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    Thermo Fisher superscript iii reverse transcriptase
    Heatmap of differentially expressed genes detected by <t>RNA-seq</t> in adult S . mansoni female and male worms treated with GSK343. The heatmaps show the hierarchical clustering of differentially expressed genes (lines) in <t>three</t> biological replicates (columns) of female (A) and male (B) adult worm samples, either for controls or for treated parasites, as indicated at the top of the heatmaps. Parasites were exposed for 48 h in vitro to vehicle (control) or to 20 μM GSK343. Gene expression levels were measured by RNA-seq and are shown as Z-scores, which are the number of standard deviations below (blue, downregulated) or above (red, upregulated) the mean expression value among treated and control samples for each gene; the expression level Z-scores are color-coded as indicated on the scale at the bottom.
    Superscript Iii Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii reverse transcriptase/product/Thermo Fisher
    Average 78 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    superscript iii reverse transcriptase - by Bioz Stars, 2020-01
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    99
    Thermo Fisher superscript iii reverse transcriptase kit
    Effects of AAs, 20E, insulin and HR3 on YPP genes. (A) Relative expression of gene—AAEL006563, Carboxypeptidase, detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (B) Relative expression of the same gene detected by qRT-PCR, in fat body tissues collected from female mosquitoes post EcR knock-down (iEcR). (C) Relative expression in tissues subjected IVFBC in culture media without (NT) and with amino acids (AA), with amino acids plus 20E (AA+20E) and after the withdrawal of 20E (20E WD). (D) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post HR3 knock-down (iHR3). (E) Relative expression of the same gene in tissues subjected to IVFBC in culture media without (NT) and with amino acids (AA), with amino acids and Insulin (AA+INS), Insulin and 20E (INS+20E), amino acids plus 20E (AA+20E), and amino acids plus 20E and Insulin (AA+20E+INS). Injecting double stranded <t>RNA</t> for the Luciferase gene (iluc) served as the control in the RNAi experiments ( B and D ). All expression calculated against housekeeping gene RPS7. Data representative of <t>three</t> biological replicates, with three technical replicates and are illustrated as average ± SD, * P
    Superscript Iii Reverse Transcriptase Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5021 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii reverse transcriptase kit/product/Thermo Fisher
    Average 99 stars, based on 5021 article reviews
    Price from $9.99 to $1999.99
    superscript iii reverse transcriptase kit - by Bioz Stars, 2020-01
    99/100 stars
      Buy from Supplier

    Image Search Results


    Heatmap of differentially expressed genes detected by RNA-seq in adult S . mansoni female and male worms treated with GSK343. The heatmaps show the hierarchical clustering of differentially expressed genes (lines) in three biological replicates (columns) of female (A) and male (B) adult worm samples, either for controls or for treated parasites, as indicated at the top of the heatmaps. Parasites were exposed for 48 h in vitro to vehicle (control) or to 20 μM GSK343. Gene expression levels were measured by RNA-seq and are shown as Z-scores, which are the number of standard deviations below (blue, downregulated) or above (red, upregulated) the mean expression value among treated and control samples for each gene; the expression level Z-scores are color-coded as indicated on the scale at the bottom.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Inhibition of histone methyltransferase EZH2 in Schistosoma mansoni in vitro by GSK343 reduces egg laying and decreases the expression of genes implicated in DNA replication and noncoding RNA metabolism

    doi: 10.1371/journal.pntd.0006873

    Figure Lengend Snippet: Heatmap of differentially expressed genes detected by RNA-seq in adult S . mansoni female and male worms treated with GSK343. The heatmaps show the hierarchical clustering of differentially expressed genes (lines) in three biological replicates (columns) of female (A) and male (B) adult worm samples, either for controls or for treated parasites, as indicated at the top of the heatmaps. Parasites were exposed for 48 h in vitro to vehicle (control) or to 20 μM GSK343. Gene expression levels were measured by RNA-seq and are shown as Z-scores, which are the number of standard deviations below (blue, downregulated) or above (red, upregulated) the mean expression value among treated and control samples for each gene; the expression level Z-scores are color-coded as indicated on the scale at the bottom.

    Article Snippet: For quantitative RT-PCR, complementary DNAs were obtained by reverse transcription (RT) of 100 ng schistosomula or adult worms total RNA using SuperScript III Reverse Transcriptase (Invitrogen) and random hexamer primers in a 20 μL volume, according to the manufacturer’s recommendations.

    Techniques: RNA Sequencing Assay, In Vitro, Expressing

    Genes related to DNA replication that were downregulated in GSK343 treated female adult worms. (A) Heatmap of 32 genes related to DNA replication that were downregulated in treated females. Genes are shown on the lines, and three biological replicates are shown in the columns, for control (turquoise bar at the top of columns) or GSK343-treated female samples (yellow bar at the top of columns). Parasites were exposed for 48 h in vitro to vehicle (control) or to 20 μM GSK343. Gene expression levels were measured by RNA-seq and are shown as Z-scores, which are the number of standard deviations below (blue, downregulated) or above (red, upregulated) the mean expression value among treated and control samples for each gene; the expression level Z-scores are color-coded as indicated on the scales at the bottom. The heatmap was obtained with unsupervised clustering of samples and genes. (B) Principal component analysis (PCA) of expression of the 32 genes from (A) that were measured by RNA-seq in control and treated females, males and schistosomula, as indicated by the legend at the bottom.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Inhibition of histone methyltransferase EZH2 in Schistosoma mansoni in vitro by GSK343 reduces egg laying and decreases the expression of genes implicated in DNA replication and noncoding RNA metabolism

    doi: 10.1371/journal.pntd.0006873

    Figure Lengend Snippet: Genes related to DNA replication that were downregulated in GSK343 treated female adult worms. (A) Heatmap of 32 genes related to DNA replication that were downregulated in treated females. Genes are shown on the lines, and three biological replicates are shown in the columns, for control (turquoise bar at the top of columns) or GSK343-treated female samples (yellow bar at the top of columns). Parasites were exposed for 48 h in vitro to vehicle (control) or to 20 μM GSK343. Gene expression levels were measured by RNA-seq and are shown as Z-scores, which are the number of standard deviations below (blue, downregulated) or above (red, upregulated) the mean expression value among treated and control samples for each gene; the expression level Z-scores are color-coded as indicated on the scales at the bottom. The heatmap was obtained with unsupervised clustering of samples and genes. (B) Principal component analysis (PCA) of expression of the 32 genes from (A) that were measured by RNA-seq in control and treated females, males and schistosomula, as indicated by the legend at the bottom.

    Article Snippet: For quantitative RT-PCR, complementary DNAs were obtained by reverse transcription (RT) of 100 ng schistosomula or adult worms total RNA using SuperScript III Reverse Transcriptase (Invitrogen) and random hexamer primers in a 20 μL volume, according to the manufacturer’s recommendations.

    Techniques: In Vitro, Expressing, RNA Sequencing Assay

    Morphological changes and increased apoptosis in schistosomula exposed to 20 μM GSK343. (A) Scanning electron microscopy of schistosomula; note that the size bar is shown within the black thin line below each image. (A, panels 1 and 2) Control schistosomula micrographs at the same magnification (Bar = 40 μm) showing the ventral sucker ( vs ) of typical schistosomula maintained for 16 h in culture medium (panel 1) or further incubated for 24 h in culture medium plus vehicle (0.1% DMSO) (panel 2); the lateral and ventral aspects are normal, and the schistosomula underwent normal development and lengthening after 24 h incubation in vehicle-containing medium (compare panels 2 and 1). (A, panel 3) Higher magnification of control schistosomulum from panel 2 showing that the entire body surface is covered by spines with no ultrastructural changes (Bar = 3 μm); (A, panels 4 and 5) Longer incubation (48 h) of control schistosomula in vehicle-containing medium shows further development and growth (panel 4, Bar = 50 μm) compared with panel 2; and a higher magnification (panel 5, Bar = 3 μm) confirms that the surface is covered by spines with no ultrastructural changes. (A, panels 6 and 7) Schistosomula micrographs at the same magnification (Bar = 30 μm) showing the ventral sucker ( vs ) of typical schistosomula maintained for 16 h in culture medium (panel 6) or further incubated for 24 h in culture medium plus 20 μM GSK343 (panel 7); after 24 h in the presence of GSK343 the schistosomulum showed no apparent development or change in size, with an extremely contracted body region and the presence of numerous folds and small blisters (panel 7). (A, panel 8) Higher magnification of schistosomulum from panel 7 is an example showing that the teguments of parasites exposed to 20 μM GSK343 for 24 h have a high degree of changes including peeling and blister formation (Bar = 3 μm). (A, panels 9 and 10) Longer incubation of schistosomula (48 h) in culture medium plus 20 μM GSK343 showed no further development (panel 9) compared with the initial schistosomulum (panel 6), with the presence of a greater number of blisters when compared with panel 7; panel 10 shows a higher magnification of the schistosomulum from panel 9, with intense peeling of tegument and loss of numerous spines (Bar = 5 μm). ( B) Total length of schistosomula incubated with vehicle (0.1% DMSO) or with 20 μM GSK343 for 24 and 48h. Mean ± SD from three replicate experiments, each with 20 schistosomula. ( C) Increase in caspases 3/7 activity after 48 h treatment with 20 μM GSK343. Mean ± SD from three replicate experiments, each with 20,000 schistosomula. (D, panels 1 to 4) TUNEL assay of schistosomula incubated for 72 h with 0.1% DMSO (panels 1 and 2) or with 20μM GSK343 (panels 3 and 4). Green parasites indicate DNA fragmentation. In panel 1, DAPI nuclear DNA staining of control parasites (0.1% DMSO). In panel 2, TUNEL staining of control parasites from panel 1 (0.1% DMSO). In panel 3, DAPI nuclear DNA staining of GSK343 treated parasites. In panel 4, TUNEL staining of GSK343 treated parasites from panel 3. ( vs ), ventral sucker; ( s) , spines; ( b ), blisters. ***p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Inhibition of histone methyltransferase EZH2 in Schistosoma mansoni in vitro by GSK343 reduces egg laying and decreases the expression of genes implicated in DNA replication and noncoding RNA metabolism

    doi: 10.1371/journal.pntd.0006873

    Figure Lengend Snippet: Morphological changes and increased apoptosis in schistosomula exposed to 20 μM GSK343. (A) Scanning electron microscopy of schistosomula; note that the size bar is shown within the black thin line below each image. (A, panels 1 and 2) Control schistosomula micrographs at the same magnification (Bar = 40 μm) showing the ventral sucker ( vs ) of typical schistosomula maintained for 16 h in culture medium (panel 1) or further incubated for 24 h in culture medium plus vehicle (0.1% DMSO) (panel 2); the lateral and ventral aspects are normal, and the schistosomula underwent normal development and lengthening after 24 h incubation in vehicle-containing medium (compare panels 2 and 1). (A, panel 3) Higher magnification of control schistosomulum from panel 2 showing that the entire body surface is covered by spines with no ultrastructural changes (Bar = 3 μm); (A, panels 4 and 5) Longer incubation (48 h) of control schistosomula in vehicle-containing medium shows further development and growth (panel 4, Bar = 50 μm) compared with panel 2; and a higher magnification (panel 5, Bar = 3 μm) confirms that the surface is covered by spines with no ultrastructural changes. (A, panels 6 and 7) Schistosomula micrographs at the same magnification (Bar = 30 μm) showing the ventral sucker ( vs ) of typical schistosomula maintained for 16 h in culture medium (panel 6) or further incubated for 24 h in culture medium plus 20 μM GSK343 (panel 7); after 24 h in the presence of GSK343 the schistosomulum showed no apparent development or change in size, with an extremely contracted body region and the presence of numerous folds and small blisters (panel 7). (A, panel 8) Higher magnification of schistosomulum from panel 7 is an example showing that the teguments of parasites exposed to 20 μM GSK343 for 24 h have a high degree of changes including peeling and blister formation (Bar = 3 μm). (A, panels 9 and 10) Longer incubation of schistosomula (48 h) in culture medium plus 20 μM GSK343 showed no further development (panel 9) compared with the initial schistosomulum (panel 6), with the presence of a greater number of blisters when compared with panel 7; panel 10 shows a higher magnification of the schistosomulum from panel 9, with intense peeling of tegument and loss of numerous spines (Bar = 5 μm). ( B) Total length of schistosomula incubated with vehicle (0.1% DMSO) or with 20 μM GSK343 for 24 and 48h. Mean ± SD from three replicate experiments, each with 20 schistosomula. ( C) Increase in caspases 3/7 activity after 48 h treatment with 20 μM GSK343. Mean ± SD from three replicate experiments, each with 20,000 schistosomula. (D, panels 1 to 4) TUNEL assay of schistosomula incubated for 72 h with 0.1% DMSO (panels 1 and 2) or with 20μM GSK343 (panels 3 and 4). Green parasites indicate DNA fragmentation. In panel 1, DAPI nuclear DNA staining of control parasites (0.1% DMSO). In panel 2, TUNEL staining of control parasites from panel 1 (0.1% DMSO). In panel 3, DAPI nuclear DNA staining of GSK343 treated parasites. In panel 4, TUNEL staining of GSK343 treated parasites from panel 3. ( vs ), ventral sucker; ( s) , spines; ( b ), blisters. ***p

    Article Snippet: For quantitative RT-PCR, complementary DNAs were obtained by reverse transcription (RT) of 100 ng schistosomula or adult worms total RNA using SuperScript III Reverse Transcriptase (Invitrogen) and random hexamer primers in a 20 μL volume, according to the manufacturer’s recommendations.

    Techniques: Electron Microscopy, Incubation, Activity Assay, TUNEL Assay, Staining

    Histone H3 post-translational modification profile in S . mansoni after treatment with GSK343. Five different histone marks in adult worms (A to E) and in schistosomula (F to J) were detected and quantified by western blotting with (A and F) anti-H3K27me3, (B and G) anti-H3K9ac, (C and H) anti-H3K27ac, (D and I) anti-H3K4me3, (E and J) anti-H3K27me1 antibodies. Each panel consists of two parts: the upper part shows the lanes of a typical western blot of nuclear extract from untreated control parasites (C) or parasites treated for 48 h with 20 μM GSK343 (T) which were developed either with the specific antibody against the indicated histone mark or with an antibody anti-H3, used as a sample loading normalizer. A molecular weight marker protein (MW, 15 kDa) is indicated. The lower part of each panel shows the mean intensity of the bands for three biological replicates, obtained by extracting the intensity values of scanned images; for each sample, the intensity of the modified histone band was normalized by the intensity of histone H3. Mean ± SD is shown; t-test was applied and statistically significant p-values are indicated: *p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Inhibition of histone methyltransferase EZH2 in Schistosoma mansoni in vitro by GSK343 reduces egg laying and decreases the expression of genes implicated in DNA replication and noncoding RNA metabolism

    doi: 10.1371/journal.pntd.0006873

    Figure Lengend Snippet: Histone H3 post-translational modification profile in S . mansoni after treatment with GSK343. Five different histone marks in adult worms (A to E) and in schistosomula (F to J) were detected and quantified by western blotting with (A and F) anti-H3K27me3, (B and G) anti-H3K9ac, (C and H) anti-H3K27ac, (D and I) anti-H3K4me3, (E and J) anti-H3K27me1 antibodies. Each panel consists of two parts: the upper part shows the lanes of a typical western blot of nuclear extract from untreated control parasites (C) or parasites treated for 48 h with 20 μM GSK343 (T) which were developed either with the specific antibody against the indicated histone mark or with an antibody anti-H3, used as a sample loading normalizer. A molecular weight marker protein (MW, 15 kDa) is indicated. The lower part of each panel shows the mean intensity of the bands for three biological replicates, obtained by extracting the intensity values of scanned images; for each sample, the intensity of the modified histone band was normalized by the intensity of histone H3. Mean ± SD is shown; t-test was applied and statistically significant p-values are indicated: *p

    Article Snippet: For quantitative RT-PCR, complementary DNAs were obtained by reverse transcription (RT) of 100 ng schistosomula or adult worms total RNA using SuperScript III Reverse Transcriptase (Invitrogen) and random hexamer primers in a 20 μL volume, according to the manufacturer’s recommendations.

    Techniques: Modification, Western Blot, Molecular Weight, Marker

    Genes related to ncRNA metabolism, egg biosynthesis and cell differentiation that were downregulated in GSK343 treated female adult worms. (A) Heatmap of 51 genes related to ncRNA metabolism that were downregulated in treated females. The heatmap was obtained with unsupervised clustering of samples and genes. (B) Principle component analysis (PCA) of expression of the 51 genes from (A) that were measured by RNA-seq in control and treated females, males and schistosomula, as indicated by the legend at the bottom. (C) Heatmap of 9 selected genes, related to egg biosynthesis and cell differentiation, whose expression was significantly affected in GSK343-treated females. Heatmap obtained with unsupervised clustering of genes. In the heatmaps of (A) and (C), genes are shown on the lines, and three biological replicates are shown in the columns, for control (turquoise bar at the top of columns) or GSK343-treated female samples (yellow bar at the top of columns). Parasites were exposed for 48 h in vitro to vehicle (control) or to 20 μM GSK343. Gene expression levels were measured by RNA-seq and are shown as Z-scores, which are the number of standard deviations below (blue, downregulated) or above (red, upregulated) the mean expression value among treated and control samples for each gene; the expression level Z-scores are color-coded as indicated on the scales at the bottom.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Inhibition of histone methyltransferase EZH2 in Schistosoma mansoni in vitro by GSK343 reduces egg laying and decreases the expression of genes implicated in DNA replication and noncoding RNA metabolism

    doi: 10.1371/journal.pntd.0006873

    Figure Lengend Snippet: Genes related to ncRNA metabolism, egg biosynthesis and cell differentiation that were downregulated in GSK343 treated female adult worms. (A) Heatmap of 51 genes related to ncRNA metabolism that were downregulated in treated females. The heatmap was obtained with unsupervised clustering of samples and genes. (B) Principle component analysis (PCA) of expression of the 51 genes from (A) that were measured by RNA-seq in control and treated females, males and schistosomula, as indicated by the legend at the bottom. (C) Heatmap of 9 selected genes, related to egg biosynthesis and cell differentiation, whose expression was significantly affected in GSK343-treated females. Heatmap obtained with unsupervised clustering of genes. In the heatmaps of (A) and (C), genes are shown on the lines, and three biological replicates are shown in the columns, for control (turquoise bar at the top of columns) or GSK343-treated female samples (yellow bar at the top of columns). Parasites were exposed for 48 h in vitro to vehicle (control) or to 20 μM GSK343. Gene expression levels were measured by RNA-seq and are shown as Z-scores, which are the number of standard deviations below (blue, downregulated) or above (red, upregulated) the mean expression value among treated and control samples for each gene; the expression level Z-scores are color-coded as indicated on the scales at the bottom.

    Article Snippet: For quantitative RT-PCR, complementary DNAs were obtained by reverse transcription (RT) of 100 ng schistosomula or adult worms total RNA using SuperScript III Reverse Transcriptase (Invitrogen) and random hexamer primers in a 20 μL volume, according to the manufacturer’s recommendations.

    Techniques: Cell Differentiation, Expressing, RNA Sequencing Assay, In Vitro

    Gene Ontology terms enrichment analysis of downregulated genes detected by RNA-seq in adult S . mansoni female and male worms treated with GSK343. Top 20 enriched GO terms for differentially expressed downregulated genes in female (A) and in male (B) adult worms. The three major GO term categories, namely Biological Process, Cellular Component and Molecular Function are separately represented in each panel. The size of the circles is proportional to the number of genes in each significantly enriched category, as indicated at the lower part scales; the colors show the statistical significance of the enrichment, as indicated by the -log10 FDR values that appear in the color-coded scales at the bottom. A GO enrichment significance cutoff of FDR ≤ 0.05 was used.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Inhibition of histone methyltransferase EZH2 in Schistosoma mansoni in vitro by GSK343 reduces egg laying and decreases the expression of genes implicated in DNA replication and noncoding RNA metabolism

    doi: 10.1371/journal.pntd.0006873

    Figure Lengend Snippet: Gene Ontology terms enrichment analysis of downregulated genes detected by RNA-seq in adult S . mansoni female and male worms treated with GSK343. Top 20 enriched GO terms for differentially expressed downregulated genes in female (A) and in male (B) adult worms. The three major GO term categories, namely Biological Process, Cellular Component and Molecular Function are separately represented in each panel. The size of the circles is proportional to the number of genes in each significantly enriched category, as indicated at the lower part scales; the colors show the statistical significance of the enrichment, as indicated by the -log10 FDR values that appear in the color-coded scales at the bottom. A GO enrichment significance cutoff of FDR ≤ 0.05 was used.

    Article Snippet: For quantitative RT-PCR, complementary DNAs were obtained by reverse transcription (RT) of 100 ng schistosomula or adult worms total RNA using SuperScript III Reverse Transcriptase (Invitrogen) and random hexamer primers in a 20 μL volume, according to the manufacturer’s recommendations.

    Techniques: RNA Sequencing Assay

    Quantitation of the decrease in viability of schistosomula caused by GSK343 treatment at different concentrations and incubation times. (A) Schistosomula treated with the indicated concentrations of GSK343 or with vehicle (0.1% DMSO) for 24, 48 and 72 h were visualized by staining with propidium iodide (marker of dead cells; 572 nm emission filter microscope) and with fluorescein diacetate (marker of living cells; 492 nm emission filter microscope). The bottom row shows a positive control, namely exposure to 70% ethanol, which kills all parasites. For each time point (indicated at the top), the left panel shows a light microscopy image and the right panel shows the image of the same field with differential fluorescence detection of PI-positive dead and FDA-positive live schistosomula by superimposition of 536nm and 494nm epifluorescence spectra. (B) Quantitation of viability of treated schistosomula. Percentage of viable schistosomula (non-stained with propidium iodide) over three days of treatment. For each condition tested, about 3600 schistosomula were used, divided into four biological replicates and three time points analyzed. Mean ± SD from four replicate experiments. (C) ATP quantitation using a luminescent assay to assess schistosomula survival under GSK343 exposure. Schistosomula (100-120/well) were incubated with the indicated concentrations of GSK343 or with vehicle (0.1% DMSO) for up to 5 days. The viability was expressed as % the luminescence values relative to the control (DMSO). Mean ± SD from three replicate experiments. *p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Inhibition of histone methyltransferase EZH2 in Schistosoma mansoni in vitro by GSK343 reduces egg laying and decreases the expression of genes implicated in DNA replication and noncoding RNA metabolism

    doi: 10.1371/journal.pntd.0006873

    Figure Lengend Snippet: Quantitation of the decrease in viability of schistosomula caused by GSK343 treatment at different concentrations and incubation times. (A) Schistosomula treated with the indicated concentrations of GSK343 or with vehicle (0.1% DMSO) for 24, 48 and 72 h were visualized by staining with propidium iodide (marker of dead cells; 572 nm emission filter microscope) and with fluorescein diacetate (marker of living cells; 492 nm emission filter microscope). The bottom row shows a positive control, namely exposure to 70% ethanol, which kills all parasites. For each time point (indicated at the top), the left panel shows a light microscopy image and the right panel shows the image of the same field with differential fluorescence detection of PI-positive dead and FDA-positive live schistosomula by superimposition of 536nm and 494nm epifluorescence spectra. (B) Quantitation of viability of treated schistosomula. Percentage of viable schistosomula (non-stained with propidium iodide) over three days of treatment. For each condition tested, about 3600 schistosomula were used, divided into four biological replicates and three time points analyzed. Mean ± SD from four replicate experiments. (C) ATP quantitation using a luminescent assay to assess schistosomula survival under GSK343 exposure. Schistosomula (100-120/well) were incubated with the indicated concentrations of GSK343 or with vehicle (0.1% DMSO) for up to 5 days. The viability was expressed as % the luminescence values relative to the control (DMSO). Mean ± SD from three replicate experiments. *p

    Article Snippet: For quantitative RT-PCR, complementary DNAs were obtained by reverse transcription (RT) of 100 ng schistosomula or adult worms total RNA using SuperScript III Reverse Transcriptase (Invitrogen) and random hexamer primers in a 20 μL volume, according to the manufacturer’s recommendations.

    Techniques: Quantitation Assay, Incubation, Staining, Marker, Microscopy, Positive Control, Light Microscopy, Fluorescence, Luminescence Assay

    Gene Ontology terms enrichment analysis of upregulated genes detected by RNA-seq in adult S . mansoni male worms treated with GSK343. Top 20 enriched GO terms for differentially expressed upregulated genes in male adult worms. The three major GO term categories, namely Biological Process, Cellular Component and Molecular Function are separately represented in each panel. The size of the circles is proportional to the number of genes in each significantly enriched category, as indicated at the lower part scales; the colors show the statistical significance of the enrichment, as indicated by the -log10 FDR values that appear in the color-coded scales at the bottom. A GO enrichment significance cutoff of FDR ≤ 0.05 was used; no significantly enriched GO terms were found among the female upregulated genes.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Inhibition of histone methyltransferase EZH2 in Schistosoma mansoni in vitro by GSK343 reduces egg laying and decreases the expression of genes implicated in DNA replication and noncoding RNA metabolism

    doi: 10.1371/journal.pntd.0006873

    Figure Lengend Snippet: Gene Ontology terms enrichment analysis of upregulated genes detected by RNA-seq in adult S . mansoni male worms treated with GSK343. Top 20 enriched GO terms for differentially expressed upregulated genes in male adult worms. The three major GO term categories, namely Biological Process, Cellular Component and Molecular Function are separately represented in each panel. The size of the circles is proportional to the number of genes in each significantly enriched category, as indicated at the lower part scales; the colors show the statistical significance of the enrichment, as indicated by the -log10 FDR values that appear in the color-coded scales at the bottom. A GO enrichment significance cutoff of FDR ≤ 0.05 was used; no significantly enriched GO terms were found among the female upregulated genes.

    Article Snippet: For quantitative RT-PCR, complementary DNAs were obtained by reverse transcription (RT) of 100 ng schistosomula or adult worms total RNA using SuperScript III Reverse Transcriptase (Invitrogen) and random hexamer primers in a 20 μL volume, according to the manufacturer’s recommendations.

    Techniques: RNA Sequencing Assay

    Gene Ontology terms enrichment analysis of differentially expressed genes detected by RNA-seq in schistosomula treated with GSK343. Enriched GO terms for differentially expressed genes in schistosomula, separated by downregulated genes (A) and upregulated genes (B) . The three major GO term categories, namely Biological Process, Cellular Component and Molecular Function are separately represented in each panel. The size of the circles is proportional to the number of genes in each significantly enriched category, as indicated at the lower part scales; the colors show the statistical significance of the enrichment, as indicated by the -log10 FDR values that appear in the color-coded scales at the bottom. A GO enrichment significance cutoff of FDR ≤ 0.05 was used.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Inhibition of histone methyltransferase EZH2 in Schistosoma mansoni in vitro by GSK343 reduces egg laying and decreases the expression of genes implicated in DNA replication and noncoding RNA metabolism

    doi: 10.1371/journal.pntd.0006873

    Figure Lengend Snippet: Gene Ontology terms enrichment analysis of differentially expressed genes detected by RNA-seq in schistosomula treated with GSK343. Enriched GO terms for differentially expressed genes in schistosomula, separated by downregulated genes (A) and upregulated genes (B) . The three major GO term categories, namely Biological Process, Cellular Component and Molecular Function are separately represented in each panel. The size of the circles is proportional to the number of genes in each significantly enriched category, as indicated at the lower part scales; the colors show the statistical significance of the enrichment, as indicated by the -log10 FDR values that appear in the color-coded scales at the bottom. A GO enrichment significance cutoff of FDR ≤ 0.05 was used.

    Article Snippet: For quantitative RT-PCR, complementary DNAs were obtained by reverse transcription (RT) of 100 ng schistosomula or adult worms total RNA using SuperScript III Reverse Transcriptase (Invitrogen) and random hexamer primers in a 20 μL volume, according to the manufacturer’s recommendations.

    Techniques: RNA Sequencing Assay

    Effect of FTA on apoptosis of PBMCs infected with BVDV. PBMCs were collected from the indicated cultures at 24 h after BVDV challenge. (A) Representative two-dimensional scatter plots of annexin V versus propidium iodide, the percentage of early apoptotic cells and the percentage of late apoptotic cells. (B) Relative mRNA expression of Bcl-xL and Bim in bovine PBMCs after simulation. Data are presented as means ± SEM of three independent experiments. * P

    Journal: PLoS ONE

    Article Title: Forsythoside A Inhibits BVDV Replication via TRAF2-Dependent CD28–4-1BB Signaling in Bovine PBMCs

    doi: 10.1371/journal.pone.0162791

    Figure Lengend Snippet: Effect of FTA on apoptosis of PBMCs infected with BVDV. PBMCs were collected from the indicated cultures at 24 h after BVDV challenge. (A) Representative two-dimensional scatter plots of annexin V versus propidium iodide, the percentage of early apoptotic cells and the percentage of late apoptotic cells. (B) Relative mRNA expression of Bcl-xL and Bim in bovine PBMCs after simulation. Data are presented as means ± SEM of three independent experiments. * P

    Article Snippet: Total RNA was extracted from cells bovine PBMCs using TRIzol reagent (Invitrogen). cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen) in a 20 μl reaction mixture.

    Techniques: Infection, Expressing

    Effect of FTA on BVDV replication and virion infectivity. (A) The number of living cell (left panels) and virus copies per cell (right panels) at 24, 48, and 72 h after simulation. The number were counted with Trypan blue stain and the virus copies per living cells were measured by absolute quantitative real-time PCR for amplifying the 5’ UTR. (B) The BVDV-E2 protein express by western blot (left panels) and ratio of E2 band intensity to that of GAPDH (right panels). BVDV E2 protein in PBMCs was collected from the indicated PBMCs cultures at 24, 48, and 72 h after stimulation. Expression of GAPDH was measured as an internal control. (C) BVDV titers in supernatants of PBMCs treated with BVDV or BVDV and FTA at 24, 48, and 72h. Data are presented as the means ± SEM of three independent experiments. * P

    Journal: PLoS ONE

    Article Title: Forsythoside A Inhibits BVDV Replication via TRAF2-Dependent CD28–4-1BB Signaling in Bovine PBMCs

    doi: 10.1371/journal.pone.0162791

    Figure Lengend Snippet: Effect of FTA on BVDV replication and virion infectivity. (A) The number of living cell (left panels) and virus copies per cell (right panels) at 24, 48, and 72 h after simulation. The number were counted with Trypan blue stain and the virus copies per living cells were measured by absolute quantitative real-time PCR for amplifying the 5’ UTR. (B) The BVDV-E2 protein express by western blot (left panels) and ratio of E2 band intensity to that of GAPDH (right panels). BVDV E2 protein in PBMCs was collected from the indicated PBMCs cultures at 24, 48, and 72 h after stimulation. Expression of GAPDH was measured as an internal control. (C) BVDV titers in supernatants of PBMCs treated with BVDV or BVDV and FTA at 24, 48, and 72h. Data are presented as the means ± SEM of three independent experiments. * P

    Article Snippet: Total RNA was extracted from cells bovine PBMCs using TRIzol reagent (Invitrogen). cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen) in a 20 μl reaction mixture.

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

    Effect of FTA on the expression of TRAF1 and TRAF2 in bovine PBMCs infected with BVDV. (A) Relative mRNA expression of TRAF-1 and TRAF-2 cultured with medium alone, FTA, BVDV, and BVDV plus FTA at 24, 48, 72 h in bovine PBMCs. (B) The TRAF-2 protein express by western blot (left panels) and ratio of TRAF-2 band intensity to that of GAPDH (right panels). TRAF-2 protein in PBMCs was collected from the indicated PBMCs cultures at 24, 48, and 72 h after stimulation. Expression of GAPDH was measured as an internal control. Data are presented as the means ± SEM of three independent experiments. * P

    Journal: PLoS ONE

    Article Title: Forsythoside A Inhibits BVDV Replication via TRAF2-Dependent CD28–4-1BB Signaling in Bovine PBMCs

    doi: 10.1371/journal.pone.0162791

    Figure Lengend Snippet: Effect of FTA on the expression of TRAF1 and TRAF2 in bovine PBMCs infected with BVDV. (A) Relative mRNA expression of TRAF-1 and TRAF-2 cultured with medium alone, FTA, BVDV, and BVDV plus FTA at 24, 48, 72 h in bovine PBMCs. (B) The TRAF-2 protein express by western blot (left panels) and ratio of TRAF-2 band intensity to that of GAPDH (right panels). TRAF-2 protein in PBMCs was collected from the indicated PBMCs cultures at 24, 48, and 72 h after stimulation. Expression of GAPDH was measured as an internal control. Data are presented as the means ± SEM of three independent experiments. * P

    Article Snippet: Total RNA was extracted from cells bovine PBMCs using TRIzol reagent (Invitrogen). cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen) in a 20 μl reaction mixture.

    Techniques: Expressing, Infection, Cell Culture, Western Blot

    Effect of FTA on the expression of CD28 and CTLA-4 in bovine PBMCs infected with BVDV. (A) Relative mRNA expression of CD28/CTLA-4 cultured with medium alone, FTA, BVDV, and BVDV plus FTA at 24, 48, 72 h in bovine PBMCs. (B) The CD28 protein express by western blot (left panels) and ratio of CD28 band intensity to that of GAPDH (right panels). CD28 protein in PBMCs was collected from the indicated PBMCs cultures at 24, 48, and 72 h after stimulation. Expression of GAPDH was measured as an internal control. Data are presented as the means ± SEM of three independent experiments. * P

    Journal: PLoS ONE

    Article Title: Forsythoside A Inhibits BVDV Replication via TRAF2-Dependent CD28–4-1BB Signaling in Bovine PBMCs

    doi: 10.1371/journal.pone.0162791

    Figure Lengend Snippet: Effect of FTA on the expression of CD28 and CTLA-4 in bovine PBMCs infected with BVDV. (A) Relative mRNA expression of CD28/CTLA-4 cultured with medium alone, FTA, BVDV, and BVDV plus FTA at 24, 48, 72 h in bovine PBMCs. (B) The CD28 protein express by western blot (left panels) and ratio of CD28 band intensity to that of GAPDH (right panels). CD28 protein in PBMCs was collected from the indicated PBMCs cultures at 24, 48, and 72 h after stimulation. Expression of GAPDH was measured as an internal control. Data are presented as the means ± SEM of three independent experiments. * P

    Article Snippet: Total RNA was extracted from cells bovine PBMCs using TRIzol reagent (Invitrogen). cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen) in a 20 μl reaction mixture.

    Techniques: Expressing, Infection, Cell Culture, Western Blot

    Effect of FTA on the expression of OX40, 4-1BB, and 4-1BBL in bovine PBMCs infected with BVDV. (A) Relative mRNA expression of OX40, 4-1BB, and 4-1BBL cultured with medium alone, FTA, BVDV, and BVDV plus FTA at 24, 48, 72 h in bovine PBMCs. (B) The 4-1BB protein express by western blot (left panels) and ratio of 4-1BB band intensity to that of GAPDH (right panels). 4-1BB protein in PBMCs was collected from the indicated PBMCs cultures at 24, 48, and 72 h after stimulation. Expression of GAPDH was measured as an internal control. Data are presented as the means ± SEM of three independent experiments. * P

    Journal: PLoS ONE

    Article Title: Forsythoside A Inhibits BVDV Replication via TRAF2-Dependent CD28–4-1BB Signaling in Bovine PBMCs

    doi: 10.1371/journal.pone.0162791

    Figure Lengend Snippet: Effect of FTA on the expression of OX40, 4-1BB, and 4-1BBL in bovine PBMCs infected with BVDV. (A) Relative mRNA expression of OX40, 4-1BB, and 4-1BBL cultured with medium alone, FTA, BVDV, and BVDV plus FTA at 24, 48, 72 h in bovine PBMCs. (B) The 4-1BB protein express by western blot (left panels) and ratio of 4-1BB band intensity to that of GAPDH (right panels). 4-1BB protein in PBMCs was collected from the indicated PBMCs cultures at 24, 48, and 72 h after stimulation. Expression of GAPDH was measured as an internal control. Data are presented as the means ± SEM of three independent experiments. * P

    Article Snippet: Total RNA was extracted from cells bovine PBMCs using TRIzol reagent (Invitrogen). cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen) in a 20 μl reaction mixture.

    Techniques: Expressing, Infection, Cell Culture, Western Blot

    Concentrations of IFN-γ, IL-2, and IgG2a in cell culture supernatants. Bovine PBMCs cultured with medium alone, FTA, BVDV, and BVDV plus FTA. Concentrations of IFN-γ (A), IL-2 (B) and IgG2a (C) were detected by ELISA. Data are presented as means ± SEM of three independent experiments. Mean values at the same time point without a common superscript ( a, b, c ) differ significantly (P

    Journal: PLoS ONE

    Article Title: Forsythoside A Inhibits BVDV Replication via TRAF2-Dependent CD28–4-1BB Signaling in Bovine PBMCs

    doi: 10.1371/journal.pone.0162791

    Figure Lengend Snippet: Concentrations of IFN-γ, IL-2, and IgG2a in cell culture supernatants. Bovine PBMCs cultured with medium alone, FTA, BVDV, and BVDV plus FTA. Concentrations of IFN-γ (A), IL-2 (B) and IgG2a (C) were detected by ELISA. Data are presented as means ± SEM of three independent experiments. Mean values at the same time point without a common superscript ( a, b, c ) differ significantly (P

    Article Snippet: Total RNA was extracted from cells bovine PBMCs using TRIzol reagent (Invitrogen). cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen) in a 20 μl reaction mixture.

    Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

    Effect of FTA on activation of T cells during BVDV infection. PBMCs were collected at 12 h after stimulation. (A) Representative flow cytometry dot plots, (B) Flow cytometry was used to determine the percentage of CD3 + CD25 + population in the bovine PBMCs. Data are presented as means ± SEM of three independent experiments. * P

    Journal: PLoS ONE

    Article Title: Forsythoside A Inhibits BVDV Replication via TRAF2-Dependent CD28–4-1BB Signaling in Bovine PBMCs

    doi: 10.1371/journal.pone.0162791

    Figure Lengend Snippet: Effect of FTA on activation of T cells during BVDV infection. PBMCs were collected at 12 h after stimulation. (A) Representative flow cytometry dot plots, (B) Flow cytometry was used to determine the percentage of CD3 + CD25 + population in the bovine PBMCs. Data are presented as means ± SEM of three independent experiments. * P

    Article Snippet: Total RNA was extracted from cells bovine PBMCs using TRIzol reagent (Invitrogen). cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen) in a 20 μl reaction mixture.

    Techniques: Activation Assay, Infection, Flow Cytometry, Cytometry

    Expression level of pri- OsamiR395h and its target genes in rice leaf and root tissues at different developmental stages. Total RNA samples were prepared from leaf and root tissues of rice harvested at indicated time points and used for RT-PCR analysis. OsaSIZ1 was used as a reference gene. Experiment was repeated three times.

    Journal: Scientific Reports

    Article Title: Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis

    doi: 10.1038/srep28791

    Figure Lengend Snippet: Expression level of pri- OsamiR395h and its target genes in rice leaf and root tissues at different developmental stages. Total RNA samples were prepared from leaf and root tissues of rice harvested at indicated time points and used for RT-PCR analysis. OsaSIZ1 was used as a reference gene. Experiment was repeated three times.

    Article Snippet: To determine the transcript level of mature miR395 , the first-strand cDNA used for stem-loop real-time PCR was synthesized following the regular SuperScript III Reverse Transcriptase (Invitrogen, USA) mediated method, except that the oligo (dT)20 was replaced with miR395 specific reverse transcription primer.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    NtamiR395 and NtaSULTR2 exhibit opposite expression patterns in tobacco roots. Real-time PCR analysis of expressions of NtaSULTR2 and mature NtamiR395 under different sulfate concentrations. Total RNA samples were prepared from ( a ) leaf tissue and ( b ) root tissue of four weeks old tobacco grown in MS medium with 0, 20, 1500 or 2000 μM (NH 4+ ) 2 SO 4 . NtaL25 was used as a reference gene. Data are presented as means of three technical replicates and two biological replicates, error bars represent SD (n = 6). The statistically significant difference between groups was determined by one-way ANOVA (F(df between , df within ) = F ration, p = p-value, where df = degrees of freedom). Means not sharing the same letter are statistically significantly different (P

    Journal: Scientific Reports

    Article Title: Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis

    doi: 10.1038/srep28791

    Figure Lengend Snippet: NtamiR395 and NtaSULTR2 exhibit opposite expression patterns in tobacco roots. Real-time PCR analysis of expressions of NtaSULTR2 and mature NtamiR395 under different sulfate concentrations. Total RNA samples were prepared from ( a ) leaf tissue and ( b ) root tissue of four weeks old tobacco grown in MS medium with 0, 20, 1500 or 2000 μM (NH 4+ ) 2 SO 4 . NtaL25 was used as a reference gene. Data are presented as means of three technical replicates and two biological replicates, error bars represent SD (n = 6). The statistically significant difference between groups was determined by one-way ANOVA (F(df between , df within ) = F ration, p = p-value, where df = degrees of freedom). Means not sharing the same letter are statistically significantly different (P

    Article Snippet: To determine the transcript level of mature miR395 , the first-strand cDNA used for stem-loop real-time PCR was synthesized following the regular SuperScript III Reverse Transcriptase (Invitrogen, USA) mediated method, except that the oligo (dT)20 was replaced with miR395 specific reverse transcription primer.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Mass Spectrometry

    Predicted target OsaSULTR1 and OsaSULTR2 exhibit opposite expression patterns to that of the OsamiR395 in rice root. ( a ) Target sites of the four putative OsamiR395 target genes in rice. The target sites were compared with the complementary sequence of mature OsamiR395h . Asterisks indicate the identical sequences. ( b ) RT-PCR analysis of expression levels of the OsamiR395 putative targets. Total RNA samples used for RT-PCR were extracted from leaf and root tissues of two weeks old rice grown in N6 medium with 0, 20, 1500 or 2000 μM (NH 4+ ) 2 SO 4 and used for RT-PCR analysis. OsaSIZ1 was used as a reference gene. Experiment was repeated three times. ( c ) Stem-loop real-time RT-PCR analysis of mature OsamiR395 and real-time RT-PCR analysis of pri-OsamiR395h . Total RNA samples were prepared from leaf and root tissues of two weeks old rice grown in regular N6 medium (+S) or N6 medium without SO 4 + (−S) and used for RT-PCR analysis. OsaSIZ1 was used as a reference gene. ( d ) Real-time RT-PCR analysis was also conducted to determine the expression levels of the OsamiR395 putative targets in rice leaves and roots. Total RNA samples were prepared as in ( c ) and used for real-time RT-PCR analysis. OsaSIZ1 was used as a reference gene. For ( c,d ), data are presented as means of two independent biological replicates and three technical replicates, error bars represent SD (n = 6). Asterisks indicate the significant differences between expression levels under −S and +S conditions. P

    Journal: Scientific Reports

    Article Title: Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis

    doi: 10.1038/srep28791

    Figure Lengend Snippet: Predicted target OsaSULTR1 and OsaSULTR2 exhibit opposite expression patterns to that of the OsamiR395 in rice root. ( a ) Target sites of the four putative OsamiR395 target genes in rice. The target sites were compared with the complementary sequence of mature OsamiR395h . Asterisks indicate the identical sequences. ( b ) RT-PCR analysis of expression levels of the OsamiR395 putative targets. Total RNA samples used for RT-PCR were extracted from leaf and root tissues of two weeks old rice grown in N6 medium with 0, 20, 1500 or 2000 μM (NH 4+ ) 2 SO 4 and used for RT-PCR analysis. OsaSIZ1 was used as a reference gene. Experiment was repeated three times. ( c ) Stem-loop real-time RT-PCR analysis of mature OsamiR395 and real-time RT-PCR analysis of pri-OsamiR395h . Total RNA samples were prepared from leaf and root tissues of two weeks old rice grown in regular N6 medium (+S) or N6 medium without SO 4 + (−S) and used for RT-PCR analysis. OsaSIZ1 was used as a reference gene. ( d ) Real-time RT-PCR analysis was also conducted to determine the expression levels of the OsamiR395 putative targets in rice leaves and roots. Total RNA samples were prepared as in ( c ) and used for real-time RT-PCR analysis. OsaSIZ1 was used as a reference gene. For ( c,d ), data are presented as means of two independent biological replicates and three technical replicates, error bars represent SD (n = 6). Asterisks indicate the significant differences between expression levels under −S and +S conditions. P

    Article Snippet: To determine the transcript level of mature miR395 , the first-strand cDNA used for stem-loop real-time PCR was synthesized following the regular SuperScript III Reverse Transcriptase (Invitrogen, USA) mediated method, except that the oligo (dT)20 was replaced with miR395 specific reverse transcription primer.

    Techniques: Expressing, Sequencing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    Heterologous expression of pri- OsamiR395h in Nicotiana tabacum . ( a ) The Schematic diagram of rice pri- OsamiR395h overexpression construct. Rice pri- OsamiR395h sequence containing stem-loop structure of OsamiR395h was cloned from rice genomic DNA and put under the control of the CaMV35S promoter. The hptII gene driven by CaMV35S promoter was used as selectable maker. The pre- OsamiR395h sequence was underlined. Sequence emphasized with red color indicates the mature miR395h . LB, Left border; RB, right border. ( b ) RT-PCR analysis of pri- OsamiR395h expression in wild type and three transgenic tobacco lines. Total RNA samples were prepared from two weeks old wild type and transgenic tobacco plants grown in MS medium. NtaL25 was used as reference gene. ( c ) Small RNA northern blotting analysis of mature miR395 transcripts in wild type and three transgenic tobacco lines. Total RNA samples were prepared from two weeks old wild type and transgenic tobacco plants grown in MS medium. rRNA was used as loading control. WT: wild type plant. OE: overexpression line.

    Journal: Scientific Reports

    Article Title: Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis

    doi: 10.1038/srep28791

    Figure Lengend Snippet: Heterologous expression of pri- OsamiR395h in Nicotiana tabacum . ( a ) The Schematic diagram of rice pri- OsamiR395h overexpression construct. Rice pri- OsamiR395h sequence containing stem-loop structure of OsamiR395h was cloned from rice genomic DNA and put under the control of the CaMV35S promoter. The hptII gene driven by CaMV35S promoter was used as selectable maker. The pre- OsamiR395h sequence was underlined. Sequence emphasized with red color indicates the mature miR395h . LB, Left border; RB, right border. ( b ) RT-PCR analysis of pri- OsamiR395h expression in wild type and three transgenic tobacco lines. Total RNA samples were prepared from two weeks old wild type and transgenic tobacco plants grown in MS medium. NtaL25 was used as reference gene. ( c ) Small RNA northern blotting analysis of mature miR395 transcripts in wild type and three transgenic tobacco lines. Total RNA samples were prepared from two weeks old wild type and transgenic tobacco plants grown in MS medium. rRNA was used as loading control. WT: wild type plant. OE: overexpression line.

    Article Snippet: To determine the transcript level of mature miR395 , the first-strand cDNA used for stem-loop real-time PCR was synthesized following the regular SuperScript III Reverse Transcriptase (Invitrogen, USA) mediated method, except that the oligo (dT)20 was replaced with miR395 specific reverse transcription primer.

    Techniques: Expressing, Over Expression, Construct, Sequencing, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Transgenic Assay, Mass Spectrometry, Northern Blot

    Sulfate deficiency induces accumulation of OsamiR395 in rice. ( a ) Small RNA northern blotting analysis of mature OsamiR395 under different sulfate concentrations. Total RNA samples were prepared from leaf and root tissues of two weeks old rice grown in N6 medium with 0, 20, 1500 or 2000 μM (NH 4+ ) 2 SO 4 and used for small RNA northern blotting analysis. Antisense oligonucleotides of OsamiR395 was labeled with γ-[ 32 P]ATP and used as probe to detect the transcript level of mature OsamiR395 . rRNA was used as a loading control. ( b ) Stem-loop real-time PCR analysis of mature OsamiR395 under different sulfate concentrations. Total RNA samples were prepared as in ( a ) and used for stem-loop real-time PCR analysis. OsaSIZ1 was used as a reference gene. Data are presented as means of three technique replicates, error bars represent SD (n = 3). ( c ) Real-time PCR analysis of rice pri- OsamiR395h under different sulfate concentrations. Total RNA samples were prepared as in ( a ) and used for real-time PCR analysis. OsaSIZ1 was used as a reference gene. Data are presented as means of three technique replicates, error bars represent SD (n = 3). The statistically significant difference between groups was determined by one-way ANOVA (F(df between , df within ) = F ration, p = p-value, where df = degrees of freedom). Means not sharing the same letter are statistically significantly different (P

    Journal: Scientific Reports

    Article Title: Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis

    doi: 10.1038/srep28791

    Figure Lengend Snippet: Sulfate deficiency induces accumulation of OsamiR395 in rice. ( a ) Small RNA northern blotting analysis of mature OsamiR395 under different sulfate concentrations. Total RNA samples were prepared from leaf and root tissues of two weeks old rice grown in N6 medium with 0, 20, 1500 or 2000 μM (NH 4+ ) 2 SO 4 and used for small RNA northern blotting analysis. Antisense oligonucleotides of OsamiR395 was labeled with γ-[ 32 P]ATP and used as probe to detect the transcript level of mature OsamiR395 . rRNA was used as a loading control. ( b ) Stem-loop real-time PCR analysis of mature OsamiR395 under different sulfate concentrations. Total RNA samples were prepared as in ( a ) and used for stem-loop real-time PCR analysis. OsaSIZ1 was used as a reference gene. Data are presented as means of three technique replicates, error bars represent SD (n = 3). ( c ) Real-time PCR analysis of rice pri- OsamiR395h under different sulfate concentrations. Total RNA samples were prepared as in ( a ) and used for real-time PCR analysis. OsaSIZ1 was used as a reference gene. Data are presented as means of three technique replicates, error bars represent SD (n = 3). The statistically significant difference between groups was determined by one-way ANOVA (F(df between , df within ) = F ration, p = p-value, where df = degrees of freedom). Means not sharing the same letter are statistically significantly different (P

    Article Snippet: To determine the transcript level of mature miR395 , the first-strand cDNA used for stem-loop real-time PCR was synthesized following the regular SuperScript III Reverse Transcriptase (Invitrogen, USA) mediated method, except that the oligo (dT)20 was replaced with miR395 specific reverse transcription primer.

    Techniques: Northern Blot, Labeling, Real-time Polymerase Chain Reaction

    Identification of a sulfate transporter gene, NtaSULTR2 , the target of miR395 in tobacco. ( a ) RT-PCR analysis of NtaSULTR2 expression in tobacco. Total RNA samples were prepared from two weeks old wild type and transgenic tobacco and used for RT-PCR analysis. NtaL25 was used as a reference gene. Experiment was repeated three times. ( b ) General structure of tobacco gene NtSULTR2 . NtaSULTR2 with a length of 1335 bp contains a sulfate transporter domain between 724 bp to 1332 bp, and a miR395 target site between 135 bp to 156 bp. The target site was compared with the complementary sequence of mature OsamIR395h and NtamiR395 . Asterisks indicate the identical sequences. ( c ) phylogenetic analysis of NtaSULTR2 protein. Protein sequences of NtaSULTR2 and 16 sulfate transporters of rice and Arabidopsis were used to establish phylogenetic tree with MEGA6. In this phylogenetic tree, NtaSULTR2 protein is classified into the second group of sulfate transporter subfamily together with AthSULTR2;1, AthSULTR2;2 and OsaSULTR2;1.

    Journal: Scientific Reports

    Article Title: Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis

    doi: 10.1038/srep28791

    Figure Lengend Snippet: Identification of a sulfate transporter gene, NtaSULTR2 , the target of miR395 in tobacco. ( a ) RT-PCR analysis of NtaSULTR2 expression in tobacco. Total RNA samples were prepared from two weeks old wild type and transgenic tobacco and used for RT-PCR analysis. NtaL25 was used as a reference gene. Experiment was repeated three times. ( b ) General structure of tobacco gene NtSULTR2 . NtaSULTR2 with a length of 1335 bp contains a sulfate transporter domain between 724 bp to 1332 bp, and a miR395 target site between 135 bp to 156 bp. The target site was compared with the complementary sequence of mature OsamIR395h and NtamiR395 . Asterisks indicate the identical sequences. ( c ) phylogenetic analysis of NtaSULTR2 protein. Protein sequences of NtaSULTR2 and 16 sulfate transporters of rice and Arabidopsis were used to establish phylogenetic tree with MEGA6. In this phylogenetic tree, NtaSULTR2 protein is classified into the second group of sulfate transporter subfamily together with AthSULTR2;1, AthSULTR2;2 and OsaSULTR2;1.

    Article Snippet: To determine the transcript level of mature miR395 , the first-strand cDNA used for stem-loop real-time PCR was synthesized following the regular SuperScript III Reverse Transcriptase (Invitrogen, USA) mediated method, except that the oligo (dT)20 was replaced with miR395 specific reverse transcription primer.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transgenic Assay, Sequencing

    R-loops and RNAi promote H3K9me2 mark over mouse β-actin terminator a . DIP performed on mouse β-actin gene in MEFs. b . RT-qPCR of total RNA from MEF cells on β-actin gene to detect antisense transcripts with region-specific forward primers. Average RT-qPCR values are +/− SD from four biological repeats. c . Ago1 ChIP performed on mouse β-actin gene in MEFs. ChIP signal is normalised to intron 1 signal. d. Left panel: Ratio of H3K9me2 ChIP signal versus H3 on mouse β-actin in MEFs. Middle panel: Normalised H3K9me3 to total H3 levels. Right panel: Ratio of H3K9me2 and H3K9me3 signal versus H3 signal on major satellites in MEFs. e . Ago1 ChIP in wild type (grey bars) and Ago2 KO (white bars) cells. Ago1 recruitment over mouse β-actin is enhanced upon Ago2 depletion. f . Left panel: Ratio of H3K9me2 ChIP signal versus total H3 on β-actin gene in wild type and G9a/GLP KO mouse ES cells. Right panel: H3K9me2/H3 ratio on the mouse major satellites in wild type and G9a/GLP KO cells. Average ChIP and DIP values are +/− SD from three biological repeats.

    Journal: Nature

    Article Title: R-loops induce repressive chromatin marks over mammalian gene terminators

    doi: 10.1038/nature13787

    Figure Lengend Snippet: R-loops and RNAi promote H3K9me2 mark over mouse β-actin terminator a . DIP performed on mouse β-actin gene in MEFs. b . RT-qPCR of total RNA from MEF cells on β-actin gene to detect antisense transcripts with region-specific forward primers. Average RT-qPCR values are +/− SD from four biological repeats. c . Ago1 ChIP performed on mouse β-actin gene in MEFs. ChIP signal is normalised to intron 1 signal. d. Left panel: Ratio of H3K9me2 ChIP signal versus H3 on mouse β-actin in MEFs. Middle panel: Normalised H3K9me3 to total H3 levels. Right panel: Ratio of H3K9me2 and H3K9me3 signal versus H3 signal on major satellites in MEFs. e . Ago1 ChIP in wild type (grey bars) and Ago2 KO (white bars) cells. Ago1 recruitment over mouse β-actin is enhanced upon Ago2 depletion. f . Left panel: Ratio of H3K9me2 ChIP signal versus total H3 on β-actin gene in wild type and G9a/GLP KO mouse ES cells. Right panel: H3K9me2/H3 ratio on the mouse major satellites in wild type and G9a/GLP KO cells. Average ChIP and DIP values are +/− SD from three biological repeats.

    Article Snippet: Total RNA was isolated using TRIzol reagent (Invitrogen) and reverse transcribed with SuperScript III Reverse Transcriptase (Invitrogen) using gene-specific primers.

    Techniques: Quantitative RT-PCR, Chromatin Immunoprecipitation, Gene Knockout

    Ago2-dependent H3K9me2 mark and R-loop formation promote efficient termination on mouse β-actin gene a,b . ChIP in WT and Ago2 KO MEFs using Ago2 and G9a antibodies respectively. c . Ratio H3K9me2 versus H3 ChIP in WT and Ago2 KO MEFs. d . Pol II ChIP with probes downstream of the PAS with extended Y axis. in WT (grey bars), WT over-expressing RNase H1 (black bars), Ago2 KO (white bars) and Ago2 KO over-expressing RNase H1 (red bars) MEFs. Full gene profile in Extended Data Fig. 7b . All ChIP values +/− SD from three to four biological repeats. e . Br-UTP NRO analysis in WT (grey bars) and Ago2 KO MEFs over-expressing RNase H1 (red bars). Nascent Br-RNA over intron 3 probe set as 1. Fold of enrichment of read-through transcripts for pause, pause2 and C calculated relative to intron 3 signal. Values +/− SD from three biological repeats.

    Journal: Nature

    Article Title: R-loops induce repressive chromatin marks over mammalian gene terminators

    doi: 10.1038/nature13787

    Figure Lengend Snippet: Ago2-dependent H3K9me2 mark and R-loop formation promote efficient termination on mouse β-actin gene a,b . ChIP in WT and Ago2 KO MEFs using Ago2 and G9a antibodies respectively. c . Ratio H3K9me2 versus H3 ChIP in WT and Ago2 KO MEFs. d . Pol II ChIP with probes downstream of the PAS with extended Y axis. in WT (grey bars), WT over-expressing RNase H1 (black bars), Ago2 KO (white bars) and Ago2 KO over-expressing RNase H1 (red bars) MEFs. Full gene profile in Extended Data Fig. 7b . All ChIP values +/− SD from three to four biological repeats. e . Br-UTP NRO analysis in WT (grey bars) and Ago2 KO MEFs over-expressing RNase H1 (red bars). Nascent Br-RNA over intron 3 probe set as 1. Fold of enrichment of read-through transcripts for pause, pause2 and C calculated relative to intron 3 signal. Values +/− SD from three biological repeats.

    Article Snippet: Total RNA was isolated using TRIzol reagent (Invitrogen) and reverse transcribed with SuperScript III Reverse Transcriptase (Invitrogen) using gene-specific primers.

    Techniques: Chromatin Immunoprecipitation, Gene Knockout, Expressing

    R-loop formation and antisense transcription are Ago2 and G9a/GLP-independent a-c DIP performed on mouse β-actin gene in wild type, Ago2 KO (a) and G9a/GLP KO (c) cells. b. Pol II ChIP in wild type (grey bars), wild type over-expressing RNase H1 (black bars), Ago2 KO (white bars) and Ago2 KO over-expressing RNase H1 (red bars) MEFs. Hatched box quantifies Pol II read-through transcription versus promoter signal. d. RT-qPCR analysis of total RNA from wild type and G9a/GLP KO cells for the mouse β-actin gene. RT reaction was performed with specific forward primers. Average DIP and RT-qPCR values are +/− SD from three biological repeats.

    Journal: Nature

    Article Title: R-loops induce repressive chromatin marks over mammalian gene terminators

    doi: 10.1038/nature13787

    Figure Lengend Snippet: R-loop formation and antisense transcription are Ago2 and G9a/GLP-independent a-c DIP performed on mouse β-actin gene in wild type, Ago2 KO (a) and G9a/GLP KO (c) cells. b. Pol II ChIP in wild type (grey bars), wild type over-expressing RNase H1 (black bars), Ago2 KO (white bars) and Ago2 KO over-expressing RNase H1 (red bars) MEFs. Hatched box quantifies Pol II read-through transcription versus promoter signal. d. RT-qPCR analysis of total RNA from wild type and G9a/GLP KO cells for the mouse β-actin gene. RT reaction was performed with specific forward primers. Average DIP and RT-qPCR values are +/− SD from three biological repeats.

    Article Snippet: Total RNA was isolated using TRIzol reagent (Invitrogen) and reverse transcribed with SuperScript III Reverse Transcriptase (Invitrogen) using gene-specific primers.

    Techniques: Gene Knockout, Chromatin Immunoprecipitation, Expressing, Quantitative RT-PCR

    Modulation of R-loop and G9a levels define mechanism of H3K9me2 formation on human α-actin terminator a . DIP with RNA:DNA hybrid antibody with/without RNase H1 over-expression. b . RT-qPCR with/without RNase H1 over-expression. c-e . ChIP analysis with/without RNase H1 over-expression using Dicer, G9a or HP1γ antibodies. f . H3K9me2 versus H3 ChIP values, +/− BIX treatment. g . DIP profile +/− BIX treatment. All ChIP and DIP values +/− SD from three biological repeats. h . Nuclear immunofluorescence of H3K9me2 with dsRNA (J2-top panel) and R-loops (S9.6-bottom panel). Arrows denote foci in close proximity. Whole cell images in Extended Data Fig. 3b . Cell numbers with > 2 J2/H3K9me2 and S9.6/H3K9me2 foci (n=100) (lower left graph). Colocalising foci of J2 and S9.6 with H3K9me2 (n=1000), based on three independent experiments (lower right graph).

    Journal: Nature

    Article Title: R-loops induce repressive chromatin marks over mammalian gene terminators

    doi: 10.1038/nature13787

    Figure Lengend Snippet: Modulation of R-loop and G9a levels define mechanism of H3K9me2 formation on human α-actin terminator a . DIP with RNA:DNA hybrid antibody with/without RNase H1 over-expression. b . RT-qPCR with/without RNase H1 over-expression. c-e . ChIP analysis with/without RNase H1 over-expression using Dicer, G9a or HP1γ antibodies. f . H3K9me2 versus H3 ChIP values, +/− BIX treatment. g . DIP profile +/− BIX treatment. All ChIP and DIP values +/− SD from three biological repeats. h . Nuclear immunofluorescence of H3K9me2 with dsRNA (J2-top panel) and R-loops (S9.6-bottom panel). Arrows denote foci in close proximity. Whole cell images in Extended Data Fig. 3b . Cell numbers with > 2 J2/H3K9me2 and S9.6/H3K9me2 foci (n=100) (lower left graph). Colocalising foci of J2 and S9.6 with H3K9me2 (n=1000), based on three independent experiments (lower right graph).

    Article Snippet: Total RNA was isolated using TRIzol reagent (Invitrogen) and reverse transcribed with SuperScript III Reverse Transcriptase (Invitrogen) using gene-specific primers.

    Techniques: Over Expression, Quantitative RT-PCR, Chromatin Immunoprecipitation, Immunofluorescence

    Ensa and Gemin7 share features of R-loop mediated pause-type termination a . DIP on Ensa and Gemin7 genes. R-loops specifically enriched over 3′ ends (grey bars), compared to promoter regions (white bars). Human β-actin gene is positive control. Values +/− SD for three biological repeats. b . RT-qPCR of total RNA from HeLa cells performed on indicated gene. RT reaction was performed with promoter or 3′ end-specific forward primer to detect antisense transcript. Average RT-qPCR values are +/− SD from four biological repeats. c . Dicer ChIP of Ensa and Gemin7 genes over promoters and termination regions. d. Left panel: Ratio of H3K9me2 ChIP signal versus H3 on Gemin7 and β-actin genes. Right panel: Ratio of H3K9me2 signal versus H3 on Ensa gene. e,f. H3K9me2 and H3 ChIP for Ensa and Gemin7 genes over promoter (white bars) and pause terminators (grey bars). β-actin gene was used as a positive control. g . HP1γ ChIP for Ensa and Gemin7 genes over intronic and 3′ end regions. ChIP values are +/− SD from three biological repeats.

    Journal: Nature

    Article Title: R-loops induce repressive chromatin marks over mammalian gene terminators

    doi: 10.1038/nature13787

    Figure Lengend Snippet: Ensa and Gemin7 share features of R-loop mediated pause-type termination a . DIP on Ensa and Gemin7 genes. R-loops specifically enriched over 3′ ends (grey bars), compared to promoter regions (white bars). Human β-actin gene is positive control. Values +/− SD for three biological repeats. b . RT-qPCR of total RNA from HeLa cells performed on indicated gene. RT reaction was performed with promoter or 3′ end-specific forward primer to detect antisense transcript. Average RT-qPCR values are +/− SD from four biological repeats. c . Dicer ChIP of Ensa and Gemin7 genes over promoters and termination regions. d. Left panel: Ratio of H3K9me2 ChIP signal versus H3 on Gemin7 and β-actin genes. Right panel: Ratio of H3K9me2 signal versus H3 on Ensa gene. e,f. H3K9me2 and H3 ChIP for Ensa and Gemin7 genes over promoter (white bars) and pause terminators (grey bars). β-actin gene was used as a positive control. g . HP1γ ChIP for Ensa and Gemin7 genes over intronic and 3′ end regions. ChIP values are +/− SD from three biological repeats.

    Article Snippet: Total RNA was isolated using TRIzol reagent (Invitrogen) and reverse transcribed with SuperScript III Reverse Transcriptase (Invitrogen) using gene-specific primers.

    Techniques: Positive Control, Quantitative RT-PCR, Chromatin Immunoprecipitation

    Genome-wide methylation analysis of pol III-bound Alu DNA. ( a ) Comparison of the fraction of methylation of the indicated Alu subfamilies between genomic DNA (input) and pol III-bound DNA, as determined by ChIP-BS-Seq. The height of a vertical line indicates the fraction methylated at a particular C within the subfamily consensus sequence. ( b ) Genome-wide comparison of pol III occupancy and DNA methylation of Alu subfamilies. ( c ) Comparison of the extent of methylation of the AluYa5 subfamily between genomic DNA (input) and pol III-bound DNA. The height of a vertical line indicates the fraction methylated at a particular C within the subfamily consensus sequence. A- and B-block promoter elements are indicated. ( d ) Pol III occupancy plotted against DNA methylation for Alu SINEs (blue) and tRNA genes (red). Data are plotted as a moving average with 25 data points per window.

    Journal: Nature Communications

    Article Title: SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation

    doi: 10.1038/ncomms7569

    Figure Lengend Snippet: Genome-wide methylation analysis of pol III-bound Alu DNA. ( a ) Comparison of the fraction of methylation of the indicated Alu subfamilies between genomic DNA (input) and pol III-bound DNA, as determined by ChIP-BS-Seq. The height of a vertical line indicates the fraction methylated at a particular C within the subfamily consensus sequence. ( b ) Genome-wide comparison of pol III occupancy and DNA methylation of Alu subfamilies. ( c ) Comparison of the extent of methylation of the AluYa5 subfamily between genomic DNA (input) and pol III-bound DNA. The height of a vertical line indicates the fraction methylated at a particular C within the subfamily consensus sequence. A- and B-block promoter elements are indicated. ( d ) Pol III occupancy plotted against DNA methylation for Alu SINEs (blue) and tRNA genes (red). Data are plotted as a moving average with 25 data points per window.

    Article Snippet: To synthesize complementary DNA for RT–PCR, SuperScript III reverse transcriptase (Invitrogen) was used as previously , with 200 ng of RNA and Hexanucleotide Mix (Roche).

    Techniques: Genome Wide, Methylation, Chromatin Immunoprecipitation, Sequencing, DNA Methylation Assay, Blocking Assay

    Pol III co-occupies methylated SINEs with MBPs. ( a ) Semiquantitative ChIP assay in A31 fibroblasts showing specific binding of TFIIIB, TFIIIC and pol III to B1 and B2 loci, as well as 7SL , but not the Apo-E gene. Histone H3 and TAF I 48 provide positive and negative controls, respectively. ( b ) Semiquantitative ChIP assay in HeLa cells showing occupancy of pol III, TFIIIB and TFIIIC at Alu loci from chromosomes 6, 10, 19 and 22, as well as 7SL and Apo-E genes. ChIPs for histone H3 and TAF I 48 provide positive and negative controls, respectively. No antibody was used for the mock sample. ( c ) Mean±s.e.m. of the percentage input bound in three independent ChIP–quantitative PCR (qPCR) assays in HeLa cells, of the indicated proteins at individual Alu loci from chromosomes 6, 10, 19 and 22, as well as 7SL and Apo-E loci and Alu PV subfamily consensus. ChIPs for histone H3 and TAF I 48 provide positive and negative controls, respectively. No antibody was used for the mock samples. P values are calculated by t -test. ( d ) Mean±s.e.m. of four independent sequential ChIP–qPCR assays in which DNA immunoprecipitated from HeLa cells using pol III antibody was reprecipitated using antibodies against pol III, TFIIIB, TAF I 48 (negative control), MBD1, MBD2 and MeCP2, as indicated. No TAF I 48 signal was detected on Alu(c6).

    Journal: Nature Communications

    Article Title: SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation

    doi: 10.1038/ncomms7569

    Figure Lengend Snippet: Pol III co-occupies methylated SINEs with MBPs. ( a ) Semiquantitative ChIP assay in A31 fibroblasts showing specific binding of TFIIIB, TFIIIC and pol III to B1 and B2 loci, as well as 7SL , but not the Apo-E gene. Histone H3 and TAF I 48 provide positive and negative controls, respectively. ( b ) Semiquantitative ChIP assay in HeLa cells showing occupancy of pol III, TFIIIB and TFIIIC at Alu loci from chromosomes 6, 10, 19 and 22, as well as 7SL and Apo-E genes. ChIPs for histone H3 and TAF I 48 provide positive and negative controls, respectively. No antibody was used for the mock sample. ( c ) Mean±s.e.m. of the percentage input bound in three independent ChIP–quantitative PCR (qPCR) assays in HeLa cells, of the indicated proteins at individual Alu loci from chromosomes 6, 10, 19 and 22, as well as 7SL and Apo-E loci and Alu PV subfamily consensus. ChIPs for histone H3 and TAF I 48 provide positive and negative controls, respectively. No antibody was used for the mock samples. P values are calculated by t -test. ( d ) Mean±s.e.m. of four independent sequential ChIP–qPCR assays in which DNA immunoprecipitated from HeLa cells using pol III antibody was reprecipitated using antibodies against pol III, TFIIIB, TAF I 48 (negative control), MBD1, MBD2 and MeCP2, as indicated. No TAF I 48 signal was detected on Alu(c6).

    Article Snippet: To synthesize complementary DNA for RT–PCR, SuperScript III reverse transcriptase (Invitrogen) was used as previously , with 200 ng of RNA and Hexanucleotide Mix (Roche).

    Techniques: Methylation, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Immunoprecipitation, Negative Control

    SINE expression is not stimulated by loss of DNA methylation. ( a ) Analysis by semiquantitative RT–PCR of expression levels of the indicated transcripts in matched Dnmt1 +/+ and Dnmt1 −/− fibroblasts. Duplicate samples are shown for both cell types. Apo-E and p53BP2 mRNAs provide controls that have been documented as being suppressed by DNA methylation. GAPDH mRNA provides a loading control. ( b ) Analysis by semiquantitative RT–PCR of expression levels of the indicated transcripts in mouse ES cells treated for 16 h with (+) or without (−) 5-azacytidine. Apo-E mRNA provides a control that has been documented as being inhibited by DNA methylation. ARPP P0 mRNA provides a loading control. ( c ) Analysis by semiquantitative RT–PCR of expression levels of the indicated transcripts in HeLa cells treated for 72 h with 5-azacytidine. Apo-E mRNA provides a control that has been documented as being inhibited by DNA methylation. ARPP P0 mRNA provides a loading control. ( d ) Analysis by primer extension of Alu transcripts in the RNA from Fig. 5c . Bracket indicates ~240 bp products that initiate at the principle pol III start site of Alu. Reverse transcriptase was omitted from the reactions in lanes 1 and 2. To confirm that the assay was not saturated, raising the amount of template RNA from 5 (lanes 5 and 6) to 10 μg (lanes 3 and 4) is shown to give a stronger signal. Alu, B1 and B2 RT–PCRs were performed with Alu, B1 and B2 consensus primers, respectively ( Supplementary Table 1 ).

    Journal: Nature Communications

    Article Title: SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation

    doi: 10.1038/ncomms7569

    Figure Lengend Snippet: SINE expression is not stimulated by loss of DNA methylation. ( a ) Analysis by semiquantitative RT–PCR of expression levels of the indicated transcripts in matched Dnmt1 +/+ and Dnmt1 −/− fibroblasts. Duplicate samples are shown for both cell types. Apo-E and p53BP2 mRNAs provide controls that have been documented as being suppressed by DNA methylation. GAPDH mRNA provides a loading control. ( b ) Analysis by semiquantitative RT–PCR of expression levels of the indicated transcripts in mouse ES cells treated for 16 h with (+) or without (−) 5-azacytidine. Apo-E mRNA provides a control that has been documented as being inhibited by DNA methylation. ARPP P0 mRNA provides a loading control. ( c ) Analysis by semiquantitative RT–PCR of expression levels of the indicated transcripts in HeLa cells treated for 72 h with 5-azacytidine. Apo-E mRNA provides a control that has been documented as being inhibited by DNA methylation. ARPP P0 mRNA provides a loading control. ( d ) Analysis by primer extension of Alu transcripts in the RNA from Fig. 5c . Bracket indicates ~240 bp products that initiate at the principle pol III start site of Alu. Reverse transcriptase was omitted from the reactions in lanes 1 and 2. To confirm that the assay was not saturated, raising the amount of template RNA from 5 (lanes 5 and 6) to 10 μg (lanes 3 and 4) is shown to give a stronger signal. Alu, B1 and B2 RT–PCRs were performed with Alu, B1 and B2 consensus primers, respectively ( Supplementary Table 1 ).

    Article Snippet: To synthesize complementary DNA for RT–PCR, SuperScript III reverse transcriptase (Invitrogen) was used as previously , with 200 ng of RNA and Hexanucleotide Mix (Roche).

    Techniques: Expressing, DNA Methylation Assay, Reverse Transcription Polymerase Chain Reaction

    DNA methylation does not prevent pol III occupancy of SINEs. ( a ) Percentage input bound in three independent ChIP–quantitative PCR (qPCR) assays with mouse ES cells treated for 16 h with (+) or without (−) 5-azacytidine, showing occupancy of MBD2, MeCP2 and pol III at 7SL, B1 and B2 loci, as well as an Alu inserted onto chromosomes 14 and 17. ChIPs for TAF I 48 and without antibody (mock) provide negative controls. ( b ) Percentage input bound in three independent ChIP–qPCR assays with HeLa cells treated for 72 h with (+) or without (−) 5-azacytidine, showing the binding of MBD2, TFIIIB, TFIIIC and pol III to DNA centred over the body of Alu(c22) or 200 bp downstream. The resolution of this assay is limited by the size of the genomic DNA fragments (~500 bp). ( c ) Percentage input bound in two independent ChIP–qPCR assays with matched Dnmt1 +/+ and Dnmt1 −/− fibroblasts showing occupancy of MBD2, TFIIIB, TFIIIC and pol III at B1 and B2 loci, as well as 7SL and Apo-E genes. ChIPs for TAF I 48 and without antibody (mock) provide negative controls. Error bars indicate s.e.m. and all P values are calculated by t -test.

    Journal: Nature Communications

    Article Title: SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation

    doi: 10.1038/ncomms7569

    Figure Lengend Snippet: DNA methylation does not prevent pol III occupancy of SINEs. ( a ) Percentage input bound in three independent ChIP–quantitative PCR (qPCR) assays with mouse ES cells treated for 16 h with (+) or without (−) 5-azacytidine, showing occupancy of MBD2, MeCP2 and pol III at 7SL, B1 and B2 loci, as well as an Alu inserted onto chromosomes 14 and 17. ChIPs for TAF I 48 and without antibody (mock) provide negative controls. ( b ) Percentage input bound in three independent ChIP–qPCR assays with HeLa cells treated for 72 h with (+) or without (−) 5-azacytidine, showing the binding of MBD2, TFIIIB, TFIIIC and pol III to DNA centred over the body of Alu(c22) or 200 bp downstream. The resolution of this assay is limited by the size of the genomic DNA fragments (~500 bp). ( c ) Percentage input bound in two independent ChIP–qPCR assays with matched Dnmt1 +/+ and Dnmt1 −/− fibroblasts showing occupancy of MBD2, TFIIIB, TFIIIC and pol III at B1 and B2 loci, as well as 7SL and Apo-E genes. ChIPs for TAF I 48 and without antibody (mock) provide negative controls. Error bars indicate s.e.m. and all P values are calculated by t -test.

    Article Snippet: To synthesize complementary DNA for RT–PCR, SuperScript III reverse transcriptase (Invitrogen) was used as previously , with 200 ng of RNA and Hexanucleotide Mix (Roche).

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

    SUV39H1 inhibits pol III loading and expression of some SINEs. ( a ) Mean±s.e.m. of the percentage input bound in two independent ChIP–quantitative PCR (qPCR) assays with HeLa cells, showing occupancy of H3, TAF I 48, H3K9me3, SUV39H1 and HP1 at Alu loci, 7SL and p21 (positive control) genes. ( b ) Mean±s.e.m. of the percentage input bound in two independent ChIP–qPCR assays with HeLa cells treated for 24 h with vehicle (−) or 100 nM chaetocin (+), showing occupancy of H3, TAF I 48, H3K9me3, pol III and TFIIIC at Alu loci, 7SL and Apo-E genes. ( c ) Semiquantitative RT–PCR analysis of expression levels of indicated transcripts in HeLa cells treated for 24 h with indicated concentrations of chaetocin. ( d ) Analysis by primer extension of expression levels of Alu transcripts initiated from pol III start site in HeLa cells treated for 24 h with indicated concentrations of chaetocin. Reverse transcriptase was omitted from reactions in lanes 5–8. Right panel shows mean±s.d. of fold change in Alu expression in two independent experiments quantified using ImageJ. All P values are calculated by t -test.

    Journal: Nature Communications

    Article Title: SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation

    doi: 10.1038/ncomms7569

    Figure Lengend Snippet: SUV39H1 inhibits pol III loading and expression of some SINEs. ( a ) Mean±s.e.m. of the percentage input bound in two independent ChIP–quantitative PCR (qPCR) assays with HeLa cells, showing occupancy of H3, TAF I 48, H3K9me3, SUV39H1 and HP1 at Alu loci, 7SL and p21 (positive control) genes. ( b ) Mean±s.e.m. of the percentage input bound in two independent ChIP–qPCR assays with HeLa cells treated for 24 h with vehicle (−) or 100 nM chaetocin (+), showing occupancy of H3, TAF I 48, H3K9me3, pol III and TFIIIC at Alu loci, 7SL and Apo-E genes. ( c ) Semiquantitative RT–PCR analysis of expression levels of indicated transcripts in HeLa cells treated for 24 h with indicated concentrations of chaetocin. ( d ) Analysis by primer extension of expression levels of Alu transcripts initiated from pol III start site in HeLa cells treated for 24 h with indicated concentrations of chaetocin. Reverse transcriptase was omitted from reactions in lanes 5–8. Right panel shows mean±s.d. of fold change in Alu expression in two independent experiments quantified using ImageJ. All P values are calculated by t -test.

    Article Snippet: To synthesize complementary DNA for RT–PCR, SuperScript III reverse transcriptase (Invitrogen) was used as previously , with 200 ng of RNA and Hexanucleotide Mix (Roche).

    Techniques: Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control, Reverse Transcription Polymerase Chain Reaction

    SINE repression by H3K9Me3 does not require DNA methylation. ( a ) Percentage input bound in three independent ChIP–quantitative PCR (qPCR) assays with Dnmt1 −/− fibroblasts assessing binding of TAF I 48, H3K9me3, MBD2 and pol III at 7SL, B1, B2 and Apo-E loci. H3K9me3 data were normalized against total H3. For clarity, Fig. 7a displays a subset of the data shown in Fig. 7c . ( b ) Percentage input bound in two independent ChIP–qPCR assays of ES cells treated for 16 h with (+) or without (−) 5-azacytidine, assessing binding of TAF I 48, MBD2, H3K9me3 and pol III at 7SL, B1 and B2 loci, as well as an Alu inserted onto chromosomes 14 and 17. H3K9me3 data were normalized against total H3. ( c ) Percentage input bound in three independent ChIP–qPCR assays of matched Dnmt1 −/− and Dnmt1 +/+ fibroblasts treated for 24 h with (+) or without (−) 100 nM chaetocin, assessing binding of TAF I 48, H3K9me3, MBD2 and pol III at 7SL, B1 and B2 and Apo-E loci. H3K9me3 data were normalized against total H3. ( d ) Semiquantitative RT–PCR analysis of expression levels of indicated transcripts in matched Dnmt1 +/+ and Dnmt1 −/− fibroblasts treated for 24 h with indicated concentrations of chaetocin. Error bars indicate s.e.m. and all P values are calculated by t -test.

    Journal: Nature Communications

    Article Title: SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation

    doi: 10.1038/ncomms7569

    Figure Lengend Snippet: SINE repression by H3K9Me3 does not require DNA methylation. ( a ) Percentage input bound in three independent ChIP–quantitative PCR (qPCR) assays with Dnmt1 −/− fibroblasts assessing binding of TAF I 48, H3K9me3, MBD2 and pol III at 7SL, B1, B2 and Apo-E loci. H3K9me3 data were normalized against total H3. For clarity, Fig. 7a displays a subset of the data shown in Fig. 7c . ( b ) Percentage input bound in two independent ChIP–qPCR assays of ES cells treated for 16 h with (+) or without (−) 5-azacytidine, assessing binding of TAF I 48, MBD2, H3K9me3 and pol III at 7SL, B1 and B2 loci, as well as an Alu inserted onto chromosomes 14 and 17. H3K9me3 data were normalized against total H3. ( c ) Percentage input bound in three independent ChIP–qPCR assays of matched Dnmt1 −/− and Dnmt1 +/+ fibroblasts treated for 24 h with (+) or without (−) 100 nM chaetocin, assessing binding of TAF I 48, H3K9me3, MBD2 and pol III at 7SL, B1 and B2 and Apo-E loci. H3K9me3 data were normalized against total H3. ( d ) Semiquantitative RT–PCR analysis of expression levels of indicated transcripts in matched Dnmt1 +/+ and Dnmt1 −/− fibroblasts treated for 24 h with indicated concentrations of chaetocin. Error bars indicate s.e.m. and all P values are calculated by t -test.

    Article Snippet: To synthesize complementary DNA for RT–PCR, SuperScript III reverse transcriptase (Invitrogen) was used as previously , with 200 ng of RNA and Hexanucleotide Mix (Roche).

    Techniques: DNA Methylation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Expressing

    Maintenance of HIV-1 Nef function in 1N and GU1N infected AGMs. a Alignment of Nef sequences derived from 1N (upper three) and GU1N (lower three) infected AGMs. The HIV-1 NA7 sequence is shown on top and dashes indicate gaps introduced to optimize the alignment. Numbers in parentheses indicate the total number of nef alleles analyzed per time point. Black indicates changes in all, blue in ≥50% and gray in at least two sequences. Sites mutated in at least three AGMs are highlighted by orange dots. b Expression of selected SIVagm and HIV-1 Nef proteins. HEK293T cells were transfected with expression plasmids encoding the indicated AU1-tagged Nefs and eGFP. GAPDH and eGFP expression levels were analyzed to control for loading and transfection efficiency, respectively. c Quantitative assessment of Nef-mediated downmodulation of the indicated cellular receptors on PBMCs (MHC-I, CD3, CD28, CXCR4, and CD74), CD4+ T cells (CD4) and THP-1 cells (CD74). Shown are mean (+SD) fluorescence intensities (MFIs) of receptor expression relative to the nef -defective control (100%) derived from three experiments. d HIV-1 Nefs did not evolve activity against AGM tetherin. HEK293T cells were cotransfected with HIV-1 NL4-3 nef-vpu- constructs, expression plasmids for the indicated nef alleles and different amounts of AGM tetherin. Infectious virus yield in the culture supernatants two dpi was determined by triplicate infection of TZM-bl reporter cells. Shown is the mean of three experiments. e Antagonism of AGM SERINC5 by SIVagm and HIV-1 Nef proteins. HEK293T cells were cotransfected with HIV-1 NL4-3 proviral constructs containing the indicated nef alleles and an empty vector control or AGM SERINC5 expression vector. Viral supernatants were obtained 3 days later and infectious HIV-1 yield determined by triplicate infection of TZM-bl cells. Shown are average values of three experiments + SD ( n = 3) relative to those obtained in the absence of SERINC5 expression vector (100%)

    Journal: Nature Communications

    Article Title: Species-specific host factors rather than virus-intrinsic virulence determine primate lentiviral pathogenicity

    doi: 10.1038/s41467-018-03762-3

    Figure Lengend Snippet: Maintenance of HIV-1 Nef function in 1N and GU1N infected AGMs. a Alignment of Nef sequences derived from 1N (upper three) and GU1N (lower three) infected AGMs. The HIV-1 NA7 sequence is shown on top and dashes indicate gaps introduced to optimize the alignment. Numbers in parentheses indicate the total number of nef alleles analyzed per time point. Black indicates changes in all, blue in ≥50% and gray in at least two sequences. Sites mutated in at least three AGMs are highlighted by orange dots. b Expression of selected SIVagm and HIV-1 Nef proteins. HEK293T cells were transfected with expression plasmids encoding the indicated AU1-tagged Nefs and eGFP. GAPDH and eGFP expression levels were analyzed to control for loading and transfection efficiency, respectively. c Quantitative assessment of Nef-mediated downmodulation of the indicated cellular receptors on PBMCs (MHC-I, CD3, CD28, CXCR4, and CD74), CD4+ T cells (CD4) and THP-1 cells (CD74). Shown are mean (+SD) fluorescence intensities (MFIs) of receptor expression relative to the nef -defective control (100%) derived from three experiments. d HIV-1 Nefs did not evolve activity against AGM tetherin. HEK293T cells were cotransfected with HIV-1 NL4-3 nef-vpu- constructs, expression plasmids for the indicated nef alleles and different amounts of AGM tetherin. Infectious virus yield in the culture supernatants two dpi was determined by triplicate infection of TZM-bl reporter cells. Shown is the mean of three experiments. e Antagonism of AGM SERINC5 by SIVagm and HIV-1 Nef proteins. HEK293T cells were cotransfected with HIV-1 NL4-3 proviral constructs containing the indicated nef alleles and an empty vector control or AGM SERINC5 expression vector. Viral supernatants were obtained 3 days later and infectious HIV-1 yield determined by triplicate infection of TZM-bl cells. Shown are average values of three experiments + SD ( n = 3) relative to those obtained in the absence of SERINC5 expression vector (100%)

    Article Snippet: Viral RNA was extracted from the plasma of infected AGMs using the QIAamp viral RNA minikit (Qiagen). cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen) and the SIVagm strain-specific primer hb3′r1 (5′-GCGAACACCCAGGCTCAAGCTG-3′).

    Techniques: Infection, Derivative Assay, Sequencing, Expressing, Transfection, Fluorescence, Activity Assay, Construct, Plasmid Preparation

    SIVagm constructs expressing functional Vpu and HIV-1 Nef. a Genomic organization of the WT SIVagm construct with the nef gene (yellow) and the GU1N derivative containing the SIVgsn vpu (blue) and the HIV-1 NA7 nef (orange). b HEK293T cells were cotransfected with a vpu -deleted HIV-1 NL4-3 construct, expression plasmids for HIV-1 or SIVgsn Vpu and a vector expressing AGM tetherin (BST-2). Infectious virus yield was measured by infection of TZM-bl cells. Curves represent the average values ( n = 3) relative to those obtained in the absence of tetherin (100%). c Levels of AGM BST-2 and human CD4, NTB-A, and CD1d in HEK293T cells cotransfected with SIVgsn or HIV-1 Vpu constructs relative to those measured in cells transfected with the eGFP control vector (100%). Symbols represent the value obtained in one of three experiments. Mean ± SEM are indicated in panels c , d , e , and j . d Effect of Vpu on NF-κB activity. HEK293T cells were cotransfected with a firefly luciferase NF-κB reporter construct, a Gaussia luciferase construct for normalization, a vector for constitutively active IKKβ and constructs expressing eGFP alone or together with Vpu. Luciferase activity was determined two dpi ( n = 3 ± SEM). CTRL represents the vector control. e Receptor downmodulation in human PBMCs or CD4+ T cells (CD4) infected with HIV-1 constructs expressing the indicated Nefs relative the nef -defective virus (100%), ( n = 3 ± SEM). f Effect of NA7 and SIVagm Nefs on virus release in the presence of AGM tetherin in transfected HEK293T cells. Curves represent averages ( n = 3) relative to those obtained in the absence of tetherin (100%). g , h Infectivity and replication of SIVagm constructs in vitro. TZM-bl and MOLT-4 clone 8 cells ( g ) or AGM PBMCs ( h ) were infected with the indicated SIVagm constructs. Virus infectivity or production was monitored by β-galactosidase assay or p27 antigen ELISA, respectively. Shown is g mean of triplicate infection or h one representative experiment. i Average infectious virus yield ( n = 3) from HEK293T cells following co-transfection with SIVagm constructs and plasmids expressing AGM tetherin relative to that detected in the absence of tetherin (100%). j Effect of SIVagm constructs on receptor expression by PBMCs from three AGM relative to those infected with the nef -defective SIVagm clone (100%). p -values were calculated using the two-tailed unpaired Student's- t -test and significant differences are indicated as: * p

    Journal: Nature Communications

    Article Title: Species-specific host factors rather than virus-intrinsic virulence determine primate lentiviral pathogenicity

    doi: 10.1038/s41467-018-03762-3

    Figure Lengend Snippet: SIVagm constructs expressing functional Vpu and HIV-1 Nef. a Genomic organization of the WT SIVagm construct with the nef gene (yellow) and the GU1N derivative containing the SIVgsn vpu (blue) and the HIV-1 NA7 nef (orange). b HEK293T cells were cotransfected with a vpu -deleted HIV-1 NL4-3 construct, expression plasmids for HIV-1 or SIVgsn Vpu and a vector expressing AGM tetherin (BST-2). Infectious virus yield was measured by infection of TZM-bl cells. Curves represent the average values ( n = 3) relative to those obtained in the absence of tetherin (100%). c Levels of AGM BST-2 and human CD4, NTB-A, and CD1d in HEK293T cells cotransfected with SIVgsn or HIV-1 Vpu constructs relative to those measured in cells transfected with the eGFP control vector (100%). Symbols represent the value obtained in one of three experiments. Mean ± SEM are indicated in panels c , d , e , and j . d Effect of Vpu on NF-κB activity. HEK293T cells were cotransfected with a firefly luciferase NF-κB reporter construct, a Gaussia luciferase construct for normalization, a vector for constitutively active IKKβ and constructs expressing eGFP alone or together with Vpu. Luciferase activity was determined two dpi ( n = 3 ± SEM). CTRL represents the vector control. e Receptor downmodulation in human PBMCs or CD4+ T cells (CD4) infected with HIV-1 constructs expressing the indicated Nefs relative the nef -defective virus (100%), ( n = 3 ± SEM). f Effect of NA7 and SIVagm Nefs on virus release in the presence of AGM tetherin in transfected HEK293T cells. Curves represent averages ( n = 3) relative to those obtained in the absence of tetherin (100%). g , h Infectivity and replication of SIVagm constructs in vitro. TZM-bl and MOLT-4 clone 8 cells ( g ) or AGM PBMCs ( h ) were infected with the indicated SIVagm constructs. Virus infectivity or production was monitored by β-galactosidase assay or p27 antigen ELISA, respectively. Shown is g mean of triplicate infection or h one representative experiment. i Average infectious virus yield ( n = 3) from HEK293T cells following co-transfection with SIVagm constructs and plasmids expressing AGM tetherin relative to that detected in the absence of tetherin (100%). j Effect of SIVagm constructs on receptor expression by PBMCs from three AGM relative to those infected with the nef -defective SIVagm clone (100%). p -values were calculated using the two-tailed unpaired Student's- t -test and significant differences are indicated as: * p

    Article Snippet: Viral RNA was extracted from the plasma of infected AGMs using the QIAamp viral RNA minikit (Qiagen). cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen) and the SIVagm strain-specific primer hb3′r1 (5′-GCGAACACCCAGGCTCAAGCTG-3′).

    Techniques: Construct, Expressing, Functional Assay, Plasmid Preparation, Infection, Transfection, Activity Assay, Luciferase, In Vitro, Enzyme-linked Immunosorbent Assay, Cotransfection, Two Tailed Test

    Replication of HIV-1-like SIVagm constructs in vivo. a Mean levels of plasma viremia in AGMs infected with the indicated SIVagm constructs. Three animals were infected with each virus and plasma RNA loads were determined as described in the Methods section. Arrows indicate time points used for single genome viral RNA analysis (SGA), determination of proviral copy numbers (vDNA), or microarray analysis (Array). Symbols refer to all panels. The limit of viral RNA detection is ~100 copies/ml of plasma. b Absolute mean CD4+ T cell counts in AGMs infected with the WT, GU, 1N, and GU1N SIVagm constructs. The prevalue (Pre) represents the average CD4+ T cell count of four measurements prior to infection. c Absolute numbers and percentages of CD4+ T cells at later time points of infection ( > 170 wpi) with mean ± SEM. Each symbol represents one measurement. d Correlation between the average vRNA loads and CD4+ T cell counts (left) and percentages (right) in the final year of follow-up. Each symbol corresponds to one AGM and represents the average value of nine measurements from 171 to 240 wpi. The open triangle represents AGM14627 that was accidentally infected. p -values were calculated using the two-tailed unpaired Student's- t -test and significant differences are indicated as: * p

    Journal: Nature Communications

    Article Title: Species-specific host factors rather than virus-intrinsic virulence determine primate lentiviral pathogenicity

    doi: 10.1038/s41467-018-03762-3

    Figure Lengend Snippet: Replication of HIV-1-like SIVagm constructs in vivo. a Mean levels of plasma viremia in AGMs infected with the indicated SIVagm constructs. Three animals were infected with each virus and plasma RNA loads were determined as described in the Methods section. Arrows indicate time points used for single genome viral RNA analysis (SGA), determination of proviral copy numbers (vDNA), or microarray analysis (Array). Symbols refer to all panels. The limit of viral RNA detection is ~100 copies/ml of plasma. b Absolute mean CD4+ T cell counts in AGMs infected with the WT, GU, 1N, and GU1N SIVagm constructs. The prevalue (Pre) represents the average CD4+ T cell count of four measurements prior to infection. c Absolute numbers and percentages of CD4+ T cells at later time points of infection ( > 170 wpi) with mean ± SEM. Each symbol represents one measurement. d Correlation between the average vRNA loads and CD4+ T cell counts (left) and percentages (right) in the final year of follow-up. Each symbol corresponds to one AGM and represents the average value of nine measurements from 171 to 240 wpi. The open triangle represents AGM14627 that was accidentally infected. p -values were calculated using the two-tailed unpaired Student's- t -test and significant differences are indicated as: * p

    Article Snippet: Viral RNA was extracted from the plasma of infected AGMs using the QIAamp viral RNA minikit (Qiagen). cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen) and the SIVagm strain-specific primer hb3′r1 (5′-GCGAACACCCAGGCTCAAGCTG-3′).

    Techniques: Construct, In Vivo, Infection, Microarray, RNA Detection, Cell Counting, Two Tailed Test

    Maintenance of HIV-1-like Vpu function in GU1N infected AGMs. a Alignment of Vpu sequences derived from GU1N-infected AGMs at 121 (2012) and 223 wpi (2014) and primary or synthetic (Sel1 and Sel2) Vpus chosen for functional analysis. Orange dots highlight mutations found in all three AGMs. Dashes indicate gaps introduced to optimize the alignment. Numbers specify the animal, year of plasma isolation, and number of the respective Vpu sequences. Black indicates changes in all, blue in ≥50% and gray in at least two sequences. b Expression of AU1-tagged SIVgsn Vpu proteins. HEK293T cells were transfected with expression plasmids encoding the indicated AU1-tagged Vpus and eGFP. Mock transfected cells were used as negative controls. GAPDH expression levels were analyzed to control for loading. c Infectious virus (left) and p24 antigen (right) yield from HEK293T cells cotransfected with an HIV-1 NL4-3 Δ vpu construct and vectors expressing the indicated vpu alleles in combination with increasing amounts of plasmids expressing AGM tetherin. Shown are average values derived from three experiments relative to those obtained in the absence of tetherin (100%). d Vpu-dependent reduction of CD4, NTB-A, CD1d, and AGM tetherin surface expression in HEK293T cells relative to those measured in cells transfected with the eGFP only control vector. Values in panels d and e represent mean (+SEM) derived from three independent experiments. e AGM-derived Vpu proteins suppress NF-κB activity. The effects were measured as described in the legend to Fig. 1c

    Journal: Nature Communications

    Article Title: Species-specific host factors rather than virus-intrinsic virulence determine primate lentiviral pathogenicity

    doi: 10.1038/s41467-018-03762-3

    Figure Lengend Snippet: Maintenance of HIV-1-like Vpu function in GU1N infected AGMs. a Alignment of Vpu sequences derived from GU1N-infected AGMs at 121 (2012) and 223 wpi (2014) and primary or synthetic (Sel1 and Sel2) Vpus chosen for functional analysis. Orange dots highlight mutations found in all three AGMs. Dashes indicate gaps introduced to optimize the alignment. Numbers specify the animal, year of plasma isolation, and number of the respective Vpu sequences. Black indicates changes in all, blue in ≥50% and gray in at least two sequences. b Expression of AU1-tagged SIVgsn Vpu proteins. HEK293T cells were transfected with expression plasmids encoding the indicated AU1-tagged Vpus and eGFP. Mock transfected cells were used as negative controls. GAPDH expression levels were analyzed to control for loading. c Infectious virus (left) and p24 antigen (right) yield from HEK293T cells cotransfected with an HIV-1 NL4-3 Δ vpu construct and vectors expressing the indicated vpu alleles in combination with increasing amounts of plasmids expressing AGM tetherin. Shown are average values derived from three experiments relative to those obtained in the absence of tetherin (100%). d Vpu-dependent reduction of CD4, NTB-A, CD1d, and AGM tetherin surface expression in HEK293T cells relative to those measured in cells transfected with the eGFP only control vector. Values in panels d and e represent mean (+SEM) derived from three independent experiments. e AGM-derived Vpu proteins suppress NF-κB activity. The effects were measured as described in the legend to Fig. 1c

    Article Snippet: Viral RNA was extracted from the plasma of infected AGMs using the QIAamp viral RNA minikit (Qiagen). cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen) and the SIVagm strain-specific primer hb3′r1 (5′-GCGAACACCCAGGCTCAAGCTG-3′).

    Techniques: Infection, Derivative Assay, Functional Assay, Isolation, Expressing, Transfection, Construct, Plasmid Preparation, Activity Assay

    Immunological findings during the first year of HIV-1-like SIVagm infection. a Increase of urinary neopterin levels at the indicated days post-infection (dpi) relative to the values measured prior to SIV infection. b , c Levels of CD16+ NK ( b ) and CCR5+ or CXCR3+/CCR5+ CD4+ (left) and CD8+ (right) T cells ( c ) at the indicated weeks post-infection (wpi) relative to the baseline values (100%). The shaded area indicates time points prior to infection. d Percentages of activated HLA-DR+ CD4+ and CD8+ T cells in duodenum prior to infection and at six wpi. Shown are mean values obtained for the three animals per group (+SEM). e Percentages of CCR5+ CD4+ T cells and activated HLA-DR+ CD4+ and CD8+ T cells in bronchoalveolar lavage obtained at 55, 64, and 72 wpi. p -values were calculated using the two-tailed unpaired Student's- t -test and significant differences are indicated as: * p

    Journal: Nature Communications

    Article Title: Species-specific host factors rather than virus-intrinsic virulence determine primate lentiviral pathogenicity

    doi: 10.1038/s41467-018-03762-3

    Figure Lengend Snippet: Immunological findings during the first year of HIV-1-like SIVagm infection. a Increase of urinary neopterin levels at the indicated days post-infection (dpi) relative to the values measured prior to SIV infection. b , c Levels of CD16+ NK ( b ) and CCR5+ or CXCR3+/CCR5+ CD4+ (left) and CD8+ (right) T cells ( c ) at the indicated weeks post-infection (wpi) relative to the baseline values (100%). The shaded area indicates time points prior to infection. d Percentages of activated HLA-DR+ CD4+ and CD8+ T cells in duodenum prior to infection and at six wpi. Shown are mean values obtained for the three animals per group (+SEM). e Percentages of CCR5+ CD4+ T cells and activated HLA-DR+ CD4+ and CD8+ T cells in bronchoalveolar lavage obtained at 55, 64, and 72 wpi. p -values were calculated using the two-tailed unpaired Student's- t -test and significant differences are indicated as: * p

    Article Snippet: Viral RNA was extracted from the plasma of infected AGMs using the QIAamp viral RNA minikit (Qiagen). cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen) and the SIVagm strain-specific primer hb3′r1 (5′-GCGAACACCCAGGCTCAAGCTG-3′).

    Techniques: Infection, Two Tailed Test

    IFN-β induction by KSHV ORF8 10420-10496 RNA is dependent on RIG-I but not on RNA Pol III. (A) HEK293 cells in 24-well plates were transfected with siRNAs targeting RIG-I or STING. At 48 h posttransfection, cells were transfected with 5′-ppp-dsRNA or poly(I·C) (positive controls), the indicated concentration (in micrograms) of ORF8 10,420-10,496 RNA, or not transfected (no-treatment control). At 24 h after ORF8 10420-10496 RNA transfection, IFN-β mRNA was measured by qRT-PCR. (B) WT RIG-I and RIG-I knockout MEF in 24-well plates were transfected with 5′-ppp-dsRNA (a positive control) or with the indicated concentration of ORF8 10420-10496 RNA or not transfected (no-treatment control). At 24 h after transfection, IFN-β mRNA was measured by qRT-PCR. (C) HEK293 cells in 24-well plates were transfected with siRNAs targeting RNA Pol III or RIG-I. At 48 h posttransfection, cells were transfected with poly(I·C) or poly(dA·dT) (positive controls) or with the indicated concentration of ORF8 10,420-10,496 RNA or not transfected (no-treatment control). At 24 h after ORF8 10420-10496 RNA transfection, IFN-β mRNA was measured by qRT-PCR. Data are presented as mean ± SD. Error bars represent the variation range of duplicate experiments. The data are representative of three independent experiments. **, P

    Journal: mBio

    Article Title: RIG-I Detects Kaposi’s Sarcoma-Associated Herpesvirus Transcripts in a RNA Polymerase III-Independent Manner

    doi: 10.1128/mBio.00823-18

    Figure Lengend Snippet: IFN-β induction by KSHV ORF8 10420-10496 RNA is dependent on RIG-I but not on RNA Pol III. (A) HEK293 cells in 24-well plates were transfected with siRNAs targeting RIG-I or STING. At 48 h posttransfection, cells were transfected with 5′-ppp-dsRNA or poly(I·C) (positive controls), the indicated concentration (in micrograms) of ORF8 10,420-10,496 RNA, or not transfected (no-treatment control). At 24 h after ORF8 10420-10496 RNA transfection, IFN-β mRNA was measured by qRT-PCR. (B) WT RIG-I and RIG-I knockout MEF in 24-well plates were transfected with 5′-ppp-dsRNA (a positive control) or with the indicated concentration of ORF8 10420-10496 RNA or not transfected (no-treatment control). At 24 h after transfection, IFN-β mRNA was measured by qRT-PCR. (C) HEK293 cells in 24-well plates were transfected with siRNAs targeting RNA Pol III or RIG-I. At 48 h posttransfection, cells were transfected with poly(I·C) or poly(dA·dT) (positive controls) or with the indicated concentration of ORF8 10,420-10,496 RNA or not transfected (no-treatment control). At 24 h after ORF8 10420-10496 RNA transfection, IFN-β mRNA was measured by qRT-PCR. Data are presented as mean ± SD. Error bars represent the variation range of duplicate experiments. The data are representative of three independent experiments. **, P

    Article Snippet: The same amount of RNA from each sample was processed for reverse transcription and quantitative PCR using SuperScript III reverse transcriptase (Invitrogen), SYBR green PCR master mix (Bio-Rad), and ABI 7300, as described previously ( ).

    Techniques: Transfection, Concentration Assay, Quantitative RT-PCR, Knock-Out, Positive Control

    Immunostimulatory activity of RNA from RIG-I and control (IgG) IPs in wild-type (WT) RIG-I and RIG-I knockout MEF cells. WT RIG-I and RIG-I knockout MEF cells in 24-well plates were transfected with four different immunoprecipitated RNA samples: (i) IgG antibody pulldown from latent iSLK.219 cells (IgG IP; No Dox); (ii) IgG antibody pulldown from lytic iSLK.219 cells reactivated with doxycycline (Dox) (IgG IP; Dox); (iii) RIG-I antibody pulldown from latent iSLK.219 cells (RIG-I; No Dox); and (iv) RIG-I antibody pulldown from lytic iSLK.219 cells (RIG-I IP; Dox). In parallel, cells were transfected with poly(I·C) or infected with VSV (positive controls). Twenty-four hours after RNA transfection or virus infection, total RNA was purified from transfected or infected cells and was treated with RNase A or not treated with RNase A, followed by qRT-PCR analysis for mouse IFN-β mRNA. (A) Enrichment of immunostimulatory RNA from RIG-I IP Dox compared with control (IgG) IP. (B) Enrichment of immunostimulatory RNA with RIG-I IP compared with total RNA (Input) from latent iSLK.219 cells (No Dox) or lytic iSLK.219 cells (Dox). (C) RNase A treatment of RIG-I-bound RNA as well as Input RNAs completely abolishes their immunostimulatory activity. (D) Mouse RIG-I mRNA in WT RIG-I and RIG-I −/− MEF by qRT-PCR analysis to confirm that there is no RIG-I in RIG-I −/− MEF. Data are presented as mean ± SD. Error bars represent the variation range of duplicate experiments. The data are representative of three independent experiments. **, P

    Journal: mBio

    Article Title: RIG-I Detects Kaposi’s Sarcoma-Associated Herpesvirus Transcripts in a RNA Polymerase III-Independent Manner

    doi: 10.1128/mBio.00823-18

    Figure Lengend Snippet: Immunostimulatory activity of RNA from RIG-I and control (IgG) IPs in wild-type (WT) RIG-I and RIG-I knockout MEF cells. WT RIG-I and RIG-I knockout MEF cells in 24-well plates were transfected with four different immunoprecipitated RNA samples: (i) IgG antibody pulldown from latent iSLK.219 cells (IgG IP; No Dox); (ii) IgG antibody pulldown from lytic iSLK.219 cells reactivated with doxycycline (Dox) (IgG IP; Dox); (iii) RIG-I antibody pulldown from latent iSLK.219 cells (RIG-I; No Dox); and (iv) RIG-I antibody pulldown from lytic iSLK.219 cells (RIG-I IP; Dox). In parallel, cells were transfected with poly(I·C) or infected with VSV (positive controls). Twenty-four hours after RNA transfection or virus infection, total RNA was purified from transfected or infected cells and was treated with RNase A or not treated with RNase A, followed by qRT-PCR analysis for mouse IFN-β mRNA. (A) Enrichment of immunostimulatory RNA from RIG-I IP Dox compared with control (IgG) IP. (B) Enrichment of immunostimulatory RNA with RIG-I IP compared with total RNA (Input) from latent iSLK.219 cells (No Dox) or lytic iSLK.219 cells (Dox). (C) RNase A treatment of RIG-I-bound RNA as well as Input RNAs completely abolishes their immunostimulatory activity. (D) Mouse RIG-I mRNA in WT RIG-I and RIG-I −/− MEF by qRT-PCR analysis to confirm that there is no RIG-I in RIG-I −/− MEF. Data are presented as mean ± SD. Error bars represent the variation range of duplicate experiments. The data are representative of three independent experiments. **, P

    Article Snippet: The same amount of RNA from each sample was processed for reverse transcription and quantitative PCR using SuperScript III reverse transcriptase (Invitrogen), SYBR green PCR master mix (Bio-Rad), and ABI 7300, as described previously ( ).

    Techniques: Activity Assay, Knock-Out, Transfection, Immunoprecipitation, Infection, Purification, Quantitative RT-PCR

    KSHV ORF8 10420-10496 RNA induces IFN-β and RIG-I in RNA-transfected cells. (A to D) HEK293 cells in 24-well plates were transfected with 5′-ppp-dsRNA or poly(I⋅C) (positive controls), or with the indicated concentration (in nanograms or micrograms) of ORF8 10420-10496 RNA or not transfected (no-treatment control). At 24 h after transfection, cells were harvested for RNA extraction. The relative mRNA levels of IFN-β (A), RIG-I (B), and STING (C) normalized to β-actin were measured by qRT-PCR. The IFN-β protein level was measured by ELISA from the culture supernatants (D). (E to G) Uninfected iSLK cells in 24-well plates were transfected with 5′-ppp-dsRNA or poly(I·C) (positive controls) or with the indicated concentration of ORF8 10420-10496 RNA or not transfected (negative control). At 24 h after transfection, cells were harvested for RNA extraction. The relative mRNA levels of IFN-β (E), RIG-I (F), and STING (G) normalized to β-actin were measured by qRT-PCR. Data are presented as mean plus SD. Error bars represent the variation range of duplicate experiments. The data are representative of three independent experiments. **, P

    Journal: mBio

    Article Title: RIG-I Detects Kaposi’s Sarcoma-Associated Herpesvirus Transcripts in a RNA Polymerase III-Independent Manner

    doi: 10.1128/mBio.00823-18

    Figure Lengend Snippet: KSHV ORF8 10420-10496 RNA induces IFN-β and RIG-I in RNA-transfected cells. (A to D) HEK293 cells in 24-well plates were transfected with 5′-ppp-dsRNA or poly(I⋅C) (positive controls), or with the indicated concentration (in nanograms or micrograms) of ORF8 10420-10496 RNA or not transfected (no-treatment control). At 24 h after transfection, cells were harvested for RNA extraction. The relative mRNA levels of IFN-β (A), RIG-I (B), and STING (C) normalized to β-actin were measured by qRT-PCR. The IFN-β protein level was measured by ELISA from the culture supernatants (D). (E to G) Uninfected iSLK cells in 24-well plates were transfected with 5′-ppp-dsRNA or poly(I·C) (positive controls) or with the indicated concentration of ORF8 10420-10496 RNA or not transfected (negative control). At 24 h after transfection, cells were harvested for RNA extraction. The relative mRNA levels of IFN-β (E), RIG-I (F), and STING (G) normalized to β-actin were measured by qRT-PCR. Data are presented as mean plus SD. Error bars represent the variation range of duplicate experiments. The data are representative of three independent experiments. **, P

    Article Snippet: The same amount of RNA from each sample was processed for reverse transcription and quantitative PCR using SuperScript III reverse transcriptase (Invitrogen), SYBR green PCR master mix (Bio-Rad), and ABI 7300, as described previously ( ).

    Techniques: Transfection, Concentration Assay, RNA Extraction, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Negative Control

    qRT-PCR analysis of RIG-I-associated RNA from RIG-I and control (IgG) IPs. (A) Schematic of qRT-PCR procedure from endogenous RIG-I immunoprecipitation (IP) in latent or lytic iSLK cells. (B) qRT-PCR products of RNAs immunoprecipitated from endogenous RIG-I IP were run on an agarose gel, revealing KSHV-specific amplifications not present in the control IgG IP. The presence (+) or absence (−) of doxycycline (Dox) and reverse transcriptase (RT) are shown above the lanes. (C) Schematic of qRT-PCR procedure from purified recombinant FLAG-RIG-I IP in latent or lytic iSLK cells. (D) qRT-PCR products of RNAs immunoprecipitated from recombinant FLAG-RIG-I were run on an agarose gel, revealing KSHV-specific amplifications not present in the control IgG IP. (E) Schematic of qRT-PCR procedure from endogenous RIG-I IP in latent and lytic BCBL1 cells. (F) qRT-PCR products from RNAs immunoprecipitated from endogenous RIG-I IP of lytic BCBL1 cells were run on an agarose gel, revealing KSHV-specific gene bands not present in the control IgG IP. The data are representative of three independent qRT-PCR experiments.

    Journal: mBio

    Article Title: RIG-I Detects Kaposi’s Sarcoma-Associated Herpesvirus Transcripts in a RNA Polymerase III-Independent Manner

    doi: 10.1128/mBio.00823-18

    Figure Lengend Snippet: qRT-PCR analysis of RIG-I-associated RNA from RIG-I and control (IgG) IPs. (A) Schematic of qRT-PCR procedure from endogenous RIG-I immunoprecipitation (IP) in latent or lytic iSLK cells. (B) qRT-PCR products of RNAs immunoprecipitated from endogenous RIG-I IP were run on an agarose gel, revealing KSHV-specific amplifications not present in the control IgG IP. The presence (+) or absence (−) of doxycycline (Dox) and reverse transcriptase (RT) are shown above the lanes. (C) Schematic of qRT-PCR procedure from purified recombinant FLAG-RIG-I IP in latent or lytic iSLK cells. (D) qRT-PCR products of RNAs immunoprecipitated from recombinant FLAG-RIG-I were run on an agarose gel, revealing KSHV-specific amplifications not present in the control IgG IP. (E) Schematic of qRT-PCR procedure from endogenous RIG-I IP in latent and lytic BCBL1 cells. (F) qRT-PCR products from RNAs immunoprecipitated from endogenous RIG-I IP of lytic BCBL1 cells were run on an agarose gel, revealing KSHV-specific gene bands not present in the control IgG IP. The data are representative of three independent qRT-PCR experiments.

    Article Snippet: The same amount of RNA from each sample was processed for reverse transcription and quantitative PCR using SuperScript III reverse transcriptase (Invitrogen), SYBR green PCR master mix (Bio-Rad), and ABI 7300, as described previously ( ).

    Techniques: Quantitative RT-PCR, Immunoprecipitation, Agarose Gel Electrophoresis, Purification, Recombinant

    RNA Pol III is not required for IFN-β expression during KSHV primary infection or reactivation. (A and B) HEK293 cells in 24-well plates were treated with the indicated concentrations of the RNA Pol III inhibitor ML-60218. Eight hours after treatment, cells were transfected with poly(dA·dT) for 6 h (A) or infection with rKSHV.219 for 48 h (B), IFN-β mRNA level was measured by qRT-PCR. (C to F) HEK293 cells in 24-well plates were transfected with siRNAs targeting RNA Pol III or RIG-I. Twenty-four hours posttransfection, cells were infected with rKSHV.219 for 48 h. The relative expression levels of IFN-β (C), RNA Pol III (D), and RIG-I (E) normalized to β-actin were measured by qRT-PCR. (F) Western blotting showing efficient knockdown of RNA Pol III or RIG-I in HEK293 cells. (G to J) iSLK.219 cells latently infected with rKSHV.219 were transfected with nontargeting control, RNA Pol III, or RIG-I siRNA. Twenty-four hours after siRNA transfection, doxycycline was added to reactivate the iSLK.219 cells. At 72 h postreactivation, the relative expression levels of IFN-β (G), RNA Pol III (H), and RIG-I (I) normalized to β-actin were measured by qRT-PCR. (J) Western blots showing efficient knockdown of RNA Pol III or RIG-I in iSLK.219 cells. Data are presented as means plus standard deviations (SD). Error bars represent the variation range of duplicate experiments. The data are representative of three independent experiments. Values that are statistically significantly different are indicated by bars and asterisks as follows: **, P

    Journal: mBio

    Article Title: RIG-I Detects Kaposi’s Sarcoma-Associated Herpesvirus Transcripts in a RNA Polymerase III-Independent Manner

    doi: 10.1128/mBio.00823-18

    Figure Lengend Snippet: RNA Pol III is not required for IFN-β expression during KSHV primary infection or reactivation. (A and B) HEK293 cells in 24-well plates were treated with the indicated concentrations of the RNA Pol III inhibitor ML-60218. Eight hours after treatment, cells were transfected with poly(dA·dT) for 6 h (A) or infection with rKSHV.219 for 48 h (B), IFN-β mRNA level was measured by qRT-PCR. (C to F) HEK293 cells in 24-well plates were transfected with siRNAs targeting RNA Pol III or RIG-I. Twenty-four hours posttransfection, cells were infected with rKSHV.219 for 48 h. The relative expression levels of IFN-β (C), RNA Pol III (D), and RIG-I (E) normalized to β-actin were measured by qRT-PCR. (F) Western blotting showing efficient knockdown of RNA Pol III or RIG-I in HEK293 cells. (G to J) iSLK.219 cells latently infected with rKSHV.219 were transfected with nontargeting control, RNA Pol III, or RIG-I siRNA. Twenty-four hours after siRNA transfection, doxycycline was added to reactivate the iSLK.219 cells. At 72 h postreactivation, the relative expression levels of IFN-β (G), RNA Pol III (H), and RIG-I (I) normalized to β-actin were measured by qRT-PCR. (J) Western blots showing efficient knockdown of RNA Pol III or RIG-I in iSLK.219 cells. Data are presented as means plus standard deviations (SD). Error bars represent the variation range of duplicate experiments. The data are representative of three independent experiments. Values that are statistically significantly different are indicated by bars and asterisks as follows: **, P

    Article Snippet: The same amount of RNA from each sample was processed for reverse transcription and quantitative PCR using SuperScript III reverse transcriptase (Invitrogen), SYBR green PCR master mix (Bio-Rad), and ABI 7300, as described previously ( ).

    Techniques: Expressing, Infection, Transfection, Quantitative RT-PCR, Western Blot

    Biochemical analysis of AUG docking and potential frame-shifting ( a ) Denaturing sequencing gel of the reverse transcription and toeprinting of wild-type (WT) and mutant IRES RNAs with the relevant part of the gel boxed and expanded to the right. Dideoxy sequencing reaction in lanes 1-4, free IRES in lanes 5, 7, 9 and 11 and IRES-80S complexes (formed by incubation in rabbit reticulocyte lysate, RRL with cycloheximide, CHX) in lanes 6, 8, 10 and 12. Nucleotide numbers are bulleted on the left, the A of the AUG is indicated by the grey arrow (+1) and the toeprint is indicated by the blue arrow (+15, +16) to the right of the expanded gel. ( b ) Graph of quantitated, normalized and background-corrected IRES-80S toeprints from panel a . +1 and +15, +16 are indicated by grey and blue lines, respectively (pseudoknot, pknot; domain IV, dIV)The location of IRES secondary structural domains are indicated beneath the graphs. ( c ) Cartoon of the uncapped, unpolyadenylated monocistronic Photinus reporter. The region of the RNA between the viral AUG and luciferase AUG (both highlighted in red) is expanded below. One or two adenosines (blue box) were added for frameshift analysis. ( d ) Graph of 90 minute translation assay for WT and ΔGCC reporters without any mutations or with the addition of one or two adenosine residues. Y-axis represents luciferase activity in relative light units (RLUs) detected and error bars represent one s.e.m of three independent triplicate experiments.

    Journal: Nature structural & molecular biology

    Article Title: HCV IRES manipulates the ribosome to promote the switch from translation initiation to elongation

    doi: 10.1038/nsmb.2465

    Figure Lengend Snippet: Biochemical analysis of AUG docking and potential frame-shifting ( a ) Denaturing sequencing gel of the reverse transcription and toeprinting of wild-type (WT) and mutant IRES RNAs with the relevant part of the gel boxed and expanded to the right. Dideoxy sequencing reaction in lanes 1-4, free IRES in lanes 5, 7, 9 and 11 and IRES-80S complexes (formed by incubation in rabbit reticulocyte lysate, RRL with cycloheximide, CHX) in lanes 6, 8, 10 and 12. Nucleotide numbers are bulleted on the left, the A of the AUG is indicated by the grey arrow (+1) and the toeprint is indicated by the blue arrow (+15, +16) to the right of the expanded gel. ( b ) Graph of quantitated, normalized and background-corrected IRES-80S toeprints from panel a . +1 and +15, +16 are indicated by grey and blue lines, respectively (pseudoknot, pknot; domain IV, dIV)The location of IRES secondary structural domains are indicated beneath the graphs. ( c ) Cartoon of the uncapped, unpolyadenylated monocistronic Photinus reporter. The region of the RNA between the viral AUG and luciferase AUG (both highlighted in red) is expanded below. One or two adenosines (blue box) were added for frameshift analysis. ( d ) Graph of 90 minute translation assay for WT and ΔGCC reporters without any mutations or with the addition of one or two adenosine residues. Y-axis represents luciferase activity in relative light units (RLUs) detected and error bars represent one s.e.m of three independent triplicate experiments.

    Article Snippet: We made the ladder used for analysis with wild type toeprint RNA reverse transcribed with SuperScript® III Reverse Transcriptase (Life Technologies) with annealing and extension temperatures at 45 °C.

    Techniques: Sequencing, Mutagenesis, Incubation, Luciferase, Activity Assay

    Wild-type (WT) and mutant IRES ribosome assembly assays and position of domain IIb ( a ) Graph of measured radiolabeled IRES RNA migration through a sucrose gradient after 15 minute incubation in rabbit reticulocyte lysate (RRL) followed by ultracentrifugation. 40S and 48S* are indistinguishable in our sucrose gradient. ( b ) Amount of 80S complex formed at time points from 0.5 to 10 min. Error bars represent one s.e.m of three independent experiments. ( c ) Top: cryo-EM reconstruction of the full-length HCV IRES RNA (purple) bound to mammalian 40S subunit (yellow) 23 . Bottom: crystal structure of 40S subunit from Tetrahymena thermophila (yellow) 32 and the NMR structure of HCV IRES domain II (dII, purple) 33 placed into the cryo-EM reconstruction (not shown). RpS5 is green and structural features are labeled. ( d ) Comparison of the orientation of E-site bound tRNA (blue) and HCV IRES dII (purple) within the decoding groove. Position of dII is based on the model shown in panel d and previously published 22 , 24 , while the E-site tRNA is from a crystal structure of the T. thermophilus 70S ribosome 55 . RpS5 (S7 in bacteria) is green, its β hairpin and the tRNA anticodon (AC) loop are indicated. ( e ) Close-up view of the position of domain IIb (dIIb, purple) near the β-hairpin of rpS5 (green). The location of the nucleotides that were mutated in this study are blue and labeled.

    Journal: Nature structural & molecular biology

    Article Title: HCV IRES manipulates the ribosome to promote the switch from translation initiation to elongation

    doi: 10.1038/nsmb.2465

    Figure Lengend Snippet: Wild-type (WT) and mutant IRES ribosome assembly assays and position of domain IIb ( a ) Graph of measured radiolabeled IRES RNA migration through a sucrose gradient after 15 minute incubation in rabbit reticulocyte lysate (RRL) followed by ultracentrifugation. 40S and 48S* are indistinguishable in our sucrose gradient. ( b ) Amount of 80S complex formed at time points from 0.5 to 10 min. Error bars represent one s.e.m of three independent experiments. ( c ) Top: cryo-EM reconstruction of the full-length HCV IRES RNA (purple) bound to mammalian 40S subunit (yellow) 23 . Bottom: crystal structure of 40S subunit from Tetrahymena thermophila (yellow) 32 and the NMR structure of HCV IRES domain II (dII, purple) 33 placed into the cryo-EM reconstruction (not shown). RpS5 is green and structural features are labeled. ( d ) Comparison of the orientation of E-site bound tRNA (blue) and HCV IRES dII (purple) within the decoding groove. Position of dII is based on the model shown in panel d and previously published 22 , 24 , while the E-site tRNA is from a crystal structure of the T. thermophilus 70S ribosome 55 . RpS5 (S7 in bacteria) is green, its β hairpin and the tRNA anticodon (AC) loop are indicated. ( e ) Close-up view of the position of domain IIb (dIIb, purple) near the β-hairpin of rpS5 (green). The location of the nucleotides that were mutated in this study are blue and labeled.

    Article Snippet: We made the ladder used for analysis with wild type toeprint RNA reverse transcribed with SuperScript® III Reverse Transcriptase (Life Technologies) with annealing and extension temperatures at 45 °C.

    Techniques: Mutagenesis, Migration, Incubation, Electron Microscopy, Nuclear Magnetic Resonance, Labeling

    Characterization of the structural changes induced by dIIb mutation ( a ) Secondary structure of the RNA sequence (previously solved 33 ) used to characterize the structural changes induced by mutating domain IIb (dIIb). Elements color-coded to match other panels. ( b ) 1-D 1 H-NMR spectra (in water) of the wild-type (WT) and dIIb mutant RNAs. This part of the spectrum contains resonances from the base imino protons with assignments for WT shown at the top. The gray boxes indicate the most shifted resonances in all three mutant spectra. ( c ) Overlaid WT and ΔapexC 2-D 1 H-NOESY NMR spectra (in water). The portion of the spectra that contains the cross-peaks between imino protons is shown with the G87 and U78 imino protons cross-peak indicated. WT spectrum is black, mutant is red. ( d ) Same overlaid spectra and color scheme as in panel c , showing the cross-peaks between imino protons and other protons. The location of the cross-peaks between the G87 imino proton and the C79 amino protons are boxed, assignments of imino proton resonances are above the spectrum matching the colors of panels a and b . ( e ) Close-up view of the tip of dIIb against the β hairpin of rpS5 (green). The C79-G87 base-pair (orange) and location of the single base deletion (C83) in ΔapexC (red) are indicated. ( f ) Same view as panel e , but showing the location of previously-reported increases in chemical modification in the ΔapexC mutant (yellow) 22 .

    Journal: Nature structural & molecular biology

    Article Title: HCV IRES manipulates the ribosome to promote the switch from translation initiation to elongation

    doi: 10.1038/nsmb.2465

    Figure Lengend Snippet: Characterization of the structural changes induced by dIIb mutation ( a ) Secondary structure of the RNA sequence (previously solved 33 ) used to characterize the structural changes induced by mutating domain IIb (dIIb). Elements color-coded to match other panels. ( b ) 1-D 1 H-NMR spectra (in water) of the wild-type (WT) and dIIb mutant RNAs. This part of the spectrum contains resonances from the base imino protons with assignments for WT shown at the top. The gray boxes indicate the most shifted resonances in all three mutant spectra. ( c ) Overlaid WT and ΔapexC 2-D 1 H-NOESY NMR spectra (in water). The portion of the spectra that contains the cross-peaks between imino protons is shown with the G87 and U78 imino protons cross-peak indicated. WT spectrum is black, mutant is red. ( d ) Same overlaid spectra and color scheme as in panel c , showing the cross-peaks between imino protons and other protons. The location of the cross-peaks between the G87 imino proton and the C79 amino protons are boxed, assignments of imino proton resonances are above the spectrum matching the colors of panels a and b . ( e ) Close-up view of the tip of dIIb against the β hairpin of rpS5 (green). The C79-G87 base-pair (orange) and location of the single base deletion (C83) in ΔapexC (red) are indicated. ( f ) Same view as panel e , but showing the location of previously-reported increases in chemical modification in the ΔapexC mutant (yellow) 22 .

    Article Snippet: We made the ladder used for analysis with wild type toeprint RNA reverse transcribed with SuperScript® III Reverse Transcriptase (Life Technologies) with annealing and extension temperatures at 45 °C.

    Techniques: Mutagenesis, Sequencing, Nuclear Magnetic Resonance, Modification

    Puromycin and toeprinting assays with antiobiotic ( a ) Structures of tyrosyl-tRNA (left) and the puromycin (right), differences in grey and blue. ( b ) Cartoon of the puromycin assay (40S subunit yellow, 60S blue) moving from left to right: IRES-40S formation, then Met-tRNA i and puromycin (puro-NH 2 ) binding in the 60S subunit P and A sites, respectively. Peptidyl transferase results in methionine bound to puromycin via a noncanonical amide linkage (met-puro), then extraction. ( c ) Quantitated and background-corrected graph of met-puro formation after 60 minutes on wild-type (WT) and mutant IRES RNAs. Error bars: one s.e.m of three independent duplicate experiments. ( d ) Yeast 80S ribosome crystal structure 56 (40S subunit yellow, 60S cyan) with approximate locations and distance between the IRES domain IIb (dIIb)-rpS5 interaction and the peptidyl transferase center (PTC) shown. ( e ) Relevant part of the toeprint gel with the dideoxy sequencing reactions in lanes 1-4, free IRES-80S complexes in rabbit reticulocyte lysate (RRL) without any antibiotic in lanes 5, 7, 9 and 11 as well as initiating and elongating IRES-80S complexes formed in RRL with hygromycin B in lanes 6, 8, 10 and 12. Black arrowhead represents initiating complexes (+15, +16) and blue arrowhead represents elongating complexes (+20) on the right, nucleotide numbers are bulleted, on the left. ( f ) Graph of quantitated, normalized, and background-corrected toeprints without antibiotic from panel e (WT IRES red, dIIb mutants grey). ( g ) Same as in panel f except graph represents toeprints with antibiotic. Error bars represent one s.d. of three independent experiments.

    Journal: Nature structural & molecular biology

    Article Title: HCV IRES manipulates the ribosome to promote the switch from translation initiation to elongation

    doi: 10.1038/nsmb.2465

    Figure Lengend Snippet: Puromycin and toeprinting assays with antiobiotic ( a ) Structures of tyrosyl-tRNA (left) and the puromycin (right), differences in grey and blue. ( b ) Cartoon of the puromycin assay (40S subunit yellow, 60S blue) moving from left to right: IRES-40S formation, then Met-tRNA i and puromycin (puro-NH 2 ) binding in the 60S subunit P and A sites, respectively. Peptidyl transferase results in methionine bound to puromycin via a noncanonical amide linkage (met-puro), then extraction. ( c ) Quantitated and background-corrected graph of met-puro formation after 60 minutes on wild-type (WT) and mutant IRES RNAs. Error bars: one s.e.m of three independent duplicate experiments. ( d ) Yeast 80S ribosome crystal structure 56 (40S subunit yellow, 60S cyan) with approximate locations and distance between the IRES domain IIb (dIIb)-rpS5 interaction and the peptidyl transferase center (PTC) shown. ( e ) Relevant part of the toeprint gel with the dideoxy sequencing reactions in lanes 1-4, free IRES-80S complexes in rabbit reticulocyte lysate (RRL) without any antibiotic in lanes 5, 7, 9 and 11 as well as initiating and elongating IRES-80S complexes formed in RRL with hygromycin B in lanes 6, 8, 10 and 12. Black arrowhead represents initiating complexes (+15, +16) and blue arrowhead represents elongating complexes (+20) on the right, nucleotide numbers are bulleted, on the left. ( f ) Graph of quantitated, normalized, and background-corrected toeprints without antibiotic from panel e (WT IRES red, dIIb mutants grey). ( g ) Same as in panel f except graph represents toeprints with antibiotic. Error bars represent one s.d. of three independent experiments.

    Article Snippet: We made the ladder used for analysis with wild type toeprint RNA reverse transcribed with SuperScript® III Reverse Transcriptase (Life Technologies) with annealing and extension temperatures at 45 °C.

    Techniques: Binding Assay, Mutagenesis, Sequencing

    In vitro translation analysis of dIIb mutations ( a ) HCV viral RNA and cellular mRNA differ in their origin and features. HCV viral RNA is delivered directly to the cytoplasm lacking a cap and poly-(A) tail, while cellular mRNA is produced and processed in the nucleus before exportation to the cytoplasm with a cap and tail. However, both are translated by the same cellular machinery, mandating different mechanisms of initiation. ( b ) Simplified diagram HCV IRES 80S ribosome assembly mechanism. The IRES first binds the 40S subunit (yellow), then eukaryotic initiation factor (eIF) 3 (green) and the ternary complex (eIF2-GTP-Met-tRNA i , red and line), and finally after GTP hydrolysis and eIF release the 60S subunit (blue) joins to form an 80S ribosome. Asterisk denotes a difference from canonical 48S complexes. ( c ) Cartoon representation of the secondary structure of the HCV IRES. The location of the start AUG is shown. Boxes areas indicate the parts of the IRES involved in different steps of the mechanism shown in panel b. ( d ) Schematic of mutations (red) made to domain IIb in the context of the uncapped and unpolyadenylated monocistronic Photinus luciferase reporter. Wild-type (WT) RNA is shown to the left. ( e ) Time course of a translation assay from 0 to 90 min as measured by produced luciferase relative light units (RLUs). ( f ) Fifteen minute translation assay with RLUs calculated as a fraction of the wild type IRES. Error bars represent one s.e.m for three independent triplicate experiments.

    Journal: Nature structural & molecular biology

    Article Title: HCV IRES manipulates the ribosome to promote the switch from translation initiation to elongation

    doi: 10.1038/nsmb.2465

    Figure Lengend Snippet: In vitro translation analysis of dIIb mutations ( a ) HCV viral RNA and cellular mRNA differ in their origin and features. HCV viral RNA is delivered directly to the cytoplasm lacking a cap and poly-(A) tail, while cellular mRNA is produced and processed in the nucleus before exportation to the cytoplasm with a cap and tail. However, both are translated by the same cellular machinery, mandating different mechanisms of initiation. ( b ) Simplified diagram HCV IRES 80S ribosome assembly mechanism. The IRES first binds the 40S subunit (yellow), then eukaryotic initiation factor (eIF) 3 (green) and the ternary complex (eIF2-GTP-Met-tRNA i , red and line), and finally after GTP hydrolysis and eIF release the 60S subunit (blue) joins to form an 80S ribosome. Asterisk denotes a difference from canonical 48S complexes. ( c ) Cartoon representation of the secondary structure of the HCV IRES. The location of the start AUG is shown. Boxes areas indicate the parts of the IRES involved in different steps of the mechanism shown in panel b. ( d ) Schematic of mutations (red) made to domain IIb in the context of the uncapped and unpolyadenylated monocistronic Photinus luciferase reporter. Wild-type (WT) RNA is shown to the left. ( e ) Time course of a translation assay from 0 to 90 min as measured by produced luciferase relative light units (RLUs). ( f ) Fifteen minute translation assay with RLUs calculated as a fraction of the wild type IRES. Error bars represent one s.e.m for three independent triplicate experiments.

    Article Snippet: We made the ladder used for analysis with wild type toeprint RNA reverse transcribed with SuperScript® III Reverse Transcriptase (Life Technologies) with annealing and extension temperatures at 45 °C.

    Techniques: In Vitro, Produced, Luciferase

    Identification of cis -acting elements within the leader sequence that are essential for (−)-strand sgmRNA synthesis. ( A ) Constructs with deletions in the leader sequence of wt sBM25A. sΔSL1: deletion of stem-loop I; sΔSL2: deletion of stem-loop II; sNL: deletion of the leader sequence. SL: stem-loop; BSL: bulged stem-loop; PK: pseudoknot; HVR: hypervariable region. ( B ) The relative efficiency of (−)-strand RNA synthesis between the constructs, as measured by RT-qPCR with (left panel) or without (right panel) head-to-tail ligation. Left panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 8 hpt and 200 ng of sBM25A transcript. Right panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: total cellular RNA from sΔSL1-transfected mock-infected cells. Control E: total cellular RNA from sΔSL2-transfected mock-infected cells. Control F: total cellular RNA from sNL-transfected mock-infected cells. ( C ) Measurements of reporter-containing sgmRNA, 18S rRNA and M sgmRNA (from helper virus) at 8 hpt of VP0 by Northern blot analysis. The values (B) represent the mean ± SD of three individual experiments. *** p

    Journal: Viruses

    Article Title: Identification of Cis-Acting Elements on Positive-Strand Subgenomic mRNA Required for the Synthesis of Negative-Strand Counterpart in Bovine Coronavirus

    doi: 10.3390/v6082938

    Figure Lengend Snippet: Identification of cis -acting elements within the leader sequence that are essential for (−)-strand sgmRNA synthesis. ( A ) Constructs with deletions in the leader sequence of wt sBM25A. sΔSL1: deletion of stem-loop I; sΔSL2: deletion of stem-loop II; sNL: deletion of the leader sequence. SL: stem-loop; BSL: bulged stem-loop; PK: pseudoknot; HVR: hypervariable region. ( B ) The relative efficiency of (−)-strand RNA synthesis between the constructs, as measured by RT-qPCR with (left panel) or without (right panel) head-to-tail ligation. Left panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 8 hpt and 200 ng of sBM25A transcript. Right panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: total cellular RNA from sΔSL1-transfected mock-infected cells. Control E: total cellular RNA from sΔSL2-transfected mock-infected cells. Control F: total cellular RNA from sNL-transfected mock-infected cells. ( C ) Measurements of reporter-containing sgmRNA, 18S rRNA and M sgmRNA (from helper virus) at 8 hpt of VP0 by Northern blot analysis. The values (B) represent the mean ± SD of three individual experiments. *** p

    Article Snippet: To assess the efficiency of (−)-strand sgmRNA synthesis from wt sBM25A and the mutants except sNL, sΔSL1 and sΔSL2, 1 µg of decapped and ligated RNA collected from BCoV-infected sgmRNA-transfected HRT-18 cells at 8 hpt was used in an RT reaction with oligonucleotide MHV3'UTR3(−) and SuperScript III reverse transcriptase (Invitrogen).

    Techniques: Sequencing, Construct, Quantitative RT-PCR, Ligation, Infection, Transfection, Northern Blot

    Mapping the cis -acting elements within 3'-terminal 55 nts that are required for (−)-strand sgmRNA synthesis. ( A ) Upper panel: schematic diagram depicting the sgmRNA. The 3'-terminal 55 nts are indicated. Lower panel: constructs of sgmRNA with deletions within the 3'-terminal 55 nts. SL: stem-loop; BSL: bulged stem-loop; PK: pseudoknot; HVR: hypervariable region. ( B ) The relative efficiency of (−)-strand RNA synthesis between the constructs, as measured by RT-qPCR with (left panel) or without (right panel) head-to-tail ligation. Left panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 8 hpt and 200 ng of sBM25A transcript. Right panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Controls C–H: total cellular RNA from mock-infected cells transfected with sBM25A, s3'Δ55–40, s3'Δ55–35, s3'Δ55–30, s3'Δ30–15 and s3'Δ15, respectively. ( C ) Measurements of reporter-containing sgmRNA, 18S rRNA and M sgmRNA (from helper virus) at 8 hpt of VP0 by Northern analysis. The values (B) represent the mean ± SD of three individual experiments. * p

    Journal: Viruses

    Article Title: Identification of Cis-Acting Elements on Positive-Strand Subgenomic mRNA Required for the Synthesis of Negative-Strand Counterpart in Bovine Coronavirus

    doi: 10.3390/v6082938

    Figure Lengend Snippet: Mapping the cis -acting elements within 3'-terminal 55 nts that are required for (−)-strand sgmRNA synthesis. ( A ) Upper panel: schematic diagram depicting the sgmRNA. The 3'-terminal 55 nts are indicated. Lower panel: constructs of sgmRNA with deletions within the 3'-terminal 55 nts. SL: stem-loop; BSL: bulged stem-loop; PK: pseudoknot; HVR: hypervariable region. ( B ) The relative efficiency of (−)-strand RNA synthesis between the constructs, as measured by RT-qPCR with (left panel) or without (right panel) head-to-tail ligation. Left panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 8 hpt and 200 ng of sBM25A transcript. Right panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Controls C–H: total cellular RNA from mock-infected cells transfected with sBM25A, s3'Δ55–40, s3'Δ55–35, s3'Δ55–30, s3'Δ30–15 and s3'Δ15, respectively. ( C ) Measurements of reporter-containing sgmRNA, 18S rRNA and M sgmRNA (from helper virus) at 8 hpt of VP0 by Northern analysis. The values (B) represent the mean ± SD of three individual experiments. * p

    Article Snippet: To assess the efficiency of (−)-strand sgmRNA synthesis from wt sBM25A and the mutants except sNL, sΔSL1 and sΔSL2, 1 µg of decapped and ligated RNA collected from BCoV-infected sgmRNA-transfected HRT-18 cells at 8 hpt was used in an RT reaction with oligonucleotide MHV3'UTR3(−) and SuperScript III reverse transcriptase (Invitrogen).

    Techniques: Construct, Quantitative RT-PCR, Ligation, Infection, Transfection, Northern Blot

    The effect of the 3'-most nt species on the synthesis of (−)-strand sgmRNA. ( A ) Upper panel: schematic diagram depicting the sgmRNA. Lower panel: constructs with nucleotide substitution at the 3'-most nt of sgmRNA. ( B ) The relative efficiency of (−)-strand RNA synthesis between the constructs, as measured by RT-qPCR with (left panel) or without (right panel) head-to-tail ligation. Left panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 8 hpt and sBM25A transcript. Right panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: total cellular RNA from sgmA’-transfected mock-infected cells. Control E: total cellular RNA from sgmU’-transfected mock-infected cells. Control F: total cellular RNA from sgmG’-transfected mock-infected cells. ( C ) Measurements of reporter-containing sgmRNA, 18S rRNA and M sgmRNA (from helper virus) at 8 hpt of VP0 by Northern analysis. Values (B) represent the mean ± SD of three individual experiment s. * p

    Journal: Viruses

    Article Title: Identification of Cis-Acting Elements on Positive-Strand Subgenomic mRNA Required for the Synthesis of Negative-Strand Counterpart in Bovine Coronavirus

    doi: 10.3390/v6082938

    Figure Lengend Snippet: The effect of the 3'-most nt species on the synthesis of (−)-strand sgmRNA. ( A ) Upper panel: schematic diagram depicting the sgmRNA. Lower panel: constructs with nucleotide substitution at the 3'-most nt of sgmRNA. ( B ) The relative efficiency of (−)-strand RNA synthesis between the constructs, as measured by RT-qPCR with (left panel) or without (right panel) head-to-tail ligation. Left panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 8 hpt and sBM25A transcript. Right panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: total cellular RNA from sgmA’-transfected mock-infected cells. Control E: total cellular RNA from sgmU’-transfected mock-infected cells. Control F: total cellular RNA from sgmG’-transfected mock-infected cells. ( C ) Measurements of reporter-containing sgmRNA, 18S rRNA and M sgmRNA (from helper virus) at 8 hpt of VP0 by Northern analysis. Values (B) represent the mean ± SD of three individual experiment s. * p

    Article Snippet: To assess the efficiency of (−)-strand sgmRNA synthesis from wt sBM25A and the mutants except sNL, sΔSL1 and sΔSL2, 1 µg of decapped and ligated RNA collected from BCoV-infected sgmRNA-transfected HRT-18 cells at 8 hpt was used in an RT reaction with oligonucleotide MHV3'UTR3(−) and SuperScript III reverse transcriptase (Invitrogen).

    Techniques: Construct, Quantitative RT-PCR, Ligation, Infection, Transfection, Northern Blot

    Comparison of the efficiency of the (−)-strand RNA synthesis between sgmRNA 7 and BCoV DI RNA with the strategy of head-to-tail ligation. ( A ) Upper panel: schematic diagram depicting the BCoV genome and BCoV DI RNA. Lower panel: constructs BM25A and sBM25Awith the 3' UTR of both constructs replaced with the MHV 3' UTR. ( B ) Strategy for detection of (−)-strand sgmRNA with head-to-tail ligation. ( C ) RT-PCR product synthesized with the strategy described in Figure 1 B. Lane 2, BM25A-transfected BCoV-infected cells; lane 3, sBM25A-transfected BCoV-infected cells; lane 4, mock-infected cells; lane 5, BCoV-infected and mock-transfected cells; lane 6, BM25A-transfected mock-infected cells; lane 7, sBM25A-transfected mock-infected cells; lane 8, a mixture of BCoV-infected cellular RNA extracted at 8 hpt and 200 ng of BM25A transcript; lane 9, a mixture of BCoV-infected cellular RNA extracted at 8 hpt and 200 ng of sBM25A transcript. RT-PCR was used to detect the potential recombination between the BCoV genome and BM25A (lane 12) or sBM25A (lane 13). A recombinant DNA of 1639 bp was produced to serve as a size marker (lane 14). M (lanes 1, 10 and 11), ds DNA size markers in nt pairs. ( D ) Left panel: the relative efficiency of (−)-strand RNA synthesis from constructs BM25A and sBM25A, as measured by RT-qPCR. Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 8 hpt and 200 ng of BM25A transcript. Right panel: measurements of reporter-containing RNA (BM25A and sBM25A), 18S rRNA and BCoV sgmRNAs at 8 hpt (VP0) by Northern blot assay. The values (D) represent the mean ± SD of three individual experiments. ** * p

    Journal: Viruses

    Article Title: Identification of Cis-Acting Elements on Positive-Strand Subgenomic mRNA Required for the Synthesis of Negative-Strand Counterpart in Bovine Coronavirus

    doi: 10.3390/v6082938

    Figure Lengend Snippet: Comparison of the efficiency of the (−)-strand RNA synthesis between sgmRNA 7 and BCoV DI RNA with the strategy of head-to-tail ligation. ( A ) Upper panel: schematic diagram depicting the BCoV genome and BCoV DI RNA. Lower panel: constructs BM25A and sBM25Awith the 3' UTR of both constructs replaced with the MHV 3' UTR. ( B ) Strategy for detection of (−)-strand sgmRNA with head-to-tail ligation. ( C ) RT-PCR product synthesized with the strategy described in Figure 1 B. Lane 2, BM25A-transfected BCoV-infected cells; lane 3, sBM25A-transfected BCoV-infected cells; lane 4, mock-infected cells; lane 5, BCoV-infected and mock-transfected cells; lane 6, BM25A-transfected mock-infected cells; lane 7, sBM25A-transfected mock-infected cells; lane 8, a mixture of BCoV-infected cellular RNA extracted at 8 hpt and 200 ng of BM25A transcript; lane 9, a mixture of BCoV-infected cellular RNA extracted at 8 hpt and 200 ng of sBM25A transcript. RT-PCR was used to detect the potential recombination between the BCoV genome and BM25A (lane 12) or sBM25A (lane 13). A recombinant DNA of 1639 bp was produced to serve as a size marker (lane 14). M (lanes 1, 10 and 11), ds DNA size markers in nt pairs. ( D ) Left panel: the relative efficiency of (−)-strand RNA synthesis from constructs BM25A and sBM25A, as measured by RT-qPCR. Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 8 hpt and 200 ng of BM25A transcript. Right panel: measurements of reporter-containing RNA (BM25A and sBM25A), 18S rRNA and BCoV sgmRNAs at 8 hpt (VP0) by Northern blot assay. The values (D) represent the mean ± SD of three individual experiments. ** * p

    Article Snippet: To assess the efficiency of (−)-strand sgmRNA synthesis from wt sBM25A and the mutants except sNL, sΔSL1 and sΔSL2, 1 µg of decapped and ligated RNA collected from BCoV-infected sgmRNA-transfected HRT-18 cells at 8 hpt was used in an RT reaction with oligonucleotide MHV3'UTR3(−) and SuperScript III reverse transcriptase (Invitrogen).

    Techniques: Ligation, Construct, Reverse Transcription Polymerase Chain Reaction, Synthesized, Transfection, Infection, Recombinant, Produced, Marker, Quantitative RT-PCR, Northern Blot

    Identification of the cis -acting elements within the 5' and 3' UTRs of sgmRNA that are required for (−)-strand sgmRNA synthesis. ( A ) Illustration of the constructs with deletions in the 5' UTR (construct sNL) and 3' UTR (constructs sΔB, sΔP, and s3'Δ55). SL: stem-loop; BSL: bulged stem-loop; PK: pseudoknot; HVR: hypervariable region. ( B ) The relative efficiency of (−)-strand sgmRNA synthesis, as measured by RT-qPCR with (left panel) or without (right panel) head-to-tail ligation. Left panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 8 hpt and 200 ng of sBM25A transcript. Right panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: total cellular RNA from sNL -transfected mock-infected cells. ( C ) The relative efficiency of (−)-strand sgmRNA synthesis, as measured by RT-qPCR with (left panel) or without (right panel) head-to-tail ligation. Left panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 8 hpt and 200 ng of sBM25A transcript. Right panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: total cellular RNA from sΔB-transfected mock-infected cells. Control E: total cellular RNA from sΔP-transfected mock-infected cells. Control F: total cellular RNA from s3'Δ55-transfected mock-infected cells. ( D ) Measurements of reporter-containing sgmRNA, 18S rRNA and M sgmRNA (from helper virus) at 8 hpt of VP0 by Northern blot assay. M sgmRNA rather than N sgmRNA was chosen to represent helper virus here and in the following figures because the reporter-containing (+)-strand sgmRNA and BCoV N sgmRNA (sgmRNA 7) migrated to the same position. The values (B) and (C) represent the mean ± SD of three individual experiments. ** p

    Journal: Viruses

    Article Title: Identification of Cis-Acting Elements on Positive-Strand Subgenomic mRNA Required for the Synthesis of Negative-Strand Counterpart in Bovine Coronavirus

    doi: 10.3390/v6082938

    Figure Lengend Snippet: Identification of the cis -acting elements within the 5' and 3' UTRs of sgmRNA that are required for (−)-strand sgmRNA synthesis. ( A ) Illustration of the constructs with deletions in the 5' UTR (construct sNL) and 3' UTR (constructs sΔB, sΔP, and s3'Δ55). SL: stem-loop; BSL: bulged stem-loop; PK: pseudoknot; HVR: hypervariable region. ( B ) The relative efficiency of (−)-strand sgmRNA synthesis, as measured by RT-qPCR with (left panel) or without (right panel) head-to-tail ligation. Left panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 8 hpt and 200 ng of sBM25A transcript. Right panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: total cellular RNA from sNL -transfected mock-infected cells. ( C ) The relative efficiency of (−)-strand sgmRNA synthesis, as measured by RT-qPCR with (left panel) or without (right panel) head-to-tail ligation. Left panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 8 hpt and 200 ng of sBM25A transcript. Right panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: total cellular RNA from sΔB-transfected mock-infected cells. Control E: total cellular RNA from sΔP-transfected mock-infected cells. Control F: total cellular RNA from s3'Δ55-transfected mock-infected cells. ( D ) Measurements of reporter-containing sgmRNA, 18S rRNA and M sgmRNA (from helper virus) at 8 hpt of VP0 by Northern blot assay. M sgmRNA rather than N sgmRNA was chosen to represent helper virus here and in the following figures because the reporter-containing (+)-strand sgmRNA and BCoV N sgmRNA (sgmRNA 7) migrated to the same position. The values (B) and (C) represent the mean ± SD of three individual experiments. ** p

    Article Snippet: To assess the efficiency of (−)-strand sgmRNA synthesis from wt sBM25A and the mutants except sNL, sΔSL1 and sΔSL2, 1 µg of decapped and ligated RNA collected from BCoV-infected sgmRNA-transfected HRT-18 cells at 8 hpt was used in an RT reaction with oligonucleotide MHV3'UTR3(−) and SuperScript III reverse transcriptase (Invitrogen).

    Techniques: Construct, Quantitative RT-PCR, Ligation, Infection, Transfection, Northern Blot

    Levels of PD-L1 transcripts increase following KSHV infection. Human monocytes (5 × 10 6 monocytes/well) were infected with KSHV (1 × 10 9 genomes/well) (A and B) or UV-inactivated KSHV (B) for 24, 48, or 72 h. Total KSHV genomes were calculated prior to infection for panel B, and different amounts of KSHV genomes per well (5.6 × 10 7 , 2.5 × 10 8 , 5.6 × 10 8 , and 1.7 × 10 9 ) were used. UV-inactivated KSHV genomes used were 1.7 × 10 9 per well. At each time point, a mock-infected control was also harvested. After harvest, RNA was isolated from cells and reverse transcribed, and cDNA levels were measured through quantitative real-time PCR to assess either PD-L1 (A) or KSHV-encoded viral interleukin-6 (vIL-6) (B) message levels. RNA levels were normalized to β-actin and are represented as fold increase over mock-infected values at each time point. The data are representative plots of single experiments but represent the general trend of 11 donors for PD-L1 (24 hpi) transcript levels and 5 donors for vIL-6 (48 hpi) transcript levels. Error bars represent standard deviations of the fold change values for three technical replicates.

    Journal: mBio

    Article Title: Kaposi’s Sarcoma-Associated Herpesvirus Increases PD-L1 and Proinflammatory Cytokine Expression in Human Monocytes

    doi: 10.1128/mBio.00917-17

    Figure Lengend Snippet: Levels of PD-L1 transcripts increase following KSHV infection. Human monocytes (5 × 10 6 monocytes/well) were infected with KSHV (1 × 10 9 genomes/well) (A and B) or UV-inactivated KSHV (B) for 24, 48, or 72 h. Total KSHV genomes were calculated prior to infection for panel B, and different amounts of KSHV genomes per well (5.6 × 10 7 , 2.5 × 10 8 , 5.6 × 10 8 , and 1.7 × 10 9 ) were used. UV-inactivated KSHV genomes used were 1.7 × 10 9 per well. At each time point, a mock-infected control was also harvested. After harvest, RNA was isolated from cells and reverse transcribed, and cDNA levels were measured through quantitative real-time PCR to assess either PD-L1 (A) or KSHV-encoded viral interleukin-6 (vIL-6) (B) message levels. RNA levels were normalized to β-actin and are represented as fold increase over mock-infected values at each time point. The data are representative plots of single experiments but represent the general trend of 11 donors for PD-L1 (24 hpi) transcript levels and 5 donors for vIL-6 (48 hpi) transcript levels. Error bars represent standard deviations of the fold change values for three technical replicates.

    Article Snippet: RNA was isolated using RNeasy Micro kit (Qiagen) and reverse transcribed with SuperScript III reverse transcriptase (Invitrogen) and oligo(dT) (Invitrogen).

    Techniques: Infection, Isolation, Real-time Polymerase Chain Reaction

    KSHV establishes lytic infection of human monocytes at 48 h postinfection. Human monocytes (5 × 10 6 monocytes/well) were infected with KSHV (8.7 × 10 7 genomes/well) for 48 h. At each time point, a mock-infected control was also harvested (A and B). (A) After harvest, RNA was isolated from cells and reverse transcribed, and cDNA levels were measured using quantitative real-time PCR to assess KSHV-encoded viral interleukin-6 (vIL-6), open reading frame 39 (ORF39), ORF57, ORF59, K8.1, viral interferon regulatory factor 1 (vIRF1), and replication and transcription activator (RTA) message levels as normalized to β-actin and represented as fold increase over mock-infected values. Error bars represent the standard deviations of fold change values for three technical replicates. (B) Immunoblot of KSHV viral protein ORF45 at 48 h postinfection. The data are representative plots of single experiments but represent the general trend of at least two (A) and four (B) independent donors and experiments.

    Journal: mBio

    Article Title: Kaposi’s Sarcoma-Associated Herpesvirus Increases PD-L1 and Proinflammatory Cytokine Expression in Human Monocytes

    doi: 10.1128/mBio.00917-17

    Figure Lengend Snippet: KSHV establishes lytic infection of human monocytes at 48 h postinfection. Human monocytes (5 × 10 6 monocytes/well) were infected with KSHV (8.7 × 10 7 genomes/well) for 48 h. At each time point, a mock-infected control was also harvested (A and B). (A) After harvest, RNA was isolated from cells and reverse transcribed, and cDNA levels were measured using quantitative real-time PCR to assess KSHV-encoded viral interleukin-6 (vIL-6), open reading frame 39 (ORF39), ORF57, ORF59, K8.1, viral interferon regulatory factor 1 (vIRF1), and replication and transcription activator (RTA) message levels as normalized to β-actin and represented as fold increase over mock-infected values. Error bars represent the standard deviations of fold change values for three technical replicates. (B) Immunoblot of KSHV viral protein ORF45 at 48 h postinfection. The data are representative plots of single experiments but represent the general trend of at least two (A) and four (B) independent donors and experiments.

    Article Snippet: RNA was isolated using RNeasy Micro kit (Qiagen) and reverse transcribed with SuperScript III reverse transcriptase (Invitrogen) and oligo(dT) (Invitrogen).

    Techniques: Infection, Isolation, Real-time Polymerase Chain Reaction

    Infection with intact KSHV results in increased PD-L1 expression. Primary human monocytes (5 × 10 6 monocytes/well) were infected with either intact or UV-inactivated KSHV (9.5 × 10 8 genomes/well) and harvested at 48 h postinfection (A and B). (A) Cells were gated on forward scatter, side scatter, and CD14 + . Histograms indicate the percentages of PD-L1-positive cells with the percentages for mock-infected cells, UV-inactivated KSHV-infected cells, and high-dose KSHV-infected cells. The results shown in panel A are representative of the results from four independent human donors. (B) RNA was isolated from cells and reverse transcribed, and cDNA levels were measured through quantitative real-time PCR to assess PD-L1 message levels. RNA levels were normalized to β-actin and represented as fold increase over mock-infected monocytes. Error bars represent standard deviations of fold change values for three technical replicates. The data are representative of the general trend of four independent donor experiments.

    Journal: mBio

    Article Title: Kaposi’s Sarcoma-Associated Herpesvirus Increases PD-L1 and Proinflammatory Cytokine Expression in Human Monocytes

    doi: 10.1128/mBio.00917-17

    Figure Lengend Snippet: Infection with intact KSHV results in increased PD-L1 expression. Primary human monocytes (5 × 10 6 monocytes/well) were infected with either intact or UV-inactivated KSHV (9.5 × 10 8 genomes/well) and harvested at 48 h postinfection (A and B). (A) Cells were gated on forward scatter, side scatter, and CD14 + . Histograms indicate the percentages of PD-L1-positive cells with the percentages for mock-infected cells, UV-inactivated KSHV-infected cells, and high-dose KSHV-infected cells. The results shown in panel A are representative of the results from four independent human donors. (B) RNA was isolated from cells and reverse transcribed, and cDNA levels were measured through quantitative real-time PCR to assess PD-L1 message levels. RNA levels were normalized to β-actin and represented as fold increase over mock-infected monocytes. Error bars represent standard deviations of fold change values for three technical replicates. The data are representative of the general trend of four independent donor experiments.

    Article Snippet: RNA was isolated using RNeasy Micro kit (Qiagen) and reverse transcribed with SuperScript III reverse transcriptase (Invitrogen) and oligo(dT) (Invitrogen).

    Techniques: Infection, Expressing, Isolation, Real-time Polymerase Chain Reaction

    Increase in PD-L1 expression is dose dependent. Primary human monocytes (5 × 10 6 monocytes/well) were infected with a low dose of KSHV (3 × 10 8 genomes/well) or a high dose of KSHV (2.1 × 10 9 genomes/well) or mock infected and harvested at 24 and 72 h postinfection. (A and B) Cells were stained for CD14 and PD-L1, and expression of these markers was measured by flow cytometry. Cells were gated on forward scatter, side scatter, and CD14 + . Histograms indicate the gate of PD-L1-positive cells with percentages for mock-infected cells, low dose of KSHV-infected cells, and high dose of KSHV-infected cells shown. Panels A and B show representative data from donors from seven independent experiments. (C) Monocytes from a donor were infected with low-dose HSV-1 (MOI of 0.02) and high-dose HSV-1 (MOI of 0.2). (D) RNA was isolated from mock- and KSHV-infected cells and reverse transcribed, and cDNA levels were measured through quantitative real-time PCR to assess PD-L1 message levels. RNA levels were normalized to β-actin and represented as fold increase over the values for mock-infected monocytes. Error bars represent standard deviations of the fold changes for three technical replicates. The data are representative of the general trend of seven independent donor experiments.

    Journal: mBio

    Article Title: Kaposi’s Sarcoma-Associated Herpesvirus Increases PD-L1 and Proinflammatory Cytokine Expression in Human Monocytes

    doi: 10.1128/mBio.00917-17

    Figure Lengend Snippet: Increase in PD-L1 expression is dose dependent. Primary human monocytes (5 × 10 6 monocytes/well) were infected with a low dose of KSHV (3 × 10 8 genomes/well) or a high dose of KSHV (2.1 × 10 9 genomes/well) or mock infected and harvested at 24 and 72 h postinfection. (A and B) Cells were stained for CD14 and PD-L1, and expression of these markers was measured by flow cytometry. Cells were gated on forward scatter, side scatter, and CD14 + . Histograms indicate the gate of PD-L1-positive cells with percentages for mock-infected cells, low dose of KSHV-infected cells, and high dose of KSHV-infected cells shown. Panels A and B show representative data from donors from seven independent experiments. (C) Monocytes from a donor were infected with low-dose HSV-1 (MOI of 0.02) and high-dose HSV-1 (MOI of 0.2). (D) RNA was isolated from mock- and KSHV-infected cells and reverse transcribed, and cDNA levels were measured through quantitative real-time PCR to assess PD-L1 message levels. RNA levels were normalized to β-actin and represented as fold increase over the values for mock-infected monocytes. Error bars represent standard deviations of the fold changes for three technical replicates. The data are representative of the general trend of seven independent donor experiments.

    Article Snippet: RNA was isolated using RNeasy Micro kit (Qiagen) and reverse transcribed with SuperScript III reverse transcriptase (Invitrogen) and oligo(dT) (Invitrogen).

    Techniques: Expressing, Infection, Staining, Flow Cytometry, Cytometry, Isolation, Real-time Polymerase Chain Reaction

    Upregulation of glycogene expression in the LV of DS hypertensive rats. (A) Relative expression levels of glycogenes selected after qPCR array in the LV of DS rats fed HS and LS diets were quantified by qPCR. Rps18 was used as an internal control. The numbers of examined rats were n = 12 and n = 15 for the HS groups at 12 and 16 weeks, respectively; n = 6 for LS groups at each period. Expression levels were normalized to that of TATA box-binding protein ( Tbp ). (B) Protein levels of glycogenes in LV extracts were analyzed by western blotting. β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as internal controls. Representative results from three rats per group are indicated. (C) Densitometry analysis of immunoblots shown in (B). Intensity of each band was normalized to that of GAPDH. Data are presented as the fold change compared with LS rats at 12 weeks (n = 6). (A,C) *, p

    Journal: PLoS ONE

    Article Title: Aberrant Glycosylation in the Left Ventricle and Plasma of Rats with Cardiac Hypertrophy and Heart Failure

    doi: 10.1371/journal.pone.0150210

    Figure Lengend Snippet: Upregulation of glycogene expression in the LV of DS hypertensive rats. (A) Relative expression levels of glycogenes selected after qPCR array in the LV of DS rats fed HS and LS diets were quantified by qPCR. Rps18 was used as an internal control. The numbers of examined rats were n = 12 and n = 15 for the HS groups at 12 and 16 weeks, respectively; n = 6 for LS groups at each period. Expression levels were normalized to that of TATA box-binding protein ( Tbp ). (B) Protein levels of glycogenes in LV extracts were analyzed by western blotting. β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as internal controls. Representative results from three rats per group are indicated. (C) Densitometry analysis of immunoblots shown in (B). Intensity of each band was normalized to that of GAPDH. Data are presented as the fold change compared with LS rats at 12 weeks (n = 6). (A,C) *, p

    Article Snippet: For qPCR with gene-specific primer pairs , cDNA was synthesized using SuperScript III reverse transcriptase (Life Technologies, Carlsbad, CA), and qPCR was performed using SYBR Premix Ex Taq II (Takara Bio) and the LightCycler 480 System.

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

    Altered O -glycosylation on CSRP3 in the LV of DS hypertensive rats. (A) ACA lectin blot analysis and SYPRO Ruby staining of fractions from sialidase-treated LV extracts. Arrow indicates the ACA-positive band, which is observed strongly in fraction 3 of HS ( H ) but weakly in that of the LS ( L ) group. (B) Two-dimensional PAGE images of sialidase-treated LV fraction 3. Proteins transferred to membranes were subjected to SYPRO Ruby staining, and then to ACA lectin blotting. Insets show magnified images of two spots used for protein identification. (C) Western blot ( WB ) and ACA lectin blot ( LB ) analyses of recombinant human CSRP3. Recombinant proteins expressed in E . coli (unglycosylated negative control) and in HEK293 cells (potentially glycosylated reference) were analyzed after treatment with sialidase and O -glycosidase. (D) Relative expression levels of Csrp3 in the LV tissues. qPCR data were normalized to Tbp expression levels. The numbers of examined rats were n = 12 and n = 15 for HS groups at 12 and 16 weeks, respectively; n = 6 for LS groups at each period. (E) Protein levels of CSRP3 in LV extracts. Densitometry analysis data of western blotting are shown (n = 6). (D,E) The data are presented as the fold change compared with LS rats at 12 weeks. (F) Western blot ( WB ) and ACA lectin blot ( LB ) analyses of CSRP3 from LV extracts of DS rats. CSRP3 in LV extracts was immunoprecipitated, denatured, separated by SDS-PAGE, and analyzed. Recombinant human CSRP3 was used as an experimental control of immunoprecipitation with anti-CSRP3 antibody (+) or normal IgG (-). Lower panel shows densitometry analysis data; the intensity of each band in LB was normalized to that in WB (n = 6). (G) Effects of glycosidases on CSRP3 dimerization. LV extracts from three HS ( H ) or LS ( L ) rats at 16 weeks were treated with three glycosidases as indicated and then analyzed by western blotting for CSRP3. Arrows indicate the bands corresponding to monomers and dimers. Lower panels show densitometry analysis from five experiments; dimer/monomer ratios are presented as the fold change compared with LS rats without glycosidase treatment. (D-G) *, p

    Journal: PLoS ONE

    Article Title: Aberrant Glycosylation in the Left Ventricle and Plasma of Rats with Cardiac Hypertrophy and Heart Failure

    doi: 10.1371/journal.pone.0150210

    Figure Lengend Snippet: Altered O -glycosylation on CSRP3 in the LV of DS hypertensive rats. (A) ACA lectin blot analysis and SYPRO Ruby staining of fractions from sialidase-treated LV extracts. Arrow indicates the ACA-positive band, which is observed strongly in fraction 3 of HS ( H ) but weakly in that of the LS ( L ) group. (B) Two-dimensional PAGE images of sialidase-treated LV fraction 3. Proteins transferred to membranes were subjected to SYPRO Ruby staining, and then to ACA lectin blotting. Insets show magnified images of two spots used for protein identification. (C) Western blot ( WB ) and ACA lectin blot ( LB ) analyses of recombinant human CSRP3. Recombinant proteins expressed in E . coli (unglycosylated negative control) and in HEK293 cells (potentially glycosylated reference) were analyzed after treatment with sialidase and O -glycosidase. (D) Relative expression levels of Csrp3 in the LV tissues. qPCR data were normalized to Tbp expression levels. The numbers of examined rats were n = 12 and n = 15 for HS groups at 12 and 16 weeks, respectively; n = 6 for LS groups at each period. (E) Protein levels of CSRP3 in LV extracts. Densitometry analysis data of western blotting are shown (n = 6). (D,E) The data are presented as the fold change compared with LS rats at 12 weeks. (F) Western blot ( WB ) and ACA lectin blot ( LB ) analyses of CSRP3 from LV extracts of DS rats. CSRP3 in LV extracts was immunoprecipitated, denatured, separated by SDS-PAGE, and analyzed. Recombinant human CSRP3 was used as an experimental control of immunoprecipitation with anti-CSRP3 antibody (+) or normal IgG (-). Lower panel shows densitometry analysis data; the intensity of each band in LB was normalized to that in WB (n = 6). (G) Effects of glycosidases on CSRP3 dimerization. LV extracts from three HS ( H ) or LS ( L ) rats at 16 weeks were treated with three glycosidases as indicated and then analyzed by western blotting for CSRP3. Arrows indicate the bands corresponding to monomers and dimers. Lower panels show densitometry analysis from five experiments; dimer/monomer ratios are presented as the fold change compared with LS rats without glycosidase treatment. (D-G) *, p

    Article Snippet: For qPCR with gene-specific primer pairs , cDNA was synthesized using SuperScript III reverse transcriptase (Life Technologies, Carlsbad, CA), and qPCR was performed using SYBR Premix Ex Taq II (Takara Bio) and the LightCycler 480 System.

    Techniques: Staining, Polyacrylamide Gel Electrophoresis, Western Blot, Recombinant, Negative Control, Expressing, Real-time Polymerase Chain Reaction, Immunoprecipitation, SDS Page

    Decrease of core fucosylation on N -glycans in DS hypertensive rats. (A) Lectin blot analysis of LV extracts using AOL. Representative images demonstrate AOL-reactive glycoproteins and SYPRO Ruby-stained proteins of three individual rats in each group. Right panel shows densitometry analysis data; intensity of each band was normalized to total protein. Data are presented as the fold change (n = 6) compared with LS rats at 12 weeks. (B) Relative expression levels of the genes responsible for core fucosylation ( Fut8 ) and defucosylation ( Fuca1 and Fuca2 ) on N -glycans were examined by qPCR; levels in the LV and liver were normalized to that of Tbp and Actb , respectively. Data are presented as the fold change compared with the LS group at 12 weeks. (C) AFU activity in the LV and liver extracts, and plasma. Data were normalized to protein content. (D) Plasma levels of FUCA1 and FUCA2. (E) Correlation of AFU activity in LV extracts shown in (C) with relative ANP expression shown in Fig 3B . (F,G) Correlation of plasma AFU activity shown in (C) with plasma NT-proANP concentration ( F ) and LV anterior wall thickness during diastole (LVAWd) (G). (B-G) The numbers of examined rats were n = 12 and n = 15 for HS groups at 12 and 16 weeks, respectively; n = 6 for LS groups at each period. (A-D) *, p

    Journal: PLoS ONE

    Article Title: Aberrant Glycosylation in the Left Ventricle and Plasma of Rats with Cardiac Hypertrophy and Heart Failure

    doi: 10.1371/journal.pone.0150210

    Figure Lengend Snippet: Decrease of core fucosylation on N -glycans in DS hypertensive rats. (A) Lectin blot analysis of LV extracts using AOL. Representative images demonstrate AOL-reactive glycoproteins and SYPRO Ruby-stained proteins of three individual rats in each group. Right panel shows densitometry analysis data; intensity of each band was normalized to total protein. Data are presented as the fold change (n = 6) compared with LS rats at 12 weeks. (B) Relative expression levels of the genes responsible for core fucosylation ( Fut8 ) and defucosylation ( Fuca1 and Fuca2 ) on N -glycans were examined by qPCR; levels in the LV and liver were normalized to that of Tbp and Actb , respectively. Data are presented as the fold change compared with the LS group at 12 weeks. (C) AFU activity in the LV and liver extracts, and plasma. Data were normalized to protein content. (D) Plasma levels of FUCA1 and FUCA2. (E) Correlation of AFU activity in LV extracts shown in (C) with relative ANP expression shown in Fig 3B . (F,G) Correlation of plasma AFU activity shown in (C) with plasma NT-proANP concentration ( F ) and LV anterior wall thickness during diastole (LVAWd) (G). (B-G) The numbers of examined rats were n = 12 and n = 15 for HS groups at 12 and 16 weeks, respectively; n = 6 for LS groups at each period. (A-D) *, p

    Article Snippet: For qPCR with gene-specific primer pairs , cDNA was synthesized using SuperScript III reverse transcriptase (Life Technologies, Carlsbad, CA), and qPCR was performed using SYBR Premix Ex Taq II (Takara Bio) and the LightCycler 480 System.

    Techniques: Staining, Expressing, Real-time Polymerase Chain Reaction, Activity Assay, Aqueous Normal-phase Chromatography, Concentration Assay

    Altered mucin-type O -glycosylation in the LV of DS hypertensive rats. (A) T-synthase activity in LV extracts. Data were normalized to protein content. (B) Correlation of T-synthase activity with ANP gene expression. ANP gene expression level was quantified by qPCR and normalized to that of Tbp . Data are presented as the fold change compared with LS rats at 12 weeks. (C) Correlation of T-synthase activity with ejection fraction. (D) Relative expression levels of glycogenes involved in the early stage of mucin-type O -glycosylation in the LV tissues of DS rats were analyzed by qPCR and normalized to that of Tbp . Data are presented as the fold change compared with LS rats at 12 weeks. (E) Schematic summary of gene expression analysis data shown in (D). Examined glycosyltransferases in the mucin-type O -glycosylation pathway are shown in red (upregulated), blue (downregulated), or black (no change) letters. Relatively rare core structures (core 5, 6, 7, and 8) synthesized from Tn are omitted. The biosynthetic pathway of disialyl-T is upregulated, as indicated with bold arrows. GalNAc, N -acetylgalactosamine; GlcNAc, N -acetylglucosamine; Gal, galactose; NeuAc, N -acetylneuraminic acid. (F) Lectin blot analysis of sialidase-treated LV extracts using ACA. Representative images demonstrate ACA-reactive glycoproteins and SYPRO Ruby-stained total proteins of three individual rats in each group. Lower panel shows densitometry analysis; intensity of each band was normalized to total protein amount. Data are presented as the fold change (n = 6) compared with sialidase-untreated LV extracts of HS rats at 12 weeks. (A,D) The numbers of examined rats were n = 12 and n = 15 for the HS groups at 12 and 16 weeks, respectively; n = 6 for LS groups at each period. (A,D,F) *, p

    Journal: PLoS ONE

    Article Title: Aberrant Glycosylation in the Left Ventricle and Plasma of Rats with Cardiac Hypertrophy and Heart Failure

    doi: 10.1371/journal.pone.0150210

    Figure Lengend Snippet: Altered mucin-type O -glycosylation in the LV of DS hypertensive rats. (A) T-synthase activity in LV extracts. Data were normalized to protein content. (B) Correlation of T-synthase activity with ANP gene expression. ANP gene expression level was quantified by qPCR and normalized to that of Tbp . Data are presented as the fold change compared with LS rats at 12 weeks. (C) Correlation of T-synthase activity with ejection fraction. (D) Relative expression levels of glycogenes involved in the early stage of mucin-type O -glycosylation in the LV tissues of DS rats were analyzed by qPCR and normalized to that of Tbp . Data are presented as the fold change compared with LS rats at 12 weeks. (E) Schematic summary of gene expression analysis data shown in (D). Examined glycosyltransferases in the mucin-type O -glycosylation pathway are shown in red (upregulated), blue (downregulated), or black (no change) letters. Relatively rare core structures (core 5, 6, 7, and 8) synthesized from Tn are omitted. The biosynthetic pathway of disialyl-T is upregulated, as indicated with bold arrows. GalNAc, N -acetylgalactosamine; GlcNAc, N -acetylglucosamine; Gal, galactose; NeuAc, N -acetylneuraminic acid. (F) Lectin blot analysis of sialidase-treated LV extracts using ACA. Representative images demonstrate ACA-reactive glycoproteins and SYPRO Ruby-stained total proteins of three individual rats in each group. Lower panel shows densitometry analysis; intensity of each band was normalized to total protein amount. Data are presented as the fold change (n = 6) compared with sialidase-untreated LV extracts of HS rats at 12 weeks. (A,D) The numbers of examined rats were n = 12 and n = 15 for the HS groups at 12 and 16 weeks, respectively; n = 6 for LS groups at each period. (A,D,F) *, p

    Article Snippet: For qPCR with gene-specific primer pairs , cDNA was synthesized using SuperScript III reverse transcriptase (Life Technologies, Carlsbad, CA), and qPCR was performed using SYBR Premix Ex Taq II (Takara Bio) and the LightCycler 480 System.

    Techniques: Activity Assay, Aqueous Normal-phase Chromatography, Expressing, Real-time Polymerase Chain Reaction, Synthesized, Staining

    Effects of miRs-21, -143, and -378e on stemness and EMT markers in triple-negative breast cancer cells BT549 cells were transfected with miRs: scr, -21, -143, and -378e, for 24h. Real Time PCR was performed to analyze oct3/4, sox2, and snail mRNA levels a . In addition, BT549 cells were transfected with these miRs alone or in combination for 48h. Western blot analysis was performed to evaluate nanog, oct3/4, sox2, and zeb protein levels b . MDA-MB-231 cells were transfected with miRs: scr, -21, -143, and -378e, for 24h. Real Time PCR was performed to analyze oct3/4, nanog, sox2, and snail mRNA levels (a). In addition, MDA-MB-231 cells were transfected with these miRs alone or in combination for 48h. Western blot analysis was performed to evaluate zeb, nanog, and oct3/4 protein levels (b). T47D cells were transfected with anti-miRs: -scr (control), -21, -143, and -378e, for 48h. Real Time PCR was performed to analyze sox2 mRNA levels c . In addition, T47D cells were transfected with anti-miRs (alone or in combination; final concentration: 200nM) for 72h. Western blot analysis was performed to evaluate e-cadherin, zeb, and slug protein levels d . Western blot analysis from representative experiments. Actin or α-tubulin were used as loading controls. The experiments were repeated at least three times (b, d). In a and c, data were obtained from three independent experiments and are presented as mean value ± SD. P-value calculated using Student's t test. * p

    Journal: Oncotarget

    Article Title: Cancer-associated fibroblasts release exosomal microRNAs that dictate an aggressive phenotype in breast cancer

    doi: 10.18632/oncotarget.14752

    Figure Lengend Snippet: Effects of miRs-21, -143, and -378e on stemness and EMT markers in triple-negative breast cancer cells BT549 cells were transfected with miRs: scr, -21, -143, and -378e, for 24h. Real Time PCR was performed to analyze oct3/4, sox2, and snail mRNA levels a . In addition, BT549 cells were transfected with these miRs alone or in combination for 48h. Western blot analysis was performed to evaluate nanog, oct3/4, sox2, and zeb protein levels b . MDA-MB-231 cells were transfected with miRs: scr, -21, -143, and -378e, for 24h. Real Time PCR was performed to analyze oct3/4, nanog, sox2, and snail mRNA levels (a). In addition, MDA-MB-231 cells were transfected with these miRs alone or in combination for 48h. Western blot analysis was performed to evaluate zeb, nanog, and oct3/4 protein levels (b). T47D cells were transfected with anti-miRs: -scr (control), -21, -143, and -378e, for 48h. Real Time PCR was performed to analyze sox2 mRNA levels c . In addition, T47D cells were transfected with anti-miRs (alone or in combination; final concentration: 200nM) for 72h. Western blot analysis was performed to evaluate e-cadherin, zeb, and slug protein levels d . Western blot analysis from representative experiments. Actin or α-tubulin were used as loading controls. The experiments were repeated at least three times (b, d). In a and c, data were obtained from three independent experiments and are presented as mean value ± SD. P-value calculated using Student's t test. * p

    Article Snippet: Reverse transcription of total RNA was performed starting from equal amounts of total RNA/sample (150/500ng) using miScript reverse Transcription Kit (Qiagen, Milan, Italy) for miR analysis, and using SuperScript® III Reverse Transcriptase (Invitrogen, Milan, Italy) for mRNA analysis.

    Techniques: Transfection, Real-time Polymerase Chain Reaction, Western Blot, Multiple Displacement Amplification, Concentration Assay

    NF exosomes transfected with miRs -21, -143, -378e, similarly to CAF exosomes, promote stemness and epithelial-mesenchymal transition phenotype NF (patients #5, and #6) were cultured for 48h. Then, exosomes were isolated from NF-conditioned media with ExoQuick-TC™ solution, and transfected using Exo-Fect™ solution with scrambled miRs (scr, control), or miRs -21, -143, -378e, alone or in combination (final concentration: 130nM). T47D cells were cultured in the presence of NF exosomes (patient #5) transfected with either scrambled miRs (control) or miRs for 48h. Cells were harvested and grown under non-adherent conditions in stem medium. After four days, the capacity of cells to form spheres was assessed a . Sphere diameter distribution for 10 representative fields b . Scale bar: 100μm c . In addition, T47D cells were cultured in the presence of NF exosomes (patients #5, and #6) transfected with either scrambled miRs (control) or miRs for 24h d or 48h e . Real Time PCR was performed to analyze nanog, sox2, and snail mRNA (d). Western blot analysis was performed to evaluate zeb, nanog, and oct3/4 protein levels. Western blot analysis from representative experiments. Actin was used as loading control. The experiments were repeated at least three times (e). In a, b, d, data were obtained from three independent experiments and are presented as mean value ± SD. P-value calculated using Student's t test. * p

    Journal: Oncotarget

    Article Title: Cancer-associated fibroblasts release exosomal microRNAs that dictate an aggressive phenotype in breast cancer

    doi: 10.18632/oncotarget.14752

    Figure Lengend Snippet: NF exosomes transfected with miRs -21, -143, -378e, similarly to CAF exosomes, promote stemness and epithelial-mesenchymal transition phenotype NF (patients #5, and #6) were cultured for 48h. Then, exosomes were isolated from NF-conditioned media with ExoQuick-TC™ solution, and transfected using Exo-Fect™ solution with scrambled miRs (scr, control), or miRs -21, -143, -378e, alone or in combination (final concentration: 130nM). T47D cells were cultured in the presence of NF exosomes (patient #5) transfected with either scrambled miRs (control) or miRs for 48h. Cells were harvested and grown under non-adherent conditions in stem medium. After four days, the capacity of cells to form spheres was assessed a . Sphere diameter distribution for 10 representative fields b . Scale bar: 100μm c . In addition, T47D cells were cultured in the presence of NF exosomes (patients #5, and #6) transfected with either scrambled miRs (control) or miRs for 24h d or 48h e . Real Time PCR was performed to analyze nanog, sox2, and snail mRNA (d). Western blot analysis was performed to evaluate zeb, nanog, and oct3/4 protein levels. Western blot analysis from representative experiments. Actin was used as loading control. The experiments were repeated at least three times (e). In a, b, d, data were obtained from three independent experiments and are presented as mean value ± SD. P-value calculated using Student's t test. * p

    Article Snippet: Reverse transcription of total RNA was performed starting from equal amounts of total RNA/sample (150/500ng) using miScript reverse Transcription Kit (Qiagen, Milan, Italy) for miR analysis, and using SuperScript® III Reverse Transcriptase (Invitrogen, Milan, Italy) for mRNA analysis.

    Techniques: Transfection, Cell Culture, Isolation, Concentration Assay, Real-time Polymerase Chain Reaction, Western Blot

    CAF exosomes promote stemness, epithelial–mesenchymal transition, and anchorage-independent cell growth T47D cells were cultured under non-adherent conditions in stem medium in the absence (control) or presence of either NF exosomes (patients #5, #6, and #10) or CAF exosomes (patients #3, #7, and #9). After four days, the capacity of cells to form spheres was assessed a . Sphere diameter distribution (control cells and T47D cells treated with either NF#6 ex or CAF#7 ex) for 10 representative fields. Scale bar: 100μm b . T47D cells were cultured in the absence (control) or presence of either NF exosomes (patients #5, #6, and #10) or CAF exosomes (patients #7, and #12). After 24h, cells were harvested and cultured in soft agar c . Scale bar: 100μm d . T47D cells were cultured in the absence (control) or presence of either NF ex (patients #5, #6, and #10) or CAF ex (patients #3, #7, #9, #12 and #13) for 24h e . or 48h f . Real Time PCR was performed to analyze oct3/4, nanog, sox2, snail and zeb mRNA levels (e). Western blot analysis was performed to evaluate nanog, sox2, oct3/4, zeb, e-cadherin and snail protein levels (f). BT549 cells were cultured in the absence (control) or presence of either NF exosomes (patient #6) or CAF exosomes (patient #7) for 48h. Western blot analysis was performed to evaluate e-cadherin and oct3/4 protein levels. MDA-MB-231 cells were cultured in the absence (control) or presence of either NF exosomes (patient #5) o CAF exosomes (patient #12) for 48h. Western blot analysis was performed to evaluate oct3/4 protein level g . Western blot from representative experiments is shown. Actin or vinculin were used as loading controls. The experiments were repeated at least three times (f, g). In a, b, c, e, data were obtained from three independent experiments and are presented as mean value ± SD. P-value was calculated using one-way ANOVA followed by Bonferroni's post hoc testing. * p

    Journal: Oncotarget

    Article Title: Cancer-associated fibroblasts release exosomal microRNAs that dictate an aggressive phenotype in breast cancer

    doi: 10.18632/oncotarget.14752

    Figure Lengend Snippet: CAF exosomes promote stemness, epithelial–mesenchymal transition, and anchorage-independent cell growth T47D cells were cultured under non-adherent conditions in stem medium in the absence (control) or presence of either NF exosomes (patients #5, #6, and #10) or CAF exosomes (patients #3, #7, and #9). After four days, the capacity of cells to form spheres was assessed a . Sphere diameter distribution (control cells and T47D cells treated with either NF#6 ex or CAF#7 ex) for 10 representative fields. Scale bar: 100μm b . T47D cells were cultured in the absence (control) or presence of either NF exosomes (patients #5, #6, and #10) or CAF exosomes (patients #7, and #12). After 24h, cells were harvested and cultured in soft agar c . Scale bar: 100μm d . T47D cells were cultured in the absence (control) or presence of either NF ex (patients #5, #6, and #10) or CAF ex (patients #3, #7, #9, #12 and #13) for 24h e . or 48h f . Real Time PCR was performed to analyze oct3/4, nanog, sox2, snail and zeb mRNA levels (e). Western blot analysis was performed to evaluate nanog, sox2, oct3/4, zeb, e-cadherin and snail protein levels (f). BT549 cells were cultured in the absence (control) or presence of either NF exosomes (patient #6) or CAF exosomes (patient #7) for 48h. Western blot analysis was performed to evaluate e-cadherin and oct3/4 protein levels. MDA-MB-231 cells were cultured in the absence (control) or presence of either NF exosomes (patient #5) o CAF exosomes (patient #12) for 48h. Western blot analysis was performed to evaluate oct3/4 protein level g . Western blot from representative experiments is shown. Actin or vinculin were used as loading controls. The experiments were repeated at least three times (f, g). In a, b, c, e, data were obtained from three independent experiments and are presented as mean value ± SD. P-value was calculated using one-way ANOVA followed by Bonferroni's post hoc testing. * p

    Article Snippet: Reverse transcription of total RNA was performed starting from equal amounts of total RNA/sample (150/500ng) using miScript reverse Transcription Kit (Qiagen, Milan, Italy) for miR analysis, and using SuperScript® III Reverse Transcriptase (Invitrogen, Milan, Italy) for mRNA analysis.

    Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Multiple Displacement Amplification

    miR-21, miR-143, and miR-378e promote stemness and epithelial–mesenchymal transition T47D cells were transfected with scrambled (scr, control) or miRs -21, -143, and -378e, alone or in combination (final concentration: 100nM). After 48h, cells were harvested and cultured under non-adherent conditions in stem medium. After four days, the capacity of cells to form spheres was assessed a . Sphere diameter distribution for 10 representative fields. Scale bar: 100μm b . T47D cells were transfected with anti-miRs (alone or in combination, final concentration: 200nM) or scrambled anti-miR (anti-miR-scr, control). After 24h, cells were harvested and cultured under non-adherent conditions in stem medium. After four days, the capacity of cells to form spheres was assessed c . Sphere diameter distribution for 10 representative fields. Scale bar: 50μm d . ALDEFLUOR assays were performed in T47D cells transfected with scrambled or miRs -21, -143, and -378e for 48h e . T47D cells were transfected with scrambled, miRs -21, -143, or -378e for 48h f . or 72h g . Real Time PCR was performed to analyze oct3/4, nanog, sox2, and snail mRNA levels (f). In addition, T47D cells were transfected with the miRs alone or in combination for 72h. Western blot analysis was performed to evaluate nanog, sox2, oct3/4, zeb, and e-cadherin protein levels (g). In a, b, c, d, e, f, data were obtained from three independent experiments and are presented as mean value ± SD. P-value was calculated using Student's t test. * p

    Journal: Oncotarget

    Article Title: Cancer-associated fibroblasts release exosomal microRNAs that dictate an aggressive phenotype in breast cancer

    doi: 10.18632/oncotarget.14752

    Figure Lengend Snippet: miR-21, miR-143, and miR-378e promote stemness and epithelial–mesenchymal transition T47D cells were transfected with scrambled (scr, control) or miRs -21, -143, and -378e, alone or in combination (final concentration: 100nM). After 48h, cells were harvested and cultured under non-adherent conditions in stem medium. After four days, the capacity of cells to form spheres was assessed a . Sphere diameter distribution for 10 representative fields. Scale bar: 100μm b . T47D cells were transfected with anti-miRs (alone or in combination, final concentration: 200nM) or scrambled anti-miR (anti-miR-scr, control). After 24h, cells were harvested and cultured under non-adherent conditions in stem medium. After four days, the capacity of cells to form spheres was assessed c . Sphere diameter distribution for 10 representative fields. Scale bar: 50μm d . ALDEFLUOR assays were performed in T47D cells transfected with scrambled or miRs -21, -143, and -378e for 48h e . T47D cells were transfected with scrambled, miRs -21, -143, or -378e for 48h f . or 72h g . Real Time PCR was performed to analyze oct3/4, nanog, sox2, and snail mRNA levels (f). In addition, T47D cells were transfected with the miRs alone or in combination for 72h. Western blot analysis was performed to evaluate nanog, sox2, oct3/4, zeb, and e-cadherin protein levels (g). In a, b, c, d, e, f, data were obtained from three independent experiments and are presented as mean value ± SD. P-value was calculated using Student's t test. * p

    Article Snippet: Reverse transcription of total RNA was performed starting from equal amounts of total RNA/sample (150/500ng) using miScript reverse Transcription Kit (Qiagen, Milan, Italy) for miR analysis, and using SuperScript® III Reverse Transcriptase (Invitrogen, Milan, Italy) for mRNA analysis.

    Techniques: Transfection, Concentration Assay, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot

    Messenger RNA expression levels of GABARAP and GABARAPL genes. Data were normalized to the geometric average of the reference genes ribosomal protein L13A (rpl13A) and ubiquitin (ubc). Data were analysed by two-way ANOVA followed by Sidak post hoc test. Open and filled columns represent three-spined sticklebacks exposed to control water ( n = 12) and high-CO 2 water ( n = 12) for 43 days. Values are shown as means + SEM.

    Journal: Conservation Physiology

    Article Title: Expression of genes involved in brain GABAergic neurotransmission in three-spined stickleback exposed to near-future CO2

    doi: 10.1093/conphys/cow068

    Figure Lengend Snippet: Messenger RNA expression levels of GABARAP and GABARAPL genes. Data were normalized to the geometric average of the reference genes ribosomal protein L13A (rpl13A) and ubiquitin (ubc). Data were analysed by two-way ANOVA followed by Sidak post hoc test. Open and filled columns represent three-spined sticklebacks exposed to control water ( n = 12) and high-CO 2 water ( n = 12) for 43 days. Values are shown as means + SEM.

    Article Snippet: Subsequently, cDNA was synthesized in duplicate from each total RNA sample using SuperScript III reverse transcriptase (Invitrogen) and oligo(dT)18 in a total reaction volume of 20 µl.

    Techniques: RNA Expression

    Messenger RNA expression levels of KCCs, NKCC1, ClC2, NDAE, AE3 ion cotransporters and CAII enzyme. Data were normalized to the geometric average of the reference genes ribosomal protein L13A (rpl13A) and ubiquitin (ubc). Data were analysed by two-way ANOVA followed by Sidak post hoc test. Open and filled columns represent three-spined sticklebacks exposed to control water ( n = 12) and high-CO 2 water ( n = 12) for 43 days. Values are shown as means + SEM.

    Journal: Conservation Physiology

    Article Title: Expression of genes involved in brain GABAergic neurotransmission in three-spined stickleback exposed to near-future CO2

    doi: 10.1093/conphys/cow068

    Figure Lengend Snippet: Messenger RNA expression levels of KCCs, NKCC1, ClC2, NDAE, AE3 ion cotransporters and CAII enzyme. Data were normalized to the geometric average of the reference genes ribosomal protein L13A (rpl13A) and ubiquitin (ubc). Data were analysed by two-way ANOVA followed by Sidak post hoc test. Open and filled columns represent three-spined sticklebacks exposed to control water ( n = 12) and high-CO 2 water ( n = 12) for 43 days. Values are shown as means + SEM.

    Article Snippet: Subsequently, cDNA was synthesized in duplicate from each total RNA sample using SuperScript III reverse transcriptase (Invitrogen) and oligo(dT)18 in a total reaction volume of 20 µl.

    Techniques: RNA Expression

    Messenger RNA expression levels of GABA A receptor subunits. Data were normalized to the geometric average of the reference genes ribosomal protein L13A (rpl13A) and ubiquitin (ubc) and grouped into four families as follows: α subunits ( A ); β subunits ( B ); γ subunits ( C ) and δ, π and ρ subunits ( D ). Each family was analysed by two-way ANOVA followed by Sidak post hoc test. Open and filled columns represent three-spined sticklebacks exposed to control water ( n = 12) and high-CO 2 water ( n = 12) for 43 days. Values are shown as means + SEM.

    Journal: Conservation Physiology

    Article Title: Expression of genes involved in brain GABAergic neurotransmission in three-spined stickleback exposed to near-future CO2

    doi: 10.1093/conphys/cow068

    Figure Lengend Snippet: Messenger RNA expression levels of GABA A receptor subunits. Data were normalized to the geometric average of the reference genes ribosomal protein L13A (rpl13A) and ubiquitin (ubc) and grouped into four families as follows: α subunits ( A ); β subunits ( B ); γ subunits ( C ) and δ, π and ρ subunits ( D ). Each family was analysed by two-way ANOVA followed by Sidak post hoc test. Open and filled columns represent three-spined sticklebacks exposed to control water ( n = 12) and high-CO 2 water ( n = 12) for 43 days. Values are shown as means + SEM.

    Article Snippet: Subsequently, cDNA was synthesized in duplicate from each total RNA sample using SuperScript III reverse transcriptase (Invitrogen) and oligo(dT)18 in a total reaction volume of 20 µl.

    Techniques: RNA Expression

    Messenger RNA expression levels of GAT, GABAT and GAD genes. Data were normalized to the geometric average of the reference genes ribosomal protein L13A (rpl13A) and ubiquitin (ubc). Data were analysed by two-way ANOVA followed by Sidak post hoc test. Open and filled columns represent three-spined sticklebacks exposed to control water ( n = 12) and high-CO 2 water ( n = 12) for 43 days. Values are shown as means + SEM.

    Journal: Conservation Physiology

    Article Title: Expression of genes involved in brain GABAergic neurotransmission in three-spined stickleback exposed to near-future CO2

    doi: 10.1093/conphys/cow068

    Figure Lengend Snippet: Messenger RNA expression levels of GAT, GABAT and GAD genes. Data were normalized to the geometric average of the reference genes ribosomal protein L13A (rpl13A) and ubiquitin (ubc). Data were analysed by two-way ANOVA followed by Sidak post hoc test. Open and filled columns represent three-spined sticklebacks exposed to control water ( n = 12) and high-CO 2 water ( n = 12) for 43 days. Values are shown as means + SEM.

    Article Snippet: Subsequently, cDNA was synthesized in duplicate from each total RNA sample using SuperScript III reverse transcriptase (Invitrogen) and oligo(dT)18 in a total reaction volume of 20 µl.

    Techniques: RNA Expression

    Full WSN HA sequence logo plot: ATF6f/XBP1s 37˚C vs. Basal 37˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon ATF6f/XBP1s induction at 37˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Journal: eLife

    Article Title: Enhanced ER proteostasis and temperature differentially impact the mutational tolerance of influenza hemagglutinin

    doi: 10.7554/eLife.38795

    Figure Lengend Snippet: Full WSN HA sequence logo plot: ATF6f/XBP1s 37˚C vs. Basal 37˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon ATF6f/XBP1s induction at 37˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Article Snippet: Viral RNA was extracted from 140 μL infectious supernatant using the QIAamp Viral RNA Mini kit and at least 106 HA molecules were reverse transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) with 5ʹ-WSN-HA and 3ʹ-WSN-HA primers ( ).

    Techniques: Hemagglutination Assay, Sequencing, Selection, Variant Assay

    Full WSN HA sequence logo plot: ATF6f/XBP1s 39˚C vs. Basal 39˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon ATF6f/XBP1s induction at 39˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Journal: eLife

    Article Title: Enhanced ER proteostasis and temperature differentially impact the mutational tolerance of influenza hemagglutinin

    doi: 10.7554/eLife.38795

    Figure Lengend Snippet: Full WSN HA sequence logo plot: ATF6f/XBP1s 39˚C vs. Basal 39˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon ATF6f/XBP1s induction at 39˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Article Snippet: Viral RNA was extracted from 140 μL infectious supernatant using the QIAamp Viral RNA Mini kit and at least 106 HA molecules were reverse transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) with 5ʹ-WSN-HA and 3ʹ-WSN-HA primers ( ).

    Techniques: Hemagglutination Assay, Sequencing, Selection, Variant Assay

    Full WSN HA sequence logo plot: XBP1s 39˚C vs. Basal 39˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon XBP1s induction at 39˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Journal: eLife

    Article Title: Enhanced ER proteostasis and temperature differentially impact the mutational tolerance of influenza hemagglutinin

    doi: 10.7554/eLife.38795

    Figure Lengend Snippet: Full WSN HA sequence logo plot: XBP1s 39˚C vs. Basal 39˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon XBP1s induction at 39˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Article Snippet: Viral RNA was extracted from 140 μL infectious supernatant using the QIAamp Viral RNA Mini kit and at least 106 HA molecules were reverse transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) with 5ʹ-WSN-HA and 3ʹ-WSN-HA primers ( ).

    Techniques: Hemagglutination Assay, Sequencing, Selection, Variant Assay

    Full WSN HA sequence logo plot: XBP1s 37˚C vs. Basal 37˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon XBP1s induction at 37˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Journal: eLife

    Article Title: Enhanced ER proteostasis and temperature differentially impact the mutational tolerance of influenza hemagglutinin

    doi: 10.7554/eLife.38795

    Figure Lengend Snippet: Full WSN HA sequence logo plot: XBP1s 37˚C vs. Basal 37˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon XBP1s induction at 37˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Article Snippet: Viral RNA was extracted from 140 μL infectious supernatant using the QIAamp Viral RNA Mini kit and at least 106 HA molecules were reverse transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) with 5ʹ-WSN-HA and 3ʹ-WSN-HA primers ( ).

    Techniques: Hemagglutination Assay, Sequencing, Selection, Variant Assay

    Full WSN HA sequence logo plot: Basal 39˚C vs. Basal 37˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched at 39˚C relative to 37˚C in a basal environment; variants below the line were depleted at 39˚C. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Journal: eLife

    Article Title: Enhanced ER proteostasis and temperature differentially impact the mutational tolerance of influenza hemagglutinin

    doi: 10.7554/eLife.38795

    Figure Lengend Snippet: Full WSN HA sequence logo plot: Basal 39˚C vs. Basal 37˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched at 39˚C relative to 37˚C in a basal environment; variants below the line were depleted at 39˚C. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Article Snippet: Viral RNA was extracted from 140 μL infectious supernatant using the QIAamp Viral RNA Mini kit and at least 106 HA molecules were reverse transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) with 5ʹ-WSN-HA and 3ʹ-WSN-HA primers ( ).

    Techniques: Hemagglutination Assay, Sequencing, Selection, Variant Assay

    TNFα induces re-localization of SIMPL to the Tnf gene promoter. ( A ) Schematic of the mouse Tnf gene indicating location of PCR primers. ( B ) WT MEFs transfected with FLAG-SIMPL or empty FLAG-vector (Flag) and were left untreated (-) or were treated with rhTNFα (10 ng/ml; 45 min). Immunocomplexing assays performed with either Flag or p65 antibody were subjected to Western analysis with SIMPL antibody to confirm the TNFα-dependence of the p65-SIMPL interaction. LC-light chain. ( C ) WT MEFs were transfected with the indicated plasmids; 72 h post-transfection, chromatin immunoprecipitation (ChIP) assays were performed as described under Materials and Methods with the indicated antibodies and primer sets specific for the mouse Tnf gene (see Panel A for primer location; Materials and Methods for primer sequence). ChIP assays and corresponding quantitative real-time PCR assays were repeated at least three times.

    Journal: PLoS ONE

    Article Title: SIMPL Enhancement of Tumor Necrosis Factor-? Dependent p65-MED1 Complex Formation Is Required for Mammalian Hematopoietic Stem and Progenitor Cell Function

    doi: 10.1371/journal.pone.0061123

    Figure Lengend Snippet: TNFα induces re-localization of SIMPL to the Tnf gene promoter. ( A ) Schematic of the mouse Tnf gene indicating location of PCR primers. ( B ) WT MEFs transfected with FLAG-SIMPL or empty FLAG-vector (Flag) and were left untreated (-) or were treated with rhTNFα (10 ng/ml; 45 min). Immunocomplexing assays performed with either Flag or p65 antibody were subjected to Western analysis with SIMPL antibody to confirm the TNFα-dependence of the p65-SIMPL interaction. LC-light chain. ( C ) WT MEFs were transfected with the indicated plasmids; 72 h post-transfection, chromatin immunoprecipitation (ChIP) assays were performed as described under Materials and Methods with the indicated antibodies and primer sets specific for the mouse Tnf gene (see Panel A for primer location; Materials and Methods for primer sequence). ChIP assays and corresponding quantitative real-time PCR assays were repeated at least three times.

    Article Snippet: RNA (2 µg) was subject to reverse transcription with random primers using SuperScript III reverse transcriptase (Invitrogen). qRT-PCR was performed with equivalent amounts of cDNA according to manufacturer's specification (LightCycler 480 DNA SYBR Green kit and LightCycler 480 system; Roche Diagnostics, Indianapolis, IN).

    Techniques: Polymerase Chain Reaction, Transfection, Plasmid Preparation, Western Blot, Liquid Chromatography, Chromatin Immunoprecipitation, Sequencing, Real-time Polymerase Chain Reaction

    SIMPL enhances the appearance of p65, MED1 and serine 5 phosphorylated RNA pol II on the Tnf gene promoter. ( A ) Schematic of the mouse Tnf gene indicating location of PCR primers. ( B and C ) MEFs derived from littermate controls (WT) or SIMPL −/− mice were left untreated (-) or were treated with rhTNFα (10 ng/ml) for 45 minutes. Chromatin immunoprecipitation (ChIP) assays were performed as described under Materials and Methods with the indicated antibodies and primer sets specific for the mouse Tnf gene (see Panel A for location of primers; Materials and Methods for sequence of primers). ChIP assays were repeated at least three times.

    Journal: PLoS ONE

    Article Title: SIMPL Enhancement of Tumor Necrosis Factor-? Dependent p65-MED1 Complex Formation Is Required for Mammalian Hematopoietic Stem and Progenitor Cell Function

    doi: 10.1371/journal.pone.0061123

    Figure Lengend Snippet: SIMPL enhances the appearance of p65, MED1 and serine 5 phosphorylated RNA pol II on the Tnf gene promoter. ( A ) Schematic of the mouse Tnf gene indicating location of PCR primers. ( B and C ) MEFs derived from littermate controls (WT) or SIMPL −/− mice were left untreated (-) or were treated with rhTNFα (10 ng/ml) for 45 minutes. Chromatin immunoprecipitation (ChIP) assays were performed as described under Materials and Methods with the indicated antibodies and primer sets specific for the mouse Tnf gene (see Panel A for location of primers; Materials and Methods for sequence of primers). ChIP assays were repeated at least three times.

    Article Snippet: RNA (2 µg) was subject to reverse transcription with random primers using SuperScript III reverse transcriptase (Invitrogen). qRT-PCR was performed with equivalent amounts of cDNA according to manufacturer's specification (LightCycler 480 DNA SYBR Green kit and LightCycler 480 system; Roche Diagnostics, Indianapolis, IN).

    Techniques: Polymerase Chain Reaction, Derivative Assay, Mouse Assay, Chromatin Immunoprecipitation, Sequencing

    SIMPL is required for initiation of Tnf gene transcription. ( A ) Schematic of mouse Tnf gene annotated with the regions amplified by primers used to distinguish initiation (+24 to +121) and elongation (+1441 to +1537). ( B ) Total cellular RNA extracted from untreated and TNFα treated (rhTNFα 10 ng/ml, 45 min) MEFs derived from littermate control (WT) or SIMPL −/− mice was subject to quantitative RT-PCR with the indicated primers. Fold-change represents a ratio of TNFα treated to untreated sample of the same genotype. Assays were repeated at least three times and a 2-tailed T-test was used to determine statistical significance, **p

    Journal: PLoS ONE

    Article Title: SIMPL Enhancement of Tumor Necrosis Factor-? Dependent p65-MED1 Complex Formation Is Required for Mammalian Hematopoietic Stem and Progenitor Cell Function

    doi: 10.1371/journal.pone.0061123

    Figure Lengend Snippet: SIMPL is required for initiation of Tnf gene transcription. ( A ) Schematic of mouse Tnf gene annotated with the regions amplified by primers used to distinguish initiation (+24 to +121) and elongation (+1441 to +1537). ( B ) Total cellular RNA extracted from untreated and TNFα treated (rhTNFα 10 ng/ml, 45 min) MEFs derived from littermate control (WT) or SIMPL −/− mice was subject to quantitative RT-PCR with the indicated primers. Fold-change represents a ratio of TNFα treated to untreated sample of the same genotype. Assays were repeated at least three times and a 2-tailed T-test was used to determine statistical significance, **p

    Article Snippet: RNA (2 µg) was subject to reverse transcription with random primers using SuperScript III reverse transcriptase (Invitrogen). qRT-PCR was performed with equivalent amounts of cDNA according to manufacturer's specification (LightCycler 480 DNA SYBR Green kit and LightCycler 480 system; Roche Diagnostics, Indianapolis, IN).

    Techniques: Amplification, Derivative Assay, Mouse Assay, Quantitative RT-PCR

    Identification of SIMPL-dependent genes. ( A ) Expression level of genes in TNFα treated as compared untreated MEFs derived from either littermate (▪) or SIMPL −/− ( ) mice. Genes are grouped according differences in TNFα responsiveness of treated SIMPL −/− derived as compared to littermate derived MEFs: complete loss (Group I); 75% reduction (Group II); 50% reduction (Group III) or a 25% reduction (Group IV). ( B ) MEFs derived from littermate (▪) or SIMPL −/− ( ) mice were treated with rhuTNFα (10 ng/ml) for 45 minutes, total RNA was isolated, converted to cDNA for analysis of indicated mRNAs by qRT-PCR.

    Journal: PLoS ONE

    Article Title: SIMPL Enhancement of Tumor Necrosis Factor-? Dependent p65-MED1 Complex Formation Is Required for Mammalian Hematopoietic Stem and Progenitor Cell Function

    doi: 10.1371/journal.pone.0061123

    Figure Lengend Snippet: Identification of SIMPL-dependent genes. ( A ) Expression level of genes in TNFα treated as compared untreated MEFs derived from either littermate (▪) or SIMPL −/− ( ) mice. Genes are grouped according differences in TNFα responsiveness of treated SIMPL −/− derived as compared to littermate derived MEFs: complete loss (Group I); 75% reduction (Group II); 50% reduction (Group III) or a 25% reduction (Group IV). ( B ) MEFs derived from littermate (▪) or SIMPL −/− ( ) mice were treated with rhuTNFα (10 ng/ml) for 45 minutes, total RNA was isolated, converted to cDNA for analysis of indicated mRNAs by qRT-PCR.

    Article Snippet: RNA (2 µg) was subject to reverse transcription with random primers using SuperScript III reverse transcriptase (Invitrogen). qRT-PCR was performed with equivalent amounts of cDNA according to manufacturer's specification (LightCycler 480 DNA SYBR Green kit and LightCycler 480 system; Roche Diagnostics, Indianapolis, IN).

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

    Effect of hnRNP C1/C2 knockdown on DENV RNA synthesis. Huh7 cells transfected with hnRNP C1/C2-specific or control siRNA were infected with DENV at MOI 0.03. Cells were collected at 0, 12, 24, 36 and 48 h post-infection and subjected to total RNA extraction and subsequent reverse transcription and real-time PCR for determination of DENV RNA expression. The amount of DENV RNA in virus-infected cells was reported as viral RNA copies per cell. Data represent mean and SEM of three independent experiments. Asterisks indicate statistically significant differences (*p

    Journal: Virology Journal

    Article Title: Role of human heterogeneous nuclear ribonucleoprotein C1/C2 in dengue virus replication

    doi: 10.1186/s12985-014-0219-7

    Figure Lengend Snippet: Effect of hnRNP C1/C2 knockdown on DENV RNA synthesis. Huh7 cells transfected with hnRNP C1/C2-specific or control siRNA were infected with DENV at MOI 0.03. Cells were collected at 0, 12, 24, 36 and 48 h post-infection and subjected to total RNA extraction and subsequent reverse transcription and real-time PCR for determination of DENV RNA expression. The amount of DENV RNA in virus-infected cells was reported as viral RNA copies per cell. Data represent mean and SEM of three independent experiments. Asterisks indicate statistically significant differences (*p

    Article Snippet: Reverse transcription was performed using 62.5 ng total RNA and SuperScript III Reverse Transcriptase (Invitrogen) or AMV Reverse Transcriptase (Promega), according to the manufacturer’s instructions with minor modifications.

    Techniques: Transfection, Infection, RNA Extraction, Real-time Polymerase Chain Reaction, RNA Expression

    siRNA-mediated knockdown of hnRNP C1/C2 mRNA and proteins. (A) Huh7 cells transfected with hnRNP C1/C2-specific or control siRNA were infected with DENV at MOI 0.03. Cells were collected at 0, 12, 24, 36 and 48 h post-infection and subjected to total RNA extraction and subsequent reverse transcription and real-time PCR for determination of hnRNP C1/C2 and β-actin (internal control) mRNA expression. Relative hnRNP C1/C2 mRNA expression was obtained by normalization with β-actin mRNA expression in siRNA-transfected cells following DENV infection. Fold change of the relative hnRNP C1/C2 mRNA expression between the specific siRNA and control siRNA-transfected samples was compared. Data represent mean and SEM of three independent experiments. Asterisks indicate statistically significant differences (*p

    Journal: Virology Journal

    Article Title: Role of human heterogeneous nuclear ribonucleoprotein C1/C2 in dengue virus replication

    doi: 10.1186/s12985-014-0219-7

    Figure Lengend Snippet: siRNA-mediated knockdown of hnRNP C1/C2 mRNA and proteins. (A) Huh7 cells transfected with hnRNP C1/C2-specific or control siRNA were infected with DENV at MOI 0.03. Cells were collected at 0, 12, 24, 36 and 48 h post-infection and subjected to total RNA extraction and subsequent reverse transcription and real-time PCR for determination of hnRNP C1/C2 and β-actin (internal control) mRNA expression. Relative hnRNP C1/C2 mRNA expression was obtained by normalization with β-actin mRNA expression in siRNA-transfected cells following DENV infection. Fold change of the relative hnRNP C1/C2 mRNA expression between the specific siRNA and control siRNA-transfected samples was compared. Data represent mean and SEM of three independent experiments. Asterisks indicate statistically significant differences (*p

    Article Snippet: Reverse transcription was performed using 62.5 ng total RNA and SuperScript III Reverse Transcriptase (Invitrogen) or AMV Reverse Transcriptase (Promega), according to the manufacturer’s instructions with minor modifications.

    Techniques: Transfection, Infection, RNA Extraction, Real-time Polymerase Chain Reaction, Expressing

    Effect of hnRNP C1/C2 knockdown on DENV protein translation. Huh7 cells transfected with hnRNP C1/C2-specific siRNA (SP) or irrelevant negative control siRNA (IR) were subjected to co-transfection with pGL3-DENV2-5′UTR-72ntC-Fluc-3′UTR construct (viral reporter) and pRL-SV40 construct (internal control reporter). At 12 h after reporter RNA transfection, cells were harvested and assessed for firefly and Renilla luciferase activities. In parallel, reporter RNA-transfected cells that were treated with DENV capsid-specific siRNA to knockdown viral reporter RNA template used for viral translation (RK) or with cycloheximide to inhibit overall protein translation (CHK) were set up as positive controls in this assay for inhibition of protein translation, compared with non-treated cells (NT). (A) Relative luciferase expression specifically indicated DENV protein translation. In each sample, relative luminescence units of firefly luciferase activity were normalized to that of Renilla luciferase activity. Normalized luciferase signals of control siRNA-transfected cells and non-treated cells were set to 1. Relative luciferase expression in hnRNP C1/C2-specific siRNA was compared to that in control siRNA-transfected cells whereas relative luciferase expression in viral reporter knockdown cells and cycloheximide-treated cells was compared to that in non-treated cells. Data represent mean and SEM of three independent experiments. Asterisks indicate statistically significant difference (***p

    Journal: Virology Journal

    Article Title: Role of human heterogeneous nuclear ribonucleoprotein C1/C2 in dengue virus replication

    doi: 10.1186/s12985-014-0219-7

    Figure Lengend Snippet: Effect of hnRNP C1/C2 knockdown on DENV protein translation. Huh7 cells transfected with hnRNP C1/C2-specific siRNA (SP) or irrelevant negative control siRNA (IR) were subjected to co-transfection with pGL3-DENV2-5′UTR-72ntC-Fluc-3′UTR construct (viral reporter) and pRL-SV40 construct (internal control reporter). At 12 h after reporter RNA transfection, cells were harvested and assessed for firefly and Renilla luciferase activities. In parallel, reporter RNA-transfected cells that were treated with DENV capsid-specific siRNA to knockdown viral reporter RNA template used for viral translation (RK) or with cycloheximide to inhibit overall protein translation (CHK) were set up as positive controls in this assay for inhibition of protein translation, compared with non-treated cells (NT). (A) Relative luciferase expression specifically indicated DENV protein translation. In each sample, relative luminescence units of firefly luciferase activity were normalized to that of Renilla luciferase activity. Normalized luciferase signals of control siRNA-transfected cells and non-treated cells were set to 1. Relative luciferase expression in hnRNP C1/C2-specific siRNA was compared to that in control siRNA-transfected cells whereas relative luciferase expression in viral reporter knockdown cells and cycloheximide-treated cells was compared to that in non-treated cells. Data represent mean and SEM of three independent experiments. Asterisks indicate statistically significant difference (***p

    Article Snippet: Reverse transcription was performed using 62.5 ng total RNA and SuperScript III Reverse Transcriptase (Invitrogen) or AMV Reverse Transcriptase (Promega), according to the manufacturer’s instructions with minor modifications.

    Techniques: Transfection, Negative Control, Cotransfection, Construct, Luciferase, Inhibition, Expressing, Activity Assay

    Association of hnRNP C1/C2 proteins with dengue viral RNA. Mock-infected (M) and DENV-infected (I) cells were subjected to immunoprecipitation using anti-hnRNPC1/C2 (αhnRNP C) monoclonal antibody and their isotype-matched control antibody (IgG1). (A) Immunoprecipitated proteins were analyzed by immunoblotting using hnRNP C1/C2-specific antibody. Mock and DENV-infected cell lysates prior to immunoprecipitation served as controls (input). (B) RNA was extracted from the immunoprecipitated samples and used as a template for RT-PCR with a primer pair specific for DENV NS1 region. PCRs performed in parallel in the absence of cDNA and in the presence of pcDNAhygro containing DENV NS1 gene were included as negative (−) and positive (+) controls, respectively. Results are representative of three independent experiments with similar outcome.

    Journal: Virology Journal

    Article Title: Role of human heterogeneous nuclear ribonucleoprotein C1/C2 in dengue virus replication

    doi: 10.1186/s12985-014-0219-7

    Figure Lengend Snippet: Association of hnRNP C1/C2 proteins with dengue viral RNA. Mock-infected (M) and DENV-infected (I) cells were subjected to immunoprecipitation using anti-hnRNPC1/C2 (αhnRNP C) monoclonal antibody and their isotype-matched control antibody (IgG1). (A) Immunoprecipitated proteins were analyzed by immunoblotting using hnRNP C1/C2-specific antibody. Mock and DENV-infected cell lysates prior to immunoprecipitation served as controls (input). (B) RNA was extracted from the immunoprecipitated samples and used as a template for RT-PCR with a primer pair specific for DENV NS1 region. PCRs performed in parallel in the absence of cDNA and in the presence of pcDNAhygro containing DENV NS1 gene were included as negative (−) and positive (+) controls, respectively. Results are representative of three independent experiments with similar outcome.

    Article Snippet: Reverse transcription was performed using 62.5 ng total RNA and SuperScript III Reverse Transcriptase (Invitrogen) or AMV Reverse Transcriptase (Promega), according to the manufacturer’s instructions with minor modifications.

    Techniques: Infection, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Identification of cis -acting RNA elements within the 3′-terminal 55 nts that are required for (−)- and (+)-strand RNA synthesis. (A) Constructs of deletion mutants within the 3′-terminal 55 nucleotides of BCoV DI RNA. Dashes denote deleted sequences. (B) The relative levels of (–)-strand DI RNA synthesis. Total cellular RNA was extracted from DI RNA-transfected BCoV-infected cells at 8 hpt. The synthesis of (–)-strand DI RNA from the deletion mutant was quantitated by qRT-PCR and was compared with that from wt BM25. Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from DI RNA-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. (C) Upper panel: the synthesis of (+)-strand DI RNA as detected by Northern blot assay. Total cellular RNA was extracted at 48 hpi of VP1 and was subjected to Northern blot assay with N sgmRNA and 18S rRNA as internal controls. Middle panel: the relative levels of the (+)-strand DI RNA synthesis. Lower panel: the sequence of the BCoV DI RNA at 48 hpi of VP1 as determined by RT-PCR and sequencing analysis. The values (B) and (C) represent the mean ±SD of three individual experiments. SD: standard deviation, wt: wild type, mx: mixed, NA: not available. *p

    Journal: PLoS ONE

    Article Title: The 3?-Terminal 55 Nucleotides of Bovine Coronavirus Defective Interfering RNA Harbor Cis-Acting Elements Required for Both Negative- and Positive-Strand RNA Synthesis

    doi: 10.1371/journal.pone.0098422

    Figure Lengend Snippet: Identification of cis -acting RNA elements within the 3′-terminal 55 nts that are required for (−)- and (+)-strand RNA synthesis. (A) Constructs of deletion mutants within the 3′-terminal 55 nucleotides of BCoV DI RNA. Dashes denote deleted sequences. (B) The relative levels of (–)-strand DI RNA synthesis. Total cellular RNA was extracted from DI RNA-transfected BCoV-infected cells at 8 hpt. The synthesis of (–)-strand DI RNA from the deletion mutant was quantitated by qRT-PCR and was compared with that from wt BM25. Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from DI RNA-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. (C) Upper panel: the synthesis of (+)-strand DI RNA as detected by Northern blot assay. Total cellular RNA was extracted at 48 hpi of VP1 and was subjected to Northern blot assay with N sgmRNA and 18S rRNA as internal controls. Middle panel: the relative levels of the (+)-strand DI RNA synthesis. Lower panel: the sequence of the BCoV DI RNA at 48 hpi of VP1 as determined by RT-PCR and sequencing analysis. The values (B) and (C) represent the mean ±SD of three individual experiments. SD: standard deviation, wt: wild type, mx: mixed, NA: not available. *p

    Article Snippet: For quantitating (–)-strand DI RNA synthesis, 1 µg of TAP-treated and ligated RNA was used for RT reaction with oligonucleotide MHV3′UTR3(–) and SuperScript III reverse transcriptase (Invitrogen).

    Techniques: Construct, Transfection, Infection, Mutagenesis, Quantitative RT-PCR, Northern Blot, Sequencing, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

    Determination of cis -acting RNA elements between the nts −4 and −40 required for (−)- and (+)-strand RNA synthesis. (A) Deletion mutants of BCoV DI RNA. Dashes denote deleted sequences. (B) The relative levels of (−)-strand DI RNA synthesis. The synthesis of (−)-strand DI RNA from the deletion mutant was quantitated by qRT-PCR and was compared with that from wt BM25. Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from DI RNA-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. (C) Upper panel: the synthesis of (+)-strand DI RNA as detected by Northern blot assay. Total cellular RNA was extracted at 48 hpi of VP1 and was subjected to Northern blot assay with N sgmRNA and 18S rRNA as internal controls. Middle panel: the relative levels of the (+)-strand DI RNA synthesis. Lower panel: the sequence of the BCoV DI RNA at 48 hpi of VP1 as determined by RT-PCR and sequencing analysis. The values (B) and (C) represent the mean ±SD of three individual experiments. SD: standard deviation, wt: wild type. **p

    Journal: PLoS ONE

    Article Title: The 3?-Terminal 55 Nucleotides of Bovine Coronavirus Defective Interfering RNA Harbor Cis-Acting Elements Required for Both Negative- and Positive-Strand RNA Synthesis

    doi: 10.1371/journal.pone.0098422

    Figure Lengend Snippet: Determination of cis -acting RNA elements between the nts −4 and −40 required for (−)- and (+)-strand RNA synthesis. (A) Deletion mutants of BCoV DI RNA. Dashes denote deleted sequences. (B) The relative levels of (−)-strand DI RNA synthesis. The synthesis of (−)-strand DI RNA from the deletion mutant was quantitated by qRT-PCR and was compared with that from wt BM25. Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from DI RNA-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. (C) Upper panel: the synthesis of (+)-strand DI RNA as detected by Northern blot assay. Total cellular RNA was extracted at 48 hpi of VP1 and was subjected to Northern blot assay with N sgmRNA and 18S rRNA as internal controls. Middle panel: the relative levels of the (+)-strand DI RNA synthesis. Lower panel: the sequence of the BCoV DI RNA at 48 hpi of VP1 as determined by RT-PCR and sequencing analysis. The values (B) and (C) represent the mean ±SD of three individual experiments. SD: standard deviation, wt: wild type. **p

    Article Snippet: For quantitating (–)-strand DI RNA synthesis, 1 µg of TAP-treated and ligated RNA was used for RT reaction with oligonucleotide MHV3′UTR3(–) and SuperScript III reverse transcriptase (Invitrogen).

    Techniques: Mutagenesis, Quantitative RT-PCR, Infection, Transfection, Northern Blot, Sequencing, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

    Effect of nucleotide species at the -1 position of 3′ terminal sequence in BCoV DI RNA on (−)- and (+)-strand RNA synthesis. (A) DI RNA constructs with nucleotide substitution (underlined) at the −1 position of 3′ terminal sequence. (B) The relative levels of (−)-strand DI RNA synthesis. BCoV-infected HRT-18 cells were transfected with the indicated DI RNA construct at 2 hpi, and the total cellular RNA was extracted at 8 hpt. The synthesis of the (−)-strand DI RNA from the substitution mutant was quantitated by qRT-PCR and compared with that from wt BM25A. Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from DI RNA-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. (C) Upper panel: the synthesis of (+)-strand DI RNA as detected by Northern blot assay with N sgmRNA and 18S rRNA as internal controls. Middle panel: the relative levels of (+)-strand DI RNA synthesis. Lower panel: the sequence of the BCoV DI RNA at 48 hpi of VP1 as determined by RT-PCR and sequencing analysis. The values (B) and (C) represent the mean ±SD of three individual experiments. SD: standard deviation, wt: wild type, mut: mutant. *p

    Journal: PLoS ONE

    Article Title: The 3?-Terminal 55 Nucleotides of Bovine Coronavirus Defective Interfering RNA Harbor Cis-Acting Elements Required for Both Negative- and Positive-Strand RNA Synthesis

    doi: 10.1371/journal.pone.0098422

    Figure Lengend Snippet: Effect of nucleotide species at the -1 position of 3′ terminal sequence in BCoV DI RNA on (−)- and (+)-strand RNA synthesis. (A) DI RNA constructs with nucleotide substitution (underlined) at the −1 position of 3′ terminal sequence. (B) The relative levels of (−)-strand DI RNA synthesis. BCoV-infected HRT-18 cells were transfected with the indicated DI RNA construct at 2 hpi, and the total cellular RNA was extracted at 8 hpt. The synthesis of the (−)-strand DI RNA from the substitution mutant was quantitated by qRT-PCR and compared with that from wt BM25A. Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from DI RNA-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. (C) Upper panel: the synthesis of (+)-strand DI RNA as detected by Northern blot assay with N sgmRNA and 18S rRNA as internal controls. Middle panel: the relative levels of (+)-strand DI RNA synthesis. Lower panel: the sequence of the BCoV DI RNA at 48 hpi of VP1 as determined by RT-PCR and sequencing analysis. The values (B) and (C) represent the mean ±SD of three individual experiments. SD: standard deviation, wt: wild type, mut: mutant. *p

    Article Snippet: For quantitating (–)-strand DI RNA synthesis, 1 µg of TAP-treated and ligated RNA was used for RT reaction with oligonucleotide MHV3′UTR3(–) and SuperScript III reverse transcriptase (Invitrogen).

    Techniques: Sequencing, Construct, Infection, Transfection, Mutagenesis, Quantitative RT-PCR, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

    Analysis of the requirement of 3′-terminal 55 nts for the synthesis of (−)-strand BCoV DI RNA. (A) Diagram of the BCoV DI RNA BM25A with the intact 3′-terminal 55 nts and the mutant construct Δ55 with the deletion of 3′-terminal 55 nts (denotes with dashes). (B) Detection of (–)-strand BCoV DI RNA with head-to-tail ligation and RT-PCR. RT-PCR products with a size of ∼150 bp were observed from BCoV-infected BM25A-transfected cells (lanes 2–8, arrowhead) but not from BCoV-infected Δ55-transfected cells (lanes 10-16). Lanes 18–21 represent the controls for RT-PCR. C1: total cellular RNA from mock-infected cells. C2: total cellular RNA from BCoV-infected cells. C3: total cellular RNA from DI RNA-transfected mock-infected cells. C4: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. (C) Sequence of the cDNA-cloned RT-PCR product with a size of ∼150 bp from lane 5 in Fig. 3B . [shown in the (+)-strand]. (D) Detection of the potential recombination between the helper virus genome and DI RNA. The primers MHV3′ UTR2(+), which anneals to the MHV 3′ UTR and was used for RT, and M3(–), which anneals to BCoV M protein gene, were used for PCR to detect potential recombination between helper virus BCoV genome and BM25A (lane 2) or Δ55 (lane 3). A recombinant DNA of 1,639 nt shown in lane 4 was created by overlap RT-PCR and was used as a size marker for the product generated with the primers MHV 3′ UTR2(+) and M3(–). (E) Left panel: the relative levels of (–)-strand DI RNA synthesis as measured by qRT-PCR. Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from DI RNA-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. Right panel: the amounts of DI RNA, helper virus N sgmRNA, and 18S rRNA from DI RNA-transfected BCoV-infected cells at 8 hpt of VP0 as measured by Northern blot assay. The values (E) represent the mean ±SD of three individual experiments. SD: standard deviation. ***p

    Journal: PLoS ONE

    Article Title: The 3?-Terminal 55 Nucleotides of Bovine Coronavirus Defective Interfering RNA Harbor Cis-Acting Elements Required for Both Negative- and Positive-Strand RNA Synthesis

    doi: 10.1371/journal.pone.0098422

    Figure Lengend Snippet: Analysis of the requirement of 3′-terminal 55 nts for the synthesis of (−)-strand BCoV DI RNA. (A) Diagram of the BCoV DI RNA BM25A with the intact 3′-terminal 55 nts and the mutant construct Δ55 with the deletion of 3′-terminal 55 nts (denotes with dashes). (B) Detection of (–)-strand BCoV DI RNA with head-to-tail ligation and RT-PCR. RT-PCR products with a size of ∼150 bp were observed from BCoV-infected BM25A-transfected cells (lanes 2–8, arrowhead) but not from BCoV-infected Δ55-transfected cells (lanes 10-16). Lanes 18–21 represent the controls for RT-PCR. C1: total cellular RNA from mock-infected cells. C2: total cellular RNA from BCoV-infected cells. C3: total cellular RNA from DI RNA-transfected mock-infected cells. C4: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. (C) Sequence of the cDNA-cloned RT-PCR product with a size of ∼150 bp from lane 5 in Fig. 3B . [shown in the (+)-strand]. (D) Detection of the potential recombination between the helper virus genome and DI RNA. The primers MHV3′ UTR2(+), which anneals to the MHV 3′ UTR and was used for RT, and M3(–), which anneals to BCoV M protein gene, were used for PCR to detect potential recombination between helper virus BCoV genome and BM25A (lane 2) or Δ55 (lane 3). A recombinant DNA of 1,639 nt shown in lane 4 was created by overlap RT-PCR and was used as a size marker for the product generated with the primers MHV 3′ UTR2(+) and M3(–). (E) Left panel: the relative levels of (–)-strand DI RNA synthesis as measured by qRT-PCR. Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from DI RNA-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. Right panel: the amounts of DI RNA, helper virus N sgmRNA, and 18S rRNA from DI RNA-transfected BCoV-infected cells at 8 hpt of VP0 as measured by Northern blot assay. The values (E) represent the mean ±SD of three individual experiments. SD: standard deviation. ***p

    Article Snippet: For quantitating (–)-strand DI RNA synthesis, 1 µg of TAP-treated and ligated RNA was used for RT reaction with oligonucleotide MHV3′UTR3(–) and SuperScript III reverse transcriptase (Invitrogen).

    Techniques: Mutagenesis, Construct, Ligation, Reverse Transcription Polymerase Chain Reaction, Infection, Transfection, Sequencing, Clone Assay, Polymerase Chain Reaction, Recombinant, Marker, Generated, Quantitative RT-PCR, Northern Blot, Standard Deviation

    Effects of AAs, 20E, insulin and HR3 on YPP genes. (A) Relative expression of gene—AAEL006563, Carboxypeptidase, detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (B) Relative expression of the same gene detected by qRT-PCR, in fat body tissues collected from female mosquitoes post EcR knock-down (iEcR). (C) Relative expression in tissues subjected IVFBC in culture media without (NT) and with amino acids (AA), with amino acids plus 20E (AA+20E) and after the withdrawal of 20E (20E WD). (D) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post HR3 knock-down (iHR3). (E) Relative expression of the same gene in tissues subjected to IVFBC in culture media without (NT) and with amino acids (AA), with amino acids and Insulin (AA+INS), Insulin and 20E (INS+20E), amino acids plus 20E (AA+20E), and amino acids plus 20E and Insulin (AA+20E+INS). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiments ( B and D ). All expression calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Journal: PLoS Genetics

    Article Title: Regulation of Gene Expression Patterns in Mosquito Reproduction

    doi: 10.1371/journal.pgen.1005450

    Figure Lengend Snippet: Effects of AAs, 20E, insulin and HR3 on YPP genes. (A) Relative expression of gene—AAEL006563, Carboxypeptidase, detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (B) Relative expression of the same gene detected by qRT-PCR, in fat body tissues collected from female mosquitoes post EcR knock-down (iEcR). (C) Relative expression in tissues subjected IVFBC in culture media without (NT) and with amino acids (AA), with amino acids plus 20E (AA+20E) and after the withdrawal of 20E (20E WD). (D) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post HR3 knock-down (iHR3). (E) Relative expression of the same gene in tissues subjected to IVFBC in culture media without (NT) and with amino acids (AA), with amino acids and Insulin (AA+INS), Insulin and 20E (INS+20E), amino acids plus 20E (AA+20E), and amino acids plus 20E and Insulin (AA+20E+INS). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiments ( B and D ). All expression calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Article Snippet: cDNAs were synthesized from 2 μg total RNA using the SuperScript III Reverse Transcriptase kit (Invitrogen).

    Techniques: Atomic Absorption Spectroscopy, Expressing, Quantitative RT-PCR, In Vitro, Luciferase

    Effects of AAs, 20E and JH on representative LGs. (A) Relative expression of gene—AALE004328 –Origin recognition complex, detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (B) Relative expression of the same gene in tissues subjected to IVFBC in culture media without (JH-) and with (JH+) juvenile hormone. (C) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post Met knock-down (iMet). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiment. All expressions calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Journal: PLoS Genetics

    Article Title: Regulation of Gene Expression Patterns in Mosquito Reproduction

    doi: 10.1371/journal.pgen.1005450

    Figure Lengend Snippet: Effects of AAs, 20E and JH on representative LGs. (A) Relative expression of gene—AALE004328 –Origin recognition complex, detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (B) Relative expression of the same gene in tissues subjected to IVFBC in culture media without (JH-) and with (JH+) juvenile hormone. (C) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post Met knock-down (iMet). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiment. All expressions calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Article Snippet: cDNAs were synthesized from 2 μg total RNA using the SuperScript III Reverse Transcriptase kit (Invitrogen).

    Techniques: Atomic Absorption Spectroscopy, Expressing, Quantitative RT-PCR, In Vitro, Luciferase

    Effects of AAs, 20E and HR3 on EMGs. (A) Relative expression of gene—AAEL001433, fgf receptor activating protein detected by qRT-PCR, in tissues subjected to in- vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (B) Relative expression of the same gene detected by qRT-PCR, in fat body tissues collected from female mosquitoes post EcR knock-down (iEcR). (C) Relative expression in tissues subjected to IVFBC in culture media without (NT) and with amino acids (AA), with amino acids plus 20E (AA+20E) and after the withdrawal of 20E (20E WD). (D) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post HR3 knock-down (iHR3). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiments ( B and D ). All expression calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Journal: PLoS Genetics

    Article Title: Regulation of Gene Expression Patterns in Mosquito Reproduction

    doi: 10.1371/journal.pgen.1005450

    Figure Lengend Snippet: Effects of AAs, 20E and HR3 on EMGs. (A) Relative expression of gene—AAEL001433, fgf receptor activating protein detected by qRT-PCR, in tissues subjected to in- vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (B) Relative expression of the same gene detected by qRT-PCR, in fat body tissues collected from female mosquitoes post EcR knock-down (iEcR). (C) Relative expression in tissues subjected to IVFBC in culture media without (NT) and with amino acids (AA), with amino acids plus 20E (AA+20E) and after the withdrawal of 20E (20E WD). (D) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post HR3 knock-down (iHR3). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiments ( B and D ). All expression calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Article Snippet: cDNAs were synthesized from 2 μg total RNA using the SuperScript III Reverse Transcriptase kit (Invitrogen).

    Techniques: Atomic Absorption Spectroscopy, Expressing, Quantitative RT-PCR, In Vitro, Luciferase

    Genes cyclically activated by JH through Met—Functional group enrichment and the effects of AAs, 20E and JH. (A) Venn diagram showing genes that are up regulated in late post eclosion (LPE) and late post blood meal (LGs) periods and that are down regulated in Met knocked-down (imet down) fat body tissues. (B-D) Comparison of functional categories viz. (B) Information storage and Processing, (C) Metabolism and (D) Cellular Processes and Signalling, that constitute the late genes (LGs) and cyclical genes (CGs) using the inNOG database. (E-F) Relative expression of gene—AAEL001171, tRNA-dihydrouridine synthase detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media, (E) without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E); (F) without (JH-) and with (JH+) juvenile hormone. (G) Relative expression of the same gene detected by qRT-PCR, in fat body tissues collected from female mosquitoes post Met knock-down (iMet); injecting double stranded RNA for the Luciferase gene (iluc) served as the control. (H) Expression profile of the gene after the first blood meal. (I) Expression profile of the gene after the completion of the first reproductive cycle (egg laying) and post second blood meal. All expression calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Journal: PLoS Genetics

    Article Title: Regulation of Gene Expression Patterns in Mosquito Reproduction

    doi: 10.1371/journal.pgen.1005450

    Figure Lengend Snippet: Genes cyclically activated by JH through Met—Functional group enrichment and the effects of AAs, 20E and JH. (A) Venn diagram showing genes that are up regulated in late post eclosion (LPE) and late post blood meal (LGs) periods and that are down regulated in Met knocked-down (imet down) fat body tissues. (B-D) Comparison of functional categories viz. (B) Information storage and Processing, (C) Metabolism and (D) Cellular Processes and Signalling, that constitute the late genes (LGs) and cyclical genes (CGs) using the inNOG database. (E-F) Relative expression of gene—AAEL001171, tRNA-dihydrouridine synthase detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media, (E) without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E); (F) without (JH-) and with (JH+) juvenile hormone. (G) Relative expression of the same gene detected by qRT-PCR, in fat body tissues collected from female mosquitoes post Met knock-down (iMet); injecting double stranded RNA for the Luciferase gene (iluc) served as the control. (H) Expression profile of the gene after the first blood meal. (I) Expression profile of the gene after the completion of the first reproductive cycle (egg laying) and post second blood meal. All expression calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Article Snippet: cDNAs were synthesized from 2 μg total RNA using the SuperScript III Reverse Transcriptase kit (Invitrogen).

    Techniques: Functional Assay, Atomic Absorption Spectroscopy, Expressing, Quantitative RT-PCR, In Vitro, Luciferase

    Effects of AAs, 20E, JH and HR3 on representative LMGs. (A) Relative expression of genes—AAEL003568, Threonine dehydratase detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (B) Relative expression of the same gene detected by qRT-PCR, in fat body tissues collected from female mosquitoes post EcR knock-down (iEcR). (C) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post HR3 knock-down (iHR3). (D) Relative expression in tissues subjected to IVFBC in culture media without (JH-) and with (JH+) juvenile hormone. (E) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post Met knock-down (iMet). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiments ( B , C and E ). All expression calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Journal: PLoS Genetics

    Article Title: Regulation of Gene Expression Patterns in Mosquito Reproduction

    doi: 10.1371/journal.pgen.1005450

    Figure Lengend Snippet: Effects of AAs, 20E, JH and HR3 on representative LMGs. (A) Relative expression of genes—AAEL003568, Threonine dehydratase detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (B) Relative expression of the same gene detected by qRT-PCR, in fat body tissues collected from female mosquitoes post EcR knock-down (iEcR). (C) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post HR3 knock-down (iHR3). (D) Relative expression in tissues subjected to IVFBC in culture media without (JH-) and with (JH+) juvenile hormone. (E) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes post Met knock-down (iMet). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiments ( B , C and E ). All expression calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Article Snippet: cDNAs were synthesized from 2 μg total RNA using the SuperScript III Reverse Transcriptase kit (Invitrogen).

    Techniques: Atomic Absorption Spectroscopy, Expressing, Quantitative RT-PCR, In Vitro, Luciferase

    Effects of AAs, 20E and JH on representative EGs. (A) Relative expression of AAEL002269, Purine nucleoside phosphorylase detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (B) Relative expression of the same gene detected by qRT-PCR, in fat body tissues collected from female mosquitoes post EcR knock-down (iEcR). (C) Relative expression of the same gene in tissues subjected to IVFBC in culture media without (JH-) and with (JH+) juvenile hormone. (D) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes after knock-down of the JH receptor Met (imet). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiments ( B and D ). All expressions calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Journal: PLoS Genetics

    Article Title: Regulation of Gene Expression Patterns in Mosquito Reproduction

    doi: 10.1371/journal.pgen.1005450

    Figure Lengend Snippet: Effects of AAs, 20E and JH on representative EGs. (A) Relative expression of AAEL002269, Purine nucleoside phosphorylase detected by qRT-PCR, in tissues subjected to in-vitro fat body culture (IVFBC) in culture media without (NT) and with amino acids (AA) and with amino acid plus 20E (AA+20E) (B) Relative expression of the same gene detected by qRT-PCR, in fat body tissues collected from female mosquitoes post EcR knock-down (iEcR). (C) Relative expression of the same gene in tissues subjected to IVFBC in culture media without (JH-) and with (JH+) juvenile hormone. (D) Relative expression detected by qRT-PCR, in fat body tissues collected from female mosquitoes after knock-down of the JH receptor Met (imet). Injecting double stranded RNA for the Luciferase gene (iluc) served as the control in the RNAi experiments ( B and D ). All expressions calculated against housekeeping gene RPS7. Data representative of three biological replicates, with three technical replicates and are illustrated as average ± SD, * P

    Article Snippet: cDNAs were synthesized from 2 μg total RNA using the SuperScript III Reverse Transcriptase kit (Invitrogen).

    Techniques: Atomic Absorption Spectroscopy, Expressing, Quantitative RT-PCR, In Vitro, Luciferase

    Ribosome bound mRNA abundance in tobacco organs. Abundance of mRNA bound to ribosomes in young leaves, fully expanded leaves, roots, flowers and floral buds of tobacco. Values represent relative polysomal RNA levels per µg of ribosome-bound RNA (Additional file 7 : Dataset 2). Data represent mean values of four biological replicates for young leaves and fully expanded leaves, three biological replicates for roots and flowers and two biological replicates for floral buds. Significance was estimated with two way ANOVA; P

    Journal: Plant Methods

    Article Title: Comparison of mitochondrial gene expression and polysome loading in different tobacco tissues

    doi: 10.1186/s13007-017-0257-4

    Figure Lengend Snippet: Ribosome bound mRNA abundance in tobacco organs. Abundance of mRNA bound to ribosomes in young leaves, fully expanded leaves, roots, flowers and floral buds of tobacco. Values represent relative polysomal RNA levels per µg of ribosome-bound RNA (Additional file 7 : Dataset 2). Data represent mean values of four biological replicates for young leaves and fully expanded leaves, three biological replicates for roots and flowers and two biological replicates for floral buds. Significance was estimated with two way ANOVA; P

    Article Snippet: Five micrograms of RNA were reverse transcribed using the Superscript III reverse transcriptase kit (Invitrogen, Carlsbad, USA).

    Techniques:

    Mitochondrial RNA abundance in tobacco organs. RNA levels of mitochondrial transcripts in young leaves, fully expanded leaves, roots, flowers and floral buds. Values represent relative transcript levels per µg of total RNA (Additional file 5 : Dataset 1). Data represent mean values of four biological replicates for young leaves and fully expanded leaves, three biological replicates for roots and flowers, and two biological replicates for floral buds. Significant changes were calculated using Two way ANOVA ( p

    Journal: Plant Methods

    Article Title: Comparison of mitochondrial gene expression and polysome loading in different tobacco tissues

    doi: 10.1186/s13007-017-0257-4

    Figure Lengend Snippet: Mitochondrial RNA abundance in tobacco organs. RNA levels of mitochondrial transcripts in young leaves, fully expanded leaves, roots, flowers and floral buds. Values represent relative transcript levels per µg of total RNA (Additional file 5 : Dataset 1). Data represent mean values of four biological replicates for young leaves and fully expanded leaves, three biological replicates for roots and flowers, and two biological replicates for floral buds. Significant changes were calculated using Two way ANOVA ( p

    Article Snippet: Five micrograms of RNA were reverse transcribed using the Superscript III reverse transcriptase kit (Invitrogen, Carlsbad, USA).

    Techniques:

    Determination of mitochondrial DNA content per nuclear DNA and the expression analysis of RpoT genes in tobacco organs using qRT-PCR analysis. Young tobacco leaves, fully expanded leaves, roots, flowers and floral buds were assayed for mitochondrial DNA content relative to nuclear DNA content ( A ). Primer combination was chosen from mitochondrial intergenic region orf112b to cob (position 34,443–40,865) and from a nuclear non-coding region from RpoTm. Data were determined by calculating the difference of mitochondrial to nuclear CT values. Bars in the figure represent standard deviations for three biological replicates. B RpoTm, and C RpoTmp messenger RNA abundance was determined by quantitative real-time PCR. Data were normalized to cytoplasmic EF1α and GAPDH levels as an internal control. RpoT gene expression is presented as 40-ΔCT values. Given values were derived from three different biological replicates; standard deviations are indicated. Different letters mark mean values that are significantly different from each other (one way ANOVA; p

    Journal: Plant Methods

    Article Title: Comparison of mitochondrial gene expression and polysome loading in different tobacco tissues

    doi: 10.1186/s13007-017-0257-4

    Figure Lengend Snippet: Determination of mitochondrial DNA content per nuclear DNA and the expression analysis of RpoT genes in tobacco organs using qRT-PCR analysis. Young tobacco leaves, fully expanded leaves, roots, flowers and floral buds were assayed for mitochondrial DNA content relative to nuclear DNA content ( A ). Primer combination was chosen from mitochondrial intergenic region orf112b to cob (position 34,443–40,865) and from a nuclear non-coding region from RpoTm. Data were determined by calculating the difference of mitochondrial to nuclear CT values. Bars in the figure represent standard deviations for three biological replicates. B RpoTm, and C RpoTmp messenger RNA abundance was determined by quantitative real-time PCR. Data were normalized to cytoplasmic EF1α and GAPDH levels as an internal control. RpoT gene expression is presented as 40-ΔCT values. Given values were derived from three different biological replicates; standard deviations are indicated. Different letters mark mean values that are significantly different from each other (one way ANOVA; p

    Article Snippet: Five micrograms of RNA were reverse transcribed using the Superscript III reverse transcriptase kit (Invitrogen, Carlsbad, USA).

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

    Northern blot analysis of mitochondrial RNA abundance in tobacco organs. a The analyzed tissues are indicated above the blots. Three mitochondrial genes (cox1, atp9 and rps10) were analyzed to confirm the pattern of mitochondrial mRNA abundance observed through mitochondrial microarray analysis. Equal loading of RNA was checked through 18S rRNA hybridization. The blots are representatives for three independent replicates. b Signal intensities relative to the 18s rRNA control from the Northern blots presented in part a of this figure. Pixel intensity values as determined using ImageJ were used to calculate the ratios

    Journal: Plant Methods

    Article Title: Comparison of mitochondrial gene expression and polysome loading in different tobacco tissues

    doi: 10.1186/s13007-017-0257-4

    Figure Lengend Snippet: Northern blot analysis of mitochondrial RNA abundance in tobacco organs. a The analyzed tissues are indicated above the blots. Three mitochondrial genes (cox1, atp9 and rps10) were analyzed to confirm the pattern of mitochondrial mRNA abundance observed through mitochondrial microarray analysis. Equal loading of RNA was checked through 18S rRNA hybridization. The blots are representatives for three independent replicates. b Signal intensities relative to the 18s rRNA control from the Northern blots presented in part a of this figure. Pixel intensity values as determined using ImageJ were used to calculate the ratios

    Article Snippet: Five micrograms of RNA were reverse transcribed using the Superscript III reverse transcriptase kit (Invitrogen, Carlsbad, USA).

    Techniques: Northern Blot, Microarray, Hybridization