18s rna  (Thermo Fisher)


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    Name:
    QuantumRNA Universal 18S Internal Standard
    Description:
    The Ambion QuantumRNA 18S Internal Standard with Competimer technology uses specially modified Competimer s of the same sequence as the normal 18S primers that cannot be extended By adjusting the ratio of 18S Competimer s to normal 18S mRNA primers the signal for 18S mRNA can be attenuated to the level of very rare messages They are used for quantitative end point RT PCR and include sufficient reagents for 100 reactions Because of its invariant expression across tissues and treatments 18S ribosomal RNA is an ideal internal control for quantitative RNA analysis The Universal 18S Internal Standards function across the broadest range of eukaryotes including animals plants and many protozoa and produce a 315 bp PCR product Accessory Products The QuantumRNA Classic 18S Internal Standard SKU AM1716 produce a different 489 bp PCR product and the QuantumRNA Classic II 18S Internal Standard SKU AM1717 produce a 324 bp PCR product
    Catalog Number:
    am1718
    Price:
    None
    Applications:
    General RT-PCR Reagents & Accessories|PCR & Real-Time PCR
    Category:
    Standards Ladders Controls
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    Structured Review

    Thermo Fisher 18s rna
    a Expression of genes Bax, Bcl - 2, Cas - 3 and TP53 in human grade IV glioma cells after treatment with 1.5 mg mL −1 Mt RV in vitro plant extract of Menyathes trifoliata . The transcript level of each gene was normalized to the expression of a reference gene ( <t>18S</t> <t>RNA</t> ). Data is presented as fold change in human grade IV glioma cells after treatment of Menyanthes trifoliata extract versus untreated human grade IV glioma cells, in which expression levels of the genes were set as 1. b Mitochondrial membrane potential in human grade IV glioma cells after treatment with 1.5 mg mL −1 Mt RV in vitro plant extract of Menyathes trifoliata . MMP is expressed as 530 nm/590 nm to 485 nm/538 nm (aggregates to monomer) fluorescence ratio, as quantified with a fluorescent plate reader after JC-1 staining). The mean values ± SD were calculated from three independent experiments. p
    The Ambion QuantumRNA 18S Internal Standard with Competimer technology uses specially modified Competimer s of the same sequence as the normal 18S primers that cannot be extended By adjusting the ratio of 18S Competimer s to normal 18S mRNA primers the signal for 18S mRNA can be attenuated to the level of very rare messages They are used for quantitative end point RT PCR and include sufficient reagents for 100 reactions Because of its invariant expression across tissues and treatments 18S ribosomal RNA is an ideal internal control for quantitative RNA analysis The Universal 18S Internal Standards function across the broadest range of eukaryotes including animals plants and many protozoa and produce a 315 bp PCR product Accessory Products The QuantumRNA Classic 18S Internal Standard SKU AM1716 produce a different 489 bp PCR product and the QuantumRNA Classic II 18S Internal Standard SKU AM1717 produce a 324 bp PCR product
    https://www.bioz.com/result/18s rna/product/Thermo Fisher
    Average 99 stars, based on 69 article reviews
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    18s rna - by Bioz Stars, 2021-01
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    Images

    1) Product Images from "Induction of apoptosis by in vitro and in vivo plant extracts derived from Menyanthes trifoliata L. in human cancer cells"

    Article Title: Induction of apoptosis by in vitro and in vivo plant extracts derived from Menyanthes trifoliata L. in human cancer cells

    Journal: Cytotechnology

    doi: 10.1007/s10616-018-0274-9

    a Expression of genes Bax, Bcl - 2, Cas - 3 and TP53 in human grade IV glioma cells after treatment with 1.5 mg mL −1 Mt RV in vitro plant extract of Menyathes trifoliata . The transcript level of each gene was normalized to the expression of a reference gene ( 18S RNA ). Data is presented as fold change in human grade IV glioma cells after treatment of Menyanthes trifoliata extract versus untreated human grade IV glioma cells, in which expression levels of the genes were set as 1. b Mitochondrial membrane potential in human grade IV glioma cells after treatment with 1.5 mg mL −1 Mt RV in vitro plant extract of Menyathes trifoliata . MMP is expressed as 530 nm/590 nm to 485 nm/538 nm (aggregates to monomer) fluorescence ratio, as quantified with a fluorescent plate reader after JC-1 staining). The mean values ± SD were calculated from three independent experiments. p
    Figure Legend Snippet: a Expression of genes Bax, Bcl - 2, Cas - 3 and TP53 in human grade IV glioma cells after treatment with 1.5 mg mL −1 Mt RV in vitro plant extract of Menyathes trifoliata . The transcript level of each gene was normalized to the expression of a reference gene ( 18S RNA ). Data is presented as fold change in human grade IV glioma cells after treatment of Menyanthes trifoliata extract versus untreated human grade IV glioma cells, in which expression levels of the genes were set as 1. b Mitochondrial membrane potential in human grade IV glioma cells after treatment with 1.5 mg mL −1 Mt RV in vitro plant extract of Menyathes trifoliata . MMP is expressed as 530 nm/590 nm to 485 nm/538 nm (aggregates to monomer) fluorescence ratio, as quantified with a fluorescent plate reader after JC-1 staining). The mean values ± SD were calculated from three independent experiments. p

    Techniques Used: Expressing, In Vitro, Fluorescence, Staining

    2) Product Images from "Estrogen receptors activate atrial natriuretic peptide in the rat heart"

    Article Title: Estrogen receptors activate atrial natriuretic peptide in the rat heart

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.201394198

    RT-PCR analysis of ERα and β transcripts in the heart chambers of newborn (4-day-old) and adult (55-day-old) female rats. Total RNA (1 μg) was reverse-transcribed, and cDNA was exponentially amplified in the presence of gene-specific primers. ( A ) A PhosphorImager scan after separation of the radioactive PCR product by electrophoresis in 1.5% agarose. Representative bands obtained from three RNA samples of the right atrium (RA) and left ventricle (LV) are shown. ( B ) Bar graphs depict ERα mRNA ( Left ) and ERβ mRNA ( Right ) levels verified by 18S RNA product used as internal standard. The results were calculated as a ratio of the signal given by radioactive bands of ERα mRNA or ERβ mRNA to the corresponding 18S band. RNA samples from the whole heart of newborn (1-day-old) rats were included in each experiment and used as a standard (100%) for calibration of ERα mRNA or ERβ mRNA levels. Values are means ± SEM for the right atrium and left ventricle, as well as for the left atrium (LA) and right ventricle (RV). n = 6; §, P
    Figure Legend Snippet: RT-PCR analysis of ERα and β transcripts in the heart chambers of newborn (4-day-old) and adult (55-day-old) female rats. Total RNA (1 μg) was reverse-transcribed, and cDNA was exponentially amplified in the presence of gene-specific primers. ( A ) A PhosphorImager scan after separation of the radioactive PCR product by electrophoresis in 1.5% agarose. Representative bands obtained from three RNA samples of the right atrium (RA) and left ventricle (LV) are shown. ( B ) Bar graphs depict ERα mRNA ( Left ) and ERβ mRNA ( Right ) levels verified by 18S RNA product used as internal standard. The results were calculated as a ratio of the signal given by radioactive bands of ERα mRNA or ERβ mRNA to the corresponding 18S band. RNA samples from the whole heart of newborn (1-day-old) rats were included in each experiment and used as a standard (100%) for calibration of ERα mRNA or ERβ mRNA levels. Values are means ± SEM for the right atrium and left ventricle, as well as for the left atrium (LA) and right ventricle (RV). n = 6; §, P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Electrophoresis

    ( A ) were subjected to SDS/PAGE, immunoblotted with an anti-ERα mAb, and visualized by the chemiluminescence technique. ( B and C ) RT-PCR analysis of ER transcripts and ANP in the left atrium ( B ) and right atrium ( C ) of sham-operated (sham), ovariectomized (ovx), and ovariectomized rats supplemented with 17-β-estradiol (ovxE). Total RNA (1 μg) was reverse-transcribed and cDNA was exponentially amplified in the presence of gene-specific primers. A PhosphorImager scan is presented after separation of the radioactive PCR product by electrophoresis in 1.5% agarose. Representative bands obtained in three RNA representative samples from sham, ovx, and ovxE rats are presented. The results were calculated as the ratio of the signal given by radioactive bands of ERα mRNA or ERβ mRNA to the corresponding 18S band. Values are means ± SEM adjusted to the values (in percentage) of sham-operated rats (100%). n = 6; *, P
    Figure Legend Snippet: ( A ) were subjected to SDS/PAGE, immunoblotted with an anti-ERα mAb, and visualized by the chemiluminescence technique. ( B and C ) RT-PCR analysis of ER transcripts and ANP in the left atrium ( B ) and right atrium ( C ) of sham-operated (sham), ovariectomized (ovx), and ovariectomized rats supplemented with 17-β-estradiol (ovxE). Total RNA (1 μg) was reverse-transcribed and cDNA was exponentially amplified in the presence of gene-specific primers. A PhosphorImager scan is presented after separation of the radioactive PCR product by electrophoresis in 1.5% agarose. Representative bands obtained in three RNA representative samples from sham, ovx, and ovxE rats are presented. The results were calculated as the ratio of the signal given by radioactive bands of ERα mRNA or ERβ mRNA to the corresponding 18S band. Values are means ± SEM adjusted to the values (in percentage) of sham-operated rats (100%). n = 6; *, P

    Techniques Used: SDS Page, Reverse Transcription Polymerase Chain Reaction, Aqueous Normal-phase Chromatography, Amplification, Polymerase Chain Reaction, Electrophoresis

    3) Product Images from "Social stress in mice induces voiding dysfunction and bladder wall remodeling"

    Article Title: Social stress in mice induces voiding dysfunction and bladder wall remodeling

    Journal:

    doi: 10.1152/ajprenal.90749.2008

    Social stress leads to increased expression of the myosin heavy chain B and A mRNA isoforms. A representative gel with normalization to 18S RNA is shown. These values represent the average ± SD from 6 control (only 4 are shown) and 6 stressed
    Figure Legend Snippet: Social stress leads to increased expression of the myosin heavy chain B and A mRNA isoforms. A representative gel with normalization to 18S RNA is shown. These values represent the average ± SD from 6 control (only 4 are shown) and 6 stressed

    Techniques Used: Expressing

    4) Product Images from "Neuronal megalin mediates synaptic plasticity—a novel mechanism underlying intellectual disabilities in megalin gene pathologies"

    Article Title: Neuronal megalin mediates synaptic plasticity—a novel mechanism underlying intellectual disabilities in megalin gene pathologies

    Journal: Brain Communications

    doi: 10.1093/braincomms/fcaa135

    TTR rescues megalin downregulation in TTR KO hippocampal neuronal cultures, in a megalin-dependent way. ( A ) Total RNA was extracted from WT ( n = 11) and TTR KO ( n = 10) hippocampal neuronal cultures (7 DIV), and megalin and 18S mRNA levels were semi-quantified through real-time PCR, showing a reduction in megalin mRNA levels in hippocampal neurons of TTR KO mice. ( B ) Megalin and tubulin protein levels were determined by western blot in TTR KO ( n = 6) and WT ( n = 8) hippocampal neuronal cultures, with a decrease in megalin protein levels observed in TTR KO neurons. TTR KO cultured hippocampal neurons were stimulated with recombinant mouse TTR [55 µg/ml (1 µM)] for 4 h (mRNA) ( C ) or 14 h (protein) ( D ). When indicated, neurons were treated with TTR, in presence or absence of anti-TTR Nanobodies 169F7 and 165C6 (2 µM), or treated only with the nanobodies ( C ). TTR treatment rescued megalin mRNA ( C , n = 5–7 independent cultures) and protein levels ( D , n = 3 independent cultures) in TTR KO neurons, and the effect was abolished in the presence of the 169F7 nanobody, specific for TTR-megalin interaction epitope ( C ). Megalin (+/−) TTR KO and TTR KO cultured hippocampal neurons (7 DIV) were stimulated with recombinant mutated forms of mouse TTR, I84S (a TTR with low affinity for its ligands) and K15N (55 µg/ml, for 4 h; a TTR mutated in the 169F7 nanobody epitope, affecting TTR-megalin interaction), and megalin and 18S mRNA levels were assessed ( E , n = 6–10 neuronal cultures). TTR-induced effect in increasing megalin levels is independent of TTR ligands but depends on TTR binding to megalin and on megalin levels. (F) WT hippocampal neuronal cultures were stimulated with recombinant mouse TTR (55 µg/ml, for 4 h) in the presence or absence of the inhibitor clathrin-mediated endocytosis Dynasore (80 μM) and megalin mRNA levels were assessed ( n = 5 neuronal cultures). No effect was observed in the presence of the inhibitor, indicating that TTR-induced increase in megalin levels does not involve receptor internalization. ( G ) LRP1 and 18S mRNA levels were determined in TTR KO hippocampal neurons ( n = 4 neuronal cultures), in the presence or absence of recombinant mouse TTR (55 µg/ml, for 4 h). LRP1 levels are not affected by TTR stimulation, indicating a specific effect for LRP2 (megalin). Statistical analysis was performed using Student’s unpaired t -test or one-way ANOVA followed by Bonferroni’s multiple comparison test. * P
    Figure Legend Snippet: TTR rescues megalin downregulation in TTR KO hippocampal neuronal cultures, in a megalin-dependent way. ( A ) Total RNA was extracted from WT ( n = 11) and TTR KO ( n = 10) hippocampal neuronal cultures (7 DIV), and megalin and 18S mRNA levels were semi-quantified through real-time PCR, showing a reduction in megalin mRNA levels in hippocampal neurons of TTR KO mice. ( B ) Megalin and tubulin protein levels were determined by western blot in TTR KO ( n = 6) and WT ( n = 8) hippocampal neuronal cultures, with a decrease in megalin protein levels observed in TTR KO neurons. TTR KO cultured hippocampal neurons were stimulated with recombinant mouse TTR [55 µg/ml (1 µM)] for 4 h (mRNA) ( C ) or 14 h (protein) ( D ). When indicated, neurons were treated with TTR, in presence or absence of anti-TTR Nanobodies 169F7 and 165C6 (2 µM), or treated only with the nanobodies ( C ). TTR treatment rescued megalin mRNA ( C , n = 5–7 independent cultures) and protein levels ( D , n = 3 independent cultures) in TTR KO neurons, and the effect was abolished in the presence of the 169F7 nanobody, specific for TTR-megalin interaction epitope ( C ). Megalin (+/−) TTR KO and TTR KO cultured hippocampal neurons (7 DIV) were stimulated with recombinant mutated forms of mouse TTR, I84S (a TTR with low affinity for its ligands) and K15N (55 µg/ml, for 4 h; a TTR mutated in the 169F7 nanobody epitope, affecting TTR-megalin interaction), and megalin and 18S mRNA levels were assessed ( E , n = 6–10 neuronal cultures). TTR-induced effect in increasing megalin levels is independent of TTR ligands but depends on TTR binding to megalin and on megalin levels. (F) WT hippocampal neuronal cultures were stimulated with recombinant mouse TTR (55 µg/ml, for 4 h) in the presence or absence of the inhibitor clathrin-mediated endocytosis Dynasore (80 μM) and megalin mRNA levels were assessed ( n = 5 neuronal cultures). No effect was observed in the presence of the inhibitor, indicating that TTR-induced increase in megalin levels does not involve receptor internalization. ( G ) LRP1 and 18S mRNA levels were determined in TTR KO hippocampal neurons ( n = 4 neuronal cultures), in the presence or absence of recombinant mouse TTR (55 µg/ml, for 4 h). LRP1 levels are not affected by TTR stimulation, indicating a specific effect for LRP2 (megalin). Statistical analysis was performed using Student’s unpaired t -test or one-way ANOVA followed by Bonferroni’s multiple comparison test. * P

    Techniques Used: Real-time Polymerase Chain Reaction, Mouse Assay, Western Blot, Cell Culture, Recombinant, Binding Assay

    5) Product Images from "RNA-Seq and differential gene expression analysis in Temora stylifera copepod females with contrasting non-feeding nauplii survival rates: an environmental transcriptomics study"

    Article Title: RNA-Seq and differential gene expression analysis in Temora stylifera copepod females with contrasting non-feeding nauplii survival rates: an environmental transcriptomics study

    Journal: BMC Genomics

    doi: 10.1186/s12864-020-07112-w

    Comparison between RNA-Seq and RT-qPCR in Temora stylifera . Relative expression ratio (log 2 -Fold-Change, FC) of 9 unigenes ( A5 , Obst , Arih1 , Ppa2 , Arsb , Hsp-70 , Vldlr , Me31b and Crebl2 ) and 3 isoforms ( Mob1b , Vasa and Pafah ) in Temora stylifera from May 23rd vs 30th samples, measured through RNA-Seq (blue bars) or RT-qPCR (yellow bars). For RT-qPCR results, bars represented mean ± SD values and data are normalized to RNA 18S and Ubi reference genes
    Figure Legend Snippet: Comparison between RNA-Seq and RT-qPCR in Temora stylifera . Relative expression ratio (log 2 -Fold-Change, FC) of 9 unigenes ( A5 , Obst , Arih1 , Ppa2 , Arsb , Hsp-70 , Vldlr , Me31b and Crebl2 ) and 3 isoforms ( Mob1b , Vasa and Pafah ) in Temora stylifera from May 23rd vs 30th samples, measured through RNA-Seq (blue bars) or RT-qPCR (yellow bars). For RT-qPCR results, bars represented mean ± SD values and data are normalized to RNA 18S and Ubi reference genes

    Techniques Used: RNA Sequencing Assay, Quantitative RT-PCR, Expressing

    6) Product Images from "Herpes Simplex Virus 1 MicroRNA miR-H28 Exported to Uninfected Cells in Exosomes Restricts Cell-to-Cell Virus Spread by Inducing Gamma Interferon mRNA"

    Article Title: Herpes Simplex Virus 1 MicroRNA miR-H28 Exported to Uninfected Cells in Exosomes Restricts Cell-to-Cell Virus Spread by Inducing Gamma Interferon mRNA

    Journal: Journal of Virology

    doi: 10.1128/JVI.01005-19

    Analyses of accumulation of IFN-γ mRNA in HEp-2 and HEK293T cells after HSV-1(F) infection. Replicate cultures of HEp-2 cells (A) and HEK293T cells (B) were mock treated or exposed to 20 PFU of HSV-1(F) per cell. The cells were harvested at the indicated times after infection, and total RNAs from cell lysates were extracted and reverse transcribed to cDNA as described in Materials and Methods. Levels of mRNA encoding IFN-γ were normalized to those of 18S RNA, and results are shown as fold changes, compared with the mRNA levels measured in uninfected HEp-2 or HEK293T cells.
    Figure Legend Snippet: Analyses of accumulation of IFN-γ mRNA in HEp-2 and HEK293T cells after HSV-1(F) infection. Replicate cultures of HEp-2 cells (A) and HEK293T cells (B) were mock treated or exposed to 20 PFU of HSV-1(F) per cell. The cells were harvested at the indicated times after infection, and total RNAs from cell lysates were extracted and reverse transcribed to cDNA as described in Materials and Methods. Levels of mRNA encoding IFN-γ were normalized to those of 18S RNA, and results are shown as fold changes, compared with the mRNA levels measured in uninfected HEp-2 or HEK293T cells.

    Techniques Used: Infection

    Accumulation of mRNAs for IFN-α, IFN-β, and IFN-γ with HEp-2 transfection of miR-H28 mimics. Replicate cultures of HEp-2 cells were mock treated or transfected with 100 nM NC, miR-H5-3p, miR-H6-3p, miR-H1-5p, miR-H26, miR-H27, miR-H28, or miR-H29 mimic. The cells were harvested at the indicated times after transfection, and total RNAs from cell lysates were extracted and reverse transcribed to cDNA as described in Materials and Methods. Levels of mRNAs for IFN-α (A), IFN-β (B), and IFN-γ (C) were normalized to those of 18S RNA, and results are shown as fold changes, compared with the mRNA levels measured in HEp-2 cells. NC, miRNA mimic negative control.
    Figure Legend Snippet: Accumulation of mRNAs for IFN-α, IFN-β, and IFN-γ with HEp-2 transfection of miR-H28 mimics. Replicate cultures of HEp-2 cells were mock treated or transfected with 100 nM NC, miR-H5-3p, miR-H6-3p, miR-H1-5p, miR-H26, miR-H27, miR-H28, or miR-H29 mimic. The cells were harvested at the indicated times after transfection, and total RNAs from cell lysates were extracted and reverse transcribed to cDNA as described in Materials and Methods. Levels of mRNAs for IFN-α (A), IFN-β (B), and IFN-γ (C) were normalized to those of 18S RNA, and results are shown as fold changes, compared with the mRNA levels measured in HEp-2 cells. NC, miRNA mimic negative control.

    Techniques Used: Transfection, Negative Control

    Temporal patterns of accumulation of mRNAs for 20 genes associated with various aspects of innate immunity. Replicate cultures of HEp-2 cells were mock treated (M) or exposed to 5 PFU of HSV-1(F) per cell. The cells were harvested at the indicated times after infection, and total RNAs were extracted and reverse transcribed to cDNA as described in Materials and Methods. Levels of mRNAs for ATF3, cGAS, CXCL10, HDAC1, HDAC4, IFI16, IFIT1, IFN-α, IFN-β, IFN- γ , IL-6, IRF3, LGP2, LSD1, MDA5, MYD88, PUM1, RIG-I, and STING were normalized to those of 18S RNA, and results are shown as fold changes, compared with the mRNA levels measured in uninfected HEp-2 cells.
    Figure Legend Snippet: Temporal patterns of accumulation of mRNAs for 20 genes associated with various aspects of innate immunity. Replicate cultures of HEp-2 cells were mock treated (M) or exposed to 5 PFU of HSV-1(F) per cell. The cells were harvested at the indicated times after infection, and total RNAs were extracted and reverse transcribed to cDNA as described in Materials and Methods. Levels of mRNAs for ATF3, cGAS, CXCL10, HDAC1, HDAC4, IFI16, IFIT1, IFN-α, IFN-β, IFN- γ , IL-6, IRF3, LGP2, LSD1, MDA5, MYD88, PUM1, RIG-I, and STING were normalized to those of 18S RNA, and results are shown as fold changes, compared with the mRNA levels measured in uninfected HEp-2 cells.

    Techniques Used: Infection

    7) Product Images from "Adipocyte-Specific Glucocorticoid Inactivation Protects Against Diet-Induced Obesity"

    Article Title: Adipocyte-Specific Glucocorticoid Inactivation Protects Against Diet-Induced Obesity

    Journal:

    doi:

    11βHSD2 and 11βHSD1 expression and activity. A : h11βHSD2 expression relative to 18S RNA in various adipose tissue depots of 24-week-old transgenic and WT mice ( n = 10 per group) using quantitative real-time PCR with human kidney
    Figure Legend Snippet: 11βHSD2 and 11βHSD1 expression and activity. A : h11βHSD2 expression relative to 18S RNA in various adipose tissue depots of 24-week-old transgenic and WT mice ( n = 10 per group) using quantitative real-time PCR with human kidney

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

    8) Product Images from "The peroxisome proliferator activated receptor ? is required for the differentiation of THP-1 monocytic cells by phorbol ester"

    Article Title: The peroxisome proliferator activated receptor ? is required for the differentiation of THP-1 monocytic cells by phorbol ester

    Journal: Nuclear Receptor

    doi: 10.1186/1478-1336-1-9

    PPARδ expression and activity is increased by phorbol ester. A: Time course of induction of PPARδ message in response to phorbol ester. THP-1 cells were plated in RPMI medium supplemented with 10% FCS and 1 nM PMA at a density of 1 × 10 6 per well. At each time point, cells were lyzed and RNA prepared, followed by cDNA synthesis. QPCR analysis was performed using PPARδ-specific primers and probe to determine the amount of message. Results are expressed as total relative to 18S and were performed in triplicate. B: Expression of PPARδ protein by THP-1 cells is up-regulated in response to phorbol ester. THP-1 cells were plated in RPMI medium supplemented with 10% FCS and either 1 nM PMA or vehicle and incubated for 48 h. Cells were then lyzed directly into SDS-PAGE sample buffer for Western blotting. Blots were probed using a rabbit polyclonal anti-serum raised against a GST-PPARδ AB domain fusion protein. C: COS-1 cells were transiently co-transfected with pFABPLuc and pCLDNhPPARδ. Transfection efficiency was controlled for by co-transfection with a plasmid encoding β-galactosidase. Cells thus transfected were treated with increasing concentrations of PMA for 24 h after which luciferase activity was measured. D: PMA-induced δ-activation is mediated through the ligand binding domain. COS-1 cells were transfected with an expression vector encoding a Gal-4/PPARδ ligand-binding domain chimera. Cells were then treated for 24 h with compound F, PMA or vehicle only and luciferase readings obtained.
    Figure Legend Snippet: PPARδ expression and activity is increased by phorbol ester. A: Time course of induction of PPARδ message in response to phorbol ester. THP-1 cells were plated in RPMI medium supplemented with 10% FCS and 1 nM PMA at a density of 1 × 10 6 per well. At each time point, cells were lyzed and RNA prepared, followed by cDNA synthesis. QPCR analysis was performed using PPARδ-specific primers and probe to determine the amount of message. Results are expressed as total relative to 18S and were performed in triplicate. B: Expression of PPARδ protein by THP-1 cells is up-regulated in response to phorbol ester. THP-1 cells were plated in RPMI medium supplemented with 10% FCS and either 1 nM PMA or vehicle and incubated for 48 h. Cells were then lyzed directly into SDS-PAGE sample buffer for Western blotting. Blots were probed using a rabbit polyclonal anti-serum raised against a GST-PPARδ AB domain fusion protein. C: COS-1 cells were transiently co-transfected with pFABPLuc and pCLDNhPPARδ. Transfection efficiency was controlled for by co-transfection with a plasmid encoding β-galactosidase. Cells thus transfected were treated with increasing concentrations of PMA for 24 h after which luciferase activity was measured. D: PMA-induced δ-activation is mediated through the ligand binding domain. COS-1 cells were transfected with an expression vector encoding a Gal-4/PPARδ ligand-binding domain chimera. Cells were then treated for 24 h with compound F, PMA or vehicle only and luciferase readings obtained.

    Techniques Used: Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Incubation, SDS Page, Western Blot, Transfection, Cotransfection, Plasmid Preparation, Luciferase, Activation Assay, Ligand Binding Assay

    9) Product Images from "Long Non-Coding RNA Hotairm1 Promotes S100A9 Support of MDSC Expansion during Sepsis"

    Article Title: Long Non-Coding RNA Hotairm1 Promotes S100A9 Support of MDSC Expansion during Sepsis

    Journal: Journal of clinical & cellular immunology

    doi:

    Knockdown of Hotairm1 in late sepsis MDSCs attenuates their immunosuppressive functions. Gr1 + CD11b + cells were isolated from the bone marrow of late septic mice by positive selection using anti-Gr1 antibody and magnetic beads. The cells were transfected with Hotairm1-specific or scramble siRNAs for 36 hr. (A) Effects of MDSCs on T cell proliferation and activation. CD4 + T cells were isolated from splenocytes of naive mice with anti-CD4 antibody and labeled with the fluorescent dye CFSE. The late sepsis Gr1 + CD11b + cells with Hotairm1 knockdown were then co-cultured (1:1 ratio). T cells were stimulated with anti-CD3 plus anti-CD28 antibodies (1 μg/ml each). After 3 days, the cells were harvested, and T cell proliferation and IFNϒ production were determined as in Figure 2 . (B and C) Effect of exosomes lacking Hotairm1 on T cells. Late sepsis Gr1 + CD11b + cells with Hotairm1 knockdown were cultured for 24 hr in serum-free media. Culture supernatants were harvested, and exosomes were purified using exoEasy Maxi kit. (B) Levels of Hotairm1 in exosomal RNA was determined by RT-qPCR. Values were normalized to 18S RNA. (C) Spleen CD4 + T cells were labeled and cultured with naive Gr1 + CD11b + cells (as described in Figure 2 ) in the presence of Hotairm1-lacking exosomes. T cell proliferation and IFNϒ production were measured as in A. Data are expressed as means ± SD of 5-6 mice/group from three experiments. * p
    Figure Legend Snippet: Knockdown of Hotairm1 in late sepsis MDSCs attenuates their immunosuppressive functions. Gr1 + CD11b + cells were isolated from the bone marrow of late septic mice by positive selection using anti-Gr1 antibody and magnetic beads. The cells were transfected with Hotairm1-specific or scramble siRNAs for 36 hr. (A) Effects of MDSCs on T cell proliferation and activation. CD4 + T cells were isolated from splenocytes of naive mice with anti-CD4 antibody and labeled with the fluorescent dye CFSE. The late sepsis Gr1 + CD11b + cells with Hotairm1 knockdown were then co-cultured (1:1 ratio). T cells were stimulated with anti-CD3 plus anti-CD28 antibodies (1 μg/ml each). After 3 days, the cells were harvested, and T cell proliferation and IFNϒ production were determined as in Figure 2 . (B and C) Effect of exosomes lacking Hotairm1 on T cells. Late sepsis Gr1 + CD11b + cells with Hotairm1 knockdown were cultured for 24 hr in serum-free media. Culture supernatants were harvested, and exosomes were purified using exoEasy Maxi kit. (B) Levels of Hotairm1 in exosomal RNA was determined by RT-qPCR. Values were normalized to 18S RNA. (C) Spleen CD4 + T cells were labeled and cultured with naive Gr1 + CD11b + cells (as described in Figure 2 ) in the presence of Hotairm1-lacking exosomes. T cell proliferation and IFNϒ production were measured as in A. Data are expressed as means ± SD of 5-6 mice/group from three experiments. * p

    Techniques Used: Isolation, Mouse Assay, Selection, Magnetic Beads, Transfection, Activation Assay, Labeling, Cell Culture, Purification, Quantitative RT-PCR

    Hotairm1 expression is increased in late sepsis MDSCs. Gr1 + CD11b + cells were isolated from the bone marrow of sham and septic mice by positive selection using anti-Gr1 antibody and magnetic beads. (A) The cells were cultured for 24 hr in serum-free media. Exosomes were purified from the culture supernatants, and exosomal RNA was extracted with exoRNeasy Starter kit. Levels of Hotairm1 were determined by RT-qPCR using RT lncRNAqPCR Assay Primers (Qiagen). Values were normalized to 18S RNA. (B) Total RNA was isolated from Gr1 + CD11b + cells using TRIzol reagent, and levels of Hotaim1 were determined as in A. Values were normalized to GAPDH RNA. (C) Gr1 + CD11b + cells isolated from naïve mice were cultured for 24 hr without or with exosomes (50 μg/well), purified from late sepsis MDSC culture. The cells were harvested, total RNA was extracted and levels of Hotairm1 were determined as in B. PCR was performed in duplicate. Data are presented relative to sham or media control (1-fold). Data in A and B are expressed as means ± SD of 6-9 mice/group from three experiments. Data in C are expressed as means ± SD of 7 cultures from two experiments. * p
    Figure Legend Snippet: Hotairm1 expression is increased in late sepsis MDSCs. Gr1 + CD11b + cells were isolated from the bone marrow of sham and septic mice by positive selection using anti-Gr1 antibody and magnetic beads. (A) The cells were cultured for 24 hr in serum-free media. Exosomes were purified from the culture supernatants, and exosomal RNA was extracted with exoRNeasy Starter kit. Levels of Hotairm1 were determined by RT-qPCR using RT lncRNAqPCR Assay Primers (Qiagen). Values were normalized to 18S RNA. (B) Total RNA was isolated from Gr1 + CD11b + cells using TRIzol reagent, and levels of Hotaim1 were determined as in A. Values were normalized to GAPDH RNA. (C) Gr1 + CD11b + cells isolated from naïve mice were cultured for 24 hr without or with exosomes (50 μg/well), purified from late sepsis MDSC culture. The cells were harvested, total RNA was extracted and levels of Hotairm1 were determined as in B. PCR was performed in duplicate. Data are presented relative to sham or media control (1-fold). Data in A and B are expressed as means ± SD of 6-9 mice/group from three experiments. Data in C are expressed as means ± SD of 7 cultures from two experiments. * p

    Techniques Used: Expressing, Isolation, Mouse Assay, Selection, Magnetic Beads, Cell Culture, Purification, Quantitative RT-PCR, Polymerase Chain Reaction

    High levels of Hotairm1 in plasma exosomes and MDSCs from late septic patients. (A) Exosomes were purified from plasma and RNA was extracted using exoRNeasy Starter kit. (B) Peripheral blood CD33 + CD11b+HLA-DR − cells were isolated by magnetic cell separation. PBMCs were first purified and depleted of the HLA-DR + cells. The HLA-DR − cell population was then subjected to positive selection with biotin-coupled anti-CD33 antibody, followed by anti-CD11b antibody. Total RNA was extracted using TRIzol reagent. Levels of Hotairm1 were determined by RT-qPCR as in Figure 3 . Values in A were normalized to 18S RNA, and values in B were normalized to GAPDH RNA. Data are expressed as means ± SD of 7-9 subjects/group and are presented relative to HC (1-fold), * p
    Figure Legend Snippet: High levels of Hotairm1 in plasma exosomes and MDSCs from late septic patients. (A) Exosomes were purified from plasma and RNA was extracted using exoRNeasy Starter kit. (B) Peripheral blood CD33 + CD11b+HLA-DR − cells were isolated by magnetic cell separation. PBMCs were first purified and depleted of the HLA-DR + cells. The HLA-DR − cell population was then subjected to positive selection with biotin-coupled anti-CD33 antibody, followed by anti-CD11b antibody. Total RNA was extracted using TRIzol reagent. Levels of Hotairm1 were determined by RT-qPCR as in Figure 3 . Values in A were normalized to 18S RNA, and values in B were normalized to GAPDH RNA. Data are expressed as means ± SD of 7-9 subjects/group and are presented relative to HC (1-fold), * p

    Techniques Used: Purification, Isolation, Magnetic Cell Separation, Selection, Quantitative RT-PCR

    10) Product Images from "Independent regulation of JNK/p38 mitogen-activated protein kinases by metabolic oxidative stress in the liver"

    Article Title: Independent regulation of JNK/p38 mitogen-activated protein kinases by metabolic oxidative stress in the liver

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Constitutive activity of p38 MAP kinase in the liver is inactivated by metabolic oxidative stress. ( A ) p38 MAP kinase inactivation in CCl 4 -treated liver extracts. The activity of p38 MAP kinase was measured by an immunecomplex protein kinase assay using [γ- 32 P]ATP and GST-ATF-2(1–109) as a substrate. The phosphorylated GST-ATF-2(1–109) was detected after SDS/PAGE by autoradiography. ( B ) p38 MAP kinase phosphorylation during oxidative stress. About 200 μg of liver extracts from control or CCl 4 -treated mice was electrophoresed on a 10% SDS/PAGE gel, blotted, and probed with antisera specific to phospho-p38 (New England Biolabs) or with antisera specific to p38. Arrows indicate specific immune complexes to either phospho-p38 or p38 protein as detected by enhanced chemiluminescence (DuPont). ( C ) Activation of MKP-1 by metabolic oxidative stress. MKP-1 mRNA levels were analyzed by RNase protection assay. MKP-1 mRNA levels were quantitated using a Molecular Dynamics PhosphorImager and normalized for 18S RNA. The mean results of two experiments are shown. ( D ) Localization of MKP-1 mRNA was determined using in situ hybridization. [ 35 S]-labeled MKP-1 antisense probe was hybridized to 7-μm sections of liver tissue from control and 4-hr CCl 4 -treated livers. The same field is shown using both bright-field and dark-field optics. P, portal vein; C, central vein. (×100.)
    Figure Legend Snippet: Constitutive activity of p38 MAP kinase in the liver is inactivated by metabolic oxidative stress. ( A ) p38 MAP kinase inactivation in CCl 4 -treated liver extracts. The activity of p38 MAP kinase was measured by an immunecomplex protein kinase assay using [γ- 32 P]ATP and GST-ATF-2(1–109) as a substrate. The phosphorylated GST-ATF-2(1–109) was detected after SDS/PAGE by autoradiography. ( B ) p38 MAP kinase phosphorylation during oxidative stress. About 200 μg of liver extracts from control or CCl 4 -treated mice was electrophoresed on a 10% SDS/PAGE gel, blotted, and probed with antisera specific to phospho-p38 (New England Biolabs) or with antisera specific to p38. Arrows indicate specific immune complexes to either phospho-p38 or p38 protein as detected by enhanced chemiluminescence (DuPont). ( C ) Activation of MKP-1 by metabolic oxidative stress. MKP-1 mRNA levels were analyzed by RNase protection assay. MKP-1 mRNA levels were quantitated using a Molecular Dynamics PhosphorImager and normalized for 18S RNA. The mean results of two experiments are shown. ( D ) Localization of MKP-1 mRNA was determined using in situ hybridization. [ 35 S]-labeled MKP-1 antisense probe was hybridized to 7-μm sections of liver tissue from control and 4-hr CCl 4 -treated livers. The same field is shown using both bright-field and dark-field optics. P, portal vein; C, central vein. (×100.)

    Techniques Used: Activity Assay, Protein Kinase Assay, SDS Page, Autoradiography, Mouse Assay, Activation Assay, Rnase Protection Assay, In Situ Hybridization, Labeling

    11) Product Images from "Actin-Related Protein 2 (ARP2) and Virus-Induced Filopodia Facilitate Human Respiratory Syncytial Virus Spread"

    Article Title: Actin-Related Protein 2 (ARP2) and Virus-Induced Filopodia Facilitate Human Respiratory Syncytial Virus Spread

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006062

    ARP2 knockdown in human respiratory epithelial A549 cells. (A) Knockdown of ARP2 protein expression. Replicate monolayers of A549 cells were transfected with siARP2, siControl, or no siRNA, as indicated. At 48 hr post-transfection, one set of monolayers was harvested. The others were infected with RSV-GFP (MOI = 1) or mock-infected, and harvested at 24, 48, and 72 hpi, as indicated. The cells were processed for Western blotting. ARP2 protein was detected using a primary rabbit mAb and an anti-rabbit IgG IRDye800 secondary Ab. Alpha-tubulin, as a loading control, was detected with a primary mouse mAb and an anti-mouse IgG IRDye680 secondary Ab. Bound antibodies were visualized by infrared fluorescence. One representative of four independent experiments is shown. (B) Knockdown of ARP2 mRNA expression. A549 cells were transfected with siARP2, siControl, or no siRNA, as indicated. Sets of monolayers were harvested 24 and 48 hr post-transfection. The remaining monolayers were infected with RSV-GFP (MOI = 1) or mock-infected, and harvested at 24, 48, and 72 hpi, as indicated. Total cell-associated ARP2 mRNA was quantified by real-time PCR using a TaqMan assay for ARP2. 18S ribosomal RNA was used as an endogenous control for normalization of each reaction, and the values of ARP2 expression are shown as fold-change relative to the no-siRNA control 24 hr post-transfection. Each sample was tested in quadruplicate by TaqMan assay and the averages are presented. (C) ARP2 knockdown did not reduce cell viability. A549 cells were transfected with siARP2, siControl or no siRNA for 48 hr. Cell viability was compared using alamarBlue and expressed relative to the no-siRNA control. Data from three independent experiments, each done in triplicate were combined for analysis. Error bars show standard deviation (SD).
    Figure Legend Snippet: ARP2 knockdown in human respiratory epithelial A549 cells. (A) Knockdown of ARP2 protein expression. Replicate monolayers of A549 cells were transfected with siARP2, siControl, or no siRNA, as indicated. At 48 hr post-transfection, one set of monolayers was harvested. The others were infected with RSV-GFP (MOI = 1) or mock-infected, and harvested at 24, 48, and 72 hpi, as indicated. The cells were processed for Western blotting. ARP2 protein was detected using a primary rabbit mAb and an anti-rabbit IgG IRDye800 secondary Ab. Alpha-tubulin, as a loading control, was detected with a primary mouse mAb and an anti-mouse IgG IRDye680 secondary Ab. Bound antibodies were visualized by infrared fluorescence. One representative of four independent experiments is shown. (B) Knockdown of ARP2 mRNA expression. A549 cells were transfected with siARP2, siControl, or no siRNA, as indicated. Sets of monolayers were harvested 24 and 48 hr post-transfection. The remaining monolayers were infected with RSV-GFP (MOI = 1) or mock-infected, and harvested at 24, 48, and 72 hpi, as indicated. Total cell-associated ARP2 mRNA was quantified by real-time PCR using a TaqMan assay for ARP2. 18S ribosomal RNA was used as an endogenous control for normalization of each reaction, and the values of ARP2 expression are shown as fold-change relative to the no-siRNA control 24 hr post-transfection. Each sample was tested in quadruplicate by TaqMan assay and the averages are presented. (C) ARP2 knockdown did not reduce cell viability. A549 cells were transfected with siARP2, siControl or no siRNA for 48 hr. Cell viability was compared using alamarBlue and expressed relative to the no-siRNA control. Data from three independent experiments, each done in triplicate were combined for analysis. Error bars show standard deviation (SD).

    Techniques Used: Expressing, Transfection, Infection, Western Blot, Fluorescence, Real-time Polymerase Chain Reaction, TaqMan Assay, Standard Deviation

    12) Product Images from "Targeted DNA oxidation by LSD1–SMAD2/3 primes TGF-β1/ EMT genes for activation or repression"

    Article Title: Targeted DNA oxidation by LSD1–SMAD2/3 primes TGF-β1/ EMT genes for activation or repression

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa599

    Nuclear G oxidation induced by TGF-β1 through the histone demethylase, LSD1. (A) Immunofluorescence detection of 8-oxo-dG by fluorescein-tagged anti-8-oxo-dG antibody in cells pretreated with 5 mM NAC or 5 μM DPI for 1 h before stimulation with 10 ng/ml TGF-β1 for 30 min. Nuclei were stained with TO-PRO-3 Iodide in red while 8-oxo-dG foci were stained in green, as described in the ‘Materials and Methods’ section. Fluorescence quantization and statistical analysis are shown in Supplementary Figures S1C and S1-Stat3–6 . (B) Immunofluorescence detection of 8-oxo-dG by fluorescein-tagged anti-8-oxo-dG antibody in cells transfected with scrambled short hairpin (shCTRL) and shLSD1 RNAs for 24 h before stimulation with 10 ng/ml TGF-β1 for 30 min. Nuclei were stained with TO-PRO-3 Iodide in red while 8-oxo-dG foci were stained in green. Colocalization was measured using ImageJ (Coloc_2 plugin) as described in the ‘Materials and Methods’ section. Fluorescence quantization and statistical analysis are shown in Supplementary Figures S1D and S1-Stat3–6 . (C) Immunofluorescence microscopy of phalloidin in red and E-cadherin in green in control or LSD1-depleted cells exposed to 10 ng/ml TGF-β1 for 72 h. (D–G) mRNA levels of genes regulated by TGF-β1 in cells treated with shLSD1 or with TCP. Results are the mean of at least three experiments performed three times ( n = 9). MANOVA was used to examine the association between the mRNA levels after TGF-β1 and LSD1 depletion; the interaction was significant (see Supplementary Figure 2-Stat.2 for panel D and Supplementary Figure 2-Stat.4 for panel E). Differences between the means of three or more groups were determined by MANOVA. Differences between three or more samples were determined by one-way ANOVA test. Pairwise comparison was performed with Student’s t -test (see Supplementary Figures -Stat). (D) mRNA levels of SNAI1, CDH2, VIM and PAI genes in cells induced for 90 min with TGF-β1 determined by qPCR. Total RNA was isolated, reverse transcribed and amplified by qPCR with specific primers ( Supplementary Table S1 ). The values were normalized to 18S RNA. RNA was isolated as described in the ‘Materials and Methods’ section. * P
    Figure Legend Snippet: Nuclear G oxidation induced by TGF-β1 through the histone demethylase, LSD1. (A) Immunofluorescence detection of 8-oxo-dG by fluorescein-tagged anti-8-oxo-dG antibody in cells pretreated with 5 mM NAC or 5 μM DPI for 1 h before stimulation with 10 ng/ml TGF-β1 for 30 min. Nuclei were stained with TO-PRO-3 Iodide in red while 8-oxo-dG foci were stained in green, as described in the ‘Materials and Methods’ section. Fluorescence quantization and statistical analysis are shown in Supplementary Figures S1C and S1-Stat3–6 . (B) Immunofluorescence detection of 8-oxo-dG by fluorescein-tagged anti-8-oxo-dG antibody in cells transfected with scrambled short hairpin (shCTRL) and shLSD1 RNAs for 24 h before stimulation with 10 ng/ml TGF-β1 for 30 min. Nuclei were stained with TO-PRO-3 Iodide in red while 8-oxo-dG foci were stained in green. Colocalization was measured using ImageJ (Coloc_2 plugin) as described in the ‘Materials and Methods’ section. Fluorescence quantization and statistical analysis are shown in Supplementary Figures S1D and S1-Stat3–6 . (C) Immunofluorescence microscopy of phalloidin in red and E-cadherin in green in control or LSD1-depleted cells exposed to 10 ng/ml TGF-β1 for 72 h. (D–G) mRNA levels of genes regulated by TGF-β1 in cells treated with shLSD1 or with TCP. Results are the mean of at least three experiments performed three times ( n = 9). MANOVA was used to examine the association between the mRNA levels after TGF-β1 and LSD1 depletion; the interaction was significant (see Supplementary Figure 2-Stat.2 for panel D and Supplementary Figure 2-Stat.4 for panel E). Differences between the means of three or more groups were determined by MANOVA. Differences between three or more samples were determined by one-way ANOVA test. Pairwise comparison was performed with Student’s t -test (see Supplementary Figures -Stat). (D) mRNA levels of SNAI1, CDH2, VIM and PAI genes in cells induced for 90 min with TGF-β1 determined by qPCR. Total RNA was isolated, reverse transcribed and amplified by qPCR with specific primers ( Supplementary Table S1 ). The values were normalized to 18S RNA. RNA was isolated as described in the ‘Materials and Methods’ section. * P

    Techniques Used: Immunofluorescence, Staining, Fluorescence, Transfection, Microscopy, Real-time Polymerase Chain Reaction, Isolation, Amplification

    13) Product Images from "Role of Muscle c-Jun NH2-Terminal Kinase 1 in Obesity-Induced Insulin Resistance ▿"

    Article Title: Role of Muscle c-Jun NH2-Terminal Kinase 1 in Obesity-Induced Insulin Resistance ▿

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01162-09

    Effect of muscle-specific JNK1 deficiency on hepatic lipogenic gene expression. Gene expression in the liver of chow-fed (ND) and HFD-fed (HF) M WT and M KO mice was measured by quantitative RT-PCR analysis of mRNA. Data for the expression of transcription factors ( C/ebp α, C/ebp β, Ppar γ, and Srebp1 ), coactivators ( Pgc1 α and Pgc1 β), fatty acid synthase ( Fas ), and microsomal triglyceride transfer protein ( Mttp ) mRNA are presented. The relative mRNA expression level was calculated by normalization of the data to the amount of 18S RNA in each sample (means ± SD) ( n = 6 to ∼8). Statistically significant differences are indicated (*, P
    Figure Legend Snippet: Effect of muscle-specific JNK1 deficiency on hepatic lipogenic gene expression. Gene expression in the liver of chow-fed (ND) and HFD-fed (HF) M WT and M KO mice was measured by quantitative RT-PCR analysis of mRNA. Data for the expression of transcription factors ( C/ebp α, C/ebp β, Ppar γ, and Srebp1 ), coactivators ( Pgc1 α and Pgc1 β), fatty acid synthase ( Fas ), and microsomal triglyceride transfer protein ( Mttp ) mRNA are presented. The relative mRNA expression level was calculated by normalization of the data to the amount of 18S RNA in each sample (means ± SD) ( n = 6 to ∼8). Statistically significant differences are indicated (*, P

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

    Effect of muscle-specific JNK1 deficiency on hepatic inflammatory gene expression. Gene expression in the liver of chow-fed (ND) and HFD-fed (HF) M WT and M KO mice fasted overnight was measured by quantitative RT-PCR analysis of mRNA (TaqMan assays). Data for the expression of Tnf α, Il6 , Ifn γ, Cd68 , Icam1 , Lyzs , and cytochrome p450 2E1 ( Cye2e1 ) mRNAs are presented. The relative mRNA expression level was calculated by normalization of the data to the amount of 18S RNA in each sample (means ± SD) ( n = 6 to ∼8). Statistically significant differences are indicated (*, P
    Figure Legend Snippet: Effect of muscle-specific JNK1 deficiency on hepatic inflammatory gene expression. Gene expression in the liver of chow-fed (ND) and HFD-fed (HF) M WT and M KO mice fasted overnight was measured by quantitative RT-PCR analysis of mRNA (TaqMan assays). Data for the expression of Tnf α, Il6 , Ifn γ, Cd68 , Icam1 , Lyzs , and cytochrome p450 2E1 ( Cye2e1 ) mRNAs are presented. The relative mRNA expression level was calculated by normalization of the data to the amount of 18S RNA in each sample (means ± SD) ( n = 6 to ∼8). Statistically significant differences are indicated (*, P

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

    14) Product Images from "Regulation of p53 by Hypoxia: Dissociation of Transcriptional Repression and Apoptosis from p53-Dependent Transactivation"

    Article Title: Regulation of p53 by Hypoxia: Dissociation of Transcriptional Repression and Apoptosis from p53-Dependent Transactivation

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.4.1297-1310.2001

    (A) TSA inhibits p53-dependent apoptosis under hypoxia. H1299 cells transfected with a p53 expression vector under the control of the tetracycline promoter were treated with IR or hypoxia. One group of cells did not receive any additional treatment (CON). TSA (25 nM) was added to a second group 1 h prior to treatment. A third group was treated with doxycycline 2 h prior to treatment, while both TSA and doxycycline were added to a fourth group prior to treatments. At the end of the treatment, cells with apoptotic morphology were visualized with Hoechst 3342 and phosphatidyl inositol staining. The total number of cells and the number of apoptotic cells in four different fields in 60-mm-diameter dishes were counted and expressed as the percentage of the total number of cells. Numbers represent the average of three independent experiments. Error bars represent SEM (B) TSA inhibits p53 transrepression under hypoxia. RKO cells were treated as described above, with the exception that cells were lysed 6 h after IR treatment and 12 h after hypoxia treatment. Northern blot analysis was performed on total RNA using a probe for human α-tubulin and 18S rRNA as described above.
    Figure Legend Snippet: (A) TSA inhibits p53-dependent apoptosis under hypoxia. H1299 cells transfected with a p53 expression vector under the control of the tetracycline promoter were treated with IR or hypoxia. One group of cells did not receive any additional treatment (CON). TSA (25 nM) was added to a second group 1 h prior to treatment. A third group was treated with doxycycline 2 h prior to treatment, while both TSA and doxycycline were added to a fourth group prior to treatments. At the end of the treatment, cells with apoptotic morphology were visualized with Hoechst 3342 and phosphatidyl inositol staining. The total number of cells and the number of apoptotic cells in four different fields in 60-mm-diameter dishes were counted and expressed as the percentage of the total number of cells. Numbers represent the average of three independent experiments. Error bars represent SEM (B) TSA inhibits p53 transrepression under hypoxia. RKO cells were treated as described above, with the exception that cells were lysed 6 h after IR treatment and 12 h after hypoxia treatment. Northern blot analysis was performed on total RNA using a probe for human α-tubulin and 18S rRNA as described above.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Staining, Northern Blot

    Hypoxia induces p53-dependent transrepression. (A) Doxycycline (200 ng/ml) induces p53 expression in H1299 cells transfected with a tet -inducible construct (clone 30) under normoxic and hypoxic conditions. The slight increase in p53 levels under hypoxia in the absence of doxycycline is probably due to stabilization of residual p53 expressed due to the “leakiness” of the construct. (B) Northern analysis of β-tubulin (β-tubul.) mRNA levels in an H1299 clone expressing a tet -inducible p53 construct and the parental clone transfected with empty vector. Cells were treated with hypoxia, doxycycline, or doxycycline for 2 h followed by treatment with hypoxia. In this cell line, hybridization with the β-tubulin probe results in two signals, probably because of alternative splicing of the β-tubulin. At the bottom, a methylene blue-stained nitrocellulose membrane, indicating levels of 18S and 28S RNA is shown. (C) Northern blot analysis of the p53-transrepressed gene coding for α-tubulin after treatment of p53 +/+ and p53 −/− MEFs with hypoxia or with 10 J of UV radiation/m 2 . Hybridization to 18S rRNA was used as a control. This is a light PhosphorImager scan that better represents the repression levels of α-tubulin. Values represent relative β-tubulin levels.
    Figure Legend Snippet: Hypoxia induces p53-dependent transrepression. (A) Doxycycline (200 ng/ml) induces p53 expression in H1299 cells transfected with a tet -inducible construct (clone 30) under normoxic and hypoxic conditions. The slight increase in p53 levels under hypoxia in the absence of doxycycline is probably due to stabilization of residual p53 expressed due to the “leakiness” of the construct. (B) Northern analysis of β-tubulin (β-tubul.) mRNA levels in an H1299 clone expressing a tet -inducible p53 construct and the parental clone transfected with empty vector. Cells were treated with hypoxia, doxycycline, or doxycycline for 2 h followed by treatment with hypoxia. In this cell line, hybridization with the β-tubulin probe results in two signals, probably because of alternative splicing of the β-tubulin. At the bottom, a methylene blue-stained nitrocellulose membrane, indicating levels of 18S and 28S RNA is shown. (C) Northern blot analysis of the p53-transrepressed gene coding for α-tubulin after treatment of p53 +/+ and p53 −/− MEFs with hypoxia or with 10 J of UV radiation/m 2 . Hybridization to 18S rRNA was used as a control. This is a light PhosphorImager scan that better represents the repression levels of α-tubulin. Values represent relative β-tubulin levels.

    Techniques Used: Expressing, Transfection, Construct, Northern Blot, Plasmid Preparation, Hybridization, Staining

    15) Product Images from "Complete maturation of the plastid protein translocation channel requires a type I signal peptidase"

    Article Title: Complete maturation of the plastid protein translocation channel requires a type I signal peptidase

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200506171

    Disruption of PLSP1 results in a seedling lethal phenotype and reduction of the development of plastid internal membranes. (A) Structure of PLSP1 . Gray boxes and solid lines indicate exons and introns, respectively. The position of the T-DNA insertion in plsp1-1 was determined experimentally and is indicated with a white arrowhead. Positions of primers used for genomic and RT-PCR are indicated with black arrows. Primers and inserted T-DNA indicated with arrows/arrowhead are not to scale. (B) Seedlings of wild-type (wt), plsp1-1 , and plsp1-1;PLSP1 plants grown on MS media supplemented with 1% sucrose for 2 wk. Bar, 1 cm. (C) Genomic PCR of wild-type and mutant A. thaliana seedlings. E, I, and C indicate reactions specific to amplify endogenous PLSP1 , the T-DNA insertion, and the transgene used to complement the mutation, respectively. Primers that were used are listed as follows: F2 and R1 for E, F2 and R3 for I, and primers from 35S promoter (forward) and nopaline synthase terminator (reverse) for C. (D) RT-PCR analyses of wild-type and mutant A. thaliana seedlings. Each reaction contained two sets of primers: one for PLSP1 cDNA (F1 and R2) that produces a 400-bp fragment and another for cDNA derived from 18S RNA that produces a 315-bp fragment. Images from different portions of the same gel are in separate boxes. (E and F) Plastids in 2-wk-old wild-type (E) and homozygous plsp1-1 (F) cotyledons. Plastoglobules are indicated with asterisks. (G and H) Plastid in 7-wk-old wild-type (G) and homozygous plsp1-1 (H) etiolated seedlings. White and black arrowheads indicate PLB and disorganized membrane structure, respectively. (E–H) Bars, 1 μm.
    Figure Legend Snippet: Disruption of PLSP1 results in a seedling lethal phenotype and reduction of the development of plastid internal membranes. (A) Structure of PLSP1 . Gray boxes and solid lines indicate exons and introns, respectively. The position of the T-DNA insertion in plsp1-1 was determined experimentally and is indicated with a white arrowhead. Positions of primers used for genomic and RT-PCR are indicated with black arrows. Primers and inserted T-DNA indicated with arrows/arrowhead are not to scale. (B) Seedlings of wild-type (wt), plsp1-1 , and plsp1-1;PLSP1 plants grown on MS media supplemented with 1% sucrose for 2 wk. Bar, 1 cm. (C) Genomic PCR of wild-type and mutant A. thaliana seedlings. E, I, and C indicate reactions specific to amplify endogenous PLSP1 , the T-DNA insertion, and the transgene used to complement the mutation, respectively. Primers that were used are listed as follows: F2 and R1 for E, F2 and R3 for I, and primers from 35S promoter (forward) and nopaline synthase terminator (reverse) for C. (D) RT-PCR analyses of wild-type and mutant A. thaliana seedlings. Each reaction contained two sets of primers: one for PLSP1 cDNA (F1 and R2) that produces a 400-bp fragment and another for cDNA derived from 18S RNA that produces a 315-bp fragment. Images from different portions of the same gel are in separate boxes. (E and F) Plastids in 2-wk-old wild-type (E) and homozygous plsp1-1 (F) cotyledons. Plastoglobules are indicated with asterisks. (G and H) Plastid in 7-wk-old wild-type (G) and homozygous plsp1-1 (H) etiolated seedlings. White and black arrowheads indicate PLB and disorganized membrane structure, respectively. (E–H) Bars, 1 μm.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Mass Spectrometry, Polymerase Chain Reaction, Mutagenesis, Derivative Assay

    16) Product Images from "Both Microtubule-Stabilizing and Microtubule-Destabilizing Drugs Inhibit Hypoxia-Inducible Factor-1α Accumulation and Activity by Disrupting Microtubule Function"

    Article Title: Both Microtubule-Stabilizing and Microtubule-Destabilizing Drugs Inhibit Hypoxia-Inducible Factor-1α Accumulation and Activity by Disrupting Microtubule Function

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-04-4095

    Microtubule-targeting drugs inhibit HIF-1α at the post-transcriptional level. A , total RNA was prepared from treated cells under normoxic and hypoxic conditions. Northern blotting was done with 32 P-labeled probes for HIF-1α and Glut-1. 18S RNA and β-actin are shown as loading controls. B , 1A9 cells were pretreated overnight with either 25 mol/L vincristine or 25 mol/L epothilone B. The cells were then cotreated with MG-132 (10 Amol/L) in the presence or absence of the drugs for the indicated time (minutes). Equal amount of proteins from each lysate was resolved by SDS-PAGE and blotted for HIF-1α protein. The same gels were stripped and reblotted for actin. Arrows , ubiquinated HIF-1α protein species. C , 1A9 cells transiently transfected with pBI-GL V6L (6xHREs of VEGF promoter) were treated with vehicle or 100 nmol/L of each indicated drug under normoxic and hypoxic conditions. Luciferase reporter activity was measured in the cellular extract 16 hours later. Luciferase activity represents arbitrary units per microgram protein in each assay point. *, P
    Figure Legend Snippet: Microtubule-targeting drugs inhibit HIF-1α at the post-transcriptional level. A , total RNA was prepared from treated cells under normoxic and hypoxic conditions. Northern blotting was done with 32 P-labeled probes for HIF-1α and Glut-1. 18S RNA and β-actin are shown as loading controls. B , 1A9 cells were pretreated overnight with either 25 mol/L vincristine or 25 mol/L epothilone B. The cells were then cotreated with MG-132 (10 Amol/L) in the presence or absence of the drugs for the indicated time (minutes). Equal amount of proteins from each lysate was resolved by SDS-PAGE and blotted for HIF-1α protein. The same gels were stripped and reblotted for actin. Arrows , ubiquinated HIF-1α protein species. C , 1A9 cells transiently transfected with pBI-GL V6L (6xHREs of VEGF promoter) were treated with vehicle or 100 nmol/L of each indicated drug under normoxic and hypoxic conditions. Luciferase reporter activity was measured in the cellular extract 16 hours later. Luciferase activity represents arbitrary units per microgram protein in each assay point. *, P

    Techniques Used: Northern Blot, Labeling, SDS Page, Transfection, Luciferase, Activity Assay

    17) Product Images from "Survey of Human Genes of Retroviral Origin: Identification and Transcriptome of the Genes with Coding Capacity for Complete Envelope Proteins"

    Article Title: Survey of Human Genes of Retroviral Origin: Identification and Transcriptome of the Genes with Coding Capacity for Complete Envelope Proteins

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.19.10414-10422.2003

    Transcript levels of the coding retroviral envelope genes of the human genome in a panel of 19 healthy human tissues, as determined by real-time quantitative PCR. Each value is expressed as the mean ± standard deviation relative to the value for a reference control plasmid assayed in each real-time PCR experiment (see Materials and Methods), after correction for total RNA content in each tissue extract (using the 18S transcript as an internal control). The code for the logarithmic gray scale is indicated on the right. PBL, peripheral blood lymphocytes.
    Figure Legend Snippet: Transcript levels of the coding retroviral envelope genes of the human genome in a panel of 19 healthy human tissues, as determined by real-time quantitative PCR. Each value is expressed as the mean ± standard deviation relative to the value for a reference control plasmid assayed in each real-time PCR experiment (see Materials and Methods), after correction for total RNA content in each tissue extract (using the 18S transcript as an internal control). The code for the logarithmic gray scale is indicated on the right. PBL, peripheral blood lymphocytes.

    Techniques Used: Real-time Polymerase Chain Reaction, Standard Deviation, Plasmid Preparation

    18) Product Images from "The Extract of Leonurus sibiricus Transgenic Roots with AtPAP1 Transcriptional Factor Induces Apoptosis via DNA Damage and Down Regulation of Selected Epigenetic Factors in Human Cancer Cells"

    Article Title: The Extract of Leonurus sibiricus Transgenic Roots with AtPAP1 Transcriptional Factor Induces Apoptosis via DNA Damage and Down Regulation of Selected Epigenetic Factors in Human Cancer Cells

    Journal: Neurochemical Research

    doi: 10.1007/s11064-018-2551-6

    Expression profiles of UHRF1 and DNMT1 genes. qRT-PCR analysis of UHRF1 and DNMT1 in glioma cells in IV grade and U87MG cells cultured for 24 h in the presence of L. sibiricus TR and AtPAP1 root extracts. Each gene was normalized to the expression of a 18S RNA- reference gene. Data is presented as fold change in glioma cells in IV grade and U87MG cells vs. untreated cells, in which expression levels of the genes were set as 1. The mean values ± SD were calculated from three independent experiments. * p
    Figure Legend Snippet: Expression profiles of UHRF1 and DNMT1 genes. qRT-PCR analysis of UHRF1 and DNMT1 in glioma cells in IV grade and U87MG cells cultured for 24 h in the presence of L. sibiricus TR and AtPAP1 root extracts. Each gene was normalized to the expression of a 18S RNA- reference gene. Data is presented as fold change in glioma cells in IV grade and U87MG cells vs. untreated cells, in which expression levels of the genes were set as 1. The mean values ± SD were calculated from three independent experiments. * p

    Techniques Used: Expressing, Quantitative RT-PCR, Cell Culture

    19) Product Images from "The elusive MAESTRO gene: Its human reproductive tissue-specific expression pattern"

    Article Title: The elusive MAESTRO gene: Its human reproductive tissue-specific expression pattern

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0174873

    Relative expression of MRO transcripts was analyzed by qPCR. (A) Schematic representation of the MRO splice variants and the position of the TaqMan probes. Overall expression detected by the pQ8/9 probe. pQ1/3 spans exons 1–3 and detects the 'b' isoforms ( MROb 1-4); pQ2/3 spans exons 2/3 and detects isoforms MROa ,c,d. The expression of MRO was normalized to 18s RNA and presented as relative a expression ratio (1/ΔCt). (B) MRO transcripts analysis in GCs and CCs from PCOS and control subjects. The overall expression, shown by exon 8/9 was higher in PCOS cells vs that of controls (* in GCs and * in CCs; p ≤0.05). Inverse expression of exon 1/3 in GCs and CCs vs exon 2/3 was detected. (C) Inverse expression of exon 2/3 vs 1/3 in kidney, liver, brain and post-menopausal ovary.
    Figure Legend Snippet: Relative expression of MRO transcripts was analyzed by qPCR. (A) Schematic representation of the MRO splice variants and the position of the TaqMan probes. Overall expression detected by the pQ8/9 probe. pQ1/3 spans exons 1–3 and detects the 'b' isoforms ( MROb 1-4); pQ2/3 spans exons 2/3 and detects isoforms MROa ,c,d. The expression of MRO was normalized to 18s RNA and presented as relative a expression ratio (1/ΔCt). (B) MRO transcripts analysis in GCs and CCs from PCOS and control subjects. The overall expression, shown by exon 8/9 was higher in PCOS cells vs that of controls (* in GCs and * in CCs; p ≤0.05). Inverse expression of exon 1/3 in GCs and CCs vs exon 2/3 was detected. (C) Inverse expression of exon 2/3 vs 1/3 in kidney, liver, brain and post-menopausal ovary.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    20) Product Images from "The Extract of Leonurus sibiricus Transgenic Roots with AtPAP1 Transcriptional Factor Induces Apoptosis via DNA Damage and Down Regulation of Selected Epigenetic Factors in Human Cancer Cells"

    Article Title: The Extract of Leonurus sibiricus Transgenic Roots with AtPAP1 Transcriptional Factor Induces Apoptosis via DNA Damage and Down Regulation of Selected Epigenetic Factors in Human Cancer Cells

    Journal: Neurochemical Research

    doi: 10.1007/s11064-018-2551-6

    Expression profiles of UHRF1 and DNMT1 genes. qRT-PCR analysis of UHRF1 and DNMT1 in glioma cells in IV grade and U87MG cells cultured for 24 h in the presence of L. sibiricus TR and AtPAP1 root extracts. Each gene was normalized to the expression of a 18S RNA- reference gene. Data is presented as fold change in glioma cells in IV grade and U87MG cells vs. untreated cells, in which expression levels of the genes were set as 1. The mean values ± SD were calculated from three independent experiments. * p
    Figure Legend Snippet: Expression profiles of UHRF1 and DNMT1 genes. qRT-PCR analysis of UHRF1 and DNMT1 in glioma cells in IV grade and U87MG cells cultured for 24 h in the presence of L. sibiricus TR and AtPAP1 root extracts. Each gene was normalized to the expression of a 18S RNA- reference gene. Data is presented as fold change in glioma cells in IV grade and U87MG cells vs. untreated cells, in which expression levels of the genes were set as 1. The mean values ± SD were calculated from three independent experiments. * p

    Techniques Used: Expressing, Quantitative RT-PCR, Cell Culture

    21) Product Images from "Disrupted synaptic development in the hypoxic newborn brain"

    Article Title: Disrupted synaptic development in the hypoxic newborn brain

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.232568799

    Hypoxia induced VEGF-regulated pathways in both neurons and the microvasculature. ( A ) Composite micrographs of representative sections of P11 brain harvested from Nx and Hx animals. VEGF and its receptor Flk-1 were increased in neurons throughout the cortex, with marked concentrations in the dentate gyrus and in the CA1 region of the hippocampus. Microarray analysis indicated a > 1.4-fold enhancement (Hx/N) at P11 for VEGF (Table 1). This same result was also obtained by conventional Northern blot analysis ( Inset : dark bars, Hx/N by blot; light bars, microarray results), by using freshly extracted mRNA standardized against 18S ribosomal RNA. ( B ) Western blot analysis, standardized against actin, of ARNT and HIF-1α. Note that although ARNT levels remain nearly unchanged, the upstream inducer of VEGF expression, HIF-1α, is up-regulated at P11.
    Figure Legend Snippet: Hypoxia induced VEGF-regulated pathways in both neurons and the microvasculature. ( A ) Composite micrographs of representative sections of P11 brain harvested from Nx and Hx animals. VEGF and its receptor Flk-1 were increased in neurons throughout the cortex, with marked concentrations in the dentate gyrus and in the CA1 region of the hippocampus. Microarray analysis indicated a > 1.4-fold enhancement (Hx/N) at P11 for VEGF (Table 1). This same result was also obtained by conventional Northern blot analysis ( Inset : dark bars, Hx/N by blot; light bars, microarray results), by using freshly extracted mRNA standardized against 18S ribosomal RNA. ( B ) Western blot analysis, standardized against actin, of ARNT and HIF-1α. Note that although ARNT levels remain nearly unchanged, the upstream inducer of VEGF expression, HIF-1α, is up-regulated at P11.

    Techniques Used: Microarray, Northern Blot, Western Blot, Expressing

    22) Product Images from "HIC1 (Hypermethylated in Cancer 1) modulates the contractile activity of prostate stromal fibroblasts and directly regulates CXCL12 expression"

    Article Title: HIC1 (Hypermethylated in Cancer 1) modulates the contractile activity of prostate stromal fibroblasts and directly regulates CXCL12 expression

    Journal: Oncotarget

    doi: 10.18632/oncotarget.27786

    RT-qPCR analyses of HIC1 expression in normal human prostate and human PCa tissues. ( A ) Profiles of HIC1 expression in 10 pairs of cancerous and matched normal prostate tissues. Total RNAs were extracted and quantitative expression of the two major HIC1 isoforms’ expression was measured by RT-qPCR analyses. Results are normalized to ALAS1 RNA [ 51 ] or 18S rRNA and are presented as means +/− S.D. from two experiments in triplicate. For each patient, the percentage of normal stroma present in the tumor samples which was used as the ranking criteria is indicated below as well as the TNM staging (see Supplementary Table 1 for details). ( B ) Analyses of HIC1 expression in prostate tumors. A cohort of 20 tumors for which the matched normal tissue was not available was analyzed by RT-qPCR analyses (black column) as described above except that the control corresponded to RNAs extracted from the prostate of a healthy young (24 year-old) donor (Biochain) as control. The 10 tumors used in panel A were also included in this study (orange column). ( C ) Quantification of HIC1 expression in the 30 prostate tumors clustered in subgroups according to their stromal content. HIC1 expression measured by RT-qPCR analyses of the 30 tumors in panel B is represented as box plots in comparison with its expression in a normal prostate tissue obtained from a young 24-years old donor. The box area corresponds to the first and third quartile. The median is shown as a horizontal line in the box. The maximum and minimum values are indicated by the whiskers above and below the box.
    Figure Legend Snippet: RT-qPCR analyses of HIC1 expression in normal human prostate and human PCa tissues. ( A ) Profiles of HIC1 expression in 10 pairs of cancerous and matched normal prostate tissues. Total RNAs were extracted and quantitative expression of the two major HIC1 isoforms’ expression was measured by RT-qPCR analyses. Results are normalized to ALAS1 RNA [ 51 ] or 18S rRNA and are presented as means +/− S.D. from two experiments in triplicate. For each patient, the percentage of normal stroma present in the tumor samples which was used as the ranking criteria is indicated below as well as the TNM staging (see Supplementary Table 1 for details). ( B ) Analyses of HIC1 expression in prostate tumors. A cohort of 20 tumors for which the matched normal tissue was not available was analyzed by RT-qPCR analyses (black column) as described above except that the control corresponded to RNAs extracted from the prostate of a healthy young (24 year-old) donor (Biochain) as control. The 10 tumors used in panel A were also included in this study (orange column). ( C ) Quantification of HIC1 expression in the 30 prostate tumors clustered in subgroups according to their stromal content. HIC1 expression measured by RT-qPCR analyses of the 30 tumors in panel B is represented as box plots in comparison with its expression in a normal prostate tissue obtained from a young 24-years old donor. The box area corresponds to the first and third quartile. The median is shown as a horizontal line in the box. The maximum and minimum values are indicated by the whiskers above and below the box.

    Techniques Used: Quantitative RT-PCR, Expressing

    23) Product Images from "Chicken MBD4 Regulates Immunoglobulin Diversification by Somatic Hypermutation"

    Article Title: Chicken MBD4 Regulates Immunoglobulin Diversification by Somatic Hypermutation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.02540

    SHM is elevated in DT40 derived MBD4 Δ/Δ clones. (A) Schematic of exons 4, 5, and 6 in the Mbd4 gene and primers F2 and R2 used for RT-PCR analysis. gRNA sequence is indicated by a line (g) in early exon 5 ( upper panel ). Semi-quantitative RT-PCR analysis of Mbd4 exons 5–6 in parental and deleted clones ( middle panel ). QRT-PCR analysis of 18S RNA was used as a loading control ( lower panel ). 18S sample values were normalized to parental values which were set to 1. Samples were assayed in triplicate and were averaged, and SEMs are shown. (B) Cell proliferation assays. Cells (1 × 10 5 /ml) were grown in culture for 96 h and live cells were counted in triplicate every 24 h. The data shown is the average and is representative of 3 independent samples. (C) IgM fluorescence loss is analyzed in parental and MBD4 Δ/Δ .14, MBD4 Δ/Δ .11, and MBD4 Δ/Δ .12 deletion clones by flow cytometry. Cells were FACS sorted at day 0 for IgM + GFP + cells and re-analyzed after 28 days in culture. Flow cytometry analyses are representative of 24 parental- and 12 subclones from each of the MBD4 Δ/Δ deletion clones. (D) IgM fluorescence loss was quantitated for each parental and MBD4 Δ/Δ deletion subclone after 28 days in culture. Each data point is representative of a single sub-clone. Bars indicate the median in each data set. P -value * p
    Figure Legend Snippet: SHM is elevated in DT40 derived MBD4 Δ/Δ clones. (A) Schematic of exons 4, 5, and 6 in the Mbd4 gene and primers F2 and R2 used for RT-PCR analysis. gRNA sequence is indicated by a line (g) in early exon 5 ( upper panel ). Semi-quantitative RT-PCR analysis of Mbd4 exons 5–6 in parental and deleted clones ( middle panel ). QRT-PCR analysis of 18S RNA was used as a loading control ( lower panel ). 18S sample values were normalized to parental values which were set to 1. Samples were assayed in triplicate and were averaged, and SEMs are shown. (B) Cell proliferation assays. Cells (1 × 10 5 /ml) were grown in culture for 96 h and live cells were counted in triplicate every 24 h. The data shown is the average and is representative of 3 independent samples. (C) IgM fluorescence loss is analyzed in parental and MBD4 Δ/Δ .14, MBD4 Δ/Δ .11, and MBD4 Δ/Δ .12 deletion clones by flow cytometry. Cells were FACS sorted at day 0 for IgM + GFP + cells and re-analyzed after 28 days in culture. Flow cytometry analyses are representative of 24 parental- and 12 subclones from each of the MBD4 Δ/Δ deletion clones. (D) IgM fluorescence loss was quantitated for each parental and MBD4 Δ/Δ deletion subclone after 28 days in culture. Each data point is representative of a single sub-clone. Bars indicate the median in each data set. P -value * p

    Techniques Used: Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Sequencing, Quantitative RT-PCR, Clone Assay, Fluorescence, Flow Cytometry, Cytometry, FACS

    24) Product Images from "Structural Optimization of 2,3-Dihydro-1H-cyclopenta[b]quinolines Targeting the Noncatalytic RVxF Site of Protein Phosphatase 1 for HIV-1 Inhibition"

    Article Title: Structural Optimization of 2,3-Dihydro-1H-cyclopenta[b]quinolines Targeting the Noncatalytic RVxF Site of Protein Phosphatase 1 for HIV-1 Inhibition

    Journal: ACS infectious diseases

    doi: 10.1021/acsinfecdis.0c00511

    Inhibition of HIV-1 IIIB infection and metabolic stability of 1E7-03 analogues. (A and B) HIV-1 gag for details). RNA was extracted from the infected cells treated with DMSO, 10 μ M 1E7-03, 10 μ M HU-1a, 10 μ M HU-1c, 10 μ M HU-1d, or 25 μ M AZT for 72 h. RNA expression was analyzed by RT-PCR using 18S mRNA for normalization. The means ± SD are shown ( n = 3 for all panels). * p
    Figure Legend Snippet: Inhibition of HIV-1 IIIB infection and metabolic stability of 1E7-03 analogues. (A and B) HIV-1 gag for details). RNA was extracted from the infected cells treated with DMSO, 10 μ M 1E7-03, 10 μ M HU-1a, 10 μ M HU-1c, 10 μ M HU-1d, or 25 μ M AZT for 72 h. RNA expression was analyzed by RT-PCR using 18S mRNA for normalization. The means ± SD are shown ( n = 3 for all panels). * p

    Techniques Used: Inhibition, Infection, RNA Expression, Reverse Transcription Polymerase Chain Reaction

    25) Product Images from "Tissue-Specific Transgenic Knockdown of Fos-Related Antigen 2 (Fra-2) Expression Mediated by Dominant Negative Fra-2"

    Article Title: Tissue-Specific Transgenic Knockdown of Fos-Related Antigen 2 (Fra-2) Expression Mediated by Dominant Negative Fra-2

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.11.3704-3713.2001

    DII is a candidate target gene for Fra-2 action. (A) Reverse Northern analysis of radiolabeled pineal (midnight) cRNA from WT and NATDNF2 rats. The immobilized plasmid vectors were as follows: 1, pUC18; 2, Fra-2; 3, c-Fos; 4, AANAT; 5, β1-adrenergic receptor; 6, c-Jun; 7, γ-phosphodiesterase; 8, α1A-adrenergic receptor; 9, α1B-adrenergic receptor; 10, preproenkephalin; 11, DII; 12, S antigen; 13, ICER; 14, RZRβ. All expression vectors contain rat genes. (B) Northern blot analysis of total pineal RNA (∼7 μg) extracted from WT and NATDNF2 animals that were sacrificed at the indicated clock times (CT) throughout the dark phase of a 24-h cycle. Blots were exposed to rat DII and 18S ribosomal probes. (C) Quantitation of the relative abundance of the DII/18S steady-state transcript levels at 22 ( n = 3 pairs of rats) and 24 ( n = 4 pairs of rats) h. Open bars, WT; closed bars, NATDNF2.
    Figure Legend Snippet: DII is a candidate target gene for Fra-2 action. (A) Reverse Northern analysis of radiolabeled pineal (midnight) cRNA from WT and NATDNF2 rats. The immobilized plasmid vectors were as follows: 1, pUC18; 2, Fra-2; 3, c-Fos; 4, AANAT; 5, β1-adrenergic receptor; 6, c-Jun; 7, γ-phosphodiesterase; 8, α1A-adrenergic receptor; 9, α1B-adrenergic receptor; 10, preproenkephalin; 11, DII; 12, S antigen; 13, ICER; 14, RZRβ. All expression vectors contain rat genes. (B) Northern blot analysis of total pineal RNA (∼7 μg) extracted from WT and NATDNF2 animals that were sacrificed at the indicated clock times (CT) throughout the dark phase of a 24-h cycle. Blots were exposed to rat DII and 18S ribosomal probes. (C) Quantitation of the relative abundance of the DII/18S steady-state transcript levels at 22 ( n = 3 pairs of rats) and 24 ( n = 4 pairs of rats) h. Open bars, WT; closed bars, NATDNF2.

    Techniques Used: Northern Blot, Plasmid Preparation, Expressing, Quantitation Assay

    Tissue survey of DNF2 expression and effects of pineal expression on the Fra-2 response in the night pineal gland. (A) Northern blot analysis performed with total RNA (∼5 μg) extracted from the indicated tissues that were harvested in the middle of the night from transgenic animals designated H17 and carrying a high copy number (estimated at 45 per haploid genome) of the NATDNF2 transgene. The membrane was probed with Fra-2 and GAPDH radiolabeled probes. Blots were exposed to X-ray film for 24 h. No DNF2 signal was observed in WT animals (not shown). (B) Northern blot analysis of total pineal RNA (∼7 μg) was used to investigate the nocturnal Fra-2 response in WT animals and in a high-copy-number transgenic line (H17). Levels of fra-2 and DNF2 mRNAs were assessed at three different time points during the night along with the levels of the 18S rRNA. Downregulation of nocturnal fra-2 mRNA expression was also observed in an independent experiment using samples derived from H16 transgenic rats. CT, clock time (hours). (C) Western blot analysis of pineal proteins prepared from WT mice or animals carrying a range of DNF2 transgene copy numbers (2 [H4], 5 [H13], 20 [H16], and 45 [H17] copies per haploid genome). Animals were sacrificed at the indicated clock times, and SDS-PAGE was run with total pineal protein samples. After electrophoretic transfer, the membrane was exposed to a pan-Fos rabbit antibody directed against the invariant M peptide between aa 129 to 153 in the mouse c-Fos sequence (SC253x). For both panels, the light-dark cycle was 12:12 with lights off at 7 p.m. Selective downregulation of Fra-2 protein was observed in two independent experiments using samples derived from additional H17 transgenic rats.
    Figure Legend Snippet: Tissue survey of DNF2 expression and effects of pineal expression on the Fra-2 response in the night pineal gland. (A) Northern blot analysis performed with total RNA (∼5 μg) extracted from the indicated tissues that were harvested in the middle of the night from transgenic animals designated H17 and carrying a high copy number (estimated at 45 per haploid genome) of the NATDNF2 transgene. The membrane was probed with Fra-2 and GAPDH radiolabeled probes. Blots were exposed to X-ray film for 24 h. No DNF2 signal was observed in WT animals (not shown). (B) Northern blot analysis of total pineal RNA (∼7 μg) was used to investigate the nocturnal Fra-2 response in WT animals and in a high-copy-number transgenic line (H17). Levels of fra-2 and DNF2 mRNAs were assessed at three different time points during the night along with the levels of the 18S rRNA. Downregulation of nocturnal fra-2 mRNA expression was also observed in an independent experiment using samples derived from H16 transgenic rats. CT, clock time (hours). (C) Western blot analysis of pineal proteins prepared from WT mice or animals carrying a range of DNF2 transgene copy numbers (2 [H4], 5 [H13], 20 [H16], and 45 [H17] copies per haploid genome). Animals were sacrificed at the indicated clock times, and SDS-PAGE was run with total pineal protein samples. After electrophoretic transfer, the membrane was exposed to a pan-Fos rabbit antibody directed against the invariant M peptide between aa 129 to 153 in the mouse c-Fos sequence (SC253x). For both panels, the light-dark cycle was 12:12 with lights off at 7 p.m. Selective downregulation of Fra-2 protein was observed in two independent experiments using samples derived from additional H17 transgenic rats.

    Techniques Used: Expressing, Northern Blot, Transgenic Assay, Derivative Assay, Western Blot, Mouse Assay, SDS Page, Sequencing

    cDNA array-based identification of nectadrin as a candidate Fra-2 target gene. (A) Representative details of duplicate rat expression arrays (Clontech's Atlas rat 1.2) probed with cDNAs derived from either transgenic (H17) or control (C17) littermate pineal glands sampled at 24 h. The nectadrin gene is located at position 1A. Following hybridization and washing, arrays were exposed to a storage phosphor screen (Kodak-K) for 3 days and visualized using a model FX molecular imager (Bio-Rad). Images were downloaded as TIFFs and montaged using Adobe Photoshop, version 4.0. The level of nectadrin mRNA is significantly reduced at midnight in the pineal gland of NATDNF2 rats. (B) Nectadrin expression displays a robust day/night rhythm in the rat pineal gland, as determined by Northern blot analysis of total pineal RNA (∼7 μg) extracted from WT and NATDNF2 animals that were sacrificed at the indicated clock times (CT) throughout the dark phase of a 24-h cycle. Blots were exposed to rat nectadrin and 18S probes. (C) Quantitation of the relative abundance of the nectadrin/18S steady-state transcript levels at 22 and 24 h ( n = 3 pairs of rats in each group). Open bars, WT; closed bars, NATDNF2. Statistical analysis for both DII and nectadrin levels of expression was performed by an unpaired t test, using corrected percentages as input values. The DII level is different from control at both 22 and 24 h, while the nectadrin is different from control at only 24 h, at a significance level of P
    Figure Legend Snippet: cDNA array-based identification of nectadrin as a candidate Fra-2 target gene. (A) Representative details of duplicate rat expression arrays (Clontech's Atlas rat 1.2) probed with cDNAs derived from either transgenic (H17) or control (C17) littermate pineal glands sampled at 24 h. The nectadrin gene is located at position 1A. Following hybridization and washing, arrays were exposed to a storage phosphor screen (Kodak-K) for 3 days and visualized using a model FX molecular imager (Bio-Rad). Images were downloaded as TIFFs and montaged using Adobe Photoshop, version 4.0. The level of nectadrin mRNA is significantly reduced at midnight in the pineal gland of NATDNF2 rats. (B) Nectadrin expression displays a robust day/night rhythm in the rat pineal gland, as determined by Northern blot analysis of total pineal RNA (∼7 μg) extracted from WT and NATDNF2 animals that were sacrificed at the indicated clock times (CT) throughout the dark phase of a 24-h cycle. Blots were exposed to rat nectadrin and 18S probes. (C) Quantitation of the relative abundance of the nectadrin/18S steady-state transcript levels at 22 and 24 h ( n = 3 pairs of rats in each group). Open bars, WT; closed bars, NATDNF2. Statistical analysis for both DII and nectadrin levels of expression was performed by an unpaired t test, using corrected percentages as input values. The DII level is different from control at both 22 and 24 h, while the nectadrin is different from control at only 24 h, at a significance level of P

    Techniques Used: Expressing, Derivative Assay, Transgenic Assay, Hybridization, Northern Blot, Quantitation Assay

    Expression of DNF2 protein has no effect on pineal AANAT expression or melatonin production. (A) Northern blot analysis of total pineal RNA (∼7 μg) extracted from WT and NATDNF2 animals that were sacrificed at the indicated clock times throughout a 24-h cycle. Blots were exposed to rat AANAT, DNF2, and 18S probes. The absence of transgene-associated changes in nocturnal AANAT mRNA level was confirmed in an independent experiment using samples derived from additional H17 transgenic rats. There were six rats in each group. (B) WT and H17 NATDNF2 rats were maintained in individual metabolic cages on a 12:12 light-dark cycle (lights off at clock time 19). Urine was collected every 6 h, starting at clock time 12, and urinary 6-sulfatoxymelatonin (expressed as total nanograms produced/period) was assessed for each group.
    Figure Legend Snippet: Expression of DNF2 protein has no effect on pineal AANAT expression or melatonin production. (A) Northern blot analysis of total pineal RNA (∼7 μg) extracted from WT and NATDNF2 animals that were sacrificed at the indicated clock times throughout a 24-h cycle. Blots were exposed to rat AANAT, DNF2, and 18S probes. The absence of transgene-associated changes in nocturnal AANAT mRNA level was confirmed in an independent experiment using samples derived from additional H17 transgenic rats. There were six rats in each group. (B) WT and H17 NATDNF2 rats were maintained in individual metabolic cages on a 12:12 light-dark cycle (lights off at clock time 19). Urine was collected every 6 h, starting at clock time 12, and urinary 6-sulfatoxymelatonin (expressed as total nanograms produced/period) was assessed for each group.

    Techniques Used: Expressing, Northern Blot, Derivative Assay, Transgenic Assay, Produced

    26) Product Images from "Altered Patterns of Cellular Gene Expression in Dermal Microvascular Endothelial Cells Infected with Kaposi's Sarcoma-Associated Herpesvirus"

    Article Title: Altered Patterns of Cellular Gene Expression in Dermal Microvascular Endothelial Cells Infected with Kaposi's Sarcoma-Associated Herpesvirus

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.7.3395-3420.2002

    Demonstration of virus-encoded latent and lytic cycle gene expression in KSHV-infected DMVEC cultures. (A) Standard nonquantitative RT-PCR products obtained for selected ORF-K1, ORF-K8, and ORF-K8.1 lytic cycle viral genes in the same set of four DMVEC RNA samples used to generate the probes for the Clontech gene array hybridization experiments (RNA batch 1). Each viral primer pair chosen encompassed known intron loci to confirm that the PCR products were derived from spliced mRNA. Primers for 18S rRNA were used as the loading control. Lane 1, control uninfected DMVEC RNA; lane 2, KSHV-infected DMVEC RNA; lane 3, uninfected DMVEC +TPA RNA; lane 4, KSHV-infected DMVEC +TPA RNA. (B) Western immunoblot comparing the expression of KSHV-encoded LANA1 (latent) and ZMP-A (K5, lytic) proteins in uninfected and infected DMVEC cultures. Lane 1, control uninfected DMVEC extract; lane 2, KSHV-infected DMVEC extract; lane 3, uninfected DMVEC +TPA extract; lane 4, KSHV-infected DMVEC +TPA extract. A β-actin loading control on the same samples is also shown in the lower panel.
    Figure Legend Snippet: Demonstration of virus-encoded latent and lytic cycle gene expression in KSHV-infected DMVEC cultures. (A) Standard nonquantitative RT-PCR products obtained for selected ORF-K1, ORF-K8, and ORF-K8.1 lytic cycle viral genes in the same set of four DMVEC RNA samples used to generate the probes for the Clontech gene array hybridization experiments (RNA batch 1). Each viral primer pair chosen encompassed known intron loci to confirm that the PCR products were derived from spliced mRNA. Primers for 18S rRNA were used as the loading control. Lane 1, control uninfected DMVEC RNA; lane 2, KSHV-infected DMVEC RNA; lane 3, uninfected DMVEC +TPA RNA; lane 4, KSHV-infected DMVEC +TPA RNA. (B) Western immunoblot comparing the expression of KSHV-encoded LANA1 (latent) and ZMP-A (K5, lytic) proteins in uninfected and infected DMVEC cultures. Lane 1, control uninfected DMVEC extract; lane 2, KSHV-infected DMVEC extract; lane 3, uninfected DMVEC +TPA extract; lane 4, KSHV-infected DMVEC +TPA extract. A β-actin loading control on the same samples is also shown in the lower panel.

    Techniques Used: Expressing, Infection, Reverse Transcription Polymerase Chain Reaction, Hybridization, Polymerase Chain Reaction, Derivative Assay, Western Blot

    27) Product Images from "Transforming Growth Factor-β1 Selectively Recruits microRNAs to the RNA-Induced Silencing Complex and Degrades CFTR mRNA under Permissive Conditions in Human Bronchial Epithelial Cells"

    Article Title: Transforming Growth Factor-β1 Selectively Recruits microRNAs to the RNA-Induced Silencing Complex and Degrades CFTR mRNA under Permissive Conditions in Human Bronchial Epithelial Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20194933

    TGF-β1 did not affect CFTR promoter activity but reduced the stability of CFTR mRNA. ( A ). Summary of the CFTR gene promoter reporter assays showing that luciferase activity was increased by expression of the vector containing 1 kb promoter pCFTR-pLuc, compared to vector control. Forskolin increased the luciferase activity of pCFTR-pLuc and TGF-β1 had no effect. Human embryonic kidney (HEK) cells were transfected with pCFTR-pLuc or vector control and 24 h later cells were treated with forskolin (10 µM), TGF-β1 (15 ng/mL), or vehicle control for 6 h. ( B ). The CFTR mRNA half-life measurements showing that TGF-β1 decreased CFTR mRNA stability in F508del HBE cells. The transcriptional inhibitor ActD (5 μg/mL) and either TGF-β1 or vehicle control were added to F508del HBE cells for 4, 8 or 24 h. Total RNA was isolated and the CFTR mRNA level at each time point was calculated by real-time quantitative reverse transcriptase PCR (qRT-PCR), using the relative quantitation method, with 18S as a reference gene. The amount of CFTR mRNA at 4 h was set to 100% ( t = 0) and mRNA half-lives were calculated from the exponential decay model, based on trend line equation C/C 0 = e −kdt (where C and C 0 are mRNA amounts at the time t and t 0 , respectively, and k d is the mRNA decay constant). The resulting curve equations were y (vehicle) = 123 −0.01x and y (TGF-β1) = 112 −0.007x . The calculated half-life of CFTR mRNA was 21.1 h and 13.7 h for the vehicle and TGF-β1-treated cells, respectively. N = 9–12 /group from 3–4 different HEK cell cultures ( A ) and N = 3 in triplicates in F508del HBE cells from three different donors ( B ). Error bars, S.E.M. **** p
    Figure Legend Snippet: TGF-β1 did not affect CFTR promoter activity but reduced the stability of CFTR mRNA. ( A ). Summary of the CFTR gene promoter reporter assays showing that luciferase activity was increased by expression of the vector containing 1 kb promoter pCFTR-pLuc, compared to vector control. Forskolin increased the luciferase activity of pCFTR-pLuc and TGF-β1 had no effect. Human embryonic kidney (HEK) cells were transfected with pCFTR-pLuc or vector control and 24 h later cells were treated with forskolin (10 µM), TGF-β1 (15 ng/mL), or vehicle control for 6 h. ( B ). The CFTR mRNA half-life measurements showing that TGF-β1 decreased CFTR mRNA stability in F508del HBE cells. The transcriptional inhibitor ActD (5 μg/mL) and either TGF-β1 or vehicle control were added to F508del HBE cells for 4, 8 or 24 h. Total RNA was isolated and the CFTR mRNA level at each time point was calculated by real-time quantitative reverse transcriptase PCR (qRT-PCR), using the relative quantitation method, with 18S as a reference gene. The amount of CFTR mRNA at 4 h was set to 100% ( t = 0) and mRNA half-lives were calculated from the exponential decay model, based on trend line equation C/C 0 = e −kdt (where C and C 0 are mRNA amounts at the time t and t 0 , respectively, and k d is the mRNA decay constant). The resulting curve equations were y (vehicle) = 123 −0.01x and y (TGF-β1) = 112 −0.007x . The calculated half-life of CFTR mRNA was 21.1 h and 13.7 h for the vehicle and TGF-β1-treated cells, respectively. N = 9–12 /group from 3–4 different HEK cell cultures ( A ) and N = 3 in triplicates in F508del HBE cells from three different donors ( B ). Error bars, S.E.M. **** p

    Techniques Used: Activity Assay, Luciferase, Expressing, Plasmid Preparation, Transfection, Isolation, Polymerase Chain Reaction, Quantitative RT-PCR, Quantitation Assay

    28) Product Images from "Constitutive Nuclear Factor-?B Activity Is Crucial for Human Retinoblastoma Cell Viability"

    Article Title: Constitutive Nuclear Factor-?B Activity Is Crucial for Human Retinoblastoma Cell Viability

    Journal: The American Journal of Pathology

    doi:

    A: Immunoblotting analysis for the apoptosis inhibitors Bcl-2, BclxL, A1, cIAP-1, cIAP-2, XIAP, and survivin, as well as the proapoptotic molecule Bax, in the Y79 cell line treated with the NF-κB inhibitor SN50 (20 μmol/L) for 3 or 6 hours. NF-κB inhibition down-regulated the protein levels of Bcl-2, A1, and cIAP-2, and up-regulated Bax expression. B and C: Quantitative RT-PCR for Bcl-2 ( B ), Bax ( C ) and 18S RNA in Y79 cells treated with or without SN50 (20 μmol/L for 3 hours). NF-κB inhibition down-regulates Bcl-2 and up-regulates Bax mRNA levels.
    Figure Legend Snippet: A: Immunoblotting analysis for the apoptosis inhibitors Bcl-2, BclxL, A1, cIAP-1, cIAP-2, XIAP, and survivin, as well as the proapoptotic molecule Bax, in the Y79 cell line treated with the NF-κB inhibitor SN50 (20 μmol/L) for 3 or 6 hours. NF-κB inhibition down-regulated the protein levels of Bcl-2, A1, and cIAP-2, and up-regulated Bax expression. B and C: Quantitative RT-PCR for Bcl-2 ( B ), Bax ( C ) and 18S RNA in Y79 cells treated with or without SN50 (20 μmol/L for 3 hours). NF-κB inhibition down-regulates Bcl-2 and up-regulates Bax mRNA levels.

    Techniques Used: Inhibition, Expressing, Quantitative RT-PCR

    29) Product Images from "Broad anti-coronaviral activity of FDA approved drugs against SARS-CoV-2 in vitro and SARS-CoV in vivo"

    Article Title: Broad anti-coronaviral activity of FDA approved drugs against SARS-CoV-2 in vitro and SARS-CoV in vivo

    Journal: bioRxiv

    doi: 10.1101/2020.03.25.008482

    Hydroxychloroquine and chloroquine inhibit production of SARS-CoV-2 N and RdRp mRNA. Vero cells were pre-treated with hydroxychloroquine sulfate (A, C and E) or chloroquine phosphate (B, D and F) at the indicated concentration (or 0.1% water as vehicle control) for 2 h prior to infection with SARS-CoV-2 (WA-1 strain) at MOI 0.1. 24 h post-infection cells were collected in TRIzol. RNA was extracted from TRIzol sample and qRT-PCR was performed for viral RdRp (A and B) or N (C and D) mRNA using WHO primers. RNA levels were normalized with 18S RNA and fold change for drug treated to vehicle control was calculated (dotted line to denote a fold change of 1 which is no change over control). Data are from 3 independent infections performed on triplicate wells, the fold change was calculated in each independent experiment and the mean fold change is plotted with error bars displaying standard deviation. Along with TRIzol samples for RNA supernatant was collected from cells and used for TCID 50 assays to determine infectious virus production following treatment with HCQ (E) or CQ (F) Data are from 3 independent infections performed on triplicate wells with the TCID 50 /ml being averaged across all wells. Error bars are the standard deviation. G) Cells were treated with 50 μM HCQ or 0.1% water as control. Drug was either added 2 h prior to infection, at the time of infection or 2 h after infection with MOI 0.1 SARS-CoV-2. After 24 h infection, supernatant was collected and used for TCID 50 assays to determine infectious virus production. Data are from 3 independent infections performed on triplicate wells with the TCID 50 /ml being averaged across all wells. Error bars are the standard deviation. H) SARS-CoV spike psuedoviruses (PV) were used for infection of BSC1 cells. The cells were treated with 10 μM of CQ or CPZ for 1 h prior to infection with PV for 3 h. The PV carry BlaM and cells were loaded with CCF2 to monitor cleavage and shift in fluorescence output for evidence of S-mediated entry into cells. Data are normalised to PV alone and are from 3 independent experiments with error bars representing standard deviation.
    Figure Legend Snippet: Hydroxychloroquine and chloroquine inhibit production of SARS-CoV-2 N and RdRp mRNA. Vero cells were pre-treated with hydroxychloroquine sulfate (A, C and E) or chloroquine phosphate (B, D and F) at the indicated concentration (or 0.1% water as vehicle control) for 2 h prior to infection with SARS-CoV-2 (WA-1 strain) at MOI 0.1. 24 h post-infection cells were collected in TRIzol. RNA was extracted from TRIzol sample and qRT-PCR was performed for viral RdRp (A and B) or N (C and D) mRNA using WHO primers. RNA levels were normalized with 18S RNA and fold change for drug treated to vehicle control was calculated (dotted line to denote a fold change of 1 which is no change over control). Data are from 3 independent infections performed on triplicate wells, the fold change was calculated in each independent experiment and the mean fold change is plotted with error bars displaying standard deviation. Along with TRIzol samples for RNA supernatant was collected from cells and used for TCID 50 assays to determine infectious virus production following treatment with HCQ (E) or CQ (F) Data are from 3 independent infections performed on triplicate wells with the TCID 50 /ml being averaged across all wells. Error bars are the standard deviation. G) Cells were treated with 50 μM HCQ or 0.1% water as control. Drug was either added 2 h prior to infection, at the time of infection or 2 h after infection with MOI 0.1 SARS-CoV-2. After 24 h infection, supernatant was collected and used for TCID 50 assays to determine infectious virus production. Data are from 3 independent infections performed on triplicate wells with the TCID 50 /ml being averaged across all wells. Error bars are the standard deviation. H) SARS-CoV spike psuedoviruses (PV) were used for infection of BSC1 cells. The cells were treated with 10 μM of CQ or CPZ for 1 h prior to infection with PV for 3 h. The PV carry BlaM and cells were loaded with CCF2 to monitor cleavage and shift in fluorescence output for evidence of S-mediated entry into cells. Data are normalised to PV alone and are from 3 independent experiments with error bars representing standard deviation.

    Techniques Used: Concentration Assay, Infection, Quantitative RT-PCR, Standard Deviation, Fluorescence

    30) Product Images from "The extracellular matrix component WIF-1 is expressed during, and can modulate, retinal development"

    Article Title: The extracellular matrix component WIF-1 is expressed during, and can modulate, retinal development

    Journal: Molecular and cellular neurosciences

    doi: 10.1016/j.mcn.2004.08.003

    RNA encoding WIF-1 is expressed in the retina during retinal differentiation. (A) RT–PCR demonstrates WIF-1 RNA in extracts of retinae from embryonic day (E) 19, postnatal day (P) 5, and adult (Ad) mice. Equal amounts of RNA were loaded in each lane as shown by RT–PCR for 18S ribosomal RNA. (B) Sections of E16 and adult retinae were hybridized with antisense RNA probes for WIF-1. WIF-1 RNA is present in the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL), with little in the neuroblast layer (NBL).
    Figure Legend Snippet: RNA encoding WIF-1 is expressed in the retina during retinal differentiation. (A) RT–PCR demonstrates WIF-1 RNA in extracts of retinae from embryonic day (E) 19, postnatal day (P) 5, and adult (Ad) mice. Equal amounts of RNA were loaded in each lane as shown by RT–PCR for 18S ribosomal RNA. (B) Sections of E16 and adult retinae were hybridized with antisense RNA probes for WIF-1. WIF-1 RNA is present in the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL), with little in the neuroblast layer (NBL).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Mouse Assay

    31) Product Images from "Targeted DNA oxidation by LSD1–SMAD2/3 primes TGF-β1/ EMT genes for activation or repression"

    Article Title: Targeted DNA oxidation by LSD1–SMAD2/3 primes TGF-β1/ EMT genes for activation or repression

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa599

    Nuclear G oxidation induced by TGF-β1 through the histone demethylase, LSD1. (A) Immunofluorescence detection of 8-oxo-dG by fluorescein-tagged anti-8-oxo-dG antibody in cells pretreated with 5 mM NAC or 5 μM DPI for 1 h before stimulation with 10 ng/ml TGF-β1 for 30 min. Nuclei were stained with TO-PRO-3 Iodide in red while 8-oxo-dG foci were stained in green, as described in the ‘Materials and Methods’ section. Fluorescence quantization and statistical analysis are shown in Supplementary Figures S1C and S1-Stat3–6 . (B) Immunofluorescence detection of 8-oxo-dG by fluorescein-tagged anti-8-oxo-dG antibody in cells transfected with scrambled short hairpin (shCTRL) and shLSD1 RNAs for 24 h before stimulation with 10 ng/ml TGF-β1 for 30 min. Nuclei were stained with TO-PRO-3 Iodide in red while 8-oxo-dG foci were stained in green. Colocalization was measured using ImageJ (Coloc_2 plugin) as described in the ‘Materials and Methods’ section. Fluorescence quantization and statistical analysis are shown in Supplementary Figures S1D and S1-Stat3–6 . (C) Immunofluorescence microscopy of phalloidin in red and E-cadherin in green in control or LSD1-depleted cells exposed to 10 ng/ml TGF-β1 for 72 h. (D–G) mRNA levels of genes regulated by TGF-β1 in cells treated with shLSD1 or with TCP. Results are the mean of at least three experiments performed three times ( n = 9). MANOVA was used to examine the association between the mRNA levels after TGF-β1 and LSD1 depletion; the interaction was significant (see Supplementary Figure 2-Stat.2 for panel D and Supplementary Figure 2-Stat.4 for panel E). Differences between the means of three or more groups were determined by MANOVA. Differences between three or more samples were determined by one-way ANOVA test. Pairwise comparison was performed with Student’s t -test (see Supplementary Figures -Stat). (D) mRNA levels of SNAI1, CDH2, VIM and PAI genes in cells induced for 90 min with TGF-β1 determined by qPCR. Total RNA was isolated, reverse transcribed and amplified by qPCR with specific primers ( Supplementary Table S1 ). The values were normalized to 18S RNA. RNA was isolated as described in the ‘Materials and Methods’ section. * P
    Figure Legend Snippet: Nuclear G oxidation induced by TGF-β1 through the histone demethylase, LSD1. (A) Immunofluorescence detection of 8-oxo-dG by fluorescein-tagged anti-8-oxo-dG antibody in cells pretreated with 5 mM NAC or 5 μM DPI for 1 h before stimulation with 10 ng/ml TGF-β1 for 30 min. Nuclei were stained with TO-PRO-3 Iodide in red while 8-oxo-dG foci were stained in green, as described in the ‘Materials and Methods’ section. Fluorescence quantization and statistical analysis are shown in Supplementary Figures S1C and S1-Stat3–6 . (B) Immunofluorescence detection of 8-oxo-dG by fluorescein-tagged anti-8-oxo-dG antibody in cells transfected with scrambled short hairpin (shCTRL) and shLSD1 RNAs for 24 h before stimulation with 10 ng/ml TGF-β1 for 30 min. Nuclei were stained with TO-PRO-3 Iodide in red while 8-oxo-dG foci were stained in green. Colocalization was measured using ImageJ (Coloc_2 plugin) as described in the ‘Materials and Methods’ section. Fluorescence quantization and statistical analysis are shown in Supplementary Figures S1D and S1-Stat3–6 . (C) Immunofluorescence microscopy of phalloidin in red and E-cadherin in green in control or LSD1-depleted cells exposed to 10 ng/ml TGF-β1 for 72 h. (D–G) mRNA levels of genes regulated by TGF-β1 in cells treated with shLSD1 or with TCP. Results are the mean of at least three experiments performed three times ( n = 9). MANOVA was used to examine the association between the mRNA levels after TGF-β1 and LSD1 depletion; the interaction was significant (see Supplementary Figure 2-Stat.2 for panel D and Supplementary Figure 2-Stat.4 for panel E). Differences between the means of three or more groups were determined by MANOVA. Differences between three or more samples were determined by one-way ANOVA test. Pairwise comparison was performed with Student’s t -test (see Supplementary Figures -Stat). (D) mRNA levels of SNAI1, CDH2, VIM and PAI genes in cells induced for 90 min with TGF-β1 determined by qPCR. Total RNA was isolated, reverse transcribed and amplified by qPCR with specific primers ( Supplementary Table S1 ). The values were normalized to 18S RNA. RNA was isolated as described in the ‘Materials and Methods’ section. * P

    Techniques Used: Immunofluorescence, Staining, Fluorescence, Transfection, Microscopy, Real-time Polymerase Chain Reaction, Isolation, Amplification

    32) Product Images from "Lytic Reactivation of the Kaposi’s sarcoma-associated herpesvirus (KSHV) is Accompanied by Major Nucleolar Alterations"

    Article Title: Lytic Reactivation of the Kaposi’s sarcoma-associated herpesvirus (KSHV) is Accompanied by Major Nucleolar Alterations

    Journal: bioRxiv

    doi: 10.1101/2020.05.15.097808

    Northern blot analysis of rRNA during viral lytic reactivation. Total RNA was extracted from uninfected SLK and BAC16-mCherry-ORF45-infected iSLK cells that were either left untreated or treated with Dox and n-Butyrate for 48-hr. Lytic reactivation was confirmed by expression of mCherry-ORF45 in ∼80% of cells. 20 µg RNA from each sample was resolved on denaturing 1.2% agarose gel, transferred to nylon membranes, and hybridized with probes representing 5.8S, 18S and 28S rRNA. No accumulation of pre-rRNA processing products was evident upon lytic reactivation.
    Figure Legend Snippet: Northern blot analysis of rRNA during viral lytic reactivation. Total RNA was extracted from uninfected SLK and BAC16-mCherry-ORF45-infected iSLK cells that were either left untreated or treated with Dox and n-Butyrate for 48-hr. Lytic reactivation was confirmed by expression of mCherry-ORF45 in ∼80% of cells. 20 µg RNA from each sample was resolved on denaturing 1.2% agarose gel, transferred to nylon membranes, and hybridized with probes representing 5.8S, 18S and 28S rRNA. No accumulation of pre-rRNA processing products was evident upon lytic reactivation.

    Techniques Used: Northern Blot, Infection, Expressing, Agarose Gel Electrophoresis

    33) Product Images from "Diterpenoids from Plectranthus spp. as Potential Chemotherapeutic Agents via Apoptosis"

    Article Title: Diterpenoids from Plectranthus spp. as Potential Chemotherapeutic Agents via Apoptosis

    Journal: Pharmaceuticals

    doi: 10.3390/ph13060123

    ( A ) Gene expression ( Bax, Bcl-2 , Cas-3 , and TP53 ) in A549 and ( B ) CCRF-CEM cell lines after 24 h treatment with all the compounds: Roy (3 µg/mL), Deroy (6.25 µg/mL), Diroy (25 µg/mL), and Parv (1.15 µg/mL) in A549 cells and Roy (0.7 µg/mL), Deroy (4.5 µg/mL), Diroy (25 µg/mL), and Parv (1.15 µg/mL) in CCRF-CEM cells. The transcript level of each gene was normalized to the expression of a reference gene ( 18S RNA ). Data are presented as a fold change in cells treated with tested compounds vs. untreated, control cells, in which expression levels of the genes were set as 1. The mean values ± SD were calculated in triplicates. The same letter (a, b, c and d) at the same genes is not significantly different at the level of p > 0.05.
    Figure Legend Snippet: ( A ) Gene expression ( Bax, Bcl-2 , Cas-3 , and TP53 ) in A549 and ( B ) CCRF-CEM cell lines after 24 h treatment with all the compounds: Roy (3 µg/mL), Deroy (6.25 µg/mL), Diroy (25 µg/mL), and Parv (1.15 µg/mL) in A549 cells and Roy (0.7 µg/mL), Deroy (4.5 µg/mL), Diroy (25 µg/mL), and Parv (1.15 µg/mL) in CCRF-CEM cells. The transcript level of each gene was normalized to the expression of a reference gene ( 18S RNA ). Data are presented as a fold change in cells treated with tested compounds vs. untreated, control cells, in which expression levels of the genes were set as 1. The mean values ± SD were calculated in triplicates. The same letter (a, b, c and d) at the same genes is not significantly different at the level of p > 0.05.

    Techniques Used: Expressing

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    Thermo Fisher 18s rrna
    Phylogenetic trees based on ribosomal 12S <t>rRNA</t> and <t>18S</t> rRNA were constructed using the Maximum Likelihood method based on the Hasegawa-Kishino-Yano model (A) and the Neighbor-Joining method based on the Kimura 2-parameter model (B), and bootstrap values are based on 5000 replicates.
    18s Rrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 604 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gene exp ucp1 mm01244861 m1
    JAK inhibition stably induces a brown-like profile in adipocytes a) bDNA quantification of <t>UCP1</t> mRNA levels over time, showing that the progressive accumulation of UCP1 induced by tofacitinib and R406 contrasts with the acute effect of THRB agonists. Values represent the mean of two biological replicates. b) Schematic illustration of experimental design for b), c), and d): PSC-WA were treated with compounds for 7 days, washed 3 times with compound-free medium, and maintained in compound-free medium for an additional 14 day-period. Images were captured at day 28 prior to addition of lysis buffer and bDNA analysis. Only JAK3i and SYKi-pre-treated cells displayed high levels of UCP1 mRNA relative to DMSO. Values represent the mean of two biological replicates. c) Bright field images showing a reduced lipid vacuole size at day 28 for tofacitinib (tofa.) and R406-pre-treated adipocytes. Scale bars, 50μm. Images are representative of two independent experiments. d) DMSO, tofacitinib (tofa.) and R406-pre-treated adipocytes were exposed to TNFα from day 26 to 28. bDNA analysis shows that pre-treatment with tofacitinib and R406 protects adipocytes from TNFα-mediated down-regulation of UCP1 expression. Values represent the mean of two biological replicates.
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    Phylogenetic trees based on ribosomal 12S rRNA and 18S rRNA were constructed using the Maximum Likelihood method based on the Hasegawa-Kishino-Yano model (A) and the Neighbor-Joining method based on the Kimura 2-parameter model (B), and bootstrap values are based on 5000 replicates.

    Journal: Helminthologia

    Article Title: Crenosoma Striatum in Lungs of European Hedgehogs (Erinaceus Europeus) from Portugal

    doi: 10.2478/helm-2020-0020

    Figure Lengend Snippet: Phylogenetic trees based on ribosomal 12S rRNA and 18S rRNA were constructed using the Maximum Likelihood method based on the Hasegawa-Kishino-Yano model (A) and the Neighbor-Joining method based on the Kimura 2-parameter model (B), and bootstrap values are based on 5000 replicates.

    Article Snippet: Phylogenetic trees based on ribosomal 12S rRNA and 18S rRNA were constructed using the Maximum Likelihood method based on the Hasegawa-Kishino-Yano model and the Neighbor-Joining method based on the.

    Techniques: Construct

    Ectopic expression of miR-148a accelerates adipocyte differentiation. miR-148a and miR-Ctrl were transfected into 3T3-L1 preadipocytes and cells differentiated. (A) Day 2 gene expression was assessed by Q-PCR and normalized to 18S rRNA (mean ±

    Journal: Molecular and Cellular Biology

    Article Title: DNMT1 Is Regulated by ATP-Citrate Lyase and Maintains Methylation Patterns during Adipocyte Differentiation

    doi: 10.1128/MCB.01495-12

    Figure Lengend Snippet: Ectopic expression of miR-148a accelerates adipocyte differentiation. miR-148a and miR-Ctrl were transfected into 3T3-L1 preadipocytes and cells differentiated. (A) Day 2 gene expression was assessed by Q-PCR and normalized to 18S rRNA (mean ±

    Article Snippet: Gene expression data were normalized to 18S rRNA or actin, as indicated.

    Techniques: Expressing, Transfection, Polymerase Chain Reaction

    Expression levels of IL-8 mRNA follow disease activity in patients with V . cholerae O1 infection. Expression levels of IL-8, CXCL9 and IL-1β mRNAs were determined in duodenal biopsies collected at acute stage (Acute) and at the convalescent stage (Convalescent) of disease caused by V . cholerae O1 infection. The mRNA expression levels were determined by a real-time qRT-PCR and normalized to the content of 18S rRNA in the sample. Results are shown as mRNA copies/18S rRNA U for IL-8 and relative quantity (RQ) calculated by using the 2 (-ΔΔct) -method and the median Δct-value at convalescent stage as reference for CXCL9 and IL-1β. Each point represents the value of an individual patient at the indicated disease stage. Lines connect the values at acute and convalescent stage of the same patient. Significance of differences between Δct-values at acute and convalescent stage was analyzed using two-sided Wilcoxon's non-parametric test for paired samples and P -values are given in the graph.

    Journal: PLoS ONE

    Article Title: Induction of immunomodulatory miR-146a and miR-155 in small intestinal epithelium of Vibrio cholerae infected patients at acute stage of cholera

    doi: 10.1371/journal.pone.0173817

    Figure Lengend Snippet: Expression levels of IL-8 mRNA follow disease activity in patients with V . cholerae O1 infection. Expression levels of IL-8, CXCL9 and IL-1β mRNAs were determined in duodenal biopsies collected at acute stage (Acute) and at the convalescent stage (Convalescent) of disease caused by V . cholerae O1 infection. The mRNA expression levels were determined by a real-time qRT-PCR and normalized to the content of 18S rRNA in the sample. Results are shown as mRNA copies/18S rRNA U for IL-8 and relative quantity (RQ) calculated by using the 2 (-ΔΔct) -method and the median Δct-value at convalescent stage as reference for CXCL9 and IL-1β. Each point represents the value of an individual patient at the indicated disease stage. Lines connect the values at acute and convalescent stage of the same patient. Significance of differences between Δct-values at acute and convalescent stage was analyzed using two-sided Wilcoxon's non-parametric test for paired samples and P -values are given in the graph.

    Article Snippet: One U was defined as the amount of 18S rRNA in 1 pg of RNA, and corresponds to approximately 1 epithelial cell [ ].

    Techniques: Expressing, Activity Assay, Infection, Quantitative RT-PCR

    Secreted products of V . cholerae O1 bacteria induce increased expression of genes involved in inflammation in T84 polarized tight monolayer cells while live bacteria do not. The same RNA samples that were analyzed for miRNA expression levels (results shown in Fig 6 ) were also analyzed for expression levels of mRNAs for the microRNA target genes IRAK1 and CARD10, the inflammasome cytokine IL-1β and the chemokines CXCL9 and IL-8. The mRNA expression levels were determined by a real-time qRT-PCR and normalized to the content of 18S rRNA in the sample. Results are shown as relative quantity (RQ) calculated by using the 2 (-ΔΔct) -method and using the median Δct-value of the sham-treated controls as reference for IRAK1, CARD10, IL-1β and CXCL9 and as mRNA copies/18S rRNA U for IL-8. Bars indicate mean + 1 SD (n = 4 in upper row and n = 3 in lower row). Statistically significant differences from the sham-treated control monolayers as determined by one-way ANOVA with Dunett's compensation for multiple comparisons, are shown. * P -value

    Journal: PLoS ONE

    Article Title: Induction of immunomodulatory miR-146a and miR-155 in small intestinal epithelium of Vibrio cholerae infected patients at acute stage of cholera

    doi: 10.1371/journal.pone.0173817

    Figure Lengend Snippet: Secreted products of V . cholerae O1 bacteria induce increased expression of genes involved in inflammation in T84 polarized tight monolayer cells while live bacteria do not. The same RNA samples that were analyzed for miRNA expression levels (results shown in Fig 6 ) were also analyzed for expression levels of mRNAs for the microRNA target genes IRAK1 and CARD10, the inflammasome cytokine IL-1β and the chemokines CXCL9 and IL-8. The mRNA expression levels were determined by a real-time qRT-PCR and normalized to the content of 18S rRNA in the sample. Results are shown as relative quantity (RQ) calculated by using the 2 (-ΔΔct) -method and using the median Δct-value of the sham-treated controls as reference for IRAK1, CARD10, IL-1β and CXCL9 and as mRNA copies/18S rRNA U for IL-8. Bars indicate mean + 1 SD (n = 4 in upper row and n = 3 in lower row). Statistically significant differences from the sham-treated control monolayers as determined by one-way ANOVA with Dunett's compensation for multiple comparisons, are shown. * P -value

    Article Snippet: One U was defined as the amount of 18S rRNA in 1 pg of RNA, and corresponds to approximately 1 epithelial cell [ ].

    Techniques: Expressing, Quantitative RT-PCR

    Expression levels of target genes for miR-146a show only marginal differences between the acute and the convalescent stage of disease in patients with V . cholerae O1 infection. Expression levels of IRAK1, TRAF6 and CARD10 mRNAs were determined in duodenal biopsies collected at acute stage (Acute) and at the convalescent stage (Convalescent) of disease caused by V . cholerae O1 infection. Expression levels were determined by a real-time qRT-PCR and normalized to the content of 18S rRNA in the sample. Results are shown as relative quantity (RQ) calculated by using the 2 (-ΔΔct) -method and the median Δct-value at convalescent stage as reference. Each point represents the value of an individual patient at the indicated disease stage. Lines connect the values at acute and convalescent stage of the same patient.

    Journal: PLoS ONE

    Article Title: Induction of immunomodulatory miR-146a and miR-155 in small intestinal epithelium of Vibrio cholerae infected patients at acute stage of cholera

    doi: 10.1371/journal.pone.0173817

    Figure Lengend Snippet: Expression levels of target genes for miR-146a show only marginal differences between the acute and the convalescent stage of disease in patients with V . cholerae O1 infection. Expression levels of IRAK1, TRAF6 and CARD10 mRNAs were determined in duodenal biopsies collected at acute stage (Acute) and at the convalescent stage (Convalescent) of disease caused by V . cholerae O1 infection. Expression levels were determined by a real-time qRT-PCR and normalized to the content of 18S rRNA in the sample. Results are shown as relative quantity (RQ) calculated by using the 2 (-ΔΔct) -method and the median Δct-value at convalescent stage as reference. Each point represents the value of an individual patient at the indicated disease stage. Lines connect the values at acute and convalescent stage of the same patient.

    Article Snippet: One U was defined as the amount of 18S rRNA in 1 pg of RNA, and corresponds to approximately 1 epithelial cell [ ].

    Techniques: Expressing, Infection, Quantitative RT-PCR

    JAK inhibition stably induces a brown-like profile in adipocytes a) bDNA quantification of UCP1 mRNA levels over time, showing that the progressive accumulation of UCP1 induced by tofacitinib and R406 contrasts with the acute effect of THRB agonists. Values represent the mean of two biological replicates. b) Schematic illustration of experimental design for b), c), and d): PSC-WA were treated with compounds for 7 days, washed 3 times with compound-free medium, and maintained in compound-free medium for an additional 14 day-period. Images were captured at day 28 prior to addition of lysis buffer and bDNA analysis. Only JAK3i and SYKi-pre-treated cells displayed high levels of UCP1 mRNA relative to DMSO. Values represent the mean of two biological replicates. c) Bright field images showing a reduced lipid vacuole size at day 28 for tofacitinib (tofa.) and R406-pre-treated adipocytes. Scale bars, 50μm. Images are representative of two independent experiments. d) DMSO, tofacitinib (tofa.) and R406-pre-treated adipocytes were exposed to TNFα from day 26 to 28. bDNA analysis shows that pre-treatment with tofacitinib and R406 protects adipocytes from TNFα-mediated down-regulation of UCP1 expression. Values represent the mean of two biological replicates.

    Journal: Nature cell biology

    Article Title: White-to-brown metabolic conversion of human adipocytes by JAK inhibition

    doi: 10.1038/ncb3075

    Figure Lengend Snippet: JAK inhibition stably induces a brown-like profile in adipocytes a) bDNA quantification of UCP1 mRNA levels over time, showing that the progressive accumulation of UCP1 induced by tofacitinib and R406 contrasts with the acute effect of THRB agonists. Values represent the mean of two biological replicates. b) Schematic illustration of experimental design for b), c), and d): PSC-WA were treated with compounds for 7 days, washed 3 times with compound-free medium, and maintained in compound-free medium for an additional 14 day-period. Images were captured at day 28 prior to addition of lysis buffer and bDNA analysis. Only JAK3i and SYKi-pre-treated cells displayed high levels of UCP1 mRNA relative to DMSO. Values represent the mean of two biological replicates. c) Bright field images showing a reduced lipid vacuole size at day 28 for tofacitinib (tofa.) and R406-pre-treated adipocytes. Scale bars, 50μm. Images are representative of two independent experiments. d) DMSO, tofacitinib (tofa.) and R406-pre-treated adipocytes were exposed to TNFα from day 26 to 28. bDNA analysis shows that pre-treatment with tofacitinib and R406 protects adipocytes from TNFα-mediated down-regulation of UCP1 expression. Values represent the mean of two biological replicates.

    Article Snippet: Taqman probes were purchased from Life Technologies (UCP1: catalog number Mm01244861_m1, 18S: catalog number Mm03928990_g1).

    Techniques: Inhibition, Stable Transfection, Lysis, Expressing

    IFN and SHH signaling contribute to adipocyte browning downstream of JAK inhibition a) Whitening of IFNγ-treated adipocytes is visible as lipid accumulation forms a single, large vacuole. Treatment with tofacitinib (tofa.) and R406 restores the formation of small lipid droplets (arrows). Bright field images representative of two independent experiments. Scale bars, 20μm. b) IFNγ treatment decreases the UCP1 / FABP4 ratio (upper graph) and increases the expression of HSL, a marker of white adipocyte (lower graph) both in WA and BA. Values represent the mean of two biological replicates. c) The SHH pathway antagonist cyclopamine fully blocks CP-6990550-mediated browning as judged by UCP1 level and partially blocks SYKi-mediated browning (left graph). Cyclopamine didn't restore R406-induced FABP4 levels, thereby decoupling the regulation of UCP1 and FABP4 by R406 (right graph). Values represent the mean of two biological replicates. d) Model of adipocyte browning by pharmacological inhibition of JAK. Tofacitinib and R406 inhibit the JAK-STAT pathway in human adipocytes, leading to down-regulation of the interferon alpha, beta and gamma responses. Sustained shut down of IFN signaling relieves inhibition of the sonic hedgehog (SHH) pathway and thereby contributes to accumulation of UCP1 . R406 acts as a pleiotropic drug with broad effects on adipocytes through activation of PPARG, BMPs and SREBF target genes. Red fonts: negative regulator of browning; Green fonts: positive regulator of browning; Arrows: activation; Flat lines: inhibition; Dash lines: hypothetical.

    Journal: Nature cell biology

    Article Title: White-to-brown metabolic conversion of human adipocytes by JAK inhibition

    doi: 10.1038/ncb3075

    Figure Lengend Snippet: IFN and SHH signaling contribute to adipocyte browning downstream of JAK inhibition a) Whitening of IFNγ-treated adipocytes is visible as lipid accumulation forms a single, large vacuole. Treatment with tofacitinib (tofa.) and R406 restores the formation of small lipid droplets (arrows). Bright field images representative of two independent experiments. Scale bars, 20μm. b) IFNγ treatment decreases the UCP1 / FABP4 ratio (upper graph) and increases the expression of HSL, a marker of white adipocyte (lower graph) both in WA and BA. Values represent the mean of two biological replicates. c) The SHH pathway antagonist cyclopamine fully blocks CP-6990550-mediated browning as judged by UCP1 level and partially blocks SYKi-mediated browning (left graph). Cyclopamine didn't restore R406-induced FABP4 levels, thereby decoupling the regulation of UCP1 and FABP4 by R406 (right graph). Values represent the mean of two biological replicates. d) Model of adipocyte browning by pharmacological inhibition of JAK. Tofacitinib and R406 inhibit the JAK-STAT pathway in human adipocytes, leading to down-regulation of the interferon alpha, beta and gamma responses. Sustained shut down of IFN signaling relieves inhibition of the sonic hedgehog (SHH) pathway and thereby contributes to accumulation of UCP1 . R406 acts as a pleiotropic drug with broad effects on adipocytes through activation of PPARG, BMPs and SREBF target genes. Red fonts: negative regulator of browning; Green fonts: positive regulator of browning; Arrows: activation; Flat lines: inhibition; Dash lines: hypothetical.

    Article Snippet: Taqman probes were purchased from Life Technologies (UCP1: catalog number Mm01244861_m1, 18S: catalog number Mm03928990_g1).

    Techniques: Inhibition, Expressing, Marker, Activation Assay

    Gene signature and cellular identity of JAK-inactivated adipocytes a-f) Adipocytes were differentiated according to scheme 1B, treated with tofacitinib and R406 at day 7, and collected at day 8 (24h time point) or day 14 (d7 time point). N= 3 biological replicates. Each independent biological replicate was pooled from two individual wells. a) Multi-dimensional scaling of RNA sequencing data revealing the white lineage identity of tofacitinib (tofa.) and R406 treated PSC-WA. b) Levels of UCP1 transcripts served as experimental control for adipocyte browning. UCP1 transcripts are higher in BA versus WA and higher in tofacitinib (tofa.) and R406-treated PSC-WA compare to DMSO-treated PSC-WA. c) Interferon targets and pro-inflammatory pathways are significantly down-regulated by both compounds at 7d in PSC-WA. Enrichment scores of 9116 gene sets are compared between two time points (24h and 7d) for both compounds. Each circle represents one gene set that is coherently regulated by an upstream pathway. Black lines indicate the change of average scores, and blue lines the change of individual pathways that are significantly reduced (|ΔES| > =2). d) Differential expression profiles of interferon targets induced by tofacitinib and R406. A substantial subset of target genes are negatively regulated in both cases, making the density curves of logFC shifts toward left and thereby forming a “red shoulder”. Compared with 24h, the expression of interferon pathway targets are repressed by both compounds at 7d ( P =2.94E-6 and 2.06E-5, respectively; one-sided Kolmogorov-Smirnov test). e) Differential expression profiles of selected interferon target genes in heatmap. f) Whole transcriptome analysis revealed that the sonic hedgehog responsive genes GLI1, SFRP5, KLHL31 and SHH were up-regulated in tofacitinib (tofa.) and R406-treated adipocytes at day 7 compare to DMSO control. Values are mean ± s.d. of n = three biological replicates and differences from DMSO are significant for * P

    Journal: Nature cell biology

    Article Title: White-to-brown metabolic conversion of human adipocytes by JAK inhibition

    doi: 10.1038/ncb3075

    Figure Lengend Snippet: Gene signature and cellular identity of JAK-inactivated adipocytes a-f) Adipocytes were differentiated according to scheme 1B, treated with tofacitinib and R406 at day 7, and collected at day 8 (24h time point) or day 14 (d7 time point). N= 3 biological replicates. Each independent biological replicate was pooled from two individual wells. a) Multi-dimensional scaling of RNA sequencing data revealing the white lineage identity of tofacitinib (tofa.) and R406 treated PSC-WA. b) Levels of UCP1 transcripts served as experimental control for adipocyte browning. UCP1 transcripts are higher in BA versus WA and higher in tofacitinib (tofa.) and R406-treated PSC-WA compare to DMSO-treated PSC-WA. c) Interferon targets and pro-inflammatory pathways are significantly down-regulated by both compounds at 7d in PSC-WA. Enrichment scores of 9116 gene sets are compared between two time points (24h and 7d) for both compounds. Each circle represents one gene set that is coherently regulated by an upstream pathway. Black lines indicate the change of average scores, and blue lines the change of individual pathways that are significantly reduced (|ΔES| > =2). d) Differential expression profiles of interferon targets induced by tofacitinib and R406. A substantial subset of target genes are negatively regulated in both cases, making the density curves of logFC shifts toward left and thereby forming a “red shoulder”. Compared with 24h, the expression of interferon pathway targets are repressed by both compounds at 7d ( P =2.94E-6 and 2.06E-5, respectively; one-sided Kolmogorov-Smirnov test). e) Differential expression profiles of selected interferon target genes in heatmap. f) Whole transcriptome analysis revealed that the sonic hedgehog responsive genes GLI1, SFRP5, KLHL31 and SHH were up-regulated in tofacitinib (tofa.) and R406-treated adipocytes at day 7 compare to DMSO control. Values are mean ± s.d. of n = three biological replicates and differences from DMSO are significant for * P

    Article Snippet: Taqman probes were purchased from Life Technologies (UCP1: catalog number Mm01244861_m1, 18S: catalog number Mm03928990_g1).

    Techniques: RNA Sequencing Assay, Expressing

    Validation of tofacitinib and R406 browning compounds in primary adipocytes a) bDNA analysis of dose response with tofacitinib and R406. At high doses, R406 increases both UCP1 and FABP4 expression but UCP1 / FABP4 remains above 2. Values represent the mean of two biological replicates. b) Western blot analysis showing that up-regulation of UCP1 and PRDM16 protein levels correlates with up-regulation of UCP1 mRNA by tofacitinib and R406. c) bDNA analysis showing that tofacitinib (tofa.) and R406 increase UCP1 expression in human primary adipocytes. ADSC: Adipose tissue-derived stromal cells. Values are mean ± s.d. of n = three biological replicates and differences from DMSO are significant for * P

    Journal: Nature cell biology

    Article Title: White-to-brown metabolic conversion of human adipocytes by JAK inhibition

    doi: 10.1038/ncb3075

    Figure Lengend Snippet: Validation of tofacitinib and R406 browning compounds in primary adipocytes a) bDNA analysis of dose response with tofacitinib and R406. At high doses, R406 increases both UCP1 and FABP4 expression but UCP1 / FABP4 remains above 2. Values represent the mean of two biological replicates. b) Western blot analysis showing that up-regulation of UCP1 and PRDM16 protein levels correlates with up-regulation of UCP1 mRNA by tofacitinib and R406. c) bDNA analysis showing that tofacitinib (tofa.) and R406 increase UCP1 expression in human primary adipocytes. ADSC: Adipose tissue-derived stromal cells. Values are mean ± s.d. of n = three biological replicates and differences from DMSO are significant for * P

    Article Snippet: Taqman probes were purchased from Life Technologies (UCP1: catalog number Mm01244861_m1, 18S: catalog number Mm03928990_g1).

    Techniques: Expressing, Western Blot, Derivative Assay

    Inhibition of STAT phosphorylation downstream of tofacitinib and R406 a) Transcript abundance in RPKM (reads per kilobase transcript per million reads) of known targets of tofacitinib and R406 indicating that the JAK kinases are predominantly represented in PSC-WA. Values are mean ± s.d. of n = three biological replicates. b) Western blot analyses of STATs, AKT and MAPKs in PSC-WA previously treated with DMSO, tofacitinib and R406 for 20 minutes or 7 days showing a pronounced inhibition of STAT phosphorylation by tofacitinib and R406 at both time points. R406 also significantly decreased phosphorylation levels of AKT and ERK1/2. The data shown is representative of two independent experiments. c) Western blot analysis of a dose-response with tofacitinib and R406 showing the correlation between UCP1 accumulation and inhibition of STAT3 phosphorylation. The data shown is representative of two independent experiments. e) bDNA analysis of PSC-WA differentiated as in figure 1b ) and treated with TNFα at day 12, indicating that the negative effect of TNFα on UCP1 expression is rescued by tofacitinib and R406. Values represent the mean of two biological replicates. f) Tofacitinib (tofa.) and R406 do not synergize during adipocyte browning. PSC-WA were treated with tofacitinib, R406 or a combination of both and analyzed for UCP1 expression by bDNA. Values are mean ± s.d. of n = three biological replicates and differences from DMSO are significant for * P

    Journal: Nature cell biology

    Article Title: White-to-brown metabolic conversion of human adipocytes by JAK inhibition

    doi: 10.1038/ncb3075

    Figure Lengend Snippet: Inhibition of STAT phosphorylation downstream of tofacitinib and R406 a) Transcript abundance in RPKM (reads per kilobase transcript per million reads) of known targets of tofacitinib and R406 indicating that the JAK kinases are predominantly represented in PSC-WA. Values are mean ± s.d. of n = three biological replicates. b) Western blot analyses of STATs, AKT and MAPKs in PSC-WA previously treated with DMSO, tofacitinib and R406 for 20 minutes or 7 days showing a pronounced inhibition of STAT phosphorylation by tofacitinib and R406 at both time points. R406 also significantly decreased phosphorylation levels of AKT and ERK1/2. The data shown is representative of two independent experiments. c) Western blot analysis of a dose-response with tofacitinib and R406 showing the correlation between UCP1 accumulation and inhibition of STAT3 phosphorylation. The data shown is representative of two independent experiments. e) bDNA analysis of PSC-WA differentiated as in figure 1b ) and treated with TNFα at day 12, indicating that the negative effect of TNFα on UCP1 expression is rescued by tofacitinib and R406. Values represent the mean of two biological replicates. f) Tofacitinib (tofa.) and R406 do not synergize during adipocyte browning. PSC-WA were treated with tofacitinib, R406 or a combination of both and analyzed for UCP1 expression by bDNA. Values are mean ± s.d. of n = three biological replicates and differences from DMSO are significant for * P

    Article Snippet: Taqman probes were purchased from Life Technologies (UCP1: catalog number Mm01244861_m1, 18S: catalog number Mm03928990_g1).

    Techniques: Inhibition, Western Blot, Expressing

    Browning screen in human stem cell-derived adipocytes a) Conceptual strategy to identify small molecules with adipocyte browning effect using human stem cells. PSC: pluripotent stem cells, EB: embryoid bodies, MPC: mesenchymal progenitor cells. b) Adipocyte browning screen, assay workflow. PPARG2 expressing-MPC (PPARG2-MPC) were maintained in adipogenic medium containing doxycycline and rosiglitazone for 3 days in order to induce adipogenesis, and differentiated in the absence of rosiglitazone for 4 days. A library of 867 compounds of known mode of action was applied to PSC-WA at day 7, 10 and 12. Total mRNA was collected at day 14, and UCP1 and FABP4 mRNA levels were quantified using the branched DNA technology. PPARG2-MPC: mesenchymal progenitor cells transduced with rtTA and doxycycline-inducible PPARG2 expression vectors. For more details see the methods section. c) Scatter plot display of browning screen results. Each data point represents the average of two biological replicates per compound, normalized on DMSO control. X axis: UCP1 mRNA level as an indicator of adipocyte browning, Y axis: FABP4 mRNA as an indicator of general adipogenesis. The color code distinguishes inactive compounds (black) from active ones: Rosiglitazone (red), Rosiglitazone-like compounds that increase UCP1 and FABP4 (blue), and potential browning compounds that induce UCP1 specifically (green). Dashed lines indicate neutral conditions, solid line delineates UCP1 induction above 2 fold. d) Validation of browning hits by bDNA analysis showing that JAK3 inhibitors, SYK inhibitors and THRB agonists scored as best UCP1/FABP4 inducers. All compounds were added at a 5 μM final concentration. X axis: Compounds are identified by target and mode of action. i= inhibitor, ag = agonist. For chemical nomenclature see the methods section. Values represent the mean of two biological replicates.

    Journal: Nature cell biology

    Article Title: White-to-brown metabolic conversion of human adipocytes by JAK inhibition

    doi: 10.1038/ncb3075

    Figure Lengend Snippet: Browning screen in human stem cell-derived adipocytes a) Conceptual strategy to identify small molecules with adipocyte browning effect using human stem cells. PSC: pluripotent stem cells, EB: embryoid bodies, MPC: mesenchymal progenitor cells. b) Adipocyte browning screen, assay workflow. PPARG2 expressing-MPC (PPARG2-MPC) were maintained in adipogenic medium containing doxycycline and rosiglitazone for 3 days in order to induce adipogenesis, and differentiated in the absence of rosiglitazone for 4 days. A library of 867 compounds of known mode of action was applied to PSC-WA at day 7, 10 and 12. Total mRNA was collected at day 14, and UCP1 and FABP4 mRNA levels were quantified using the branched DNA technology. PPARG2-MPC: mesenchymal progenitor cells transduced with rtTA and doxycycline-inducible PPARG2 expression vectors. For more details see the methods section. c) Scatter plot display of browning screen results. Each data point represents the average of two biological replicates per compound, normalized on DMSO control. X axis: UCP1 mRNA level as an indicator of adipocyte browning, Y axis: FABP4 mRNA as an indicator of general adipogenesis. The color code distinguishes inactive compounds (black) from active ones: Rosiglitazone (red), Rosiglitazone-like compounds that increase UCP1 and FABP4 (blue), and potential browning compounds that induce UCP1 specifically (green). Dashed lines indicate neutral conditions, solid line delineates UCP1 induction above 2 fold. d) Validation of browning hits by bDNA analysis showing that JAK3 inhibitors, SYK inhibitors and THRB agonists scored as best UCP1/FABP4 inducers. All compounds were added at a 5 μM final concentration. X axis: Compounds are identified by target and mode of action. i= inhibitor, ag = agonist. For chemical nomenclature see the methods section. Values represent the mean of two biological replicates.

    Article Snippet: Taqman probes were purchased from Life Technologies (UCP1: catalog number Mm01244861_m1, 18S: catalog number Mm03928990_g1).

    Techniques: Derivative Assay, Expressing, Transduction, Concentration Assay