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  • 96
    Thermo Fisher ribonucleic acid rna
    Effects of PSK and docetaxel treatments on mRNA expression of markers of antitumor immune response. Groups of mice (n=8 control; n=5 docetaxel; n= 6 PSK; n=4 PSK + docetaxel) bearing established TRAMP-C2 tumors (12 days) were treated with either oral saline, subtherapeutic dose of docetaxel (5 mg/kg twice weekly), oral gavage of PSK (300 mg/kg), or a combination of docetaxel and PSK. <t>RNA</t> was extracted from isolated tumors and real-time RT-PCR performed using primers and probes for several different cytokines and antitumor immune response markers. Shown are the effects of the different treatment groups on mRNA expression of <t>IFN-γ</t> (A), IL-2 (B), and TNF-α (C). The values shown are relative cytokine mRNA per 1,000 copies of β-actin. Mice treated with PSK, either alone (p=0.04) or with docetaxel (p=0.01) significantly induced IFN-γ mRNA expression in TRAMP-C2 tumors compared to saline control.
    Ribonucleic Acid Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 365 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore rna isolation
    PXR enhances expression of apoptotic genes while reduces expression of cell-cycle regulatory genes in hepatic cancer cells A. Real-time PCR analysis was performed for Bcl-xl , Bcl-2 , Cdk2 and Cdk4 with total <t>RNA</t> extracted from HepG2, human HepXR and mouse HepXR cell lines for various cell cycle and apoptosis regulatory genes. β-Actin served as an internal control. B. Higher cellular expression of PXR increased the doubling time in human HepXR and mouse HepXR cells in comparison to HepG2 cells. The experiments were performed three times with three samples of each cell lines and the values are represented as the mean ±SE. The P-value represents the significance in human HepXR and mouse HepXR cell lines as compared to the control HepG2 cell line. P-value
    Rna Isolation, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6830 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore tri rna isolation reagent
    PXR enhances expression of apoptotic genes while reduces expression of cell-cycle regulatory genes in hepatic cancer cells A. Real-time PCR analysis was performed for Bcl-xl , Bcl-2 , Cdk2 and Cdk4 with total <t>RNA</t> extracted from HepG2, human HepXR and mouse HepXR cell lines for various cell cycle and apoptosis regulatory genes. β-Actin served as an internal control. B. Higher cellular expression of PXR increased the doubling time in human HepXR and mouse HepXR cells in comparison to HepG2 cells. The experiments were performed three times with three samples of each cell lines and the values are represented as the mean ±SE. The P-value represents the significance in human HepXR and mouse HepXR cell lines as compared to the control HepG2 cell line. P-value
    Tri Rna Isolation Reagent, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rna
    Prediction of <t>RNA</t> motifs in cell cycle co-regulated gene clusters. Left panel: Heatmap representation of the expression of 305-cell cycle genes. The gene expression values (log 2 normalized read counts) were subjected to a clustering analysis in the GENE-E software (Broad Institute). Both columns (samples) and rows (genes) were clustered by Pearson correlation. Right panel: the gene expression profile in the three sequenced cell cycle phases was plotted for each cluster. Consensus sequence motifs found exclusively at the 3’ UTRs are presented right to each cluster, and the coverage and the p-value obtained from the DREME search is indicated. Consensus structural motifs found with CMfinder are presented on the right most column, and their coverage, specificity and free energy are indicated. The structural motifs were all found at the 5 ‘UTR except for the motif found in cluster 7. None of the motifs found in cluster 6 reach the thresholds. The best motif for each cluster is depicted.
    Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 177224 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher total ribonucleic acid rna extraction kit
    Prediction of <t>RNA</t> motifs in cell cycle co-regulated gene clusters. Left panel: Heatmap representation of the expression of 305-cell cycle genes. The gene expression values (log 2 normalized read counts) were subjected to a clustering analysis in the GENE-E software (Broad Institute). Both columns (samples) and rows (genes) were clustered by Pearson correlation. Right panel: the gene expression profile in the three sequenced cell cycle phases was plotted for each cluster. Consensus sequence motifs found exclusively at the 3’ UTRs are presented right to each cluster, and the coverage and the p-value obtained from the DREME search is indicated. Consensus structural motifs found with CMfinder are presented on the right most column, and their coverage, specificity and free energy are indicated. The structural motifs were all found at the 5 ‘UTR except for the motif found in cluster 7. None of the motifs found in cluster 6 reach the thresholds. The best motif for each cluster is depicted.
    Total Ribonucleic Acid Rna Extraction Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Promega rna isolation reagents
    Prediction of <t>RNA</t> motifs in cell cycle co-regulated gene clusters. Left panel: Heatmap representation of the expression of 305-cell cycle genes. The gene expression values (log 2 normalized read counts) were subjected to a clustering analysis in the GENE-E software (Broad Institute). Both columns (samples) and rows (genes) were clustered by Pearson correlation. Right panel: the gene expression profile in the three sequenced cell cycle phases was plotted for each cluster. Consensus sequence motifs found exclusively at the 3’ UTRs are presented right to each cluster, and the coverage and the p-value obtained from the DREME search is indicated. Consensus structural motifs found with CMfinder are presented on the right most column, and their coverage, specificity and free energy are indicated. The structural motifs were all found at the 5 ‘UTR except for the motif found in cluster 7. None of the motifs found in cluster 6 reach the thresholds. The best motif for each cluster is depicted.
    Rna Isolation Reagents, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Fisher Scientific trizol rna isolation reagents
    Prediction of <t>RNA</t> motifs in cell cycle co-regulated gene clusters. Left panel: Heatmap representation of the expression of 305-cell cycle genes. The gene expression values (log 2 normalized read counts) were subjected to a clustering analysis in the GENE-E software (Broad Institute). Both columns (samples) and rows (genes) were clustered by Pearson correlation. Right panel: the gene expression profile in the three sequenced cell cycle phases was plotted for each cluster. Consensus sequence motifs found exclusively at the 3’ UTRs are presented right to each cluster, and the coverage and the p-value obtained from the DREME search is indicated. Consensus structural motifs found with CMfinder are presented on the right most column, and their coverage, specificity and free energy are indicated. The structural motifs were all found at the 5 ‘UTR except for the motif found in cluster 7. None of the motifs found in cluster 6 reach the thresholds. The best motif for each cluster is depicted.
    Trizol Rna Isolation Reagents, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Meridian Life Science isolate ii rna isolation reagents
    Prediction of <t>RNA</t> motifs in cell cycle co-regulated gene clusters. Left panel: Heatmap representation of the expression of 305-cell cycle genes. The gene expression values (log 2 normalized read counts) were subjected to a clustering analysis in the GENE-E software (Broad Institute). Both columns (samples) and rows (genes) were clustered by Pearson correlation. Right panel: the gene expression profile in the three sequenced cell cycle phases was plotted for each cluster. Consensus sequence motifs found exclusively at the 3’ UTRs are presented right to each cluster, and the coverage and the p-value obtained from the DREME search is indicated. Consensus structural motifs found with CMfinder are presented on the right most column, and their coverage, specificity and free energy are indicated. The structural motifs were all found at the 5 ‘UTR except for the motif found in cluster 7. None of the motifs found in cluster 6 reach the thresholds. The best motif for each cluster is depicted.
    Isolate Ii Rna Isolation Reagents, supplied by Meridian Life Science, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effects of PSK and docetaxel treatments on mRNA expression of markers of antitumor immune response. Groups of mice (n=8 control; n=5 docetaxel; n= 6 PSK; n=4 PSK + docetaxel) bearing established TRAMP-C2 tumors (12 days) were treated with either oral saline, subtherapeutic dose of docetaxel (5 mg/kg twice weekly), oral gavage of PSK (300 mg/kg), or a combination of docetaxel and PSK. RNA was extracted from isolated tumors and real-time RT-PCR performed using primers and probes for several different cytokines and antitumor immune response markers. Shown are the effects of the different treatment groups on mRNA expression of IFN-γ (A), IL-2 (B), and TNF-α (C). The values shown are relative cytokine mRNA per 1,000 copies of β-actin. Mice treated with PSK, either alone (p=0.04) or with docetaxel (p=0.01) significantly induced IFN-γ mRNA expression in TRAMP-C2 tumors compared to saline control.

    Journal: International Journal of Oncology

    Article Title: Polysaccharide-K augments docetaxel-induced tumor suppression and antitumor immune response in an immunocompetent murine model of human prostate cancer

    doi: 10.3892/ijo.2011.1292

    Figure Lengend Snippet: Effects of PSK and docetaxel treatments on mRNA expression of markers of antitumor immune response. Groups of mice (n=8 control; n=5 docetaxel; n= 6 PSK; n=4 PSK + docetaxel) bearing established TRAMP-C2 tumors (12 days) were treated with either oral saline, subtherapeutic dose of docetaxel (5 mg/kg twice weekly), oral gavage of PSK (300 mg/kg), or a combination of docetaxel and PSK. RNA was extracted from isolated tumors and real-time RT-PCR performed using primers and probes for several different cytokines and antitumor immune response markers. Shown are the effects of the different treatment groups on mRNA expression of IFN-γ (A), IL-2 (B), and TNF-α (C). The values shown are relative cytokine mRNA per 1,000 copies of β-actin. Mice treated with PSK, either alone (p=0.04) or with docetaxel (p=0.01) significantly induced IFN-γ mRNA expression in TRAMP-C2 tumors compared to saline control.

    Article Snippet: Cytokine and cytolytic gene and Foxp3 mRNA expression To assess expression of IFN-γ, IL-2, TNF-α, TGF-β, perforin, granzyme B and FoxP3 genes, ribonucleic acid (RNA) was isolated from tumors using the RNAqueous4PCR kit (Applied Biosystems/Ambion, Austin, TX).

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

    PXR enhances expression of apoptotic genes while reduces expression of cell-cycle regulatory genes in hepatic cancer cells A. Real-time PCR analysis was performed for Bcl-xl , Bcl-2 , Cdk2 and Cdk4 with total RNA extracted from HepG2, human HepXR and mouse HepXR cell lines for various cell cycle and apoptosis regulatory genes. β-Actin served as an internal control. B. Higher cellular expression of PXR increased the doubling time in human HepXR and mouse HepXR cells in comparison to HepG2 cells. The experiments were performed three times with three samples of each cell lines and the values are represented as the mean ±SE. The P-value represents the significance in human HepXR and mouse HepXR cell lines as compared to the control HepG2 cell line. P-value

    Journal: PLoS ONE

    Article Title: Role of PXR in Hepatic Cancer: Its Influences on Liver Detoxification Capacity and Cancer Progression

    doi: 10.1371/journal.pone.0164087

    Figure Lengend Snippet: PXR enhances expression of apoptotic genes while reduces expression of cell-cycle regulatory genes in hepatic cancer cells A. Real-time PCR analysis was performed for Bcl-xl , Bcl-2 , Cdk2 and Cdk4 with total RNA extracted from HepG2, human HepXR and mouse HepXR cell lines for various cell cycle and apoptosis regulatory genes. β-Actin served as an internal control. B. Higher cellular expression of PXR increased the doubling time in human HepXR and mouse HepXR cells in comparison to HepG2 cells. The experiments were performed three times with three samples of each cell lines and the values are represented as the mean ±SE. The P-value represents the significance in human HepXR and mouse HepXR cell lines as compared to the control HepG2 cell line. P-value

    Article Snippet: Chemicals TRI-reagent for RNA isolation, plasmid/DNA transfection reagents Escort III/Lipofectamine, DEN (Di-ethyl-nitrosamine), Dulbecco’s Modified Eagle’s Medium (DMEM) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Assessment of expression levels of nuclear receptor PXR, CAR and RXR-α in DEN-induced hepatic cancer. A. Real-time PCR analysis was performed for PXR, CAR and RXR-α genes with the total RNA extracted from mouse liver tissues of saline-treated control and DEN-induced hepatic cancer mice. GAPDH served as an internal control. B. Cell lysate of control and hepatic cancerous mice liver tissues were electrophoresed (50 μg per sample) on a 10% SDS-PAGE. The proteins were transferred onto the methanol-activated PVDF membrane and probed with anti-mouse PXR antibody, anti-mouse CAR antibody and anti-mouse RXR-α antibody. β-Actin antibody served as control; C. The relative endogenous protein expression levels of PXR, CAR and RXR-α in control and DEN-induced hepatic cancer were quantified by densitometry. The experiments were performed with six samples of each control and DEN-induced cancerous mice. The values are represented the mean ±SE. The P-value represents the significance in DEN-induced hepatic cancer mice as compared to the control mice. P-value

    Journal: PLoS ONE

    Article Title: Role of PXR in Hepatic Cancer: Its Influences on Liver Detoxification Capacity and Cancer Progression

    doi: 10.1371/journal.pone.0164087

    Figure Lengend Snippet: Assessment of expression levels of nuclear receptor PXR, CAR and RXR-α in DEN-induced hepatic cancer. A. Real-time PCR analysis was performed for PXR, CAR and RXR-α genes with the total RNA extracted from mouse liver tissues of saline-treated control and DEN-induced hepatic cancer mice. GAPDH served as an internal control. B. Cell lysate of control and hepatic cancerous mice liver tissues were electrophoresed (50 μg per sample) on a 10% SDS-PAGE. The proteins were transferred onto the methanol-activated PVDF membrane and probed with anti-mouse PXR antibody, anti-mouse CAR antibody and anti-mouse RXR-α antibody. β-Actin antibody served as control; C. The relative endogenous protein expression levels of PXR, CAR and RXR-α in control and DEN-induced hepatic cancer were quantified by densitometry. The experiments were performed with six samples of each control and DEN-induced cancerous mice. The values are represented the mean ±SE. The P-value represents the significance in DEN-induced hepatic cancer mice as compared to the control mice. P-value

    Article Snippet: Chemicals TRI-reagent for RNA isolation, plasmid/DNA transfection reagents Escort III/Lipofectamine, DEN (Di-ethyl-nitrosamine), Dulbecco’s Modified Eagle’s Medium (DMEM) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

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

    Levels of inflammatory proteins are enhanced in DEN-induced hepatic cancer. A. Real-time PCR analysis was performed for Tnf-α , p65 , and Stat3 with total RNA extracted from mouse liver tissues of saline-treated control and DEN-induced hepatic cancer mice. GAPDH served as an internal control. B. Cell extracts of control and DEN-induced hepatic cancerous mice liver tissues were electrophoresed (50 μg per sample) on a 10% SDS-PAGE. The proteins were transferred onto the methanol-activated PVDF membrane and probed with anti-mouse TNF-α, anti-mouse P65, and anti-mouse IL-6 antibody. Bands of human and mouse P65 were detected at 65 KDa and 60 KDa respectively. β-Actin antibody served as control; C. The relative endogenous TNF-α, P65 and IL-6 protein expressions in control and DEN-induced hepatic cancer were quantified by densitometry. The experiments were performed with seven samples of each control and DEN-induced cancerous mice, and the values are represented as the mean ±SE. The P-value represents the significance in DEN-induced hepatic cancer mice as compared to the control mice. P-value

    Journal: PLoS ONE

    Article Title: Role of PXR in Hepatic Cancer: Its Influences on Liver Detoxification Capacity and Cancer Progression

    doi: 10.1371/journal.pone.0164087

    Figure Lengend Snippet: Levels of inflammatory proteins are enhanced in DEN-induced hepatic cancer. A. Real-time PCR analysis was performed for Tnf-α , p65 , and Stat3 with total RNA extracted from mouse liver tissues of saline-treated control and DEN-induced hepatic cancer mice. GAPDH served as an internal control. B. Cell extracts of control and DEN-induced hepatic cancerous mice liver tissues were electrophoresed (50 μg per sample) on a 10% SDS-PAGE. The proteins were transferred onto the methanol-activated PVDF membrane and probed with anti-mouse TNF-α, anti-mouse P65, and anti-mouse IL-6 antibody. Bands of human and mouse P65 were detected at 65 KDa and 60 KDa respectively. β-Actin antibody served as control; C. The relative endogenous TNF-α, P65 and IL-6 protein expressions in control and DEN-induced hepatic cancer were quantified by densitometry. The experiments were performed with seven samples of each control and DEN-induced cancerous mice, and the values are represented as the mean ±SE. The P-value represents the significance in DEN-induced hepatic cancer mice as compared to the control mice. P-value

    Article Snippet: Chemicals TRI-reagent for RNA isolation, plasmid/DNA transfection reagents Escort III/Lipofectamine, DEN (Di-ethyl-nitrosamine), Dulbecco’s Modified Eagle’s Medium (DMEM) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Real-time Polymerase Chain Reaction, Mouse Assay, SDS Page

    Altered expression of PXR-regulated genes in DEN-induced hepatic cancer. A. Total RNA was extracted from mouse liver tissues of saline-treated control and DEN-induced hepatic cancer mice. Real-time PCR analysis was performed for Cyp3A11 , Gsta2 and Mrp3 . For Mdr1 semi-quantitative PCR was performed. GAPDH served as an internal control. B. Cell lysates of control and hepatic cancerous mice liver tissues were electrophoresed (50 μg per sample) on a 10% SDS-PAGE. The proteins were transferred onto the methanol-activated PVDF membrane and probed with anti-mouse Cyp3a11 antibody (upper panel). β-Actin antibody served as control (lower panel); C. The relative endogenous Cyp3a11 protein expression in control and DEN-induced hepatic cancer was quantified by densitometry. The experiments were performed with six samples of each control and DEN-induced cancerous mice, and the values are represented as the mean ±SE. The P-value represents the significance in DEN-induced hepatic cancer mice as compared to the control mice. The P-value

    Journal: PLoS ONE

    Article Title: Role of PXR in Hepatic Cancer: Its Influences on Liver Detoxification Capacity and Cancer Progression

    doi: 10.1371/journal.pone.0164087

    Figure Lengend Snippet: Altered expression of PXR-regulated genes in DEN-induced hepatic cancer. A. Total RNA was extracted from mouse liver tissues of saline-treated control and DEN-induced hepatic cancer mice. Real-time PCR analysis was performed for Cyp3A11 , Gsta2 and Mrp3 . For Mdr1 semi-quantitative PCR was performed. GAPDH served as an internal control. B. Cell lysates of control and hepatic cancerous mice liver tissues were electrophoresed (50 μg per sample) on a 10% SDS-PAGE. The proteins were transferred onto the methanol-activated PVDF membrane and probed with anti-mouse Cyp3a11 antibody (upper panel). β-Actin antibody served as control (lower panel); C. The relative endogenous Cyp3a11 protein expression in control and DEN-induced hepatic cancer was quantified by densitometry. The experiments were performed with six samples of each control and DEN-induced cancerous mice, and the values are represented as the mean ±SE. The P-value represents the significance in DEN-induced hepatic cancer mice as compared to the control mice. The P-value

    Article Snippet: Chemicals TRI-reagent for RNA isolation, plasmid/DNA transfection reagents Escort III/Lipofectamine, DEN (Di-ethyl-nitrosamine), Dulbecco’s Modified Eagle’s Medium (DMEM) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

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

    Transient enforced Mina elevation impairs Il4 expression in CD4 + T cells. CD4 + T cells isolated from three independent Mina transgenic lines (Lines #6, #38 and #21; closed symbols, 2 mice each) and their corresponding littermate controls (open symbols, 2 mice each) were stimulated with antibodies to the TCR and CD28. RNA harvested at the indicated times post-stimulation was analyzed for Mina , Il4 , Il2 and Ifng by quantitative real-time RT-PCR. Data, expressed as relative expression ( Actb normalized, arbitrary units), are from two independent experiments. Each trace represents an individual mouse.

    Journal: Nature immunology

    Article Title: MINA, an IL-4 repressor controls TH2 bias

    doi: 10.1038/ni.1747

    Figure Lengend Snippet: Transient enforced Mina elevation impairs Il4 expression in CD4 + T cells. CD4 + T cells isolated from three independent Mina transgenic lines (Lines #6, #38 and #21; closed symbols, 2 mice each) and their corresponding littermate controls (open symbols, 2 mice each) were stimulated with antibodies to the TCR and CD28. RNA harvested at the indicated times post-stimulation was analyzed for Mina , Il4 , Il2 and Ifng by quantitative real-time RT-PCR. Data, expressed as relative expression ( Actb normalized, arbitrary units), are from two independent experiments. Each trace represents an individual mouse.

    Article Snippet: Splenic CD4+ T cells, isolated by MACS (CD4+ T cell Isolation kit, Miltenyi), were stimulated with plate-bound anti-TCRβ (1 μg/ml) and anti-CD28 (10 μg/ml) antibodies for 16 h prior to harvesting RNA (Tri reagent, Sigma-Aldrich) and determining Il4 and Hprt1 expression by reverse transcriptase real time PCR, as described , .

    Techniques: Expressing, Isolation, Transgenic Assay, Mouse Assay, Quantitative RT-PCR

    Germ-free mice exhibit low numbers of intestinal mast cells, low expression of Cxcr2 and its ligands, and low levels of Mast cell protease-1 after OVA-sensitization and challenge. (A) Histological staining of jejunal sections for mastocytosis by hematoxylin/pararosaniline was performed on samples from control conventional (CV/ctrl) and germ-free (GF/ctrl), and from OVA-treated CV (CV/OVA) and germ-free (GF/OVA) mice (scale bars, 100 μm; inset scale bars, 25 μm) (B) , Quantification of mast cells per villus in jejunal sections (CV/ctrl n = 3, CV/OVA n = 8, GF/ctrl n = 5, GF/OVA n = 5 mice per group). Messenger RNA expression of (C) , Cxcr2 (D) , Cxcl1 , and (E) , Cxcl2 (in the jejunal tissues was determined by real-time PCR. Relative expression to β-actin is shown (CV/ctrl n = 6, CV/OVA n = 7, GF/ctrl n = 6, GF/OVA n = 5 mice per group). Mast cell protease-1 (MCPT-1) levels were determined in (F) , jejunal homogenates and in (G) , sera of control conventional (CV; gray bars) and germ-free (GF; dashed bars) mice, and in OVA-treated CV (black bars), and GF (white bars) mice by ELISA. Data are plotted as mean values ± SEM. Pooled values of at least two independent experiments (CV/ctrl n = 8, CV/OVA n = 14, GF/ctrl n = 9, GF/OVA n = 11 mice per group) are shown. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

    Journal: Frontiers in Immunology

    Article Title: Germ-Free Mice Exhibit Mast Cells With Impaired Functionality and Gut Homing and Do Not Develop Food Allergy

    doi: 10.3389/fimmu.2019.00205

    Figure Lengend Snippet: Germ-free mice exhibit low numbers of intestinal mast cells, low expression of Cxcr2 and its ligands, and low levels of Mast cell protease-1 after OVA-sensitization and challenge. (A) Histological staining of jejunal sections for mastocytosis by hematoxylin/pararosaniline was performed on samples from control conventional (CV/ctrl) and germ-free (GF/ctrl), and from OVA-treated CV (CV/OVA) and germ-free (GF/OVA) mice (scale bars, 100 μm; inset scale bars, 25 μm) (B) , Quantification of mast cells per villus in jejunal sections (CV/ctrl n = 3, CV/OVA n = 8, GF/ctrl n = 5, GF/OVA n = 5 mice per group). Messenger RNA expression of (C) , Cxcr2 (D) , Cxcl1 , and (E) , Cxcl2 (in the jejunal tissues was determined by real-time PCR. Relative expression to β-actin is shown (CV/ctrl n = 6, CV/OVA n = 7, GF/ctrl n = 6, GF/OVA n = 5 mice per group). Mast cell protease-1 (MCPT-1) levels were determined in (F) , jejunal homogenates and in (G) , sera of control conventional (CV; gray bars) and germ-free (GF; dashed bars) mice, and in OVA-treated CV (black bars), and GF (white bars) mice by ELISA. Data are plotted as mean values ± SEM. Pooled values of at least two independent experiments (CV/ctrl n = 8, CV/OVA n = 14, GF/ctrl n = 9, GF/OVA n = 11 mice per group) are shown. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

    Article Snippet: RNA Isolation and Real-Time PCR Jejunal tissues were stored in RNA-later reagent (Sigma-Aldrich, USA) overnight at 4°C and kept at −80°C until processed.

    Techniques: Mouse Assay, Expressing, Staining, RNA Expression, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Germ-free mice have less mature mast cells and these cells are functionally impaired. (A) Messenger RNA expression of Stem cell factor ( Scf ) in the jejunal tissues was determined by real-time PCR. Relative expression to β-actin is shown. CV/ctrl (gray bars) n = 5, GF/ctrl (dashed bars) n = 6, CV/OVA (black bars) n = 5, GF/OVA (white bars) n = 5 mice per group. (B) Mean fluorescence intensity of CD117 was determined by flow cytometry on CD45+FcεRIα+ cells isolated from small intestine. CV (gray bars) n = 3, GF (dashed bars) n = 3 mice per group. (C) GF and CV mice were injected with degranulation-inducing compound 48/80 or PBS and edema was recorded. Data are presented as paw width after subtraction of baseline values, GF 48/80 (black circles) n = 6, CV 48/80 (open circles) n = 8, GF PBS (open squares) n = 5, CV PBS (gray squares) n = 8 mice per group. Data are plotted as mean values ± SEM. * P ≤ 0.05, ** P ≤ 0.01, and **** P ≤ 0.0001.

    Journal: Frontiers in Immunology

    Article Title: Germ-Free Mice Exhibit Mast Cells With Impaired Functionality and Gut Homing and Do Not Develop Food Allergy

    doi: 10.3389/fimmu.2019.00205

    Figure Lengend Snippet: Germ-free mice have less mature mast cells and these cells are functionally impaired. (A) Messenger RNA expression of Stem cell factor ( Scf ) in the jejunal tissues was determined by real-time PCR. Relative expression to β-actin is shown. CV/ctrl (gray bars) n = 5, GF/ctrl (dashed bars) n = 6, CV/OVA (black bars) n = 5, GF/OVA (white bars) n = 5 mice per group. (B) Mean fluorescence intensity of CD117 was determined by flow cytometry on CD45+FcεRIα+ cells isolated from small intestine. CV (gray bars) n = 3, GF (dashed bars) n = 3 mice per group. (C) GF and CV mice were injected with degranulation-inducing compound 48/80 or PBS and edema was recorded. Data are presented as paw width after subtraction of baseline values, GF 48/80 (black circles) n = 6, CV 48/80 (open circles) n = 8, GF PBS (open squares) n = 5, CV PBS (gray squares) n = 8 mice per group. Data are plotted as mean values ± SEM. * P ≤ 0.05, ** P ≤ 0.01, and **** P ≤ 0.0001.

    Article Snippet: RNA Isolation and Real-Time PCR Jejunal tissues were stored in RNA-later reagent (Sigma-Aldrich, USA) overnight at 4°C and kept at −80°C until processed.

    Techniques: Mouse Assay, RNA Expression, Real-time Polymerase Chain Reaction, Expressing, Fluorescence, Flow Cytometry, Cytometry, Isolation, Injection

    FZ alters glucose uptake and impairs enzymatic activity of HKII in NSCLC cells. ( a ) A549 or H460 cells were treated with 1 uM FZ for 4 h and uptake of the fluorescent glucose derivative 2-NBDG was examined thereafter by fluorescence microscopy as described. Representative images of cells from three independent experiments are shown. ( b ) Human NSCLC H460 cells were exposed to increasing doses of FZ for 24 h. Culture supernatants were then used to assess glucose consumption by glucose oxidation assay using GO assay kit from Sigma. ( c ) H460 cells were left untreated or treated with 1 uM FZ and the lactate levels in culture supernatants were assessed after the indicated time points using Lactate Assay Kit from BioVision. ( d ) Human H460 cells were exposed to 1 uM FZ for 24 or 48 h as indicated, total RNA was isolated and RT-PCR was performed using primers specific for the indicated genes. ( e ) (i) H460 cells were left untreated or treated with 1 uM FZ for 24 h and the cell extracts were then processed for a spectophotometric assay for SDH (A 630nm ). (ii) A549 cells were left untreated or treated with 1 uM FZ for 20 h following which they were processed for histochemical assay to assess SDH activity. Cells were then observed under a microscope and images were acquired at 40X magnification. ( f ) H460 cells were left untreated or treated with 1 uM FZ for 24 h. HK enzymatic activity was then determined spectrophotometrically as described under “Materials and Methods”. ( g ) H460 and A549 cell lysates were incubated with DMSO or FZ for 15 min prior to initiation of reaction. HK enzymatic activity was then determined spectrophotometrically as described under “Materials and Methods”. ( h ) Purified HKII from S. cerevisae was incubated with increasing doses of FZ and HK activity was then measured spectrophotometrically. (* p

    Journal: Scientific Reports

    Article Title: Fenbendazole acts as a moderate microtubule destabilizing agent and causes cancer cell death by modulating multiple cellular pathways

    doi: 10.1038/s41598-018-30158-6

    Figure Lengend Snippet: FZ alters glucose uptake and impairs enzymatic activity of HKII in NSCLC cells. ( a ) A549 or H460 cells were treated with 1 uM FZ for 4 h and uptake of the fluorescent glucose derivative 2-NBDG was examined thereafter by fluorescence microscopy as described. Representative images of cells from three independent experiments are shown. ( b ) Human NSCLC H460 cells were exposed to increasing doses of FZ for 24 h. Culture supernatants were then used to assess glucose consumption by glucose oxidation assay using GO assay kit from Sigma. ( c ) H460 cells were left untreated or treated with 1 uM FZ and the lactate levels in culture supernatants were assessed after the indicated time points using Lactate Assay Kit from BioVision. ( d ) Human H460 cells were exposed to 1 uM FZ for 24 or 48 h as indicated, total RNA was isolated and RT-PCR was performed using primers specific for the indicated genes. ( e ) (i) H460 cells were left untreated or treated with 1 uM FZ for 24 h and the cell extracts were then processed for a spectophotometric assay for SDH (A 630nm ). (ii) A549 cells were left untreated or treated with 1 uM FZ for 20 h following which they were processed for histochemical assay to assess SDH activity. Cells were then observed under a microscope and images were acquired at 40X magnification. ( f ) H460 cells were left untreated or treated with 1 uM FZ for 24 h. HK enzymatic activity was then determined spectrophotometrically as described under “Materials and Methods”. ( g ) H460 and A549 cell lysates were incubated with DMSO or FZ for 15 min prior to initiation of reaction. HK enzymatic activity was then determined spectrophotometrically as described under “Materials and Methods”. ( h ) Purified HKII from S. cerevisae was incubated with increasing doses of FZ and HK activity was then measured spectrophotometrically. (* p

    Article Snippet: RT-PCR and qPCR Cells were treated with FZ as indicated and total RNA was extracted using TRI reagent from Sigma.

    Techniques: Activity Assay, Fluorescence, Microscopy, Oxidation Assay, Lactate Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Incubation, Purification

    Prediction of RNA motifs in cell cycle co-regulated gene clusters. Left panel: Heatmap representation of the expression of 305-cell cycle genes. The gene expression values (log 2 normalized read counts) were subjected to a clustering analysis in the GENE-E software (Broad Institute). Both columns (samples) and rows (genes) were clustered by Pearson correlation. Right panel: the gene expression profile in the three sequenced cell cycle phases was plotted for each cluster. Consensus sequence motifs found exclusively at the 3’ UTRs are presented right to each cluster, and the coverage and the p-value obtained from the DREME search is indicated. Consensus structural motifs found with CMfinder are presented on the right most column, and their coverage, specificity and free energy are indicated. The structural motifs were all found at the 5 ‘UTR except for the motif found in cluster 7. None of the motifs found in cluster 6 reach the thresholds. The best motif for each cluster is depicted.

    Journal: PLoS ONE

    Article Title: Transcriptome-wide analysis of the Trypanosoma cruzi proliferative cycle identifies the periodically expressed mRNAs and their multiple levels of control

    doi: 10.1371/journal.pone.0188441

    Figure Lengend Snippet: Prediction of RNA motifs in cell cycle co-regulated gene clusters. Left panel: Heatmap representation of the expression of 305-cell cycle genes. The gene expression values (log 2 normalized read counts) were subjected to a clustering analysis in the GENE-E software (Broad Institute). Both columns (samples) and rows (genes) were clustered by Pearson correlation. Right panel: the gene expression profile in the three sequenced cell cycle phases was plotted for each cluster. Consensus sequence motifs found exclusively at the 3’ UTRs are presented right to each cluster, and the coverage and the p-value obtained from the DREME search is indicated. Consensus structural motifs found with CMfinder are presented on the right most column, and their coverage, specificity and free energy are indicated. The structural motifs were all found at the 5 ‘UTR except for the motif found in cluster 7. None of the motifs found in cluster 6 reach the thresholds. The best motif for each cluster is depicted.

    Article Snippet: The obtained RNA was treated with DNAse, according to manufacturer’s protocol (DNA-free, Ambion) and quantified in a Nanodrop spectrometer (Thermo Scientific, USA). cDNA was synthetized from 1 μg of total RNA using Superscript™ III first strand synthesis kit (Invitrogen™) and random primers.

    Techniques: Expressing, Software, Sequencing

    Comparative analysis of the samples and the resulting RNA-sequencing datasets. A. Principal Component Analysis of the individual replicates (ΔΔCt values relative to tubulin, obtained by RT-qPCR) and the pooled samples used for sequencing (nCounts normalized by tubulin, obtained by RNA-Seq), based on the quantification of 21 selected differentially expressed genes (see S2 Table ). B. Phase specific gene expression values compared in a ternary plot. Gene expression was denoted as the gene expression value in each cell cycle stage relative to the total additive expression of the three stages. Thus, each gene is represented by three fractions, one per stage, which together adds 1. The values were plotted in R using the ggtern library. Pearson correlation coefficients (r 2 ) were calculated for every set of 2 cell cycle phases and are presented next to the corresponding connecting arrow.

    Journal: PLoS ONE

    Article Title: Transcriptome-wide analysis of the Trypanosoma cruzi proliferative cycle identifies the periodically expressed mRNAs and their multiple levels of control

    doi: 10.1371/journal.pone.0188441

    Figure Lengend Snippet: Comparative analysis of the samples and the resulting RNA-sequencing datasets. A. Principal Component Analysis of the individual replicates (ΔΔCt values relative to tubulin, obtained by RT-qPCR) and the pooled samples used for sequencing (nCounts normalized by tubulin, obtained by RNA-Seq), based on the quantification of 21 selected differentially expressed genes (see S2 Table ). B. Phase specific gene expression values compared in a ternary plot. Gene expression was denoted as the gene expression value in each cell cycle stage relative to the total additive expression of the three stages. Thus, each gene is represented by three fractions, one per stage, which together adds 1. The values were plotted in R using the ggtern library. Pearson correlation coefficients (r 2 ) were calculated for every set of 2 cell cycle phases and are presented next to the corresponding connecting arrow.

    Article Snippet: The obtained RNA was treated with DNAse, according to manufacturer’s protocol (DNA-free, Ambion) and quantified in a Nanodrop spectrometer (Thermo Scientific, USA). cDNA was synthetized from 1 μg of total RNA using Superscript™ III first strand synthesis kit (Invitrogen™) and random primers.

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