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  • 99
    Thermo Fisher mirvana qrt pcr mirna detection kit
    CircZFR directly interacted with miR-578 by binding to miR-578. a Venn diagrams representing the putative <t>miRNAs</t> that bind to circZFR identified by CircInteractome and CircBANK online algorithms. b , c The levels of miR-578, miR-944 and miR-532-3p by <t>qRT-PCR</t> in sh-NC-infected or sh-circZFR-transduced MCF7 and BT-549 cell lines. d qRT-PCR for miR-578 expression in the two BC cell lines transfected with miR-NC mimic or miR-578 mimic. e Schematic of the miR-578-binding sites within circZFR and the mutation of the seed sequence. f Relative luciferase activity in MCF7 and BT-549 cell lines cotransfected with circZFR-WT or circZFR-MUT and miR-NC mimic or miR-578 mimic. g qRT-PCR for circZFR level in cell lysates incubated with Bio-NC or Bio-miR-578 and streptavidin beads. * P
    Mirvana Qrt Pcr Mirna Detection Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 981 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Bio-Rad qpcr reactions
    Expression of mature and <t>pre-mRNA</t> Arc transcripts in hippocampus and cerebral cortex of sleep deprived animals. (A) Arc transcript structure and quantitative polymerase chain reaction <t>(qPCR)</t> primer design. To quantify de novo Arc transcription, Arc primers were designed to target either the transcript’s open reading frame (green) or the first intron on its 3′ UTR (yellow). These primer sets were aimed at amplifying mature and pre-mRNA, respectively. (B) Expression of Arc mRNA and pre-mRNA in samples of whole hippocampus or whole cerebral cortex, normalized to expression of gamma actin (Actg1). Gene expression data in samples taken from mice after 3 h of ad lib sleep (Sleep) and sleep deprivation (SD) were normalized as a fold change relative to mean values from the Sleep group. Values indicate mean ± SEM; n = 5 mice/group; ** indicate p
    Qpcr Reactions, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 6924 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad qpcr reaction
    MethyLight duplex <t>dPCR.</t> (A) Duplex p14 dPCR assay showing data for p14_M and p14_M2 assays separately and with estimated targets for both assays combined. (B) dPCR heatmap showing distribution of p14_M (red) and p14_M2 (blue) positive chambers in a duplex reaction showing three example panels of a dPCR plate. (C) Correlation between MethyLight <t>qPCR</t> vs . duplex dPCR (estimated targets for both assays combined). All correlations were significant at p
    Qpcr Reaction, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 2028 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene qpcr reactions
    β-catenin mRNA production is not affected in vimentin (vim) −/− mice undergoing unilateral ureteral obstruction (UUO). <t>qPCR</t> primers for vim, β-catenin, and keratin 8 were designed and utilized to probe wild-type (WT) and vim −/− kidneys 1, 2, and 4 wk following UUO. qPCR readouts were normalized to ribosome 18 s <t>(Rn18s)</t> to account for variability in loading and then expressed as fold changes of the internal control, nonligated right kidney, for each individual mouse that was analyzed. Relative mRNA expression to Rn18s shows increase in vim expression at 1 and 2 wk following UUO in WT kidneys, whereas vim expression is detected relative to Rn18s 4 wk following UUO in the unligated WT control kidney ( A ). β-catenin RNA expression is not statistically significant among all kidneys 1, 2, and 4 wk following UUO ( B ). Keratin 8 mRNA levels are also increased 1 wk post-UUO in WT ligated kidneys. At 4 wk, there is an increase of keratin 8 when compared with the other samples ( C ). mRNA levels were then normalized against nonligated right kidney (taken as 1). Histogram shows the mRNA levels of ligated left kidney. Vim expression was detected 1 and 2 wk following UUO and decreased sharply 4 wk post-UUO ( D ). There is no statistical significance in the detection of β-catenin mRNA 1, 2, and 4 wk post-UUO between WT and vim −/− mice ( E ). Keratin 8 mRNA levels are slightly higher in vim −/− mice 2 and 4 wk post-UUO ( F ). Data are representative of n = 4 independent experiments. Error bars = Standard error; * P
    Qpcr Reactions, supplied by Stratagene, used in various techniques. Bioz Stars score: 93/100, based on 608 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    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: 94/100, based on 678 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad quantitative real time pcr reactions
    CXCL1 and CCL3 are produced independently of TRIF in Kupffer cells, HSCs, and hepatocytes. ( A and B ) Primary Kupffer cells, ( C and D ) HSCs, and ( E and F ) hepatocytes were isolated from wild-type (WT) mice, ( A , C , and E ) TRIF -/- mice, and ( B , D , and F ) MyD88 -/- mice. Cells then were treated with LPS for 6 hours. Messenger <t>RNA</t> (mRNA) expression of CXCL1, CCL5, CCL3, IL10, Bambi, and Timp-1 was determined by quantitative real-time <t>PCR.</t> Similar results were obtained in 3 independent experiments. A representative result is shown. White square , wild-type mice; black square , TRIF -/- mice; gray square , MyD88 -/- mice. * P
    Quantitative Real Time Pcr Reactions, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 802 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Agilent technologies qpcr reactions
    Inhibition of miR-217 and miR-576-3p affects OROV replication. HuH-7 cells were transfected with 75 nM negative control inhibitor, miR-217 inhibitor, miR-576-3p inhibitor or both. 3 h post transfection, cells were infected with OROV at MOI 1 and <t>RT-qPCR</t> was performed to measure <t>miRNA,</t> target genes and OROV RNA levels. ( A ) Fold change of miRNA expression levels 6 h post-infection. MiRNA levels were normalized by U6 levels and infected cells were compared to uninfected cells, both pre-transfected with the respective inhibitor for each condition. Cells transfected with negative inhibitor followed by OROV infection were compared to uninfected cells transfected with the same inhibitor and considered as positive control (set as 1, y-axis) for comparison. Black columns represent miR-217 expression levels and gray columns represent miR-576-3p levels. NQ–not quantified. ( B ) Fold change of target genes RNA levels 18 h post infection. Target genes expression was normalized by GAPDH RNA levels and deregulation was measured relative to the same target in uninfected cells. Black columns represent DCP2 RNA levels and gray columns represent STING RNA levels. ( C ) Intracellular OROV segment S RNA levels 18 h post-infection. Positive control (cells transfected with negative inhibitor and infected with OROV) levels were set as 1 (y-axis) for comparison. ( D ) Viral titration of virus supernatants from experiments performed in C . Viruses in the supernatant were quantified by plaque assay 18 h post-infection and plotted as pfu/ml x 10 6 (y-axis). Error bars represent SD of triplicates for two independent experiments. NS = non-significant; * = p ≤ 0.05; ** = p ≤ 0.01.
    Qpcr Reactions, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 532 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher qpcr reactions
    Effects, on recipient cells, of miR-134-enriched <t>EVs</t> expelled from miR-134-over-expressing Hs578Ts(i) 8 cells A. <t>qPCR</t> analysis confirmed that miR-134 is enriched in EVs derived from miR-134-overexpressing Hs578Ts(i) 8 cells when compared to levels in EVs from NC-mimic-transfected Hs578Ts(i) 8 cells. B. These miR-134-enriched EVs (or NC mimic-EVs) were incubated with Hs578Ts(i) 8 parent cells resulting in significantly reduced expression of C(i). STAT5B and C(ii). Hsp90, as determined by immunoblot analysis followed by densitometry. This observation was associated with significantly reduced cellular D. migration and E. invasion of the recipient parental Hs578Ts(i) 8 cells. Graphs represent triplicate biological repeats, each including three technical repeats and are displayed as mean ± SEM, where * p
    Qpcr Reactions, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 15274 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Bio-Rad polymerase chain reactions qpcr
    Interaction networks of up-regulated genes and species-specific <t>qPCR</t> in xenogenic co-culture of bPCs and hMSCs. (A) The predicted interaction networks of the 180 up-regulated genes/proteins were shown. (B) 80% of human MSCs and 20% bovine chondrocytes were co-cultured for 2 days. <t>RNA</t> was then isolated. Expression of bovine genes was examined by real-time RT-PCR using bovine specific primers that did not cross-react with human genes. Data are expressed as fold change relative to the expression in chondrocyte monocultures and represent the mean of three independent experiments±S.D. (C) Expression of human genes was also demonstrated by real-time RT-PCR using human specific primers that did not cross-react with bovine genes. Data are expressed as fold change relative to the expression in MSC monocultures and represent the mean of three independent experiments±S.D. (D)
    Polymerase Chain Reactions Qpcr, 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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    93
    Quanta Biosciences qpcr reactions
    Transcriptional analysis of ct271/ftsL and ct764/ftsQ in Chlamydia . RNA was collected from infected cells at the indicated timepoints and assayed for transcripts by <t>RT-qPCR.</t> Transcript levels were normalized to genomic DNA (ng <t>cDNA/gDNA).</t> (A) Representative early stage ( euo ), (B) mid cycle division-related ( mreB, rodZ, ftsK ), and (C) late-stage ( omcB ) transcripts are shown for comparison. (D) ct271 and ct764 transcription are consistent with a mid-cycle profile. Data are representative of a minimum of two experiments. Standard deviations were typically less than 5% of the mean.
    Qpcr Reactions, supplied by Quanta Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 291 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher maxima probe qpcr master mix 2x
    Transcriptional analysis of ct271/ftsL and ct764/ftsQ in Chlamydia . RNA was collected from infected cells at the indicated timepoints and assayed for transcripts by <t>RT-qPCR.</t> Transcript levels were normalized to genomic DNA (ng <t>cDNA/gDNA).</t> (A) Representative early stage ( euo ), (B) mid cycle division-related ( mreB, rodZ, ftsK ), and (C) late-stage ( omcB ) transcripts are shown for comparison. (D) ct271 and ct764 transcription are consistent with a mid-cycle profile. Data are representative of a minimum of two experiments. Standard deviations were typically less than 5% of the mean.
    Maxima Probe Qpcr Master Mix 2x, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 114 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher qpcr reaction
    BRD4 expression is sufficient to transform immortalized ovarian surface epithelial cells. (A) Analysis of BRD4 copy-number from 1042 cell lines (CCLE). (B) BRD4 long and short-isoform expression in BRD4-transduced IOSE cells, and several BRD4-non-amplified (OV0419F, OV110F, OV2022F and OV0452F) and BRD4-amplified (HOXF062 and OV0857F) patient derived xenograft models. BRD4 copy-number (CN) in different PDX models were determined using <t>TaqMan</t> ® Copy-number Assay by <t>qPCR.</t> (C) Colony formation potential of IOSE cells expressing BRD4 -/+ 500 nM JQ1 was assessed using a soft-agar assay. (D) Proliferation of BRD4 expressing IOSE cells was examined by measuring cell confluence over time via live cell imaging. (E) Cell cycle analysis of long and short BRD4-isoform expressing IOSE cells.
    Qpcr Reaction, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5887 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene qpcr reaction
    BRD4 expression is sufficient to transform immortalized ovarian surface epithelial cells. (A) Analysis of BRD4 copy-number from 1042 cell lines (CCLE). (B) BRD4 long and short-isoform expression in BRD4-transduced IOSE cells, and several BRD4-non-amplified (OV0419F, OV110F, OV2022F and OV0452F) and BRD4-amplified (HOXF062 and OV0857F) patient derived xenograft models. BRD4 copy-number (CN) in different PDX models were determined using <t>TaqMan</t> ® Copy-number Assay by <t>qPCR.</t> (C) Colony formation potential of IOSE cells expressing BRD4 -/+ 500 nM JQ1 was assessed using a soft-agar assay. (D) Proliferation of BRD4 expressing IOSE cells was examined by measuring cell confluence over time via live cell imaging. (E) Cell cycle analysis of long and short BRD4-isoform expressing IOSE cells.
    Qpcr Reaction, supplied by Stratagene, used in various techniques. Bioz Stars score: 93/100, based on 220 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Bio-Rad real time pcr qpcr reactions
    Quantitative <t>PCR</t> <t>(qPCR)</t> analysis. (A) Relative expression levels of Mmp20, Klk4, and Ctrc normalized to β-actin during secretory, early-mid maturation, and mid-late maturation stages of amelogenesis. The expression patterns of Mmp20 and Klk4 are
    Real Time Pcr Qpcr Reactions, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 89/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher reverse transcription quantitative polymerase chain reaction rt qpcr trizol reagent
    Quantitative <t>PCR</t> <t>(qPCR)</t> analysis. (A) Relative expression levels of Mmp20, Klk4, and Ctrc normalized to β-actin during secretory, early-mid maturation, and mid-late maturation stages of amelogenesis. The expression patterns of Mmp20 and Klk4 are
    Reverse Transcription Quantitative Polymerase Chain Reaction Rt Qpcr Trizol Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 144 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    CircZFR directly interacted with miR-578 by binding to miR-578. a Venn diagrams representing the putative miRNAs that bind to circZFR identified by CircInteractome and CircBANK online algorithms. b , c The levels of miR-578, miR-944 and miR-532-3p by qRT-PCR in sh-NC-infected or sh-circZFR-transduced MCF7 and BT-549 cell lines. d qRT-PCR for miR-578 expression in the two BC cell lines transfected with miR-NC mimic or miR-578 mimic. e Schematic of the miR-578-binding sites within circZFR and the mutation of the seed sequence. f Relative luciferase activity in MCF7 and BT-549 cell lines cotransfected with circZFR-WT or circZFR-MUT and miR-NC mimic or miR-578 mimic. g qRT-PCR for circZFR level in cell lysates incubated with Bio-NC or Bio-miR-578 and streptavidin beads. * P

    Journal: Cancer Cell International

    Article Title: CircZFR functions as a sponge of miR-578 to promote breast cancer progression by regulating HIF1A expression

    doi: 10.1186/s12935-020-01492-5

    Figure Lengend Snippet: CircZFR directly interacted with miR-578 by binding to miR-578. a Venn diagrams representing the putative miRNAs that bind to circZFR identified by CircInteractome and CircBANK online algorithms. b , c The levels of miR-578, miR-944 and miR-532-3p by qRT-PCR in sh-NC-infected or sh-circZFR-transduced MCF7 and BT-549 cell lines. d qRT-PCR for miR-578 expression in the two BC cell lines transfected with miR-NC mimic or miR-578 mimic. e Schematic of the miR-578-binding sites within circZFR and the mutation of the seed sequence. f Relative luciferase activity in MCF7 and BT-549 cell lines cotransfected with circZFR-WT or circZFR-MUT and miR-NC mimic or miR-578 mimic. g qRT-PCR for circZFR level in cell lysates incubated with Bio-NC or Bio-miR-578 and streptavidin beads. * P

    Article Snippet: Quantitative real-time polymerase chain reaction (qRT-PCR) The expression levels of circZFR, HIF1A and miRNAs were gauged by qRT-PCR.

    Techniques: Binding Assay, Quantitative RT-PCR, Infection, Expressing, Transfection, Mutagenesis, Sequencing, Luciferase, Activity Assay, Incubation

    Expression of mature and pre-mRNA Arc transcripts in hippocampus and cerebral cortex of sleep deprived animals. (A) Arc transcript structure and quantitative polymerase chain reaction (qPCR) primer design. To quantify de novo Arc transcription, Arc primers were designed to target either the transcript’s open reading frame (green) or the first intron on its 3′ UTR (yellow). These primer sets were aimed at amplifying mature and pre-mRNA, respectively. (B) Expression of Arc mRNA and pre-mRNA in samples of whole hippocampus or whole cerebral cortex, normalized to expression of gamma actin (Actg1). Gene expression data in samples taken from mice after 3 h of ad lib sleep (Sleep) and sleep deprivation (SD) were normalized as a fold change relative to mean values from the Sleep group. Values indicate mean ± SEM; n = 5 mice/group; ** indicate p

    Journal: Neurobiology of learning and memory

    Article Title: Sleep loss disrupts Arc expression in dentate gyrus neurons

    doi: 10.1016/j.nlm.2018.04.006

    Figure Lengend Snippet: Expression of mature and pre-mRNA Arc transcripts in hippocampus and cerebral cortex of sleep deprived animals. (A) Arc transcript structure and quantitative polymerase chain reaction (qPCR) primer design. To quantify de novo Arc transcription, Arc primers were designed to target either the transcript’s open reading frame (green) or the first intron on its 3′ UTR (yellow). These primer sets were aimed at amplifying mature and pre-mRNA, respectively. (B) Expression of Arc mRNA and pre-mRNA in samples of whole hippocampus or whole cerebral cortex, normalized to expression of gamma actin (Actg1). Gene expression data in samples taken from mice after 3 h of ad lib sleep (Sleep) and sleep deprivation (SD) were normalized as a fold change relative to mean values from the Sleep group. Values indicate mean ± SEM; n = 5 mice/group; ** indicate p

    Article Snippet: 0.5 μg of RNA was used to synthesize cDNA using iScript cDNA Synthesis Kit (Bio-Rad) and cDNA was diluted 1:10 for mature Arc mRNA quantification and, due to the low expression of premature Arc mRNA transcript, cDNA was diluted 1:5. qPCR reactions were measured using a CFX96 Real-Time System, in 96-well reaction plates (Bio-Rad).

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

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

    Journal: Frontiers in Immunology

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

    doi: 10.3389/fimmu.2018.01667

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

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

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

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

    Journal: Frontiers in Immunology

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

    doi: 10.3389/fimmu.2018.01667

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

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

    Techniques: Real-time Polymerase Chain Reaction

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

    Journal: Frontiers in Immunology

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

    doi: 10.3389/fimmu.2018.01667

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

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

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

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

    Journal: Frontiers in Immunology

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

    doi: 10.3389/fimmu.2018.01667

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

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

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

    ChIP-qPCR of the DNA binding for PXR and RNA-Pol-II to the Cyp3a gene loci, as well as PPAR α and RNA-Pol-II to the Cyp4a gene loci. For PXR, sites 1–5 were selected based on the reanalysis of a published PXR-ChIP sequencing experiment

    Journal: Drug Metabolism and Disposition

    Article Title:

    doi: 10.1124/dmd.115.067504

    Figure Lengend Snippet: ChIP-qPCR of the DNA binding for PXR and RNA-Pol-II to the Cyp3a gene loci, as well as PPAR α and RNA-Pol-II to the Cyp4a gene loci. For PXR, sites 1–5 were selected based on the reanalysis of a published PXR-ChIP sequencing experiment

    Article Snippet: Real-time qPCR reactions of the ChIP DNA were performed using SsoAdvanced Universal SYBR Green Supermix in a Bio-Rad CFX384 Real-Time PCR Detection System.

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

    Fold decrements of template DNA of the β-globin and rhodopsin genes after MNase digestion of chromatin samples quantified by qPCR. Dark gray, phosphorylated samples. Light gray, unphosphorylated samples. ( A, C ) Results for the β-globin gene. ( B, D ) Results for the rhodopsin gene. Fold decrements were calculated as 2 ΔCt , using the Ct value of the initial undigested chromatin sample as reference for each gene. Error bars correspond to the interval 2 ΔCt±SD .

    Journal: Nucleic Acids Research

    Article Title: Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation

    doi: 10.1093/nar/gkv304

    Figure Lengend Snippet: Fold decrements of template DNA of the β-globin and rhodopsin genes after MNase digestion of chromatin samples quantified by qPCR. Dark gray, phosphorylated samples. Light gray, unphosphorylated samples. ( A, C ) Results for the β-globin gene. ( B, D ) Results for the rhodopsin gene. Fold decrements were calculated as 2 ΔCt , using the Ct value of the initial undigested chromatin sample as reference for each gene. Error bars correspond to the interval 2 ΔCt±SD .

    Article Snippet: Purified DNA was used as a template for the amplification of β-globin and rhodopsin genes by Quantitative Polymerase Chain Reaction (qPCR), following MIQE guidelines, in a C1000TM thermal cycler (BioRad).

    Techniques: Real-time Polymerase Chain Reaction

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-019-09148-3

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

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

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

    Gnas expression by BPA exposure and age. Based on BPA-related DHMR annotated to the Gnas gene, real-time quantitative polymerase chain reaction (RT-qPCR) was used to investigate longitudinal blood Gnas mRNA expression levels. RNA was isolated from the same longitudinal Control ( n = 6 per age group) and BPA-exposed ( n = 6 per age group) mouse blood samples used for DNA hydroxymethylation analyses. RT-qPCR was performed on the Gnas gene in triplicate. Three housekeeping genes— β -actin , 18S, and Gapdh—were included as internal controls in all RT-qPCR runs. In addition to housekeeping genes, an inter-plate calibrator control of brain cDNA was included for calculation of relative gene expression across multiple plates; all expression values are shown relative to this inter-plate calibrator. Expression levels were calculated following the 2 − Δ Δ Ct method. a Mean Gnas expression in BPA-exposed mouse blood showed a significant increase between 2 and 10 months of age ( p = 0.05 ); this pattern was not found in Control samples. b Mean Gnas expression was significantly lower in Control blood than BPA-exposed blood at 10 months of age ( p = 0.01 ).

    Journal: Environmental Health Perspectives

    Article Title: Longitudinal Effects of Developmental Bisphenol A Exposure on Epigenome-Wide DNA Hydroxymethylation at Imprinted Loci in Mouse Blood

    doi: 10.1289/EHP3441

    Figure Lengend Snippet: Gnas expression by BPA exposure and age. Based on BPA-related DHMR annotated to the Gnas gene, real-time quantitative polymerase chain reaction (RT-qPCR) was used to investigate longitudinal blood Gnas mRNA expression levels. RNA was isolated from the same longitudinal Control ( n = 6 per age group) and BPA-exposed ( n = 6 per age group) mouse blood samples used for DNA hydroxymethylation analyses. RT-qPCR was performed on the Gnas gene in triplicate. Three housekeeping genes— β -actin , 18S, and Gapdh—were included as internal controls in all RT-qPCR runs. In addition to housekeeping genes, an inter-plate calibrator control of brain cDNA was included for calculation of relative gene expression across multiple plates; all expression values are shown relative to this inter-plate calibrator. Expression levels were calculated following the 2 − Δ Δ Ct method. a Mean Gnas expression in BPA-exposed mouse blood showed a significant increase between 2 and 10 months of age ( p = 0.05 ); this pattern was not found in Control samples. b Mean Gnas expression was significantly lower in Control blood than BPA-exposed blood at 10 months of age ( p = 0.01 ).

    Article Snippet: In preparation for real-time quantitative polymerase chain reaction (RT-qPCR), cDNA samples were diluted 1:2.5 in RNase-free water, then mixed with 10 μ M forward/reverse primers, nuclease-free water, and Bio-Rad iQ SYBR Green Supermix (Catalog #1,708,880).

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

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

    Journal: BMC Genomics

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

    doi: 10.1186/1471-2164-15-1174

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

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

    Techniques: Digital PCR, Real-time Polymerase Chain Reaction

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

    Journal: BMC Genomics

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

    doi: 10.1186/1471-2164-15-1174

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

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

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

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

    Journal: BMC Genomics

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

    doi: 10.1186/1471-2164-15-1174

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

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

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

    β-catenin mRNA production is not affected in vimentin (vim) −/− mice undergoing unilateral ureteral obstruction (UUO). qPCR primers for vim, β-catenin, and keratin 8 were designed and utilized to probe wild-type (WT) and vim −/− kidneys 1, 2, and 4 wk following UUO. qPCR readouts were normalized to ribosome 18 s (Rn18s) to account for variability in loading and then expressed as fold changes of the internal control, nonligated right kidney, for each individual mouse that was analyzed. Relative mRNA expression to Rn18s shows increase in vim expression at 1 and 2 wk following UUO in WT kidneys, whereas vim expression is detected relative to Rn18s 4 wk following UUO in the unligated WT control kidney ( A ). β-catenin RNA expression is not statistically significant among all kidneys 1, 2, and 4 wk following UUO ( B ). Keratin 8 mRNA levels are also increased 1 wk post-UUO in WT ligated kidneys. At 4 wk, there is an increase of keratin 8 when compared with the other samples ( C ). mRNA levels were then normalized against nonligated right kidney (taken as 1). Histogram shows the mRNA levels of ligated left kidney. Vim expression was detected 1 and 2 wk following UUO and decreased sharply 4 wk post-UUO ( D ). There is no statistical significance in the detection of β-catenin mRNA 1, 2, and 4 wk post-UUO between WT and vim −/− mice ( E ). Keratin 8 mRNA levels are slightly higher in vim −/− mice 2 and 4 wk post-UUO ( F ). Data are representative of n = 4 independent experiments. Error bars = Standard error; * P

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Mechanism and Treatment of Renal Fibrosis: Vimentin expression is required for the development of EMT-related renal fibrosis following unilateral ureteral obstruction in mice

    doi: 10.1152/ajprenal.00340.2017

    Figure Lengend Snippet: β-catenin mRNA production is not affected in vimentin (vim) −/− mice undergoing unilateral ureteral obstruction (UUO). qPCR primers for vim, β-catenin, and keratin 8 were designed and utilized to probe wild-type (WT) and vim −/− kidneys 1, 2, and 4 wk following UUO. qPCR readouts were normalized to ribosome 18 s (Rn18s) to account for variability in loading and then expressed as fold changes of the internal control, nonligated right kidney, for each individual mouse that was analyzed. Relative mRNA expression to Rn18s shows increase in vim expression at 1 and 2 wk following UUO in WT kidneys, whereas vim expression is detected relative to Rn18s 4 wk following UUO in the unligated WT control kidney ( A ). β-catenin RNA expression is not statistically significant among all kidneys 1, 2, and 4 wk following UUO ( B ). Keratin 8 mRNA levels are also increased 1 wk post-UUO in WT ligated kidneys. At 4 wk, there is an increase of keratin 8 when compared with the other samples ( C ). mRNA levels were then normalized against nonligated right kidney (taken as 1). Histogram shows the mRNA levels of ligated left kidney. Vim expression was detected 1 and 2 wk following UUO and decreased sharply 4 wk post-UUO ( D ). There is no statistical significance in the detection of β-catenin mRNA 1, 2, and 4 wk post-UUO between WT and vim −/− mice ( E ). Keratin 8 mRNA levels are slightly higher in vim −/− mice 2 and 4 wk post-UUO ( F ). Data are representative of n = 4 independent experiments. Error bars = Standard error; * P

    Article Snippet: Primer set for mouse ribosome 18 S (Rn18s) was generous gift from Dr. Jourd’heuil’s laboratory. qPCR reactions were performed on Stratagene Mc3005P (Agilent Technologies, Santa Clara, CA) using PerfecCTa SYBR Green FastMix kit (95074-012, Quanta BioSciences, Beverly, MA). qPCR data were analyzed by the software Strategene MxPro (Agilent Technologies).

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

    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

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

    Journal: Cellular and Molecular Gastroenterology and Hepatology

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

    doi: 10.1016/j.jcmgh.2016.12.004

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

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

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

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

    Journal: Cellular and Molecular Gastroenterology and Hepatology

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

    doi: 10.1016/j.jcmgh.2016.12.004

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

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

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

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

    Journal: Cellular and Molecular Gastroenterology and Hepatology

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

    doi: 10.1016/j.jcmgh.2016.12.004

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

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

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

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

    Journal: Cellular and Molecular Gastroenterology and Hepatology

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

    doi: 10.1016/j.jcmgh.2016.12.004

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

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

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

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

    Journal: Cellular and Molecular Gastroenterology and Hepatology

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

    doi: 10.1016/j.jcmgh.2016.12.004

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

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

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

    Deletion of β-catenin abolishes the impact of Wnt3a on STAT3 and proinflammatory cytokine expression in macrophages. A and B , Quantitative real-time polymerase chain reaction (QPCR) analysis of STAT3, STAT1, and STAT3 target genes ( A ) and proinflammatory genes ( B ) in bone marrow–derived macrophages (BMM) isolated from β-catenin F/F LDLR −/− and β-catenin ΔMye LDLR −/− mice treated with vehicle control or Wnt 3a (* P

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    Article Title: Myeloid β-Catenin Deficiency Exacerbates Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient Mice

    doi: 10.1161/ATVBAHA.118.311059

    Figure Lengend Snippet: Deletion of β-catenin abolishes the impact of Wnt3a on STAT3 and proinflammatory cytokine expression in macrophages. A and B , Quantitative real-time polymerase chain reaction (QPCR) analysis of STAT3, STAT1, and STAT3 target genes ( A ) and proinflammatory genes ( B ) in bone marrow–derived macrophages (BMM) isolated from β-catenin F/F LDLR −/− and β-catenin ΔMye LDLR −/− mice treated with vehicle control or Wnt 3a (* P

    Article Snippet: RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction Analysis Total RNA was isolated from mouse tissues or cells using TRIzol Reagent (Life Technologies, 15596018), and quantitative real-time polymerase chain reaction was performed using gene-specific primers and SYBR Green PCR kit (BioRad, 1725125) as previously described.

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

    Ablation of myeloid β-catenin increases macrophage and systemic inflammation in β-catenin ΔMye LDLR −/− mice. A , Quantitative real-time polymerase chain reaction (QPCR) analysis of proinflammatory cytokine expression in peritoneal macrophages (PM) of β-catenin F/F LDLR −/− and β-catenin ΔMye LDLR −/− mice (** P

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    Article Title: Myeloid β-Catenin Deficiency Exacerbates Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient Mice

    doi: 10.1161/ATVBAHA.118.311059

    Figure Lengend Snippet: Ablation of myeloid β-catenin increases macrophage and systemic inflammation in β-catenin ΔMye LDLR −/− mice. A , Quantitative real-time polymerase chain reaction (QPCR) analysis of proinflammatory cytokine expression in peritoneal macrophages (PM) of β-catenin F/F LDLR −/− and β-catenin ΔMye LDLR −/− mice (** P

    Article Snippet: RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction Analysis Total RNA was isolated from mouse tissues or cells using TRIzol Reagent (Life Technologies, 15596018), and quantitative real-time polymerase chain reaction was performed using gene-specific primers and SYBR Green PCR kit (BioRad, 1725125) as previously described.

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

    β-Catenin deficiency affects STAT3 expression and STAT1 activity in macrophages. A, Quantitative real-time polymerase chain reaction (QPCR) analysis of STAT3, STAT1, and STAT3 target genes in peritoneal macrophages (PM) of β-catenin F/F LDLR −/− and β-catenin ΔMye LDLR −/− mice (** P

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    Article Title: Myeloid β-Catenin Deficiency Exacerbates Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient Mice

    doi: 10.1161/ATVBAHA.118.311059

    Figure Lengend Snippet: β-Catenin deficiency affects STAT3 expression and STAT1 activity in macrophages. A, Quantitative real-time polymerase chain reaction (QPCR) analysis of STAT3, STAT1, and STAT3 target genes in peritoneal macrophages (PM) of β-catenin F/F LDLR −/− and β-catenin ΔMye LDLR −/− mice (** P

    Article Snippet: RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction Analysis Total RNA was isolated from mouse tissues or cells using TRIzol Reagent (Life Technologies, 15596018), and quantitative real-time polymerase chain reaction was performed using gene-specific primers and SYBR Green PCR kit (BioRad, 1725125) as previously described.

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

    Inhibition of miR-217 and miR-576-3p affects OROV replication. HuH-7 cells were transfected with 75 nM negative control inhibitor, miR-217 inhibitor, miR-576-3p inhibitor or both. 3 h post transfection, cells were infected with OROV at MOI 1 and RT-qPCR was performed to measure miRNA, target genes and OROV RNA levels. ( A ) Fold change of miRNA expression levels 6 h post-infection. MiRNA levels were normalized by U6 levels and infected cells were compared to uninfected cells, both pre-transfected with the respective inhibitor for each condition. Cells transfected with negative inhibitor followed by OROV infection were compared to uninfected cells transfected with the same inhibitor and considered as positive control (set as 1, y-axis) for comparison. Black columns represent miR-217 expression levels and gray columns represent miR-576-3p levels. NQ–not quantified. ( B ) Fold change of target genes RNA levels 18 h post infection. Target genes expression was normalized by GAPDH RNA levels and deregulation was measured relative to the same target in uninfected cells. Black columns represent DCP2 RNA levels and gray columns represent STING RNA levels. ( C ) Intracellular OROV segment S RNA levels 18 h post-infection. Positive control (cells transfected with negative inhibitor and infected with OROV) levels were set as 1 (y-axis) for comparison. ( D ) Viral titration of virus supernatants from experiments performed in C . Viruses in the supernatant were quantified by plaque assay 18 h post-infection and plotted as pfu/ml x 10 6 (y-axis). Error bars represent SD of triplicates for two independent experiments. NS = non-significant; * = p ≤ 0.05; ** = p ≤ 0.01.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: MicroRNA and cellular targets profiling reveal miR-217 and miR-576-3p as proviral factors during Oropouche infection

    doi: 10.1371/journal.pntd.0006508

    Figure Lengend Snippet: Inhibition of miR-217 and miR-576-3p affects OROV replication. HuH-7 cells were transfected with 75 nM negative control inhibitor, miR-217 inhibitor, miR-576-3p inhibitor or both. 3 h post transfection, cells were infected with OROV at MOI 1 and RT-qPCR was performed to measure miRNA, target genes and OROV RNA levels. ( A ) Fold change of miRNA expression levels 6 h post-infection. MiRNA levels were normalized by U6 levels and infected cells were compared to uninfected cells, both pre-transfected with the respective inhibitor for each condition. Cells transfected with negative inhibitor followed by OROV infection were compared to uninfected cells transfected with the same inhibitor and considered as positive control (set as 1, y-axis) for comparison. Black columns represent miR-217 expression levels and gray columns represent miR-576-3p levels. NQ–not quantified. ( B ) Fold change of target genes RNA levels 18 h post infection. Target genes expression was normalized by GAPDH RNA levels and deregulation was measured relative to the same target in uninfected cells. Black columns represent DCP2 RNA levels and gray columns represent STING RNA levels. ( C ) Intracellular OROV segment S RNA levels 18 h post-infection. Positive control (cells transfected with negative inhibitor and infected with OROV) levels were set as 1 (y-axis) for comparison. ( D ) Viral titration of virus supernatants from experiments performed in C . Viruses in the supernatant were quantified by plaque assay 18 h post-infection and plotted as pfu/ml x 10 6 (y-axis). Error bars represent SD of triplicates for two independent experiments. NS = non-significant; * = p ≤ 0.05; ** = p ≤ 0.01.

    Article Snippet: Validation of miRNA through primer designed RT-qPCR Reverse transcription was performed using miRNA 1st-Strand cDNA Synthesis Kit (Agilent Technologies ) and qPCR reactions were made with High-Specificity miRNA QPCR Core Kit (Agilent Technologies ) and forward specific primers for each miRNA investigated.

    Techniques: Inhibition, Transfection, Negative Control, Infection, Quantitative RT-PCR, Expressing, Positive Control, Titration, Plaque Assay

    Validation of individual miRNAs expression. RT-qPCR with specific primers for individual miRNAs. Mean fold change of expression levels in OROV infected cells relative to uninfected cells for ( A ) miR-217 and ( B ) miR-576-3p. RT-qPCR was performed at 3, 6, 12 h post-infection. ( C ) RT-qPCR for miR-26a-1-3p, miR-26a-2-3p and miR-92a-1-5p was performed only at 12 h post-infection time point. MicroRNAs expression was normalized by U6 RNA endogenous levels. Error bars represent SD of triplicates of three independent experiments. Asterisks represent significant values compared to non-infected cells. * = p ≤ 0.05; ** = p ≤ 0.01.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: MicroRNA and cellular targets profiling reveal miR-217 and miR-576-3p as proviral factors during Oropouche infection

    doi: 10.1371/journal.pntd.0006508

    Figure Lengend Snippet: Validation of individual miRNAs expression. RT-qPCR with specific primers for individual miRNAs. Mean fold change of expression levels in OROV infected cells relative to uninfected cells for ( A ) miR-217 and ( B ) miR-576-3p. RT-qPCR was performed at 3, 6, 12 h post-infection. ( C ) RT-qPCR for miR-26a-1-3p, miR-26a-2-3p and miR-92a-1-5p was performed only at 12 h post-infection time point. MicroRNAs expression was normalized by U6 RNA endogenous levels. Error bars represent SD of triplicates of three independent experiments. Asterisks represent significant values compared to non-infected cells. * = p ≤ 0.05; ** = p ≤ 0.01.

    Article Snippet: Validation of miRNA through primer designed RT-qPCR Reverse transcription was performed using miRNA 1st-Strand cDNA Synthesis Kit (Agilent Technologies ) and qPCR reactions were made with High-Specificity miRNA QPCR Core Kit (Agilent Technologies ) and forward specific primers for each miRNA investigated.

    Techniques: Expressing, Quantitative RT-PCR, Infection

    Effects, on recipient cells, of miR-134-enriched EVs expelled from miR-134-over-expressing Hs578Ts(i) 8 cells A. qPCR analysis confirmed that miR-134 is enriched in EVs derived from miR-134-overexpressing Hs578Ts(i) 8 cells when compared to levels in EVs from NC-mimic-transfected Hs578Ts(i) 8 cells. B. These miR-134-enriched EVs (or NC mimic-EVs) were incubated with Hs578Ts(i) 8 parent cells resulting in significantly reduced expression of C(i). STAT5B and C(ii). Hsp90, as determined by immunoblot analysis followed by densitometry. This observation was associated with significantly reduced cellular D. migration and E. invasion of the recipient parental Hs578Ts(i) 8 cells. Graphs represent triplicate biological repeats, each including three technical repeats and are displayed as mean ± SEM, where * p

    Journal: Oncotarget

    Article Title: miR-134 in extracellular vesicles reduces triple-negative breast cancer aggression and increases drug sensitivity

    doi:

    Figure Lengend Snippet: Effects, on recipient cells, of miR-134-enriched EVs expelled from miR-134-over-expressing Hs578Ts(i) 8 cells A. qPCR analysis confirmed that miR-134 is enriched in EVs derived from miR-134-overexpressing Hs578Ts(i) 8 cells when compared to levels in EVs from NC-mimic-transfected Hs578Ts(i) 8 cells. B. These miR-134-enriched EVs (or NC mimic-EVs) were incubated with Hs578Ts(i) 8 parent cells resulting in significantly reduced expression of C(i). STAT5B and C(ii). Hsp90, as determined by immunoblot analysis followed by densitometry. This observation was associated with significantly reduced cellular D. migration and E. invasion of the recipient parental Hs578Ts(i) 8 cells. Graphs represent triplicate biological repeats, each including three technical repeats and are displayed as mean ± SEM, where * p

    Article Snippet: miRNA profiling miRNA profiling (384 miRNAs) was performed on biological triplicates of Hs578T and Hs578Ts(i)8 cells and their derived EVs using TaqMan Low-Density Assays (TLDA). qPCR reactions were performed according to the manufacturer's instructions (Applied Biosystems, Foster City, CA, USA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Transfection, Incubation, Migration

    Interaction networks of up-regulated genes and species-specific qPCR in xenogenic co-culture of bPCs and hMSCs. (A) The predicted interaction networks of the 180 up-regulated genes/proteins were shown. (B) 80% of human MSCs and 20% bovine chondrocytes were co-cultured for 2 days. RNA was then isolated. Expression of bovine genes was examined by real-time RT-PCR using bovine specific primers that did not cross-react with human genes. Data are expressed as fold change relative to the expression in chondrocyte monocultures and represent the mean of three independent experiments±S.D. (C) Expression of human genes was also demonstrated by real-time RT-PCR using human specific primers that did not cross-react with bovine genes. Data are expressed as fold change relative to the expression in MSC monocultures and represent the mean of three independent experiments±S.D. (D)

    Journal: Stem Cells and Development

    Article Title: Fibroblast Growth Factor-1 Is a Mesenchymal Stromal Cell-Secreted Factor Stimulating Proliferation of Osteoarthritic Chondrocytes in Co-Culture

    doi: 10.1089/scd.2013.0118

    Figure Lengend Snippet: Interaction networks of up-regulated genes and species-specific qPCR in xenogenic co-culture of bPCs and hMSCs. (A) The predicted interaction networks of the 180 up-regulated genes/proteins were shown. (B) 80% of human MSCs and 20% bovine chondrocytes were co-cultured for 2 days. RNA was then isolated. Expression of bovine genes was examined by real-time RT-PCR using bovine specific primers that did not cross-react with human genes. Data are expressed as fold change relative to the expression in chondrocyte monocultures and represent the mean of three independent experiments±S.D. (C) Expression of human genes was also demonstrated by real-time RT-PCR using human specific primers that did not cross-react with bovine genes. Data are expressed as fold change relative to the expression in MSC monocultures and represent the mean of three independent experiments±S.D. (D)

    Article Snippet: For quantitative polymerase chain reactions (qPCR), 1 μg of total RNA was reverse transcribed into cDNA using the iScript cDNA Synthesis kit (Bio-Rad). qPCR was performed on cDNA samples by using the iQ SYBR Green Supermix (Bio-Rad).

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

    Transcriptional analysis of ct271/ftsL and ct764/ftsQ in Chlamydia . RNA was collected from infected cells at the indicated timepoints and assayed for transcripts by RT-qPCR. Transcript levels were normalized to genomic DNA (ng cDNA/gDNA). (A) Representative early stage ( euo ), (B) mid cycle division-related ( mreB, rodZ, ftsK ), and (C) late-stage ( omcB ) transcripts are shown for comparison. (D) ct271 and ct764 transcription are consistent with a mid-cycle profile. Data are representative of a minimum of two experiments. Standard deviations were typically less than 5% of the mean.

    Journal: Frontiers in Microbiology

    Article Title: Identification and Partial Characterization of Potential FtsL and FtsQ Homologs of Chlamydia

    doi: 10.3389/fmicb.2015.01264

    Figure Lengend Snippet: Transcriptional analysis of ct271/ftsL and ct764/ftsQ in Chlamydia . RNA was collected from infected cells at the indicated timepoints and assayed for transcripts by RT-qPCR. Transcript levels were normalized to genomic DNA (ng cDNA/gDNA). (A) Representative early stage ( euo ), (B) mid cycle division-related ( mreB, rodZ, ftsK ), and (C) late-stage ( omcB ) transcripts are shown for comparison. (D) ct271 and ct764 transcription are consistent with a mid-cycle profile. Data are representative of a minimum of two experiments. Standard deviations were typically less than 5% of the mean.

    Article Snippet: Equal volumes of cDNA were used in qPCR reactions with SYBR Green (Quanta Biosciences, Gaithersburg, MD, USA) and measured on an ABI 7300 system [Applied Biosystems (Life Technologies)].

    Techniques: Infection, Quantitative RT-PCR

    BRD4 expression is sufficient to transform immortalized ovarian surface epithelial cells. (A) Analysis of BRD4 copy-number from 1042 cell lines (CCLE). (B) BRD4 long and short-isoform expression in BRD4-transduced IOSE cells, and several BRD4-non-amplified (OV0419F, OV110F, OV2022F and OV0452F) and BRD4-amplified (HOXF062 and OV0857F) patient derived xenograft models. BRD4 copy-number (CN) in different PDX models were determined using TaqMan ® Copy-number Assay by qPCR. (C) Colony formation potential of IOSE cells expressing BRD4 -/+ 500 nM JQ1 was assessed using a soft-agar assay. (D) Proliferation of BRD4 expressing IOSE cells was examined by measuring cell confluence over time via live cell imaging. (E) Cell cycle analysis of long and short BRD4-isoform expressing IOSE cells.

    Journal: PLoS ONE

    Article Title: BRD4 amplification facilitates an oncogenic gene expression program in high-grade serous ovarian cancer and confers sensitivity to BET inhibitors

    doi: 10.1371/journal.pone.0200826

    Figure Lengend Snippet: BRD4 expression is sufficient to transform immortalized ovarian surface epithelial cells. (A) Analysis of BRD4 copy-number from 1042 cell lines (CCLE). (B) BRD4 long and short-isoform expression in BRD4-transduced IOSE cells, and several BRD4-non-amplified (OV0419F, OV110F, OV2022F and OV0452F) and BRD4-amplified (HOXF062 and OV0857F) patient derived xenograft models. BRD4 copy-number (CN) in different PDX models were determined using TaqMan ® Copy-number Assay by qPCR. (C) Colony formation potential of IOSE cells expressing BRD4 -/+ 500 nM JQ1 was assessed using a soft-agar assay. (D) Proliferation of BRD4 expressing IOSE cells was examined by measuring cell confluence over time via live cell imaging. (E) Cell cycle analysis of long and short BRD4-isoform expressing IOSE cells.

    Article Snippet: 2 μg RNA was converted to cDNA with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems #4368814). qPCR reaction was set up with TaqMan® Gene Expression Master Mix (Thermo Fisher Scientific #4369016) and carried out on an Applied Biosystems 7500 HT instrument.

    Techniques: Expressing, Amplification, Derivative Assay, TaqMan Copy Number Assay, Real-time Polymerase Chain Reaction, Soft Agar Assay, Live Cell Imaging, Cell Cycle Assay

    Quantitative PCR (qPCR) analysis. (A) Relative expression levels of Mmp20, Klk4, and Ctrc normalized to β-actin during secretory, early-mid maturation, and mid-late maturation stages of amelogenesis. The expression patterns of Mmp20 and Klk4 are

    Journal: Journal of Dental Research

    Article Title: Chymotrypsin C (Caldecrin) Is Associated with Enamel Development

    doi: 10.1177/0022034511418231

    Figure Lengend Snippet: Quantitative PCR (qPCR) analysis. (A) Relative expression levels of Mmp20, Klk4, and Ctrc normalized to β-actin during secretory, early-mid maturation, and mid-late maturation stages of amelogenesis. The expression patterns of Mmp20 and Klk4 are

    Article Snippet: Real time-PCR (qPCR) reactions were performed with iQTM SYBR® Green Supermix (Bio-Rad) with rat-specific primer pairs as follows: Actb (β-actin; GenBank # , forward 5′-AGTGTGAC GTTGACATCCGTA-3′ and reverse 5′-GCCAGGGCAGTAA TCTCCTTCT-3′, product 112 bp); Klk4 (GenBank # NM_00 1004101, forward 5′-GGGATACCTGCCTAGTTTCTGG-3′ and reverse 5′-AGGTGGTACACGGGGTCATAC-3′, product 130 bp); Mmp20 (GenBank # , forward 5′-GGCGAG ATGGTGGCAAGAG-3′ and reverse 5′-CTGGGAAGAGGC GGTAGTT-3′, product 166 bp); and Ctrc (GenBank # NM_ 001077649, forward 5′- AGGACTATCCCTGCTATGTCA-3′ and reverse 5′- ACCACCAGTCCAACCTGGA-3′, product 126 bp).

    Techniques: Real-time Polymerase Chain Reaction, Expressing