs have no effect on the MACC1 mRNA expression in these colorectal tumors. B ) SW480, DLD1, HCT116 colorectal cancer cells were transfected with pcDNA3.1/MACC1/wt, pcDNA3.1/MACC1/L31V, pcDNA3.1/MACC1/S515L and pcDNA3.1/MACC1/R804T. The MACC1 mRNA expression in these cell clones was measured by qRT-PCR and normalized to the MACC1 mRNA expression of parental cells. Additionally, MACC1 protein expression was determined. β-tubulin was used as a loading control. SNPs have no effect on the MACC1 mRNA and protein expression in SW480, DLD1 and HCT116 colorectal cancer cells.
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    <t>QRT-PCR</t> and Western blot assays showed no significant changes in the mRNA ( A ) or protein levels ( B ) of SMAD2 in overexpressed or knocked down circSMAD2 HepG2 cells. Note: Data are presented as mean ± SEM. Abbreviations: QRT-PCR, quantitative real-time polymerase chain reaction; circSMAD2, circRNA SMAD2; SEM, standard error of the mean; NC, negative control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
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    PUM1 regulates ATXN1 levels via a highly conserved binding motif (A) Schematic representation of Human ATXN1 -3′UTR showing three putative PUM1 binding motifs (gray and red boxes) and their conservation in different species. The numbers indicate positions of PUM1 motifs in the human ATXN1 -3′UTR. (B) ATXN1 mRNA quantification by <t>qRT-PCR</t> in HEK293T cells upon overexpression (left panel) or knockdown (si PUM1 -81 and -82) (right panel) of PUM1 . The destination-cloning vector (Control), scrambled siRNAs (siScr.) and cel-miR-67 were used as negative controls. The housekeeping gene GAPDH was used to normalize the expression of genes in all the qRT-PCR experiments. (C) Luciferase assay in HEK293 cells overexpressing the reporter construct harboring the full length ATXN1 -3′UTR. In these conditions, we overexpressed (left panel) or decreased expression (right panel) of PUM1 . (D) Luciferase assay in HEK293T cells transfected with single WT and mutant (MUT) putative PUM1 binding site on the ATXN1 -3′UTR. The position of cloned regions for each PUM1 binding motif are indicated. The mutagenized nucleotides are highlighted in blue. In panels C and D the destination-vector (control), RNAi scramble (siScramble) and cel-miR-67 were used as negative controls; miR-101a was used as positive control. (RL) Renilla, (FL) Firefly luciferase. All the experiments were performed in triplicate (data represent mean ± S.E.M.). P values were calculated by Student’s t-test. Statistical significance is indicated as follows: * P
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    PUM1 regulates ATXN1 levels via a highly conserved binding motif (A) Schematic representation of Human ATXN1 -3′UTR showing three putative PUM1 binding motifs (gray and red boxes) and their conservation in different species. The numbers indicate positions of PUM1 motifs in the human ATXN1 -3′UTR. (B) ATXN1 mRNA quantification by <t>qRT-PCR</t> in HEK293T cells upon overexpression (left panel) or knockdown (si PUM1 -81 and -82) (right panel) of PUM1 . The destination-cloning vector (Control), scrambled siRNAs (siScr.) and cel-miR-67 were used as negative controls. The housekeeping gene GAPDH was used to normalize the expression of genes in all the qRT-PCR experiments. (C) Luciferase assay in HEK293 cells overexpressing the reporter construct harboring the full length ATXN1 -3′UTR. In these conditions, we overexpressed (left panel) or decreased expression (right panel) of PUM1 . (D) Luciferase assay in HEK293T cells transfected with single WT and mutant (MUT) putative PUM1 binding site on the ATXN1 -3′UTR. The position of cloned regions for each PUM1 binding motif are indicated. The mutagenized nucleotides are highlighted in blue. In panels C and D the destination-vector (control), RNAi scramble (siScramble) and cel-miR-67 were used as negative controls; miR-101a was used as positive control. (RL) Renilla, (FL) Firefly luciferase. All the experiments were performed in triplicate (data represent mean ± S.E.M.). P values were calculated by Student’s t-test. Statistical significance is indicated as follows: * P
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    PUM1 regulates ATXN1 levels via a highly conserved binding motif (A) Schematic representation of Human ATXN1 -3′UTR showing three putative PUM1 binding motifs (gray and red boxes) and their conservation in different species. The numbers indicate positions of PUM1 motifs in the human ATXN1 -3′UTR. (B) ATXN1 mRNA quantification by <t>qRT-PCR</t> in HEK293T cells upon overexpression (left panel) or knockdown (si PUM1 -81 and -82) (right panel) of PUM1 . The destination-cloning vector (Control), scrambled siRNAs (siScr.) and cel-miR-67 were used as negative controls. The housekeeping gene GAPDH was used to normalize the expression of genes in all the qRT-PCR experiments. (C) Luciferase assay in HEK293 cells overexpressing the reporter construct harboring the full length ATXN1 -3′UTR. In these conditions, we overexpressed (left panel) or decreased expression (right panel) of PUM1 . (D) Luciferase assay in HEK293T cells transfected with single WT and mutant (MUT) putative PUM1 binding site on the ATXN1 -3′UTR. The position of cloned regions for each PUM1 binding motif are indicated. The mutagenized nucleotides are highlighted in blue. In panels C and D the destination-vector (control), RNAi scramble (siScramble) and cel-miR-67 were used as negative controls; miR-101a was used as positive control. (RL) Renilla, (FL) Firefly luciferase. All the experiments were performed in triplicate (data represent mean ± S.E.M.). P values were calculated by Student’s t-test. Statistical significance is indicated as follows: * P
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    miR-628 suppressed the mRNA and protein expression of SOS1 . Notes: The breast CSCs of MDA-MB-231 ( A ) and MCF-7 ( B ) cells were transfected with the miR-628 mimic and miR-628 inhibitor, and SOS1 mRNA level was determined by <t>qRT-PCR.</t> ( C ) The breast CSCs of MDA-MB-231 and MCF-7 cells were transfected with miR-628 mimic and miR-628 inhibitor, and SOS1 protein level was analyzed by Western blotting. The protein expression levels were statistically analyzed by quantitating the intensity of the protein bands relative to that of the internal loading control (α-tubulin). Values indicate mean ± SD; * P
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    Image Search Results


    HIF-1α regulated the expression of CCL2 in PDAC cells, PC, N and H indicate positive control, normoxia, and hypoxia, respectively. ( A ) HPDE6-C7,BxPC3 and MiaPaca-2 cells were transfected with siHIF-1α (50 nmol/L) and pcDNA3.1-HIF-1α plasmids (4 µg) for 48 h, respectively; the mRNA expression levels of HIF-1α and CCL2 were assessed by qRT-PCR. The experiments were performed thrice independently. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Hypoxia Inducible Factor 1 (HIF-1) Recruits Macrophage to Activate Pancreatic Stellate Cells in Pancreatic Ductal Adenocarcinoma

    doi: 10.3390/ijms17060799

    Figure Lengend Snippet: HIF-1α regulated the expression of CCL2 in PDAC cells, PC, N and H indicate positive control, normoxia, and hypoxia, respectively. ( A ) HPDE6-C7,BxPC3 and MiaPaca-2 cells were transfected with siHIF-1α (50 nmol/L) and pcDNA3.1-HIF-1α plasmids (4 µg) for 48 h, respectively; the mRNA expression levels of HIF-1α and CCL2 were assessed by qRT-PCR. The experiments were performed thrice independently. * p

    Article Snippet: The reverse transcription polymerase chain reaction (qRT-PCR) system (TaKaRa Bio Group, Shiga, Japan) was utilized to obtain complementary DNA (cDNA).

    Techniques: Expressing, Positive Control, Transfection, Quantitative RT-PCR

    CDKI expression in human terminal erythroid differentiation. (A) RNA-seq data showing CDKI members’ expression (fragments per kilobase of transcript per million) at each distinct stage of human terminal erythroid differentiation. Baso-E, basophilic erythroblast; ortho-E, orthochromatic erythroblast; poly-E, polychromatic erythroblast; pro-E, proerythroblast. (B) Representative image of western blotting of p19 INK4d , p18 INK4c , and p27 KIP1 expression in whole-cell lysates prepared from erythroblasts cultured for different times (left). Quantitative analysis of data from 3 independent experiments of protein expression levels (right). GAPDH was used as a loading control. (C) qRT-PCR results showing p19 INK4d expression in erythroblasts infected with lentivirus containing control (Lucif shRNA) or p19 INK4d shRNA on day (D) 7, D 11, and D 15 of culture. The results were normalized to GAPDH mRNA. (D) Representative images of western blotting showing p19 INK4d expression levels in erythroblasts infected with Lucif shRNA or p19 INK4d shRNA (left). Quantitative analysis of protein expression data from 3 independent experiments (right). The results were normalized to GAPDH protein. Statistical analysis of data of 3 independent experiments and bar plot represents mean ± SD of triplicate samples. *** P

    Journal: Blood

    Article Title: Unexpected role for p19INK4d in posttranscriptional regulation of GATA1 and modulation of human terminal erythropoiesis

    doi: 10.1182/blood-2016-09-739268

    Figure Lengend Snippet: CDKI expression in human terminal erythroid differentiation. (A) RNA-seq data showing CDKI members’ expression (fragments per kilobase of transcript per million) at each distinct stage of human terminal erythroid differentiation. Baso-E, basophilic erythroblast; ortho-E, orthochromatic erythroblast; poly-E, polychromatic erythroblast; pro-E, proerythroblast. (B) Representative image of western blotting of p19 INK4d , p18 INK4c , and p27 KIP1 expression in whole-cell lysates prepared from erythroblasts cultured for different times (left). Quantitative analysis of data from 3 independent experiments of protein expression levels (right). GAPDH was used as a loading control. (C) qRT-PCR results showing p19 INK4d expression in erythroblasts infected with lentivirus containing control (Lucif shRNA) or p19 INK4d shRNA on day (D) 7, D 11, and D 15 of culture. The results were normalized to GAPDH mRNA. (D) Representative images of western blotting showing p19 INK4d expression levels in erythroblasts infected with Lucif shRNA or p19 INK4d shRNA (left). Quantitative analysis of protein expression data from 3 independent experiments (right). The results were normalized to GAPDH protein. Statistical analysis of data of 3 independent experiments and bar plot represents mean ± SD of triplicate samples. *** P

    Article Snippet: RNA extracted from primary cultured human erythroid cells using TRIzol reagent (Invitrogen) was reverse transcripted with SuperScript first-strand synthesis system (Invitrogen) and amplified by quantitative polymerase chain reaction (qRT-PCR) with the Cycler (Bio-Rad) and appropriate primer pairs.

    Techniques: Expressing, RNA Sequencing Assay, Western Blot, Cell Culture, Quantitative RT-PCR, Infection, shRNA

    p19 INK4d knockdown decreased GATA1 protein expression and ectopic GATA1 expression rescues the differentiation delay. (A) Representative images of western blotting analysis showing GATA1, 4.1R, tropomodulin (Tmod), HBG, p18 INK4c , KLF1, and CD44 expression in erythroblasts infected with Lucif shRNA or p19 INK4d shRNA (day 11 cells) (left). Quantitative analysis of protein expression data from 3 independent experiments (right). GAPDH was used as a loading control and the results were normalized to GAPDH protein expression. (B) qRT-PCR analysis of GATA1 mRNA levels in erythroblasts infected with Lucif shRNA or p19 INK4d shRNA. The results were normalized to GAPDH mRNA. (C) Representative data of flow cytometry analysis of erythroblasts infected with Lucif shRNA, p19 INK4d shRNA, and p19 INK4d shRNA combined with GATA1 overexpression. GPA expression was monitored on D 9 cells (left) and expression of band 3 and α4-integrin on D 15 cells (right). The red numbers indicate statistical analysis of GPA-positive rate from 3 independent experiments. (D) Representative images of western blotting showing GATA1 levels in whole cell lysates prepared from erythroblasts (left). Quantitative analysis of expression data from 3 independent experiments (right). GAPDH was used as a loading control. Statistical analysis of data from 3 independent experiments and bar plot represents mean ± SD of triplicate samples. * P

    Journal: Blood

    Article Title: Unexpected role for p19INK4d in posttranscriptional regulation of GATA1 and modulation of human terminal erythropoiesis

    doi: 10.1182/blood-2016-09-739268

    Figure Lengend Snippet: p19 INK4d knockdown decreased GATA1 protein expression and ectopic GATA1 expression rescues the differentiation delay. (A) Representative images of western blotting analysis showing GATA1, 4.1R, tropomodulin (Tmod), HBG, p18 INK4c , KLF1, and CD44 expression in erythroblasts infected with Lucif shRNA or p19 INK4d shRNA (day 11 cells) (left). Quantitative analysis of protein expression data from 3 independent experiments (right). GAPDH was used as a loading control and the results were normalized to GAPDH protein expression. (B) qRT-PCR analysis of GATA1 mRNA levels in erythroblasts infected with Lucif shRNA or p19 INK4d shRNA. The results were normalized to GAPDH mRNA. (C) Representative data of flow cytometry analysis of erythroblasts infected with Lucif shRNA, p19 INK4d shRNA, and p19 INK4d shRNA combined with GATA1 overexpression. GPA expression was monitored on D 9 cells (left) and expression of band 3 and α4-integrin on D 15 cells (right). The red numbers indicate statistical analysis of GPA-positive rate from 3 independent experiments. (D) Representative images of western blotting showing GATA1 levels in whole cell lysates prepared from erythroblasts (left). Quantitative analysis of expression data from 3 independent experiments (right). GAPDH was used as a loading control. Statistical analysis of data from 3 independent experiments and bar plot represents mean ± SD of triplicate samples. * P

    Article Snippet: RNA extracted from primary cultured human erythroid cells using TRIzol reagent (Invitrogen) was reverse transcripted with SuperScript first-strand synthesis system (Invitrogen) and amplified by quantitative polymerase chain reaction (qRT-PCR) with the Cycler (Bio-Rad) and appropriate primer pairs.

    Techniques: Expressing, Western Blot, Infection, shRNA, Quantitative RT-PCR, Flow Cytometry, Cytometry, Over Expression

    Comparison of microarray and qRT-PCR data for glycan-related transcripts from mouse liver and kidney. RMA values from the GLYCOv2 gene chip microarray analysis are plotted against relative transcript abundance data from qRT-PCR analysis (normalized to Rpl4 ) for 149 glycan-related genes (plotted on a log 10 scale on both axes). Data obtained from mouse liver ( upper panel ) and kidney ( lower panel ) are shown. Transcripts determined as present in both methods (see “Experimental Procedures”) are shown as filled circles . Transcripts that were called as absent in the microarray data set are shown as open circles, and transcripts that were not detected by either method are shown as triangles . The correlation coefficient ( solid line ) for transcripts scored as present in both analyses were R 2 = 0.39 for liver ( upper panel ) and R 2 = 0.24 for kidney ( lower panel ). The dashed horizontal line represents a best-fit line for the transcripts called as absent in the microarray analysis indicating a lower limit of detection in this method. The dotted vertical line represents the lower limit of detection for qRT-PCR analysis.

    Journal: The Journal of Biological Chemistry

    Article Title: Regulation of Glycan Structures in Animal Tissues

    doi: 10.1074/jbc.M801964200

    Figure Lengend Snippet: Comparison of microarray and qRT-PCR data for glycan-related transcripts from mouse liver and kidney. RMA values from the GLYCOv2 gene chip microarray analysis are plotted against relative transcript abundance data from qRT-PCR analysis (normalized to Rpl4 ) for 149 glycan-related genes (plotted on a log 10 scale on both axes). Data obtained from mouse liver ( upper panel ) and kidney ( lower panel ) are shown. Transcripts determined as present in both methods (see “Experimental Procedures”) are shown as filled circles . Transcripts that were called as absent in the microarray data set are shown as open circles, and transcripts that were not detected by either method are shown as triangles . The correlation coefficient ( solid line ) for transcripts scored as present in both analyses were R 2 = 0.39 for liver ( upper panel ) and R 2 = 0.24 for kidney ( lower panel ). The dashed horizontal line represents a best-fit line for the transcripts called as absent in the microarray analysis indicating a lower limit of detection in this method. The dotted vertical line represents the lower limit of detection for qRT-PCR analysis.

    Article Snippet: Following the amplification and melt curve analysis, data were set to a common threshold, and the efficiency of the primer pair was determined from the slope of the standard curve using software supplied with the qRT-PCR instrumentation (Bio-Rad).

    Techniques: Microarray, Quantitative RT-PCR, Chromatin Immunoprecipitation

    IL-1-mediated enhancement of secretion and expression of IL-9 is inhibited by sialoL (A) Naïve CD4 + T cells from BALB/c mice were stimulated (anti-CD3/CD28) under Th9-skewing conditions in the absence or presence of sialoL (3 μM) or IL-1β (10ng/ml) and a combination of both. IL-9 was determined after 72h by ELISA. (B) Naïve CD4 + T cells from BALB/c mice were stimulated (anti-CD3/CD28) under Th9-skewing conditions. After five days T cells were restimulated in the absence or presence of sialoL (3 μM) or IL-1β (10ng/ml) and a combination of both. IL-9 was determined after 48h by ELISA. (C) Naïve CD4 + T cells from BALB/c mice were stimulated (anti-CD3/CD28) under Th9-skewing conditions. After five days T cells were restimulated in the absence or presence of sialoL (3 μM) or IL-1β (10ng/ml) and a combination of both. Expression of Il9 was quantified after 48h by qRT-PCR. Similar results were obtained in four independent experiments.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: The tick salivary protein sialostatin L inhibits the Th9-derived production of the asthma-promoting cytokine interleukin-9 and is effective in the prevention of experimental asthma

    doi: 10.4049/jimmunol.1100529

    Figure Lengend Snippet: IL-1-mediated enhancement of secretion and expression of IL-9 is inhibited by sialoL (A) Naïve CD4 + T cells from BALB/c mice were stimulated (anti-CD3/CD28) under Th9-skewing conditions in the absence or presence of sialoL (3 μM) or IL-1β (10ng/ml) and a combination of both. IL-9 was determined after 72h by ELISA. (B) Naïve CD4 + T cells from BALB/c mice were stimulated (anti-CD3/CD28) under Th9-skewing conditions. After five days T cells were restimulated in the absence or presence of sialoL (3 μM) or IL-1β (10ng/ml) and a combination of both. IL-9 was determined after 48h by ELISA. (C) Naïve CD4 + T cells from BALB/c mice were stimulated (anti-CD3/CD28) under Th9-skewing conditions. After five days T cells were restimulated in the absence or presence of sialoL (3 μM) or IL-1β (10ng/ml) and a combination of both. Expression of Il9 was quantified after 48h by qRT-PCR. Similar results were obtained in four independent experiments.

    Article Snippet: Quantitative qRT-PCRs were performed using the following oligonucleotides: Murine HGPRT forward: 5′-GTT GGA TAC AGG CCA GAC TTT GTT G-3′ Murine HGPRT reverse: 5′GAG GGT AGG CTG GCC TAT AGG CT-3′ Murine IL-9 forward: 5′-CTG ATG ATT GTA CCA CAC CGT GC-3′ Murine IL-9 reverse: 5′-GCC TTT GCA TCT CTG TCT TCT GG-3′ Murine IL-13 forward: 5'-GGA GCT GAG CAA CAT CAC ACA-3' Murine IL-13 reverse: 5'-GGT CCT GTA GAT GGC ATT GCA-3' Oligonucleotides were chosen to span at least one intron at the level of genomic DNA. qRT-PCR analyses were performed in triplicates on an iCycler (Bio-Rad,) using the SYBR GreenER qPCR Supermix (Invitrogen).

    Techniques: Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

    SialoL impairs primary T cell-derived production of IL-9 but not T cell activation and proliferation Naïve CD4 + T cells from BALB/c mice were stimulated (anti-CD3/CD28) in the presence and absence of different concentrations of sialoL (0.75 μM, 1.5 μM, 3 μM) under Th9-skewing conditions. Production of IL-9 was determined by (A) ELISA, (B) FACS, and (C) qRT-PCR. (D) T cell activation was determined by FACS analyses of CD25 and CD122 cell surface expression. Shown is each time one representative from three independent experiments ± SD. (E) T cell proliferation was determined by counting the cell numbers on day five after primary activation.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: The tick salivary protein sialostatin L inhibits the Th9-derived production of the asthma-promoting cytokine interleukin-9 and is effective in the prevention of experimental asthma

    doi: 10.4049/jimmunol.1100529

    Figure Lengend Snippet: SialoL impairs primary T cell-derived production of IL-9 but not T cell activation and proliferation Naïve CD4 + T cells from BALB/c mice were stimulated (anti-CD3/CD28) in the presence and absence of different concentrations of sialoL (0.75 μM, 1.5 μM, 3 μM) under Th9-skewing conditions. Production of IL-9 was determined by (A) ELISA, (B) FACS, and (C) qRT-PCR. (D) T cell activation was determined by FACS analyses of CD25 and CD122 cell surface expression. Shown is each time one representative from three independent experiments ± SD. (E) T cell proliferation was determined by counting the cell numbers on day five after primary activation.

    Article Snippet: Quantitative qRT-PCRs were performed using the following oligonucleotides: Murine HGPRT forward: 5′-GTT GGA TAC AGG CCA GAC TTT GTT G-3′ Murine HGPRT reverse: 5′GAG GGT AGG CTG GCC TAT AGG CT-3′ Murine IL-9 forward: 5′-CTG ATG ATT GTA CCA CAC CGT GC-3′ Murine IL-9 reverse: 5′-GCC TTT GCA TCT CTG TCT TCT GG-3′ Murine IL-13 forward: 5'-GGA GCT GAG CAA CAT CAC ACA-3' Murine IL-13 reverse: 5'-GGT CCT GTA GAT GGC ATT GCA-3' Oligonucleotides were chosen to span at least one intron at the level of genomic DNA. qRT-PCR analyses were performed in triplicates on an iCycler (Bio-Rad,) using the SYBR GreenER qPCR Supermix (Invitrogen).

    Techniques: Derivative Assay, Activation Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, FACS, Quantitative RT-PCR, Expressing

    LPS / ATP activate NLRP 3 inflammasomes in VSMC s. Human primary VSMC s were treated with vehicle or the indicated concentrations of LPS for 16 hours and/or ATP for 1 hour. Some cells were transfected with control or NLRP 3‐specific si RNA or treated with vehicle DMSO or ZYVAD ‐ FMK , followed by treatment with LPS and/or ATP . The relative levels of NLRP 3 and cleaved caspase‐1 expression were determined by qRT ‐ PCR and Western blot. The distribution of HMGB 1 in the different groups of cells and their cultured supernatants were determined by Western blot and ELISA . Data are representative images or expressed as the mean± SD of each group from 4 separated experiments (Western blot) or 3 separated experiments with 4 duplicated wells ( qRT ‐ PCR and ELISA ). A, The levels of NLRP 3 mRNA transcripts. B, The levels of NLRP 3 proteins. C, The levels of cleaved caspase‐1. D, The levels of total HMGB 1. E, The levels of nuclear HMGB 1. F, The levels of cytoplasmic HMGB 1. G, The levels of HMGB 1 in the supernatants. * P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Activation of NLRP3 Inflammasome Promotes Foam Cell Formation in Vascular Smooth Muscle Cells and Atherogenesis Via HMGB1

    doi: 10.1161/JAHA.118.008596

    Figure Lengend Snippet: LPS / ATP activate NLRP 3 inflammasomes in VSMC s. Human primary VSMC s were treated with vehicle or the indicated concentrations of LPS for 16 hours and/or ATP for 1 hour. Some cells were transfected with control or NLRP 3‐specific si RNA or treated with vehicle DMSO or ZYVAD ‐ FMK , followed by treatment with LPS and/or ATP . The relative levels of NLRP 3 and cleaved caspase‐1 expression were determined by qRT ‐ PCR and Western blot. The distribution of HMGB 1 in the different groups of cells and their cultured supernatants were determined by Western blot and ELISA . Data are representative images or expressed as the mean± SD of each group from 4 separated experiments (Western blot) or 3 separated experiments with 4 duplicated wells ( qRT ‐ PCR and ELISA ). A, The levels of NLRP 3 mRNA transcripts. B, The levels of NLRP 3 proteins. C, The levels of cleaved caspase‐1. D, The levels of total HMGB 1. E, The levels of nuclear HMGB 1. F, The levels of cytoplasmic HMGB 1. G, The levels of HMGB 1 in the supernatants. * P

    Article Snippet: The relative levels of target gene mRNA transcripts were determined by quantitative real‐time polymerase chain reaction (qRT‐PCR) using the FastStart Universal SYBR Green Master (Roche Applied Sciences) and specific primers on a Roche LightCycler 480 Real Time PCR System.

    Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

    NLRP 3 inflammasome activation reduces LXR α and ABCA 1 expression in VSMC s. Following treatment with the indicated drugs and LPS / ATP stimulation, the relative levels of LXR α and ABCA 1 expression were determined qRT ‐ PCR and Western blot. Data are representative images or expressed as the mean± SD of each group from 3 separated experiments (Western blot) or 3 separated experiments with 3 duplicated wells ( qRT ‐ PCR ). A, qRT ‐ PCR analysis of LXR α and ABCA 1 mRNA transcripts. B, Western blot analysis of LXR α and ABCA 1 proteins. C through F, Treatment with ZYVAD ‐ FMK or NLRP 3 silencing mitigated the LPS / ATP ‐reduced LXR α and ABCA 1 expression. G and H, Treatment with T091317, the LXR α agonist enhanced ABCA 1 expression and mitigated the LPS / ATP ‐reduced ABCA 1 expression. * P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Activation of NLRP3 Inflammasome Promotes Foam Cell Formation in Vascular Smooth Muscle Cells and Atherogenesis Via HMGB1

    doi: 10.1161/JAHA.118.008596

    Figure Lengend Snippet: NLRP 3 inflammasome activation reduces LXR α and ABCA 1 expression in VSMC s. Following treatment with the indicated drugs and LPS / ATP stimulation, the relative levels of LXR α and ABCA 1 expression were determined qRT ‐ PCR and Western blot. Data are representative images or expressed as the mean± SD of each group from 3 separated experiments (Western blot) or 3 separated experiments with 3 duplicated wells ( qRT ‐ PCR ). A, qRT ‐ PCR analysis of LXR α and ABCA 1 mRNA transcripts. B, Western blot analysis of LXR α and ABCA 1 proteins. C through F, Treatment with ZYVAD ‐ FMK or NLRP 3 silencing mitigated the LPS / ATP ‐reduced LXR α and ABCA 1 expression. G and H, Treatment with T091317, the LXR α agonist enhanced ABCA 1 expression and mitigated the LPS / ATP ‐reduced ABCA 1 expression. * P

    Article Snippet: The relative levels of target gene mRNA transcripts were determined by quantitative real‐time polymerase chain reaction (qRT‐PCR) using the FastStart Universal SYBR Green Master (Roche Applied Sciences) and specific primers on a Roche LightCycler 480 Real Time PCR System.

    Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Western Blot

    HMGB 1 reduces LXR α and ABCA 1 expression in VSMC s. Following pretreatment with HMGB 1 and/or glycyrrhizin the HMGB 1 inhibitor and LPS / ATP stimulation, the relative levels of LXR α and ABCA 1 expression were determined by qRT ‐ PCR and Western blot. Data are representative images or expressed as the mean± SD of each group from 3 separated experiments (Western blot) or 3 separated experiments with 3 duplicated wells ( qRT ‐ PCR ). A and B, inhibition of HMGB 1 mitigated the LPS / ATP ‐reduced LXR α and ABCA 1 expression. C and D, HMGB 1 reduces the levels of LXR α and ABCA 1 expression in a dose‐dependent manner. E and F, Treatment with T091317 mitigated the HMGB 1‐reduced ABCA 1 expression. * P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Activation of NLRP3 Inflammasome Promotes Foam Cell Formation in Vascular Smooth Muscle Cells and Atherogenesis Via HMGB1

    doi: 10.1161/JAHA.118.008596

    Figure Lengend Snippet: HMGB 1 reduces LXR α and ABCA 1 expression in VSMC s. Following pretreatment with HMGB 1 and/or glycyrrhizin the HMGB 1 inhibitor and LPS / ATP stimulation, the relative levels of LXR α and ABCA 1 expression were determined by qRT ‐ PCR and Western blot. Data are representative images or expressed as the mean± SD of each group from 3 separated experiments (Western blot) or 3 separated experiments with 3 duplicated wells ( qRT ‐ PCR ). A and B, inhibition of HMGB 1 mitigated the LPS / ATP ‐reduced LXR α and ABCA 1 expression. C and D, HMGB 1 reduces the levels of LXR α and ABCA 1 expression in a dose‐dependent manner. E and F, Treatment with T091317 mitigated the HMGB 1‐reduced ABCA 1 expression. * P

    Article Snippet: The relative levels of target gene mRNA transcripts were determined by quantitative real‐time polymerase chain reaction (qRT‐PCR) using the FastStart Universal SYBR Green Master (Roche Applied Sciences) and specific primers on a Roche LightCycler 480 Real Time PCR System.

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

    NLRP 3 inflammasome‐dependent HMGB 1 secretion reduces LXR α and ABCA 1 expression through binding to RAGE in VSMC s. Following NLRP 3 silencing, pretreatment with RAP , a RAGE antagonist peptide and LPS / ATP stimulation in the presence or absence of Chol:Mβ CD , the cholesterol accumulation in VSMC s was characterized and the relative levels of LXR α and ABCA 1 expression were determined by qRT ‐ PCR and Western blot. Data are representative images or expressed as the mean± SD of each group from 3 separated experiments (Western blot) or 3 separated experiments with 3 duplicated wells ( qRT ‐ PCR and Cholesterol Efflux Fluorometric Assay). A through C, The cholesterol accumulation in VSMC s. D, The cholesterol efflux in VSMC s. E through H, The relative levels of LXR α and ABCA 1 expression in the different groups of VSMC s. * P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Activation of NLRP3 Inflammasome Promotes Foam Cell Formation in Vascular Smooth Muscle Cells and Atherogenesis Via HMGB1

    doi: 10.1161/JAHA.118.008596

    Figure Lengend Snippet: NLRP 3 inflammasome‐dependent HMGB 1 secretion reduces LXR α and ABCA 1 expression through binding to RAGE in VSMC s. Following NLRP 3 silencing, pretreatment with RAP , a RAGE antagonist peptide and LPS / ATP stimulation in the presence or absence of Chol:Mβ CD , the cholesterol accumulation in VSMC s was characterized and the relative levels of LXR α and ABCA 1 expression were determined by qRT ‐ PCR and Western blot. Data are representative images or expressed as the mean± SD of each group from 3 separated experiments (Western blot) or 3 separated experiments with 3 duplicated wells ( qRT ‐ PCR and Cholesterol Efflux Fluorometric Assay). A through C, The cholesterol accumulation in VSMC s. D, The cholesterol efflux in VSMC s. E through H, The relative levels of LXR α and ABCA 1 expression in the different groups of VSMC s. * P

    Article Snippet: The relative levels of target gene mRNA transcripts were determined by quantitative real‐time polymerase chain reaction (qRT‐PCR) using the FastStart Universal SYBR Green Master (Roche Applied Sciences) and specific primers on a Roche LightCycler 480 Real Time PCR System.

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

    Primer Design and qRT-PCR

    Journal: Plastic and reconstructive surgery

    Article Title: Beta-catenin-dependent Wnt signaling: a pathway in acute cutaneous wounding

    doi: 10.1097/PRS.0000000000004170

    Figure Lengend Snippet: Primer Design and qRT-PCR

    Article Snippet: All other reagents for quantitative real-time polymerase chain reaction (qRT-PCR) were purchased from Applied Biosystems (Foster City, CA). rmWnt3a was from R & D Systems (Minneapolis, MN).

    Techniques: Quantitative RT-PCR

    POT1 mRNA expression as determined by qRT-PCR (a) and POT1 protein expression as determined by western blot analysis (b) in shRNA-infected human ovarian cancer SK-OV3 cells. POT1-KD significantly decreased cell proliferation (c) and colony formation (d) in the early POT1-KD SK-OV3 cells. “Negative control” indicates negative control SK-OV3 cells that express a nonspecific shRNA, and “POT1-KD” represents POT1-knockdown SK-OV3 cells that express POT1-specific shRNA. (c) shows the cPDs of the SK-OV3 cells. The cPDs of the negative control cells are depicted with black dots and lines, while the cPDs of the immediate effect POT1-KD cells are depicted with black squares and lines. (d) shows images from the soft agar colony formation assay that used SK-OV3 cells. The dark dots represent the colonies. Images of the negative control cells and the immediate effect POT1-KD cells were captured via digital phase-contrast microscopy at 4x magnification; scale bar: 1000 μ m. ∗∗ P

    Journal: BioMed Research International

    Article Title: Time-Dependent Effects of POT1 Knockdown on Proliferation, Tumorigenicity, and HDACi Response of SK-OV3 Ovarian Cancer Cells

    doi: 10.1155/2018/7184253

    Figure Lengend Snippet: POT1 mRNA expression as determined by qRT-PCR (a) and POT1 protein expression as determined by western blot analysis (b) in shRNA-infected human ovarian cancer SK-OV3 cells. POT1-KD significantly decreased cell proliferation (c) and colony formation (d) in the early POT1-KD SK-OV3 cells. “Negative control” indicates negative control SK-OV3 cells that express a nonspecific shRNA, and “POT1-KD” represents POT1-knockdown SK-OV3 cells that express POT1-specific shRNA. (c) shows the cPDs of the SK-OV3 cells. The cPDs of the negative control cells are depicted with black dots and lines, while the cPDs of the immediate effect POT1-KD cells are depicted with black squares and lines. (d) shows images from the soft agar colony formation assay that used SK-OV3 cells. The dark dots represent the colonies. Images of the negative control cells and the immediate effect POT1-KD cells were captured via digital phase-contrast microscopy at 4x magnification; scale bar: 1000 μ m. ∗∗ P

    Article Snippet: The primers indicated below, which were specific for hTERT mRNA and the TaqMan probe [ ], were also used in the qRT-PCR assays (Roche, Indianapolis, IN, USA): forward 5′-TGACACCTCACCTCACCCAC-3′ and reverse 5′-CACTGTCTTCCGCAAGTTCAC-3′ and TaqMan probe 5′-ACCCTGGTCCGAGGTGTCCCTGAG-3′.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, shRNA, Infection, Negative Control, Soft Agar Assay, Microscopy

    Elevated levels of Mg 2+ in APP/PS1 transgenic mice decrease the expression of IL-1β. The APP/PS1 transgenic mice at the age of 4 months were administered Mg 2+ (100 mg/kg/d) for 2 months before collecting the brain ( a ). In select experiments, the brains of APP/PS1 transgenic mice at the age of 3 months were harvested and sectioned (400 μm) using a cryostat ( b ). In separate experiments, the left cerebral ventricle was injected with Mg 2+ (2 μg/5 μl) or vehicle (PBS) and the right cerebral ventricle was injected (i.c.v.) with D1A cells, which was pre-transfected with IL-1β promoter in the right cerebral ventricle ( c ). In distinct experiments, the left cerebral ventricle was injected with Mg 2+ (2 μg/5 μl) or vehicle (PBS) before staining with IL-1β antibody and scanning under two-photon microscopy ( d ). The immunoreactivity of IL-1β was determined by immunohistochemistry using an anti-IL-1β antibody (a left panel, b). These images are representative of six independent experiments, all with similar results. IL-1β protein and mRNA levels were determined by qRT-PCR and IL-1β enzyme immunoassay kits, respectively (a right panel). The total amounts of GAPDH and protein served as an internal control. The experimental cartoon and real surgery images are shown (c, d upper panel). Luciferase activities from the different groups of mice were measured using a live animal imaging system (c lower panel). The immunofluorescence of IL-1β was scanned using a two-photon microscope (d lower panel). The data represent the means ± S.E. of three independent experiments. * p

    Journal: Cellular and Molecular Immunology

    Article Title: Magnesium ion influx reduces neuroinflammation in Aβ precursor protein/Presenilin 1 transgenic mice by suppressing the expression of interleukin-1β

    doi: 10.1038/cmi.2015.93

    Figure Lengend Snippet: Elevated levels of Mg 2+ in APP/PS1 transgenic mice decrease the expression of IL-1β. The APP/PS1 transgenic mice at the age of 4 months were administered Mg 2+ (100 mg/kg/d) for 2 months before collecting the brain ( a ). In select experiments, the brains of APP/PS1 transgenic mice at the age of 3 months were harvested and sectioned (400 μm) using a cryostat ( b ). In separate experiments, the left cerebral ventricle was injected with Mg 2+ (2 μg/5 μl) or vehicle (PBS) and the right cerebral ventricle was injected (i.c.v.) with D1A cells, which was pre-transfected with IL-1β promoter in the right cerebral ventricle ( c ). In distinct experiments, the left cerebral ventricle was injected with Mg 2+ (2 μg/5 μl) or vehicle (PBS) before staining with IL-1β antibody and scanning under two-photon microscopy ( d ). The immunoreactivity of IL-1β was determined by immunohistochemistry using an anti-IL-1β antibody (a left panel, b). These images are representative of six independent experiments, all with similar results. IL-1β protein and mRNA levels were determined by qRT-PCR and IL-1β enzyme immunoassay kits, respectively (a right panel). The total amounts of GAPDH and protein served as an internal control. The experimental cartoon and real surgery images are shown (c, d upper panel). Luciferase activities from the different groups of mice were measured using a live animal imaging system (c lower panel). The immunofluorescence of IL-1β was scanned using a two-photon microscope (d lower panel). The data represent the means ± S.E. of three independent experiments. * p

    Article Snippet: All reagents for the quantitative real time polymerase chain reaction (qRT-PCR) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiments were purchased from Bio-Rad Laboratories (California, USA).

    Techniques: Transgenic Assay, Mouse Assay, Expressing, Injection, Transfection, Staining, Microscopy, Immunohistochemistry, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Luciferase, Imaging, Immunofluorescence

    MgT treatment attenuated the effects of Aβ oligomers in the CSF for inducing the expression of IL-1β in the cerebral cortex. The WT C57BL/6 mice at the age of 3 months were injected (i.c.v) with Aβ oligomers (2 μg/5 μl) in the absence or presence of Mg 2+ (2 μg/5 μl). The brains were then collected and sectioned after 24 h ( a and b ). In select experiments, the brains of WT C57BL/6 mice at the age of 3 months were harvested and freshly sectioned (400 μm) before treatment with Aβ oligomers (1 μM) in the absence or presence of MgT (50 μM) for 24 h ( c ). In distinct experiments, A172 cells were treated with Aβ oligomers (1 μM) in the absence or presence of MgT (50 μM) for 24 h ( d ). The immunoreactivity of IL-1β was determined by immunohistochemistry using an anti-IL-1β antibody ( a and c ). These images are representative of six independent experiments, all with similar results. IL-1β mRNA and protein levels were determined by qRT-PCR and IL-1β enzyme immunoassay kits, respectively (b and d). The total amounts of GAPDH and protein served as an internal control. The data represent the means ± SE of three independent experiments. * P

    Journal: Cellular and Molecular Immunology

    Article Title: Magnesium ion influx reduces neuroinflammation in Aβ precursor protein/Presenilin 1 transgenic mice by suppressing the expression of interleukin-1β

    doi: 10.1038/cmi.2015.93

    Figure Lengend Snippet: MgT treatment attenuated the effects of Aβ oligomers in the CSF for inducing the expression of IL-1β in the cerebral cortex. The WT C57BL/6 mice at the age of 3 months were injected (i.c.v) with Aβ oligomers (2 μg/5 μl) in the absence or presence of Mg 2+ (2 μg/5 μl). The brains were then collected and sectioned after 24 h ( a and b ). In select experiments, the brains of WT C57BL/6 mice at the age of 3 months were harvested and freshly sectioned (400 μm) before treatment with Aβ oligomers (1 μM) in the absence or presence of MgT (50 μM) for 24 h ( c ). In distinct experiments, A172 cells were treated with Aβ oligomers (1 μM) in the absence or presence of MgT (50 μM) for 24 h ( d ). The immunoreactivity of IL-1β was determined by immunohistochemistry using an anti-IL-1β antibody ( a and c ). These images are representative of six independent experiments, all with similar results. IL-1β mRNA and protein levels were determined by qRT-PCR and IL-1β enzyme immunoassay kits, respectively (b and d). The total amounts of GAPDH and protein served as an internal control. The data represent the means ± SE of three independent experiments. * P

    Article Snippet: All reagents for the quantitative real time polymerase chain reaction (qRT-PCR) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiments were purchased from Bio-Rad Laboratories (California, USA).

    Techniques: Expressing, Mouse Assay, Injection, Immunohistochemistry, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Involvement of ERK1/2 and PPARγ pathways in regulating the expression of IL-1β in MgT-treated A172 or D1A cells. Human glioblastoma A172 ( a ) or mouse astrocytes/microglia D1A cells ( b ) were treated with MgT (50 μM) for 48 h. In select experiments, A172 cells were treated with PD98059 (10 μM) in the absence or presence of MgT (50 μM) for 48 h ( c and e ). In separate experiments, the cells were transfected with ERK1/2 ( d ) or PPARγ siRNA ( f ) before incubation with MgT (50 μM) for 48 h. In distinct experiments, A172 cells were treated with GW9662 (1 μM) in the absence or presence of MgT (50 μM) for 48 h ( e '). In other experiments, primary cultured astrocytes were treated with MgT (50 μM) in the absence or presence of PD98059 (10 μM) (g) or GW9662 (1 μM) for 48 h (h). Total ERK1/2 ( c , d and g upper panel), phosphorylated ERK1/2 levels ( c and g upper panel), total PPARγ ( e and f upper panel), and phosphorylated PPARγ ( e ) were detected by immunoblotting using specific Abs. Equal lane loading is demonstrated by the similar intensities of total β-actin. IL-1β protein and mRNA levels were determined by IL-1β enzyme immunoassay kits and qRT-PCR, respectively. The total amounts of protein and GAPDH served as internal controls. The data represent the means ± SE of three independent experiments. * P

    Journal: Cellular and Molecular Immunology

    Article Title: Magnesium ion influx reduces neuroinflammation in Aβ precursor protein/Presenilin 1 transgenic mice by suppressing the expression of interleukin-1β

    doi: 10.1038/cmi.2015.93

    Figure Lengend Snippet: Involvement of ERK1/2 and PPARγ pathways in regulating the expression of IL-1β in MgT-treated A172 or D1A cells. Human glioblastoma A172 ( a ) or mouse astrocytes/microglia D1A cells ( b ) were treated with MgT (50 μM) for 48 h. In select experiments, A172 cells were treated with PD98059 (10 μM) in the absence or presence of MgT (50 μM) for 48 h ( c and e ). In separate experiments, the cells were transfected with ERK1/2 ( d ) or PPARγ siRNA ( f ) before incubation with MgT (50 μM) for 48 h. In distinct experiments, A172 cells were treated with GW9662 (1 μM) in the absence or presence of MgT (50 μM) for 48 h ( e '). In other experiments, primary cultured astrocytes were treated with MgT (50 μM) in the absence or presence of PD98059 (10 μM) (g) or GW9662 (1 μM) for 48 h (h). Total ERK1/2 ( c , d and g upper panel), phosphorylated ERK1/2 levels ( c and g upper panel), total PPARγ ( e and f upper panel), and phosphorylated PPARγ ( e ) were detected by immunoblotting using specific Abs. Equal lane loading is demonstrated by the similar intensities of total β-actin. IL-1β protein and mRNA levels were determined by IL-1β enzyme immunoassay kits and qRT-PCR, respectively. The total amounts of protein and GAPDH served as internal controls. The data represent the means ± SE of three independent experiments. * P

    Article Snippet: All reagents for the quantitative real time polymerase chain reaction (qRT-PCR) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiments were purchased from Bio-Rad Laboratories (California, USA).

    Techniques: Expressing, Transfection, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

    Aβ oligomers in the CSF of APP/PS1 mice have the ability to stimulate the expression of IL-1β in the cerebral cortex of WT mice. The APP/PS1 Tg mice at the age of 4 months were treated with Mg 2+ (100 mg/kg/d) for 5 months before collecting the CSF and brains ( a-c ). Cerebrospinal fluid (CSF) was then injected into the wild type C57BL/6 mice in the absence or presence of Aβ antibody (1 μg/5 μl) for two weeks before scarifice ( d-f ). The production of Aβ 1-42 was determined by ELISA kits, and the total amount of protein served as an internal control ( a ). The immunoreactivity of Aβ and IL-1β was determined by immunohistochemistry using an anti-Aβ or IL-1β antibody ( c, d ). These images are representative of six independent experiments, all with similar results. The number of AP/fields was calculated according to the images of IHC ( b ). IL-1β mRNA and protein levels were determined by qRT-PCR and IL-1β enzyme immunoassay kits, respectively ( e, f ). The total amounts of GAPDH and protein served as an internal control. * p

    Journal: Cellular and Molecular Immunology

    Article Title: Magnesium ion influx reduces neuroinflammation in Aβ precursor protein/Presenilin 1 transgenic mice by suppressing the expression of interleukin-1β

    doi: 10.1038/cmi.2015.93

    Figure Lengend Snippet: Aβ oligomers in the CSF of APP/PS1 mice have the ability to stimulate the expression of IL-1β in the cerebral cortex of WT mice. The APP/PS1 Tg mice at the age of 4 months were treated with Mg 2+ (100 mg/kg/d) for 5 months before collecting the CSF and brains ( a-c ). Cerebrospinal fluid (CSF) was then injected into the wild type C57BL/6 mice in the absence or presence of Aβ antibody (1 μg/5 μl) for two weeks before scarifice ( d-f ). The production of Aβ 1-42 was determined by ELISA kits, and the total amount of protein served as an internal control ( a ). The immunoreactivity of Aβ and IL-1β was determined by immunohistochemistry using an anti-Aβ or IL-1β antibody ( c, d ). These images are representative of six independent experiments, all with similar results. The number of AP/fields was calculated according to the images of IHC ( b ). IL-1β mRNA and protein levels were determined by qRT-PCR and IL-1β enzyme immunoassay kits, respectively ( e, f ). The total amounts of GAPDH and protein served as an internal control. * p

    Article Snippet: All reagents for the quantitative real time polymerase chain reaction (qRT-PCR) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiments were purchased from Bio-Rad Laboratories (California, USA).

    Techniques: Mouse Assay, Expressing, Injection, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Quantitative RT-PCR

    IL-1β was upregulated in AD patients and APP/PS1 transgenic mice. The tissue blocks of human brains at different stages of AD were collected from the New York Brain Bank at Columbia University. From 40 μm free-floating slices were prepared using a cryostat. ( a ) In select experiments, the brains of 3-month-old APP/PS1 transgenic mice were collected after anesthesia and perfusion. ( b – d ) The immunoreactivity of IL-1β was determined by immunohistochemistry using an anti-IL-1β antibody ( a and b ). These images are representative of six independent experiments, all with similar results. IL-1β mRNA levels were determined by qRT-PCR, and total amounts of GAPDH served as an internal control ( c and d upper panels). The production of IL-1β in culture medium was determined using IL-1β enzyme immunoassay kits ( c and d lower panels). The data represent the means ± SE of three independent experiments. * P

    Journal: Cellular and Molecular Immunology

    Article Title: Magnesium ion influx reduces neuroinflammation in Aβ precursor protein/Presenilin 1 transgenic mice by suppressing the expression of interleukin-1β

    doi: 10.1038/cmi.2015.93

    Figure Lengend Snippet: IL-1β was upregulated in AD patients and APP/PS1 transgenic mice. The tissue blocks of human brains at different stages of AD were collected from the New York Brain Bank at Columbia University. From 40 μm free-floating slices were prepared using a cryostat. ( a ) In select experiments, the brains of 3-month-old APP/PS1 transgenic mice were collected after anesthesia and perfusion. ( b – d ) The immunoreactivity of IL-1β was determined by immunohistochemistry using an anti-IL-1β antibody ( a and b ). These images are representative of six independent experiments, all with similar results. IL-1β mRNA levels were determined by qRT-PCR, and total amounts of GAPDH served as an internal control ( c and d upper panels). The production of IL-1β in culture medium was determined using IL-1β enzyme immunoassay kits ( c and d lower panels). The data represent the means ± SE of three independent experiments. * P

    Article Snippet: All reagents for the quantitative real time polymerase chain reaction (qRT-PCR) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiments were purchased from Bio-Rad Laboratories (California, USA).

    Techniques: Transgenic Assay, Mouse Assay, Immunohistochemistry, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    MgT treatment diminished the effects of IL-1β in the CSF with respect to inducing the expression of IL-1β in the cerebral cortex. WT C57BL/6 mice at the age of 3 months were intracerebroventricularly injected with IL-1β (0.5 μg/5 μl) in the absence or presence of Mg 2+ (2 μg/5 μl). The brains were then collected and sectioned after 24 h ( a and b ). In select experiments, the brains of WT C57BL/6 mice at the age of 3 months were harvested and freshly sectioned (400 μm) before treatment with IL-1β (100 ng ml –1 ) in the absence or presence of MgT (50 μM) for 24 h ( c ). In separate experiments, A172 cells were treated with IL-1β (100 ng ml –1 ) in the absence or presence of MgT (50 μM) for 24 h ( d ). The immunoreactivity of IL-1β was determined by immunohistochemistry using an anti-IL-1β or -APH-1 antibody ( a and c ). These images are representative of six independent experiments, all with similar results. IL-1β mRNA and protein levels were determined by qRT-PCR and IL-1β enzyme immunoassay kits, respectively ( b and d ). The total amounts of GAPDH and protein served as an internal control. The data represent the means ± SE of three independent experiments. * P

    Journal: Cellular and Molecular Immunology

    Article Title: Magnesium ion influx reduces neuroinflammation in Aβ precursor protein/Presenilin 1 transgenic mice by suppressing the expression of interleukin-1β

    doi: 10.1038/cmi.2015.93

    Figure Lengend Snippet: MgT treatment diminished the effects of IL-1β in the CSF with respect to inducing the expression of IL-1β in the cerebral cortex. WT C57BL/6 mice at the age of 3 months were intracerebroventricularly injected with IL-1β (0.5 μg/5 μl) in the absence or presence of Mg 2+ (2 μg/5 μl). The brains were then collected and sectioned after 24 h ( a and b ). In select experiments, the brains of WT C57BL/6 mice at the age of 3 months were harvested and freshly sectioned (400 μm) before treatment with IL-1β (100 ng ml –1 ) in the absence or presence of MgT (50 μM) for 24 h ( c ). In separate experiments, A172 cells were treated with IL-1β (100 ng ml –1 ) in the absence or presence of MgT (50 μM) for 24 h ( d ). The immunoreactivity of IL-1β was determined by immunohistochemistry using an anti-IL-1β or -APH-1 antibody ( a and c ). These images are representative of six independent experiments, all with similar results. IL-1β mRNA and protein levels were determined by qRT-PCR and IL-1β enzyme immunoassay kits, respectively ( b and d ). The total amounts of GAPDH and protein served as an internal control. The data represent the means ± SE of three independent experiments. * P

    Article Snippet: All reagents for the quantitative real time polymerase chain reaction (qRT-PCR) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiments were purchased from Bio-Rad Laboratories (California, USA).

    Techniques: Expressing, Mouse Assay, Injection, Immunohistochemistry, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Relative expression of MabZIP genes in BX and FJ by qRT-PCR. ( A – D ) expression patterns of MabZIP61, MabZIP75, MabZIP25 and MabZIP11 in different organs. The mRNA fold difference was relative to that of BX-root samples used as calibrator. ( E – H ) expression patterns of MabZIP61, MabZIP75, MabZIP25 and MabZIP11 in different stages of fruit development and ripening. The mRNA fold difference was relative to that of BX-0DAF samples used as calibrator. ( I – L ) expression patterns of MabZIP3, MabZIP93, MabZIP40 and MabZIP101 in response to cold, salt and osmotic stresses. The mRNA fold difference was relative to that of untreated samples used as calibrator. Data are means ± SD of n = 3 biological replicates.

    Journal: Scientific Reports

    Article Title: Genome-wide analyses of the bZIP family reveal their involvement in the development, ripening and abiotic stress response in banana

    doi: 10.1038/srep30203

    Figure Lengend Snippet: Relative expression of MabZIP genes in BX and FJ by qRT-PCR. ( A – D ) expression patterns of MabZIP61, MabZIP75, MabZIP25 and MabZIP11 in different organs. The mRNA fold difference was relative to that of BX-root samples used as calibrator. ( E – H ) expression patterns of MabZIP61, MabZIP75, MabZIP25 and MabZIP11 in different stages of fruit development and ripening. The mRNA fold difference was relative to that of BX-0DAF samples used as calibrator. ( I – L ) expression patterns of MabZIP3, MabZIP93, MabZIP40 and MabZIP101 in response to cold, salt and osmotic stresses. The mRNA fold difference was relative to that of untreated samples used as calibrator. Data are means ± SD of n = 3 biological replicates.

    Article Snippet: qRT-PCR Analysis Changes in the expression of MabZIP genes in different organs, different stages of fruit development and ripening, and response to abiotic stresses of cold, salt and osmotic were validated by quantitative real-time polymerase chain reaction (qRT-PCR) using SYBR® Premix Ex Taq™ (TaKaRa, Shiga, Japan) chemistry on a Stratagene Mx3000P (Stratagene, CA, USA) instrument.

    Techniques: Expressing, Quantitative RT-PCR

    qRT-PCR analysis of PTPC transcript levels in developing and germinating sorghum seeds. To normalize the values, 18S RNA was used as internal control in each sample. Data were normalized to stage VI for developing seeds (A, C, E), or 14h post-imbibition germinating seeds (B, D, F). Results are means ±SE of at least three independent experiments. Mean values that are not significantly different ( P

    Journal: Journal of Experimental Botany

    Article Title: New insights into the post-translational modification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination during sorghum seed development and germination

    doi: 10.1093/jxb/erw186

    Figure Lengend Snippet: qRT-PCR analysis of PTPC transcript levels in developing and germinating sorghum seeds. To normalize the values, 18S RNA was used as internal control in each sample. Data were normalized to stage VI for developing seeds (A, C, E), or 14h post-imbibition germinating seeds (B, D, F). Results are means ±SE of at least three independent experiments. Mean values that are not significantly different ( P

    Article Snippet: qRT-PCR Quantitative polymerase chain reaction (qRT-PCR) was performed in a final volume of 20 µl consisting of 1 µl of the cDNA, 10 µl of SensiFAST SYBR No-ROX kit (Roche) and 15 µM of the gene-specific primers pairs as follows: SbPPC3 (forward 5′–TGTTGAACAGTTTCTGGAACCTCTT-3′, reverse 5′-GCTTCA CAAGGGCAAGCCCAAAG-3′), SbPPC2 (forward 5′-CCGCCT CGCAACACCTGAAACA-3′, reverse 5′-ACCGGGAGGTGGAA CCGTGT-3′), SbPPC4 (forward 5′-TGAGCTTCGGGCACAAGC AGATG-3′, reverse 5′-GCTCCAAAGGCTCTAAGAACTGCT C-3′), and 18 S rRNA (forward 5′-GGGGAAACTTACCA GGTCCA-3′, reverse 5′-GGATGGCTCCGCATAGCTA-3′).

    Techniques: Quantitative RT-PCR

    ( A ) Hierarchal clustering of differentially expressed microRNAs. Forty-six differentially expressed microRNAs (for up- and down-regulation, respectively) compared to the negative control group in at least one sample were clustered hierarchically. Each rows represent individual microRNAs. The expression ratio representing colour ranges from green (low) to red (high), as indicated by the scale bar. The column bar chart shows the relative expression level of selected microRNAs by qRT-PCR (right column, white) as compared to the fold changes of the corresponding microRNA (left column, grey); ( B ) MicroRNAs expression of Group B versus Group A on the first day of treatment; ( C ) Group B versus Group A on the third day of treatment; ( D ) MicroRNAs expression of on the fifth day of treatment. U87 was used as housekeeping gene. Bars represent the mean ± standard error of three independent experiments; and ( E ) qRT-PCR product was confirmed by gel electrophoresis at 90 V for 40 min.

    Journal: International Journal of Molecular Sciences

    Article Title: MicroRNA Expression Profile of Neural Progenitor-Like Cells Derived from Rat Bone Marrow Mesenchymal Stem Cells under the Influence of IGF-1, bFGF and EGF

    doi: 10.3390/ijms16059693

    Figure Lengend Snippet: ( A ) Hierarchal clustering of differentially expressed microRNAs. Forty-six differentially expressed microRNAs (for up- and down-regulation, respectively) compared to the negative control group in at least one sample were clustered hierarchically. Each rows represent individual microRNAs. The expression ratio representing colour ranges from green (low) to red (high), as indicated by the scale bar. The column bar chart shows the relative expression level of selected microRNAs by qRT-PCR (right column, white) as compared to the fold changes of the corresponding microRNA (left column, grey); ( B ) MicroRNAs expression of Group B versus Group A on the first day of treatment; ( C ) Group B versus Group A on the third day of treatment; ( D ) MicroRNAs expression of on the fifth day of treatment. U87 was used as housekeeping gene. Bars represent the mean ± standard error of three independent experiments; and ( E ) qRT-PCR product was confirmed by gel electrophoresis at 90 V for 40 min.

    Article Snippet: MicroRNA-qPCR Assay MicroRNA expressions were validated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) according to the manufacturer’s protocol (all from Applied Biosystems, Life Technologies Co.; Waltham, MA, USA).

    Techniques: Negative Control, Expressing, Quantitative RT-PCR, Nucleic Acid Electrophoresis

    FA2H expression among breast cancer cell lines. (A) FA2H gene expression among different breast cancer cell lines by qRT-PCR. (B) FA2H protein expression among different breast cancer cell lines by Western-blotting. (C) FA2H protein expression quantified using the ImageJ software. (D) FA2H gene expression across 56 breast cancer cell lines ( 26 ).

    Journal: Frontiers in Oncology

    Article Title: FA2H Exhibits Tumor Suppressive Roles on Breast Cancers via Cancer Stemness Control

    doi: 10.3389/fonc.2019.01089

    Figure Lengend Snippet: FA2H expression among breast cancer cell lines. (A) FA2H gene expression among different breast cancer cell lines by qRT-PCR. (B) FA2H protein expression among different breast cancer cell lines by Western-blotting. (C) FA2H protein expression quantified using the ImageJ software. (D) FA2H gene expression across 56 breast cancer cell lines ( 26 ).

    Article Snippet: Primers designed for candidate genes and used for quantitative reverse transcription polymerase chain reaction (qRT-PCR) validation (ordered from GENEWIZ) were listed in .

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

    QRT-PCR for 27-hydroxylase and ABCA1 message in NS398-treated THP-1 cells exposed to prostaglandins. (a) 27-Hydroxylase message is decreased by the COX-2 inhibitor NS398 and this decrease is reversed by prostaglandins E 1 , E 2 , and D 2 . THP-1 human monocytes were exposed to the following conditions represented by the six bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE 1 (0.1 μM) + NS398 (50 μM), (4) PGE 2 (0.1 μM) + NS398 (50 μM), (5) PGE 1 (0.1 μM) + PGE 2 (0.1 μM) + NS398 (50 μM), and (6) PGD 2 (14 μM) + NS398 (50 μM) (all 18-hour exposures). Cells were extracted for total RNA and were evaluated for 27-hydroxylase mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) ABCA1 message is decreased by the COX-2 inhibitor NS398 and this decrease is reversed by prostaglandins E 1 , E 2 , and D 2 . THP-1 human monocytes were exposed to the following conditions represented by the six bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE 1 (0.1 μM) + NS398 (50 μM), (4) PGE 2 (0.1 μM) + NS398 (50 μM), (5) PGE 1 (0.1 μM) + PGE 2 (0.1 μM) + NS398 (50 μM), and (6) PGD 2 (14 μM) + NS398 (50 μM) (all 18-hour exposures). Cells were extracted for total RNA and were evaluated for ABCA1 mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. ** p

    Journal: Arthritis Research & Therapy

    Article Title: Effect of cyclooxygenase inhibition on cholesterol efflux proteins and atheromatous foam cell transformation in THP-1 human macrophages: a possible mechanism for increased cardiovascular risk

    doi: 10.1186/ar2109

    Figure Lengend Snippet: QRT-PCR for 27-hydroxylase and ABCA1 message in NS398-treated THP-1 cells exposed to prostaglandins. (a) 27-Hydroxylase message is decreased by the COX-2 inhibitor NS398 and this decrease is reversed by prostaglandins E 1 , E 2 , and D 2 . THP-1 human monocytes were exposed to the following conditions represented by the six bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE 1 (0.1 μM) + NS398 (50 μM), (4) PGE 2 (0.1 μM) + NS398 (50 μM), (5) PGE 1 (0.1 μM) + PGE 2 (0.1 μM) + NS398 (50 μM), and (6) PGD 2 (14 μM) + NS398 (50 μM) (all 18-hour exposures). Cells were extracted for total RNA and were evaluated for 27-hydroxylase mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) ABCA1 message is decreased by the COX-2 inhibitor NS398 and this decrease is reversed by prostaglandins E 1 , E 2 , and D 2 . THP-1 human monocytes were exposed to the following conditions represented by the six bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE 1 (0.1 μM) + NS398 (50 μM), (4) PGE 2 (0.1 μM) + NS398 (50 μM), (5) PGE 1 (0.1 μM) + PGE 2 (0.1 μM) + NS398 (50 μM), and (6) PGD 2 (14 μM) + NS398 (50 μM) (all 18-hour exposures). Cells were extracted for total RNA and were evaluated for ABCA1 mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. ** p

    Article Snippet: All reagents for reverse transcription and quantitative real-time polymerase chain reaction (QRT-PCR) were purchased from Applied Biosystems (Foster City, CA, USA).

    Techniques: Quantitative RT-PCR, Expressing, Amplification

    QRT-PCR for 27-hydroxylase and ABCA1 message in indomethacin-treated THP-1 cells. (a) 27-Hydroxylase mRNA expression is decreased by the non-specific COX inhibitor indomethacin in a dose-dependent fashion in THP-1 monocytes. Cultured THP-1 monocytic cells were untreated or exposed to increasing doses of indomethacin for 18 hours. After isolation of total RNA, the RNA was reverse-transcribed and the cDNA amplified by QRT-PCR as described. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) ABCA1 mRNA expression is decreased by the non-specific COX inhibitor indomethacin in a dose-dependent fashion in THP-1 monocytes. Cultured THP-1 monocytic cells were untreated or exposed to increasing doses of indomethacin for 18 hours. After isolation of total RNA, the RNA was reverse-transcribed and the cDNA amplified by QRT-PCR as described. Signals obtained from the amplification of GAPDH message were used as internal controls. At 50 mM indomethacin concentration, cell death was statistically significant (16.8% ± 1.0%). ABCA1, ATP-binding cassette transporter A1; COX, cyclooxygenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; QRT-PCR, quantitative real-time polymerase chain reaction. * p

    Journal: Arthritis Research & Therapy

    Article Title: Effect of cyclooxygenase inhibition on cholesterol efflux proteins and atheromatous foam cell transformation in THP-1 human macrophages: a possible mechanism for increased cardiovascular risk

    doi: 10.1186/ar2109

    Figure Lengend Snippet: QRT-PCR for 27-hydroxylase and ABCA1 message in indomethacin-treated THP-1 cells. (a) 27-Hydroxylase mRNA expression is decreased by the non-specific COX inhibitor indomethacin in a dose-dependent fashion in THP-1 monocytes. Cultured THP-1 monocytic cells were untreated or exposed to increasing doses of indomethacin for 18 hours. After isolation of total RNA, the RNA was reverse-transcribed and the cDNA amplified by QRT-PCR as described. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) ABCA1 mRNA expression is decreased by the non-specific COX inhibitor indomethacin in a dose-dependent fashion in THP-1 monocytes. Cultured THP-1 monocytic cells were untreated or exposed to increasing doses of indomethacin for 18 hours. After isolation of total RNA, the RNA was reverse-transcribed and the cDNA amplified by QRT-PCR as described. Signals obtained from the amplification of GAPDH message were used as internal controls. At 50 mM indomethacin concentration, cell death was statistically significant (16.8% ± 1.0%). ABCA1, ATP-binding cassette transporter A1; COX, cyclooxygenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; QRT-PCR, quantitative real-time polymerase chain reaction. * p

    Article Snippet: All reagents for reverse transcription and quantitative real-time polymerase chain reaction (QRT-PCR) were purchased from Applied Biosystems (Foster City, CA, USA).

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

    QRT-PCR for 27-hydroxylase and ABCA1 message in NS398-treated THP-1 macrophages exposed to prostaglandins or TXA 2 . (a) 27-Hydroxylase message in THP-1 macrophages is decreased by the COX-2 inhibitor NS398 and this decrease is reversed by prostaglandins E 2 and D 2 , but not TXA 2 . THP-1 human macrophages were exposed to the following conditions represented by the five bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE 2 (0.1 μM) + NS398 (50 μM), (4) PGD 2 (14 μM) + NS398 (50 μM), and (5) TXA 2 (3 μM) + NS398 (50 μM) (24-hour exposures to NS398 alone followed by addition of indicated PG or TXA 2 for a further 24 hours). Cells were extracted for total RNA and were evaluated for 27-hydroxylase mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) ABCA1 message is decreased by the COX-2 inhibitor NS398 in THP-1 macrophages and this decrease is reversed by prostaglandins E 2 and D 2 , but not TXA 2 . THP-1 human macrophages were exposed to the following conditions represented by the five bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE 2 (0.1 μM) + NS398 (50 μM), (4) PGD 2 (14 μM) + NS398 (50 μM), and (5) TXA 2 (3 μM) + NS398 (50 μM) (24-hour exposures to NS398 alone followed by addition of indicated PG or TXA 2 for a further 24 hours). Cells were extracted for total RNA and were evaluated for ABCA1 mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. * p

    Journal: Arthritis Research & Therapy

    Article Title: Effect of cyclooxygenase inhibition on cholesterol efflux proteins and atheromatous foam cell transformation in THP-1 human macrophages: a possible mechanism for increased cardiovascular risk

    doi: 10.1186/ar2109

    Figure Lengend Snippet: QRT-PCR for 27-hydroxylase and ABCA1 message in NS398-treated THP-1 macrophages exposed to prostaglandins or TXA 2 . (a) 27-Hydroxylase message in THP-1 macrophages is decreased by the COX-2 inhibitor NS398 and this decrease is reversed by prostaglandins E 2 and D 2 , but not TXA 2 . THP-1 human macrophages were exposed to the following conditions represented by the five bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE 2 (0.1 μM) + NS398 (50 μM), (4) PGD 2 (14 μM) + NS398 (50 μM), and (5) TXA 2 (3 μM) + NS398 (50 μM) (24-hour exposures to NS398 alone followed by addition of indicated PG or TXA 2 for a further 24 hours). Cells were extracted for total RNA and were evaluated for 27-hydroxylase mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) ABCA1 message is decreased by the COX-2 inhibitor NS398 in THP-1 macrophages and this decrease is reversed by prostaglandins E 2 and D 2 , but not TXA 2 . THP-1 human macrophages were exposed to the following conditions represented by the five bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE 2 (0.1 μM) + NS398 (50 μM), (4) PGD 2 (14 μM) + NS398 (50 μM), and (5) TXA 2 (3 μM) + NS398 (50 μM) (24-hour exposures to NS398 alone followed by addition of indicated PG or TXA 2 for a further 24 hours). Cells were extracted for total RNA and were evaluated for ABCA1 mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. * p

    Article Snippet: All reagents for reverse transcription and quantitative real-time polymerase chain reaction (QRT-PCR) were purchased from Applied Biosystems (Foster City, CA, USA).

    Techniques: Quantitative RT-PCR, Expressing, Amplification

    KMP 1 specifically recognized CD 44 epitope located on the cell surface in bladder cancer. (A) To investigate the epitope recognized by KMP 1, the supernatants after EJ cell lysis were applied to protein A‐Sepharose column with KMP 1. Two peaks were obtained from the eluates by Sephacryl S200. (B) Antigen–antibody complex peak and antibody excess peak obtained from the eluates were resolved by 10% SDS – PAGE and stained with silver. (C) qRT – PCR analysis of CD 44 expression in EJ cells that were transfected with pS uper‐puro‐ CD 44‐sh RNA (sh CD 44‐ EJ ) or the empty pS uper‐puro vector (shCtrl‐ EJ ). (D) Western blot imagines indicated that the band using KMP 1 as a primary antibody was markedly thinned in the CD 44‐silenced EJ cells versus the shCtrl‐ EJ cells. (E) sh CD 44‐ EJ and shCtrl‐ EJ cells were analyzed by flow cytometry after staining with KMP 1, and nontransfected EJ cells were stained with mIgG as a negative control. (F) ELISA analysis of KMP 1 recognized CD 44 levels in shCtrl or sh CD 44‐ EJ cells. Each bar is expressed as the mean ± standard deviation of three experiments. * P

    Journal: Cancer Medicine

    Article Title: A novel monoclonal antibody KMP1 has potential antitumor activity of bladder cancer by blocking CD44 in vivo and in vitro

    doi: 10.1002/cam4.1446

    Figure Lengend Snippet: KMP 1 specifically recognized CD 44 epitope located on the cell surface in bladder cancer. (A) To investigate the epitope recognized by KMP 1, the supernatants after EJ cell lysis were applied to protein A‐Sepharose column with KMP 1. Two peaks were obtained from the eluates by Sephacryl S200. (B) Antigen–antibody complex peak and antibody excess peak obtained from the eluates were resolved by 10% SDS – PAGE and stained with silver. (C) qRT – PCR analysis of CD 44 expression in EJ cells that were transfected with pS uper‐puro‐ CD 44‐sh RNA (sh CD 44‐ EJ ) or the empty pS uper‐puro vector (shCtrl‐ EJ ). (D) Western blot imagines indicated that the band using KMP 1 as a primary antibody was markedly thinned in the CD 44‐silenced EJ cells versus the shCtrl‐ EJ cells. (E) sh CD 44‐ EJ and shCtrl‐ EJ cells were analyzed by flow cytometry after staining with KMP 1, and nontransfected EJ cells were stained with mIgG as a negative control. (F) ELISA analysis of KMP 1 recognized CD 44 levels in shCtrl or sh CD 44‐ EJ cells. Each bar is expressed as the mean ± standard deviation of three experiments. * P

    Article Snippet: The efficiency of CD44 silencing after transfection was examined on a 7900HT quantitative real‐time polymerase chain reaction (qRT–PCR) System (Applied Biosystems, Foster City, CA, USA). β ‐Actin (Sigma) was used as the internal control.

    Techniques: Lysis, SDS Page, Staining, Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation, Western Blot, Flow Cytometry, Cytometry, Negative Control, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Downregulation of SIRT4 expression correlates with clinicopathological factors and poor prognosis in GC patients. ( A ) The expression of SIRT4 in tumor tissues and adjacent normal tissues was determined by qRT-PCR and Western blotting, respectively. Tumor tissues vs normal adjacent tissues, * p

    Journal: OncoTargets and therapy

    Article Title: SIRT4 acts as a tumor suppressor in gastric cancer by inhibiting cell proliferation, migration, and invasion

    doi: 10.2147/OTT.S156143

    Figure Lengend Snippet: Downregulation of SIRT4 expression correlates with clinicopathological factors and poor prognosis in GC patients. ( A ) The expression of SIRT4 in tumor tissues and adjacent normal tissues was determined by qRT-PCR and Western blotting, respectively. Tumor tissues vs normal adjacent tissues, * p

    Article Snippet: RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) TRIzol reagent (Invitrogen) was used to extract total RNA from tissues or cells according to the manufacturer’s instructions.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    SIRT4 suppresses EMT through promoting E-cadherin expression. ( A ) SIRT4 was overexpressed or knocked down in MKN-45 or HGC-27 cells. After transfection for 48 h, the mRNA level of E-cadherin was detected using qRT-PCR. SIRT4 vs vector and siSIRT4 vs SCR, * p

    Journal: OncoTargets and therapy

    Article Title: SIRT4 acts as a tumor suppressor in gastric cancer by inhibiting cell proliferation, migration, and invasion

    doi: 10.2147/OTT.S156143

    Figure Lengend Snippet: SIRT4 suppresses EMT through promoting E-cadherin expression. ( A ) SIRT4 was overexpressed or knocked down in MKN-45 or HGC-27 cells. After transfection for 48 h, the mRNA level of E-cadherin was detected using qRT-PCR. SIRT4 vs vector and siSIRT4 vs SCR, * p

    Article Snippet: RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) TRIzol reagent (Invitrogen) was used to extract total RNA from tissues or cells according to the manufacturer’s instructions.

    Techniques: Expressing, Transfection, Quantitative RT-PCR, Plasmid Preparation

    SIRT4 suppresses GC cell proliferation. ( A ) MKN-45 and HGC-27 were transfected with vector or SIRT4, scramble siRNA (SCR), or SIRT4 siRNA (siSIRT4). After transfection for 48 h, the mRNA level of SIRT4 was determined using qRT-PCR. SIRT4 vs vector and siSIRT4 vs SCR, * p

    Journal: OncoTargets and therapy

    Article Title: SIRT4 acts as a tumor suppressor in gastric cancer by inhibiting cell proliferation, migration, and invasion

    doi: 10.2147/OTT.S156143

    Figure Lengend Snippet: SIRT4 suppresses GC cell proliferation. ( A ) MKN-45 and HGC-27 were transfected with vector or SIRT4, scramble siRNA (SCR), or SIRT4 siRNA (siSIRT4). After transfection for 48 h, the mRNA level of SIRT4 was determined using qRT-PCR. SIRT4 vs vector and siSIRT4 vs SCR, * p

    Article Snippet: RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) TRIzol reagent (Invitrogen) was used to extract total RNA from tissues or cells according to the manufacturer’s instructions.

    Techniques: Transfection, Plasmid Preparation, Quantitative RT-PCR

    HFD feeding increased the translocation to the nucleus of p65 and expression of NFκB‐related genes in Irf3 −/− mice. Male C57BL/6, Irf3 −/− , and Irf3 S1/S1 mice were fed an HFD or chow for 12 weeks. (A) Expressions of phospho‐IKKα∕β, IKKβ, phospho‐p65, p65, and IκBα were assessed by western blot and normalized to GAPDH, n = 4 per genotype. (B) Formalin‐fixed paraffin‐embedded sections of liver were deparaffinized, and localization of phospho‐p65 was assessed by immunohistochemistry. Images were acquired at 40×. White arrows indicate nonparenchymal cells, and black arrows indicate hepatocytes. Images are representative of triplicate images from eight mice per treatment group. Values represent means ± SEM. (C) Nuclear fractions were isolated from livers, and equal amounts of protein were assessed by western blot and probed against phosphor‐Ser 536 p65‐NFκB. Lamin A/C was used as a nuclei marker, and GAPDH was used as a cytosolic marker. (D) Expressions of CCL5, CXCL10, CXCL2, and CCL3 were analyzed by qRT‐PCR. Values were normalized to 18S and are shown as fold increase over the chow‐fed C57BL/6 control group, n = 12 per genotype. Values with different superscripts are significantly different from each other, P

    Journal: Hepatology Communications

    Article Title: Nontranscriptional Activity of Interferon Regulatory Factor 3 Protects Mice From High‐Fat Diet‐Induced Liver Injury

    doi: 10.1002/hep4.1441

    Figure Lengend Snippet: HFD feeding increased the translocation to the nucleus of p65 and expression of NFκB‐related genes in Irf3 −/− mice. Male C57BL/6, Irf3 −/− , and Irf3 S1/S1 mice were fed an HFD or chow for 12 weeks. (A) Expressions of phospho‐IKKα∕β, IKKβ, phospho‐p65, p65, and IκBα were assessed by western blot and normalized to GAPDH, n = 4 per genotype. (B) Formalin‐fixed paraffin‐embedded sections of liver were deparaffinized, and localization of phospho‐p65 was assessed by immunohistochemistry. Images were acquired at 40×. White arrows indicate nonparenchymal cells, and black arrows indicate hepatocytes. Images are representative of triplicate images from eight mice per treatment group. Values represent means ± SEM. (C) Nuclear fractions were isolated from livers, and equal amounts of protein were assessed by western blot and probed against phosphor‐Ser 536 p65‐NFκB. Lamin A/C was used as a nuclei marker, and GAPDH was used as a cytosolic marker. (D) Expressions of CCL5, CXCL10, CXCL2, and CCL3 were analyzed by qRT‐PCR. Values were normalized to 18S and are shown as fold increase over the chow‐fed C57BL/6 control group, n = 12 per genotype. Values with different superscripts are significantly different from each other, P

    Article Snippet: Two micrograms of liver RNA was reverse transcribed and analyzed with PowerSYBR quantitative real‐time polymerase chain reaction (qRT‐PCR) kits (Applied Biosystems, Foster City, CA) on a QuantStudio5 RT‐PCR Machine (Applied Biosystems).

    Techniques: Translocation Assay, Expressing, Mouse Assay, Western Blot, Formalin-fixed Paraffin-Embedded, Immunohistochemistry, Isolation, Marker, Quantitative RT-PCR

    Irf3 S1/S1 mice reduced HFD‐induced liver injury, steatosis, and markers of inflammation. Male C57BL/6, Irf3 −/− , and Irf3 S1/S1 mice were fed an HFD or chow for 12 weeks. (A,B) ALT and AST activities were measured in plasma, n = 12 per genotype. (C) Hepatic triglyceride content was measured in whole‐liver homogenates, n = 12 per genotype. (D) Paraffin‐embedded liver sections were stained with hematoxylin and eosin. Images were acquired using 10× and 40× objectives, n = 4 per genotype. Expressions of (E) TNFα, (F) IL‐1β, and (G) MCP‐1 mRNA were analyzed by qRT‐PCR. Values were normalized to 18S and are shown as fold increase over the chow‐fed C57BL/6 control group, n = 12 per genotype. Values represent means ± SEM. Values with different alphabetical superscripts are significantly different, P

    Journal: Hepatology Communications

    Article Title: Nontranscriptional Activity of Interferon Regulatory Factor 3 Protects Mice From High‐Fat Diet‐Induced Liver Injury

    doi: 10.1002/hep4.1441

    Figure Lengend Snippet: Irf3 S1/S1 mice reduced HFD‐induced liver injury, steatosis, and markers of inflammation. Male C57BL/6, Irf3 −/− , and Irf3 S1/S1 mice were fed an HFD or chow for 12 weeks. (A,B) ALT and AST activities were measured in plasma, n = 12 per genotype. (C) Hepatic triglyceride content was measured in whole‐liver homogenates, n = 12 per genotype. (D) Paraffin‐embedded liver sections were stained with hematoxylin and eosin. Images were acquired using 10× and 40× objectives, n = 4 per genotype. Expressions of (E) TNFα, (F) IL‐1β, and (G) MCP‐1 mRNA were analyzed by qRT‐PCR. Values were normalized to 18S and are shown as fold increase over the chow‐fed C57BL/6 control group, n = 12 per genotype. Values represent means ± SEM. Values with different alphabetical superscripts are significantly different, P

    Article Snippet: Two micrograms of liver RNA was reverse transcribed and analyzed with PowerSYBR quantitative real‐time polymerase chain reaction (qRT‐PCR) kits (Applied Biosystems, Foster City, CA) on a QuantStudio5 RT‐PCR Machine (Applied Biosystems).

    Techniques: Mouse Assay, AST Assay, Staining, Quantitative RT-PCR

    HFD enhanced CD45 + infiltration in Irf3 −/− but not in wild‐type and Irf3 S1/S1 livers. Male C57BL/6, Irf3 −/− , and Irf3 S1/S1 mice were fed an HFD or chow for 12 weeks. (A) Expression of CD45 mRNA was analyzed by qRT‐PCR. Values were normalized to 18S and are shown as fold increase over the chow‐fed C57BL/6 control group, n = 8 per genotype. (B) Hepatic CD45 + leukocytes were gated in isolated liver NPCs by flow cytometry, and total number of cells were quantified in isolated liver NPCs, n = 4 per genotype. (C) Paraffin‐embedded liver sections were stained for CD45. Images were acquired using a 20× objective, n = 4 per genotype. Values represent means ± SEM. Values with different alphabetical superscripts are significantly different, P

    Journal: Hepatology Communications

    Article Title: Nontranscriptional Activity of Interferon Regulatory Factor 3 Protects Mice From High‐Fat Diet‐Induced Liver Injury

    doi: 10.1002/hep4.1441

    Figure Lengend Snippet: HFD enhanced CD45 + infiltration in Irf3 −/− but not in wild‐type and Irf3 S1/S1 livers. Male C57BL/6, Irf3 −/− , and Irf3 S1/S1 mice were fed an HFD or chow for 12 weeks. (A) Expression of CD45 mRNA was analyzed by qRT‐PCR. Values were normalized to 18S and are shown as fold increase over the chow‐fed C57BL/6 control group, n = 8 per genotype. (B) Hepatic CD45 + leukocytes were gated in isolated liver NPCs by flow cytometry, and total number of cells were quantified in isolated liver NPCs, n = 4 per genotype. (C) Paraffin‐embedded liver sections were stained for CD45. Images were acquired using a 20× objective, n = 4 per genotype. Values represent means ± SEM. Values with different alphabetical superscripts are significantly different, P

    Article Snippet: Two micrograms of liver RNA was reverse transcribed and analyzed with PowerSYBR quantitative real‐time polymerase chain reaction (qRT‐PCR) kits (Applied Biosystems, Foster City, CA) on a QuantStudio5 RT‐PCR Machine (Applied Biosystems).

    Techniques: Mouse Assay, Expressing, Quantitative RT-PCR, Isolation, Flow Cytometry, Cytometry, Staining

    Enhanced markers of hepatocellular death and fibrosis in Irf3 −/− mice compared with wild‐type and Irf3 S1/S1 mice after HFD feeding. Male C57BL/6, Irf3 −/− , and Irf3 S1/S1 mice were fed an HFD or chow for 12 weeks. (A,B) Paraffin‐embedded liver sections were stained for M30, a caspase‐dependent cleavage product of cytokeratin 18. Images were acquired using 10× and 40× objectives, and the positive area was quantified, n = 4 per genotype. (C,D) Paraffin‐embedded liver sections were stained with sirius red. Images were acquired using a 40× objective, and the positive area was quantified, n = 4 per genotype. (E) Expression of Col1α1 mRNA was analyzed by qRT‐PCR. Values were normalized to 18S and are shown as fold increase over the chow‐fed C57BL/6 control group, n = 8 per genotype. Values represent means ± SEM. Values with different alphabetical superscripts are significantly different, P

    Journal: Hepatology Communications

    Article Title: Nontranscriptional Activity of Interferon Regulatory Factor 3 Protects Mice From High‐Fat Diet‐Induced Liver Injury

    doi: 10.1002/hep4.1441

    Figure Lengend Snippet: Enhanced markers of hepatocellular death and fibrosis in Irf3 −/− mice compared with wild‐type and Irf3 S1/S1 mice after HFD feeding. Male C57BL/6, Irf3 −/− , and Irf3 S1/S1 mice were fed an HFD or chow for 12 weeks. (A,B) Paraffin‐embedded liver sections were stained for M30, a caspase‐dependent cleavage product of cytokeratin 18. Images were acquired using 10× and 40× objectives, and the positive area was quantified, n = 4 per genotype. (C,D) Paraffin‐embedded liver sections were stained with sirius red. Images were acquired using a 40× objective, and the positive area was quantified, n = 4 per genotype. (E) Expression of Col1α1 mRNA was analyzed by qRT‐PCR. Values were normalized to 18S and are shown as fold increase over the chow‐fed C57BL/6 control group, n = 8 per genotype. Values represent means ± SEM. Values with different alphabetical superscripts are significantly different, P

    Article Snippet: Two micrograms of liver RNA was reverse transcribed and analyzed with PowerSYBR quantitative real‐time polymerase chain reaction (qRT‐PCR) kits (Applied Biosystems, Foster City, CA) on a QuantStudio5 RT‐PCR Machine (Applied Biosystems).

    Techniques: Mouse Assay, Staining, Expressing, Quantitative RT-PCR

    Hepatic IRF3 activation after HFD feeding. Male C57BL/6, Irf3 −/− , and Irf3 S1/S1 mice were fed an HFD or chow for 12 weeks. (A) Expression of IFIT1 and IFIT3 mRNA was analyzed by qRT‐PCR. Values were normalized to 18S and are shown as fold increase over the chow‐fed C57BL/6 control group, n = 12 per genotype. (B) Expression of IRF3 was assessed by western blot and normalized to GAPDH, n = 4 per genotype. (C) Expression of IRF3 mRNA was analyzed by qRT‐PCR. Values were normalized to 18S and are shown as fold increase over the chow‐fed C57BL/6 control group, n = 12 per genotype. Values represent means ± SEM. Values with different alphabetical superscripts are significantly different, P

    Journal: Hepatology Communications

    Article Title: Nontranscriptional Activity of Interferon Regulatory Factor 3 Protects Mice From High‐Fat Diet‐Induced Liver Injury

    doi: 10.1002/hep4.1441

    Figure Lengend Snippet: Hepatic IRF3 activation after HFD feeding. Male C57BL/6, Irf3 −/− , and Irf3 S1/S1 mice were fed an HFD or chow for 12 weeks. (A) Expression of IFIT1 and IFIT3 mRNA was analyzed by qRT‐PCR. Values were normalized to 18S and are shown as fold increase over the chow‐fed C57BL/6 control group, n = 12 per genotype. (B) Expression of IRF3 was assessed by western blot and normalized to GAPDH, n = 4 per genotype. (C) Expression of IRF3 mRNA was analyzed by qRT‐PCR. Values were normalized to 18S and are shown as fold increase over the chow‐fed C57BL/6 control group, n = 12 per genotype. Values represent means ± SEM. Values with different alphabetical superscripts are significantly different, P

    Article Snippet: Two micrograms of liver RNA was reverse transcribed and analyzed with PowerSYBR quantitative real‐time polymerase chain reaction (qRT‐PCR) kits (Applied Biosystems, Foster City, CA) on a QuantStudio5 RT‐PCR Machine (Applied Biosystems).

    Techniques: Activation Assay, Mouse Assay, Expressing, Quantitative RT-PCR, Western Blot

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

    Journal: Aging Cell

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

    doi: 10.1111/acel.12446

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

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

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

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

    Journal: Aging Cell

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

    doi: 10.1111/acel.12446

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

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

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

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

    Journal: Aging Cell

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

    doi: 10.1111/acel.12446

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

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

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

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

    Journal: Aging Cell

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

    doi: 10.1111/acel.12446

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

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

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

    Modulation of endogenous Nrf2 expression. a After transfected with Nrf2-shRNA (shRNA-867, shRNA-1118, shRNA-1757, or shRNA-2019) or control shRNA (shNC), expression levels of Nrf2 mRNA in Bel-7402 cells were detected by qRT-PCR; b - c After transfected with Nrf2-shRNA (shRNA-867, shRNA-1118, shRNA-1757, or shRNA-2019) or control shRNA (shNC), expression levels of Nrf2 protein in Bel-7402 cells were detected by western blot; d After transfected with Nrf2 expression plasmid (pEFGP-Nrf2-1 or pEFGP-Nrf2-2) or mock pEGFP plasmid (pEGFP-NC), expression levels of Nrf2 mRNA in HepG2 cells were detected by qRT-PCR; e - f After transfected with Nrf2 expression plasmid (pEFGP-Nrf2-1 or pEFGP-Nrf2-2) or mock pEGFP plasmid (pEGFP-NC), expression levels of Nrf2 protein in HepG2 cells were detected by western blot. *P

    Journal: BMC Cancer

    Article Title: Nrf2 is a potential prognostic marker and promotes proliferation and invasion in human hepatocellular carcinoma

    doi: 10.1186/s12885-015-1541-1

    Figure Lengend Snippet: Modulation of endogenous Nrf2 expression. a After transfected with Nrf2-shRNA (shRNA-867, shRNA-1118, shRNA-1757, or shRNA-2019) or control shRNA (shNC), expression levels of Nrf2 mRNA in Bel-7402 cells were detected by qRT-PCR; b - c After transfected with Nrf2-shRNA (shRNA-867, shRNA-1118, shRNA-1757, or shRNA-2019) or control shRNA (shNC), expression levels of Nrf2 protein in Bel-7402 cells were detected by western blot; d After transfected with Nrf2 expression plasmid (pEFGP-Nrf2-1 or pEFGP-Nrf2-2) or mock pEGFP plasmid (pEGFP-NC), expression levels of Nrf2 mRNA in HepG2 cells were detected by qRT-PCR; e - f After transfected with Nrf2 expression plasmid (pEFGP-Nrf2-1 or pEFGP-Nrf2-2) or mock pEGFP plasmid (pEGFP-NC), expression levels of Nrf2 protein in HepG2 cells were detected by western blot. *P

    Article Snippet: Quantitative real time polymerase chain reaction (qRT-PCR) qRT-PCR assay was carried out by a BioRad iQ5 Real-Time PCR Detection System to analyze the mRNA levels of Nrf2.

    Techniques: Expressing, Transfection, shRNA, Quantitative RT-PCR, Western Blot, Plasmid Preparation

    Expression profile of selected miRNAs from qRT-PCR analysis

    Journal: BMC Plant Biology

    Article Title: Computational prediction of miRNAs and their targets in Phaseolus vulgaris using simple sequence repeat signatures

    doi: 10.1186/s12870-015-0516-3

    Figure Lengend Snippet: Expression profile of selected miRNAs from qRT-PCR analysis

    Article Snippet: Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) qRT-PCR reactions were carried out for the five selected miRNAs in a Bio-Rad CFX96 Real-Time PCR system using Bio-Rad iQ SYBR green supermix.

    Techniques: Expressing, Quantitative RT-PCR

    Inhibition of LINC01116 suppressed cell proliferation and migration in NPC cells. A, qRT‐PCR result of LINC01116 expression in NPC cell lines and the normal HNEpC cells. B, qRT‐PCR result of LINC01116 expression in CNE2 and 5‐8F cells under the transfection of shCtrl or two shRNAs against LINC01116. C, CCK‐8 assay was performed to evaluate cell viability in above cells. D, Cell proliferation was examined by EdU assay. E, Transwell assay for the assessment of cell migration in NPC cells with or without LINC01116 inhibition. * P

    Journal: Cancer Medicine

    Article Title: LINC01116 accelerates nasopharyngeal carcinoma progression based on its enhancement on MYC transcription activity. LINC01116 accelerates nasopharyngeal carcinoma progression based on its enhancement on MYC transcription activity

    doi: 10.1002/cam4.2624

    Figure Lengend Snippet: Inhibition of LINC01116 suppressed cell proliferation and migration in NPC cells. A, qRT‐PCR result of LINC01116 expression in NPC cell lines and the normal HNEpC cells. B, qRT‐PCR result of LINC01116 expression in CNE2 and 5‐8F cells under the transfection of shCtrl or two shRNAs against LINC01116. C, CCK‐8 assay was performed to evaluate cell viability in above cells. D, Cell proliferation was examined by EdU assay. E, Transwell assay for the assessment of cell migration in NPC cells with or without LINC01116 inhibition. * P

    Article Snippet: 2.3 Quantitative real‐time polymerase chain reaction (QRT‐PCR) TRIzol reagent from Invitrogen was utilized to isolate total RNAs from cultured NPC cells.

    Techniques: Inhibition, Migration, Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay, EdU Assay, Transwell Assay

    MYC activated LINC01116 at transcriptional level in NPC. A‐D, The expression of MYC or LINC01116 in indicated CNE2 and 5‐8F cells was tested by qRT‐PCR. E, ChIP assay and AGE analysis verified the binding of MYC protein to LINC01116 promoter. F,G, Luciferase reporter assay confirmed that LINC01116 was transcriptionally activated by MYC in both CNE2 and 5‐8F cells. * P

    Journal: Cancer Medicine

    Article Title: LINC01116 accelerates nasopharyngeal carcinoma progression based on its enhancement on MYC transcription activity. LINC01116 accelerates nasopharyngeal carcinoma progression based on its enhancement on MYC transcription activity

    doi: 10.1002/cam4.2624

    Figure Lengend Snippet: MYC activated LINC01116 at transcriptional level in NPC. A‐D, The expression of MYC or LINC01116 in indicated CNE2 and 5‐8F cells was tested by qRT‐PCR. E, ChIP assay and AGE analysis verified the binding of MYC protein to LINC01116 promoter. F,G, Luciferase reporter assay confirmed that LINC01116 was transcriptionally activated by MYC in both CNE2 and 5‐8F cells. * P

    Article Snippet: 2.3 Quantitative real‐time polymerase chain reaction (QRT‐PCR) TRIzol reagent from Invitrogen was utilized to isolate total RNAs from cultured NPC cells.

    Techniques: Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Luciferase, Reporter Assay

    LINC01116 mainly located in the cytoplasm of NPC cells and interacted with the 5ʹUTR of MYC mRNA. A, LINC01116 was predicted by lncLocator as a cytoplasmic lncRNA. B, Subcellular fractionation plus qRT‐PCR validated that LINC01116 was mainly distributed in the cytoplasm of NPC cells. C, The online IntaRNA tool suggested that LINC01116 could bind to the 5'UTR of MYC mRNA. D, The interaction between LINC01116 and MYC mRNA in CNE2 and 5‐8F cells was confirmed by RNA pull down assay. ** P

    Journal: Cancer Medicine

    Article Title: LINC01116 accelerates nasopharyngeal carcinoma progression based on its enhancement on MYC transcription activity. LINC01116 accelerates nasopharyngeal carcinoma progression based on its enhancement on MYC transcription activity

    doi: 10.1002/cam4.2624

    Figure Lengend Snippet: LINC01116 mainly located in the cytoplasm of NPC cells and interacted with the 5ʹUTR of MYC mRNA. A, LINC01116 was predicted by lncLocator as a cytoplasmic lncRNA. B, Subcellular fractionation plus qRT‐PCR validated that LINC01116 was mainly distributed in the cytoplasm of NPC cells. C, The online IntaRNA tool suggested that LINC01116 could bind to the 5'UTR of MYC mRNA. D, The interaction between LINC01116 and MYC mRNA in CNE2 and 5‐8F cells was confirmed by RNA pull down assay. ** P

    Article Snippet: 2.3 Quantitative real‐time polymerase chain reaction (QRT‐PCR) TRIzol reagent from Invitrogen was utilized to isolate total RNAs from cultured NPC cells.

    Techniques: Fractionation, Quantitative RT-PCR, Pull Down Assay

    LINC01116 enhanced MYC protein level so as to promote the transcription of MYC targets. A,B, The impact of LINC01116 on the mRNA and protein levels of MYC was estimated by qRT‐PCR (A) and western blot (B). C, Changes on the transcription of three MYC targets (CDK4, CCND2, and BMI1) in the context of MYC overexpression or together with LINC01116 suppression were evaluated by luciferase reporter assays. D‐E, qRT‐PCR results of the expression of CDK4, CCND2, and BMI1 in LINC01116‐silenced NPC cells. F, The influence of LINC01116 per se on the transcription of CDK4, CCND2, and BMI1 in NPC cells was determined by luciferase reporter assay. * P

    Journal: Cancer Medicine

    Article Title: LINC01116 accelerates nasopharyngeal carcinoma progression based on its enhancement on MYC transcription activity. LINC01116 accelerates nasopharyngeal carcinoma progression based on its enhancement on MYC transcription activity

    doi: 10.1002/cam4.2624

    Figure Lengend Snippet: LINC01116 enhanced MYC protein level so as to promote the transcription of MYC targets. A,B, The impact of LINC01116 on the mRNA and protein levels of MYC was estimated by qRT‐PCR (A) and western blot (B). C, Changes on the transcription of three MYC targets (CDK4, CCND2, and BMI1) in the context of MYC overexpression or together with LINC01116 suppression were evaluated by luciferase reporter assays. D‐E, qRT‐PCR results of the expression of CDK4, CCND2, and BMI1 in LINC01116‐silenced NPC cells. F, The influence of LINC01116 per se on the transcription of CDK4, CCND2, and BMI1 in NPC cells was determined by luciferase reporter assay. * P

    Article Snippet: 2.3 Quantitative real‐time polymerase chain reaction (QRT‐PCR) TRIzol reagent from Invitrogen was utilized to isolate total RNAs from cultured NPC cells.

    Techniques: Quantitative RT-PCR, Western Blot, Over Expression, Luciferase, Expressing, Reporter Assay

    Overexpression of MYC rescued LINC01116 depletion‐restrained proliferation and migration in NPC cells. A, The expression of MYC at both mRNA and protein levels in indicated cells was detected via qRT‐PCR and western blot. B‐D, The viability, proliferation, and migration in 5‐8F cells under different conditions were respectively evaluated through CCK‐8 (B), EdU (C), and transwell assays (D). * P

    Journal: Cancer Medicine

    Article Title: LINC01116 accelerates nasopharyngeal carcinoma progression based on its enhancement on MYC transcription activity. LINC01116 accelerates nasopharyngeal carcinoma progression based on its enhancement on MYC transcription activity

    doi: 10.1002/cam4.2624

    Figure Lengend Snippet: Overexpression of MYC rescued LINC01116 depletion‐restrained proliferation and migration in NPC cells. A, The expression of MYC at both mRNA and protein levels in indicated cells was detected via qRT‐PCR and western blot. B‐D, The viability, proliferation, and migration in 5‐8F cells under different conditions were respectively evaluated through CCK‐8 (B), EdU (C), and transwell assays (D). * P

    Article Snippet: 2.3 Quantitative real‐time polymerase chain reaction (QRT‐PCR) TRIzol reagent from Invitrogen was utilized to isolate total RNAs from cultured NPC cells.

    Techniques: Over Expression, Migration, Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay

    Graphic representation of NF-κB mRNA expression levels via qRT-PCR in the three groups. NF-κB mRNA expression levels in the model and propofol groups are significantly increased compared with those in the control group (**P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Propofol protects against endotoxin-induced myocardial injury by inhibiting NF-κB-mediated inflammation

    doi: 10.3892/etm.2017.5605

    Figure Lengend Snippet: Graphic representation of NF-κB mRNA expression levels via qRT-PCR in the three groups. NF-κB mRNA expression levels in the model and propofol groups are significantly increased compared with those in the control group (**P

    Article Snippet: Next, reverse transcription and qPCR were performed following the instructions in the reverse transcription, and quantitative real-time polymerase chain reaction (qRT-PCR) kits (Takara, Dalian, China).

    Techniques: Expressing, Quantitative RT-PCR

    Site-specific contribution to differential gene expression in KD. (A) Comparison of gene expression between laser-captured dermal tissue ( in situ ) and fibroblasts for both keloid and normal skin. qRT-PCR graph for both TGFβ1 and CTGF (additional examples found in S2A Fig ). All data are mean ± SEM for at least three independent experiments. B) qRT-PCR for TGFβ1 and interleukin-8 ( IL-8 ) showing relative contributions of different keloid sites to overall expression and comparison with normal skin (additional genes available in S2B Fig ). Data are mean ± SEM where * p-value

    Journal: PLoS ONE

    Article Title: Site-specific gene expression profiling as a novel strategy for unravelling keloid disease pathobiology

    doi: 10.1371/journal.pone.0172955

    Figure Lengend Snippet: Site-specific contribution to differential gene expression in KD. (A) Comparison of gene expression between laser-captured dermal tissue ( in situ ) and fibroblasts for both keloid and normal skin. qRT-PCR graph for both TGFβ1 and CTGF (additional examples found in S2A Fig ). All data are mean ± SEM for at least three independent experiments. B) qRT-PCR for TGFβ1 and interleukin-8 ( IL-8 ) showing relative contributions of different keloid sites to overall expression and comparison with normal skin (additional genes available in S2B Fig ). Data are mean ± SEM where * p-value

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) qRT-PCR was performed using the Lightcycler® 480 II platform (Roche Diagnostics, UK) as previously described [ ].

    Techniques: Expressing, In Situ, Quantitative RT-PCR

    qRT-PCR validation of candidate genes. Four candidate genes were chosen from each of the epidermis and dermis for validation by qRT-PCR. The bar graphs represent the qRT-PCR data for the microdissected keloid sites and normal skin and the line graph represents the associated microarray fold change in gene expression. In all cases the line graph follows the trend of the bar graph indicating the PCR reflects the microarray, thus validating the data. Data are presented as mean ± SEM and are from at least three independent experiments. For some of the genes there was no expression in normal skin and therefore for those genes no fold change for the qRT-PCR could be generated. In the interest of standardisation of all of the graphs they were then presented with the two axes. ADAM, a disintegrin and metalloproteinase; ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; BMP2, bone morphogenetic protein 2; CD36, cluster of differentiation 36; COMP, cartilage oligomeric protein; NOTCH4, notch 4; WDR66, WD repeat domain 66.

    Journal: PLoS ONE

    Article Title: Site-specific gene expression profiling as a novel strategy for unravelling keloid disease pathobiology

    doi: 10.1371/journal.pone.0172955

    Figure Lengend Snippet: qRT-PCR validation of candidate genes. Four candidate genes were chosen from each of the epidermis and dermis for validation by qRT-PCR. The bar graphs represent the qRT-PCR data for the microdissected keloid sites and normal skin and the line graph represents the associated microarray fold change in gene expression. In all cases the line graph follows the trend of the bar graph indicating the PCR reflects the microarray, thus validating the data. Data are presented as mean ± SEM and are from at least three independent experiments. For some of the genes there was no expression in normal skin and therefore for those genes no fold change for the qRT-PCR could be generated. In the interest of standardisation of all of the graphs they were then presented with the two axes. ADAM, a disintegrin and metalloproteinase; ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; BMP2, bone morphogenetic protein 2; CD36, cluster of differentiation 36; COMP, cartilage oligomeric protein; NOTCH4, notch 4; WDR66, WD repeat domain 66.

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) qRT-PCR was performed using the Lightcycler® 480 II platform (Roche Diagnostics, UK) as previously described [ ].

    Techniques: Quantitative RT-PCR, Microarray, Expressing, Polymerase Chain Reaction, Generated

    Expression of NF-κB1 and Bcl-2 detection in glioma tissues by qRT-PCR. Notes: The expression of NF-κB1 in glioma was significantly higher than that in nonneoplastic brain tissues ( P

    Journal: OncoTargets and therapy

    Article Title: Nuclear factor-kappa B1 inhibits early apoptosis of glioma cells by promoting the expression of Bcl-2

    doi: 10.2147/OTT.S144014

    Figure Lengend Snippet: Expression of NF-κB1 and Bcl-2 detection in glioma tissues by qRT-PCR. Notes: The expression of NF-κB1 in glioma was significantly higher than that in nonneoplastic brain tissues ( P

    Article Snippet: Relative levels of NF-κB1 and Bcl-2 mRNA were examined using SYBR green quantitative real-time polymerase chain reaction (qRT-PCR) (LightCycler® 480; Hoffman-La Roche Ltd., Basel, Switzerland) and were normalized to levels of GAPDH mRNA.

    Techniques: Expressing, Quantitative RT-PCR

    MACC1 SNPs L31V, S515L, and R804T and MACC1 expression in colorectal cells and tumors A). MACC1 mRNA expression was determined in 60 colorectal tumors by qRT-PCR. Box plots show the mRNA expression compared to MACC1 genotypes. SNPs have no effect on the MACC1 mRNA expression in these colorectal tumors. B ) SW480, DLD1, HCT116 colorectal cancer cells were transfected with pcDNA3.1/MACC1/wt, pcDNA3.1/MACC1/L31V, pcDNA3.1/MACC1/S515L and pcDNA3.1/MACC1/R804T. The MACC1 mRNA expression in these cell clones was measured by qRT-PCR and normalized to the MACC1 mRNA expression of parental cells. Additionally, MACC1 protein expression was determined. β-tubulin was used as a loading control. SNPs have no effect on the MACC1 mRNA and protein expression in SW480, DLD1 and HCT116 colorectal cancer cells.

    Journal: Molecular Cancer

    Article Title: SNPs in the coding region of the metastasis-inducing gene MACC1 and clinical outcome in colorectal cancer

    doi: 10.1186/1476-4598-11-49

    Figure Lengend Snippet: MACC1 SNPs L31V, S515L, and R804T and MACC1 expression in colorectal cells and tumors A). MACC1 mRNA expression was determined in 60 colorectal tumors by qRT-PCR. Box plots show the mRNA expression compared to MACC1 genotypes. SNPs have no effect on the MACC1 mRNA expression in these colorectal tumors. B ) SW480, DLD1, HCT116 colorectal cancer cells were transfected with pcDNA3.1/MACC1/wt, pcDNA3.1/MACC1/L31V, pcDNA3.1/MACC1/S515L and pcDNA3.1/MACC1/R804T. The MACC1 mRNA expression in these cell clones was measured by qRT-PCR and normalized to the MACC1 mRNA expression of parental cells. Additionally, MACC1 protein expression was determined. β-tubulin was used as a loading control. SNPs have no effect on the MACC1 mRNA and protein expression in SW480, DLD1 and HCT116 colorectal cancer cells.

    Article Snippet: Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) QRT-PCR was carried out using the LightCycler480 (Roche) as described previously [ ].

    Techniques: Expressing, Quantitative RT-PCR, Transfection, Clone Assay

    QRT-PCR and Western blot assays showed no significant changes in the mRNA ( A ) or protein levels ( B ) of SMAD2 in overexpressed or knocked down circSMAD2 HepG2 cells. Note: Data are presented as mean ± SEM. Abbreviations: QRT-PCR, quantitative real-time polymerase chain reaction; circSMAD2, circRNA SMAD2; SEM, standard error of the mean; NC, negative control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

    Journal: OncoTargets and therapy

    Article Title: circSMAD2 inhibits the epithelial–mesenchymal transition by targeting miR-629 in hepatocellular carcinoma

    doi: 10.2147/OTT.S158008

    Figure Lengend Snippet: QRT-PCR and Western blot assays showed no significant changes in the mRNA ( A ) or protein levels ( B ) of SMAD2 in overexpressed or knocked down circSMAD2 HepG2 cells. Note: Data are presented as mean ± SEM. Abbreviations: QRT-PCR, quantitative real-time polymerase chain reaction; circSMAD2, circRNA SMAD2; SEM, standard error of the mean; NC, negative control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

    Article Snippet: Quantitative real-time polymerase chain reaction (qRT-PCR) qRT-PCR was performed with SYBR® Premix Ex Taq™ II (Takara) on the Bio-Rad CFX96 (Bio-Rad Laboratories Inc., Hercules, CA, USA) according to the manufacturer’s instructions.

    Techniques: Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction, Negative Control

    PUM1 regulates ATXN1 levels via a highly conserved binding motif (A) Schematic representation of Human ATXN1 -3′UTR showing three putative PUM1 binding motifs (gray and red boxes) and their conservation in different species. The numbers indicate positions of PUM1 motifs in the human ATXN1 -3′UTR. (B) ATXN1 mRNA quantification by qRT-PCR in HEK293T cells upon overexpression (left panel) or knockdown (si PUM1 -81 and -82) (right panel) of PUM1 . The destination-cloning vector (Control), scrambled siRNAs (siScr.) and cel-miR-67 were used as negative controls. The housekeeping gene GAPDH was used to normalize the expression of genes in all the qRT-PCR experiments. (C) Luciferase assay in HEK293 cells overexpressing the reporter construct harboring the full length ATXN1 -3′UTR. In these conditions, we overexpressed (left panel) or decreased expression (right panel) of PUM1 . (D) Luciferase assay in HEK293T cells transfected with single WT and mutant (MUT) putative PUM1 binding site on the ATXN1 -3′UTR. The position of cloned regions for each PUM1 binding motif are indicated. The mutagenized nucleotides are highlighted in blue. In panels C and D the destination-vector (control), RNAi scramble (siScramble) and cel-miR-67 were used as negative controls; miR-101a was used as positive control. (RL) Renilla, (FL) Firefly luciferase. All the experiments were performed in triplicate (data represent mean ± S.E.M.). P values were calculated by Student’s t-test. Statistical significance is indicated as follows: * P

    Journal: Cell

    Article Title: Pumilio1 Haploinsufficiency Leads to SCA1-like Neurodegeneration by Increasing Wild-Type Ataxin1 Levels

    doi: 10.1016/j.cell.2015.02.012

    Figure Lengend Snippet: PUM1 regulates ATXN1 levels via a highly conserved binding motif (A) Schematic representation of Human ATXN1 -3′UTR showing three putative PUM1 binding motifs (gray and red boxes) and their conservation in different species. The numbers indicate positions of PUM1 motifs in the human ATXN1 -3′UTR. (B) ATXN1 mRNA quantification by qRT-PCR in HEK293T cells upon overexpression (left panel) or knockdown (si PUM1 -81 and -82) (right panel) of PUM1 . The destination-cloning vector (Control), scrambled siRNAs (siScr.) and cel-miR-67 were used as negative controls. The housekeeping gene GAPDH was used to normalize the expression of genes in all the qRT-PCR experiments. (C) Luciferase assay in HEK293 cells overexpressing the reporter construct harboring the full length ATXN1 -3′UTR. In these conditions, we overexpressed (left panel) or decreased expression (right panel) of PUM1 . (D) Luciferase assay in HEK293T cells transfected with single WT and mutant (MUT) putative PUM1 binding site on the ATXN1 -3′UTR. The position of cloned regions for each PUM1 binding motif are indicated. The mutagenized nucleotides are highlighted in blue. In panels C and D the destination-vector (control), RNAi scramble (siScramble) and cel-miR-67 were used as negative controls; miR-101a was used as positive control. (RL) Renilla, (FL) Firefly luciferase. All the experiments were performed in triplicate (data represent mean ± S.E.M.). P values were calculated by Student’s t-test. Statistical significance is indicated as follows: * P

    Article Snippet: Quantitative RT-polymerase chain reaction (qRT-PCR) experiments were performed using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories) with PerfeCta SYBR Green FastMix, ROX (Quanta Biosciences).

    Techniques: Binding Assay, Quantitative RT-PCR, Over Expression, Clone Assay, Plasmid Preparation, Expressing, Luciferase, Construct, Transfection, Mutagenesis, Positive Control

    Topology-dependent transcription of P tc cat is dependent on its chromosomal location. Transcriptional response to DNA relaxation by novobiocin measured by qRT−PCR. Exponentially growing cultures of the R6-CAT strains at OD 620nm = 0.4 were treated with novobiocin at 10× MIC. Total RNA was isolated; cDNA was synthesized and subjected to qRT−PCR. To normalize the three independent replicate samples, values were divided by those obtained of an internal fragment of the 16SrDNA gene. These normalized values were made relative to those obtained at time 0 min. Fold change represented the log2 mean of qRT-PCR values of three independent replicates ± SEM.

    Journal: PLoS ONE

    Article Title: Role of Global and Local Topology in the Regulation of Gene Expression in Streptococcus pneumoniae

    doi: 10.1371/journal.pone.0101574

    Figure Lengend Snippet: Topology-dependent transcription of P tc cat is dependent on its chromosomal location. Transcriptional response to DNA relaxation by novobiocin measured by qRT−PCR. Exponentially growing cultures of the R6-CAT strains at OD 620nm = 0.4 were treated with novobiocin at 10× MIC. Total RNA was isolated; cDNA was synthesized and subjected to qRT−PCR. To normalize the three independent replicate samples, values were divided by those obtained of an internal fragment of the 16SrDNA gene. These normalized values were made relative to those obtained at time 0 min. Fold change represented the log2 mean of qRT-PCR values of three independent replicates ± SEM.

    Article Snippet: RNA techniques Synthesis of cDNAs from 5 µg of total RNA was performed as previously described . cDNAs obtained were subjected to quantitative qRT-PCR (Chromo 4, BioRad) in 20 µl reactions containing 2 µl of cDNA, 0.4 µM of each specific primer, and 10 µl of LightCycler FastStart Universal A SYBR Green Master (Roche).

    Techniques: Quantitative RT-PCR, Isolation, Synthesized

    Relaxation-dependent transcription of P topA and P gyrB is different in their chromosomal location and in a replicating plasmid and similar for P parE . (A) A culture of R6 carrying a plasmid with a P topA gfp fusion was grown until OD 620nm = 0.4 and treated with novobiocin at 0.5× MIC and 10× MIC. Samples were processed as described in Figure 3 . Results obtained from qRT-PCR analysis at the two novobiocin concentrations indicated are shown. (B) Results obtained with two cultures of R6 one carrying a plasmid with a P gyrB gfp fusion and the other a plasmid with a P gyrB cat grown and treated as in A. (C) Results obtained with a culture of R6 carrying a plasmid with a P parE cat fusion grown and treated as in A. The expression from the promoters in their chromosomal locations ( topA , gyrB, parE ) was also determined in the same cultures. Relative values (log2 mean of three independent replicates ± SEM) are represented. To normalize the three independent replicate samples, values were divided by those obtained from internal fragments of the 16S rDNA. These normalized values were made relative to those obtained at time 0 min. The nucleotide sequences of the promoter regions present in the plasmids carrying the fusions are indicated in each case. The −35 and −10 boxes, the +1 mRNA, the ribosome-binding site (RBS), and the ATG initiation codon are indicated in upper case and underlined. Letters in cursive are those present in the pAST vector used for cloning.

    Journal: PLoS ONE

    Article Title: Role of Global and Local Topology in the Regulation of Gene Expression in Streptococcus pneumoniae

    doi: 10.1371/journal.pone.0101574

    Figure Lengend Snippet: Relaxation-dependent transcription of P topA and P gyrB is different in their chromosomal location and in a replicating plasmid and similar for P parE . (A) A culture of R6 carrying a plasmid with a P topA gfp fusion was grown until OD 620nm = 0.4 and treated with novobiocin at 0.5× MIC and 10× MIC. Samples were processed as described in Figure 3 . Results obtained from qRT-PCR analysis at the two novobiocin concentrations indicated are shown. (B) Results obtained with two cultures of R6 one carrying a plasmid with a P gyrB gfp fusion and the other a plasmid with a P gyrB cat grown and treated as in A. (C) Results obtained with a culture of R6 carrying a plasmid with a P parE cat fusion grown and treated as in A. The expression from the promoters in their chromosomal locations ( topA , gyrB, parE ) was also determined in the same cultures. Relative values (log2 mean of three independent replicates ± SEM) are represented. To normalize the three independent replicate samples, values were divided by those obtained from internal fragments of the 16S rDNA. These normalized values were made relative to those obtained at time 0 min. The nucleotide sequences of the promoter regions present in the plasmids carrying the fusions are indicated in each case. The −35 and −10 boxes, the +1 mRNA, the ribosome-binding site (RBS), and the ATG initiation codon are indicated in upper case and underlined. Letters in cursive are those present in the pAST vector used for cloning.

    Article Snippet: RNA techniques Synthesis of cDNAs from 5 µg of total RNA was performed as previously described . cDNAs obtained were subjected to quantitative qRT-PCR (Chromo 4, BioRad) in 20 µl reactions containing 2 µl of cDNA, 0.4 µM of each specific primer, and 10 µl of LightCycler FastStart Universal A SYBR Green Master (Roche).

    Techniques: Plasmid Preparation, Quantitative RT-PCR, Expressing, Binding Assay, Clone Assay

    The relaxation up-regulation of P gyrA depends on a bending. (A) Sequences of wild-type P gyrA , P gyrA 126Pae and P gyrA 121Pae derivatives. The −35 and extended −10 boxes, the nucleotide in which transcription is initiated (+1), the center of the intrinsic DNA curvature (diamond), and the location of the inserted GATC sequence that creates a PaeI restriction site are indicated. The 5 nucleotides deleted in P gyrA 121Pae are into brackets. (B) Curvature prediction in P gyrA and P gyrA Pae by using the model.it program at http// www.icgeb.trieste.it/dna program [46] . (C) Mobility of 232-bp (P gyrA ), 237-bp (P gyrA 126Pae ) and 232-bp (P gyrA 121Pae ) fragments in acrylamide gels at 60°C and 6°C to detect DNA curvature. (D) Transcription from P gyrA , P gyrA 126Pae , and P gyrA 121Pae . Results obtained from qRT-PCR analysis at 0.5× MIC and 10× MIC novobiocin concentrations are indicated. Cultures were grown and samples processed as described in Figure 3 . Relative values (mean of three independent replicates ± SEM) are represented and made relative to those obtained at time 0. Normalization of values was made dividing by those obtained from internal fragments of 16SrDNA gene. € CAT activity measurements detected as described in material and methods . Values represented are the mean of three independent replicates ± SEM. Specific activity, SA, is expressed as nmol acetylated chloramphenicol/mg of protein.

    Journal: PLoS ONE

    Article Title: Role of Global and Local Topology in the Regulation of Gene Expression in Streptococcus pneumoniae

    doi: 10.1371/journal.pone.0101574

    Figure Lengend Snippet: The relaxation up-regulation of P gyrA depends on a bending. (A) Sequences of wild-type P gyrA , P gyrA 126Pae and P gyrA 121Pae derivatives. The −35 and extended −10 boxes, the nucleotide in which transcription is initiated (+1), the center of the intrinsic DNA curvature (diamond), and the location of the inserted GATC sequence that creates a PaeI restriction site are indicated. The 5 nucleotides deleted in P gyrA 121Pae are into brackets. (B) Curvature prediction in P gyrA and P gyrA Pae by using the model.it program at http// www.icgeb.trieste.it/dna program [46] . (C) Mobility of 232-bp (P gyrA ), 237-bp (P gyrA 126Pae ) and 232-bp (P gyrA 121Pae ) fragments in acrylamide gels at 60°C and 6°C to detect DNA curvature. (D) Transcription from P gyrA , P gyrA 126Pae , and P gyrA 121Pae . Results obtained from qRT-PCR analysis at 0.5× MIC and 10× MIC novobiocin concentrations are indicated. Cultures were grown and samples processed as described in Figure 3 . Relative values (mean of three independent replicates ± SEM) are represented and made relative to those obtained at time 0. Normalization of values was made dividing by those obtained from internal fragments of 16SrDNA gene. € CAT activity measurements detected as described in material and methods . Values represented are the mean of three independent replicates ± SEM. Specific activity, SA, is expressed as nmol acetylated chloramphenicol/mg of protein.

    Article Snippet: RNA techniques Synthesis of cDNAs from 5 µg of total RNA was performed as previously described . cDNAs obtained were subjected to quantitative qRT-PCR (Chromo 4, BioRad) in 20 µl reactions containing 2 µl of cDNA, 0.4 µM of each specific primer, and 10 µl of LightCycler FastStart Universal A SYBR Green Master (Roche).

    Techniques: Sequencing, Quantitative RT-PCR, Activity Assay

    miR-628 suppressed the mRNA and protein expression of SOS1 . Notes: The breast CSCs of MDA-MB-231 ( A ) and MCF-7 ( B ) cells were transfected with the miR-628 mimic and miR-628 inhibitor, and SOS1 mRNA level was determined by qRT-PCR. ( C ) The breast CSCs of MDA-MB-231 and MCF-7 cells were transfected with miR-628 mimic and miR-628 inhibitor, and SOS1 protein level was analyzed by Western blotting. The protein expression levels were statistically analyzed by quantitating the intensity of the protein bands relative to that of the internal loading control (α-tubulin). Values indicate mean ± SD; * P

    Journal: OncoTargets and therapy

    Article Title: MicroRNA 628 suppresses migration and invasion of breast cancer stem cells through targeting SOS1

    doi: 10.2147/OTT.S164575

    Figure Lengend Snippet: miR-628 suppressed the mRNA and protein expression of SOS1 . Notes: The breast CSCs of MDA-MB-231 ( A ) and MCF-7 ( B ) cells were transfected with the miR-628 mimic and miR-628 inhibitor, and SOS1 mRNA level was determined by qRT-PCR. ( C ) The breast CSCs of MDA-MB-231 and MCF-7 cells were transfected with miR-628 mimic and miR-628 inhibitor, and SOS1 protein level was analyzed by Western blotting. The protein expression levels were statistically analyzed by quantitating the intensity of the protein bands relative to that of the internal loading control (α-tubulin). Values indicate mean ± SD; * P

    Article Snippet: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA following the manufacturer’s instructions.

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

    Effect of miR-628 overexpression on migration and invasion of the breast CSC subpopulation in MCF-7 and MDA-MB-231 cell lines. Notes: ( A ) miR-628 expression after miR-628 mimic was transfected into CSCs of MCF-7 and MDA-MB-231 cells was determined by qRT-PCR. ( B – D ) Effects of miR-628 overexpression on migration ( B , C ) and invasion ( D , E ) of MCF-7 and MDA-MB-231 CSCs. ( F ) Cells were transfected with the miR-628 mimic. The effect of miR-628 mimic transfection on the protein levels of vimentin, Snail, and E-cadherin was analyzed by Western blotting. ( G ) Protein expression levels were statistically analyzed by quantitating the intensity of the protein bands relative to that of the internal loading control (α-tubulin). *Values indicate mean ± SD; P

    Journal: OncoTargets and therapy

    Article Title: MicroRNA 628 suppresses migration and invasion of breast cancer stem cells through targeting SOS1

    doi: 10.2147/OTT.S164575

    Figure Lengend Snippet: Effect of miR-628 overexpression on migration and invasion of the breast CSC subpopulation in MCF-7 and MDA-MB-231 cell lines. Notes: ( A ) miR-628 expression after miR-628 mimic was transfected into CSCs of MCF-7 and MDA-MB-231 cells was determined by qRT-PCR. ( B – D ) Effects of miR-628 overexpression on migration ( B , C ) and invasion ( D , E ) of MCF-7 and MDA-MB-231 CSCs. ( F ) Cells were transfected with the miR-628 mimic. The effect of miR-628 mimic transfection on the protein levels of vimentin, Snail, and E-cadherin was analyzed by Western blotting. ( G ) Protein expression levels were statistically analyzed by quantitating the intensity of the protein bands relative to that of the internal loading control (α-tubulin). *Values indicate mean ± SD; P

    Article Snippet: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA following the manufacturer’s instructions.

    Techniques: Over Expression, Migration, Multiple Displacement Amplification, Expressing, Transfection, Quantitative RT-PCR, Western Blot