superscript iii reverse transcriptase  (Thermo Fisher)


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    Structured Review

    Thermo Fisher superscript iii reverse transcriptase
    The saxE gene was required for Pst growth in leaf extracts and in planta . A. A schematic diagram of the complementary tests pinpointing the minimal region required for rescuing the growth of 25D12 mutant in plant leaf extracts. Filled arrows indicate genes annotated by the sequencing project marked with locus tags on the top. The filled triangle shows the insertion site of Ω‐Km transposon in mutant 25D12 . Genomic <t>DNA</t> fragments downstream the transposon insertion site were tested for the ability to confer tolerance to leaf extracts. B. The minimal region that protected 25D12 in leaf extracts also rescued the growth of the mutant on Arabidopsis plant. Bacterial inocula were infiltrated into Arabidopsis leaves at OD 600 = 0.001. Samples were taken at 0 and 3 dpi for colony counts. The saxE orf was required for bacterial virulence on Arabidopsis (C) and tomato (D) plants. Wild‐type Pst , Pst ▵saxE and the complemented strain Pst ▵saxE ‐ saxEhis were infiltrated into Arabidopsis (C) and tomato (D) leaves at OD 600 = 0.001. Samples were taken at 0 and 3 dpi for colony counts. All experiments were repeated at least <t>three</t> times, and similar results were observed. Data shown are means ± SD. ** indicates significant difference ( t ‐test, P
    Superscript Iii Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Pseudomonas bacteriocin syringacin M released upon desiccation suppresses the growth of sensitive bacteria in plant necrotic lesions"

    Article Title: Pseudomonas bacteriocin syringacin M released upon desiccation suppresses the growth of sensitive bacteria in plant necrotic lesions

    Journal: Microbial Biotechnology

    doi: 10.1111/1751-7915.13367

    The saxE gene was required for Pst growth in leaf extracts and in planta . A. A schematic diagram of the complementary tests pinpointing the minimal region required for rescuing the growth of 25D12 mutant in plant leaf extracts. Filled arrows indicate genes annotated by the sequencing project marked with locus tags on the top. The filled triangle shows the insertion site of Ω‐Km transposon in mutant 25D12 . Genomic DNA fragments downstream the transposon insertion site were tested for the ability to confer tolerance to leaf extracts. B. The minimal region that protected 25D12 in leaf extracts also rescued the growth of the mutant on Arabidopsis plant. Bacterial inocula were infiltrated into Arabidopsis leaves at OD 600 = 0.001. Samples were taken at 0 and 3 dpi for colony counts. The saxE orf was required for bacterial virulence on Arabidopsis (C) and tomato (D) plants. Wild‐type Pst , Pst ▵saxE and the complemented strain Pst ▵saxE ‐ saxEhis were infiltrated into Arabidopsis (C) and tomato (D) leaves at OD 600 = 0.001. Samples were taken at 0 and 3 dpi for colony counts. All experiments were repeated at least three times, and similar results were observed. Data shown are means ± SD. ** indicates significant difference ( t ‐test, P
    Figure Legend Snippet: The saxE gene was required for Pst growth in leaf extracts and in planta . A. A schematic diagram of the complementary tests pinpointing the minimal region required for rescuing the growth of 25D12 mutant in plant leaf extracts. Filled arrows indicate genes annotated by the sequencing project marked with locus tags on the top. The filled triangle shows the insertion site of Ω‐Km transposon in mutant 25D12 . Genomic DNA fragments downstream the transposon insertion site were tested for the ability to confer tolerance to leaf extracts. B. The minimal region that protected 25D12 in leaf extracts also rescued the growth of the mutant on Arabidopsis plant. Bacterial inocula were infiltrated into Arabidopsis leaves at OD 600 = 0.001. Samples were taken at 0 and 3 dpi for colony counts. The saxE orf was required for bacterial virulence on Arabidopsis (C) and tomato (D) plants. Wild‐type Pst , Pst ▵saxE and the complemented strain Pst ▵saxE ‐ saxEhis were infiltrated into Arabidopsis (C) and tomato (D) leaves at OD 600 = 0.001. Samples were taken at 0 and 3 dpi for colony counts. All experiments were repeated at least three times, and similar results were observed. Data shown are means ± SD. ** indicates significant difference ( t ‐test, P

    Techniques Used: Mutagenesis, Sequencing

    2) Product Images from "A Conserved Noncoding Locus Regulates Random Monoallelic Xist Expression across a Topological Boundary"

    Article Title: A Conserved Noncoding Locus Regulates Random Monoallelic Xist Expression across a Topological Boundary

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2019.10.030

    Linx -Related Topological Features Are Not Implicated in Xist Regulation (A) The Linx locus, CTCF binding, and orientation of CTCF motifs associated with CTCF chromatin immunoprecipitation sequencing (ChIP-seq) peaks. Orientation of CTCF motifs within the Tsix-TAD is represented above. The targeted deletions ΔLinxCBS (∼25 kb) and ΔLinx-int1 (∼51 kb) are indicated. See STAR Methods for sources of CTCF, DNaseI, and H3K27Ac datasets. (B and C) 5C profiles of the Tsix-TAD (B) and the two Xic TADs (C); pooled data from two biological replicates for each genotype. Differential map is corrected for deletion (see STAR Methods ). Gray pixels represent either the deleted region or filtered contacts. (D) Left: cross used for analysis of RNA allelic ratios in female hybrid embryos. Right: Xist RNA allelic ratios; each black dot corresponds to a single female embryo. Statistical analysis was performed using a two-tailed t test. The table summarizes the number of embryos collected. Analysis of Atp7a RNA allelic ratios and reverse cross is shown in Figures S5 A and S5B. (E and F) Capture-C profiles for LinxP (E) and Xist (F) viewpoints, at different time points of differentiation of XX (Pgk12.1) mESCs. Data represent one replicate; two or three replicates for each time point were performed and are identical to the one shown (data available in GEO). Profiles represent number of contacts for each DpnII fragment per 10,000 total contacts within a specified region (see STAR Methods ). CTCF ChIP-seq on male mESCs is represented below ( Nora et al., 2017 ). (G–I) 5C differential maps for mutant male mESCs: ΔLinxE (G), ΔLinxP (H) and LinxP-inv (I); pooled data from two biological replicates for each genotype. 5C profiles for each genotype are shown in Figure S5 D. Gray pixels correspond to either deleted regions or filtered contacts. (J) Quantification of 5C inter-TAD contacts (see Figure S5 E for details). Bars represent the average of the calculated proportions of four (E14 and ΔLinxP) or two (ΔLinxE and LinxP-inv) independent replicates. Statistical analysis was performed using a two-tailed t test ( ∗∗ p
    Figure Legend Snippet: Linx -Related Topological Features Are Not Implicated in Xist Regulation (A) The Linx locus, CTCF binding, and orientation of CTCF motifs associated with CTCF chromatin immunoprecipitation sequencing (ChIP-seq) peaks. Orientation of CTCF motifs within the Tsix-TAD is represented above. The targeted deletions ΔLinxCBS (∼25 kb) and ΔLinx-int1 (∼51 kb) are indicated. See STAR Methods for sources of CTCF, DNaseI, and H3K27Ac datasets. (B and C) 5C profiles of the Tsix-TAD (B) and the two Xic TADs (C); pooled data from two biological replicates for each genotype. Differential map is corrected for deletion (see STAR Methods ). Gray pixels represent either the deleted region or filtered contacts. (D) Left: cross used for analysis of RNA allelic ratios in female hybrid embryos. Right: Xist RNA allelic ratios; each black dot corresponds to a single female embryo. Statistical analysis was performed using a two-tailed t test. The table summarizes the number of embryos collected. Analysis of Atp7a RNA allelic ratios and reverse cross is shown in Figures S5 A and S5B. (E and F) Capture-C profiles for LinxP (E) and Xist (F) viewpoints, at different time points of differentiation of XX (Pgk12.1) mESCs. Data represent one replicate; two or three replicates for each time point were performed and are identical to the one shown (data available in GEO). Profiles represent number of contacts for each DpnII fragment per 10,000 total contacts within a specified region (see STAR Methods ). CTCF ChIP-seq on male mESCs is represented below ( Nora et al., 2017 ). (G–I) 5C differential maps for mutant male mESCs: ΔLinxE (G), ΔLinxP (H) and LinxP-inv (I); pooled data from two biological replicates for each genotype. 5C profiles for each genotype are shown in Figure S5 D. Gray pixels correspond to either deleted regions or filtered contacts. (J) Quantification of 5C inter-TAD contacts (see Figure S5 E for details). Bars represent the average of the calculated proportions of four (E14 and ΔLinxP) or two (ΔLinxE and LinxP-inv) independent replicates. Statistical analysis was performed using a two-tailed t test ( ∗∗ p

    Techniques Used: Binding Assay, ChIP-sequencing, Chromatin Immunoprecipitation, Two Tailed Test, Capture-C, Mutagenesis

    LinxP Enhances Xist Expression In cis When Knocked In to the Xist-TAD (A and B) (Top) Location of the two knock-in cassettes, in between Jpx and Ftx (A) or in between Ftx and Xpct (B). (Bottom) Allelic quantification of Xist RNA at differentiation time points day 0, day 2, and day 4. Note that for each clone, the cassette was knocked in one allele only, and allelic ratios are shown for each clone relative to the knock-in allele. Data are presented as means, and error bars represent SEM (three biological replicates each). Statistical analysis was performed using a two-tailed paired t test ( ∗ p
    Figure Legend Snippet: LinxP Enhances Xist Expression In cis When Knocked In to the Xist-TAD (A and B) (Top) Location of the two knock-in cassettes, in between Jpx and Ftx (A) or in between Ftx and Xpct (B). (Bottom) Allelic quantification of Xist RNA at differentiation time points day 0, day 2, and day 4. Note that for each clone, the cassette was knocked in one allele only, and allelic ratios are shown for each clone relative to the knock-in allele. Data are presented as means, and error bars represent SEM (three biological replicates each). Statistical analysis was performed using a two-tailed paired t test ( ∗ p

    Techniques Used: Expressing, Knock-In, Two Tailed Test

    3) Product Images from "A symmetric toggle switch explains the onset of random X inactivation in different mammals"

    Article Title: A symmetric toggle switch explains the onset of random X inactivation in different mammals

    Journal: Nature structural & molecular biology

    doi: 10.1038/s41594-019-0214-1

    Predicted cis -acting feedback can be mediated by antisense transcription (a) Schematic representation of the model were Tsix acts as the predicted cis -acting repressor: RNA Pol II complexes can bind to the Tsix (blue) and Xist (green) promoters and then move along the gene in a convergent fashion. Mutual repression occurs at three levels: (1) Silencing of the Tsix promoter by Xist RNA, (2) repression of the Xist promoter by antisense transcription and (3) random removal of one Pol II complex, if two antisense Pol II complexes occupy the same DNA element. Black dotted lines indicate interactions removed in the reduced models in (d). Lighter colors and dotted nascent RNA indicate potential interruption of transcription through TI. (b-c) Stochastic simulation of Xist up-regulation for one example parameter set for the model shown in (a), showing three individual cells (b) and a population of 100 cells (c). Light and dark green in (b) represent Xist levels expressed from the two X chromosomes, light and dark grey in (c) represent mono- and bi-allelic Xist expression, as indicated. (d) Testing of model simplifications for the network in (a), where Xist and Tsix interact through one or two of the three repressive mechanisms as indicated. The percentage of parameter sets that can maintain the XaXi state (top) and that can initiate mono-allelic Xist up-regulation (bottom) in a stochastic simulation for each model are shown. Mono-allelic up-regulation was only tested for parameter sets that could maintain the XaXi state (others n.d.). Source data for panel d are available online.
    Figure Legend Snippet: Predicted cis -acting feedback can be mediated by antisense transcription (a) Schematic representation of the model were Tsix acts as the predicted cis -acting repressor: RNA Pol II complexes can bind to the Tsix (blue) and Xist (green) promoters and then move along the gene in a convergent fashion. Mutual repression occurs at three levels: (1) Silencing of the Tsix promoter by Xist RNA, (2) repression of the Xist promoter by antisense transcription and (3) random removal of one Pol II complex, if two antisense Pol II complexes occupy the same DNA element. Black dotted lines indicate interactions removed in the reduced models in (d). Lighter colors and dotted nascent RNA indicate potential interruption of transcription through TI. (b-c) Stochastic simulation of Xist up-regulation for one example parameter set for the model shown in (a), showing three individual cells (b) and a population of 100 cells (c). Light and dark green in (b) represent Xist levels expressed from the two X chromosomes, light and dark grey in (c) represent mono- and bi-allelic Xist expression, as indicated. (d) Testing of model simplifications for the network in (a), where Xist and Tsix interact through one or two of the three repressive mechanisms as indicated. The percentage of parameter sets that can maintain the XaXi state (top) and that can initiate mono-allelic Xist up-regulation (bottom) in a stochastic simulation for each model are shown. Mono-allelic up-regulation was only tested for parameter sets that could maintain the XaXi state (others n.d.). Source data for panel d are available online.

    Techniques Used: Expressing

    Bi-allelic Xist up-regulation is reversible (a) Schematic representation of the cell line used (top) and treatment performed (bottom) in (b-c). (b-c) In a simulation (b) and in an experiment (c) cells were treated with doxycycline one day before differentiation and the percentage of cells showing mono-allelic (left) and bi-allelic Xist up-regulation (right) is shown. (b) The simulation for one example parameter set is shown; the results for all tested parameter sets can be found in Fig. S3a . (c) Xist patterns were assessed by RNA FISH. Mean and s.d. of n=3 independent experiments are shown ( > 80 cells/replicate, for details see source data ). (d-i) Bi-allelic Xist up-regulation is artificially induced by treating TX1072dT cells (d) with doxycycline after 48h of differentiation. (e) The model predicts Xist down-regulation from the Cast chromosome, potentially with a transition through an Xa* state, where H3K27me3 (red) is still enriched, while Xist (green) has already been down-regulated. (f) Xist expression pattern at different time points after doxycycline addition as assessed by RNA FISH. Mean and s.d. of n=3 independent experiments are shown ( > 100 cells/replicate, for details see source data ). (g) Xist expression levels from the B6 and Cast alleles at different time points after doxycycline treatment as predicted by the simulation (top) and measured experimentally by allele-specific amplicon sequencing (bottom). In the simulation one example parameter set is shown; results for all other tested parameter sets can be found in supplementary Fig. S3b . (h-i) Immunofluorescence followed by RNA FISH to detect Xist and H3K27me3 48h after doxycycline induction. (i) Three states were quantified (XaXi, Xa*Xi, XiXi) as shown in the example image, scale bar indicates 5μm (h). Mean and s.d. of n=3 independent experiments are shown ( > 120 cells/replicate). *p
    Figure Legend Snippet: Bi-allelic Xist up-regulation is reversible (a) Schematic representation of the cell line used (top) and treatment performed (bottom) in (b-c). (b-c) In a simulation (b) and in an experiment (c) cells were treated with doxycycline one day before differentiation and the percentage of cells showing mono-allelic (left) and bi-allelic Xist up-regulation (right) is shown. (b) The simulation for one example parameter set is shown; the results for all tested parameter sets can be found in Fig. S3a . (c) Xist patterns were assessed by RNA FISH. Mean and s.d. of n=3 independent experiments are shown ( > 80 cells/replicate, for details see source data ). (d-i) Bi-allelic Xist up-regulation is artificially induced by treating TX1072dT cells (d) with doxycycline after 48h of differentiation. (e) The model predicts Xist down-regulation from the Cast chromosome, potentially with a transition through an Xa* state, where H3K27me3 (red) is still enriched, while Xist (green) has already been down-regulated. (f) Xist expression pattern at different time points after doxycycline addition as assessed by RNA FISH. Mean and s.d. of n=3 independent experiments are shown ( > 100 cells/replicate, for details see source data ). (g) Xist expression levels from the B6 and Cast alleles at different time points after doxycycline treatment as predicted by the simulation (top) and measured experimentally by allele-specific amplicon sequencing (bottom). In the simulation one example parameter set is shown; results for all other tested parameter sets can be found in supplementary Fig. S3b . (h-i) Immunofluorescence followed by RNA FISH to detect Xist and H3K27me3 48h after doxycycline induction. (i) Three states were quantified (XaXi, Xa*Xi, XiXi) as shown in the example image, scale bar indicates 5μm (h). Mean and s.d. of n=3 independent experiments are shown ( > 120 cells/replicate). *p

    Techniques Used: Fluorescence In Situ Hybridization, Expressing, Amplification, Sequencing, Immunofluorescence

    4) Product Images from "Hepatitis Delta Virus histone mimicry drives the recruitment of chromatin remodelers for viral RNA replication"

    Article Title: Hepatitis Delta Virus histone mimicry drives the recruitment of chromatin remodelers for viral RNA replication

    Journal: Nature Communications

    doi: 10.1038/s41467-020-14299-9

    The chromatin remodeling complexes BRF-1 and BRF-5 interact with the HDV RNP. a Interaction of wt S-HDAg with endogenous SNF2L, SNF2H, and BAZ2B. Nuclear extracts from Huh7 cells stably expressing wt S-HDAg or Huh7 cells were subjected to immunoprecipitation (IP) with α-HDAg (IP-HDAg), α-SNF2L (IP-SNF2L), α-SNF2H (IP-SNF2H), and αBAZ2B (IP-BAZ2B). Nonspecific antibody (IP-IgG) was used as a negative immunoprecipitation control. Input and elutions were analyzed by immunoblotting (IB) using the indicated antibodies. Input and HDAg lanes represent 10% and 20% volume compared with the other lanes, respectively. b Amplification of SNF2L variants from hepatocyte-derived cell lines. Left: PCR products demonstrating that both SNF2L and SNF2L(ex13) isoforms were observed in each cell line. Right: schematic representation of PCR products in the presence or absence of exon 13. Arrows represent primer positions. c , d HDV RNA immunoprecipitation assays (RIP). Glutaraldehyde-crosslinked nuclei from Huh7 cells transfected with the replication competent pSVLD3 plasmid ( c ) and PHHs infected with HDV (m.o.i. = 10) ( d ) were immunoprecipitated with antibodies directed against HDAg, RNA Pol II, SNF2L, SNF2H, and BAZ2B. HDV RNA was detected by qRT-PCR using HDV-specific primers. Histone H3 served as a negative RIP control. Error bars indicate SEMs from at least three independent experiments. e , f Genomic (g) or antigenomic (ag) HDV RIP assay. Glutaraldehyde-crosslinked nuclei from PHHs infected with HDV (m.o.i. = 10) were immunoprecipitated with antibodies directed against HDAg, RNA Pol II, SNF2L, SNF2H, BAZ2B, and the negative control H3. Genomic HDV ( e ) or ag HDV ( f ) RNA were detected by the biotinylated magnetic beads-based qRT-PCR assay using specific biotinylated ag HDV or g HDV primers, respectively. Values represent the mean ± SEM ( n = 4 with the exception of n = 2 for Pol II RIP). Source data are provided as a Source Data file.
    Figure Legend Snippet: The chromatin remodeling complexes BRF-1 and BRF-5 interact with the HDV RNP. a Interaction of wt S-HDAg with endogenous SNF2L, SNF2H, and BAZ2B. Nuclear extracts from Huh7 cells stably expressing wt S-HDAg or Huh7 cells were subjected to immunoprecipitation (IP) with α-HDAg (IP-HDAg), α-SNF2L (IP-SNF2L), α-SNF2H (IP-SNF2H), and αBAZ2B (IP-BAZ2B). Nonspecific antibody (IP-IgG) was used as a negative immunoprecipitation control. Input and elutions were analyzed by immunoblotting (IB) using the indicated antibodies. Input and HDAg lanes represent 10% and 20% volume compared with the other lanes, respectively. b Amplification of SNF2L variants from hepatocyte-derived cell lines. Left: PCR products demonstrating that both SNF2L and SNF2L(ex13) isoforms were observed in each cell line. Right: schematic representation of PCR products in the presence or absence of exon 13. Arrows represent primer positions. c , d HDV RNA immunoprecipitation assays (RIP). Glutaraldehyde-crosslinked nuclei from Huh7 cells transfected with the replication competent pSVLD3 plasmid ( c ) and PHHs infected with HDV (m.o.i. = 10) ( d ) were immunoprecipitated with antibodies directed against HDAg, RNA Pol II, SNF2L, SNF2H, and BAZ2B. HDV RNA was detected by qRT-PCR using HDV-specific primers. Histone H3 served as a negative RIP control. Error bars indicate SEMs from at least three independent experiments. e , f Genomic (g) or antigenomic (ag) HDV RIP assay. Glutaraldehyde-crosslinked nuclei from PHHs infected with HDV (m.o.i. = 10) were immunoprecipitated with antibodies directed against HDAg, RNA Pol II, SNF2L, SNF2H, BAZ2B, and the negative control H3. Genomic HDV ( e ) or ag HDV ( f ) RNA were detected by the biotinylated magnetic beads-based qRT-PCR assay using specific biotinylated ag HDV or g HDV primers, respectively. Values represent the mean ± SEM ( n = 4 with the exception of n = 2 for Pol II RIP). Source data are provided as a Source Data file.

    Techniques Used: Stable Transfection, Expressing, Immunoprecipitation, Amplification, Derivative Assay, Polymerase Chain Reaction, Transfection, Plasmid Preparation, Infection, Quantitative RT-PCR, Negative Control, Magnetic Beads

    5) Product Images from "Transcriptome alteration spectrum in rat lung induced by radiotherapy"

    Article Title: Transcriptome alteration spectrum in rat lung induced by radiotherapy

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-56027-4

    Data analysis process and quality control. ( A ) The rats were divided into three groups: radiotherapy for 4 weeks (nCon = 4, nexp = 5); radiotherapy for 8 weeks (nCon = 5, nexp = 4); radiotherapy for 16 weeks (nCon = 5, nexp = 5). Then rats’ lung tissues were processed with RNA extract, sequencing and function analysis; ( B ) The distribution of RNA-seq data in intron, exon and intergenic regions; ( C ) The distribution of gene expression values quantified by FPKM algorithm; ( D ) Heatmap of global mRNAs and lncRNAs in 28 clinical samples.
    Figure Legend Snippet: Data analysis process and quality control. ( A ) The rats were divided into three groups: radiotherapy for 4 weeks (nCon = 4, nexp = 5); radiotherapy for 8 weeks (nCon = 5, nexp = 4); radiotherapy for 16 weeks (nCon = 5, nexp = 5). Then rats’ lung tissues were processed with RNA extract, sequencing and function analysis; ( B ) The distribution of RNA-seq data in intron, exon and intergenic regions; ( C ) The distribution of gene expression values quantified by FPKM algorithm; ( D ) Heatmap of global mRNAs and lncRNAs in 28 clinical samples.

    Techniques Used: Sequencing, RNA Sequencing Assay, Expressing

    6) Product Images from "Alternatively spliced isoforms reveal a novel type of PTB domain in CCM2 protein"

    Article Title: Alternatively spliced isoforms reveal a novel type of PTB domain in CCM2 protein

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-52386-0

    The relative expression level and cellular stability between A and B group isoforms. ( A ) Comparison of endogenous expression levels between CCM2 isoform pairs among various tissues. The relative mRNA expression levels of paired-CCM2 isoforms (2 −∆CT ) were presented with bar plots, in which light grey bars represent A group isoforms, dark grey bars represent their respective counterparts, B group isoforms. For experimental design, the left three panels represent expression levels between CCM2 isoform pairs with primer set, CCM2-A100 and CCM2-B200; the right three panels for CCM2 isoform pairs with primer set, CCM2-A101 and CCM2-B201. For tissue location and cell lines, upper two panels represent the expression levels of paired-CCM2 isoforms among major tissues (see Fig. 2A ), middle two panels for various brain tissues, and lower two panels for multiple cell lines (see Suppl. Fig. 1 ) Middle and lower panels are further described in supplemental Fig. 1 . One-way ANOVA was also performed for the comparison between A and B groups of isoforms among different tissues and cells; it was found there is a very significant difference (P
    Figure Legend Snippet: The relative expression level and cellular stability between A and B group isoforms. ( A ) Comparison of endogenous expression levels between CCM2 isoform pairs among various tissues. The relative mRNA expression levels of paired-CCM2 isoforms (2 −∆CT ) were presented with bar plots, in which light grey bars represent A group isoforms, dark grey bars represent their respective counterparts, B group isoforms. For experimental design, the left three panels represent expression levels between CCM2 isoform pairs with primer set, CCM2-A100 and CCM2-B200; the right three panels for CCM2 isoform pairs with primer set, CCM2-A101 and CCM2-B201. For tissue location and cell lines, upper two panels represent the expression levels of paired-CCM2 isoforms among major tissues (see Fig. 2A ), middle two panels for various brain tissues, and lower two panels for multiple cell lines (see Suppl. Fig. 1 ) Middle and lower panels are further described in supplemental Fig. 1 . One-way ANOVA was also performed for the comparison between A and B groups of isoforms among different tissues and cells; it was found there is a very significant difference (P

    Techniques Used: Expressing

    Molecular interactions defined with yeast two-hybrid system. ( A ) Interactions between various NPXY-motif containing protein fragments and CCM2 PTB-less isoforms (CCM2-116, CCM2-107, and CCM2-212). CCM2-101 serves as positive control, while CCM2-1209 as negative control. pGAD-T with p53 is a system control. ( B ) Interactions between wild type (W) and mutated (M) three NPXY motifs of CCM1 (K2, K5, and K8) and a CCM2 PTB-less isoform (CCM2-116). CCM2-102 and full-length CCM2 PTB domain serves as positive control, while CCM2-1209 as negative control. ( C ) Interactions between protein fragments containing various number of NPXY-motifs and CCM2 PTB-less isoforms (CCM2-107, and CCM2-212). CCM2-206 serves as positive control, while CCM2-1209 as negative control. ( D ) Interactions between various NPXY-motif containing protein fragments and CCM2 exons (6, 6A, and 6B). CCM2-PTB serves as positive control, while pGAD as negative control. pGAD-T with p53 is a system control. ( E ) Interactions between wild type (W) and mutated (M) three NPXY motifs of CCM1 (K2, K5, and K8) and CCM2 exons (6, 6A, and 6B). Large-T serves as system control. ( F ) Interactions between wild type (W) and mutated (M) three NPXY motifs of CCM1 (K2, K5, and K8) and CCM2 exons (6, 6A, and 6B) and duplicate forms (2 × 6 and 2 × 6A). Large-T serves as system control. ( G ) Interactions between CCM3 protein and CCM2 exons (6, 6A, and 6B). Large-T with p53 serves as system control. ( H ) Competition assays between CCM3 protein with either CCM1-HK5 (containing 1 st NPXY motif) (upper panel) or CCM1-THK (containing 2 nd and 3 rd NPXY motif) (lower panel) binding to CCM2 exons (6, 6A, and 6B). β -galactosidase activity of each transformant was measured, normalized, and converted to relative β -galactosidase activity (RBGA). The normalized data were represented with means and standard deviations (M ± SD) generated from at least three independent assays (n = 3). RBGA +++ , ++ : significantly higher than that observed in any negative controls (P
    Figure Legend Snippet: Molecular interactions defined with yeast two-hybrid system. ( A ) Interactions between various NPXY-motif containing protein fragments and CCM2 PTB-less isoforms (CCM2-116, CCM2-107, and CCM2-212). CCM2-101 serves as positive control, while CCM2-1209 as negative control. pGAD-T with p53 is a system control. ( B ) Interactions between wild type (W) and mutated (M) three NPXY motifs of CCM1 (K2, K5, and K8) and a CCM2 PTB-less isoform (CCM2-116). CCM2-102 and full-length CCM2 PTB domain serves as positive control, while CCM2-1209 as negative control. ( C ) Interactions between protein fragments containing various number of NPXY-motifs and CCM2 PTB-less isoforms (CCM2-107, and CCM2-212). CCM2-206 serves as positive control, while CCM2-1209 as negative control. ( D ) Interactions between various NPXY-motif containing protein fragments and CCM2 exons (6, 6A, and 6B). CCM2-PTB serves as positive control, while pGAD as negative control. pGAD-T with p53 is a system control. ( E ) Interactions between wild type (W) and mutated (M) three NPXY motifs of CCM1 (K2, K5, and K8) and CCM2 exons (6, 6A, and 6B). Large-T serves as system control. ( F ) Interactions between wild type (W) and mutated (M) three NPXY motifs of CCM1 (K2, K5, and K8) and CCM2 exons (6, 6A, and 6B) and duplicate forms (2 × 6 and 2 × 6A). Large-T serves as system control. ( G ) Interactions between CCM3 protein and CCM2 exons (6, 6A, and 6B). Large-T with p53 serves as system control. ( H ) Competition assays between CCM3 protein with either CCM1-HK5 (containing 1 st NPXY motif) (upper panel) or CCM1-THK (containing 2 nd and 3 rd NPXY motif) (lower panel) binding to CCM2 exons (6, 6A, and 6B). β -galactosidase activity of each transformant was measured, normalized, and converted to relative β -galactosidase activity (RBGA). The normalized data were represented with means and standard deviations (M ± SD) generated from at least three independent assays (n = 3). RBGA +++ , ++ : significantly higher than that observed in any negative controls (P

    Techniques Used: Positive Control, Negative Control, Binding Assay, Activity Assay, Generated

    Genomic structure, conservation, and variability among alterative start-codon exons and promotors of CCM2. ( A ) The complex promoter regions of human CCM2 locus were defined with bioinformatics (promoter prediction software from top to bottom: Cister, promotor2.0, Softberry, MEME, CTCFBSDB, BDGP/NNPP and Genscan as indicated by different colors). Symbols on top of DNA templates are on positive strand, below are on negative strand. The single promoter for the original bonafide start-codon exon, exon 1, simply lies immediately upstream of the transcription start site for exon 1, as P0. The promoter region for exon 1A is much more complicated. Although a seemingly weak promoter, P1 lies immediately upstream of its transcription-start site; in addition, there are three relatively strong promoters (P2-P4) upstream adjacent to P1 promoter. Three exons (exon 1B, 1D, 1E) with the transcription start site driven by these three promoters (P2-P4), respectively, usually skip exon 1A (coding exon), presumably to down-regulate the transcription level of group B CCM2 isoforms. Genomic structure of 5′ region of the human CCM2 locus is schematically summarized in this map. Noncoding region within a transcription-start exon labeled as white box while coding region within the exon labeled as black box. ( B ) Multiple-alignment between two alterative start codon exons (exon 1 and exon 1A) across species reveals a vertebrate-specific exon 1 and a mammalian-specific exon 1A and their evolutional relationship. Exon 1A is evolutionarily evolved from exon1 with its C-terminus homolog to the N-terminus of exon1. ( C ) Phylogenetic relationships between exon 1A and exon 1 among CCM2 isoforms across species based on neighbor joining (NJ) method which hypothesizes a stochastic process in different lineages during evolution.
    Figure Legend Snippet: Genomic structure, conservation, and variability among alterative start-codon exons and promotors of CCM2. ( A ) The complex promoter regions of human CCM2 locus were defined with bioinformatics (promoter prediction software from top to bottom: Cister, promotor2.0, Softberry, MEME, CTCFBSDB, BDGP/NNPP and Genscan as indicated by different colors). Symbols on top of DNA templates are on positive strand, below are on negative strand. The single promoter for the original bonafide start-codon exon, exon 1, simply lies immediately upstream of the transcription start site for exon 1, as P0. The promoter region for exon 1A is much more complicated. Although a seemingly weak promoter, P1 lies immediately upstream of its transcription-start site; in addition, there are three relatively strong promoters (P2-P4) upstream adjacent to P1 promoter. Three exons (exon 1B, 1D, 1E) with the transcription start site driven by these three promoters (P2-P4), respectively, usually skip exon 1A (coding exon), presumably to down-regulate the transcription level of group B CCM2 isoforms. Genomic structure of 5′ region of the human CCM2 locus is schematically summarized in this map. Noncoding region within a transcription-start exon labeled as white box while coding region within the exon labeled as black box. ( B ) Multiple-alignment between two alterative start codon exons (exon 1 and exon 1A) across species reveals a vertebrate-specific exon 1 and a mammalian-specific exon 1A and their evolutional relationship. Exon 1A is evolutionarily evolved from exon1 with its C-terminus homolog to the N-terminus of exon1. ( C ) Phylogenetic relationships between exon 1A and exon 1 among CCM2 isoforms across species based on neighbor joining (NJ) method which hypothesizes a stochastic process in different lineages during evolution.

    Techniques Used: Software, Labeling

    7) Product Images from "Reactive oxygen species drive herpes simplex virus (HSV)-1-induced proinflammatory cytokine production by murine microglia"

    Article Title: Reactive oxygen species drive herpes simplex virus (HSV)-1-induced proinflammatory cytokine production by murine microglia

    Journal: Journal of Neuroinflammation

    doi: 10.1186/1742-2094-8-123

    ROS drive cytokine and chemokine mRNA expression in virus-infected microglia . Microglial cell cultures were pre-treated with the NADPH oxidase inhibitors DPI or APO for 1 h prior to a 5 h exposure to HSV. Following viral infection, RNA was extracted and cDNA synthesized to assess mRNA expression through quantitative real-time PCR for A) TNF-α; B) IL-1β; C) CCL2; and D) CXCL10. mRNA levels were normalized to the housekeeping gene HPRT and are presented as fold induction over uninfected controls. Data shown are representative of three individual experiments using microglial cells obtained from different animals.
    Figure Legend Snippet: ROS drive cytokine and chemokine mRNA expression in virus-infected microglia . Microglial cell cultures were pre-treated with the NADPH oxidase inhibitors DPI or APO for 1 h prior to a 5 h exposure to HSV. Following viral infection, RNA was extracted and cDNA synthesized to assess mRNA expression through quantitative real-time PCR for A) TNF-α; B) IL-1β; C) CCL2; and D) CXCL10. mRNA levels were normalized to the housekeeping gene HPRT and are presented as fold induction over uninfected controls. Data shown are representative of three individual experiments using microglial cells obtained from different animals.

    Techniques Used: Expressing, Infection, Synthesized, Real-time Polymerase Chain Reaction

    8) Product Images from "TET enzymes augment activation-induced deaminase (AID) expression via 5-hydroxymethylcytosine modifications at the Aicda superenhancer"

    Article Title: TET enzymes augment activation-induced deaminase (AID) expression via 5-hydroxymethylcytosine modifications at the Aicda superenhancer

    Journal: Science immunology

    doi: 10.1126/sciimmunol.aau7523

    TET proteins facilitate CSR. ( A ) Flowchart of in vivo experiment. f.p., footpad. ( B ) Top: Flow cytometric analysis of CD19 + GL7 + Fas + GC B cells at the draining popliteal lymph nodes from WT and Tet2/3 DKO mice after NP-OVA immunization as in (A). Bottom: Decreased IgG1-switched cells among GC B cells in Tet2/3 DKO (YFP + GC B–gated) compared with WT mice (GC B–gated). ( C to F ) Quantification of experiments shown in (B). Data shown are aggregated results from two independent experiments. Means and SEs are shown. WT, n = 11; DKO, n = 12. ( G ) Flowchart of in vitro IgG1 switching. Cells were labeled with Cell trace violet and activated for 4 days with LPS and IL-4. ( H and I ) Flow cytometry plots (H) and quantification (I) of IgG1-switched B cells in Tet2/3 DKO ( n = 4) and WT ( n = 4) mice. Representative of at least three independent experiments. ( J ) Circular γ1 transcript was quantified by qRT-PCR and normalized to Gapdh and then to the level of WT. Representative experiment of two independent experiments with three technical replicates. ( K ) Flowchart of in vitro IgA switching. Cells were activated for 5 days with anti-CD40, rmIL-4, rmIL-5, and rhTGFβ. ( L to N ) Flow cytometry plots (L) and quantification (M and N) of IgG1-switched (M) and IgA-switched cells (N). Representative of three independent experiments with three technical replicates. Statistical significance was calculated using unpaired two-tailed t test. * P
    Figure Legend Snippet: TET proteins facilitate CSR. ( A ) Flowchart of in vivo experiment. f.p., footpad. ( B ) Top: Flow cytometric analysis of CD19 + GL7 + Fas + GC B cells at the draining popliteal lymph nodes from WT and Tet2/3 DKO mice after NP-OVA immunization as in (A). Bottom: Decreased IgG1-switched cells among GC B cells in Tet2/3 DKO (YFP + GC B–gated) compared with WT mice (GC B–gated). ( C to F ) Quantification of experiments shown in (B). Data shown are aggregated results from two independent experiments. Means and SEs are shown. WT, n = 11; DKO, n = 12. ( G ) Flowchart of in vitro IgG1 switching. Cells were labeled with Cell trace violet and activated for 4 days with LPS and IL-4. ( H and I ) Flow cytometry plots (H) and quantification (I) of IgG1-switched B cells in Tet2/3 DKO ( n = 4) and WT ( n = 4) mice. Representative of at least three independent experiments. ( J ) Circular γ1 transcript was quantified by qRT-PCR and normalized to Gapdh and then to the level of WT. Representative experiment of two independent experiments with three technical replicates. ( K ) Flowchart of in vitro IgA switching. Cells were activated for 5 days with anti-CD40, rmIL-4, rmIL-5, and rhTGFβ. ( L to N ) Flow cytometry plots (L) and quantification (M and N) of IgG1-switched (M) and IgA-switched cells (N). Representative of three independent experiments with three technical replicates. Statistical significance was calculated using unpaired two-tailed t test. * P

    Techniques Used: In Vivo, Flow Cytometry, Mouse Assay, In Vitro, Labeling, Cytometry, Quantitative RT-PCR, Two Tailed Test

    TET2/3 facilitate CSR by regulating expression of the cytidine deaminase AID. ( A ) qRT-PCR analysis of Aicda mRNA expression in WT and Tet2/3 DKO B cells activated 4 days with LPS and IL-4. Aicda expression was normalized to Gapdh and then to the level in WT. Data shown are representative of two independent experiments with three technical replicates. * P
    Figure Legend Snippet: TET2/3 facilitate CSR by regulating expression of the cytidine deaminase AID. ( A ) qRT-PCR analysis of Aicda mRNA expression in WT and Tet2/3 DKO B cells activated 4 days with LPS and IL-4. Aicda expression was normalized to Gapdh and then to the level in WT. Data shown are representative of two independent experiments with three technical replicates. * P

    Techniques Used: Expressing, Quantitative RT-PCR

    9) Product Images from "Hepatitis Delta Virus histone mimicry drives the recruitment of chromatin remodelers for viral RNA replication"

    Article Title: Hepatitis Delta Virus histone mimicry drives the recruitment of chromatin remodelers for viral RNA replication

    Journal: Nature Communications

    doi: 10.1038/s41467-020-14299-9

    The chromatin remodeling complexes BRF-1 and BRF-5 interact with the HDV RNP. a Interaction of wt S-HDAg with endogenous SNF2L, SNF2H, and BAZ2B. Nuclear extracts from Huh7 cells stably expressing wt S-HDAg or Huh7 cells were subjected to immunoprecipitation (IP) with α-HDAg (IP-HDAg), α-SNF2L (IP-SNF2L), α-SNF2H (IP-SNF2H), and αBAZ2B (IP-BAZ2B). Nonspecific antibody (IP-IgG) was used as a negative immunoprecipitation control. Input and elutions were analyzed by immunoblotting (IB) using the indicated antibodies. Input and HDAg lanes represent 10% and 20% volume compared with the other lanes, respectively. b Amplification of SNF2L variants from hepatocyte-derived cell lines. Left: PCR products demonstrating that both SNF2L and SNF2L(ex13) isoforms were observed in each cell line. Right: schematic representation of PCR products in the presence or absence of exon 13. Arrows represent primer positions. c , d HDV RNA immunoprecipitation assays (RIP). Glutaraldehyde-crosslinked nuclei from Huh7 cells transfected with the replication competent pSVLD3 plasmid ( c ) and PHHs infected with HDV (m.o.i. = 10) ( d ) were immunoprecipitated with antibodies directed against HDAg, RNA Pol II, SNF2L, SNF2H, and BAZ2B. HDV RNA was detected by qRT-PCR using HDV-specific primers. Histone H3 served as a negative RIP control. Error bars indicate SEMs from at least three independent experiments. e , f Genomic (g) or antigenomic (ag) HDV RIP assay. Glutaraldehyde-crosslinked nuclei from PHHs infected with HDV (m.o.i. = 10) were immunoprecipitated with antibodies directed against HDAg, RNA Pol II, SNF2L, SNF2H, BAZ2B, and the negative control H3. Genomic HDV ( e ) or ag HDV ( f ) RNA were detected by the biotinylated magnetic beads-based qRT-PCR assay using specific biotinylated ag HDV or g HDV primers, respectively. Values represent the mean ± SEM ( n = 4 with the exception of n = 2 for Pol II RIP). Source data are provided as a Source Data file.
    Figure Legend Snippet: The chromatin remodeling complexes BRF-1 and BRF-5 interact with the HDV RNP. a Interaction of wt S-HDAg with endogenous SNF2L, SNF2H, and BAZ2B. Nuclear extracts from Huh7 cells stably expressing wt S-HDAg or Huh7 cells were subjected to immunoprecipitation (IP) with α-HDAg (IP-HDAg), α-SNF2L (IP-SNF2L), α-SNF2H (IP-SNF2H), and αBAZ2B (IP-BAZ2B). Nonspecific antibody (IP-IgG) was used as a negative immunoprecipitation control. Input and elutions were analyzed by immunoblotting (IB) using the indicated antibodies. Input and HDAg lanes represent 10% and 20% volume compared with the other lanes, respectively. b Amplification of SNF2L variants from hepatocyte-derived cell lines. Left: PCR products demonstrating that both SNF2L and SNF2L(ex13) isoforms were observed in each cell line. Right: schematic representation of PCR products in the presence or absence of exon 13. Arrows represent primer positions. c , d HDV RNA immunoprecipitation assays (RIP). Glutaraldehyde-crosslinked nuclei from Huh7 cells transfected with the replication competent pSVLD3 plasmid ( c ) and PHHs infected with HDV (m.o.i. = 10) ( d ) were immunoprecipitated with antibodies directed against HDAg, RNA Pol II, SNF2L, SNF2H, and BAZ2B. HDV RNA was detected by qRT-PCR using HDV-specific primers. Histone H3 served as a negative RIP control. Error bars indicate SEMs from at least three independent experiments. e , f Genomic (g) or antigenomic (ag) HDV RIP assay. Glutaraldehyde-crosslinked nuclei from PHHs infected with HDV (m.o.i. = 10) were immunoprecipitated with antibodies directed against HDAg, RNA Pol II, SNF2L, SNF2H, BAZ2B, and the negative control H3. Genomic HDV ( e ) or ag HDV ( f ) RNA were detected by the biotinylated magnetic beads-based qRT-PCR assay using specific biotinylated ag HDV or g HDV primers, respectively. Values represent the mean ± SEM ( n = 4 with the exception of n = 2 for Pol II RIP). Source data are provided as a Source Data file.

    Techniques Used: Stable Transfection, Expressing, Immunoprecipitation, Amplification, Derivative Assay, Polymerase Chain Reaction, Transfection, Plasmid Preparation, Infection, Quantitative RT-PCR, Negative Control, Magnetic Beads

    10) Product Images from "Functional Complementation of sir2Δ Yeast Mutation by the Human Orthologous Gene SIRT1"

    Article Title: Functional Complementation of sir2Δ Yeast Mutation by the Human Orthologous Gene SIRT1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0083114

    SIRT1 overexpression partially restores the transcriptional repression within specific loci in sir2Δ mutant. RT-PCR transcriptional analysis in WT and sir2Δ strains transformed with empty plasmid (-), SIRT1 construct (+) or SIRT1 - H363Y mutant construct (+*) both in repression (glucose) and induction (galactose) conditions. (A) rDNA locus: NTS1r and NTS2 ; (B) TEL VI locus: YFR057W and IRC7 ; (C) HM loci: HMLalpha1 . Histograms indicate averages and Std. Dev. bars from at least three independent biological replicates. Two−tailed t−test was applied for statistical analysis. Asterisks indicate statistically significant differences between sir2Δ - and sir2Δ + or between sir2Δ +* and sir2Δ + in galactose medium; α = 0.05. (Percentages of p−value: *p
    Figure Legend Snippet: SIRT1 overexpression partially restores the transcriptional repression within specific loci in sir2Δ mutant. RT-PCR transcriptional analysis in WT and sir2Δ strains transformed with empty plasmid (-), SIRT1 construct (+) or SIRT1 - H363Y mutant construct (+*) both in repression (glucose) and induction (galactose) conditions. (A) rDNA locus: NTS1r and NTS2 ; (B) TEL VI locus: YFR057W and IRC7 ; (C) HM loci: HMLalpha1 . Histograms indicate averages and Std. Dev. bars from at least three independent biological replicates. Two−tailed t−test was applied for statistical analysis. Asterisks indicate statistically significant differences between sir2Δ - and sir2Δ + or between sir2Δ +* and sir2Δ + in galactose medium; α = 0.05. (Percentages of p−value: *p

    Techniques Used: Over Expression, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Plasmid Preparation, Construct, Two Tailed Test

    11) Product Images from "MicroRNA-26b inhibits the immune response to Mycobacterium tuberculosis (M.tb) infection in THP-1 cells via targeting TGFβ-activated kinase-1 (TAK1), a promoter of the NF-κB pathway"

    Article Title: MicroRNA-26b inhibits the immune response to Mycobacterium tuberculosis (M.tb) infection in THP-1 cells via targeting TGFβ-activated kinase-1 (TAK1), a promoter of the NF-κB pathway

    Journal: International Journal of Clinical and Experimental Pathology

    doi:

    miR-26b is markedly down-regulated in M.tb infected THP-1. (A-D) THP-1 cells were infected with H37Rv at MOI 10. Supernatant was harvested at different time point and ELISA assay was conducted for measuring IFN-γ (A), IL-1β (B), IL-6 (C) and TNF-α (D) secretion. (E) Heat-map of microRNA array. (F-I) Cells were treated as (A-D) and were harvested at indicated time for RNA extraction. Real-time PCR assay was conducted to measure the levels of miR-26b (F), miR-124 (G), miR-16 (H) and miR-451 (I). Results represent of three independent experiments (mean ± SD). * P
    Figure Legend Snippet: miR-26b is markedly down-regulated in M.tb infected THP-1. (A-D) THP-1 cells were infected with H37Rv at MOI 10. Supernatant was harvested at different time point and ELISA assay was conducted for measuring IFN-γ (A), IL-1β (B), IL-6 (C) and TNF-α (D) secretion. (E) Heat-map of microRNA array. (F-I) Cells were treated as (A-D) and were harvested at indicated time for RNA extraction. Real-time PCR assay was conducted to measure the levels of miR-26b (F), miR-124 (G), miR-16 (H) and miR-451 (I). Results represent of three independent experiments (mean ± SD). * P

    Techniques Used: Infection, Enzyme-linked Immunosorbent Assay, RNA Extraction, Real-time Polymerase Chain Reaction

    12) Product Images from "MicroRNA-mRNA expression profiles and their potential role in cadmium stress response in Brassica napus"

    Article Title: MicroRNA-mRNA expression profiles and their potential role in cadmium stress response in Brassica napus

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-019-2189-9

    Lengths of small RNAs (sRNAs) and first base .s of mature miRNAs in 12 libraries (three biological replicates) constructed from shoot and root tissues of Brassica napus seedlings under 0 or 10 mg / mL cadmium stress for 10 days. a The proportion of sRNAs of different lengths in the 12 sequencing libraries. The Y-axis displays the number of sRNAs of a certain length, while the X-axis represents sRNAs of different lengths. b The first base preference of mature miRNAs in the 12 libraries. The Y-axis displays the proportion of mature miRNAs with a certain base type as the first base, and the X-axis represents the length classification of the sRNAs
    Figure Legend Snippet: Lengths of small RNAs (sRNAs) and first base .s of mature miRNAs in 12 libraries (three biological replicates) constructed from shoot and root tissues of Brassica napus seedlings under 0 or 10 mg / mL cadmium stress for 10 days. a The proportion of sRNAs of different lengths in the 12 sequencing libraries. The Y-axis displays the number of sRNAs of a certain length, while the X-axis represents sRNAs of different lengths. b The first base preference of mature miRNAs in the 12 libraries. The Y-axis displays the proportion of mature miRNAs with a certain base type as the first base, and the X-axis represents the length classification of the sRNAs

    Techniques Used: Construct, Sequencing

    13) Product Images from "Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts"

    Article Title: Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts

    Journal: Nature biotechnology

    doi: 10.1038/s41587-019-0090-6

    Abundant circRNA expression in different cell lines with fluorogenic aptamers. a , Broccoli is more readily detected by fluorescence microscopy when expressed as a circular RNA using the Tornado expression system. In HEK293T cells, the Tornado-expressed circular Broccoli signal is readily detected. At the same short exposure time, tricRNA Broccoli exhibits substantially lower fluorescence and linear Broccoli is barely detected. Representative cells showing fluorescence from linear or tricRNA Broccoli are highlighted with white arrows. Scale bar is 25 μm. b , The Tornado expression system enables efficient expression of the Corn aptamer in a circular form. HEK293T cells expressing Corn in a tRNA scaffold using a linear promoter, the tricY system, and the Tornado expression system were imaged using fluorescence microscopy after incubation of cells with the Corn-binding fluorogenic dye, DFHO. Scale bar is 25 μm. c , Quantification of Tornado-expressed circular RNA concentration in transfected cells. After three days of expressing Broccoli using Tornado (Tornado-Broccoli), total RNA from HepG2, HeLa, and HEK293T cells were harvested and separated by 10% PAGE alongside circular Broccoli standards (100, 10, 1, 0.1 ng). Normalizing for transfection efficiency, intracellular circular Broccoli concentrations were estimated to 1.6 μM for HepG2, 16 μM for HeLa, and 21 μM for HEK293T cells (see Methods ). d , The Tornado expression system results in efficient circularization of an RNA aptamer in a variety of cell lines. Three days after transfection with plasmids encoding linear Broccoli or circular Broccoli with the tricY system or with the Tornado expression system, cells (HepG2, HeLa, and COS-7) were imaged by fluorescence microscopy with DFHBI-1T. Cell nuclei are labeled with Hoechst stain. In all cell types observed, green fluorescence from Broccoli is readily detected only when expressed using the Tornado expression system. Scale bar is 25 μm. e , Tornado-expressed circular Broccoli is highly abundant. Broccoli was expressed using the Tornade expression system and other methods for generating circular in HEK293T cells. After separation of total RNA on a 6% PAGE gel, Broccoli-containing RNAs are detected by a DFHBI-1T gel stain, while total RNA was detected by SYBR Gold. Circular Broccoli bands are substantially increased when expressed using the Tornado expression system compared to all other methods. The Tornado-generated circular Broccoli band detected by SYBR Gold is as abundant as the 5.8S, 5S, and tRNA bands.
    Figure Legend Snippet: Abundant circRNA expression in different cell lines with fluorogenic aptamers. a , Broccoli is more readily detected by fluorescence microscopy when expressed as a circular RNA using the Tornado expression system. In HEK293T cells, the Tornado-expressed circular Broccoli signal is readily detected. At the same short exposure time, tricRNA Broccoli exhibits substantially lower fluorescence and linear Broccoli is barely detected. Representative cells showing fluorescence from linear or tricRNA Broccoli are highlighted with white arrows. Scale bar is 25 μm. b , The Tornado expression system enables efficient expression of the Corn aptamer in a circular form. HEK293T cells expressing Corn in a tRNA scaffold using a linear promoter, the tricY system, and the Tornado expression system were imaged using fluorescence microscopy after incubation of cells with the Corn-binding fluorogenic dye, DFHO. Scale bar is 25 μm. c , Quantification of Tornado-expressed circular RNA concentration in transfected cells. After three days of expressing Broccoli using Tornado (Tornado-Broccoli), total RNA from HepG2, HeLa, and HEK293T cells were harvested and separated by 10% PAGE alongside circular Broccoli standards (100, 10, 1, 0.1 ng). Normalizing for transfection efficiency, intracellular circular Broccoli concentrations were estimated to 1.6 μM for HepG2, 16 μM for HeLa, and 21 μM for HEK293T cells (see Methods ). d , The Tornado expression system results in efficient circularization of an RNA aptamer in a variety of cell lines. Three days after transfection with plasmids encoding linear Broccoli or circular Broccoli with the tricY system or with the Tornado expression system, cells (HepG2, HeLa, and COS-7) were imaged by fluorescence microscopy with DFHBI-1T. Cell nuclei are labeled with Hoechst stain. In all cell types observed, green fluorescence from Broccoli is readily detected only when expressed using the Tornado expression system. Scale bar is 25 μm. e , Tornado-expressed circular Broccoli is highly abundant. Broccoli was expressed using the Tornade expression system and other methods for generating circular in HEK293T cells. After separation of total RNA on a 6% PAGE gel, Broccoli-containing RNAs are detected by a DFHBI-1T gel stain, while total RNA was detected by SYBR Gold. Circular Broccoli bands are substantially increased when expressed using the Tornado expression system compared to all other methods. The Tornado-generated circular Broccoli band detected by SYBR Gold is as abundant as the 5.8S, 5S, and tRNA bands.

    Techniques Used: Expressing, Fluorescence, Microscopy, Incubation, Binding Assay, Concentration Assay, Transfection, Polyacrylamide Gel Electrophoresis, Labeling, Staining, Generated

    14) Product Images from "Primary microRNA transcripts are processed co-transcriptionally"

    Article Title: Primary microRNA transcripts are processed co-transcriptionally

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.1475

    Chromatin association of the Microprocessor complex. ( a ) using four different miRNA-specific probes and negative control probes for GAPDH and tRNA genes. Error bars show s.e.m. based on three independent experiments. ( b ) ChIP analysis of two endogenous intronic miR-330 and miR-25 miRNA sequences using Drosha and Pol II antibodies in HeLa cells. Maps of host EML2 and MCM7 genes are shown above the quantitative ChIP analysis. ( c ) Analysis of co-transcriptional recruitment of Drosha to the endogenous miR-223 gene following transcriptional activation by retinoic acid (RA) in NB4 cells. Northern blot analysis shows the progressive appearance of miR-223 following RA treatment. U2 snRNA was used as a positive control. This profile is consistent with increasing ChIP signals for Pol II and Drosha chromatin association. ( d ) miR-223 pcDNA plasmid constructs driven by the miR-23a promoter with (WT) or without (Δ) the 5′ single-stranded region of miR-223 (left) were transfected into HeLa cells, and total RNA was analyzed by northern blot (middle) or chromatin by Drosha ChIP (right). U2 and miR-23a probes provide positive controls.
    Figure Legend Snippet: Chromatin association of the Microprocessor complex. ( a ) using four different miRNA-specific probes and negative control probes for GAPDH and tRNA genes. Error bars show s.e.m. based on three independent experiments. ( b ) ChIP analysis of two endogenous intronic miR-330 and miR-25 miRNA sequences using Drosha and Pol II antibodies in HeLa cells. Maps of host EML2 and MCM7 genes are shown above the quantitative ChIP analysis. ( c ) Analysis of co-transcriptional recruitment of Drosha to the endogenous miR-223 gene following transcriptional activation by retinoic acid (RA) in NB4 cells. Northern blot analysis shows the progressive appearance of miR-223 following RA treatment. U2 snRNA was used as a positive control. This profile is consistent with increasing ChIP signals for Pol II and Drosha chromatin association. ( d ) miR-223 pcDNA plasmid constructs driven by the miR-23a promoter with (WT) or without (Δ) the 5′ single-stranded region of miR-223 (left) were transfected into HeLa cells, and total RNA was analyzed by northern blot (middle) or chromatin by Drosha ChIP (right). U2 and miR-23a probes provide positive controls.

    Techniques Used: Negative Control, Chromatin Immunoprecipitation, Activation Assay, Northern Blot, Positive Control, Plasmid Preparation, Construct, Transfection

    15) Product Images from "Functional and structural characterization of the chikungunya virus translational recoding signals"

    Article Title: Functional and structural characterization of the chikungunya virus translational recoding signals

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.005606

    Structural analyses of the CHIKV recoding signals. A–C , stimulatory elements for Af/As and Carib CHIKV TCR and −1 PRF signals resolved through SHAPE. RNA templates containing the translational recoding sequences were transcribed from corresponding Dual-Luciferase reporter vectors and treated with NMIA. Untreated RNAs were used as negative controls. γ- 32 P-radiolabeled cDNA products were separated by 8% urea-PAGE and visualized via a Fujifilm phosphorimaging system. Autoradiograms are annotated to indicate the respective sequencing lanes (G, A, U, and C), an untreated control lane (−), and the NMIA-labeled experimental lane (+). Circles denote the relative reactivity of bases where white is the most unreactive and black is the most reactive. For added visual clarity of the CHIKV TCR gels, a longer run of the samples has been provided to further separate the 5′ sequence information. D and F , structures of CHIKV recoding signals derived from the above SHAPE data. Circles correspond to the previously described nucleotide reactivity scale. Polymorphisms between the Af/As and Carib consensus sequences are indicated in red. E and G , three-dimensional models of the CHIKV TCR and −1 PRF signals were generated by molecular dynamics simulations and visualized in PyMOL.
    Figure Legend Snippet: Structural analyses of the CHIKV recoding signals. A–C , stimulatory elements for Af/As and Carib CHIKV TCR and −1 PRF signals resolved through SHAPE. RNA templates containing the translational recoding sequences were transcribed from corresponding Dual-Luciferase reporter vectors and treated with NMIA. Untreated RNAs were used as negative controls. γ- 32 P-radiolabeled cDNA products were separated by 8% urea-PAGE and visualized via a Fujifilm phosphorimaging system. Autoradiograms are annotated to indicate the respective sequencing lanes (G, A, U, and C), an untreated control lane (−), and the NMIA-labeled experimental lane (+). Circles denote the relative reactivity of bases where white is the most unreactive and black is the most reactive. For added visual clarity of the CHIKV TCR gels, a longer run of the samples has been provided to further separate the 5′ sequence information. D and F , structures of CHIKV recoding signals derived from the above SHAPE data. Circles correspond to the previously described nucleotide reactivity scale. Polymorphisms between the Af/As and Carib consensus sequences are indicated in red. E and G , three-dimensional models of the CHIKV TCR and −1 PRF signals were generated by molecular dynamics simulations and visualized in PyMOL.

    Techniques Used: Luciferase, Polyacrylamide Gel Electrophoresis, Sequencing, Labeling, Derivative Assay, Generated

    16) Product Images from "OsDCL1a activation impairs phytoalexin biosynthesis and compromises disease resistance in rice"

    Article Title: OsDCL1a activation impairs phytoalexin biosynthesis and compromises disease resistance in rice

    Journal: Annals of Botany

    doi: 10.1093/aob/mcy141

    Expression of OsDCL genes during infection with M. oryzae and treatment with fungal elicitors. (A) OsDCL1a expression at different times after inoculation with M. oryzae spores (1 × 10 5 spores mL –1 ) (left panel) or treatment with elicitors from this fungus (300 μg mL –1 ) (right panel) in wild-type plants. Black and red bars correspond to mock-inoculated and M. oryzae -inoculated (or elicitor-treated) plants, respectively. The expression level in mock-inoculated plants was set to 1.0. Three independent experiments (each with 24 plants per condition) were performed with similar results. Data are mean ± s.d. (* P ≤ 0.05; ** P ≤ 0.01 by ANOVA). (B) Expression of OsDCL family members at 72 h post-inoculation (hpi) with M. oryzae spores.
    Figure Legend Snippet: Expression of OsDCL genes during infection with M. oryzae and treatment with fungal elicitors. (A) OsDCL1a expression at different times after inoculation with M. oryzae spores (1 × 10 5 spores mL –1 ) (left panel) or treatment with elicitors from this fungus (300 μg mL –1 ) (right panel) in wild-type plants. Black and red bars correspond to mock-inoculated and M. oryzae -inoculated (or elicitor-treated) plants, respectively. The expression level in mock-inoculated plants was set to 1.0. Three independent experiments (each with 24 plants per condition) were performed with similar results. Data are mean ± s.d. (* P ≤ 0.05; ** P ≤ 0.01 by ANOVA). (B) Expression of OsDCL family members at 72 h post-inoculation (hpi) with M. oryzae spores.

    Techniques Used: Expressing, Infection

    17) Product Images from "Construction of Corynebacterium glutamicum cells as containers encapsulating dsRNA overexpressed for agricultural pest control"

    Article Title: Construction of Corynebacterium glutamicum cells as containers encapsulating dsRNA overexpressed for agricultural pest control

    Journal: Applied Microbiology and Biotechnology

    doi: 10.1007/s00253-019-10113-9

    Identification of diap1* -dsRNA produced in C. glutamicum . Structural analysis of the produced RNA with RNase A ( a ) and RNase III ( b ). Total RNA from C. glutamicum strain 2256LΔ rnc harboring pVC7H2 or pVH2-HvIap-1 was prepared, and then, each RNA sample treated with RNase (plus sign) or not treated (minus sign) was subjected to 6% PAGE. Prominent RNA bands corresponding to diap1* -dsRNA are indicated with black arrows. Size marker of dsRNAs is also shown
    Figure Legend Snippet: Identification of diap1* -dsRNA produced in C. glutamicum . Structural analysis of the produced RNA with RNase A ( a ) and RNase III ( b ). Total RNA from C. glutamicum strain 2256LΔ rnc harboring pVC7H2 or pVH2-HvIap-1 was prepared, and then, each RNA sample treated with RNase (plus sign) or not treated (minus sign) was subjected to 6% PAGE. Prominent RNA bands corresponding to diap1* -dsRNA are indicated with black arrows. Size marker of dsRNAs is also shown

    Techniques Used: Produced, Polyacrylamide Gel Electrophoresis, Marker

    Production of diap1* -dsRNA by C. glutamicum in batch fermentation. a Growth of C. glutamicum 2256LΔ rnc harboring pVC7H2 as a control or pVH2-HvIap-1. Average values and standard deviations (SDs) for three independent experiments are shown, although the variation range is too small to distinguish the SDs on the graph. b PAGE analysis of total RNA prepared from each C. glutamicum strain during the fermentation. Lane M shows dsRNA marker, and each culture time (h) is indicated at the top of the gel. The arrow and asterisks indicate the positions of diap1* -dsRNA and its possible degradation products, respectively. The result presents one representative diap1* -dsRNA production experiment in a jar fermentor
    Figure Legend Snippet: Production of diap1* -dsRNA by C. glutamicum in batch fermentation. a Growth of C. glutamicum 2256LΔ rnc harboring pVC7H2 as a control or pVH2-HvIap-1. Average values and standard deviations (SDs) for three independent experiments are shown, although the variation range is too small to distinguish the SDs on the graph. b PAGE analysis of total RNA prepared from each C. glutamicum strain during the fermentation. Lane M shows dsRNA marker, and each culture time (h) is indicated at the top of the gel. The arrow and asterisks indicate the positions of diap1* -dsRNA and its possible degradation products, respectively. The result presents one representative diap1* -dsRNA production experiment in a jar fermentor

    Techniques Used: Polyacrylamide Gel Electrophoresis, Marker

    18) Product Images from "KIS, a Kinase Associated with Microtubule Regulators, Enhances Translation of AMPA Receptors and Stimulates Dendritic Spine Remodeling"

    Article Title: KIS, a Kinase Associated with Microtubule Regulators, Enhances Translation of AMPA Receptors and Stimulates Dendritic Spine Remodeling

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1573-14.2014

    KIS promotes CPEB3 dissociation from GluR2 3′UTR and regulates its polyadenylation. A , Luciferase reporter analysis of the GluR2 3′UTR. HEK293T cells were transfected with the Gaussia luciferase ORF fused to the 3′UTR of GluR2, in combination with plasmid vectors expressing KIS, CPEB3, or CPEB3 + KIS. Values were made relative to those obtained from an empty vector (Ctrl). Mean values ( n = 6) and confidence limits (α = 0.05) for the mean are represented. Obtained p values for relevant pairwise t tests are shown. B , Effect of RNase treatment on the association of KIS to CPEB3. Total extracts (input) from HEK293T cells expressing CPEB3 and FLAG or FLAG-KIS were incubated with (+) or without (−) RNase prior to immunoprecipitation (IP), and the corresponding αFLAG immunoprecipitates (FLAG IP), were analyzed by immunoblotting to detect CPEB3 and FLAG-KIS proteins. C , RNA immunoprecipitation and quantitative PCR. HEK293T cells were transfected with the firefly luciferase ORF fused to the 3′UTR of GluR2, in combination with vectors expressing FLAG, FLAG-CPEB3, or FLAG-CPEB3 + KIS. Cell lysates were immunoprecipitated with FLAG IgG, and precipitated mRNAs were reverse transcribed and quantified by RT-qPCR. Luc-GluR2 3′UTR mRNA levels were made relative to GADPH mRNA. Mean values from three independent experiments and confidence limits (α = 0.05) for the mean are plotted. Obtained p values for relevant pairwise t tests are shown. D , Polyadenylation assays of the GluR2 3′UTR. Cell extracts from HEK293T cells as in C were used to amplify poly(A) (top) and total (bottom) Luc-GluR2 3′UTR mRNAs, and the resulting products were separated by agarose electrophoresis. E , Cell extracts as in D were analyzed by immunoblotting to detect CPEB3 and KIS proteins. F , Polyadenylation profiles obtained by densitometric analysis of samples as in D . G , Quantification of relative polyadenylation levels from profiles in F as poly(A)/nonpoly(A) ratios. Mean values ( n = 3) and confidence limits (α = 0.05) for the mean are represented. Obtained p values for relevant pairwise t tests are shown.
    Figure Legend Snippet: KIS promotes CPEB3 dissociation from GluR2 3′UTR and regulates its polyadenylation. A , Luciferase reporter analysis of the GluR2 3′UTR. HEK293T cells were transfected with the Gaussia luciferase ORF fused to the 3′UTR of GluR2, in combination with plasmid vectors expressing KIS, CPEB3, or CPEB3 + KIS. Values were made relative to those obtained from an empty vector (Ctrl). Mean values ( n = 6) and confidence limits (α = 0.05) for the mean are represented. Obtained p values for relevant pairwise t tests are shown. B , Effect of RNase treatment on the association of KIS to CPEB3. Total extracts (input) from HEK293T cells expressing CPEB3 and FLAG or FLAG-KIS were incubated with (+) or without (−) RNase prior to immunoprecipitation (IP), and the corresponding αFLAG immunoprecipitates (FLAG IP), were analyzed by immunoblotting to detect CPEB3 and FLAG-KIS proteins. C , RNA immunoprecipitation and quantitative PCR. HEK293T cells were transfected with the firefly luciferase ORF fused to the 3′UTR of GluR2, in combination with vectors expressing FLAG, FLAG-CPEB3, or FLAG-CPEB3 + KIS. Cell lysates were immunoprecipitated with FLAG IgG, and precipitated mRNAs were reverse transcribed and quantified by RT-qPCR. Luc-GluR2 3′UTR mRNA levels were made relative to GADPH mRNA. Mean values from three independent experiments and confidence limits (α = 0.05) for the mean are plotted. Obtained p values for relevant pairwise t tests are shown. D , Polyadenylation assays of the GluR2 3′UTR. Cell extracts from HEK293T cells as in C were used to amplify poly(A) (top) and total (bottom) Luc-GluR2 3′UTR mRNAs, and the resulting products were separated by agarose electrophoresis. E , Cell extracts as in D were analyzed by immunoblotting to detect CPEB3 and KIS proteins. F , Polyadenylation profiles obtained by densitometric analysis of samples as in D . G , Quantification of relative polyadenylation levels from profiles in F as poly(A)/nonpoly(A) ratios. Mean values ( n = 3) and confidence limits (α = 0.05) for the mean are represented. Obtained p values for relevant pairwise t tests are shown.

    Techniques Used: Luciferase, Transfection, Plasmid Preparation, Expressing, Incubation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Electrophoresis

    19) Product Images from "Primary microRNA transcripts are processed co-transcriptionally"

    Article Title: Primary microRNA transcripts are processed co-transcriptionally

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.1475

    Chromatin association of the Microprocessor complex. ( a ) using four different miRNA-specific probes and negative control probes for GAPDH and tRNA genes. Error bars show s.e.m. based on three independent experiments. ( b ) ChIP analysis of two endogenous intronic miR-330 and miR-25 miRNA sequences using Drosha and Pol II antibodies in HeLa cells. Maps of host EML2 and MCM7 genes are shown above the quantitative ChIP analysis. ( c ) Analysis of co-transcriptional recruitment of Drosha to the endogenous miR-223 gene following transcriptional activation by retinoic acid (RA) in NB4 cells. Northern blot analysis shows the progressive appearance of miR-223 following RA treatment. U2 snRNA was used as a positive control. This profile is consistent with increasing ChIP signals for Pol II and Drosha chromatin association. ( d ) miR-223 pcDNA plasmid constructs driven by the miR-23a promoter with (WT) or without (Δ) the 5′ single-stranded region of miR-223 (left) were transfected into HeLa cells, and total RNA was analyzed by northern blot (middle) or chromatin by Drosha ChIP (right). U2 and miR-23a probes provide positive controls.
    Figure Legend Snippet: Chromatin association of the Microprocessor complex. ( a ) using four different miRNA-specific probes and negative control probes for GAPDH and tRNA genes. Error bars show s.e.m. based on three independent experiments. ( b ) ChIP analysis of two endogenous intronic miR-330 and miR-25 miRNA sequences using Drosha and Pol II antibodies in HeLa cells. Maps of host EML2 and MCM7 genes are shown above the quantitative ChIP analysis. ( c ) Analysis of co-transcriptional recruitment of Drosha to the endogenous miR-223 gene following transcriptional activation by retinoic acid (RA) in NB4 cells. Northern blot analysis shows the progressive appearance of miR-223 following RA treatment. U2 snRNA was used as a positive control. This profile is consistent with increasing ChIP signals for Pol II and Drosha chromatin association. ( d ) miR-223 pcDNA plasmid constructs driven by the miR-23a promoter with (WT) or without (Δ) the 5′ single-stranded region of miR-223 (left) were transfected into HeLa cells, and total RNA was analyzed by northern blot (middle) or chromatin by Drosha ChIP (right). U2 and miR-23a probes provide positive controls.

    Techniques Used: Negative Control, Chromatin Immunoprecipitation, Activation Assay, Northern Blot, Positive Control, Plasmid Preparation, Construct, Transfection

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    Centrifugation:

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    SYBR Green Assay:

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    Incubation:

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    Expressing:

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    Modification:

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    Cell Culture:

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    Reverse Transcription Polymerase Chain Reaction:

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    Sequencing:

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    Recombinant:

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    RNA Sequencing Assay:

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    Isolation:

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    Article Snippet: Quantitative real‐time PCR (qRT‐PCR) assay of syrM gene expression Total RNA was isolated from bacteria on paper discs with TRIzol reagent (Invitrogen) following the manufacturer's instructions. .. Recombinant RNase‐free DNase I (Takara, Kusatsu, Japan) and SuperScript III Reverse Transcriptase (Invitrogen) were used to remove genomic DNA and synthesize the first‐strand cDNA respectively.

    Article Title: Functional Complementation of sir2Δ Yeast Mutation by the Human Orthologous Gene SIRT1
    Article Snippet: .. RT-PCR RNA from logarithmically growing cultures was isolated as previously described [ ].A 1.5-μg amount of DNase I–treated RNA was subjected to cDNA synthesis, starting from 2.5 μM oligo(dT) for evaluation of SIRT1, NTS1r, YFR057W, IRC7 and HMLα1 mRNA expression levels (50ng/μl Random hexamers at 25°C 10min for NTS2 ), by incubation with 30 U of SuperScript III Reverse Transcriptase (Invitrogen, Cat.No. ..

    Article Title: Alternatively spliced isoforms reveal a novel type of PTB domain in CCM2 protein
    Article Snippet: .. Isolation of novel exons, open reading frame, isoforms, and conserved domains in CCM2 gene Multi-tissue reference RNAs pool from four different suppliers (Super Array, BiotaQ, Biochain, NTomics) were used to amplify CCM2 cDNA fragments with SuperScript® III reverse transcriptase (Invitrogen). .. Rapid amplification of cDNA end (RACE) was applied for using either human brain Marathon-Ready cDNA or SMART RACE kit reagents (BD Clontech) to define full-length cDNA.

    Article Title: Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts
    Article Snippet: .. Isolated RNAs were reverse transcribed using SuperScript™ III (Thermo Fisher 18080093) and subsequently amplified by Taq DNA polymerase (New England Biolabs M02373) using convergent primers. .. Amplified DNA was visualized as a ladder of bands on agarose gels and specific bands were excised (see ).

    Negative Control:

    Article Title: Reactive oxygen species drive herpes simplex virus (HSV)-1-induced proinflammatory cytokine production by murine microglia
    Article Snippet: .. Reagents The following reagents were purchased from the indicated sources: Dulbecco's modified Eagle's medium (DMEM), Hanks' balanced salts (HBSS), penicillin, streptomycin, trypsin, Tween 20, phosphate buffered saline (PBS), poly-L-lysine, Tris, bovine serum albumin (BSA), diphenylene iodonium (DPI), apocynin (APO, Sigma-Aldrich, St. Louis, MO); Iba1 (ionized calcium binding adaptor molecule 1) and Mac-1 antibodies (BD Biosceneces, San Diego, CA); acrylamide/bis-acrylamide gel (Bio-Rad, Hercules, CA); CDP-Star substrate (Applied Biosystems, Foster City, CA); K-Blue substrate (Neogen, Lexington, KY); heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT); anti-p38 and -extracellular signal-regulated kinase 1 and 2 (ERK1/2 or p44/42) MAPK antibodies (Cell Signaling, Beverly, MA); recombinant murine interleukin (IL)-1β, tumor necrosis factor (TNF)-α, CCL2 CXCL10, anti-murine TNF-α, IL-1β, CCL2 and CXCL10 antibodies (R & D Systems, Minneapolis, MN); RNase inhibitor, SuperScript™ III reverse transcriptase (Invitrogen, Carlsbad, CA); DNase (Ambion, Austin, TX); random hexmer, and oligo (dT)12-18 (Gene Link, Hawthorne, NY); SYBR® Advantage® qPCR premix (ClonTech, Mountain View, CA); dNTPs (GE Healthcare, Piscataway, NJ); 2', 7' -dichlorodihydrofluorescein diacetate (H2 DCFDA), SB203580 (an inhibitor of p38 MAPK), SB202474 (a negative control for SB203580), U0126 (an inhibitor of MAP kinase kinase [MEK]1/2, upstream of ERK1/2), and U0124 (a negative control for U0126) (EMD Chemicals, Gibbstown, NJ). .. Animals Female and male BALB/c mice, 8 to 10 weeks old, were purchased from Charles River (Wilmington, MA).

    Purification:

    Article Title: Transcriptome alteration spectrum in rat lung induced by radiotherapy
    Article Snippet: Then 1 μg of total RNA was used with the TruSeq RNA library preparation kit (Illumina) in accordance with low-throughput protocol, except that SuperScript III reverse transcriptase (Invitrogen) was used to synthesize first strand cDNA. .. After PCR enrichment and purification of adapter-ligated fragments, the concentration of DNA with adapters was determined with quantitative PCR (Applied Biosystems 7,500).

    Article Title: Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts
    Article Snippet: Sequencing of expressed racRNAs RacRNAs were harvested from HEK239T cells with TRIzol™ LS Reagent (Invitrogen 10296010) and purified by excision from denaturing acrylamide gels. .. Isolated RNAs were reverse transcribed using SuperScript™ III (Thermo Fisher 18080093) and subsequently amplified by Taq DNA polymerase (New England Biolabs M02373) using convergent primers.

    Article Title: A symmetric toggle switch explains the onset of random X inactivation in different mammals
    Article Snippet: RNA was then purified according to the manufacturer's recommendations, including on-column DNAse digestion. .. For quantitative PCR (qPCR), 1ug RNA was reverse transcribed using Superscript III Reverse Transcriptase (Invitrogen).

    Polymerase Chain Reaction:

    Article Title: Transcriptome alteration spectrum in rat lung induced by radiotherapy
    Article Snippet: Then 1 μg of total RNA was used with the TruSeq RNA library preparation kit (Illumina) in accordance with low-throughput protocol, except that SuperScript III reverse transcriptase (Invitrogen) was used to synthesize first strand cDNA. .. After PCR enrichment and purification of adapter-ligated fragments, the concentration of DNA with adapters was determined with quantitative PCR (Applied Biosystems 7,500).

    Article Title: Hepatitis Delta Virus histone mimicry drives the recruitment of chromatin remodelers for viral RNA replication
    Article Snippet: .. SNF2L splice variant PCR assay Total RNA was reverse-transcribed to cDNA using Superscript™ III reverse transcriptase (Invitrogen) as per the manufacturer’s instructions. .. PCRs were performed by using specific primers (Supplementary Table ) and Q5® High-Fidelity DNA Polymerase (NEB, Cat#: M0491S).

    Article Title: Functional Complementation of sir2Δ Yeast Mutation by the Human Orthologous Gene SIRT1
    Article Snippet: RT-PCR RNA from logarithmically growing cultures was isolated as previously described [ ].A 1.5-μg amount of DNase I–treated RNA was subjected to cDNA synthesis, starting from 2.5 μM oligo(dT) for evaluation of SIRT1, NTS1r, YFR057W, IRC7 and HMLα1 mRNA expression levels (50ng/μl Random hexamers at 25°C 10min for NTS2 ), by incubation with 30 U of SuperScript III Reverse Transcriptase (Invitrogen, Cat.No. .. The resulting cDNAs were amplified by PCR co-amplification using the following primer pairs: SIRT1-F/SIRT1-R, NTS2-F/NTS2-R each with ACT1-450-F/ACT1-450-R; NTS1r-F/NTS1r-R, YFR057W-F/ YFR057W-R, IRC7-F/IRC7-R, HML1α-F/ HML1α-R each co-amplified with ACT1-182-F/ACT1-182-R. PCR was performed under the following conditions: 95°C for 30 s, 60°C for 30 s, and 68°C for 1 min, with 18 cycles for ACT1 , 24 cycles for SIRT1 , NTS1r and NTS2 , 27 cycles for YFR057W , IRC7 and HMLα1 .

    Article Title: Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts
    Article Snippet: Isolated RNAs were reverse transcribed using SuperScript™ III (Thermo Fisher 18080093) and subsequently amplified by Taq DNA polymerase (New England Biolabs M02373) using convergent primers. .. Excised DNA was cloned by TA cloning into the pCR™4-TOPO™ vector (Thermo Fisher K457502), and individual clones were sequenced.

    RNA Expression:

    Article Title: Transcriptome alteration spectrum in rat lung induced by radiotherapy
    Article Snippet: 2500 platform to obtain RNA expression profile data. .. Then 1 μg of total RNA was used with the TruSeq RNA library preparation kit (Illumina) in accordance with low-throughput protocol, except that SuperScript III reverse transcriptase (Invitrogen) was used to synthesize first strand cDNA.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Functional Complementation of sir2Δ Yeast Mutation by the Human Orthologous Gene SIRT1
    Article Snippet: .. RT-PCR RNA from logarithmically growing cultures was isolated as previously described [ ].A 1.5-μg amount of DNase I–treated RNA was subjected to cDNA synthesis, starting from 2.5 μM oligo(dT) for evaluation of SIRT1, NTS1r, YFR057W, IRC7 and HMLα1 mRNA expression levels (50ng/μl Random hexamers at 25°C 10min for NTS2 ), by incubation with 30 U of SuperScript III Reverse Transcriptase (Invitrogen, Cat.No. ..

    Plasmid Preparation:

    Article Title: Alternatively spliced isoforms reveal a novel type of PTB domain in CCM2 protein
    Article Snippet: Isolation of novel exons, open reading frame, isoforms, and conserved domains in CCM2 gene Multi-tissue reference RNAs pool from four different suppliers (Super Array, BiotaQ, Biochain, NTomics) were used to amplify CCM2 cDNA fragments with SuperScript® III reverse transcriptase (Invitrogen). .. The RACE and RT-PCR resulting products were sequenced and further analyzed with Vector NTI (Invitrogen) and CLC genomic workbench (Qiagen) to define the potential novel fragments and open reading frames (ORFs) as described before .

    Article Title: Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts
    Article Snippet: Isolated RNAs were reverse transcribed using SuperScript™ III (Thermo Fisher 18080093) and subsequently amplified by Taq DNA polymerase (New England Biolabs M02373) using convergent primers. .. Excised DNA was cloned by TA cloning into the pCR™4-TOPO™ vector (Thermo Fisher K457502), and individual clones were sequenced.

    Real-time Polymerase Chain Reaction:

    Article Title: Reactive oxygen species drive herpes simplex virus (HSV)-1-induced proinflammatory cytokine production by murine microglia
    Article Snippet: .. Reagents The following reagents were purchased from the indicated sources: Dulbecco's modified Eagle's medium (DMEM), Hanks' balanced salts (HBSS), penicillin, streptomycin, trypsin, Tween 20, phosphate buffered saline (PBS), poly-L-lysine, Tris, bovine serum albumin (BSA), diphenylene iodonium (DPI), apocynin (APO, Sigma-Aldrich, St. Louis, MO); Iba1 (ionized calcium binding adaptor molecule 1) and Mac-1 antibodies (BD Biosceneces, San Diego, CA); acrylamide/bis-acrylamide gel (Bio-Rad, Hercules, CA); CDP-Star substrate (Applied Biosystems, Foster City, CA); K-Blue substrate (Neogen, Lexington, KY); heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT); anti-p38 and -extracellular signal-regulated kinase 1 and 2 (ERK1/2 or p44/42) MAPK antibodies (Cell Signaling, Beverly, MA); recombinant murine interleukin (IL)-1β, tumor necrosis factor (TNF)-α, CCL2 CXCL10, anti-murine TNF-α, IL-1β, CCL2 and CXCL10 antibodies (R & D Systems, Minneapolis, MN); RNase inhibitor, SuperScript™ III reverse transcriptase (Invitrogen, Carlsbad, CA); DNase (Ambion, Austin, TX); random hexmer, and oligo (dT)12-18 (Gene Link, Hawthorne, NY); SYBR® Advantage® qPCR premix (ClonTech, Mountain View, CA); dNTPs (GE Healthcare, Piscataway, NJ); 2', 7' -dichlorodihydrofluorescein diacetate (H2 DCFDA), SB203580 (an inhibitor of p38 MAPK), SB202474 (a negative control for SB203580), U0126 (an inhibitor of MAP kinase kinase [MEK]1/2, upstream of ERK1/2), and U0124 (a negative control for U0126) (EMD Chemicals, Gibbstown, NJ). .. Animals Female and male BALB/c mice, 8 to 10 weeks old, were purchased from Charles River (Wilmington, MA).

    Article Title: Hepatitis Delta Virus histone mimicry drives the recruitment of chromatin remodelers for viral RNA replication
    Article Snippet: Total RNA (1 µg) was reverse-transcribed using Superscript III (Invitrogen, Cat#: 18080044) according to the manufacturer’s recommendations. .. Quantification of HDV cDNA was carried out by quantitative PCR (qPCR) with the PowerUp™ SYBR® Green Master mix (Applied Biosystems, Cat#: A25779 ) in the Roche LightCycler® 480 instrument.

    Article Title: A Conserved Noncoding Locus Regulates Random Monoallelic Xist Expression across a Topological Boundary
    Article Snippet: Reverse transcription cDNA was synthesized from 0.5 μg of RNA using SuperScript III Reverse Transcriptase and random primers (both Invitrogen) according to the manufacturer’s recommendations. .. Two independent reverse transcription experiments were carried out for each sample, pooled at the end and diluted 25-fold prior to qPCR or allelic expression analysis.

    Article Title: Transcriptome alteration spectrum in rat lung induced by radiotherapy
    Article Snippet: Then 1 μg of total RNA was used with the TruSeq RNA library preparation kit (Illumina) in accordance with low-throughput protocol, except that SuperScript III reverse transcriptase (Invitrogen) was used to synthesize first strand cDNA. .. After PCR enrichment and purification of adapter-ligated fragments, the concentration of DNA with adapters was determined with quantitative PCR (Applied Biosystems 7,500).

    Article Title: TET enzymes augment activation-induced deaminase (AID) expression via 5-hydroxymethylcytosine modifications at the Aicda superenhancer
    Article Snippet: .. Total RNA was isolated with RNeasy Plus Kit (Qiagen, Germany) or with Trizol (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturers’ instructions. cDNA was synthesized using SuperScript III reverse transcriptase (Thermo Fisher Scientific), and qRT-PCR was performed using FastStart Universal SYBR Green Master Mix (Roche, Germany) on a StepOnePlus Real-time PCR system (Thermo Fisher Scientific). .. Gene expression was normalized to Gapdh .

    Article Title: Pseudomonas bacteriocin syringacin M released upon desiccation suppresses the growth of sensitive bacteria in plant necrotic lesions
    Article Snippet: Paragraph title: Quantitative real‐time PCR (qRT‐PCR) assay of syrM gene expression ... Recombinant RNase‐free DNase I (Takara, Kusatsu, Japan) and SuperScript III Reverse Transcriptase (Invitrogen) were used to remove genomic DNA and synthesize the first‐strand cDNA respectively.

    Article Title: MicroRNA-26b inhibits the immune response to Mycobacterium tuberculosis (M.tb) infection in THP-1 cells via targeting TGFβ-activated kinase-1 (TAK1), a promoter of the NF-κB pathway
    Article Snippet: Paragraph title: Real-time PCR ... To quantify the level of gene expression, total RNAs were extracted using TRIzol (Invitrogen), according to the manufacturer’s instruction. cDNA was reverse transcribed from 1 μg total RNA by SuperScript III Reverse Transcriptase (Invitrogen).

    Article Title: A symmetric toggle switch explains the onset of random X inactivation in different mammals
    Article Snippet: .. For quantitative PCR (qPCR), 1ug RNA was reverse transcribed using Superscript III Reverse Transcriptase (Invitrogen). .. Expression levels were quantified using 2x SybRGreen Master Mix (Applied Biosystems) and a ViiA7 system (Applied biosystems) with ~8ng cDNA and the primers given in table S1.

    RNA Extraction:

    Article Title: TET enzymes augment activation-induced deaminase (AID) expression via 5-hydroxymethylcytosine modifications at the Aicda superenhancer
    Article Snippet: Paragraph title: RNA extraction, cDNA synthesis, and qRT-PCR ... Total RNA was isolated with RNeasy Plus Kit (Qiagen, Germany) or with Trizol (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturers’ instructions. cDNA was synthesized using SuperScript III reverse transcriptase (Thermo Fisher Scientific), and qRT-PCR was performed using FastStart Universal SYBR Green Master Mix (Roche, Germany) on a StepOnePlus Real-time PCR system (Thermo Fisher Scientific).

    Article Title: A symmetric toggle switch explains the onset of random X inactivation in different mammals
    Article Snippet: Paragraph title: RNA extraction, reverse transcription, qPCR ... For quantitative PCR (qPCR), 1ug RNA was reverse transcribed using Superscript III Reverse Transcriptase (Invitrogen).

    Binding Assay:

    Article Title: Reactive oxygen species drive herpes simplex virus (HSV)-1-induced proinflammatory cytokine production by murine microglia
    Article Snippet: .. Reagents The following reagents were purchased from the indicated sources: Dulbecco's modified Eagle's medium (DMEM), Hanks' balanced salts (HBSS), penicillin, streptomycin, trypsin, Tween 20, phosphate buffered saline (PBS), poly-L-lysine, Tris, bovine serum albumin (BSA), diphenylene iodonium (DPI), apocynin (APO, Sigma-Aldrich, St. Louis, MO); Iba1 (ionized calcium binding adaptor molecule 1) and Mac-1 antibodies (BD Biosceneces, San Diego, CA); acrylamide/bis-acrylamide gel (Bio-Rad, Hercules, CA); CDP-Star substrate (Applied Biosystems, Foster City, CA); K-Blue substrate (Neogen, Lexington, KY); heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT); anti-p38 and -extracellular signal-regulated kinase 1 and 2 (ERK1/2 or p44/42) MAPK antibodies (Cell Signaling, Beverly, MA); recombinant murine interleukin (IL)-1β, tumor necrosis factor (TNF)-α, CCL2 CXCL10, anti-murine TNF-α, IL-1β, CCL2 and CXCL10 antibodies (R & D Systems, Minneapolis, MN); RNase inhibitor, SuperScript™ III reverse transcriptase (Invitrogen, Carlsbad, CA); DNase (Ambion, Austin, TX); random hexmer, and oligo (dT)12-18 (Gene Link, Hawthorne, NY); SYBR® Advantage® qPCR premix (ClonTech, Mountain View, CA); dNTPs (GE Healthcare, Piscataway, NJ); 2', 7' -dichlorodihydrofluorescein diacetate (H2 DCFDA), SB203580 (an inhibitor of p38 MAPK), SB202474 (a negative control for SB203580), U0126 (an inhibitor of MAP kinase kinase [MEK]1/2, upstream of ERK1/2), and U0124 (a negative control for U0126) (EMD Chemicals, Gibbstown, NJ). .. Animals Female and male BALB/c mice, 8 to 10 weeks old, were purchased from Charles River (Wilmington, MA).

    Agarose Gel Electrophoresis:

    Article Title: Hepatitis Delta Virus histone mimicry drives the recruitment of chromatin remodelers for viral RNA replication
    Article Snippet: The concentration and purity of the RNA were verified using the Nanodrop spectrophotometer and ethidium bromide stained 18S/28S RNA bands were visualized using an ultraviolet trans -illuminator after agarose gel electrophoresis. .. Total RNA (1 µg) was reverse-transcribed using Superscript III (Invitrogen, Cat#: 18080044) according to the manufacturer’s recommendations.

    Article Title: Transcriptome alteration spectrum in rat lung induced by radiotherapy
    Article Snippet: The RNA was quantified using NanoDrop ND-1000 and assessed using a standard denaturing agarose gel electrophoresis assay. .. Then 1 μg of total RNA was used with the TruSeq RNA library preparation kit (Illumina) in accordance with low-throughput protocol, except that SuperScript III reverse transcriptase (Invitrogen) was used to synthesize first strand cDNA.

    Spectrophotometry:

    Article Title: Hepatitis Delta Virus histone mimicry drives the recruitment of chromatin remodelers for viral RNA replication
    Article Snippet: The concentration and purity of the RNA were verified using the Nanodrop spectrophotometer and ethidium bromide stained 18S/28S RNA bands were visualized using an ultraviolet trans -illuminator after agarose gel electrophoresis. .. Total RNA (1 µg) was reverse-transcribed using Superscript III (Invitrogen, Cat#: 18080044) according to the manufacturer’s recommendations.

    Concentration Assay:

    Article Title: Hepatitis Delta Virus histone mimicry drives the recruitment of chromatin remodelers for viral RNA replication
    Article Snippet: The concentration and purity of the RNA were verified using the Nanodrop spectrophotometer and ethidium bromide stained 18S/28S RNA bands were visualized using an ultraviolet trans -illuminator after agarose gel electrophoresis. .. Total RNA (1 µg) was reverse-transcribed using Superscript III (Invitrogen, Cat#: 18080044) according to the manufacturer’s recommendations.

    Article Title: Transcriptome alteration spectrum in rat lung induced by radiotherapy
    Article Snippet: Then 1 μg of total RNA was used with the TruSeq RNA library preparation kit (Illumina) in accordance with low-throughput protocol, except that SuperScript III reverse transcriptase (Invitrogen) was used to synthesize first strand cDNA. .. After PCR enrichment and purification of adapter-ligated fragments, the concentration of DNA with adapters was determined with quantitative PCR (Applied Biosystems 7,500).

    High Throughput Screening Assay:

    Article Title: Transcriptome alteration spectrum in rat lung induced by radiotherapy
    Article Snippet: RNA sequencing In order to explore the effects of radiotherapy in gene expression, rats’ total RNA was extracted and subjected to high-throughput sequencing based on Illumina HiSeq. .. Then 1 μg of total RNA was used with the TruSeq RNA library preparation kit (Illumina) in accordance with low-throughput protocol, except that SuperScript III reverse transcriptase (Invitrogen) was used to synthesize first strand cDNA.

    Staining:

    Article Title: Hepatitis Delta Virus histone mimicry drives the recruitment of chromatin remodelers for viral RNA replication
    Article Snippet: The concentration and purity of the RNA were verified using the Nanodrop spectrophotometer and ethidium bromide stained 18S/28S RNA bands were visualized using an ultraviolet trans -illuminator after agarose gel electrophoresis. .. Total RNA (1 µg) was reverse-transcribed using Superscript III (Invitrogen, Cat#: 18080044) according to the manufacturer’s recommendations.

    Variant Assay:

    Article Title: Hepatitis Delta Virus histone mimicry drives the recruitment of chromatin remodelers for viral RNA replication
    Article Snippet: .. SNF2L splice variant PCR assay Total RNA was reverse-transcribed to cDNA using Superscript™ III reverse transcriptase (Invitrogen) as per the manufacturer’s instructions. .. PCRs were performed by using specific primers (Supplementary Table ) and Q5® High-Fidelity DNA Polymerase (NEB, Cat#: M0491S).

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    Thermo Fisher superscript iii rt
    Construction and characterization of synZIKV-R2A reporter virus genomes. ( A ) Schematic representation of the synZIKV-R2A reporter virus genomes. For both strains the R2A reporter cassette (light red) was inserted into the wild-type pFK-synZIKV plasmids via MLuI/KpnI restriction sites. The NotI/NruI sites flanking the RLuc gene allow for the exchange of the reporter gene. ( B ) Immunofluorescence analysis of VeroE6 cells transfected with synZIKV-R2A in vitro transcripts. Cells were grown on coverslips, fixed 72 h and 96 h after transfection and stained with E-specific antibody (green). Nuclear DNA was counterstained with DAPI (grey). Scale bar = 15 μm. ( C ) Replication kinetics of the synZIKV-R2A reporter viruses in VeroE6 cells. After electroporation (EPO) cells were harvested at given time points and RLuc activity was determined. Values were normalized to the 4 h-value reflecting transfection efficiency. Mean ± SEM of <t>three</t> independent experiments is shown. Replication deficient mutants containing two mutations affecting the active site of the <t>RNA-dependent-RNA</t> polymerase in NS5 (GAA) served as negative controls. ( D ) VeroE6 cells were transfected with synZIKV-R2A RNAs, cell culture supernatants were collected 72 h post- transfection (P0) and passaged three times by infection of VeroE6 cells (P1-P3) in 72 h intervals. Culture supernatants obtained from each passage were used to inoculate Huh7 cells. In the case of supernatant obtained directly from transfected VeroE6 cells (P0), Huh7 cells were inoculated with undiluted (undil) or 1:10 diluted supernatant. After 72 h cells were harvested and RLuc activity in cell lysates was determined. Mean ± SEM from two independent experiments is shown. ( E ) Virus titres as determined by plaque assay for each synZIKV-R2A passage; values are mean ± SEM of two independent experiments. ( F ) Stability of the reporter gene. SynZIKV-R2A viruses released into culture supernatants were harvested after each passage as described in panel D, RNA was isolated and the region encompassing the RLuc coding sequence was amplified by using random hexamer primers for reverse transcription and specific primers for subsequent PCR. The ~1350 bp long DNA fragment in the P0 virus sample corresponds to the reporter gene, while the ~250 bp long fragment corresponds to the WT sequence. ( G ) Antiviral assay using synZIKV-R2A viruses. VeroE6 cells were inoculated with a 1:10 dilution of a P0 stock and one hour later the medium was replaced with DMEM containing the indicated amount of 2′CMC. RLuc activity was measured in cell lysates 72 h post-infection. Mean ± SEM from two independent experiments is shown.
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    Construction and characterization of synZIKV-R2A reporter virus genomes. ( A ) Schematic representation of the synZIKV-R2A reporter virus genomes. For both strains the R2A reporter cassette (light red) was inserted into the wild-type pFK-synZIKV plasmids via MLuI/KpnI restriction sites. The NotI/NruI sites flanking the RLuc gene allow for the exchange of the reporter gene. ( B ) Immunofluorescence analysis of VeroE6 cells transfected with synZIKV-R2A in vitro transcripts. Cells were grown on coverslips, fixed 72 h and 96 h after transfection and stained with E-specific antibody (green). Nuclear DNA was counterstained with DAPI (grey). Scale bar = 15 μm. ( C ) Replication kinetics of the synZIKV-R2A reporter viruses in VeroE6 cells. After electroporation (EPO) cells were harvested at given time points and RLuc activity was determined. Values were normalized to the 4 h-value reflecting transfection efficiency. Mean ± SEM of three independent experiments is shown. Replication deficient mutants containing two mutations affecting the active site of the RNA-dependent-RNA polymerase in NS5 (GAA) served as negative controls. ( D ) VeroE6 cells were transfected with synZIKV-R2A RNAs, cell culture supernatants were collected 72 h post- transfection (P0) and passaged three times by infection of VeroE6 cells (P1-P3) in 72 h intervals. Culture supernatants obtained from each passage were used to inoculate Huh7 cells. In the case of supernatant obtained directly from transfected VeroE6 cells (P0), Huh7 cells were inoculated with undiluted (undil) or 1:10 diluted supernatant. After 72 h cells were harvested and RLuc activity in cell lysates was determined. Mean ± SEM from two independent experiments is shown. ( E ) Virus titres as determined by plaque assay for each synZIKV-R2A passage; values are mean ± SEM of two independent experiments. ( F ) Stability of the reporter gene. SynZIKV-R2A viruses released into culture supernatants were harvested after each passage as described in panel D, RNA was isolated and the region encompassing the RLuc coding sequence was amplified by using random hexamer primers for reverse transcription and specific primers for subsequent PCR. The ~1350 bp long DNA fragment in the P0 virus sample corresponds to the reporter gene, while the ~250 bp long fragment corresponds to the WT sequence. ( G ) Antiviral assay using synZIKV-R2A viruses. VeroE6 cells were inoculated with a 1:10 dilution of a P0 stock and one hour later the medium was replaced with DMEM containing the indicated amount of 2′CMC. RLuc activity was measured in cell lysates 72 h post-infection. Mean ± SEM from two independent experiments is shown.

    Journal: Viruses

    Article Title: A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy

    doi: 10.3390/v10070368

    Figure Lengend Snippet: Construction and characterization of synZIKV-R2A reporter virus genomes. ( A ) Schematic representation of the synZIKV-R2A reporter virus genomes. For both strains the R2A reporter cassette (light red) was inserted into the wild-type pFK-synZIKV plasmids via MLuI/KpnI restriction sites. The NotI/NruI sites flanking the RLuc gene allow for the exchange of the reporter gene. ( B ) Immunofluorescence analysis of VeroE6 cells transfected with synZIKV-R2A in vitro transcripts. Cells were grown on coverslips, fixed 72 h and 96 h after transfection and stained with E-specific antibody (green). Nuclear DNA was counterstained with DAPI (grey). Scale bar = 15 μm. ( C ) Replication kinetics of the synZIKV-R2A reporter viruses in VeroE6 cells. After electroporation (EPO) cells were harvested at given time points and RLuc activity was determined. Values were normalized to the 4 h-value reflecting transfection efficiency. Mean ± SEM of three independent experiments is shown. Replication deficient mutants containing two mutations affecting the active site of the RNA-dependent-RNA polymerase in NS5 (GAA) served as negative controls. ( D ) VeroE6 cells were transfected with synZIKV-R2A RNAs, cell culture supernatants were collected 72 h post- transfection (P0) and passaged three times by infection of VeroE6 cells (P1-P3) in 72 h intervals. Culture supernatants obtained from each passage were used to inoculate Huh7 cells. In the case of supernatant obtained directly from transfected VeroE6 cells (P0), Huh7 cells were inoculated with undiluted (undil) or 1:10 diluted supernatant. After 72 h cells were harvested and RLuc activity in cell lysates was determined. Mean ± SEM from two independent experiments is shown. ( E ) Virus titres as determined by plaque assay for each synZIKV-R2A passage; values are mean ± SEM of two independent experiments. ( F ) Stability of the reporter gene. SynZIKV-R2A viruses released into culture supernatants were harvested after each passage as described in panel D, RNA was isolated and the region encompassing the RLuc coding sequence was amplified by using random hexamer primers for reverse transcription and specific primers for subsequent PCR. The ~1350 bp long DNA fragment in the P0 virus sample corresponds to the reporter gene, while the ~250 bp long fragment corresponds to the WT sequence. ( G ) Antiviral assay using synZIKV-R2A viruses. VeroE6 cells were inoculated with a 1:10 dilution of a P0 stock and one hour later the medium was replaced with DMEM containing the indicated amount of 2′CMC. RLuc activity was measured in cell lysates 72 h post-infection. Mean ± SEM from two independent experiments is shown.

    Article Snippet: Viral RNA was isolated from cell lysates using the NucleoSpin RNA II kit (Machery-Nagel, Düren, Germany) and reverse transcribed using SuperScript III RT (Thermo Fisher Scientific Waltham, MA, USA). cDNA was amplified by PCR and amplicons were sequenced by Sanger sequencing (GATC Biotech, Constance, Germany) using primers spanning the complete ZIKV genome.

    Techniques: Immunofluorescence, Transfection, In Vitro, Staining, Electroporation, Activity Assay, Cell Culture, Infection, Plaque Assay, Isolation, Sequencing, Amplification, Random Hexamer Labeling, Polymerase Chain Reaction, Antiviral Assay

    Quantification of transcript amounts of jhp0832 in H. pylori strains J99, J99-mut and the J99 mutants with point mutations within the GCGC motifs. qPCR results are represented in the right panel, three different biological replicates were performed. Statistics: One-way ANOVA, ** P

    Journal: Nucleic Acids Research

    Article Title: The core genome m5C methyltransferase JHP1050 (M.Hpy99III) plays an important role in orchestrating gene expression in Helicobacter pylori

    doi: 10.1093/nar/gky1307

    Figure Lengend Snippet: Quantification of transcript amounts of jhp0832 in H. pylori strains J99, J99-mut and the J99 mutants with point mutations within the GCGC motifs. qPCR results are represented in the right panel, three different biological replicates were performed. Statistics: One-way ANOVA, ** P

    Article Snippet: Quantitative PCR (qPCR) One μg of RNA was used for cDNA synthesis using the SuperScript™ III Reverse Transcriptase (Thermo Fisher Scientific, Darmstadt, Germany) as described before ( ). qPCR was performed with gene specific primers ( ) and SYBR Green Master Mix (Qiagen, Hilden, Germany).

    Techniques: Real-time Polymerase Chain Reaction

    SGMS1 is transcriptionally up-regulated in K562 cells. A , B ) SGMS1 ) were used to quantify hnRNA expression, normalized to β-actin and expressed as MNE. Primer locations are indicated on the SGMS1 locus (white boxes represent untranslated exons, black boxes represent translated exons, and lines represent introns). Negative control for cDNA synthesis (without superscript III) in K562 showed no amplification, thus verifying the absence of genomic DNA. Results from 3 independent experiments are shown. C ) K562 and HL-60 cells were treated with vehicle (H 2 O) or actinomycin D (5 μg/ml) over a 2-h time course. SGMS1 ) to quantify the percent of remaining mRNA. D ) K562 cells were treated with imatinib (1 μM) for 8 h. Cells were harvested and lysates were prepared for Western blot analysis. Total STAT5 and pSTAT5 levels were measured in control and treated cells. E ) In control and imatinib-treated cells, RNA was extracted to measure hnRNA levels of SGMS1. Intron VII and exon 8–specific RT-PCR primers were used. Results represent 3 independent experiments. ND, not detectable; SSIII, superscript III. * P

    Journal: The FASEB Journal

    Article Title: Bcr-Abl regulation of sphingomyelin synthase 1 reveals a novel oncogenic-driven mechanism of protein up-regulation

    doi: 10.1096/fj.201701016R

    Figure Lengend Snippet: SGMS1 is transcriptionally up-regulated in K562 cells. A , B ) SGMS1 ) were used to quantify hnRNA expression, normalized to β-actin and expressed as MNE. Primer locations are indicated on the SGMS1 locus (white boxes represent untranslated exons, black boxes represent translated exons, and lines represent introns). Negative control for cDNA synthesis (without superscript III) in K562 showed no amplification, thus verifying the absence of genomic DNA. Results from 3 independent experiments are shown. C ) K562 and HL-60 cells were treated with vehicle (H 2 O) or actinomycin D (5 μg/ml) over a 2-h time course. SGMS1 ) to quantify the percent of remaining mRNA. D ) K562 cells were treated with imatinib (1 μM) for 8 h. Cells were harvested and lysates were prepared for Western blot analysis. Total STAT5 and pSTAT5 levels were measured in control and treated cells. E ) In control and imatinib-treated cells, RNA was extracted to measure hnRNA levels of SGMS1. Intron VII and exon 8–specific RT-PCR primers were used. Results represent 3 independent experiments. ND, not detectable; SSIII, superscript III. * P

    Article Snippet: Dephosphorylated and uncapped mRNA was added to the GeneRacer RNA Oligo (provided in the kit) and incubated with T4 RNA ligase at 37°C for 1 h. The uncapped, full-length mRNA that was ligated to the GeneRacer RNA oligo (provided in the kit) was used for reverse transcription of mRNA into cDNA using Superscript III reverse transcriptase and random primers according to the standard protocol (Thermo Fisher Scientific).

    Techniques: Expressing, Negative Control, Amplification, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Overexpression of wild-type or inactive ClpP proteins has varied effects on Chlamydia species. C. trachomatis serovar L2 was transformed with aTc-inducible shuttle vectors encoding either wild-type or active-site mutants of each ClpP paralog with a 6×His tag at the C terminus. HEp2 cells were infected with each transformant, and expression was induced at 10 h postinfection (hpi). (A) ClpP1 wild-type and S92A mutant overexpression assay. Overexpression of inactive ClpP1 has a negative impact on the bacteria. (B) ClpP2 wild-type and S98A mutant overexpression assay. Parameters were the same as those described above. Overexpression does not appear to negatively affect Chlamydia species. Samples were stained for major outer membrane protein (MOMP; green), 6×His-tagged ClpP protein of interest (red), and DNA (blue). Representative images of three independent experiments are presented. Scale bars are equal to 10 μm. Images were acquired on a Zeiss LSM 800 laser scanning confocal microscope with a 60× objective and a 3× digital magnification. (C) Inclusion-forming unit (IFU) assay measuring the effect of increasing levels of ClpP protein induction on chlamydial growth. Values are averages of the results from three independent experiments and are reported as a percentage of the respective uninduced sample. *, P

    Journal: Journal of Bacteriology

    Article Title: Initial Characterization of the Two ClpP Paralogs of Chlamydia trachomatis Suggests Unique Functionality for Each

    doi: 10.1128/JB.00635-18

    Figure Lengend Snippet: Overexpression of wild-type or inactive ClpP proteins has varied effects on Chlamydia species. C. trachomatis serovar L2 was transformed with aTc-inducible shuttle vectors encoding either wild-type or active-site mutants of each ClpP paralog with a 6×His tag at the C terminus. HEp2 cells were infected with each transformant, and expression was induced at 10 h postinfection (hpi). (A) ClpP1 wild-type and S92A mutant overexpression assay. Overexpression of inactive ClpP1 has a negative impact on the bacteria. (B) ClpP2 wild-type and S98A mutant overexpression assay. Parameters were the same as those described above. Overexpression does not appear to negatively affect Chlamydia species. Samples were stained for major outer membrane protein (MOMP; green), 6×His-tagged ClpP protein of interest (red), and DNA (blue). Representative images of three independent experiments are presented. Scale bars are equal to 10 μm. Images were acquired on a Zeiss LSM 800 laser scanning confocal microscope with a 60× objective and a 3× digital magnification. (C) Inclusion-forming unit (IFU) assay measuring the effect of increasing levels of ClpP protein induction on chlamydial growth. Values are averages of the results from three independent experiments and are reported as a percentage of the respective uninduced sample. *, P

    Article Snippet: DNA was removed from total RNA by rigorous DNA-free treatment (Thermo Fisher) before 1 µg was reverse transcribed with Superscript III reverse transcriptase (RT; Thermo Fisher).

    Techniques: Over Expression, Transformation Assay, Infection, Expressing, Mutagenesis, Staining, Microscopy