target mrna  (Thermo Fisher)


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    Structured Review

    Thermo Fisher target mrna
    Effect of oral Lactobacillus chungangensis CAU 28 cream cheese administration on <t>mRNA</t> expression. Target mRNA expression levels including those of ( a ) IL-4, ( b ) IL-5, ( c ) IL-10, (d) IL-12, (e) IFN-γ, (f) TNF-α, and (g) <t>IL-1β</t> were determined via real-time polymerase chain reaction. Significance marks (* p
    Target Mrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/target mrna/product/Thermo Fisher
    Average 95 stars, based on 89 article reviews
    Price from $9.99 to $1999.99
    target mrna - by Bioz Stars, 2020-05
    95/100 stars

    Images

    1) Product Images from "Cream Cheese-Derived Lactococcus chungangensis CAU 28 Modulates the Gut Microbiota and Alleviates Atopic Dermatitis in BALB/c Mice"

    Article Title: Cream Cheese-Derived Lactococcus chungangensis CAU 28 Modulates the Gut Microbiota and Alleviates Atopic Dermatitis in BALB/c Mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36864-5

    Effect of oral Lactobacillus chungangensis CAU 28 cream cheese administration on mRNA expression. Target mRNA expression levels including those of ( a ) IL-4, ( b ) IL-5, ( c ) IL-10, (d) IL-12, (e) IFN-γ, (f) TNF-α, and (g) IL-1β were determined via real-time polymerase chain reaction. Significance marks (* p
    Figure Legend Snippet: Effect of oral Lactobacillus chungangensis CAU 28 cream cheese administration on mRNA expression. Target mRNA expression levels including those of ( a ) IL-4, ( b ) IL-5, ( c ) IL-10, (d) IL-12, (e) IFN-γ, (f) TNF-α, and (g) IL-1β were determined via real-time polymerase chain reaction. Significance marks (* p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    2) Product Images from "Promyelocytic leukemia zinc finger triggers ATP-binding cassette subfamily E member 1-mediated growth inhibition in breast cancer cells"

    Article Title: Promyelocytic leukemia zinc finger triggers ATP-binding cassette subfamily E member 1-mediated growth inhibition in breast cancer cells

    Journal: Oncology Letters

    doi: 10.3892/ol.2018.9207

    Reverse transcription-quantitative polymerase chain reaction assay of PLZF mRNA expression in breast cancer cells. Results are normalized to 18S expression, and are reported as a fold-change relative to the levels of PLZF mRNA in 293 cells and are presented as the mean ± standard deviation (n=3). *P
    Figure Legend Snippet: Reverse transcription-quantitative polymerase chain reaction assay of PLZF mRNA expression in breast cancer cells. Results are normalized to 18S expression, and are reported as a fold-change relative to the levels of PLZF mRNA in 293 cells and are presented as the mean ± standard deviation (n=3). *P

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

    3) Product Images from "MiR-30c/PGC-1β protects against diabetic cardiomyopathy via PPARα"

    Article Title: MiR-30c/PGC-1β protects against diabetic cardiomyopathy via PPARα

    Journal: Cardiovascular Diabetology

    doi: 10.1186/s12933-019-0811-7

    PGC-1β expression was reduced by miR-30c. a Venn diagram showing the overlap number of microRNA targeting PGC-1β predicted by TargetScan, PicTar and TarBase websites. b Sequence alignment of miR-30c on the 3′ UTR of PGC-1β from different organisms. c miR-30c expression was determined by RT-PCR in the heart tissues from 28-week old male db/db diabetic mice and their C57 littermates (n = 3). d miR-30c expression was determined by RT-PCR in H9c2 cells with or without palmitate (1 mmol/L) treatment. e Animals (db/db mice and C57 control mice) were injected with the corresponding rAAVs at 8–10 weeks of age and sacrificed at 28 weeks of age (n = 8–10). f H9c2 cells were transfected with miR-30c mimics/inhibitors (or their control) and then subjected to palmitate (1 mmol/L) stimulation. Representative Western blots and quantitative analysis of PGC-1β in the cardiac tissues ( e ) and H9c2 cells ( f ). β-actin was used as an internal control. g PGC-1β mRNA level was determined by RT-PCR in the whole RNA and the RNA of the anti-ago2 co-IP complex from H9c2 cells treated with miR-30c mimics or miR-control. h Schematic diagram of luciferase reporter plasmids pMIR PGC-1β 3′ UTR and mutant PGC-1β 3′ UTR, and the potential (or mutant) seed sequence of miR-30c targeting PGC-1β 3′ UTR are shown in red. i Regulation of PGC-1β via miR-30c targeting its 3′ UTR was determined with luciferase reporter assays in HEK293T cells. For d , f , g and i , n = three independent experiments. Data are expressed as mean ± SEM, *p
    Figure Legend Snippet: PGC-1β expression was reduced by miR-30c. a Venn diagram showing the overlap number of microRNA targeting PGC-1β predicted by TargetScan, PicTar and TarBase websites. b Sequence alignment of miR-30c on the 3′ UTR of PGC-1β from different organisms. c miR-30c expression was determined by RT-PCR in the heart tissues from 28-week old male db/db diabetic mice and their C57 littermates (n = 3). d miR-30c expression was determined by RT-PCR in H9c2 cells with or without palmitate (1 mmol/L) treatment. e Animals (db/db mice and C57 control mice) were injected with the corresponding rAAVs at 8–10 weeks of age and sacrificed at 28 weeks of age (n = 8–10). f H9c2 cells were transfected with miR-30c mimics/inhibitors (or their control) and then subjected to palmitate (1 mmol/L) stimulation. Representative Western blots and quantitative analysis of PGC-1β in the cardiac tissues ( e ) and H9c2 cells ( f ). β-actin was used as an internal control. g PGC-1β mRNA level was determined by RT-PCR in the whole RNA and the RNA of the anti-ago2 co-IP complex from H9c2 cells treated with miR-30c mimics or miR-control. h Schematic diagram of luciferase reporter plasmids pMIR PGC-1β 3′ UTR and mutant PGC-1β 3′ UTR, and the potential (or mutant) seed sequence of miR-30c targeting PGC-1β 3′ UTR are shown in red. i Regulation of PGC-1β via miR-30c targeting its 3′ UTR was determined with luciferase reporter assays in HEK293T cells. For d , f , g and i , n = three independent experiments. Data are expressed as mean ± SEM, *p

    Techniques Used: Pyrolysis Gas Chromatography, Expressing, Sequencing, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Injection, Transfection, Western Blot, Co-Immunoprecipitation Assay, Luciferase, Mutagenesis

    4) Product Images from "Effects of β-sitosterol derived from Artemisia capillaris on the activated human hepatic stellate cells and dimethylnitrosamine-induced mouse liver fibrosis"

    Article Title: Effects of β-sitosterol derived from Artemisia capillaris on the activated human hepatic stellate cells and dimethylnitrosamine-induced mouse liver fibrosis

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/1472-6882-14-363

    Effects of TGF-β treatment on the activation of HSCs. Relatively expressed MMP1 (A) , MMP2 (B) , COL1A1 (C) , ACTA2 (D) , and GFAP (E) mRNA levels were measured by real-time quantitative PCR. Experiments were carried out at least twice performed in triplicate. Statistical significance determined by one-way ANOVA; values are means ± SEM; *, p
    Figure Legend Snippet: Effects of TGF-β treatment on the activation of HSCs. Relatively expressed MMP1 (A) , MMP2 (B) , COL1A1 (C) , ACTA2 (D) , and GFAP (E) mRNA levels were measured by real-time quantitative PCR. Experiments were carried out at least twice performed in triplicate. Statistical significance determined by one-way ANOVA; values are means ± SEM; *, p

    Techniques Used: Activation Assay, Real-time Polymerase Chain Reaction

    Effects of β -sitosterol on collagen-1 and α -SMA mRNA expression in DMN-induced mouse liver fibrosis. Relatively expressed Col1a1 (A) and Acta2 (B) levels were measured by real-time quantitative PCR. Experiments were carried out in triplicate. Statistical significance determined by one-way ANOVA; values are means ± SEM; ***, p
    Figure Legend Snippet: Effects of β -sitosterol on collagen-1 and α -SMA mRNA expression in DMN-induced mouse liver fibrosis. Relatively expressed Col1a1 (A) and Acta2 (B) levels were measured by real-time quantitative PCR. Experiments were carried out in triplicate. Statistical significance determined by one-way ANOVA; values are means ± SEM; ***, p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Effects of β -sitosterol on the collagen-1 and α-SMA mRNA expressions in activated HSCs. Relatively expressed COL1A1 (A) and ACTA2 (B) levels were measured by real-time quantitative PCR. Experiments were carried out at least twice performed in triplicate. Statistical significance determined by one-way ANOVA; values are means ± SEM; ***, p
    Figure Legend Snippet: Effects of β -sitosterol on the collagen-1 and α-SMA mRNA expressions in activated HSCs. Relatively expressed COL1A1 (A) and ACTA2 (B) levels were measured by real-time quantitative PCR. Experiments were carried out at least twice performed in triplicate. Statistical significance determined by one-way ANOVA; values are means ± SEM; ***, p

    Techniques Used: Real-time Polymerase Chain Reaction

    5) Product Images from "PDGF-AA Promotes Osteogenic Differentiation and Migration of Mesenchymal Stem Cell by Down-Regulating PDGFRα and Derepressing BMP-Smad1/5/8 Signaling"

    Article Title: PDGF-AA Promotes Osteogenic Differentiation and Migration of Mesenchymal Stem Cell by Down-Regulating PDGFRα and Derepressing BMP-Smad1/5/8 Signaling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0113785

    PDGF-AA promotes MSC migration via BMP-Smad1/5/8 signaling. A. Transwell experiments and quantitation data. Migration of MSCs was evaluated using a transwell chamber equipped with an 8-µm-pore filter membrane. Cells were plated at 5×10 4 cells/well in serum-free media onto the upper compartment of the chamber and cultured in the absence or presence of PDGF-AA for 24 h. The filter membrane was removed, fixed with methanol, and stained with 0.1% crystal violet. Lower panel: the number of cells that had migrated to the lower surface of the filter membrane was counted in 5 random fields under a light scope (×200). B. PDGF-AA promotes expression of BMP-Smad1/5/8-controlled genes that regulate cell migration. Primary mouse MSC cells were cultured with or without PDGF-AA for 7 days. To test the effect of BMPRI activation, LDN-193189 was included in the culture medium. Total RNA was isolated from the cells and realtime-PCR was carried out to determine the mRNA levels of genes that are involved in regulating cell migration. Data are means±s.e.m. (n = 3). * p
    Figure Legend Snippet: PDGF-AA promotes MSC migration via BMP-Smad1/5/8 signaling. A. Transwell experiments and quantitation data. Migration of MSCs was evaluated using a transwell chamber equipped with an 8-µm-pore filter membrane. Cells were plated at 5×10 4 cells/well in serum-free media onto the upper compartment of the chamber and cultured in the absence or presence of PDGF-AA for 24 h. The filter membrane was removed, fixed with methanol, and stained with 0.1% crystal violet. Lower panel: the number of cells that had migrated to the lower surface of the filter membrane was counted in 5 random fields under a light scope (×200). B. PDGF-AA promotes expression of BMP-Smad1/5/8-controlled genes that regulate cell migration. Primary mouse MSC cells were cultured with or without PDGF-AA for 7 days. To test the effect of BMPRI activation, LDN-193189 was included in the culture medium. Total RNA was isolated from the cells and realtime-PCR was carried out to determine the mRNA levels of genes that are involved in regulating cell migration. Data are means±s.e.m. (n = 3). * p

    Techniques Used: Migration, Quantitation Assay, Cell Culture, Staining, Expressing, Activation Assay, Isolation, Polymerase Chain Reaction

    PDGF-AA promotes MSC osteogenic differentiation. A. PDGF-AA promotes ALP expression in MSC, which requires BMP-Smad1/5/8 signaling. Primary mouse MSC cells were cultured with or without PDGF-AA for 7 or 14 days. To test the effect of BMPRI activation, LDN-193189 was included in the culture medium. ALP was stained 7 or 14 days after culture. B. Quantitation data for ALP activities that were normalized to the total protein levels of each culture. C. PDGF-AA promotes bone mineralization in MSC, which requires BMP-Smad1/5/8 signaling. Primary mouse MSC cells were cultured in differentiation medium with or without PDGF-AA (changed every 3 days) for 28 days. To test the effect of BMPRI activation, LDN-193189 was included in the culture medium. Mineralization was stained with Von Kossa and Alizarin red method. D. PDGF-AA up-regulated the mRNA levels of Runx2, Atf4, and Osx via the BMP-Smad1/5/8 signaling, without affecting the mRNA levels of Sox9, C/EBPα, or PPARγ. Primary MSC cells were cultured with or without PDGF-AA for 7 days. To test the effect of BMPRI activation, LDN-193189 was included in the culture medium. Total RNA was isolated from the cells and realtime PCR was carried out to determine the mRNA levels of the six transcription factors. Data are means ±s.e.m. (n = 3 for all panels). * p
    Figure Legend Snippet: PDGF-AA promotes MSC osteogenic differentiation. A. PDGF-AA promotes ALP expression in MSC, which requires BMP-Smad1/5/8 signaling. Primary mouse MSC cells were cultured with or without PDGF-AA for 7 or 14 days. To test the effect of BMPRI activation, LDN-193189 was included in the culture medium. ALP was stained 7 or 14 days after culture. B. Quantitation data for ALP activities that were normalized to the total protein levels of each culture. C. PDGF-AA promotes bone mineralization in MSC, which requires BMP-Smad1/5/8 signaling. Primary mouse MSC cells were cultured in differentiation medium with or without PDGF-AA (changed every 3 days) for 28 days. To test the effect of BMPRI activation, LDN-193189 was included in the culture medium. Mineralization was stained with Von Kossa and Alizarin red method. D. PDGF-AA up-regulated the mRNA levels of Runx2, Atf4, and Osx via the BMP-Smad1/5/8 signaling, without affecting the mRNA levels of Sox9, C/EBPα, or PPARγ. Primary MSC cells were cultured with or without PDGF-AA for 7 days. To test the effect of BMPRI activation, LDN-193189 was included in the culture medium. Total RNA was isolated from the cells and realtime PCR was carried out to determine the mRNA levels of the six transcription factors. Data are means ±s.e.m. (n = 3 for all panels). * p

    Techniques Used: ALP Assay, Expressing, Cell Culture, Activation Assay, Staining, Quantitation Assay, Isolation, Polymerase Chain Reaction

    6) Product Images from "Impact of target mRNA structure on siRNA silencing efficiency: a large-scale study"

    Article Title: Impact of target mRNA structure on siRNA silencing efficiency: a large-scale study

    Journal:

    doi: 10.1002/bit.21798

    mRNA predicted structures for siRNAs targeting EGFP
    Figure Legend Snippet: mRNA predicted structures for siRNAs targeting EGFP

    Techniques Used:

    siRNA-mRNA interaction energies were calculated by the RNAup algorithm from the Vienna RNA Package
    Figure Legend Snippet: siRNA-mRNA interaction energies were calculated by the RNAup algorithm from the Vienna RNA Package

    Techniques Used:

    7) Product Images from "Longitudinal transcriptomic dysregulation in the peripheral blood of transgenic Huntington's disease monkeys"

    Article Title: Longitudinal transcriptomic dysregulation in the peripheral blood of transgenic Huntington's disease monkeys

    Journal: BMC Neuroscience

    doi: 10.1186/1471-2202-14-88

    Extended examination of HD-associated mRNAs by qPCR from blood of HD monkeys. Five of the selected HD-associated mRNA transcripts with strong divergent trends ( FZD8 , NDUFA5 , ALOX12 , LMNA , LXN; panels A - E ) were further subjected to evaluation by qPCR at extended developmental timespans of 29, 32, and 39 months of age from all control and HD monkeys. Additionally, the 5, 11, 17, and 23 month results timepoints for those same 5 candidates, initially examined by microarray, were also validated by qPCR. White blood cells were collected at each timepoint, followed by RNA extraction and real-time PCR (qPCR) analysis with gene specific Taqman assays (Applied Biosystems). The samples for qPCR were repeated in duplicates for each monkey. The values for all 4 controls were analyzed together and the values for all 4 HD monkeys were analyzed together; one blood sample was analyzed per monkey per timepoint. All qPCR results were normalized with the geometric mean of two endogenous controls, beta-actin ( ACTB ) and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ). A table of stats, including P values and FDRs for disease-association and age-effect by Two-Way ANOVA with repeated measures are outlined in panel F .
    Figure Legend Snippet: Extended examination of HD-associated mRNAs by qPCR from blood of HD monkeys. Five of the selected HD-associated mRNA transcripts with strong divergent trends ( FZD8 , NDUFA5 , ALOX12 , LMNA , LXN; panels A - E ) were further subjected to evaluation by qPCR at extended developmental timespans of 29, 32, and 39 months of age from all control and HD monkeys. Additionally, the 5, 11, 17, and 23 month results timepoints for those same 5 candidates, initially examined by microarray, were also validated by qPCR. White blood cells were collected at each timepoint, followed by RNA extraction and real-time PCR (qPCR) analysis with gene specific Taqman assays (Applied Biosystems). The samples for qPCR were repeated in duplicates for each monkey. The values for all 4 controls were analyzed together and the values for all 4 HD monkeys were analyzed together; one blood sample was analyzed per monkey per timepoint. All qPCR results were normalized with the geometric mean of two endogenous controls, beta-actin ( ACTB ) and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ). A table of stats, including P values and FDRs for disease-association and age-effect by Two-Way ANOVA with repeated measures are outlined in panel F .

    Techniques Used: Real-time Polymerase Chain Reaction, Microarray, RNA Extraction

    8) Product Images from "Evaluation of Exogenous siRNA Addition as a Metabolic Engineering Tool for Modifying Biopharmaceuticals"

    Article Title: Evaluation of Exogenous siRNA Addition as a Metabolic Engineering Tool for Modifying Biopharmaceuticals

    Journal: Biotechnology progress

    doi: 10.1002/btpr.1667

    Fut8 and gmds silencing is dose dependent. A) Fut8 mRNA time course profile shows a decrease in fut8 expression with increasing siRNA concentration. mRNA concentrations were determined via quantitative PCR and normalized based on respective daily cells
    Figure Legend Snippet: Fut8 and gmds silencing is dose dependent. A) Fut8 mRNA time course profile shows a decrease in fut8 expression with increasing siRNA concentration. mRNA concentrations were determined via quantitative PCR and normalized based on respective daily cells

    Techniques Used: Expressing, Concentration Assay, Real-time Polymerase Chain Reaction

    siRNA dosed in a bioreactor results in drastic fut8 and gmds mRNA knockdown with minimal effects on growth. A) Integral viable cell concentration profiles for control and transfected bioreactors show minimal effects of exogenously added siRNA on growth.
    Figure Legend Snippet: siRNA dosed in a bioreactor results in drastic fut8 and gmds mRNA knockdown with minimal effects on growth. A) Integral viable cell concentration profiles for control and transfected bioreactors show minimal effects of exogenously added siRNA on growth.

    Techniques Used: Concentration Assay, Transfection

    9) Product Images from "Intestinal cell kinase, a MAP kinase-related kinase, regulates proliferation and G1 cell cycle progression of intestinal epithelial cells"

    Article Title: Intestinal cell kinase, a MAP kinase-related kinase, regulates proliferation and G1 cell cycle progression of intestinal epithelial cells

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    doi: 10.1152/ajpgi.00066.2009

    ICK deficiency in Caco-2 cells induces a significant increase in the mRNA level of sucrase isomaltase (SI), a marker for enterocytic differentiation. A : mRNA levels of ICK and SI were determined in Caco-2 cultures at different stages of confluence by quantitative RT-PCR; means ± SD, n = 3, * P
    Figure Legend Snippet: ICK deficiency in Caco-2 cells induces a significant increase in the mRNA level of sucrase isomaltase (SI), a marker for enterocytic differentiation. A : mRNA levels of ICK and SI were determined in Caco-2 cultures at different stages of confluence by quantitative RT-PCR; means ± SD, n = 3, * P

    Techniques Used: Marker, Quantitative RT-PCR

    10) Product Images from "Profiling the expression domains of a rice-specific microRNA under stress"

    Article Title: Profiling the expression domains of a rice-specific microRNA under stress

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2015.00333

    Cleavage site mapping of Osa-miR820 target DNA cytosine-5-methyltransferase (OsDRM2). The 5′ end of the cleaved product determined by 5′ RACE followed by cloning and sequencing. The cleavage site is indicated by an arrow in the miRNA:mRNA base-pairing diagram. The numbers indicate the clones in which the cleavage site was mapped, among the total clones analyzed.
    Figure Legend Snippet: Cleavage site mapping of Osa-miR820 target DNA cytosine-5-methyltransferase (OsDRM2). The 5′ end of the cleaved product determined by 5′ RACE followed by cloning and sequencing. The cleavage site is indicated by an arrow in the miRNA:mRNA base-pairing diagram. The numbers indicate the clones in which the cleavage site was mapped, among the total clones analyzed.

    Techniques Used: Clone Assay, Sequencing

    11) Product Images from "Nrf2/Keap1 system regulates vascular smooth muscle cell apoptosis for vascular homeostasis: role in neointimal formation after vascular injury"

    Article Title: Nrf2/Keap1 system regulates vascular smooth muscle cell apoptosis for vascular homeostasis: role in neointimal formation after vascular injury

    Journal: Scientific Reports

    doi: 10.1038/srep26291

    Endogenous Keap1 knockdown by siRNA and expression of Nrf2 and its target genes, including Nqo1 and Hmox1 in VSMCs. ( a,b ) RASMCs were transiently transfected with Keap1 or control siRNA for 48 h. ( a ) Nuclear and cytoplasmic protein levels were analyzed by Western blotting. Cytoplasmic and nuclear proteins were semi-quantified by normalizing with actin and lamin B protein, respectively. ( b ) mRNA levels were analyzed by real-time PCR. Data are expressed as mean ± SEM of three independent experiments. * P
    Figure Legend Snippet: Endogenous Keap1 knockdown by siRNA and expression of Nrf2 and its target genes, including Nqo1 and Hmox1 in VSMCs. ( a,b ) RASMCs were transiently transfected with Keap1 or control siRNA for 48 h. ( a ) Nuclear and cytoplasmic protein levels were analyzed by Western blotting. Cytoplasmic and nuclear proteins were semi-quantified by normalizing with actin and lamin B protein, respectively. ( b ) mRNA levels were analyzed by real-time PCR. Data are expressed as mean ± SEM of three independent experiments. * P

    Techniques Used: Expressing, Transfection, Western Blot, Real-time Polymerase Chain Reaction

    VSMC apoptosis in the middle stages of neointimal expansion after the vascular injury is associated with a high expression of Nrf2, decrease in Keap1, and caspase-3 activation. Femoral arteries uninjured or injured (14 days later) co-stained with TUNEL (green), DAPI (blue), and anti-αSMA (red; ( a )) or anti-Nrf2 (red; ( b )) antibodies for immunofluorescence analysis, or stained with anti-cleaved caspase-3 antibody ( c ) for immunohistochemical analysis. Fluorescence images were taken by confocal microscopy under fixed exposure conditions. Arrowheads indicate cleaved caspase-3; white arrows indicate internal elastic lamina. Keap1 mRNA ( d ) and protein ( e ) levels of uninjured or injured (7 or 14 days later) femoral artery were analyzed using real-time PCR and Western blotting, respectively. Data are expressed as mean ± SEM of four vessels. * P
    Figure Legend Snippet: VSMC apoptosis in the middle stages of neointimal expansion after the vascular injury is associated with a high expression of Nrf2, decrease in Keap1, and caspase-3 activation. Femoral arteries uninjured or injured (14 days later) co-stained with TUNEL (green), DAPI (blue), and anti-αSMA (red; ( a )) or anti-Nrf2 (red; ( b )) antibodies for immunofluorescence analysis, or stained with anti-cleaved caspase-3 antibody ( c ) for immunohistochemical analysis. Fluorescence images were taken by confocal microscopy under fixed exposure conditions. Arrowheads indicate cleaved caspase-3; white arrows indicate internal elastic lamina. Keap1 mRNA ( d ) and protein ( e ) levels of uninjured or injured (7 or 14 days later) femoral artery were analyzed using real-time PCR and Western blotting, respectively. Data are expressed as mean ± SEM of four vessels. * P

    Techniques Used: Expressing, Activation Assay, Staining, TUNEL Assay, Immunofluorescence, Immunohistochemistry, Fluorescence, Confocal Microscopy, Real-time Polymerase Chain Reaction, Western Blot

    12) Product Images from "Expression and Regulation of Toll-Like Receptor 2 in Rheumatoid Arthritis Synovium"

    Article Title: Expression and Regulation of Toll-Like Receptor 2 in Rheumatoid Arthritis Synovium

    Journal: The American Journal of Pathology

    doi:

    Effects of stimulation on the expression of TLR2 and TLR4 mRNA in RA ( n = 6) and OA ( n = 7) SFs. The ordinate shows the relative increase of specific mRNA compared to nonstimulated cells. Each symbol indicates one cell culture from one RA patient examined. The bars indicate the medians. The increase of TLR2 mRNA in response to all four stimuli reached statistical significance (*, Wilcoxon paired-sample test; tied Z values from −2.37 to −2.02, P values from 0.02 to 0.04). Levels of TLR4 mRNA expression of stimulated cells did not differ significantly from nonstimulated cells apart from stimulation of RA SFs with TNF-α, and from stimulation of OA SFs with IL-1β or sBLP (**, Wilcoxon paired-sample test; tied Z values from−2.2 to −2.36, P values from 0.02 to 0.04).
    Figure Legend Snippet: Effects of stimulation on the expression of TLR2 and TLR4 mRNA in RA ( n = 6) and OA ( n = 7) SFs. The ordinate shows the relative increase of specific mRNA compared to nonstimulated cells. Each symbol indicates one cell culture from one RA patient examined. The bars indicate the medians. The increase of TLR2 mRNA in response to all four stimuli reached statistical significance (*, Wilcoxon paired-sample test; tied Z values from −2.37 to −2.02, P values from 0.02 to 0.04). Levels of TLR4 mRNA expression of stimulated cells did not differ significantly from nonstimulated cells apart from stimulation of RA SFs with TNF-α, and from stimulation of OA SFs with IL-1β or sBLP (**, Wilcoxon paired-sample test; tied Z values from−2.2 to −2.36, P values from 0.02 to 0.04).

    Techniques Used: Expressing, Cell Culture

    Control and double stainings. A: One representative section from OA tissues of four patients stained for TLR2 mRNA. B: One representative tissue section from one normal subject of two stained for TLR2 mRNA. Representative tissue section derived from RA-synovium, stained with anti-CD3 antibodies (specific for T cells) after in situ hybridization with a probe specific for TLR2 mRNA. TLR2 mRNA presents as purple/brown color, T cells are red. C: Expression of TLR2 mRNA and CD3 is detectable in distinct cells. Representative tissue sections derived from RA-synovium at sites of synovial invasion into cartilage. The sections are stained with anti-CD68 antibodies (specific for macrophages) after in situ hybridization with probes specific for TLR2 mRNA. TLR2 mRNA presents as purple color, macrophages are red. D–F: Most of the cells expressing TLR2 mRNA are not expressing CD68. Representative tissue section derived from RA synovium at sites of synovial invasion into cartilage. The sections are stained with antibodies against vimentin ( G–H ) or prolyl-4-hydroxylase ( I ) after in situ hybridization with probes specific for TLR2 mRNA. TLR2 presents as purple/black color, antibodies are visible as orange. A great portion of cells expressing TLR2 is positive for the fibroblast markers. Original magnifications: ×100 ( A , B ); ×200 ( C ); ×630 ( D , E , G , I ); ×400 ( F ).
    Figure Legend Snippet: Control and double stainings. A: One representative section from OA tissues of four patients stained for TLR2 mRNA. B: One representative tissue section from one normal subject of two stained for TLR2 mRNA. Representative tissue section derived from RA-synovium, stained with anti-CD3 antibodies (specific for T cells) after in situ hybridization with a probe specific for TLR2 mRNA. TLR2 mRNA presents as purple/brown color, T cells are red. C: Expression of TLR2 mRNA and CD3 is detectable in distinct cells. Representative tissue sections derived from RA-synovium at sites of synovial invasion into cartilage. The sections are stained with anti-CD68 antibodies (specific for macrophages) after in situ hybridization with probes specific for TLR2 mRNA. TLR2 mRNA presents as purple color, macrophages are red. D–F: Most of the cells expressing TLR2 mRNA are not expressing CD68. Representative tissue section derived from RA synovium at sites of synovial invasion into cartilage. The sections are stained with antibodies against vimentin ( G–H ) or prolyl-4-hydroxylase ( I ) after in situ hybridization with probes specific for TLR2 mRNA. TLR2 presents as purple/black color, antibodies are visible as orange. A great portion of cells expressing TLR2 is positive for the fibroblast markers. Original magnifications: ×100 ( A , B ); ×200 ( C ); ×630 ( D , E , G , I ); ×400 ( F ).

    Techniques Used: Staining, Derivative Assay, In Situ Hybridization, Expressing

    TLR2 in situ hybridizations of RA synovial tissues. One representative section of RA synovial tissue of seven patients, hybridized in situ with specific RNA probes for TLR2 mRNA ( A , C , E ). Cells positive for TLR2 mRNA are dark purple. As negative controls, corresponding tissue sections were hybridized with the sense probes. All tissues hybridized with the sense control probes show no specific signals ( B , D , F ). Cells expressing detectable levels of TLR2 mRNA are located in areas of adhesion and invasion into bone or cartilage ( A ; negative control, B ), around small vessels ( C ; negative control, D ) and in areas of lymphocyte infiltrations ( E ; negative control, F ). Original magnifications: ×200 ( A , B , C , E ); ×100 ( D , F ).
    Figure Legend Snippet: TLR2 in situ hybridizations of RA synovial tissues. One representative section of RA synovial tissue of seven patients, hybridized in situ with specific RNA probes for TLR2 mRNA ( A , C , E ). Cells positive for TLR2 mRNA are dark purple. As negative controls, corresponding tissue sections were hybridized with the sense probes. All tissues hybridized with the sense control probes show no specific signals ( B , D , F ). Cells expressing detectable levels of TLR2 mRNA are located in areas of adhesion and invasion into bone or cartilage ( A ; negative control, B ), around small vessels ( C ; negative control, D ) and in areas of lymphocyte infiltrations ( E ; negative control, F ). Original magnifications: ×200 ( A , B , C , E ); ×100 ( D , F ).

    Techniques Used: In Situ, Expressing, Negative Control

    13) Product Images from "Lung Epithelial Cells and Extracellular Matrix Components Induce Expression of Pneumocystis carinii STE20, a Gene Complementing the Mating and Pseudohyphal Growth Defects of ste20 Mutant Yeast "

    Article Title: Lung Epithelial Cells and Extracellular Matrix Components Induce Expression of Pneumocystis carinii STE20, a Gene Complementing the Mating and Pseudohyphal Growth Defects of ste20 Mutant Yeast

    Journal: Infection and Immunity

    doi: 10.1128/IAI.71.11.6463-6471.2003

    Adherence of P. carinii to A549 alveolar epithelial cells specifically induces expression of PCSTE20 and PCMAPK , which participate in fungal mating pathways . To assess whether lung epithelial cells also promote expression of PCSTE20 , P. carinii was cultured either directly adherent to A549 lung cells ( P. carinii on A549's) or separated from A549 cell monolayers by uncoated Transwell membranes ( P. carinii over A549's) for 2 h. (A) Direct adherence of P. carinii to A549 lung cells enhanced PCSTE20 mRNA expression in the organism. In contrast, those organisms cocultured on uncoated tissue culture membranes separating them from the A549 cell layers did not exhibit enhanced expression of PCSTE20 . RNA derived from A549 cells cultured without P. carinii exhibited no cross-reactivity to the PCSTE20 probe. Relative RNA loading was again verified by ethidium bromide staining of rRNA. (B) In a similar manner, PCMAPK , a kinase gene also implicated in fungal mating, showed enhanced expression following direct interaction with A549 lung cells. (C) In contrast, a distinct P. carinii kinase gene, PCMKP1 , a gene involved in maintaining cell wall integrity, was expressed at a lower level and was not substantially altered by incubation of the organism on lung epithelial cells. (D and E) Furthermore, the P. carinii cyclin-dependent kinase gene PCCDC2 (D) and the cognate cyclin gene PCCDC13 (E) were also not induced following the binding of P. carinii to A549 lung cells.
    Figure Legend Snippet: Adherence of P. carinii to A549 alveolar epithelial cells specifically induces expression of PCSTE20 and PCMAPK , which participate in fungal mating pathways . To assess whether lung epithelial cells also promote expression of PCSTE20 , P. carinii was cultured either directly adherent to A549 lung cells ( P. carinii on A549's) or separated from A549 cell monolayers by uncoated Transwell membranes ( P. carinii over A549's) for 2 h. (A) Direct adherence of P. carinii to A549 lung cells enhanced PCSTE20 mRNA expression in the organism. In contrast, those organisms cocultured on uncoated tissue culture membranes separating them from the A549 cell layers did not exhibit enhanced expression of PCSTE20 . RNA derived from A549 cells cultured without P. carinii exhibited no cross-reactivity to the PCSTE20 probe. Relative RNA loading was again verified by ethidium bromide staining of rRNA. (B) In a similar manner, PCMAPK , a kinase gene also implicated in fungal mating, showed enhanced expression following direct interaction with A549 lung cells. (C) In contrast, a distinct P. carinii kinase gene, PCMKP1 , a gene involved in maintaining cell wall integrity, was expressed at a lower level and was not substantially altered by incubation of the organism on lung epithelial cells. (D and E) Furthermore, the P. carinii cyclin-dependent kinase gene PCCDC2 (D) and the cognate cyclin gene PCCDC13 (E) were also not induced following the binding of P. carinii to A549 lung cells.

    Techniques Used: Expressing, Cell Culture, Derivative Assay, Staining, Incubation, Binding Assay

    14) Product Images from "Cream Cheese-Derived Lactococcus chungangensis CAU 28 Modulates the Gut Microbiota and Alleviates Atopic Dermatitis in BALB/c Mice"

    Article Title: Cream Cheese-Derived Lactococcus chungangensis CAU 28 Modulates the Gut Microbiota and Alleviates Atopic Dermatitis in BALB/c Mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36864-5

    Effect of oral Lactobacillus chungangensis CAU 28 cream cheese administration on mRNA expression. Target mRNA expression levels including those of ( a ) IL-4, ( b ) IL-5, ( c ) IL-10, (d) IL-12, (e) IFN-γ, (f) TNF-α, and (g) IL-1β were determined via real-time polymerase chain reaction. Significance marks (* p
    Figure Legend Snippet: Effect of oral Lactobacillus chungangensis CAU 28 cream cheese administration on mRNA expression. Target mRNA expression levels including those of ( a ) IL-4, ( b ) IL-5, ( c ) IL-10, (d) IL-12, (e) IFN-γ, (f) TNF-α, and (g) IL-1β were determined via real-time polymerase chain reaction. Significance marks (* p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    15) Product Images from "Target-dependent biogenesis of cognate microRNAs in human cells"

    Article Title: Target-dependent biogenesis of cognate microRNAs in human cells

    Journal: Nature Communications

    doi: 10.1038/ncomms12200

    Effect of target mRNA concentration on substrate-dependent miRNA increase in human cells. ( a ) Amount of mature miR-122 formed per unit of target mRNA in HEK293 cells transfected with pmiR-122 and respective reporter plasmids. Values were calculated by normalizing the amount of mature miR-122 against the amount of respective target mRNA level and plotted. ( b ) Effect of increasing concentration of target mRNA on mature miRNA levels. HEK293 cells expressing pre-miR-122 were transfected with increasing amounts of in vitro -transcribed mRNA (RL-con or RL-3 × bulge-miR-122) and mature miR-122 and pre-miR-122 levels were quantified 6 h post transfection. In the left panel, changes in relative level of mature miR-122 has been plotted for experiments done with RL-con or RL-3 × bulge-miR-122. Relative change of mature and pre-miR-122 in the presence of different amounts of RL-3 × bulge-miR-122 was plotted (right panel). Values obtained with 100 ng of transcript to transfect 2 × 10 5 cells were considered as 1. ( c ) IRE-RL-3 × bulge-miR-122 mRNA with Ferritin IRE element in 5′-UTR is schematically depicted. ( d ) Cells transfected with pre-miR-122 and IRE-RL-3 × bulge-miR-122 were split 24 h post transfection and iron chelator DFMO (100 μM) or Fe 2+ source Hemin (50 μM) was added after an additional 6 h. Cells were harvested after 16 h post Hemin or DFMO addition for analysis. Polysomal enrichment of IRE-RL-3 × bulge-miR-122 was estimated by normalizing polysomal mRNA content by total mRNA level. ( e ) Target mRNA and mature miR-122 level were measured in cells treated with either Hemin or DFMO. Paired two-tailed Student's t -tests were used for all comparisons. * P
    Figure Legend Snippet: Effect of target mRNA concentration on substrate-dependent miRNA increase in human cells. ( a ) Amount of mature miR-122 formed per unit of target mRNA in HEK293 cells transfected with pmiR-122 and respective reporter plasmids. Values were calculated by normalizing the amount of mature miR-122 against the amount of respective target mRNA level and plotted. ( b ) Effect of increasing concentration of target mRNA on mature miRNA levels. HEK293 cells expressing pre-miR-122 were transfected with increasing amounts of in vitro -transcribed mRNA (RL-con or RL-3 × bulge-miR-122) and mature miR-122 and pre-miR-122 levels were quantified 6 h post transfection. In the left panel, changes in relative level of mature miR-122 has been plotted for experiments done with RL-con or RL-3 × bulge-miR-122. Relative change of mature and pre-miR-122 in the presence of different amounts of RL-3 × bulge-miR-122 was plotted (right panel). Values obtained with 100 ng of transcript to transfect 2 × 10 5 cells were considered as 1. ( c ) IRE-RL-3 × bulge-miR-122 mRNA with Ferritin IRE element in 5′-UTR is schematically depicted. ( d ) Cells transfected with pre-miR-122 and IRE-RL-3 × bulge-miR-122 were split 24 h post transfection and iron chelator DFMO (100 μM) or Fe 2+ source Hemin (50 μM) was added after an additional 6 h. Cells were harvested after 16 h post Hemin or DFMO addition for analysis. Polysomal enrichment of IRE-RL-3 × bulge-miR-122 was estimated by normalizing polysomal mRNA content by total mRNA level. ( e ) Target mRNA and mature miR-122 level were measured in cells treated with either Hemin or DFMO. Paired two-tailed Student's t -tests were used for all comparisons. * P

    Techniques Used: Concentration Assay, Transfection, Expressing, In Vitro, Two Tailed Test

    Target mRNA-dependent increase of mature miR-122 in human cells. ( a ) Scheme of the different target mRNAs used. Positions of the miR-122-binding sites are indicated. ( b ) Effect of RL-3 × bulge-miR-122 on mature miR-122 level in cells transfected with pre-miR-122 and reporter plasmids or in vitro -transcribed mRNAs. In experiment described in the right panel, HEK293 cells co-transfected with plasmid encoding pre-miR-122 (pmiR-122) and RL reporters were used. Total RNA was extracted and northern blotted for mature miR-122, for all the experiments. U6 snRNA was used as loading control. ( c ) Mature miR-122, pre-miR-122 and target mRNA levels were quantified by quantitative reverse transcriptase–PCR in HEK293 cells expressing target mRNAs and co-transfected with plasmid encoding pre-miR-122. ( d ) Effect of modification of 5′ or 3′ miR-122-binding site on target mRNA-driven miRNA elevation. Relative quantification of mature miR-122 level increase in the presence of target RL-3 × bulge-miR-122 mRNA and in the presence of mRNAs with weak 5′- region (W5′) or weak 3′-region (W3′). Relative levels were normalized against respective target mRNA levels. ( e ) In vitro RISC cleavage assay done with protein equivalent amounts of affinity-purified FH-AGO2 isolated from pre-miR-122-transfected FH-AGO2 stable HEK293 cells expressing RL-3 × bulge-miR-122 or RL-con. @, Cleaved product of RISC assay; radio labeled 21-nt band serves as a marker. Paired two-tailed Student's t -tests were used for all comparisons. * P
    Figure Legend Snippet: Target mRNA-dependent increase of mature miR-122 in human cells. ( a ) Scheme of the different target mRNAs used. Positions of the miR-122-binding sites are indicated. ( b ) Effect of RL-3 × bulge-miR-122 on mature miR-122 level in cells transfected with pre-miR-122 and reporter plasmids or in vitro -transcribed mRNAs. In experiment described in the right panel, HEK293 cells co-transfected with plasmid encoding pre-miR-122 (pmiR-122) and RL reporters were used. Total RNA was extracted and northern blotted for mature miR-122, for all the experiments. U6 snRNA was used as loading control. ( c ) Mature miR-122, pre-miR-122 and target mRNA levels were quantified by quantitative reverse transcriptase–PCR in HEK293 cells expressing target mRNAs and co-transfected with plasmid encoding pre-miR-122. ( d ) Effect of modification of 5′ or 3′ miR-122-binding site on target mRNA-driven miRNA elevation. Relative quantification of mature miR-122 level increase in the presence of target RL-3 × bulge-miR-122 mRNA and in the presence of mRNAs with weak 5′- region (W5′) or weak 3′-region (W3′). Relative levels were normalized against respective target mRNA levels. ( e ) In vitro RISC cleavage assay done with protein equivalent amounts of affinity-purified FH-AGO2 isolated from pre-miR-122-transfected FH-AGO2 stable HEK293 cells expressing RL-3 × bulge-miR-122 or RL-con. @, Cleaved product of RISC assay; radio labeled 21-nt band serves as a marker. Paired two-tailed Student's t -tests were used for all comparisons. * P

    Techniques Used: Binding Assay, Transfection, In Vitro, Plasmid Preparation, Northern Blot, Polymerase Chain Reaction, Expressing, Modification, Cleavage Assay, Affinity Purification, Isolation, Labeling, Marker, Two Tailed Test

    Increased activity of AGO2-associated DICER1 contributes to the target mRNA-driven miRNA production. ( a – c ) Increased DICER1 activity in the presence of target mRNA contributes to enhanced miRNA production from pre-miRNA in vitro. Scheme of the in vitro RISC loading assay has been depicted in the upper panel. Immunoprecipitated FH-AGO2 isolated from HEK293 cells stably expressing the protein was subjected to loading assay with 10 nM pre-miR-122 and 25 ng μl −1 of respective target mRNAs. Quantification was done either by densitometry ( a ) or quantitative reverse transcriptase PCR (qRT-PCR) ( b ). The amount of mature miR-122 formed was normalized to the amount of AGO2 immunoprecipitated for quantification. Immunoprecipitation of FH-AGO2 and associated endogenous DICER1 was confirmed by western blotting. Increased DICER1 activity in the presence of target mRNA RL-3 × bulge-let-7a contributes to enhanced miRNA production from pre-let-7a in vitro ( c ). ( d ) Removal of AGO2-associated DICER1 impairs target-driven miRNA biogenesis. Scheme of experiment has been shown. Lysate of FH-AGO2-stable HEK293 cells were treated with SLA and FH-AGO2 were immunoprecipitated for in vitro loading assay. Quantification was done by qRT-PCR. Paired two-tailed Student's t -tests were used for all comparisons. * P
    Figure Legend Snippet: Increased activity of AGO2-associated DICER1 contributes to the target mRNA-driven miRNA production. ( a – c ) Increased DICER1 activity in the presence of target mRNA contributes to enhanced miRNA production from pre-miRNA in vitro. Scheme of the in vitro RISC loading assay has been depicted in the upper panel. Immunoprecipitated FH-AGO2 isolated from HEK293 cells stably expressing the protein was subjected to loading assay with 10 nM pre-miR-122 and 25 ng μl −1 of respective target mRNAs. Quantification was done either by densitometry ( a ) or quantitative reverse transcriptase PCR (qRT-PCR) ( b ). The amount of mature miR-122 formed was normalized to the amount of AGO2 immunoprecipitated for quantification. Immunoprecipitation of FH-AGO2 and associated endogenous DICER1 was confirmed by western blotting. Increased DICER1 activity in the presence of target mRNA RL-3 × bulge-let-7a contributes to enhanced miRNA production from pre-let-7a in vitro ( c ). ( d ) Removal of AGO2-associated DICER1 impairs target-driven miRNA biogenesis. Scheme of experiment has been shown. Lysate of FH-AGO2-stable HEK293 cells were treated with SLA and FH-AGO2 were immunoprecipitated for in vitro loading assay. Quantification was done by qRT-PCR. Paired two-tailed Student's t -tests were used for all comparisons. * P

    Techniques Used: Activity Assay, In Vitro, Immunoprecipitation, Isolation, Stable Transfection, Expressing, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Two Tailed Test

    Target mRNA drives increased biogenesis of mature miRNA from pre-miRNA. ( a ) De novo synthesis of mature miR-122 in the presence of target mRNA is accompanied by a simultaneous drop in pre-miR-122 level. Experimental format is illustrated in the left panel. Tet-ON HEK293 cells were induced for specific time points with doxycycline to synthesize pre-miR-122 from a plasmid with Tet-response element. Cells were harvested after 14 and 24 h, and mature and pre-miR-122 levels quantified. To measure the relative changes at 24 h, values at 14 h are taken as the unit. ( b ) Target mRNA-induced increase of miRNA levels does not occur due to enhanced stability of a preformed miRNP in the presence of target mRNA. Cells were transfected with 1 μM synthetic pre-miR-122. After 48 h, cells were again transfected with RL-con or RL-3 × bulge-miR-122 plasmids. This was followed by RNA isolation after 24, 48 and 72 h, and mature miR-122 levels quantified to plot the decay rate of mature miR-122. Relative changes in levels of target RNAs over time have been plotted. ( c ) FH-AGO2 was immunoprecipitated from FH-AGO2-stable HEK293 cells transfected with pre-miR-122 plasmid and FH-AGO2 beads corresponding to ∼2 × 10 6 cells were incubated with 500 ng in vitro -transcribed RL-con or RL-3 × bulge-miR-122 mRNA in a 20 μl reaction for increasing time. The supernatant was removed and on-bead RISC cleavage assay was subsequently performed to quantify the amount of miR-122 retained with AGO2 post interaction with target mRNA. Paired two-tailed Student's t -tests were used for all comparisons. * P
    Figure Legend Snippet: Target mRNA drives increased biogenesis of mature miRNA from pre-miRNA. ( a ) De novo synthesis of mature miR-122 in the presence of target mRNA is accompanied by a simultaneous drop in pre-miR-122 level. Experimental format is illustrated in the left panel. Tet-ON HEK293 cells were induced for specific time points with doxycycline to synthesize pre-miR-122 from a plasmid with Tet-response element. Cells were harvested after 14 and 24 h, and mature and pre-miR-122 levels quantified. To measure the relative changes at 24 h, values at 14 h are taken as the unit. ( b ) Target mRNA-induced increase of miRNA levels does not occur due to enhanced stability of a preformed miRNP in the presence of target mRNA. Cells were transfected with 1 μM synthetic pre-miR-122. After 48 h, cells were again transfected with RL-con or RL-3 × bulge-miR-122 plasmids. This was followed by RNA isolation after 24, 48 and 72 h, and mature miR-122 levels quantified to plot the decay rate of mature miR-122. Relative changes in levels of target RNAs over time have been plotted. ( c ) FH-AGO2 was immunoprecipitated from FH-AGO2-stable HEK293 cells transfected with pre-miR-122 plasmid and FH-AGO2 beads corresponding to ∼2 × 10 6 cells were incubated with 500 ng in vitro -transcribed RL-con or RL-3 × bulge-miR-122 mRNA in a 20 μl reaction for increasing time. The supernatant was removed and on-bead RISC cleavage assay was subsequently performed to quantify the amount of miR-122 retained with AGO2 post interaction with target mRNA. Paired two-tailed Student's t -tests were used for all comparisons. * P

    Techniques Used: Plasmid Preparation, Transfection, Isolation, Immunoprecipitation, Incubation, In Vitro, Cleavage Assay, Two Tailed Test

    Increased processivity of AGO2-associated DICER1 in the presence of target mRNA. ( a ) In vitro pre-miRNA processing assay with rAGO2 and rDICER1 reconfirmed target mRNA-driven increase in AGO2-associated DICER1 activity. In vitro pre-miRNA processing assay with rDICER1 and rAGO2 (native or heat-denatured) to quantify miR-122 biogenesis in the presence of target mRNA. Heat denaturation of rAGO2 was carried out at 95 °C for 5 min followed by rapid chilling. ( b ) In vitro pre-miRNA processing assay of pre-let-7a with rDICER1 and increasing concentrations of rAGO2 (10, 25 and 50 ng) in the presence of RL-con or RL-3 × bulge-let-7a (25 ng ml −1 ). Mature let-7a levels were measured and plotted. ( c ) Schematic representation of RL-3 × bulge-let-7a_5BoxB mRNA used in the in vitro assays. In vitro pre-miRNA processing assay of pre-let-7a with rDICER1 and 50 ng rAGO2 in the presence of RL-3 × bulge-let-7a or RL-3 × bulge-let-7a_5BoxB mRNA (both at 25 ng μl −1 ). Mature let-7a levels after the reaction were measured and plotted. ( d ) In vitro assay to measure the association of AGO2 and DICER1 along the 3′-UTR of target mRNAs. FH-AGO2 immunoprecipitated from HEK293 cells transiently expressing NHA-DICER1 was subjected to in vitro pre-miRNA processing assay with pre-miR-122 and RL-3 × bulge-miR-122 as described earlier, followed by immunoprecipitation of AGO2 and DICER1 with antibodies specific to endogenous proteins. Quantitative reverse transcriptase PCR (qRT-PCR) was done with indicated primers. ( e ) In vitro assay to measure processivity of DICER1. Immunopurified AGO2 (let-7a miRISC) incubated with 25 ng ml −1 RL-con or RL-3 × bulge-let-7a in the presence of pre-miR-122 (10 nM) at 37 °C for 30 min followed by RNA isolation and quantification of mature miR-122 formed by qRT-PCR. Paired two-tailed Student's t -tests were used for all comparisons. * P
    Figure Legend Snippet: Increased processivity of AGO2-associated DICER1 in the presence of target mRNA. ( a ) In vitro pre-miRNA processing assay with rAGO2 and rDICER1 reconfirmed target mRNA-driven increase in AGO2-associated DICER1 activity. In vitro pre-miRNA processing assay with rDICER1 and rAGO2 (native or heat-denatured) to quantify miR-122 biogenesis in the presence of target mRNA. Heat denaturation of rAGO2 was carried out at 95 °C for 5 min followed by rapid chilling. ( b ) In vitro pre-miRNA processing assay of pre-let-7a with rDICER1 and increasing concentrations of rAGO2 (10, 25 and 50 ng) in the presence of RL-con or RL-3 × bulge-let-7a (25 ng ml −1 ). Mature let-7a levels were measured and plotted. ( c ) Schematic representation of RL-3 × bulge-let-7a_5BoxB mRNA used in the in vitro assays. In vitro pre-miRNA processing assay of pre-let-7a with rDICER1 and 50 ng rAGO2 in the presence of RL-3 × bulge-let-7a or RL-3 × bulge-let-7a_5BoxB mRNA (both at 25 ng μl −1 ). Mature let-7a levels after the reaction were measured and plotted. ( d ) In vitro assay to measure the association of AGO2 and DICER1 along the 3′-UTR of target mRNAs. FH-AGO2 immunoprecipitated from HEK293 cells transiently expressing NHA-DICER1 was subjected to in vitro pre-miRNA processing assay with pre-miR-122 and RL-3 × bulge-miR-122 as described earlier, followed by immunoprecipitation of AGO2 and DICER1 with antibodies specific to endogenous proteins. Quantitative reverse transcriptase PCR (qRT-PCR) was done with indicated primers. ( e ) In vitro assay to measure processivity of DICER1. Immunopurified AGO2 (let-7a miRISC) incubated with 25 ng ml −1 RL-con or RL-3 × bulge-let-7a in the presence of pre-miR-122 (10 nM) at 37 °C for 30 min followed by RNA isolation and quantification of mature miR-122 formed by qRT-PCR. Paired two-tailed Student's t -tests were used for all comparisons. * P

    Techniques Used: In Vitro, Activity Assay, Immunoprecipitation, Expressing, Polymerase Chain Reaction, Quantitative RT-PCR, Incubation, Isolation, Two Tailed Test

    Reversal of amino acid-starvation-induced stress increases miR-122 biogenesis in Huh7 cells. ( a , b ) Outline of the experimental set-up used ( a ). Relative level of CAT-1 mRNA in Huh7 cells either fed or starved for 4 h and re-fed with media containing amino acids for another 2 h. CAT-1 mRNA levels were quantified by quantitative reverse transcriptase-PCR with levels in Fed cells taken as 1 ( b ). ( c ) Effect of starvation and re-feeding on mature miR-122 levels in Huh7 cells. Total RNA was extracted and 8 μg RNA was used for northern blotting of mature miR-122, let-7a and miR-16. U6 snRNA was used as loading control. ( d ) Copy number/number of molecules of mature miR-122 and CAT-1 mRNA per Huh7 cell calculated in fed, starved and re-fed Huh7 cells. Estimations were done by real-time-based methods. ( e ) Increase in mature miR-122 but not that of other non-relevant miRNAs, miR-16, miR-21, miR-24 and miR-125b on relief of starvation. Real-time PCR-based quantification of mature miRNA levels in Huh7 cells starved for amino acids (4 h) and subsequently re-fed (2 h). ( f ) Increase in mature miR-122 is accompanied by a concomitant decrease in pre-miR-122 on re-feeding the starved cells for 2 h. Cellular small RNA population was isolated by mirVANA kit to minimize possible contamination of pri-miR-122 and real-time-based assays were carried out to quantify pre-miR-122. Pre-miR-122 detected by northern blotting with 15 μg total RNA. Synthetic pre-miR-122 was used as a size marker to determine the position of the pre-miR-122 in the northern blotting. ( g ) Increase of mature miR-122 on re-feeding of starved cells is reduced in DICER1 knockdown Huh7 cells. miR-16 and miR-21 did not show any significant change. siDICER1-mediated knockdown of DICER1 is confirmed by western blotting. Paired two-tailed Student's t -tests were used for all comparisons. * P
    Figure Legend Snippet: Reversal of amino acid-starvation-induced stress increases miR-122 biogenesis in Huh7 cells. ( a , b ) Outline of the experimental set-up used ( a ). Relative level of CAT-1 mRNA in Huh7 cells either fed or starved for 4 h and re-fed with media containing amino acids for another 2 h. CAT-1 mRNA levels were quantified by quantitative reverse transcriptase-PCR with levels in Fed cells taken as 1 ( b ). ( c ) Effect of starvation and re-feeding on mature miR-122 levels in Huh7 cells. Total RNA was extracted and 8 μg RNA was used for northern blotting of mature miR-122, let-7a and miR-16. U6 snRNA was used as loading control. ( d ) Copy number/number of molecules of mature miR-122 and CAT-1 mRNA per Huh7 cell calculated in fed, starved and re-fed Huh7 cells. Estimations were done by real-time-based methods. ( e ) Increase in mature miR-122 but not that of other non-relevant miRNAs, miR-16, miR-21, miR-24 and miR-125b on relief of starvation. Real-time PCR-based quantification of mature miRNA levels in Huh7 cells starved for amino acids (4 h) and subsequently re-fed (2 h). ( f ) Increase in mature miR-122 is accompanied by a concomitant decrease in pre-miR-122 on re-feeding the starved cells for 2 h. Cellular small RNA population was isolated by mirVANA kit to minimize possible contamination of pri-miR-122 and real-time-based assays were carried out to quantify pre-miR-122. Pre-miR-122 detected by northern blotting with 15 μg total RNA. Synthetic pre-miR-122 was used as a size marker to determine the position of the pre-miR-122 in the northern blotting. ( g ) Increase of mature miR-122 on re-feeding of starved cells is reduced in DICER1 knockdown Huh7 cells. miR-16 and miR-21 did not show any significant change. siDICER1-mediated knockdown of DICER1 is confirmed by western blotting. Paired two-tailed Student's t -tests were used for all comparisons. * P

    Techniques Used: Polymerase Chain Reaction, Northern Blot, Real-time Polymerase Chain Reaction, Isolation, Marker, Western Blot, Two Tailed Test

    16) Product Images from "Increased Expression of NAPDH Oxidase 4 (NOX4) in Systemic Sclerosis Dermal Fibroblasts: Regulation by Transforming Growth Factor β"

    Article Title: Increased Expression of NAPDH Oxidase 4 (NOX4) in Systemic Sclerosis Dermal Fibroblasts: Regulation by Transforming Growth Factor β

    Journal: Arthritis & rheumatology (Hoboken, N.J.)

    doi: 10.1002/art.39242

    A . NOX4 mRNA expression levels assessed in triplicate by Real Time PCR analysis in four normal dermal fibroblast cell lines under treatment with TGF-β1 and rottlerin. GAPDH was used as endogenous control. Lower panel shows the effect of TGF-β1 and rottlerin on three SSc cell lines. B . Representative Western blot analysis of NOX4 and β-actin (as protein control) using cell extracts from four normal dermal fibroblast cell lines treated with TGF-β1 alone or TGF-β1 plus rottlerin. Bar graph represents NOX4 protein levels following correction for the intensity of the β actin band. C . Bar graphs of PKC-δ and NOX4 mRNA expression by normal dermal fibroblasts following treatment with specific siRNA against PKC-δ. The data shown are the average of the siRNA effect on three normal dermal fibroblast cell lines. D . Bar graph of PKC-δ expression by normal dermal fibroblasts following treatment with TGF-β1 and a specific siRNA against PKC-δ. The data shown are the average of duplicate experiments examining the PKC-δ siRNA effect on two normal dermal fibroblast cell lines.
    Figure Legend Snippet: A . NOX4 mRNA expression levels assessed in triplicate by Real Time PCR analysis in four normal dermal fibroblast cell lines under treatment with TGF-β1 and rottlerin. GAPDH was used as endogenous control. Lower panel shows the effect of TGF-β1 and rottlerin on three SSc cell lines. B . Representative Western blot analysis of NOX4 and β-actin (as protein control) using cell extracts from four normal dermal fibroblast cell lines treated with TGF-β1 alone or TGF-β1 plus rottlerin. Bar graph represents NOX4 protein levels following correction for the intensity of the β actin band. C . Bar graphs of PKC-δ and NOX4 mRNA expression by normal dermal fibroblasts following treatment with specific siRNA against PKC-δ. The data shown are the average of the siRNA effect on three normal dermal fibroblast cell lines. D . Bar graph of PKC-δ expression by normal dermal fibroblasts following treatment with TGF-β1 and a specific siRNA against PKC-δ. The data shown are the average of duplicate experiments examining the PKC-δ siRNA effect on two normal dermal fibroblast cell lines.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot

    17) Product Images from "Rapid and Specific Detection of tdh, trh1, and trh2 mRNA of Vibrio parahaemolyticus by Transcription-Reverse Transcription Concerted Reaction with an Automated System"

    Article Title: Rapid and Specific Detection of tdh, trh1, and trh2 mRNA of Vibrio parahaemolyticus by Transcription-Reverse Transcription Concerted Reaction with an Automated System

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.42.9.4284-4292.2004

    Real-time monitoring of the TRC reactions for the RNA calibrators. (A, C, and E) Fluorescent profile of the TRC reaction for the RNA calibrators (A, tdh RNA; C, trh 1 RNA; and E, trh 2 RNA); the initial copies of the calibrator mRNA (0, 10 3 , 10 4 , 10 5 , 10 6 , and 10 7 ) are indicated. (B, D, and F) Correlation between the initial copies of the mRNA calibrators and their detection times (B, tdh RNA; D, trh 1 RNA; F, trh 2 RNA).
    Figure Legend Snippet: Real-time monitoring of the TRC reactions for the RNA calibrators. (A, C, and E) Fluorescent profile of the TRC reaction for the RNA calibrators (A, tdh RNA; C, trh 1 RNA; and E, trh 2 RNA); the initial copies of the calibrator mRNA (0, 10 3 , 10 4 , 10 5 , 10 6 , and 10 7 ) are indicated. (B, D, and F) Correlation between the initial copies of the mRNA calibrators and their detection times (B, tdh RNA; D, trh 1 RNA; F, trh 2 RNA).

    Techniques Used:

    Real-time monitoring of TRC reactions for RNA preparations of control strains of V. parahaemolyticus by using TRC assays to detect tdh , trh 1, and trh 2 mRNAs. The profiles of the TRC reactions for detection of the tdh mRNA (A), the trh 1 mRNA (B), and the trh 2 mRNA (C) are shown. The total RNA extracted from AQ3815 (red dots; tdh 1 positive, tdh 2 positive, trh 1 negative, and trh 2 negative), AQ3776 (blue dots; tdh 3 positive, tdh 4 positive, trh 1 positive, and trh 2 negative), AQ3860 (green dots; tdh 5 positive, trh 1 positive, and trh 2 negative), AQ4037 (pink dots; tdh negative, trh 1 positive, and trh 2 negative), AT4 (pale-blue dots; tdh negative, trh 1 negative, and trh 2 positive), and DOH272 (gray dots; tdh negative, trh 1 negative, and trh 2 negative) were examined.
    Figure Legend Snippet: Real-time monitoring of TRC reactions for RNA preparations of control strains of V. parahaemolyticus by using TRC assays to detect tdh , trh 1, and trh 2 mRNAs. The profiles of the TRC reactions for detection of the tdh mRNA (A), the trh 1 mRNA (B), and the trh 2 mRNA (C) are shown. The total RNA extracted from AQ3815 (red dots; tdh 1 positive, tdh 2 positive, trh 1 negative, and trh 2 negative), AQ3776 (blue dots; tdh 3 positive, tdh 4 positive, trh 1 positive, and trh 2 negative), AQ3860 (green dots; tdh 5 positive, trh 1 positive, and trh 2 negative), AQ4037 (pink dots; tdh negative, trh 1 positive, and trh 2 negative), AT4 (pale-blue dots; tdh negative, trh 1 negative, and trh 2 positive), and DOH272 (gray dots; tdh negative, trh 1 negative, and trh 2 negative) were examined.

    Techniques Used:

    18) Product Images from "Human Synovia Contains Trefoil Factor Family (TFF) Peptides 1–3 Although Synovial Membrane Only Produces TFF3: Implications in Osteoarthritis and Rheumatoid Arthritis"

    Article Title: Human Synovia Contains Trefoil Factor Family (TFF) Peptides 1–3 Although Synovial Membrane Only Produces TFF3: Implications in Osteoarthritis and Rheumatoid Arthritis

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20236105

    Presence and localization of TFFs in synovial membrane (SM). ( A ) Semi-quantitative PCR analysis of TFFs mRNA in human SM of five healthy (28, 48, 54, 78, and 92 years (Lines 1, 2, 3, 4, and 5)), rheumatoid arthritis (RA) (Lines 6 and 7) and osteoarthritis (OA) (Lines 8 and 9) samples. Line 10 represents the negative control (without cDNA template), and human stomach serves as positive control (Line 11). Beta-actin (β-actin) was used as the loading control. ( B ) Immunohistochemical analysis of TFF1, -2 and -3 in human SM of 22 ( a , b , c ), 48 ( d , e , f ), and 83 ( g , h , i ) year-old healthy donors. TFF3 is present in each examined sample ( c , f , i ). TFF1 ( a , d , g ) and TFF2 ( b , e , h ) reveal negative results irrespective of the donor’s age. Insets show magnifications. Scale bars: 100 μm. Red staining indicates positive antibody reaction.
    Figure Legend Snippet: Presence and localization of TFFs in synovial membrane (SM). ( A ) Semi-quantitative PCR analysis of TFFs mRNA in human SM of five healthy (28, 48, 54, 78, and 92 years (Lines 1, 2, 3, 4, and 5)), rheumatoid arthritis (RA) (Lines 6 and 7) and osteoarthritis (OA) (Lines 8 and 9) samples. Line 10 represents the negative control (without cDNA template), and human stomach serves as positive control (Line 11). Beta-actin (β-actin) was used as the loading control. ( B ) Immunohistochemical analysis of TFF1, -2 and -3 in human SM of 22 ( a , b , c ), 48 ( d , e , f ), and 83 ( g , h , i ) year-old healthy donors. TFF3 is present in each examined sample ( c , f , i ). TFF1 ( a , d , g ) and TFF2 ( b , e , h ) reveal negative results irrespective of the donor’s age. Insets show magnifications. Scale bars: 100 μm. Red staining indicates positive antibody reaction.

    Techniques Used: Real-time Polymerase Chain Reaction, Negative Control, Positive Control, Immunohistochemistry, Staining

    19) Product Images from "Increased Early RNA Replication by Chimeric West Nile Virus W956IC Leads to IPS-1-Mediated Activation of NF-?B and Insufficient Virus-Mediated Counteraction of the Resulting Canonical Type I Interferon Signaling"

    Article Title: Increased Early RNA Replication by Chimeric West Nile Virus W956IC Leads to IPS-1-Mediated Activation of NF-?B and Insufficient Virus-Mediated Counteraction of the Resulting Canonical Type I Interferon Signaling

    Journal: Journal of Virology

    doi: 10.1128/JVI.02842-12

    Induction and activation of PKR in MEFs after infection with WNV Eg101 or W956IC. (A) C3H/He MEFs were infected with WNV Eg101 or W956IC at an MOI of 1. Changes in Eif2ak2 (PKR) mRNA levels were assessed by real-time qRT-PCR at the indicated times after
    Figure Legend Snippet: Induction and activation of PKR in MEFs after infection with WNV Eg101 or W956IC. (A) C3H/He MEFs were infected with WNV Eg101 or W956IC at an MOI of 1. Changes in Eif2ak2 (PKR) mRNA levels were assessed by real-time qRT-PCR at the indicated times after

    Techniques Used: Activation Assay, Infection, Quantitative RT-PCR

    20) Product Images from "Usefulness of the MRP2 promoter to overcome the chemoresistance of gastrointestinal and liver tumors by enhancing the expression of the drug transporter OATP1B1"

    Article Title: Usefulness of the MRP2 promoter to overcome the chemoresistance of gastrointestinal and liver tumors by enhancing the expression of the drug transporter OATP1B1

    Journal: Oncotarget

    doi: 10.18632/oncotarget.16119

    (A) Time-course of OATP1B1 expression in Alexander cells after transfection with a plasmid containing Z3-MRP2pr-OATP1B1-V5 or an empty vector (Mock). 48 h after transfection, OATP1B1 expressing cells were treated with 100 nM dexamethasone (Dex) or the vehicle. (B) Representative Western blot of OATP1B1-V5 in lysates from Alexander cells transfected with Z3-MRP2pr-OATP1B1-V5, treated with 100 nM Dex or the vehicle for 24 h. As a positive control of V5 expression a plasmid containing the V5-tagged ORF of chloramphenicol acetyl transferase (CAT) under the control of CMV promoter was used. An empty vector (Mock) was used as a negative control. Western blots were carried out with an antibody against the V5 epitope. GAPDH was used as loading protein normalizer in each lane. (C) Representative fluorescence confocal microscopy pictures of Alexander and LS174T/R cells expressing V5-tagged OATP1B1 or CAT, which were detected by using a monoclonal antibody against the V5 epitope (green). Nuclei were counterstained with DAPI (blue). (D) Time-course of OATP1B1 expression in human LS174T cells from colorectal adenocarcinoma and in the chemoresistant subline LS174T/R after transfection with a plasmid containing Z3-MRP2pr-OATP1B1-V5 or an empty vector (Mock). The amount of mRNA was determined by RT-QPCR and expressed as a percentage of that found in human liver. Values are expressed as mean±SD from 3 independent experiments performed in triplicate. *, p
    Figure Legend Snippet: (A) Time-course of OATP1B1 expression in Alexander cells after transfection with a plasmid containing Z3-MRP2pr-OATP1B1-V5 or an empty vector (Mock). 48 h after transfection, OATP1B1 expressing cells were treated with 100 nM dexamethasone (Dex) or the vehicle. (B) Representative Western blot of OATP1B1-V5 in lysates from Alexander cells transfected with Z3-MRP2pr-OATP1B1-V5, treated with 100 nM Dex or the vehicle for 24 h. As a positive control of V5 expression a plasmid containing the V5-tagged ORF of chloramphenicol acetyl transferase (CAT) under the control of CMV promoter was used. An empty vector (Mock) was used as a negative control. Western blots were carried out with an antibody against the V5 epitope. GAPDH was used as loading protein normalizer in each lane. (C) Representative fluorescence confocal microscopy pictures of Alexander and LS174T/R cells expressing V5-tagged OATP1B1 or CAT, which were detected by using a monoclonal antibody against the V5 epitope (green). Nuclei were counterstained with DAPI (blue). (D) Time-course of OATP1B1 expression in human LS174T cells from colorectal adenocarcinoma and in the chemoresistant subline LS174T/R after transfection with a plasmid containing Z3-MRP2pr-OATP1B1-V5 or an empty vector (Mock). The amount of mRNA was determined by RT-QPCR and expressed as a percentage of that found in human liver. Values are expressed as mean±SD from 3 independent experiments performed in triplicate. *, p

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Western Blot, Positive Control, Chloramphenicol Acetyltransferase Assay, Negative Control, Fluorescence, Confocal Microscopy, Quantitative RT-PCR

    21) Product Images from "Tissue- and Time-Dependent Upregulation of Cytokine mRNA in a Murine Model for the Multiple Organ Dysfunction Syndrome"

    Article Title: Tissue- and Time-Dependent Upregulation of Cytokine mRNA in a Murine Model for the Multiple Organ Dysfunction Syndrome

    Journal: Annals of Surgery

    doi: 10.1097/01.sla.0000130725.52373.e7

    FIGURE 4. Cytokine mRNA expression in the liver. Individual values of mRNA for TNF-α (•) and IL-1β (○), quantitated by a real-time RT-PCR, in livers from untreated control mice and zymosan-treated animals.
    Figure Legend Snippet: FIGURE 4. Cytokine mRNA expression in the liver. Individual values of mRNA for TNF-α (•) and IL-1β (○), quantitated by a real-time RT-PCR, in livers from untreated control mice and zymosan-treated animals.

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

    22) Product Images from "Regulation of PLK1 through competition between hnRNPK, miR-149-3p and miR-193b-5p"

    Article Title: Regulation of PLK1 through competition between hnRNPK, miR-149-3p and miR-193b-5p

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2017.106

    miR-149-3p and miR-193b-5p suppress PLK1 expression through direct interactions with the 3′UTR of PLK1 mRNA. ( a , b ) HeLa cells were transfected with control miRNA or pre-miR-149-3p. After 48 h post-transfection, PLK1 protein and mRNA levels were determined by western blot and RT–qPCR, respectively. hnRNPK and the loading control, GAPHD, were examined by western blot. ( c ) We constructed two GFP vectors to investigate whether a direct interaction with the 3′UTR PLK1 mRNA is required for downregulation of PLK1 by miR-149-3p. Based on vectors expressing chimeric RNAs spanning the GFP ( A ) and fragment #3 of the 3′UTR PLK1 mRNA ( B ), we constructed vectors ( C ) containing mutated sequences of the miR-149-3p-binding sites in the 3′UTR PLK1 mRNA (fragment #3). After overexpression of miR-149-3p, cells were transfected with the indicated GFP vectors: blank, wild-type (WT), or mutated type (MT). ( d – f ) Similar to our investigation of miR-149-3p, we tested the effect of miR-193b-5p on PLK1 expression as described above. All experiments were performed more than three times and data represent mean±S.D.
    Figure Legend Snippet: miR-149-3p and miR-193b-5p suppress PLK1 expression through direct interactions with the 3′UTR of PLK1 mRNA. ( a , b ) HeLa cells were transfected with control miRNA or pre-miR-149-3p. After 48 h post-transfection, PLK1 protein and mRNA levels were determined by western blot and RT–qPCR, respectively. hnRNPK and the loading control, GAPHD, were examined by western blot. ( c ) We constructed two GFP vectors to investigate whether a direct interaction with the 3′UTR PLK1 mRNA is required for downregulation of PLK1 by miR-149-3p. Based on vectors expressing chimeric RNAs spanning the GFP ( A ) and fragment #3 of the 3′UTR PLK1 mRNA ( B ), we constructed vectors ( C ) containing mutated sequences of the miR-149-3p-binding sites in the 3′UTR PLK1 mRNA (fragment #3). After overexpression of miR-149-3p, cells were transfected with the indicated GFP vectors: blank, wild-type (WT), or mutated type (MT). ( d – f ) Similar to our investigation of miR-149-3p, we tested the effect of miR-193b-5p on PLK1 expression as described above. All experiments were performed more than three times and data represent mean±S.D.

    Techniques Used: Expressing, Transfection, Western Blot, Quantitative RT-PCR, Construct, Binding Assay, Over Expression

    Competitive regulation of PLK1 by hnRNPK and miR-149-3p/193b-5p. ( a ) Cytoplasmic lysates were obtained from miR-149-3p- or miR-193b-5p-overexpressing cells and immunoprecipitated (IP) with Ago2-specific antibody. Enrichment of PLK1 mRNA in Ago2 IP was assessed by RT–qPCR. ( b,c ) To investigate the effect of hnRNPK on the interaction between PLK1 mRNA and an miRNA-loaded RISC complex, enrichment of PLK1 mRNA in Ago2 IP was examined using cytoplasmic lysates obtained from hnRNPK-silenced ( b ) or -overexpressing ( c ) HeLa cells. The level of PLK1 mRNA in Ago2 IP was assessed by RT–qPCR. ( d ) To test whether hnRNPK affects expression of miR-149-3p and miR-193b-5p, cells were transfected with control or hnRNPK siRNA. After 48 h post-transfection, miRNA expression levels were determined by RT–qPCR. ( e ) To examine the effect of miRNA mimics on GFP expression, GFP reporters were generated in which GFP was linked to fragment #3 harboring or lacking the binding sequence for hnRNPK and the miRNAs (GFP vector B and E in the schematic). GFP expression was assessed by western blot. ( f ) The interaction between hnRNPK and GFP chimeric mRNAs was examined. Cells were transfected with the previously described GFP vectors and cytoplasmic lysates were prepared. GFP mRNA enrichment was measured by RNP IP using hnRNPK antibody followed by RT–qPCR. ( g ) To test whether hnRNPK affects GFP expression in the absence of an miRNA-binding sequence, GFP reporters described in e were used. The expression levels of hnRNPK and GFP were assessed by western blot. ( h ) To determine whether hnRNPK restored PLK1 expression, cells were transfected with miRNA mimics (pre-miR-149-3p and miR-193b-5p) and an hnRNPK vector (FLAG-hnRNPK). Expression of hnRNPK and PLK1 was assessed by western blot. All experiments were performed more than three times and data represent mean±S.D.
    Figure Legend Snippet: Competitive regulation of PLK1 by hnRNPK and miR-149-3p/193b-5p. ( a ) Cytoplasmic lysates were obtained from miR-149-3p- or miR-193b-5p-overexpressing cells and immunoprecipitated (IP) with Ago2-specific antibody. Enrichment of PLK1 mRNA in Ago2 IP was assessed by RT–qPCR. ( b,c ) To investigate the effect of hnRNPK on the interaction between PLK1 mRNA and an miRNA-loaded RISC complex, enrichment of PLK1 mRNA in Ago2 IP was examined using cytoplasmic lysates obtained from hnRNPK-silenced ( b ) or -overexpressing ( c ) HeLa cells. The level of PLK1 mRNA in Ago2 IP was assessed by RT–qPCR. ( d ) To test whether hnRNPK affects expression of miR-149-3p and miR-193b-5p, cells were transfected with control or hnRNPK siRNA. After 48 h post-transfection, miRNA expression levels were determined by RT–qPCR. ( e ) To examine the effect of miRNA mimics on GFP expression, GFP reporters were generated in which GFP was linked to fragment #3 harboring or lacking the binding sequence for hnRNPK and the miRNAs (GFP vector B and E in the schematic). GFP expression was assessed by western blot. ( f ) The interaction between hnRNPK and GFP chimeric mRNAs was examined. Cells were transfected with the previously described GFP vectors and cytoplasmic lysates were prepared. GFP mRNA enrichment was measured by RNP IP using hnRNPK antibody followed by RT–qPCR. ( g ) To test whether hnRNPK affects GFP expression in the absence of an miRNA-binding sequence, GFP reporters described in e were used. The expression levels of hnRNPK and GFP were assessed by western blot. ( h ) To determine whether hnRNPK restored PLK1 expression, cells were transfected with miRNA mimics (pre-miR-149-3p and miR-193b-5p) and an hnRNPK vector (FLAG-hnRNPK). Expression of hnRNPK and PLK1 was assessed by western blot. All experiments were performed more than three times and data represent mean±S.D.

    Techniques Used: Immunoprecipitation, Quantitative RT-PCR, Expressing, Transfection, Generated, Binding Assay, Sequencing, Plasmid Preparation, Western Blot

    hnRNPK regulates PLK1 expression. ( a , b ) HeLa cells were transfected with control or hnRNPK-specific siRNA. After 48 h post-transfection, protein and mRNA levels of hnRNPK and PLK1 were determined by western blot and RT–qPCR, respectively. ( c , d ) Cells were transfected with FLAG or FLAG-hnRNPK vector. Protein and mRNA levels of hnRNPK and PLK1 were assessed as described above. ( e ) To verify that hnRNPK regulates PLK1, we designed a specific siRNA targeting the 3′UTR of hnRNPK ). HeLa cells were simultaneously transfected with siRNAs (control or hnRNPK 3′UTR siRNA) and with plasmid DNA (FLAG or FLAG-hnRNPK vector). After 48 h post-transfection, the level of endogenous and ectopic hnRNPK, PLK1, and the loading control, GAPDH, was assessed by western blot. All experiments were performed more than three times and data represent mean±S.D.
    Figure Legend Snippet: hnRNPK regulates PLK1 expression. ( a , b ) HeLa cells were transfected with control or hnRNPK-specific siRNA. After 48 h post-transfection, protein and mRNA levels of hnRNPK and PLK1 were determined by western blot and RT–qPCR, respectively. ( c , d ) Cells were transfected with FLAG or FLAG-hnRNPK vector. Protein and mRNA levels of hnRNPK and PLK1 were assessed as described above. ( e ) To verify that hnRNPK regulates PLK1, we designed a specific siRNA targeting the 3′UTR of hnRNPK ). HeLa cells were simultaneously transfected with siRNAs (control or hnRNPK 3′UTR siRNA) and with plasmid DNA (FLAG or FLAG-hnRNPK vector). After 48 h post-transfection, the level of endogenous and ectopic hnRNPK, PLK1, and the loading control, GAPDH, was assessed by western blot. All experiments were performed more than three times and data represent mean±S.D.

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

    Proposed action mechanism underlying hnRNPK-mediated PLK1 regulation. Under the condition of high hnRNPK, the interaction between PLK1 mRNA 3′UTR and miRNA-loaded RISC is disrupted by hnRNPK, which results in increase of PLK1 expression. Conversely, in the presence of low hnRNPK, miRNA-loaded RISC easily interacts with the 3′UTR of PLK1 mRNA, in turn lowering the PLK1 expression
    Figure Legend Snippet: Proposed action mechanism underlying hnRNPK-mediated PLK1 regulation. Under the condition of high hnRNPK, the interaction between PLK1 mRNA 3′UTR and miRNA-loaded RISC is disrupted by hnRNPK, which results in increase of PLK1 expression. Conversely, in the presence of low hnRNPK, miRNA-loaded RISC easily interacts with the 3′UTR of PLK1 mRNA, in turn lowering the PLK1 expression

    Techniques Used: Expressing

    hnRNPK directly interacts with the 3′UTR of PLK1 mRNA through its KH1 and KH2 domains. ( a ) To determine whether hnRNPK binds to the 3′UTR of PLK1 mRNA, cytoplasmic lysates were prepared as described in the Materials and Methods, and immunoprecipitated (IP) using hnRNPK antibody-coated beads. RNA was isolated from IP materials and the level of PLK1 mRNA was determined by RT–qPCR. Western blot was performed to confirm IP efficiency. ( b ) As an RNA-binding protein, hnRNPK has three KH domains: KH1, KH2, and KH3. To evaluate which domain or domains are responsible for the interaction between hnRNPK and PLK1 ). HeLa cells were transfected with FLAG vectors containing the wild-type (full length, FL) and the four deletion mutants. Cytoplasmic lysates were prepared and IP was performed using FLAG antibody. Identical expression levels of ectopic hnRNPK (FLAG-hnRNPK) in input were determined by western blot, and the level of enriched PLK1 mRNA in FLAG-IP was assessed by RT–qPCR. ( c ) Schematic depiction of the biotinylated RNAs of the 3′UTR of PLK1 mRNA used for biotin pull-down analysis. The level of hnRNPK in biotin pull-down samples was determined by western blot. ( d ) We constructed vectors expressing chimeric RNAs spanning the GFP ( A ) and fragment #3 of the 3′UTR PLK1 mRNA ( B ). HeLa cells were first transfected with control or hnRNPK siRNA. After 24 h post-transfection, cells were resuspended into six-well plates followed by transfection with GFP vectors (blank or fragment #3 of the 3′UTR PLK1 mRNA). The level of hnRNPK, GFP, and the loading control, GAPDH, was assessed by western blot. All experiments were performed more than three times and data represent mean±S.D.
    Figure Legend Snippet: hnRNPK directly interacts with the 3′UTR of PLK1 mRNA through its KH1 and KH2 domains. ( a ) To determine whether hnRNPK binds to the 3′UTR of PLK1 mRNA, cytoplasmic lysates were prepared as described in the Materials and Methods, and immunoprecipitated (IP) using hnRNPK antibody-coated beads. RNA was isolated from IP materials and the level of PLK1 mRNA was determined by RT–qPCR. Western blot was performed to confirm IP efficiency. ( b ) As an RNA-binding protein, hnRNPK has three KH domains: KH1, KH2, and KH3. To evaluate which domain or domains are responsible for the interaction between hnRNPK and PLK1 ). HeLa cells were transfected with FLAG vectors containing the wild-type (full length, FL) and the four deletion mutants. Cytoplasmic lysates were prepared and IP was performed using FLAG antibody. Identical expression levels of ectopic hnRNPK (FLAG-hnRNPK) in input were determined by western blot, and the level of enriched PLK1 mRNA in FLAG-IP was assessed by RT–qPCR. ( c ) Schematic depiction of the biotinylated RNAs of the 3′UTR of PLK1 mRNA used for biotin pull-down analysis. The level of hnRNPK in biotin pull-down samples was determined by western blot. ( d ) We constructed vectors expressing chimeric RNAs spanning the GFP ( A ) and fragment #3 of the 3′UTR PLK1 mRNA ( B ). HeLa cells were first transfected with control or hnRNPK siRNA. After 24 h post-transfection, cells were resuspended into six-well plates followed by transfection with GFP vectors (blank or fragment #3 of the 3′UTR PLK1 mRNA). The level of hnRNPK, GFP, and the loading control, GAPDH, was assessed by western blot. All experiments were performed more than three times and data represent mean±S.D.

    Techniques Used: Immunoprecipitation, Isolation, Quantitative RT-PCR, Western Blot, RNA Binding Assay, Transfection, Expressing, Construct

    Positive correlation between hnRNPK and PLK1 in several types of cancer cells. ( a ) To examine whether hnRNPK is involved in PLK1 expression, cells from the following cancer cell lines were transfected with hnRNPK-specific siRNA: lung adenocarcinoma (A549 and H322), glioblastoma (LN229 and T98G), renal cell carcinoma (Caki1 and 786-O), colon cancer (HCT116), osteosarcoma (U2OS), and hepatocellular carcinoma (HepG2). The level of hnRNPK and PLK1 was determined by western blot. As a loading control, the level of GAPDH was examined. ( b , c ) To validate positive correlations, the mRNA level of hnRNPK and PLK1 in lung squamous cell carcinoma ( b ) and glioblastoma ( c ) was obtained from the TCGA database
    Figure Legend Snippet: Positive correlation between hnRNPK and PLK1 in several types of cancer cells. ( a ) To examine whether hnRNPK is involved in PLK1 expression, cells from the following cancer cell lines were transfected with hnRNPK-specific siRNA: lung adenocarcinoma (A549 and H322), glioblastoma (LN229 and T98G), renal cell carcinoma (Caki1 and 786-O), colon cancer (HCT116), osteosarcoma (U2OS), and hepatocellular carcinoma (HepG2). The level of hnRNPK and PLK1 was determined by western blot. As a loading control, the level of GAPDH was examined. ( b , c ) To validate positive correlations, the mRNA level of hnRNPK and PLK1 in lung squamous cell carcinoma ( b ) and glioblastoma ( c ) was obtained from the TCGA database

    Techniques Used: Expressing, Transfection, Western Blot

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    Plasmid Preparation:

    Article Title: MiR-30c/PGC-1β protects against diabetic cardiomyopathy via PPARα
    Article Snippet: .. To evaluate the binding of miRNA and target mRNA, the sequence containing predicted miR-30c binding site of the human PGC-1β 3′ UTR and corresponding mutant sequence (Additional file : Table S1) were synthesized and inserted into pMIR-REPORT luciferase vector (Ambion, Thermo Fisher Scientific, Waltham, MA). .. To determine whether miR-30c targeting 3′ UTR of PGC-1β, HEK293T cells were co-transfected with appropriate pMIR construct (1 μg/mL), pRL-TK plasmid (0.1 μg/mL) with miR-30c mimics or negative controls (100 nM).

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    Thermo Fisher target mrna
    Reverse transcription-quantitative polymerase chain reaction assay of PLZF <t>mRNA</t> expression in breast cancer cells. Results are normalized to <t>18S</t> expression, and are reported as a fold-change relative to the levels of PLZF mRNA in 293 cells and are presented as the mean ± standard deviation (n=3). *P
    Target Mrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/target mrna/product/Thermo Fisher
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    92
    Thermo Fisher target mrna rt qpcr analysis
    Relative expression levels of selected target mRNAs predicted for miR-217 and miR-576-3p. HuH-7 cells were infected with OROV at MOI 1 and <t>RT-qPCR</t> was done 12 h post infection ( A ) or 24 h post infection ( B ). ( A ) Data denotes mean fold change (y-axis) of infected cells relative to uninfected cells for 18 deregulated <t>mRNA</t> out of 95 selected targets. Gene expression was normalized by endogenous 18S, GAPDH, HPRT1 and GUSB levels. Deregulated target genes are depicted in x-axis. Black columns represent predicted targets for miR-576-3p and gray columns represent predicted targets for miR-217, respectively. Error bars represent Standard Error Mean (SEM) for 6 independent samples. ns = non-significant (0.05 ≤ p ≤ 0.1); * = p ≤ 0.05; ** = p ≤ 0.01. ( B ) Data denotes mean fold change (y-axis) of infected cells relative to uninfected cells for 6 selected targets 24 h post infection. Gene expression was normalized by endogenous GAPDH levels. Black columns represent predicted targets for miR-576-3p and gray columns represent predicted targets for miR-217, respectively. Error bars represent SD for four independent samples. * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001.
    Target Mrna Rt Qpcr Analysis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher sirna transfection sirna targeting gpr30 mrna
    <t>GPR30</t> knockdown eliminated IP-induced PI3K/AKT pathway activation of hPDLCs. The <t>siRNA</t> <t>transfection</t> efficiency of hPDLCs was tested by using the positive control with fluorescence. ( A ) Western blot ( B ) and PCR ( C ) results of GPR30 knockdown of hPDLCs. The expression of AKT and p-AKT was assessed by Western blot. ( D ) Quantitative analysis of Western blot band from ( D ) was performed. ( E ) * P
    Sirna Transfection Sirna Targeting Gpr30 Mrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher target gene mrna transcripts
    STAT3 is important to IL-10 expression induced by CD69. 1 × 10 6 /ml CD69 − overexpressing EL4 cells were treated with STAT3 siRNA or STAT5 siRNA for the indicated time. The relative levels of c-Maf, IL-10, and TGF-β1 expression in the different groups of EL4 cells were determined by real time <t>PCR</t> and ELISA. a The <t>mRNA</t> level of c-Maf in pMX and pMX-CD69 transduced EL4 cells following treatment with STAT3 siRNA or STAT5 siRNA. b – e The mRNA and protein levels IL-10 and TGF-β1 expression in pMX and pMX-CD69 EL4 cells following treatment with STAT3 siRNA or STAT5 siRNA. f , h 1 × 10 6 /ml EL4 cells were transfected with control retrovirus pMX or pMX-CD69 to induce CD69 over-expression, GFP positive cells were harvested and the activation of STAT3 and STAT5 in CD69 over-expressed or pMX EL4 cells were measured by western blot. g , i CD4 + Foxp3 + CD69 + and CD4 + Foxp3 + CD69 − Tregs were fixed, permeabilized and intracellularly stained with anti-p-STAT3 or anti-p-STAT5 antibodies or isotype controls, then analyzed by flow cytometry. j , k ChIP analysis of STAT3 or STAT5 binding to the c-Maf promoter in CD69 over-expressing or control EL4 cells. Date are representative images or expressed as the mean ± SD of three independent experiments. * P
    Target Gene Mrna Transcripts, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Reverse transcription-quantitative polymerase chain reaction assay of PLZF mRNA expression in breast cancer cells. Results are normalized to 18S expression, and are reported as a fold-change relative to the levels of PLZF mRNA in 293 cells and are presented as the mean ± standard deviation (n=3). *P

    Journal: Oncology Letters

    Article Title: Promyelocytic leukemia zinc finger triggers ATP-binding cassette subfamily E member 1-mediated growth inhibition in breast cancer cells

    doi: 10.3892/ol.2018.9207

    Figure Lengend Snippet: Reverse transcription-quantitative polymerase chain reaction assay of PLZF mRNA expression in breast cancer cells. Results are normalized to 18S expression, and are reported as a fold-change relative to the levels of PLZF mRNA in 293 cells and are presented as the mean ± standard deviation (n=3). *P

    Article Snippet: The levels of target mRNA were normalized to that of 18S (reference ID: Hs03003631_g1; Thermo Fisher Scientific, Inc.) and were charted using the 2−ΔΔCq method ( ).

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Standard Deviation

    Relative expression levels of selected target mRNAs predicted for miR-217 and miR-576-3p. HuH-7 cells were infected with OROV at MOI 1 and RT-qPCR was done 12 h post infection ( A ) or 24 h post infection ( B ). ( A ) Data denotes mean fold change (y-axis) of infected cells relative to uninfected cells for 18 deregulated mRNA out of 95 selected targets. Gene expression was normalized by endogenous 18S, GAPDH, HPRT1 and GUSB levels. Deregulated target genes are depicted in x-axis. Black columns represent predicted targets for miR-576-3p and gray columns represent predicted targets for miR-217, respectively. Error bars represent Standard Error Mean (SEM) for 6 independent samples. ns = non-significant (0.05 ≤ p ≤ 0.1); * = p ≤ 0.05; ** = p ≤ 0.01. ( B ) Data denotes mean fold change (y-axis) of infected cells relative to uninfected cells for 6 selected targets 24 h post infection. Gene expression was normalized by endogenous GAPDH levels. Black columns represent predicted targets for miR-576-3p and gray columns represent predicted targets for miR-217, respectively. Error bars represent SD for four independent samples. * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001.

    Journal: PLoS Neglected Tropical Diseases

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

    doi: 10.1371/journal.pntd.0006508

    Figure Lengend Snippet: Relative expression levels of selected target mRNAs predicted for miR-217 and miR-576-3p. HuH-7 cells were infected with OROV at MOI 1 and RT-qPCR was done 12 h post infection ( A ) or 24 h post infection ( B ). ( A ) Data denotes mean fold change (y-axis) of infected cells relative to uninfected cells for 18 deregulated mRNA out of 95 selected targets. Gene expression was normalized by endogenous 18S, GAPDH, HPRT1 and GUSB levels. Deregulated target genes are depicted in x-axis. Black columns represent predicted targets for miR-576-3p and gray columns represent predicted targets for miR-217, respectively. Error bars represent Standard Error Mean (SEM) for 6 independent samples. ns = non-significant (0.05 ≤ p ≤ 0.1); * = p ≤ 0.05; ** = p ≤ 0.01. ( B ) Data denotes mean fold change (y-axis) of infected cells relative to uninfected cells for 6 selected targets 24 h post infection. Gene expression was normalized by endogenous GAPDH levels. Black columns represent predicted targets for miR-576-3p and gray columns represent predicted targets for miR-217, respectively. Error bars represent SD for four independent samples. * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001.

    Article Snippet: RNA isolation and quality assessment Total cellular RNA for microarray and target mRNA RT-qPCR analysis was isolated using MirVana kit (Thermo Fisher Scientific ) according to manufacturer’s instructions.

    Techniques: Expressing, Infection, Quantitative RT-PCR

    IFN-β antiviral response is attenuated during infection. HuH-7 cells were infected with OROV at MOI 1 and RT-qPCR was done at indicated post-infection time points. ( A ) Normalized expression of IFN-β mRNA during infection. HuH-7 cells were infected with OROV at MOI 1 and IFN-β levels were measured at indicated post-infection time points. Error bars represent SD for four independent infections. Data denotes mean fold change (y-axis) of infected cells relative to uninfected cells for ( B ) STING and ( C ) TRAF3 RNA levels. Gene expression was normalized by endogenous GAPDH levels. Error bars represent SD for four independent samples. ns = non-significant (p ≤ 0.1); * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001. ( D ) Schematic representation of IFN-β and miR-576-3p interplay in antiviral response during OROV infection. Values are relative to uninfected cells. miR-576-3p, IFN-β, STING and TRAF3 transcripts levels are represented by green, blue, red and yellow lines, respectively.

    Journal: PLoS Neglected Tropical Diseases

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

    doi: 10.1371/journal.pntd.0006508

    Figure Lengend Snippet: IFN-β antiviral response is attenuated during infection. HuH-7 cells were infected with OROV at MOI 1 and RT-qPCR was done at indicated post-infection time points. ( A ) Normalized expression of IFN-β mRNA during infection. HuH-7 cells were infected with OROV at MOI 1 and IFN-β levels were measured at indicated post-infection time points. Error bars represent SD for four independent infections. Data denotes mean fold change (y-axis) of infected cells relative to uninfected cells for ( B ) STING and ( C ) TRAF3 RNA levels. Gene expression was normalized by endogenous GAPDH levels. Error bars represent SD for four independent samples. ns = non-significant (p ≤ 0.1); * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001. ( D ) Schematic representation of IFN-β and miR-576-3p interplay in antiviral response during OROV infection. Values are relative to uninfected cells. miR-576-3p, IFN-β, STING and TRAF3 transcripts levels are represented by green, blue, red and yellow lines, respectively.

    Article Snippet: RNA isolation and quality assessment Total cellular RNA for microarray and target mRNA RT-qPCR analysis was isolated using MirVana kit (Thermo Fisher Scientific ) according to manufacturer’s instructions.

    Techniques: Infection, Quantitative RT-PCR, Expressing

    GPR30 knockdown eliminated IP-induced PI3K/AKT pathway activation of hPDLCs. The siRNA transfection efficiency of hPDLCs was tested by using the positive control with fluorescence. ( A ) Western blot ( B ) and PCR ( C ) results of GPR30 knockdown of hPDLCs. The expression of AKT and p-AKT was assessed by Western blot. ( D ) Quantitative analysis of Western blot band from ( D ) was performed. ( E ) * P

    Journal: Drug Design, Development and Therapy

    Article Title: Ipriflavone promotes proliferation and osteogenic differentiation of periodontal ligament cells by activating GPR30/PI3K/AKT signaling pathway

    doi: 10.2147/DDDT.S148457

    Figure Lengend Snippet: GPR30 knockdown eliminated IP-induced PI3K/AKT pathway activation of hPDLCs. The siRNA transfection efficiency of hPDLCs was tested by using the positive control with fluorescence. ( A ) Western blot ( B ) and PCR ( C ) results of GPR30 knockdown of hPDLCs. The expression of AKT and p-AKT was assessed by Western blot. ( D ) Quantitative analysis of Western blot band from ( D ) was performed. ( E ) * P

    Article Snippet: siRNA transfection siRNA targeting GPR30 mRNA (siGPR30) and siRNA-NC were synthesized by Thermo Fisher Scientific.

    Techniques: Activation Assay, Transfection, Positive Control, Fluorescence, Western Blot, Polymerase Chain Reaction, Expressing

    STAT3 is important to IL-10 expression induced by CD69. 1 × 10 6 /ml CD69 − overexpressing EL4 cells were treated with STAT3 siRNA or STAT5 siRNA for the indicated time. The relative levels of c-Maf, IL-10, and TGF-β1 expression in the different groups of EL4 cells were determined by real time PCR and ELISA. a The mRNA level of c-Maf in pMX and pMX-CD69 transduced EL4 cells following treatment with STAT3 siRNA or STAT5 siRNA. b – e The mRNA and protein levels IL-10 and TGF-β1 expression in pMX and pMX-CD69 EL4 cells following treatment with STAT3 siRNA or STAT5 siRNA. f , h 1 × 10 6 /ml EL4 cells were transfected with control retrovirus pMX or pMX-CD69 to induce CD69 over-expression, GFP positive cells were harvested and the activation of STAT3 and STAT5 in CD69 over-expressed or pMX EL4 cells were measured by western blot. g , i CD4 + Foxp3 + CD69 + and CD4 + Foxp3 + CD69 − Tregs were fixed, permeabilized and intracellularly stained with anti-p-STAT3 or anti-p-STAT5 antibodies or isotype controls, then analyzed by flow cytometry. j , k ChIP analysis of STAT3 or STAT5 binding to the c-Maf promoter in CD69 over-expressing or control EL4 cells. Date are representative images or expressed as the mean ± SD of three independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: CD69 enhances immunosuppressive function of regulatory T-cells and attenuates colitis by prompting IL-10 production

    doi: 10.1038/s41419-018-0927-9

    Figure Lengend Snippet: STAT3 is important to IL-10 expression induced by CD69. 1 × 10 6 /ml CD69 − overexpressing EL4 cells were treated with STAT3 siRNA or STAT5 siRNA for the indicated time. The relative levels of c-Maf, IL-10, and TGF-β1 expression in the different groups of EL4 cells were determined by real time PCR and ELISA. a The mRNA level of c-Maf in pMX and pMX-CD69 transduced EL4 cells following treatment with STAT3 siRNA or STAT5 siRNA. b – e The mRNA and protein levels IL-10 and TGF-β1 expression in pMX and pMX-CD69 EL4 cells following treatment with STAT3 siRNA or STAT5 siRNA. f , h 1 × 10 6 /ml EL4 cells were transfected with control retrovirus pMX or pMX-CD69 to induce CD69 over-expression, GFP positive cells were harvested and the activation of STAT3 and STAT5 in CD69 over-expressed or pMX EL4 cells were measured by western blot. g , i CD4 + Foxp3 + CD69 + and CD4 + Foxp3 + CD69 − Tregs were fixed, permeabilized and intracellularly stained with anti-p-STAT3 or anti-p-STAT5 antibodies or isotype controls, then analyzed by flow cytometry. j , k ChIP analysis of STAT3 or STAT5 binding to the c-Maf promoter in CD69 over-expressing or control EL4 cells. Date are representative images or expressed as the mean ± SD of three independent experiments. * P

    Article Snippet: The relative levels of target gene mRNA transcripts were determined by real-time PCR using the SYBR Green Master Mix Kit (Thermo fisher).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Transfection, Over Expression, Activation Assay, Western Blot, Staining, Flow Cytometry, Cytometry, Chromatin Immunoprecipitation, Binding Assay

    Characterization of CD4 + Foxp3 + CD69 + Tregs. a Density plots show CD69 expression in gated CD4 + Foxp3 + cells from freshly isolated Spl, PLN and MLN, PPs, IEL, and colonic LPL in Foxp3 GFP knock-in mice. b Real-time PCR was performed to assess mRNA expression of T-bet, GATA3, RORγt and Foxp3 genes in CD69 + Treg and CD69 − Tregs. β-actin mRNA was used for the normalization. c 1 × 10 6 /ml naïve CD4 + T, CD69 + Treg or CD69 − Treg cells were cultured under Th1-cell-differentiation conditions for four days. The CD4 + naïve T-cells treatment without IL-12 were regarded as negative control. Each group of T-cells were stimulated with the cell stimulation cocktail for 6 h and then stained with anti-mouse CD4 and IFN-γ antibodies, followed by flow cytometry (left). The levels of IFN-γ in the supernatants of cultured T-cells were detected by ELISA (right). d BMDCs co-cultured respectively with 1 × 10 6 /ml naïve CD4 + T, CD69 + Treg, or CD69 − Treg cells at a ratio of 1:5 under Th17-differentiation conditions for four days. The CD4+ naïve T-cells treatment without IL-6 and TGF-β1 were regarded as negative control. Each group of T-cells were stimulated with the cell stimulation cocktail for 6 h and then stained with anti-mouse CD4 and IL-17 antibodies, followed by flow cytometry (left). The levels of IL-17 in the supernatants of cultured T-cells were detected by ELISA (right). e The expression levels of immunosuppressive markers in CD69 + Treg and CD69 − Tregs were analyzed using flow cytometry with indicated antibodies. Data are representative images or expressed as the mean ± SD of three independent experiments ( n = 5). * P

    Journal: Cell Death & Disease

    Article Title: CD69 enhances immunosuppressive function of regulatory T-cells and attenuates colitis by prompting IL-10 production

    doi: 10.1038/s41419-018-0927-9

    Figure Lengend Snippet: Characterization of CD4 + Foxp3 + CD69 + Tregs. a Density plots show CD69 expression in gated CD4 + Foxp3 + cells from freshly isolated Spl, PLN and MLN, PPs, IEL, and colonic LPL in Foxp3 GFP knock-in mice. b Real-time PCR was performed to assess mRNA expression of T-bet, GATA3, RORγt and Foxp3 genes in CD69 + Treg and CD69 − Tregs. β-actin mRNA was used for the normalization. c 1 × 10 6 /ml naïve CD4 + T, CD69 + Treg or CD69 − Treg cells were cultured under Th1-cell-differentiation conditions for four days. The CD4 + naïve T-cells treatment without IL-12 were regarded as negative control. Each group of T-cells were stimulated with the cell stimulation cocktail for 6 h and then stained with anti-mouse CD4 and IFN-γ antibodies, followed by flow cytometry (left). The levels of IFN-γ in the supernatants of cultured T-cells were detected by ELISA (right). d BMDCs co-cultured respectively with 1 × 10 6 /ml naïve CD4 + T, CD69 + Treg, or CD69 − Treg cells at a ratio of 1:5 under Th17-differentiation conditions for four days. The CD4+ naïve T-cells treatment without IL-6 and TGF-β1 were regarded as negative control. Each group of T-cells were stimulated with the cell stimulation cocktail for 6 h and then stained with anti-mouse CD4 and IL-17 antibodies, followed by flow cytometry (left). The levels of IL-17 in the supernatants of cultured T-cells were detected by ELISA (right). e The expression levels of immunosuppressive markers in CD69 + Treg and CD69 − Tregs were analyzed using flow cytometry with indicated antibodies. Data are representative images or expressed as the mean ± SD of three independent experiments ( n = 5). * P

    Article Snippet: The relative levels of target gene mRNA transcripts were determined by real-time PCR using the SYBR Green Master Mix Kit (Thermo fisher).

    Techniques: Expressing, Isolation, Knock-In, Mouse Assay, Real-time Polymerase Chain Reaction, Cell Culture, Cell Differentiation, Negative Control, Cell Stimulation, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    c-Maf is critical for CD69-induced IL-10 expression. a , b CD69 + Tregs, CD69 − Tregs and CD4 + Foxp3 − T cells were isolated from Foxp3 GFP knock-in mice and the relative mRNA and protein levels of c-Maf in were analyzed by real-time RCR and western blot. c , d 1 × 10 6 /ml EL4 cells were infected with control retrovirus pMX or pMX-CD69 at MOI of 50 for 48 h to induce CD69 over-expression and the GFP positive cells were harvested. The relative mRNA and protein levels of c-Maf were analyzed by real time RCR and western blot. e – i The levels of c-Maf, IL-10, and TGF-β1 expression in CD69 overexpressing EL4 cells transfected with c-Maf siRNA or control siRNA were detected by real-time PCR and ELISA. Date are representative images or expressed as the mean ± SD of three independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: CD69 enhances immunosuppressive function of regulatory T-cells and attenuates colitis by prompting IL-10 production

    doi: 10.1038/s41419-018-0927-9

    Figure Lengend Snippet: c-Maf is critical for CD69-induced IL-10 expression. a , b CD69 + Tregs, CD69 − Tregs and CD4 + Foxp3 − T cells were isolated from Foxp3 GFP knock-in mice and the relative mRNA and protein levels of c-Maf in were analyzed by real-time RCR and western blot. c , d 1 × 10 6 /ml EL4 cells were infected with control retrovirus pMX or pMX-CD69 at MOI of 50 for 48 h to induce CD69 over-expression and the GFP positive cells were harvested. The relative mRNA and protein levels of c-Maf were analyzed by real time RCR and western blot. e – i The levels of c-Maf, IL-10, and TGF-β1 expression in CD69 overexpressing EL4 cells transfected with c-Maf siRNA or control siRNA were detected by real-time PCR and ELISA. Date are representative images or expressed as the mean ± SD of three independent experiments. * P

    Article Snippet: The relative levels of target gene mRNA transcripts were determined by real-time PCR using the SYBR Green Master Mix Kit (Thermo fisher).

    Techniques: Expressing, Isolation, Knock-In, Mouse Assay, Western Blot, Infection, Over Expression, Transfection, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay