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  • 93
    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: 93/100, based on 389 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad quantitative rt polymerase chain reaction qrt pcr reactions
    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 Rt Polymerase Chain Reaction Qrt Pcr Reactions, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 89/100, based on 194 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad real time polymerase chain reactions
    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
    Real Time Polymerase Chain Reactions, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 90/100, based on 97 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad real time polymerase chain reaction qrt pcr detection system
    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
    Real Time Polymerase Chain Reaction Qrt Pcr Detection System, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad quantitative reverse polymerase chain reaction qrt pcr
    Genome-wide analysis for cryptic SVs identifies disruption of further neurodevelopmental disease candidate genes and demonstrates reduced expression of ZNF804A in patient cells. (A,B) Graphical representation of the three genes disrupted by an ∼10 kB deletion on chr 19 [del(19q13.4)] (A) and the cryptic paracentric inversion inv(2)(p32.1q32.1) in the patient. Sites of breakpoints are denoted by arrows. (C) Graphical representation of anomalous-read (red dots) fusion positions for the cryptic 2.49 Mb paracentric inversion on chromosome 2. Mate-pair library sequencing-predicted breakpoints 5′ and 3′ of the inversion were amplified with breakpoint-specific primers and validated at base-pair level by <t>PCR</t> and capillary sequencing. (D) mRNA-levels of ZNF804A and the housekeeping gene RPL19 were quantified by <t>qRT-PCR</t> from total RNA isolated from fibroblasts of the patient or a healthy male control and normalized to expression of beta-actin.
    Quantitative Reverse Polymerase Chain Reaction Qrt Pcr, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad reverse transcription polymerase chain reaction qrt pcr
    The Numb -abated osteoblasts enhanced RANKL/OPG via the Hedgehog pathway. a <t>qRT-PCR</t> results of Pthrp and Pth transcriptional changes. qRT-PCRs were normalized to β-actin, and the data represent the mean ± SEM, * P
    Reverse Transcription Polymerase Chain Reaction Qrt Pcr, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 91/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad real time polymerase chain reactions qrt pcr
    SIRT1 is strongly upregulated in the BCa tissues compared with both paracancerous tissues and normal bladder tissues . (a) <t>qRT-PCR</t> analysis exhibited that the gene expression of SIRT1 in bladder cancer tissues was significantly higher than the matched paracancerous tissues. The value of GAPDH was used as an internal control. ∗ p
    Real Time Polymerase Chain Reactions Qrt Pcr, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 88/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad real time pcr
    SIRT1 is strongly upregulated in the BCa tissues compared with both paracancerous tissues and normal bladder tissues . (a) <t>qRT-PCR</t> analysis exhibited that the gene expression of SIRT1 in bladder cancer tissues was significantly higher than the matched paracancerous tissues. The value of GAPDH was used as an internal control. ∗ p
    Real Time Pcr, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 12747 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad quantitative reverse transcription polymerase chain reaction qrt pcr reactions
    SIRT1 is strongly upregulated in the BCa tissues compared with both paracancerous tissues and normal bladder tissues . (a) <t>qRT-PCR</t> analysis exhibited that the gene expression of SIRT1 in bladder cancer tissues was significantly higher than the matched paracancerous tissues. The value of GAPDH was used as an internal control. ∗ p
    Quantitative Reverse Transcription Polymerase Chain Reaction Qrt Pcr Reactions, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad real time polymerase chain reaction qrt pcr choromo 4 detector system
    SIRT1 is strongly upregulated in the BCa tissues compared with both paracancerous tissues and normal bladder tissues . (a) <t>qRT-PCR</t> analysis exhibited that the gene expression of SIRT1 in bladder cancer tissues was significantly higher than the matched paracancerous tissues. The value of GAPDH was used as an internal control. ∗ p
    Real Time Polymerase Chain Reaction Qrt Pcr Choromo 4 Detector System, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad quantitative real time polymerase chain reactions qrt pcr
    THC, CBD and CFZ inhibit cell migration in U266 cell line A. U266 cells were treated with CBD 12.5 μM, THC 12.5 μM, CFZ 100 nM alone or in combination for 24 h. CXCR4 and CD147 mRNA levels were determined by <t>qRT-PCR.</t> GAPDH was used for normalization. Data are expressed as relative fold with respect to vehicle treated cells used as control. Data are expressed as mean ± SD. *p
    Quantitative Real Time Polymerase Chain Reactions Qrt Pcr, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 90/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad quantitative real time polymerase chain reaction
    THC, CBD and CFZ inhibit cell migration in U266 cell line A. U266 cells were treated with CBD 12.5 μM, THC 12.5 μM, CFZ 100 nM alone or in combination for 24 h. CXCR4 and CD147 mRNA levels were determined by <t>qRT-PCR.</t> GAPDH was used for normalization. Data are expressed as relative fold with respect to vehicle treated cells used as control. Data are expressed as mean ± SD. *p
    Quantitative Real Time Polymerase Chain Reaction, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 241 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    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

    Synergistic effect of PGSM on IFN- α -induced anti-HCV activity in HCV replicon cells. HCV subgenomic replicon cells (genotype 1b) were treated with the indicated amounts of either PGSM or IFN- α alone or both as indicated. Culture media containing fresh compounds were replaced every three days. At the indicated time points after treatments, intracellular HCV RNA levels were determined by qRT-PCR. The copy number of HCV RNA was calculated from cells treated with compound as compared to that for control. Control indicates that cells were treated with 0.2% DMSO (vehicle).

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Triterpenoid Saponins Isolated from Platycodon grandiflorum Inhibit Hepatitis C Virus Replication

    doi: 10.1155/2013/560417

    Figure Lengend Snippet: Synergistic effect of PGSM on IFN- α -induced anti-HCV activity in HCV replicon cells. HCV subgenomic replicon cells (genotype 1b) were treated with the indicated amounts of either PGSM or IFN- α alone or both as indicated. Culture media containing fresh compounds were replaced every three days. At the indicated time points after treatments, intracellular HCV RNA levels were determined by qRT-PCR. The copy number of HCV RNA was calculated from cells treated with compound as compared to that for control. Control indicates that cells were treated with 0.2% DMSO (vehicle).

    Article Snippet: The HCV RNA levels were quantified by a quantitative real-time polymerase chain reaction (qRT-PCR) assay using IQ5 real-time PCR detection system (Bio-Rad, Hercules, CA, USA) with HCV-specific primers (5′-GAC ACT CCA CCA TAG ATC ACT C-3′ and 5′-CCC AAC ACT ACT CGG CTA G-3′) and probe (5′-FAM-CCC AAA TCT CCA GGC ATT GAG CGG-3′ BHQ-1).

    Techniques: Activity Assay, Quantitative RT-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 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

    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

    Vitamin D3 (VitD3) upregulates endogenous osteopontin (OPN) -A and -C isomers via a VDR-dependent pathway after subarachnoid hemorrhage (SAH). (a) Effect of VDR siRNA on the VDR mRNA expression. Pre-SAH + VitD3, intranasal treatment 30 ng/kg/day VitD3 24 h before SAH; siRNA, small interfering ribonucleic acid. (b) Structure of OPN isomers and qRT-PCR universal primer design. (c) VitD3 upregulates endogenous OPN-A and -C isomers. * P

    Journal: Journal of Cerebral Blood Flow & Metabolism

    Article Title: Intranasal administration of vitamin D attenuates blood–brain barrier disruption through endogenous upregulation of osteopontin and activation of CD44/P-gp glycosylation signaling after subarachnoid hemorrhage in rats

    doi: 10.1177/0271678X16671147

    Figure Lengend Snippet: Vitamin D3 (VitD3) upregulates endogenous osteopontin (OPN) -A and -C isomers via a VDR-dependent pathway after subarachnoid hemorrhage (SAH). (a) Effect of VDR siRNA on the VDR mRNA expression. Pre-SAH + VitD3, intranasal treatment 30 ng/kg/day VitD3 24 h before SAH; siRNA, small interfering ribonucleic acid. (b) Structure of OPN isomers and qRT-PCR universal primer design. (c) VitD3 upregulates endogenous OPN-A and -C isomers. * P

    Article Snippet: Quantitative real-time polymerase chain reaction (qRT-PCR) reactions were conducted using the SYBR Green detection reagent in Bio-Rad iQ5 system (Hercules, California).

    Techniques: Expressing, Quantitative RT-PCR

    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

    Comparison of circRNA expression levels between sequencing and qRT-PCR results. The Y-axis of the columns in the chart represents the log2-transformed fold changes computed from the sequencing and qRT-PCR data

    Journal: Anatolian Journal of Cardiology

    Article Title: Circular RNA expression profiles of persistent atrial fibrillation in patients with rheumatic heart disease

    doi: 10.14744/AnatolJCardiol.2018.35902

    Figure Lengend Snippet: Comparison of circRNA expression levels between sequencing and qRT-PCR results. The Y-axis of the columns in the chart represents the log2-transformed fold changes computed from the sequencing and qRT-PCR data

    Article Snippet: Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using an iCycler iQ system (Bio-Rad, CA, USA) as described previously ( ).

    Techniques: Expressing, Sequencing, Quantitative RT-PCR, Transformation Assay

    Genome-wide analysis for cryptic SVs identifies disruption of further neurodevelopmental disease candidate genes and demonstrates reduced expression of ZNF804A in patient cells. (A,B) Graphical representation of the three genes disrupted by an ∼10 kB deletion on chr 19 [del(19q13.4)] (A) and the cryptic paracentric inversion inv(2)(p32.1q32.1) in the patient. Sites of breakpoints are denoted by arrows. (C) Graphical representation of anomalous-read (red dots) fusion positions for the cryptic 2.49 Mb paracentric inversion on chromosome 2. Mate-pair library sequencing-predicted breakpoints 5′ and 3′ of the inversion were amplified with breakpoint-specific primers and validated at base-pair level by PCR and capillary sequencing. (D) mRNA-levels of ZNF804A and the housekeeping gene RPL19 were quantified by qRT-PCR from total RNA isolated from fibroblasts of the patient or a healthy male control and normalized to expression of beta-actin.

    Journal: PLoS ONE

    Article Title: Sequencing of a Patient with Balanced Chromosome Abnormalities and Neurodevelopmental Disease Identifies Disruption of Multiple High Risk Loci by Structural Variation

    doi: 10.1371/journal.pone.0090894

    Figure Lengend Snippet: Genome-wide analysis for cryptic SVs identifies disruption of further neurodevelopmental disease candidate genes and demonstrates reduced expression of ZNF804A in patient cells. (A,B) Graphical representation of the three genes disrupted by an ∼10 kB deletion on chr 19 [del(19q13.4)] (A) and the cryptic paracentric inversion inv(2)(p32.1q32.1) in the patient. Sites of breakpoints are denoted by arrows. (C) Graphical representation of anomalous-read (red dots) fusion positions for the cryptic 2.49 Mb paracentric inversion on chromosome 2. Mate-pair library sequencing-predicted breakpoints 5′ and 3′ of the inversion were amplified with breakpoint-specific primers and validated at base-pair level by PCR and capillary sequencing. (D) mRNA-levels of ZNF804A and the housekeeping gene RPL19 were quantified by qRT-PCR from total RNA isolated from fibroblasts of the patient or a healthy male control and normalized to expression of beta-actin.

    Article Snippet: Presence of abnormal gene products was validated by amplifying proposed fusion mRNAs isolated from patient and control lymphoblasts by quantitative-reverse polymerase chain reaction (qRT-PCR) using SYBR Green Supermix (Bio-Rad, Hercules, CA) according to established protocols.

    Techniques: Genome Wide, Expressing, Sequencing, Amplification, Polymerase Chain Reaction, Quantitative RT-PCR, Isolation

    The Numb -abated osteoblasts enhanced RANKL/OPG via the Hedgehog pathway. a qRT-PCR results of Pthrp and Pth transcriptional changes. qRT-PCRs were normalized to β-actin, and the data represent the mean ± SEM, * P

    Journal: Bone Research

    Article Title: NUMB maintains bone mass by promoting degradation of PTEN and GLI1 via ubiquitination in osteoblasts

    doi: 10.1038/s41413-018-0030-y

    Figure Lengend Snippet: The Numb -abated osteoblasts enhanced RANKL/OPG via the Hedgehog pathway. a qRT-PCR results of Pthrp and Pth transcriptional changes. qRT-PCRs were normalized to β-actin, and the data represent the mean ± SEM, * P

    Article Snippet: The concentration, purity, and integrity of the RNA samples were identified using the NanoDrop ND-2000 (Thermo Scientific) and by electrophoresis using denaturing formaldehyde gels. cDNA was synthesized using the QuantiTect Reverse Transcription kit (Qiagen), and a quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed in a CFX96 Real-Time System (Bio-Rad) using the QuantiTect SYBR Green PCR Kit (Qiagen). β-actin was chosen as an internal control, and relative quantification was used for calculating the results (Ct).

    Techniques: Quantitative RT-PCR

    SIRT1 is strongly upregulated in the BCa tissues compared with both paracancerous tissues and normal bladder tissues . (a) qRT-PCR analysis exhibited that the gene expression of SIRT1 in bladder cancer tissues was significantly higher than the matched paracancerous tissues. The value of GAPDH was used as an internal control. ∗ p

    Journal: BioMed Research International

    Article Title: Knockdown of SIRT1 Suppresses Bladder Cancer Cell Proliferation and Migration and Induces Cell Cycle Arrest and Antioxidant Response through FOXO3a-Mediated Pathways

    doi: 10.1155/2017/3781904

    Figure Lengend Snippet: SIRT1 is strongly upregulated in the BCa tissues compared with both paracancerous tissues and normal bladder tissues . (a) qRT-PCR analysis exhibited that the gene expression of SIRT1 in bladder cancer tissues was significantly higher than the matched paracancerous tissues. The value of GAPDH was used as an internal control. ∗ p

    Article Snippet: Real-time polymerase chain reactions (qRT-PCR) were performed with iQTM SYBR® Green Supermix (Bio-Rad Ltd., China) in a reaction system of 20 μ l total volume using 1 μ g of cDNA.

    Techniques: BIA-KA, Quantitative RT-PCR, Expressing

    Knockdown of SIRT1 in BCa cells induced alteration of ROS and related proteins . (a) qRT-PCR was used to testify the KD efficiency by using different siRNA to knockdown SIRT1 in T24 (A) and EJ (B) (contaminated by T24 as per “ http://iclac.org/databases/cross-contaminations/ ”) bladder cancer cells. All shown values were mean ± SD of three measurements and repeated three or more times, ∗ p

    Journal: BioMed Research International

    Article Title: Knockdown of SIRT1 Suppresses Bladder Cancer Cell Proliferation and Migration and Induces Cell Cycle Arrest and Antioxidant Response through FOXO3a-Mediated Pathways

    doi: 10.1155/2017/3781904

    Figure Lengend Snippet: Knockdown of SIRT1 in BCa cells induced alteration of ROS and related proteins . (a) qRT-PCR was used to testify the KD efficiency by using different siRNA to knockdown SIRT1 in T24 (A) and EJ (B) (contaminated by T24 as per “ http://iclac.org/databases/cross-contaminations/ ”) bladder cancer cells. All shown values were mean ± SD of three measurements and repeated three or more times, ∗ p

    Article Snippet: Real-time polymerase chain reactions (qRT-PCR) were performed with iQTM SYBR® Green Supermix (Bio-Rad Ltd., China) in a reaction system of 20 μ l total volume using 1 μ g of cDNA.

    Techniques: BIA-KA, Quantitative RT-PCR

    THC, CBD and CFZ inhibit cell migration in U266 cell line A. U266 cells were treated with CBD 12.5 μM, THC 12.5 μM, CFZ 100 nM alone or in combination for 24 h. CXCR4 and CD147 mRNA levels were determined by qRT-PCR. GAPDH was used for normalization. Data are expressed as relative fold with respect to vehicle treated cells used as control. Data are expressed as mean ± SD. *p

    Journal: Oncotarget

    Article Title: Cannabinoids synergize with carfilzomib, reducing multiple myeloma cells viability and migration

    doi: 10.18632/oncotarget.12721

    Figure Lengend Snippet: THC, CBD and CFZ inhibit cell migration in U266 cell line A. U266 cells were treated with CBD 12.5 μM, THC 12.5 μM, CFZ 100 nM alone or in combination for 24 h. CXCR4 and CD147 mRNA levels were determined by qRT-PCR. GAPDH was used for normalization. Data are expressed as relative fold with respect to vehicle treated cells used as control. Data are expressed as mean ± SD. *p

    Article Snippet: Quantitative real-time polymerase chain reactions (qRT-PCR) for iβ5, CXCR4 and CD147 were performed using the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad, Hercules, CA).

    Techniques: Migration, Quantitative RT-PCR

    Regulation of the β5i subunit by THC and CBD in MM cell lines stimulated with IFN-γ U266 and RPMI cells were treated with IFN-γ 100 U/ml for 24 h. Then cells were treated with THC (12.5 μM), CBD (12.5 μM) alone and in combination for additional 24 h. A. The β5i mRNA levels were determined by qRT-PCR. GAPDH was used for normalization. Data are expressed as relative fold with respect to vehicle treated cells used as the control. Data are expressed as mean ± SD. *p

    Journal: Oncotarget

    Article Title: Cannabinoids synergize with carfilzomib, reducing multiple myeloma cells viability and migration

    doi: 10.18632/oncotarget.12721

    Figure Lengend Snippet: Regulation of the β5i subunit by THC and CBD in MM cell lines stimulated with IFN-γ U266 and RPMI cells were treated with IFN-γ 100 U/ml for 24 h. Then cells were treated with THC (12.5 μM), CBD (12.5 μM) alone and in combination for additional 24 h. A. The β5i mRNA levels were determined by qRT-PCR. GAPDH was used for normalization. Data are expressed as relative fold with respect to vehicle treated cells used as the control. Data are expressed as mean ± SD. *p

    Article Snippet: Quantitative real-time polymerase chain reactions (qRT-PCR) for iβ5, CXCR4 and CD147 were performed using the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad, Hercules, CA).

    Techniques: Quantitative RT-PCR