rna  (Qiagen)

 
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    Name:
    RNase Free DNase Set
    Description:
    For DNase digestion during RNA purification Kit contents Qiagen RNase free DNase Set 50 preps For DNase Digestion During RNA Purification Silica gel Membrane Spin column Technology Efficiently Removes the Majority of the DNA Without DNase Treatment The Buffer is Also Well suited for Efficient DNase Digestion in Solution Includes 1500U RNase free DNase I RNase free Buffer RDD and RNase free Water
    Catalog Number:
    79254
    Price:
    108
    Category:
    RNase Free DNase Set
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    Structured Review

    Qiagen rna
    RNase Free DNase Set
    For DNase digestion during RNA purification Kit contents Qiagen RNase free DNase Set 50 preps For DNase Digestion During RNA Purification Silica gel Membrane Spin column Technology Efficiently Removes the Majority of the DNA Without DNase Treatment The Buffer is Also Well suited for Efficient DNase Digestion in Solution Includes 1500U RNase free DNase I RNase free Buffer RDD and RNase free Water
    https://www.bioz.com/result/rna/product/Qiagen
    Average 96 stars, based on 44499 article reviews
    Price from $9.99 to $1999.99
    rna - by Bioz Stars, 2020-08
    96/100 stars

    Images

    1) Product Images from "Mlc Is a Transcriptional Activator with a Key Role in Integrating Cyclic AMP Receptor Protein and Integration Host Factor Regulation of Leukotoxin RNA Synthesis in Aggregatibacter actinomycetemcomitans"

    Article Title: Mlc Is a Transcriptional Activator with a Key Role in Integrating Cyclic AMP Receptor Protein and Integration Host Factor Regulation of Leukotoxin RNA Synthesis in Aggregatibacter actinomycetemcomitans

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.02144-12

    An mlc mutant makes less leukotoxin protein and RNA. Samples were taken at log phase from aerobically (+) and anaerobically (−) grown cultures of the parent strain (JP2) and Δ mlc (AAM155) cells as well as the deletion strain with a plasmid carrying mlc + (the Δ mlc / mlc + strain). Cells were harvested by centrifugation, resuspended in SDS-PAGE loading buffer, boiled, and centrifuged, and the soluble material was electrophoresed on duplicate gels. (A) Gel stained with Coomassie brilliant blue. (B) Western blot analysis of total cell protein from the indicated strains using rabbit antileukotoxin antibody as the primary antibody. The arrows mark the position of leukotoxin. The numbers show the positions of molecular size markers (not shown) in kDa. (C) For each sample, total cell RNA was prepared from anaerobically grown log phase cells, and cDNA was synthesized with random hexamer primers and then used in quantitative RT-PCR. The level of ltxA mRNA in each sample is normalized to the level of pdxY mRNA in the same sample. The data are the average of the expression ratios from three biological replicates. The error bars are standard error, and the difference between the two samples is statistically significant ( P ≤ 0.05, by Student's t test).
    Figure Legend Snippet: An mlc mutant makes less leukotoxin protein and RNA. Samples were taken at log phase from aerobically (+) and anaerobically (−) grown cultures of the parent strain (JP2) and Δ mlc (AAM155) cells as well as the deletion strain with a plasmid carrying mlc + (the Δ mlc / mlc + strain). Cells were harvested by centrifugation, resuspended in SDS-PAGE loading buffer, boiled, and centrifuged, and the soluble material was electrophoresed on duplicate gels. (A) Gel stained with Coomassie brilliant blue. (B) Western blot analysis of total cell protein from the indicated strains using rabbit antileukotoxin antibody as the primary antibody. The arrows mark the position of leukotoxin. The numbers show the positions of molecular size markers (not shown) in kDa. (C) For each sample, total cell RNA was prepared from anaerobically grown log phase cells, and cDNA was synthesized with random hexamer primers and then used in quantitative RT-PCR. The level of ltxA mRNA in each sample is normalized to the level of pdxY mRNA in the same sample. The data are the average of the expression ratios from three biological replicates. The error bars are standard error, and the difference between the two samples is statistically significant ( P ≤ 0.05, by Student's t test).

    Techniques Used: Mutagenesis, Plasmid Preparation, Centrifugation, SDS Page, Staining, Western Blot, Synthesized, Random Hexamer Labeling, Quantitative RT-PCR, Expressing

    The genetic interactions of IHF, CRP, and Mlc in altering leukotoxin transcription. After each sample was grown to log phase, total cell RNA was isolated, and cDNA was prepared with random hexamer primers and then used in quantitative RT-PCR. The level of ltxA mRNA in each sample is normalized to the level of pdxY mRNA in the same sample. The data are the average of the expression ratios from three biological replicates. The error bars are standard error. A single asterisk indicates samples for which the ratio of ltxA mRNA to pdxY mRNA is statistically significantly ( P
    Figure Legend Snippet: The genetic interactions of IHF, CRP, and Mlc in altering leukotoxin transcription. After each sample was grown to log phase, total cell RNA was isolated, and cDNA was prepared with random hexamer primers and then used in quantitative RT-PCR. The level of ltxA mRNA in each sample is normalized to the level of pdxY mRNA in the same sample. The data are the average of the expression ratios from three biological replicates. The error bars are standard error. A single asterisk indicates samples for which the ratio of ltxA mRNA to pdxY mRNA is statistically significantly ( P

    Techniques Used: Immunohistofluorescence, Isolation, Random Hexamer Labeling, Quantitative RT-PCR, Expressing

    Quantitative RT-PCR of BamHI mutations define key regulatory elements within the leukotoxin promoter. Each of the indicated strains was grown anaerobically (A) or aerobically (B) to log phase, total RNA was isolated, and cDNA was prepared with random hexamer primers and then used in quantitative RT-PCR with ltxA - and pdxY -specific primers. The levels of ltxA mRNA in a single sample were normalized to pdxY (internal control) levels in the same sample. The value for a given strain is the average of the ratios of the ltxA mRNA to pdxY mRNA levels from three biological replicates (samples) of that strain. The error bars are the sample standard deviation. A single asterisk indicates a sample for which the ratio of ltxA mRNA to pdxY mRNA is statistically significantly ( P
    Figure Legend Snippet: Quantitative RT-PCR of BamHI mutations define key regulatory elements within the leukotoxin promoter. Each of the indicated strains was grown anaerobically (A) or aerobically (B) to log phase, total RNA was isolated, and cDNA was prepared with random hexamer primers and then used in quantitative RT-PCR with ltxA - and pdxY -specific primers. The levels of ltxA mRNA in a single sample were normalized to pdxY (internal control) levels in the same sample. The value for a given strain is the average of the ratios of the ltxA mRNA to pdxY mRNA levels from three biological replicates (samples) of that strain. The error bars are the sample standard deviation. A single asterisk indicates a sample for which the ratio of ltxA mRNA to pdxY mRNA is statistically significantly ( P

    Techniques Used: Quantitative RT-PCR, Isolation, Random Hexamer Labeling, Standard Deviation

    2) Product Images from "Characterization of human plasma-derived exosomal RNAs by deep sequencing"

    Article Title: Characterization of human plasma-derived exosomal RNAs by deep sequencing

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-14-319

    Other RNA species that were detected in the exosomal RNA libraries. ( A ) Pie chart of RNA species and their distributions in the plasma-derived exosomes. Misc RNAs are the RNA sequences that mapped to the human genome but not in any of the categories listed. The DNA category represents the novel transcripts that have no annotation in the human RNA database. ( B ) Graphic and statistics of a representative novel miRNA predicted by miRDeep2. Both star and mature strands were detected and integrated. Lower left table shows information about the sample and the miRDeep2 scores, along with the read count for each component of the putative miRNA. mm, number of mismatches. Mismatched nucleotides are indicated by uppercase letters.
    Figure Legend Snippet: Other RNA species that were detected in the exosomal RNA libraries. ( A ) Pie chart of RNA species and their distributions in the plasma-derived exosomes. Misc RNAs are the RNA sequences that mapped to the human genome but not in any of the categories listed. The DNA category represents the novel transcripts that have no annotation in the human RNA database. ( B ) Graphic and statistics of a representative novel miRNA predicted by miRDeep2. Both star and mature strands were detected and integrated. Lower left table shows information about the sample and the miRDeep2 scores, along with the read count for each component of the putative miRNA. mm, number of mismatches. Mismatched nucleotides are indicated by uppercase letters.

    Techniques Used: Derivative Assay

    3) Product Images from "Comparative Genomics of Emerging Human Ehrlichiosis Agents "

    Article Title: Comparative Genomics of Emerging Human Ehrlichiosis Agents

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.0020021

    Synteny of the Rickettsiales Regions of conserved synteny were identified using the ortholog clusters (see Materials and Methods ) and visualized with Sybil. The genes along each ordered chromosome were colored on a gradient from yellow to blue. The ortholog clusters for each query genome were then plotted relative to the order of the reference genome. Regions of synteny are then seen as continuous gradients across large regions of the genome. Above the synteny gradient display is the atypical nucleotide composition. Below the gradient display are the predicted coding regions on the plus strand and the minus strand, and the GC-skew. Representatives of all the Rickettsiales (A) and representative Ehrlichia spp. and Anaplasma spp. (B) were compared separately. AMA, A. marginale St. Maries; APH, A. phagocytophilum HZ; ECH, E. chaffeensis Arkansas; ERU, E. ruminantium Welgevonden; NES, N. sennetsu Miyayama; RPR, R. prowazekii Madrid E; WOL, W. pipientis w Mel.
    Figure Legend Snippet: Synteny of the Rickettsiales Regions of conserved synteny were identified using the ortholog clusters (see Materials and Methods ) and visualized with Sybil. The genes along each ordered chromosome were colored on a gradient from yellow to blue. The ortholog clusters for each query genome were then plotted relative to the order of the reference genome. Regions of synteny are then seen as continuous gradients across large regions of the genome. Above the synteny gradient display is the atypical nucleotide composition. Below the gradient display are the predicted coding regions on the plus strand and the minus strand, and the GC-skew. Representatives of all the Rickettsiales (A) and representative Ehrlichia spp. and Anaplasma spp. (B) were compared separately. AMA, A. marginale St. Maries; APH, A. phagocytophilum HZ; ECH, E. chaffeensis Arkansas; ERU, E. ruminantium Welgevonden; NES, N. sennetsu Miyayama; RPR, R. prowazekii Madrid E; WOL, W. pipientis w Mel.

    Techniques Used:

    Comparison of the Rickettsiales Gene Sets The composition of ortholog clusters (see Materials and Methods ) of representative Rickettsiales (A), Ehrlichia spp. (B), and Anaplasma spp. (C) were compared. Numbers within the intersections of different ovals indicate ortholog clusters shared by 2, 3, 4, or 5 organisms. Species compared are indicated in diagram intersections as follows. A, R. prowazekii; B, N. sennetsu; C, W. pipientis; D, A. phagocytophilum; E, E. chaffeensis; F, A. marginale; G, E. ruminantium Gardel; and H, E. ruminantium Welgevonden.
    Figure Legend Snippet: Comparison of the Rickettsiales Gene Sets The composition of ortholog clusters (see Materials and Methods ) of representative Rickettsiales (A), Ehrlichia spp. (B), and Anaplasma spp. (C) were compared. Numbers within the intersections of different ovals indicate ortholog clusters shared by 2, 3, 4, or 5 organisms. Species compared are indicated in diagram intersections as follows. A, R. prowazekii; B, N. sennetsu; C, W. pipientis; D, A. phagocytophilum; E, E. chaffeensis; F, A. marginale; G, E. ruminantium Gardel; and H, E. ruminantium Welgevonden.

    Techniques Used:

    Comparative Metabolic Potential of Select Rickettsiales Metabolic pathways of E. chaffeensis (magenta arrows), A. phagocytophilum (green arrows), N. sennetsu (gold arrows), W. pipientis (lavender arrows), and R. prowazekii (cyan arrows) were reconstructed and compared. The networks of some of the more important pathways are shown with metabolites color coded: red and purple, central and intermediary metabolites; blue, cofactors; green, amino acids; and black, cell structures. Transporters are shown in the membrane and are grouped by predicted substrate specificity: green, inorganic cations; magenta, inorganic anions; red, carbohydrates and carboxylates; blue, amino acids/peptides/amines; yellow, nucleotides/nucleosides; and black, drug/polysaccharide efflux or unknown.
    Figure Legend Snippet: Comparative Metabolic Potential of Select Rickettsiales Metabolic pathways of E. chaffeensis (magenta arrows), A. phagocytophilum (green arrows), N. sennetsu (gold arrows), W. pipientis (lavender arrows), and R. prowazekii (cyan arrows) were reconstructed and compared. The networks of some of the more important pathways are shown with metabolites color coded: red and purple, central and intermediary metabolites; blue, cofactors; green, amino acids; and black, cell structures. Transporters are shown in the membrane and are grouped by predicted substrate specificity: green, inorganic cations; magenta, inorganic anions; red, carbohydrates and carboxylates; blue, amino acids/peptides/amines; yellow, nucleotides/nucleosides; and black, drug/polysaccharide efflux or unknown.

    Techniques Used:

    Synteny between Anaplasma spp. and Ehrlichia spp. Anaplasma spp. and Ehrlichia spp. share conserved gene order (synteny) across their chromosomes. E. ruminantium and E. chaffeensis have a single symmetrical inversion near two duplicate Rho termination factors (approximate positions shown in pink). Genomic rearrangements between these Rho termination factors are also apparent in A. marginale (pink). In addition to the synteny breaks near the Rho termination factors, A. marginale has rearrangements located near the msp2- and msp3- expression locus and pseudogenes (approximate positions shown in light blue). Likewise, in A. phagocytophilum, numerous changes in genome arrangement are located near the homologous p44 expression locus and silent genes (approximate positions shown in lavender).
    Figure Legend Snippet: Synteny between Anaplasma spp. and Ehrlichia spp. Anaplasma spp. and Ehrlichia spp. share conserved gene order (synteny) across their chromosomes. E. ruminantium and E. chaffeensis have a single symmetrical inversion near two duplicate Rho termination factors (approximate positions shown in pink). Genomic rearrangements between these Rho termination factors are also apparent in A. marginale (pink). In addition to the synteny breaks near the Rho termination factors, A. marginale has rearrangements located near the msp2- and msp3- expression locus and pseudogenes (approximate positions shown in light blue). Likewise, in A. phagocytophilum, numerous changes in genome arrangement are located near the homologous p44 expression locus and silent genes (approximate positions shown in lavender).

    Techniques Used: Expressing

    4) Product Images from "Malignant germ cell tumours of the testis express interferon-γ, but are resistant to endogenous interferon-γ"

    Article Title: Malignant germ cell tumours of the testis express interferon-γ, but are resistant to endogenous interferon-γ

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6601209

    Expression of IFN γ R proteins in TGCT cell lines ( A – D ). Production of IFN γ R α and IFN γ R β was examined by RT–PCR ( A ), Western blot ( B ) and FACS analysis ( C ). IFN γ R α and IFN γ R β mRNA ( A ) and proteins ( B and C ) are expressed in both TGCT cell lines NCCIT and NTERA. For control, human monocytes (Mo) were treated with IFN γ , as described in Materials and Methods, and proved to express IFN γ R α and IFN γ R β mRNA ( A ) and proteins ( B and C ). In part C, cells were stained by indirect immunofluorescence with anti-IFN γ R α or anti-IFN γ R β antibody (filled histograms) or with isotype control IgG (open histograms).
    Figure Legend Snippet: Expression of IFN γ R proteins in TGCT cell lines ( A – D ). Production of IFN γ R α and IFN γ R β was examined by RT–PCR ( A ), Western blot ( B ) and FACS analysis ( C ). IFN γ R α and IFN γ R β mRNA ( A ) and proteins ( B and C ) are expressed in both TGCT cell lines NCCIT and NTERA. For control, human monocytes (Mo) were treated with IFN γ , as described in Materials and Methods, and proved to express IFN γ R α and IFN γ R β mRNA ( A ) and proteins ( B and C ). In part C, cells were stained by indirect immunofluorescence with anti-IFN γ R α or anti-IFN γ R β antibody (filled histograms) or with isotype control IgG (open histograms).

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, FACS, Staining, Immunofluorescence

    5) Product Images from "Intracytoplasmic Copper Homeostasis Controls Cytochrome c Oxidase Production"

    Article Title: Intracytoplasmic Copper Homeostasis Controls Cytochrome c Oxidase Production

    Journal: mBio

    doi: 10.1128/mBio.01055-13

    Transcriptional response of copA , ccoI , and RCC01042 genes to Cu 2+ supplementation. RT-PCR data using total RNA extracted from a wild-type R. capsulatus strain (MT1131) grown under respiratory conditions in MPYE medium not supplemented with CuSO 4 (− Cu) or supplemented with 1 µM CuSO 4 (+ Cu) are shown. copA (RCC01180), RCC01042, and ccoI expression are shown together with the 16S rRNA expression, which is unaffected by the addition of Cu 2+ . Note that copA expression is increased, whereas ccoI expression is decreased by Cu supplementation.
    Figure Legend Snippet: Transcriptional response of copA , ccoI , and RCC01042 genes to Cu 2+ supplementation. RT-PCR data using total RNA extracted from a wild-type R. capsulatus strain (MT1131) grown under respiratory conditions in MPYE medium not supplemented with CuSO 4 (− Cu) or supplemented with 1 µM CuSO 4 (+ Cu) are shown. copA (RCC01180), RCC01042, and ccoI expression are shown together with the 16S rRNA expression, which is unaffected by the addition of Cu 2+ . Note that copA expression is increased, whereas ccoI expression is decreased by Cu supplementation.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

    6) Product Images from "Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses"

    Article Title: Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2016.01588

    Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including DNase digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.
    Figure Legend Snippet: Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including DNase digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.

    Techniques Used: Environmental Sampling, Purification, Real-time Polymerase Chain Reaction, Lysis

    7) Product Images from "Metagenomic quorum quenching enzymes affect biofilm formation of Candida albicans and Staphylococcus epidermidis"

    Article Title: Metagenomic quorum quenching enzymes affect biofilm formation of Candida albicans and Staphylococcus epidermidis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0211366

    Transcript levels of icaR , lrgA and rnaIII in S . epidermidis in the presence of MBP-QQ-7. S . epidermidis (3 x 10 8 cells/mL in 3 mL TSB) were incubated in the presence of 500 μg/mL MBP-QQ-7 without shaking. After 3 h incubation, RNA was isolated and the relative transcript levels of icaR , lrgA and rnaIII were determined by quantitative RT-PCR analysis. Three biological replicates each with three technical replicates were performed to determine the standard deviation of the calculated 2 -ΔΔCt values. Fold changes in transcript abundances were determined by comparison with the threshold cycle (Ct) of transcripts of the control gene gyrB .
    Figure Legend Snippet: Transcript levels of icaR , lrgA and rnaIII in S . epidermidis in the presence of MBP-QQ-7. S . epidermidis (3 x 10 8 cells/mL in 3 mL TSB) were incubated in the presence of 500 μg/mL MBP-QQ-7 without shaking. After 3 h incubation, RNA was isolated and the relative transcript levels of icaR , lrgA and rnaIII were determined by quantitative RT-PCR analysis. Three biological replicates each with three technical replicates were performed to determine the standard deviation of the calculated 2 -ΔΔCt values. Fold changes in transcript abundances were determined by comparison with the threshold cycle (Ct) of transcripts of the control gene gyrB .

    Techniques Used: Incubation, Isolation, Quantitative RT-PCR, Standard Deviation

    8) Product Images from "Induction of immunomodulatory miR-146a and miR-155 in small intestinal epithelium of Vibrio cholerae infected patients at acute stage of cholera"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0173817

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

    Techniques Used: Expressing, Quantitative RT-PCR

    9) Product Images from "Nucleoside analog studies indicate mechanistic differences between RNA-editing adenosine deaminases"

    Article Title: Nucleoside analog studies indicate mechanistic differences between RNA-editing adenosine deaminases

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks752

    Known interactions with adenine N7 in different enzymes that catalyze adenosine deamination. ( A ) tRNA adenosine deaminase (TadA), ( B ) ADA and ( C ) AMPDA make hydrogen bonding interactions with adenosine. (A) Staphylococcus aureus TadA makes a hydrogen bond to N7 of nebularine through a network of ordered water molecules ( 73 ). (B) D296 of murine ADA makes a hydrogen bond to N7 of 1-deazaadenosine ( 77 ). (C) D736 of Arabidopsis AMPDA ligates the catalytic zinc, but is also within hydrogen bonding distance of N7 of coformycin 5′-phosphate ( 76 ).
    Figure Legend Snippet: Known interactions with adenine N7 in different enzymes that catalyze adenosine deamination. ( A ) tRNA adenosine deaminase (TadA), ( B ) ADA and ( C ) AMPDA make hydrogen bonding interactions with adenosine. (A) Staphylococcus aureus TadA makes a hydrogen bond to N7 of nebularine through a network of ordered water molecules ( 73 ). (B) D296 of murine ADA makes a hydrogen bond to N7 of 1-deazaadenosine ( 77 ). (C) D736 of Arabidopsis AMPDA ligates the catalytic zinc, but is also within hydrogen bonding distance of N7 of coformycin 5′-phosphate ( 76 ).

    Techniques Used:

    10) Product Images from "Vitamin D Elicits Anti-Inflammatory Response, Inhibits Contractile-Associated Proteins, and Modulates Toll-Like Receptors in Human Myometrial Cells"

    Article Title: Vitamin D Elicits Anti-Inflammatory Response, Inhibits Contractile-Associated Proteins, and Modulates Toll-Like Receptors in Human Myometrial Cells

    Journal: Reproductive Sciences

    doi: 10.1177/1933719112459225

    Quantitative RT-PCR analysis of contractile-associated proteins in immortalized human myometrial (UtSM) cells treated with vitamin D. The UtSM cells were treated with different concentrations of vitamin D for 12 hours and the RNA obtained was subjected
    Figure Legend Snippet: Quantitative RT-PCR analysis of contractile-associated proteins in immortalized human myometrial (UtSM) cells treated with vitamin D. The UtSM cells were treated with different concentrations of vitamin D for 12 hours and the RNA obtained was subjected

    Techniques Used: Quantitative RT-PCR

    The effects of vitamin D on LPS-induced mRNA expression of cytokines, chemokines, and contractile-associated proteins in immortalized human myometrial (UtSM) cells. The UtSM cells were treated with different concentrations of vitamin D for 12 hours in
    Figure Legend Snippet: The effects of vitamin D on LPS-induced mRNA expression of cytokines, chemokines, and contractile-associated proteins in immortalized human myometrial (UtSM) cells. The UtSM cells were treated with different concentrations of vitamin D for 12 hours in

    Techniques Used: Expressing

    The quantitative RT-PCR analysis of the expression of inflammatory markers and contractile-associated factors in immortalized human myometrial (UtSM) cells treated with vitamin D in the presence of IL-1β. The UtSM cells were treated with different
    Figure Legend Snippet: The quantitative RT-PCR analysis of the expression of inflammatory markers and contractile-associated factors in immortalized human myometrial (UtSM) cells treated with vitamin D in the presence of IL-1β. The UtSM cells were treated with different

    Techniques Used: Quantitative RT-PCR, Expressing

    The effects of vitamin D on the protein expression of connexin 43 in immortalized human myometrial (UtSM) cells treated with interleukin 1β. The UtSM cells were treated with 100 nmol/L of vitamin D for 24 and 48 hours in the presence of interleukin
    Figure Legend Snippet: The effects of vitamin D on the protein expression of connexin 43 in immortalized human myometrial (UtSM) cells treated with interleukin 1β. The UtSM cells were treated with 100 nmol/L of vitamin D for 24 and 48 hours in the presence of interleukin

    Techniques Used: Expressing

    The effects of vitamin D on LPS-induced protein expression of MCP-1, TLR-4, and connexin 43 in immortalized human myometrial (UtSM) cells. The UtSM cells were treated with 100 nmol/L of vitamin D for 48 hours in the presence of LPS (1 μg/mL).
    Figure Legend Snippet: The effects of vitamin D on LPS-induced protein expression of MCP-1, TLR-4, and connexin 43 in immortalized human myometrial (UtSM) cells. The UtSM cells were treated with 100 nmol/L of vitamin D for 48 hours in the presence of LPS (1 μg/mL).

    Techniques Used: Expressing

    The dose effect of lipopolysaccharide (LPS) on mRNA expression of cytokines and contractile-associated proteins in immortalized human myometrial (UtSM) cells. The UtSM cells were treated with different concentrations of LPS for 12 hours, and the effects
    Figure Legend Snippet: The dose effect of lipopolysaccharide (LPS) on mRNA expression of cytokines and contractile-associated proteins in immortalized human myometrial (UtSM) cells. The UtSM cells were treated with different concentrations of LPS for 12 hours, and the effects

    Techniques Used: Expressing

    The effects of vitamin D on the mRNA expression of cytokine and chemokine genes in immortalized human myometrial (UtSM) cells treated with vitamin D for 12 hours. The UtSM cells were treated with different concentrations of vitamin D and the effects on
    Figure Legend Snippet: The effects of vitamin D on the mRNA expression of cytokine and chemokine genes in immortalized human myometrial (UtSM) cells treated with vitamin D for 12 hours. The UtSM cells were treated with different concentrations of vitamin D and the effects on

    Techniques Used: Expressing

    11) Product Images from "Tumor Suppressor PDCD4 Represses Internal Ribosome Entry Site-Mediated Translation of Antiapoptotic Proteins and Is Regulated by S6 Kinase 2"

    Article Title: Tumor Suppressor PDCD4 Represses Internal Ribosome Entry Site-Mediated Translation of Antiapoptotic Proteins and Is Regulated by S6 Kinase 2

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.06317-11

    Activation of S6K2 leads to PDCD4 degradation, with a concomitant increase in XIAP and Bcl-x L protein levels. (A) TOKAS6K2-stable or control cells (transiently transfected with an inducible pTripz-Kate plasmid) were treated with doxycycline (Dox) (1 μg/ml)
    Figure Legend Snippet: Activation of S6K2 leads to PDCD4 degradation, with a concomitant increase in XIAP and Bcl-x L protein levels. (A) TOKAS6K2-stable or control cells (transiently transfected with an inducible pTripz-Kate plasmid) were treated with doxycycline (Dox) (1 μg/ml)

    Techniques Used: Activation Assay, Transfection, Plasmid Preparation

    Loss of PDCD4 correlates with an increase in XIAP and Bcl-x L translation through their respective IRES elements. (A) HEK293T cells were treated with PDCD4 siRNA or control (CTRL), nontargeting siRNA. Cell lysates were harvested and subjected to Western
    Figure Legend Snippet: Loss of PDCD4 correlates with an increase in XIAP and Bcl-x L translation through their respective IRES elements. (A) HEK293T cells were treated with PDCD4 siRNA or control (CTRL), nontargeting siRNA. Cell lysates were harvested and subjected to Western

    Techniques Used: Western Blot

    PDCD4 specifically binds to XIAP and Bcl-x L IRES RNA both in vitro and in vivo . (A) Recombinant His-PDCD4 was incubated in the presence of 32 P-labeled, in vitro -transcribed RNA and subjected to UV cross-linking. RNA-protein complexes were separated by
    Figure Legend Snippet: PDCD4 specifically binds to XIAP and Bcl-x L IRES RNA both in vitro and in vivo . (A) Recombinant His-PDCD4 was incubated in the presence of 32 P-labeled, in vitro -transcribed RNA and subjected to UV cross-linking. RNA-protein complexes were separated by

    Techniques Used: In Vitro, In Vivo, Recombinant, Incubation, Labeling

    12) Product Images from "Vitamin D Elicits Anti-Inflammatory Response, Inhibits Contractile-Associated Proteins, and Modulates Toll-Like Receptors in Human Myometrial Cells"

    Article Title: Vitamin D Elicits Anti-Inflammatory Response, Inhibits Contractile-Associated Proteins, and Modulates Toll-Like Receptors in Human Myometrial Cells

    Journal: Reproductive Sciences

    doi: 10.1177/1933719112459225

    Quantitative RT-PCR analysis of contractile-associated proteins in immortalized human myometrial (UtSM) cells treated with vitamin D. The UtSM cells were treated with different concentrations of vitamin D for 12 hours and the RNA obtained was subjected
    Figure Legend Snippet: Quantitative RT-PCR analysis of contractile-associated proteins in immortalized human myometrial (UtSM) cells treated with vitamin D. The UtSM cells were treated with different concentrations of vitamin D for 12 hours and the RNA obtained was subjected

    Techniques Used: Quantitative RT-PCR

    The effects of vitamin D on LPS-induced mRNA expression of cytokines, chemokines, and contractile-associated proteins in immortalized human myometrial (UtSM) cells. The UtSM cells were treated with different concentrations of vitamin D for 12 hours in
    Figure Legend Snippet: The effects of vitamin D on LPS-induced mRNA expression of cytokines, chemokines, and contractile-associated proteins in immortalized human myometrial (UtSM) cells. The UtSM cells were treated with different concentrations of vitamin D for 12 hours in

    Techniques Used: Expressing

    Differential mRNA expression of Toll-like receptor 10 in term human myometrial tissues. Myometrial tissues were obtained from term pregnant women in labor and not in labor. The complementary DNA obtained was subjected to qPCR analysis using primers specific
    Figure Legend Snippet: Differential mRNA expression of Toll-like receptor 10 in term human myometrial tissues. Myometrial tissues were obtained from term pregnant women in labor and not in labor. The complementary DNA obtained was subjected to qPCR analysis using primers specific

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    The quantitative RT-PCR analysis of the expression of inflammatory markers and contractile-associated factors in immortalized human myometrial (UtSM) cells treated with vitamin D in the presence of IL-1β. The UtSM cells were treated with different
    Figure Legend Snippet: The quantitative RT-PCR analysis of the expression of inflammatory markers and contractile-associated factors in immortalized human myometrial (UtSM) cells treated with vitamin D in the presence of IL-1β. The UtSM cells were treated with different

    Techniques Used: Quantitative RT-PCR, Expressing

    The effects of vitamin D on the protein expression of connexin 43 in immortalized human myometrial (UtSM) cells treated with interleukin 1β. The UtSM cells were treated with 100 nmol/L of vitamin D for 24 and 48 hours in the presence of interleukin
    Figure Legend Snippet: The effects of vitamin D on the protein expression of connexin 43 in immortalized human myometrial (UtSM) cells treated with interleukin 1β. The UtSM cells were treated with 100 nmol/L of vitamin D for 24 and 48 hours in the presence of interleukin

    Techniques Used: Expressing

    The effects of vitamin D on LPS-induced protein expression of MCP-1, TLR-4, and connexin 43 in immortalized human myometrial (UtSM) cells. The UtSM cells were treated with 100 nmol/L of vitamin D for 48 hours in the presence of LPS (1 μg/mL).
    Figure Legend Snippet: The effects of vitamin D on LPS-induced protein expression of MCP-1, TLR-4, and connexin 43 in immortalized human myometrial (UtSM) cells. The UtSM cells were treated with 100 nmol/L of vitamin D for 48 hours in the presence of LPS (1 μg/mL).

    Techniques Used: Expressing

    The dose effect of lipopolysaccharide (LPS) on mRNA expression of cytokines and contractile-associated proteins in immortalized human myometrial (UtSM) cells. The UtSM cells were treated with different concentrations of LPS for 12 hours, and the effects
    Figure Legend Snippet: The dose effect of lipopolysaccharide (LPS) on mRNA expression of cytokines and contractile-associated proteins in immortalized human myometrial (UtSM) cells. The UtSM cells were treated with different concentrations of LPS for 12 hours, and the effects

    Techniques Used: Expressing

    The effects of vitamin D on the mRNA expression of cytokine and chemokine genes in immortalized human myometrial (UtSM) cells treated with vitamin D for 12 hours. The UtSM cells were treated with different concentrations of vitamin D and the effects on
    Figure Legend Snippet: The effects of vitamin D on the mRNA expression of cytokine and chemokine genes in immortalized human myometrial (UtSM) cells treated with vitamin D for 12 hours. The UtSM cells were treated with different concentrations of vitamin D and the effects on

    Techniques Used: Expressing

    13) Product Images from "Selective Targeting of Leukemic Cell Growth in Vivo and in Vitro Using a Gene Silencing Approach to DiminishS-Adenosylmethionine Synthesis *-Adenosylmethionine Synthesis * S⃞"

    Article Title: Selective Targeting of Leukemic Cell Growth in Vivo and in Vitro Using a Gene Silencing Approach to DiminishS-Adenosylmethionine Synthesis *-Adenosylmethionine Synthesis * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M804159200

    MAT-IIβ ablation diminishes leukemic cell growth in vivo . Hyperimmune-deficient NOD/Scid IL-2Rγ null mice were irradiated with 3.5GY 24 h prior to intraperitoneal transplant with 15 × 10 6 of the indicated cells. Controls included irradiated but not injected mice and V1302 transplanted mice. Tumor engraftment and growth were monitored by measuring % human CD3 + /GFP + cells in mice spleens and bone marrow at 4 and 5 weeks post-transplant with either control V1302 or V1110 cells. Flow cytometry histograms of CD3 + /GFP + in spleen ( A ) or bone marrow ( B ) 5 weeks post-transplant. C , GFP expression in whole spleens of same mice prior to processing their splenocytes. D , number of mice engrafted with V1302 or V1110 leukemic cells at 4 and 5 weeks post-transplant. E and F show % CD3 + /GFP + cells in spleens ( E ) or bone marrow ( F ) of mice transplanted with either V1302 or V1110 leukemic cells, 5 weeks post-transplant. The statistical differences were calculated using a Mann-Whitney test. * , p ≤ 0.05; ** , p ≤ 0.01; *** , p ≤ 0.001.
    Figure Legend Snippet: MAT-IIβ ablation diminishes leukemic cell growth in vivo . Hyperimmune-deficient NOD/Scid IL-2Rγ null mice were irradiated with 3.5GY 24 h prior to intraperitoneal transplant with 15 × 10 6 of the indicated cells. Controls included irradiated but not injected mice and V1302 transplanted mice. Tumor engraftment and growth were monitored by measuring % human CD3 + /GFP + cells in mice spleens and bone marrow at 4 and 5 weeks post-transplant with either control V1302 or V1110 cells. Flow cytometry histograms of CD3 + /GFP + in spleen ( A ) or bone marrow ( B ) 5 weeks post-transplant. C , GFP expression in whole spleens of same mice prior to processing their splenocytes. D , number of mice engrafted with V1302 or V1110 leukemic cells at 4 and 5 weeks post-transplant. E and F show % CD3 + /GFP + cells in spleens ( E ) or bone marrow ( F ) of mice transplanted with either V1302 or V1110 leukemic cells, 5 weeks post-transplant. The statistical differences were calculated using a Mann-Whitney test. * , p ≤ 0.05; ** , p ≤ 0.01; *** , p ≤ 0.001.

    Techniques Used: In Vivo, Mouse Assay, Irradiation, Injection, Flow Cytometry, Cytometry, Expressing, MANN-WHITNEY

    14) Product Images from "Meglumine Antimoniate and Miltefosine Combined With Allopurinol Sustain Pro-inflammatory Immune Environments During Canine Leishmaniosis Treatment"

    Article Title: Meglumine Antimoniate and Miltefosine Combined With Allopurinol Sustain Pro-inflammatory Immune Environments During Canine Leishmaniosis Treatment

    Journal: Frontiers in Veterinary Science

    doi: 10.3389/fvets.2019.00362

    Pro-inflammatory cytokine gene expression in dogs treated with either MT+A or MG+A protocol along all time-points. IL-2 (A–C) , IL-12 (D–F) , TNF-α (G–I) , and IFN-γ (J–L) mRNA in PBMC (A,D,G,J) , lymph node (B,E,H,K) and bone marrow (C,F,I,L) cells of dogs from MT+A, MG+A, and Control Group (CG) was evaluated by qPCR. Results of 17 dogs and three replicates per sample are represented by box and whisker plot, median, minimum, and maximum values. The non-parametric Kruskal-Wallis Test (one-way ANOVA on ranks) with Dunn's post hoc test was used for statistical comparisons between treatments groups and the CG. The Repeated Measures ANOVA Test with Tukey's post hoc test was used for statistical comparisons inside each treatment group. * p
    Figure Legend Snippet: Pro-inflammatory cytokine gene expression in dogs treated with either MT+A or MG+A protocol along all time-points. IL-2 (A–C) , IL-12 (D–F) , TNF-α (G–I) , and IFN-γ (J–L) mRNA in PBMC (A,D,G,J) , lymph node (B,E,H,K) and bone marrow (C,F,I,L) cells of dogs from MT+A, MG+A, and Control Group (CG) was evaluated by qPCR. Results of 17 dogs and three replicates per sample are represented by box and whisker plot, median, minimum, and maximum values. The non-parametric Kruskal-Wallis Test (one-way ANOVA on ranks) with Dunn's post hoc test was used for statistical comparisons between treatments groups and the CG. The Repeated Measures ANOVA Test with Tukey's post hoc test was used for statistical comparisons inside each treatment group. * p

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

    Regulatory cytokine gene expression in dogs treated with either MT+A or MG+A protocol along all time-points. IL-10 (A–C) and TGF-β (D–F) mRNA in PBMC (A,D) , lymph node (B,E) , and bone marrow (C,F) cells of dogs from MT+A, MG+A, and Control Group (CG) was evaluated by qPCR. Results of 17 dogs and three replicates per sample are represented by box and whisker plot, median, minimum, and maximum values. The non-parametric Kruskal-Wallis Test (one-way ANOVA on ranks) with Dunn's post hoc test was used for statistical comparisons between treatments groups and the CG. The Repeated Measures ANOVA Test with Tukey's post hoc test was used for statistical comparisons inside each treatment group. * p
    Figure Legend Snippet: Regulatory cytokine gene expression in dogs treated with either MT+A or MG+A protocol along all time-points. IL-10 (A–C) and TGF-β (D–F) mRNA in PBMC (A,D) , lymph node (B,E) , and bone marrow (C,F) cells of dogs from MT+A, MG+A, and Control Group (CG) was evaluated by qPCR. Results of 17 dogs and three replicates per sample are represented by box and whisker plot, median, minimum, and maximum values. The non-parametric Kruskal-Wallis Test (one-way ANOVA on ranks) with Dunn's post hoc test was used for statistical comparisons between treatments groups and the CG. The Repeated Measures ANOVA Test with Tukey's post hoc test was used for statistical comparisons inside each treatment group. * p

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

    Anti-inflammatory cytokine gene expression in dogs treated with either MT+A or MG+A protocol along all time-points. IL-4 (A–C) and IL-5 (D–F) mRNA in PBMC (A,D) , lymph node (B,E) , and bone marrow (C,F) cells of dogs from MT+A, MG+A, and Control Group (CG) was evaluated by qPCR. Results of 17 dogs and three replicates per sample are represented by box and whisker plot, median, minimum, and maximum values. The non-parametric Kruskal-Wallis Test (one-way ANOVA on ranks) with Dunn's post hoc test was used for statistical comparisons between treatments groups and the CG. The Repeated Measures ANOVA Test with Tukey's post hoc test was used for statistical comparisons inside each treatment group. * p
    Figure Legend Snippet: Anti-inflammatory cytokine gene expression in dogs treated with either MT+A or MG+A protocol along all time-points. IL-4 (A–C) and IL-5 (D–F) mRNA in PBMC (A,D) , lymph node (B,E) , and bone marrow (C,F) cells of dogs from MT+A, MG+A, and Control Group (CG) was evaluated by qPCR. Results of 17 dogs and three replicates per sample are represented by box and whisker plot, median, minimum, and maximum values. The non-parametric Kruskal-Wallis Test (one-way ANOVA on ranks) with Dunn's post hoc test was used for statistical comparisons between treatments groups and the CG. The Repeated Measures ANOVA Test with Tukey's post hoc test was used for statistical comparisons inside each treatment group. * p

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

    15) Product Images from "Characterization of human plasma-derived exosomal RNAs by deep sequencing"

    Article Title: Characterization of human plasma-derived exosomal RNAs by deep sequencing

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-14-319

    Exosome isolation and exosomal small RNA quantification. ( A ) Representative histogram of exosome size distribution. ( B ) Exosomal RNAs determined using the Agilent Small RNA Chip. The small RNAs were dominant in the exosomal RNAs. ( C ) Exosomal RNAs determined by the Agilent RNA Pico Chip. Exosomal RNA samples A , B and C contained no detectable 18S and 28S rRNAs. Cellular RNA from HEK293 was loaded as positive control for 18S and 28S rRNAs ( D ) Isolated exosomal RNAs treated either with DNase I or RNase A. ( E ) Plasma and control small RNAs treated under various conditions.
    Figure Legend Snippet: Exosome isolation and exosomal small RNA quantification. ( A ) Representative histogram of exosome size distribution. ( B ) Exosomal RNAs determined using the Agilent Small RNA Chip. The small RNAs were dominant in the exosomal RNAs. ( C ) Exosomal RNAs determined by the Agilent RNA Pico Chip. Exosomal RNA samples A , B and C contained no detectable 18S and 28S rRNAs. Cellular RNA from HEK293 was loaded as positive control for 18S and 28S rRNAs ( D ) Isolated exosomal RNAs treated either with DNase I or RNase A. ( E ) Plasma and control small RNAs treated under various conditions.

    Techniques Used: Isolation, Chromatin Immunoprecipitation, Positive Control

    16) Product Images from "NF-κB Signaling Pathway in Controlling Intervertebral Disk Cell Response to Inflammatory and Mechanical Stressors"

    Article Title: NF-κB Signaling Pathway in Controlling Intervertebral Disk Cell Response to Inflammatory and Mechanical Stressors

    Journal: Physical Therapy

    doi: 10.2522/ptj.20150045

    Relative change in messenger RNA (mRNA) gene expression of matrix metalloproteinase-3 (MMP-3), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) as measured by real-time polymerase chain reaction shows increased matrix breakdown and
    Figure Legend Snippet: Relative change in messenger RNA (mRNA) gene expression of matrix metalloproteinase-3 (MMP-3), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) as measured by real-time polymerase chain reaction shows increased matrix breakdown and

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    17) Product Images from "In Vivo Occupancy of Mitochondrial Single-Stranded DNA Binding Protein Supports the Strand Displacement Mode of DNA Replication"

    Article Title: In Vivo Occupancy of Mitochondrial Single-Stranded DNA Binding Protein Supports the Strand Displacement Mode of DNA Replication

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004832

    Neutral 2D-AGE analysis of mtDNA replication products. ( A ) Extracted DNA analyzed on 1% Agarose. ( B ) Purified DNA cut with HincII was analysed using 2D-AGE. A fragment (mtDNA 13636-1006 bp) spanning the OriH region was visualized with probe located in CYTB (14641-15590 bp). Upper panel; untreated DNA (containing both RNA and DNA), Middle panel; RNase A and RNase H treated DNA (containing only DNA); Lower panel, DNA treated with RNaseA and RNaseH remixed with the RNA still present after DNase I treatment. ( C ) Schematic illustration of how Y and bubble arcs are expected to run in 2D-AGE. The bubble arc observed here is dependent on RNA (indicated in red).
    Figure Legend Snippet: Neutral 2D-AGE analysis of mtDNA replication products. ( A ) Extracted DNA analyzed on 1% Agarose. ( B ) Purified DNA cut with HincII was analysed using 2D-AGE. A fragment (mtDNA 13636-1006 bp) spanning the OriH region was visualized with probe located in CYTB (14641-15590 bp). Upper panel; untreated DNA (containing both RNA and DNA), Middle panel; RNase A and RNase H treated DNA (containing only DNA); Lower panel, DNA treated with RNaseA and RNaseH remixed with the RNA still present after DNase I treatment. ( C ) Schematic illustration of how Y and bubble arcs are expected to run in 2D-AGE. The bubble arc observed here is dependent on RNA (indicated in red).

    Techniques Used: Purification

    18) Product Images from "Metagenomic quorum quenching enzymes affect biofilm formation of Candida albicans and Staphylococcus epidermidis"

    Article Title: Metagenomic quorum quenching enzymes affect biofilm formation of Candida albicans and Staphylococcus epidermidis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0211366

    Transcript levels of icaR , lrgA and rnaIII in S . epidermidis in the presence of MBP-QQ-7. S . epidermidis (3 x 10 8 cells/mL in 3 mL TSB) were incubated in the presence of 500 μg/mL MBP-QQ-7 without shaking. After 3 h incubation, RNA was isolated and the relative transcript levels of icaR , lrgA and rnaIII were determined by quantitative RT-PCR analysis. Three biological replicates each with three technical replicates were performed to determine the standard deviation of the calculated 2 -ΔΔCt values. Fold changes in transcript abundances were determined by comparison with the threshold cycle (Ct) of transcripts of the control gene gyrB .
    Figure Legend Snippet: Transcript levels of icaR , lrgA and rnaIII in S . epidermidis in the presence of MBP-QQ-7. S . epidermidis (3 x 10 8 cells/mL in 3 mL TSB) were incubated in the presence of 500 μg/mL MBP-QQ-7 without shaking. After 3 h incubation, RNA was isolated and the relative transcript levels of icaR , lrgA and rnaIII were determined by quantitative RT-PCR analysis. Three biological replicates each with three technical replicates were performed to determine the standard deviation of the calculated 2 -ΔΔCt values. Fold changes in transcript abundances were determined by comparison with the threshold cycle (Ct) of transcripts of the control gene gyrB .

    Techniques Used: Incubation, Isolation, Quantitative RT-PCR, Standard Deviation

    S . epidermidis biofilm formation under static conditions in presence of MBP-QQ-5, MBP-QQ-7 and MBP. S . epidermidis was grown in 96 well plates in 200 μL culture medium in presence of purified MBP-QQ proteins MBP-QQ-5 (light grey bars) and MBP-QQ-7 (dark grey bars) at final concentrations of 10 μg, 50 μg and 100 μg. Purified MBP (black bar) was added as control to a final concentration of 100 μg and the determined absorbance set to 100%. Biofilm formation was quantified via crystal violet assay. Means of standard deviations of at least 4 independent biological replicates are indicated. Statistics (unpaired t test) were performed with GraphPad Prism 6 software with differences *p
    Figure Legend Snippet: S . epidermidis biofilm formation under static conditions in presence of MBP-QQ-5, MBP-QQ-7 and MBP. S . epidermidis was grown in 96 well plates in 200 μL culture medium in presence of purified MBP-QQ proteins MBP-QQ-5 (light grey bars) and MBP-QQ-7 (dark grey bars) at final concentrations of 10 μg, 50 μg and 100 μg. Purified MBP (black bar) was added as control to a final concentration of 100 μg and the determined absorbance set to 100%. Biofilm formation was quantified via crystal violet assay. Means of standard deviations of at least 4 independent biological replicates are indicated. Statistics (unpaired t test) were performed with GraphPad Prism 6 software with differences *p

    Techniques Used: Purification, Concentration Assay, Crystal Violet Assay, Software

    Excluding potential effects of QQ proteins on growth of S . epidermidis RP6A. S . epidermidis RP6A was grown in Tryptic Soy Broth medium at 37°C and 120 rpm in 20 mL. Cultures were grown complemented with 100 μg MBP, QQ-5 or QQ-7 as well as without any supplement. The results represent a mean of three independent experiments. Error bars indicate standard deviations.
    Figure Legend Snippet: Excluding potential effects of QQ proteins on growth of S . epidermidis RP6A. S . epidermidis RP6A was grown in Tryptic Soy Broth medium at 37°C and 120 rpm in 20 mL. Cultures were grown complemented with 100 μg MBP, QQ-5 or QQ-7 as well as without any supplement. The results represent a mean of three independent experiments. Error bars indicate standard deviations.

    Techniques Used:

    Impact of immobilized protein MBP-QQ-7 on S . epidermidis initial attachment. Proteins MBP-QQ-7 and MBP as control were immobilized on coverslips, which were inserted into flow chambers with a total volume of 1.3 mL. After 1 h adhesion, flow chambers were incubated for 3 h (short-term experiment) at a flow rate of 20 mL/h with 3% TSB medium. Adhered cells were stained with Syto9 and propidium iodide and visualized using ( A ) CLSM and ( B ) SEM.
    Figure Legend Snippet: Impact of immobilized protein MBP-QQ-7 on S . epidermidis initial attachment. Proteins MBP-QQ-7 and MBP as control were immobilized on coverslips, which were inserted into flow chambers with a total volume of 1.3 mL. After 1 h adhesion, flow chambers were incubated for 3 h (short-term experiment) at a flow rate of 20 mL/h with 3% TSB medium. Adhered cells were stained with Syto9 and propidium iodide and visualized using ( A ) CLSM and ( B ) SEM.

    Techniques Used: Flow Cytometry, Incubation, Staining, Confocal Laser Scanning Microscopy

    Decreased S . epidermidis biofilm formation on immobilized proteins MBP-QQ-5 and MBP-QQ-7. Coverslips coated with proteins MBP-QQ-5, MBP-QQ-7and MBP as control were used in flow chambers (total volume of 1.3 mL) for S . epidermidis biofilm formation (3 x 10 8 cells/ mL). After 1 h adhesion, flow chambers were incubated for 20 h (long-term experiment) with a flow rate of 20 mL/h using 3% TSB medium. Biofilms were stained with Syto9 and propidium iodide. Effects on biofilm formation were visualized using ( A ) CLSM and ( B ) SEM. ( C ) Biofilm characteristics thickness and volume were quantified using IMARIS software. Means of 2 independent biological replicates, each with 3 technical replicates are indicated. Unpaired t test was performed with GraphPad Prism 6 software with differences *p
    Figure Legend Snippet: Decreased S . epidermidis biofilm formation on immobilized proteins MBP-QQ-5 and MBP-QQ-7. Coverslips coated with proteins MBP-QQ-5, MBP-QQ-7and MBP as control were used in flow chambers (total volume of 1.3 mL) for S . epidermidis biofilm formation (3 x 10 8 cells/ mL). After 1 h adhesion, flow chambers were incubated for 20 h (long-term experiment) with a flow rate of 20 mL/h using 3% TSB medium. Biofilms were stained with Syto9 and propidium iodide. Effects on biofilm formation were visualized using ( A ) CLSM and ( B ) SEM. ( C ) Biofilm characteristics thickness and volume were quantified using IMARIS software. Means of 2 independent biological replicates, each with 3 technical replicates are indicated. Unpaired t test was performed with GraphPad Prism 6 software with differences *p

    Techniques Used: Flow Cytometry, Incubation, Staining, Confocal Laser Scanning Microscopy, Software

    19) Product Images from "Role of the Single Regulator MrsR1 and the Two-Component System MrsR2/K2 in the Regulation of Mersacidin Production and Immunity"

    Article Title: Role of the Single Regulator MrsR1 and the Two-Component System MrsR2/K2 in the Regulation of Mersacidin Production and Immunity

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.68.1.106-113.2002

    RT-PCR of total RNA of Bacillus sp. strain TTΔ mrsR2/K2 and the wild-type strain. Cells were harvested after 8 (A) or 16 (B) h of incubation. Slots 3, 5, 8, and 10 contain control reactions for the samples presented in slots 2, 4, 7, and 9, respectively. These controls were performed to rule out contamination with chromosomal DNA by introducing parallel samples into the thermal cycler during the 95°C step, thereby ensuring immediate denaturation of the reverse transcriptase enzyme. Slots 1 and 11, GeneRuler DNA ladder mix; slot 2, RT-PCR of the mrsEFG transcript in the wild-type Bacillus sp. strain HIL Y-85,54728 with RT1/RT2; slot 3, control; slot 4, RT-PCR of the mrsEFG -transcript in Bacillus sp. strain TTΔ mrsR2/K2 ; slot 5, control; 6, GeneRuler 100-bp DNA ladder mix (1.031, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, and 0.2 kb); slot 7, RT-PCR of the mrsA transcript in Bacillus sp. strain HIL Y-85,54728 using RT4/RT5; slot 8, control; slot 9, RT-PCR of the mrsA -transcript in Bacillus sp. strain TTΔ mrsR2/K2 ; slot 10, control.
    Figure Legend Snippet: RT-PCR of total RNA of Bacillus sp. strain TTΔ mrsR2/K2 and the wild-type strain. Cells were harvested after 8 (A) or 16 (B) h of incubation. Slots 3, 5, 8, and 10 contain control reactions for the samples presented in slots 2, 4, 7, and 9, respectively. These controls were performed to rule out contamination with chromosomal DNA by introducing parallel samples into the thermal cycler during the 95°C step, thereby ensuring immediate denaturation of the reverse transcriptase enzyme. Slots 1 and 11, GeneRuler DNA ladder mix; slot 2, RT-PCR of the mrsEFG transcript in the wild-type Bacillus sp. strain HIL Y-85,54728 with RT1/RT2; slot 3, control; slot 4, RT-PCR of the mrsEFG -transcript in Bacillus sp. strain TTΔ mrsR2/K2 ; slot 5, control; 6, GeneRuler 100-bp DNA ladder mix (1.031, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, and 0.2 kb); slot 7, RT-PCR of the mrsA transcript in Bacillus sp. strain HIL Y-85,54728 using RT4/RT5; slot 8, control; slot 9, RT-PCR of the mrsA -transcript in Bacillus sp. strain TTΔ mrsR2/K2 ; slot 10, control.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Incubation

    20) Product Images from "Induction of immunomodulatory miR-146a and miR-155 in small intestinal epithelium of Vibrio cholerae infected patients at acute stage of cholera"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0173817

    V . cholerae O1 bacteria, but not their secreted products, induce increased expression of microRNAs in T84 polarized tight monolayer cells. Tight monolayers of polarized T84 cells were challenged at the apical side with 10 5 bacteria of the V . cholerae O1 wild type strain C6706 and two V . cholerae O1 clinical isolates, one from patient VC08 (VC08) and one from patient VC09 (VC09), that had been grown in LB broth overnight (upper row) or with culture supernatants from overnight cultures of the same bacterial strains in LB (lower row). Levels of miR-146a, miR-155 and miR-375 were determined by real-time qRT-PCR and expressed as relative quantity (RQ) compared to the median Δct of tight monolayers incubated in parallel to challenged monolayers with tissue culture medium without added bacteria (Ctr, upper row) or fresh LB (Ctr, lower row). Bars indicate mean RQ + 1 SD of 4 tight monolayers for each challenge in the upper row and 3 in the lower row. Statistically significant differences from the control monolayers as determined by one-way ANOVA with Dunett's compensation for multiple comparisons, are shown. ** P -value
    Figure Legend Snippet: V . cholerae O1 bacteria, but not their secreted products, induce increased expression of microRNAs in T84 polarized tight monolayer cells. Tight monolayers of polarized T84 cells were challenged at the apical side with 10 5 bacteria of the V . cholerae O1 wild type strain C6706 and two V . cholerae O1 clinical isolates, one from patient VC08 (VC08) and one from patient VC09 (VC09), that had been grown in LB broth overnight (upper row) or with culture supernatants from overnight cultures of the same bacterial strains in LB (lower row). Levels of miR-146a, miR-155 and miR-375 were determined by real-time qRT-PCR and expressed as relative quantity (RQ) compared to the median Δct of tight monolayers incubated in parallel to challenged monolayers with tissue culture medium without added bacteria (Ctr, upper row) or fresh LB (Ctr, lower row). Bars indicate mean RQ + 1 SD of 4 tight monolayers for each challenge in the upper row and 3 in the lower row. Statistically significant differences from the control monolayers as determined by one-way ANOVA with Dunett's compensation for multiple comparisons, are shown. ** P -value

    Techniques Used: Expressing, Quantitative RT-PCR, Incubation

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

    Techniques Used: Expressing, Quantitative RT-PCR

    21) Product Images from "Mutations in Genes Involved in the Flagellar Export Apparatus of the Solvent-Tolerant Pseudomonas putida DOT-T1E Strain Impair Motility and Lead to Hypersensitivity to Toluene Shocks"

    Article Title: Mutations in Genes Involved in the Flagellar Export Apparatus of the Solvent-Tolerant Pseudomonas putida DOT-T1E Strain Impair Motility and Lead to Hypersensitivity to Toluene Shocks

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.183.14.4127-4133.2001

    Physical map of fli-flh gene cluster in the solvent-tolerant P. putida DOT-T1E strain. (a) Gene order. The arrows indicate the direction of transcription. Small arrows indicate the positions of the primers used in RT-PCR. (b) Summary of RT-PCR results obtained with total RNA isolated from the indicated strains growing on LB. Symbols: +, RT-PCR yielded a fragment of the expected size when primers based on the indicated genes were used; −, RT-PCR failed to amplify RNA with the indicated primers; n.d., not determined. (c) Specific RT-PCR results with primers based on fliP and fliQ and on fliR and flhB . Total RNA was isolated from P. putida DOT-T1E and DOT-T1E-42, and RT-PCRs were done with primers based on fliP and fliQ that yielded a 325-nucleotide (nt) band (lane 1) and fliR and flhB that yielded a 240-nt band (lane 2). Lane 3, negative control using primers based on fliP and fliQ but in the absence of reverse transcriptase. (d) Specific RT-PCR with primers based on fliR and flhB and on fliL and fliM . RNA was isolated from P. putida DOT-T1E and DOT-T1-PS23 and RT-PCRs were done with primers based on fliR and flhB that yielded a 240-nt band (lane 1) and fliL and fliM that yielded a 220-nt band (lane 3). Lane 2, negative control using primers based on fliR and flhB but in the absence of reverse transcriptase.
    Figure Legend Snippet: Physical map of fli-flh gene cluster in the solvent-tolerant P. putida DOT-T1E strain. (a) Gene order. The arrows indicate the direction of transcription. Small arrows indicate the positions of the primers used in RT-PCR. (b) Summary of RT-PCR results obtained with total RNA isolated from the indicated strains growing on LB. Symbols: +, RT-PCR yielded a fragment of the expected size when primers based on the indicated genes were used; −, RT-PCR failed to amplify RNA with the indicated primers; n.d., not determined. (c) Specific RT-PCR results with primers based on fliP and fliQ and on fliR and flhB . Total RNA was isolated from P. putida DOT-T1E and DOT-T1E-42, and RT-PCRs were done with primers based on fliP and fliQ that yielded a 325-nucleotide (nt) band (lane 1) and fliR and flhB that yielded a 240-nt band (lane 2). Lane 3, negative control using primers based on fliP and fliQ but in the absence of reverse transcriptase. (d) Specific RT-PCR with primers based on fliR and flhB and on fliL and fliM . RNA was isolated from P. putida DOT-T1E and DOT-T1-PS23 and RT-PCRs were done with primers based on fliR and flhB that yielded a 240-nt band (lane 1) and fliL and fliM that yielded a 220-nt band (lane 3). Lane 2, negative control using primers based on fliR and flhB but in the absence of reverse transcriptase.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation, Negative Control

    22) Product Images from "RNA:DNA hybrids are a novel molecular pattern sensed by TLR9"

    Article Title: RNA:DNA hybrids are a novel molecular pattern sensed by TLR9

    Journal: The EMBO Journal

    doi: 10.1002/embj.201386117

    Viral RNA:DNA hybrids accumulate in the cytoplasm and endosomes of MMLV infected cells. A, B Viral RNA:DNA hybrids are present in the cytoplasm of MMLV infected cells. (A) MMLV-specific PCR detects RNA:DNA hybrids immunoprecipitated from cytoplasmic nucleic acids. As PCR detects both MMLV DNA and RNA:DNA hybrids, specificity of the S9.6 pull down for hybrids is confirmed by the absence of PCR product in control samples processed with either S9.6 antibody omitted or when cytoplasmic nucleic acids were pretreated with RNase H. Labels: H 2 O, PCR without template added; Input, cytoplasmic nucleic acids prior to immunopreciptation (equal in proportion to post-pull down template); Uninfected, cytoplasmic nucleic acids from uninfected B3T3 cells. B Quantification of MMLV RNA:DNA hybrids immunoprecipitated by S9.6 by qPCR using two independent MMLV primer sets. S9.6 immunoprecipitate contains 4.1 ± 1.1% of input molecules. RNase H treatment reduces the amount of MMLV-DNA pulled down by S9.6 by > 90%. Data shown are the mean of three independent IP/qPCR experiments ± s.d. * P = 0.0366 (MMLV-1), * P = 0.231 (MMLV-2). C Validation of endosomal fractionation: the early endosome marker Rab5 is enriched in endosomal preparations. Western blotting of cytoplasmic and endosomal fractions shows the presence of the endosomal marker Rab5 in both endosomal and cytoplasmic fractions, whereas GAPDH is only present in cytoplasmic fractions. Densitometry measurements show that relative Rab5 enrichment is > 22-fold relative to GAPDH. D Viral RNA:DNA hybrids are present in endosomal fractions of MMLV-infected cells. MMLV DNA was detected by PCR after S9.6 pull-down of hybrids from endosomal nucleic acids, but not in beads only or RNase H treated controls.
    Figure Legend Snippet: Viral RNA:DNA hybrids accumulate in the cytoplasm and endosomes of MMLV infected cells. A, B Viral RNA:DNA hybrids are present in the cytoplasm of MMLV infected cells. (A) MMLV-specific PCR detects RNA:DNA hybrids immunoprecipitated from cytoplasmic nucleic acids. As PCR detects both MMLV DNA and RNA:DNA hybrids, specificity of the S9.6 pull down for hybrids is confirmed by the absence of PCR product in control samples processed with either S9.6 antibody omitted or when cytoplasmic nucleic acids were pretreated with RNase H. Labels: H 2 O, PCR without template added; Input, cytoplasmic nucleic acids prior to immunopreciptation (equal in proportion to post-pull down template); Uninfected, cytoplasmic nucleic acids from uninfected B3T3 cells. B Quantification of MMLV RNA:DNA hybrids immunoprecipitated by S9.6 by qPCR using two independent MMLV primer sets. S9.6 immunoprecipitate contains 4.1 ± 1.1% of input molecules. RNase H treatment reduces the amount of MMLV-DNA pulled down by S9.6 by > 90%. Data shown are the mean of three independent IP/qPCR experiments ± s.d. * P = 0.0366 (MMLV-1), * P = 0.231 (MMLV-2). C Validation of endosomal fractionation: the early endosome marker Rab5 is enriched in endosomal preparations. Western blotting of cytoplasmic and endosomal fractions shows the presence of the endosomal marker Rab5 in both endosomal and cytoplasmic fractions, whereas GAPDH is only present in cytoplasmic fractions. Densitometry measurements show that relative Rab5 enrichment is > 22-fold relative to GAPDH. D Viral RNA:DNA hybrids are present in endosomal fractions of MMLV-infected cells. MMLV DNA was detected by PCR after S9.6 pull-down of hybrids from endosomal nucleic acids, but not in beads only or RNase H treated controls.

    Techniques Used: Infection, Polymerase Chain Reaction, Immunoprecipitation, Real-time Polymerase Chain Reaction, Fractionation, Marker, Western Blot

    TLR9 is activated by intact RNA-DNA hybrids. A, B The cytokine response (IL-6 (A), IFN-α (B)) of FLDCs to R:D60 is distinct from that of its single-stranded nucleic acid components. Day 8-FLDC cultures were FACS-sorted into pDC and cDC populations and transfected with R:D60 or equimolar amounts of ssRNA60 and ssDNA60. Data shown are the mean of three independent experiments ± s.e.m. C Cytokine induction by R:D60 but not ssRNA60 is TLR9-dependent. FLDCs derived from Tlr9 −/− and C57BL/6 mice were transfected as described for (A) and (B). Data shown are from two independent experiments ± s.d. D, E The cytokine response of FLDCs to R:D60 is not due to immunostimulatory contaminants remaining after FPLC. Nucleic acid hybridisation reactions were generated as outlined in Fig 1 A omitting one of the two oligonucleotides and fractionated using identical FPLC conditions to those used to purify annealed RNA:DNA hybrids. Upper panel: nucleic acids from mock hybridization reactions (minus ssRNA (D), minus ssDNA (E)) eluted later than the established elution position of R:D60 (arrow). The fractions (12.9 ml – 13.7 ml) corresponding to those that would usually contain the hybrid (“R:D fraction”) and fractions containing the nucleic acids at the indicated peaks, were concentrated by ethanol precipitation. Lower panels: 200 ng of FPLC-purified R:D60, 200 ng of hybridisation reaction column input (labelled “minus ssRNA” and “minus ssDNA” respectively), along with equal volumes of each fraction, were analysed by native PAGE. F Immunostimulatory contaminants are absent from the R:D60 fraction. FLDCs transfected with FPLC-purified R:D60 and the equivalent volume of R:D fractions from the hybridisations shown in (D) and (E) using Lipofectamine 2000. Supernatant concentrations of IL-6 (upper panel) were quantified 18 h post-transfection by ELISA and Ifna1 transcript levels (lower panel) normalised to Actb expression quantified 6 h post-transfection by qRT-PCR (fold mRNA induction from medium alone samples). Data shown are the mean of three experiments ± s.e.m. (IL-6, ** P = 0.0028) and PCR triplicates ± s.d. ( Ifna1 , ** P = 0.0001).
    Figure Legend Snippet: TLR9 is activated by intact RNA-DNA hybrids. A, B The cytokine response (IL-6 (A), IFN-α (B)) of FLDCs to R:D60 is distinct from that of its single-stranded nucleic acid components. Day 8-FLDC cultures were FACS-sorted into pDC and cDC populations and transfected with R:D60 or equimolar amounts of ssRNA60 and ssDNA60. Data shown are the mean of three independent experiments ± s.e.m. C Cytokine induction by R:D60 but not ssRNA60 is TLR9-dependent. FLDCs derived from Tlr9 −/− and C57BL/6 mice were transfected as described for (A) and (B). Data shown are from two independent experiments ± s.d. D, E The cytokine response of FLDCs to R:D60 is not due to immunostimulatory contaminants remaining after FPLC. Nucleic acid hybridisation reactions were generated as outlined in Fig 1 A omitting one of the two oligonucleotides and fractionated using identical FPLC conditions to those used to purify annealed RNA:DNA hybrids. Upper panel: nucleic acids from mock hybridization reactions (minus ssRNA (D), minus ssDNA (E)) eluted later than the established elution position of R:D60 (arrow). The fractions (12.9 ml – 13.7 ml) corresponding to those that would usually contain the hybrid (“R:D fraction”) and fractions containing the nucleic acids at the indicated peaks, were concentrated by ethanol precipitation. Lower panels: 200 ng of FPLC-purified R:D60, 200 ng of hybridisation reaction column input (labelled “minus ssRNA” and “minus ssDNA” respectively), along with equal volumes of each fraction, were analysed by native PAGE. F Immunostimulatory contaminants are absent from the R:D60 fraction. FLDCs transfected with FPLC-purified R:D60 and the equivalent volume of R:D fractions from the hybridisations shown in (D) and (E) using Lipofectamine 2000. Supernatant concentrations of IL-6 (upper panel) were quantified 18 h post-transfection by ELISA and Ifna1 transcript levels (lower panel) normalised to Actb expression quantified 6 h post-transfection by qRT-PCR (fold mRNA induction from medium alone samples). Data shown are the mean of three experiments ± s.e.m. (IL-6, ** P = 0.0028) and PCR triplicates ± s.d. ( Ifna1 , ** P = 0.0001).

    Techniques Used: FACS, Transfection, Derivative Assay, Mouse Assay, Fast Protein Liquid Chromatography, Hybridization, Generated, Ethanol Precipitation, Purification, Clear Native PAGE, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Polymerase Chain Reaction

    23) Product Images from "Phloem small RNAs, nutrient stress responses, and systemic mobility"

    Article Title: Phloem small RNAs, nutrient stress responses, and systemic mobility

    Journal: BMC Plant Biology

    doi: 10.1186/1471-2229-10-64

    WT/ hen1-1 grafting experiments . Analysis of mature miR395, miR399 and miR171 by RNA gel blot analysis in scions and rootstocks of reciprocal hen1-1 /WT and WT/ hen1-1 grafts under sulfate and phosphate deficiency. A : miRNAs 395 and 399 were translocated from WT scions to hen1-1 rootstocks but not in the opposite direction, miR171 was immobile. One representative result is shown for WT, and three replications for hen1-1 roots and shoots. The hen1-1 graft parts kept their growth retardation phenotype, indicating that not all necessary miRNAs could be transferred. The 5.8 ribosomal RNA band served as a loading control. B : Control of miR395 expression in WT and hen1-1 mutant plants. In WT plants miR395 was induced by sulfate deficiency in shoots and roots, while no signal was detected in hen1-1 mutants under both conditions.
    Figure Legend Snippet: WT/ hen1-1 grafting experiments . Analysis of mature miR395, miR399 and miR171 by RNA gel blot analysis in scions and rootstocks of reciprocal hen1-1 /WT and WT/ hen1-1 grafts under sulfate and phosphate deficiency. A : miRNAs 395 and 399 were translocated from WT scions to hen1-1 rootstocks but not in the opposite direction, miR171 was immobile. One representative result is shown for WT, and three replications for hen1-1 roots and shoots. The hen1-1 graft parts kept their growth retardation phenotype, indicating that not all necessary miRNAs could be transferred. The 5.8 ribosomal RNA band served as a loading control. B : Control of miR395 expression in WT and hen1-1 mutant plants. In WT plants miR395 was induced by sulfate deficiency in shoots and roots, while no signal was detected in hen1-1 mutants under both conditions.

    Techniques Used: Western Blot, Expressing, Mutagenesis

    24) Product Images from "The NAC Transcription Factor Gene OsY37 (ONAC011) Promotes Leaf Senescence and Accelerates Heading Time in Rice"

    Article Title: The NAC Transcription Factor Gene OsY37 (ONAC011) Promotes Leaf Senescence and Accelerates Heading Time in Rice

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18102165

    Transcriptional gene expression determined by reverse transcription polymerase chain reaction (RT-PCR) analysis. Total RNA was extracted from flag leaves at the indicated days after heading. Semi-quantititative RT-PCR was performed using OsY37 primers Osy2 and Osy752 which give rise to the amplification of 754 DNA fragment ( a – d ). To amplify OsY37 SRDX transcript, OsY37- specific forward primer; OsyPri1 and SRDX-specific reverse primer; SRDX-R were used . Actin gene expression analysis was carried out similarly with forward; OsActinF318 and reverse; OsActinR675 primers for internal standard. Wild-type and OE lines of Nipponbare ( a ); Kinmaze ( b ); RNAi lines of Nipponbare ( c ); Kinmaze ( d ); and SRDX lines of Nipponbare ( e ). Names of plant lines are shown in Figure 4 .
    Figure Legend Snippet: Transcriptional gene expression determined by reverse transcription polymerase chain reaction (RT-PCR) analysis. Total RNA was extracted from flag leaves at the indicated days after heading. Semi-quantititative RT-PCR was performed using OsY37 primers Osy2 and Osy752 which give rise to the amplification of 754 DNA fragment ( a – d ). To amplify OsY37 SRDX transcript, OsY37- specific forward primer; OsyPri1 and SRDX-specific reverse primer; SRDX-R were used . Actin gene expression analysis was carried out similarly with forward; OsActinF318 and reverse; OsActinR675 primers for internal standard. Wild-type and OE lines of Nipponbare ( a ); Kinmaze ( b ); RNAi lines of Nipponbare ( c ); Kinmaze ( d ); and SRDX lines of Nipponbare ( e ). Names of plant lines are shown in Figure 4 .

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

    25) Product Images from "An Orphan Seven-Transmembrane Domain Receptor Expressed Widely in the Brain Functions as a Coreceptor for Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus"

    Article Title: An Orphan Seven-Transmembrane Domain Receptor Expressed Widely in the Brain Functions as a Coreceptor for Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus

    Journal: Journal of Virology

    doi:

    Expression of APJ in primary cells and in cell lines. RNA from the indicated cells was used in one-tube RT-PCRs and 10 μl of each 25-μl reaction mixture was run on a 2% agarose gel. Alternatively, Superscript was used to generate cDNA from DNase-treated RNA obtained from microglia, oligodendrocytes (OLIGO), NT2 cells, and NT2N cells. The size of the predicted APJ band is 481 bp. Both plasmid DNA and U87-APJ RNA are included as positive controls; water was used as template for a negative control. MONO, monocytes; MAC, macrophages.
    Figure Legend Snippet: Expression of APJ in primary cells and in cell lines. RNA from the indicated cells was used in one-tube RT-PCRs and 10 μl of each 25-μl reaction mixture was run on a 2% agarose gel. Alternatively, Superscript was used to generate cDNA from DNase-treated RNA obtained from microglia, oligodendrocytes (OLIGO), NT2 cells, and NT2N cells. The size of the predicted APJ band is 481 bp. Both plasmid DNA and U87-APJ RNA are included as positive controls; water was used as template for a negative control. MONO, monocytes; MAC, macrophages.

    Techniques Used: Expressing, Agarose Gel Electrophoresis, Plasmid Preparation, Negative Control

    26) Product Images from "Programmed Cell Death in the Leaves of the Arabidopsis Spontaneous Necrotic Spots (sns-D) Mutant Correlates with Increased Expression of the Eukaryotic Translation Initiation Factor eIF4B2"

    Article Title: Programmed Cell Death in the Leaves of the Arabidopsis Spontaneous Necrotic Spots (sns-D) Mutant Correlates with Increased Expression of the Eukaryotic Translation Initiation Factor eIF4B2

    Journal: Frontiers in plant science

    doi: 10.3389/fpls.2011.00009

    Expression levels of genes flanking the T-DNA/vector insert . (A) Relative expression levels of At1g13020 (dark gray bars) and At1g21550 (light gray bars) in wild-type plants (WT) and in sns-D mutants with weak and strong phenotypes as determined via Q-RT-PCR. The expression level in the wild-type was set on 1 and the SD in the mutants was indicated by error bars. (B) Northern blot analysis of At1g13020 in the wild-type (W) and the sns-D mutant (S) at different growth stages. G, just germinated; 1, 1 week after germination; 2, 2 weeks after germination; 3, 3 weeks after germination; 4, 4 weeks after germination; 5, 5 weeks after germination; Sw, sns-D plants with weak phenotype; Ss, sns-D plants with strong phenotype. Loading of RNA was determined by quantification of the bands after hybridization with At4g38740 (cyclophilin). Relative expression of At1g13020 in the mutant plants was shown for each developmental stage. The expression in the wild-type was set on 1.
    Figure Legend Snippet: Expression levels of genes flanking the T-DNA/vector insert . (A) Relative expression levels of At1g13020 (dark gray bars) and At1g21550 (light gray bars) in wild-type plants (WT) and in sns-D mutants with weak and strong phenotypes as determined via Q-RT-PCR. The expression level in the wild-type was set on 1 and the SD in the mutants was indicated by error bars. (B) Northern blot analysis of At1g13020 in the wild-type (W) and the sns-D mutant (S) at different growth stages. G, just germinated; 1, 1 week after germination; 2, 2 weeks after germination; 3, 3 weeks after germination; 4, 4 weeks after germination; 5, 5 weeks after germination; Sw, sns-D plants with weak phenotype; Ss, sns-D plants with strong phenotype. Loading of RNA was determined by quantification of the bands after hybridization with At4g38740 (cyclophilin). Relative expression of At1g13020 in the mutant plants was shown for each developmental stage. The expression in the wild-type was set on 1.

    Techniques Used: Expressing, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Mutagenesis, Hybridization

    27) Product Images from "Programmed Cell Death in the Leaves of the Arabidopsis Spontaneous Necrotic Spots (sns-D) Mutant Correlates with Increased Expression of the Eukaryotic Translation Initiation Factor eIF4B2"

    Article Title: Programmed Cell Death in the Leaves of the Arabidopsis Spontaneous Necrotic Spots (sns-D) Mutant Correlates with Increased Expression of the Eukaryotic Translation Initiation Factor eIF4B2

    Journal: Frontiers in plant science

    doi: 10.3389/fpls.2011.00009

    Expression levels of genes flanking the T-DNA/vector insert . (A) Relative expression levels of At1g13020 (dark gray bars) and At1g21550 (light gray bars) in wild-type plants (WT) and in sns-D mutants with weak and strong phenotypes as determined via Q-RT-PCR. The expression level in the wild-type was set on 1 and the SD in the mutants was indicated by error bars. (B) Northern blot analysis of At1g13020 in the wild-type (W) and the sns-D mutant (S) at different growth stages. G, just germinated; 1, 1 week after germination; 2, 2 weeks after germination; 3, 3 weeks after germination; 4, 4 weeks after germination; 5, 5 weeks after germination; Sw, sns-D plants with weak phenotype; Ss, sns-D plants with strong phenotype. Loading of RNA was determined by quantification of the bands after hybridization with At4g38740 (cyclophilin). Relative expression of At1g13020 in the mutant plants was shown for each developmental stage. The expression in the wild-type was set on 1.
    Figure Legend Snippet: Expression levels of genes flanking the T-DNA/vector insert . (A) Relative expression levels of At1g13020 (dark gray bars) and At1g21550 (light gray bars) in wild-type plants (WT) and in sns-D mutants with weak and strong phenotypes as determined via Q-RT-PCR. The expression level in the wild-type was set on 1 and the SD in the mutants was indicated by error bars. (B) Northern blot analysis of At1g13020 in the wild-type (W) and the sns-D mutant (S) at different growth stages. G, just germinated; 1, 1 week after germination; 2, 2 weeks after germination; 3, 3 weeks after germination; 4, 4 weeks after germination; 5, 5 weeks after germination; Sw, sns-D plants with weak phenotype; Ss, sns-D plants with strong phenotype. Loading of RNA was determined by quantification of the bands after hybridization with At4g38740 (cyclophilin). Relative expression of At1g13020 in the mutant plants was shown for each developmental stage. The expression in the wild-type was set on 1.

    Techniques Used: Expressing, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Mutagenesis, Hybridization

    28) Product Images from "Role of the Single Regulator MrsR1 and the Two-Component System MrsR2/K2 in the Regulation of Mersacidin Production and Immunity"

    Article Title: Role of the Single Regulator MrsR1 and the Two-Component System MrsR2/K2 in the Regulation of Mersacidin Production and Immunity

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.68.1.106-113.2002

    RT-PCR of total RNA of Bacillus sp. strain TTΔ mrsR2/K2 and the wild-type strain. Cells were harvested after 8 (A) or 16 (B) h of incubation. Slots 3, 5, 8, and 10 contain control reactions for the samples presented in slots 2, 4, 7, and 9, respectively. These controls were performed to rule out contamination with chromosomal DNA by introducing parallel samples into the thermal cycler during the 95°C step, thereby ensuring immediate denaturation of the reverse transcriptase enzyme. Slots 1 and 11, GeneRuler DNA ladder mix; slot 2, RT-PCR of the mrsEFG transcript in the wild-type Bacillus sp. strain HIL Y-85,54728 with RT1/RT2; slot 3, control; slot 4, RT-PCR of the mrsEFG -transcript in Bacillus sp. strain TTΔ mrsR2/K2 ; slot 5, control; 6, GeneRuler 100-bp DNA ladder mix (1.031, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, and 0.2 kb); slot 7, RT-PCR of the mrsA transcript in Bacillus sp. strain HIL Y-85,54728 using RT4/RT5; slot 8, control; slot 9, RT-PCR of the mrsA -transcript in Bacillus sp. strain TTΔ mrsR2/K2 ; slot 10, control.
    Figure Legend Snippet: RT-PCR of total RNA of Bacillus sp. strain TTΔ mrsR2/K2 and the wild-type strain. Cells were harvested after 8 (A) or 16 (B) h of incubation. Slots 3, 5, 8, and 10 contain control reactions for the samples presented in slots 2, 4, 7, and 9, respectively. These controls were performed to rule out contamination with chromosomal DNA by introducing parallel samples into the thermal cycler during the 95°C step, thereby ensuring immediate denaturation of the reverse transcriptase enzyme. Slots 1 and 11, GeneRuler DNA ladder mix; slot 2, RT-PCR of the mrsEFG transcript in the wild-type Bacillus sp. strain HIL Y-85,54728 with RT1/RT2; slot 3, control; slot 4, RT-PCR of the mrsEFG -transcript in Bacillus sp. strain TTΔ mrsR2/K2 ; slot 5, control; 6, GeneRuler 100-bp DNA ladder mix (1.031, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, and 0.2 kb); slot 7, RT-PCR of the mrsA transcript in Bacillus sp. strain HIL Y-85,54728 using RT4/RT5; slot 8, control; slot 9, RT-PCR of the mrsA -transcript in Bacillus sp. strain TTΔ mrsR2/K2 ; slot 10, control.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Incubation

    29) Product Images from "Ghrelin stimulates neurogenesis in the dorsal motor nucleus of the vagus"

    Article Title: Ghrelin stimulates neurogenesis in the dorsal motor nucleus of the vagus

    Journal: The Journal of Physiology

    doi: 10.1113/jphysiol.2004.064121

    Expression of ghrelin receptor in DMNV A , RT-PCR products of RNA extracted from 3- to 5-day-old rat DMNV tissues corresponding to ghrelin receptor coding sequences (116 bp) are shown in lanes numbered 1 to 5 (each lane represents result from one individual rat, the numbers correspond to each rat). Lanes M show molecular size markers. Other lanes represent the positive control (+) from hypothalamus and the negative control (–) (RT-PCR performed without RNA reverse transcription). The upper panel represents ghrelin receptor mRNA, whereas the lower panel shows β-actin mRNA (181 bp). B , ghrelin receptor immunoreactivity was localized in DMNV neurones by a specific antibody against the 330–366 amino acid sequence of the human ghrelin receptor 1a (upper panel). Control tissue incubated with rabbit IgG showed no significant staining (lower panel). Scale bar represents 100 μm.
    Figure Legend Snippet: Expression of ghrelin receptor in DMNV A , RT-PCR products of RNA extracted from 3- to 5-day-old rat DMNV tissues corresponding to ghrelin receptor coding sequences (116 bp) are shown in lanes numbered 1 to 5 (each lane represents result from one individual rat, the numbers correspond to each rat). Lanes M show molecular size markers. Other lanes represent the positive control (+) from hypothalamus and the negative control (–) (RT-PCR performed without RNA reverse transcription). The upper panel represents ghrelin receptor mRNA, whereas the lower panel shows β-actin mRNA (181 bp). B , ghrelin receptor immunoreactivity was localized in DMNV neurones by a specific antibody against the 330–366 amino acid sequence of the human ghrelin receptor 1a (upper panel). Control tissue incubated with rabbit IgG showed no significant staining (lower panel). Scale bar represents 100 μm.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Sequencing, Incubation, Staining

    30) Product Images from "Persistent effects of Libby amphibole and amosite asbestos following subchronic inhalation in rats"

    Article Title: Persistent effects of Libby amphibole and amosite asbestos following subchronic inhalation in rats

    Journal: Particle and Fibre Toxicology

    doi: 10.1186/s12989-016-0130-z

    Short-term inhalation study: transcriptional markers of apoptosis and inflammation in lung samples after final exposure to AM or LA for 10 days. Results show relative mean values ± SE of lung tissue mRNA for inflammasome pathway components ( Nlrp3 , Pycard , Casp1 ), downstream cytokines ( Il1b and Il18 ), and other pro-inflammatory cytokines ( Il6 , Tnfa , Cxcl2 ) as determined by RT-qPCR ( n = 7 rats per group). P
    Figure Legend Snippet: Short-term inhalation study: transcriptional markers of apoptosis and inflammation in lung samples after final exposure to AM or LA for 10 days. Results show relative mean values ± SE of lung tissue mRNA for inflammasome pathway components ( Nlrp3 , Pycard , Casp1 ), downstream cytokines ( Il1b and Il18 ), and other pro-inflammatory cytokines ( Il6 , Tnfa , Cxcl2 ) as determined by RT-qPCR ( n = 7 rats per group). P

    Techniques Used: Quantitative RT-PCR

    31) Product Images from "Phloem small RNAs, nutrient stress responses, and systemic mobility"

    Article Title: Phloem small RNAs, nutrient stress responses, and systemic mobility

    Journal: BMC Plant Biology

    doi: 10.1186/1471-2229-10-64

    List of nutrient-responsive sRNAs . List of sRNAs that showed a strong positive reaction to S, Cu or Fe deprivation, respectively, shown as log2 values of stressed vs. FN samples. Only sRNAs that fulfilled the criteria described in the Methods section (positive response, log2 > 2 in one of the stress treatments, signal value > 100 in FN or deprived sample) in at least one of the comparisons are listed. The insets show results obtained by miRNA sqRT-PCR (after 25 cycles) from an independent experiment. To allow a better overview, values for known nutrient starvation-responsive miRNAs (398 and 857 for -Cu and 2111 for -P) were included, although they only showed a negative response or were not detectable. Arrows indicate directions of changes obtained in a second, independent -Cu experiment. n.d.: not detectable (both, FN and stress, signal values
    Figure Legend Snippet: List of nutrient-responsive sRNAs . List of sRNAs that showed a strong positive reaction to S, Cu or Fe deprivation, respectively, shown as log2 values of stressed vs. FN samples. Only sRNAs that fulfilled the criteria described in the Methods section (positive response, log2 > 2 in one of the stress treatments, signal value > 100 in FN or deprived sample) in at least one of the comparisons are listed. The insets show results obtained by miRNA sqRT-PCR (after 25 cycles) from an independent experiment. To allow a better overview, values for known nutrient starvation-responsive miRNAs (398 and 857 for -Cu and 2111 for -P) were included, although they only showed a negative response or were not detectable. Arrows indicate directions of changes obtained in a second, independent -Cu experiment. n.d.: not detectable (both, FN and stress, signal values

    Techniques Used: Polymerase Chain Reaction

    32) Product Images from "Phloem small RNAs, nutrient stress responses, and systemic mobility"

    Article Title: Phloem small RNAs, nutrient stress responses, and systemic mobility

    Journal: BMC Plant Biology

    doi: 10.1186/1471-2229-10-64

    List of nutrient-responsive sRNAs . List of sRNAs that showed a strong positive reaction to S, Cu or Fe deprivation, respectively, shown as log2 values of stressed vs. FN samples. Only sRNAs that fulfilled the criteria described in the Methods section (positive response, log2 > 2 in one of the stress treatments, signal value > 100 in FN or deprived sample) in at least one of the comparisons are listed. The insets show results obtained by miRNA sqRT-PCR (after 25 cycles) from an independent experiment. To allow a better overview, values for known nutrient starvation-responsive miRNAs (398 and 857 for -Cu and 2111 for -P) were included, although they only showed a negative response or were not detectable. Arrows indicate directions of changes obtained in a second, independent -Cu experiment. n.d.: not detectable (both, FN and stress, signal values
    Figure Legend Snippet: List of nutrient-responsive sRNAs . List of sRNAs that showed a strong positive reaction to S, Cu or Fe deprivation, respectively, shown as log2 values of stressed vs. FN samples. Only sRNAs that fulfilled the criteria described in the Methods section (positive response, log2 > 2 in one of the stress treatments, signal value > 100 in FN or deprived sample) in at least one of the comparisons are listed. The insets show results obtained by miRNA sqRT-PCR (after 25 cycles) from an independent experiment. To allow a better overview, values for known nutrient starvation-responsive miRNAs (398 and 857 for -Cu and 2111 for -P) were included, although they only showed a negative response or were not detectable. Arrows indicate directions of changes obtained in a second, independent -Cu experiment. n.d.: not detectable (both, FN and stress, signal values

    Techniques Used: Polymerase Chain Reaction

    33) Product Images from "An Orphan Seven-Transmembrane Domain Receptor Expressed Widely in the Brain Functions as a Coreceptor for Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus"

    Article Title: An Orphan Seven-Transmembrane Domain Receptor Expressed Widely in the Brain Functions as a Coreceptor for Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus

    Journal: Journal of Virology

    doi:

    Expression of APJ in primary cells and in cell lines. RNA from the indicated cells was used in one-tube RT-PCRs and 10 μl of each 25-μl reaction mixture was run on a 2% agarose gel. Alternatively, Superscript was used to generate cDNA from DNase-treated RNA obtained from microglia, oligodendrocytes (OLIGO), NT2 cells, and NT2N cells. The size of the predicted APJ band is 481 bp. Both plasmid DNA and U87-APJ RNA are included as positive controls; water was used as template for a negative control. MONO, monocytes; MAC, macrophages.
    Figure Legend Snippet: Expression of APJ in primary cells and in cell lines. RNA from the indicated cells was used in one-tube RT-PCRs and 10 μl of each 25-μl reaction mixture was run on a 2% agarose gel. Alternatively, Superscript was used to generate cDNA from DNase-treated RNA obtained from microglia, oligodendrocytes (OLIGO), NT2 cells, and NT2N cells. The size of the predicted APJ band is 481 bp. Both plasmid DNA and U87-APJ RNA are included as positive controls; water was used as template for a negative control. MONO, monocytes; MAC, macrophages.

    Techniques Used: Expressing, Agarose Gel Electrophoresis, Plasmid Preparation, Negative Control

    34) Product Images from "Tumor Suppressor PDCD4 Represses Internal Ribosome Entry Site-Mediated Translation of Antiapoptotic Proteins and Is Regulated by S6 Kinase 2"

    Article Title: Tumor Suppressor PDCD4 Represses Internal Ribosome Entry Site-Mediated Translation of Antiapoptotic Proteins and Is Regulated by S6 Kinase 2

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.06317-11

    Activation of S6K2 leads to PDCD4 degradation, with a concomitant increase in XIAP and Bcl-x L protein levels. (A) TOKAS6K2-stable or control cells (transiently transfected with an inducible pTripz-Kate plasmid) were treated with doxycycline (Dox) (1 μg/ml)
    Figure Legend Snippet: Activation of S6K2 leads to PDCD4 degradation, with a concomitant increase in XIAP and Bcl-x L protein levels. (A) TOKAS6K2-stable or control cells (transiently transfected with an inducible pTripz-Kate plasmid) were treated with doxycycline (Dox) (1 μg/ml)

    Techniques Used: Activation Assay, Transfection, Plasmid Preparation

    Loss of PDCD4 correlates with an increase in XIAP and Bcl-x L translation through their respective IRES elements. (A) HEK293T cells were treated with PDCD4 siRNA or control (CTRL), nontargeting siRNA. Cell lysates were harvested and subjected to Western
    Figure Legend Snippet: Loss of PDCD4 correlates with an increase in XIAP and Bcl-x L translation through their respective IRES elements. (A) HEK293T cells were treated with PDCD4 siRNA or control (CTRL), nontargeting siRNA. Cell lysates were harvested and subjected to Western

    Techniques Used: Western Blot

    PDCD4 specifically binds to XIAP and Bcl-x L IRES RNA both in vitro and in vivo . (A) Recombinant His-PDCD4 was incubated in the presence of 32 P-labeled, in vitro -transcribed RNA and subjected to UV cross-linking. RNA-protein complexes were separated by
    Figure Legend Snippet: PDCD4 specifically binds to XIAP and Bcl-x L IRES RNA both in vitro and in vivo . (A) Recombinant His-PDCD4 was incubated in the presence of 32 P-labeled, in vitro -transcribed RNA and subjected to UV cross-linking. RNA-protein complexes were separated by

    Techniques Used: In Vitro, In Vivo, Recombinant, Incubation, Labeling

    35) Product Images from "RNA aptamers to initiation factor 4A helicase hinder cap-dependent translation by blocking ATP hydrolysis"

    Article Title: RNA aptamers to initiation factor 4A helicase hinder cap-dependent translation by blocking ATP hydrolysis

    Journal: RNA

    doi: 10.1261/rna.2161303

    RNA-binding specificity of wild-type and mutant eIF4A proteins. Shown mostly is the percentage of input RNA that bound to the nitrocellulose filter with various concentrations of the selected RNAs. Experiments were performed independently at least three times and the values are expressed with or without standard deviations. ( A ) Nitrocellulose filter-binding assays of selected RNAs for wild-type eIF4A. (×) N40 random pool; (▪) no. 1; (•) no. 11; (▴) no. 20; (▾) no. 21; (♦) no. 30. ( B ) Binding affinity of RNA no. 20 in the absence (•) or presence of ATP (▪), ADP (▴) and ATPγS (▾). ( C ) Sensorgrams of RNA no. 21 binding to eIF4A. Each RNA sample with the indicated concentration was injected to the flow cells immobilized with wild-type eIF4A as well as to the control cell. Experimental details are described in Materials and Methods. ( D ) Nitrocellulose filter-binding assays of RNA no. 20 for eIF4A fragments. eIF4A samples: (×) wild-type; (▪) amino-terminal fragment N1–235; (•) carboxy-terminal fragment C216–406; (▴) N1–235 + C216–406. ( E ) Nitrocellulose filter-binding assays of RNA no. 20 for eIF4A mutants. eIF4A proteins: (×) wild-type; (▪) R362Q; (•) DQAD; and (▴) PRRVAA. ( F ) Pull-down assay of eIF4A and eIF4G613–1560 binary complex. Ni-NTA agarose resin conjugated with eIF4A (0.4 μM) was mixed with Flag-tagged eIF4G613–1560 (0.4 μM) in the presence of N40 and selected RNAs, and formed complexes were spun down. Bound eIF4G613–1560 and eIF4A were detected by Western blotting using anti-Flag antibody (upper panel) and by Coomassie staining ( lower panel), respectively. Experimental details are described in Materials and Methods. Proteins: (lane 1 ) eIF4A alone; (lane 2 ) eIF4G613–1560 alone; (lanes 3–9 ) eIF4A plus eIF4G613–1560. RNAs: (lanes 1–3 ) none; (lane 4 ) N40 (2 μM); (lane 5 ) N40 (10 μM); (lane 6 ) no. 1 (2 μM); (lane 7 ) no. 1 (10 μM); (lane 8 ) no. 20 (2 μM); (lane 9 ) no. 20 (10 μM).
    Figure Legend Snippet: RNA-binding specificity of wild-type and mutant eIF4A proteins. Shown mostly is the percentage of input RNA that bound to the nitrocellulose filter with various concentrations of the selected RNAs. Experiments were performed independently at least three times and the values are expressed with or without standard deviations. ( A ) Nitrocellulose filter-binding assays of selected RNAs for wild-type eIF4A. (×) N40 random pool; (▪) no. 1; (•) no. 11; (▴) no. 20; (▾) no. 21; (♦) no. 30. ( B ) Binding affinity of RNA no. 20 in the absence (•) or presence of ATP (▪), ADP (▴) and ATPγS (▾). ( C ) Sensorgrams of RNA no. 21 binding to eIF4A. Each RNA sample with the indicated concentration was injected to the flow cells immobilized with wild-type eIF4A as well as to the control cell. Experimental details are described in Materials and Methods. ( D ) Nitrocellulose filter-binding assays of RNA no. 20 for eIF4A fragments. eIF4A samples: (×) wild-type; (▪) amino-terminal fragment N1–235; (•) carboxy-terminal fragment C216–406; (▴) N1–235 + C216–406. ( E ) Nitrocellulose filter-binding assays of RNA no. 20 for eIF4A mutants. eIF4A proteins: (×) wild-type; (▪) R362Q; (•) DQAD; and (▴) PRRVAA. ( F ) Pull-down assay of eIF4A and eIF4G613–1560 binary complex. Ni-NTA agarose resin conjugated with eIF4A (0.4 μM) was mixed with Flag-tagged eIF4G613–1560 (0.4 μM) in the presence of N40 and selected RNAs, and formed complexes were spun down. Bound eIF4G613–1560 and eIF4A were detected by Western blotting using anti-Flag antibody (upper panel) and by Coomassie staining ( lower panel), respectively. Experimental details are described in Materials and Methods. Proteins: (lane 1 ) eIF4A alone; (lane 2 ) eIF4G613–1560 alone; (lanes 3–9 ) eIF4A plus eIF4G613–1560. RNAs: (lanes 1–3 ) none; (lane 4 ) N40 (2 μM); (lane 5 ) N40 (10 μM); (lane 6 ) no. 1 (2 μM); (lane 7 ) no. 1 (10 μM); (lane 8 ) no. 20 (2 μM); (lane 9 ) no. 20 (10 μM).

    Techniques Used: RNA Binding Assay, Mutagenesis, Binding Assay, Concentration Assay, Injection, Flow Cytometry, Pull Down Assay, Western Blot, Staining

    Selected RNA sequences with affinity for eIF4A. ( A ) Representative RNA sequences selected from randomized RNA libraries using different selection procedures. In selection I(+) and I(−), RNAs were selected from N40 pool in the presence and absence of ATP, respectively, via affinity precipitation with Ni-NTA agarose. Selection II and III used N40 and N30 pools, respectively, and RNAs were selected in the absence of ATP via nitrocellulose membrane trapping. The frequency of each sequence in these selections is shown as numbers of each clone found in 48 independent isolates. Boxed sequences indicate the consensus elements I, II, and III. According to consensus elements, RNAs in selection I are classified into two groups, group I and II. ( B ) Predicted secondary structures of certain RNA ligands selected by eIF4A. Selected sequences are shown in capital letters. Lowercase letters indicate the constant sequence regions flanking the random sequence. Conserved elements I, II, and III are shaded.
    Figure Legend Snippet: Selected RNA sequences with affinity for eIF4A. ( A ) Representative RNA sequences selected from randomized RNA libraries using different selection procedures. In selection I(+) and I(−), RNAs were selected from N40 pool in the presence and absence of ATP, respectively, via affinity precipitation with Ni-NTA agarose. Selection II and III used N40 and N30 pools, respectively, and RNAs were selected in the absence of ATP via nitrocellulose membrane trapping. The frequency of each sequence in these selections is shown as numbers of each clone found in 48 independent isolates. Boxed sequences indicate the consensus elements I, II, and III. According to consensus elements, RNAs in selection I are classified into two groups, group I and II. ( B ) Predicted secondary structures of certain RNA ligands selected by eIF4A. Selected sequences are shown in capital letters. Lowercase letters indicate the constant sequence regions flanking the random sequence. Conserved elements I, II, and III are shaded.

    Techniques Used: Selection, Affinity Precipitation, Sequencing

    36) Product Images from "An Integrated Polysome Profiling and Ribosome Profiling Method to Investigate In Vivo Translatome"

    Article Title: An Integrated Polysome Profiling and Ribosome Profiling Method to Investigate In Vivo Translatome

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-4939-7514-3_1

    Size selection of ribosome footprints from RNase I treated monosome fractions. Representative gel images before and after gel cutting are shown. Brackets indicate the location of excised gel bands
    Figure Legend Snippet: Size selection of ribosome footprints from RNase I treated monosome fractions. Representative gel images before and after gel cutting are shown. Brackets indicate the location of excised gel bands

    Techniques Used: Selection

    37) Product Images from "Ghrelin stimulates neurogenesis in the dorsal motor nucleus of the vagus"

    Article Title: Ghrelin stimulates neurogenesis in the dorsal motor nucleus of the vagus

    Journal: The Journal of Physiology

    doi: 10.1113/jphysiol.2004.064121

    Expression of ghrelin receptor in DMNV A , RT-PCR products of RNA extracted from 3- to 5-day-old rat DMNV tissues corresponding to ghrelin receptor coding sequences (116 bp) are shown in lanes numbered 1 to 5 (each lane represents result from one individual rat, the numbers correspond to each rat). Lanes M show molecular size markers. Other lanes represent the positive control (+) from hypothalamus and the negative control (–) (RT-PCR performed without RNA reverse transcription). The upper panel represents ghrelin receptor mRNA, whereas the lower panel shows β-actin mRNA (181 bp). B , ghrelin receptor immunoreactivity was localized in DMNV neurones by a specific antibody against the 330–366 amino acid sequence of the human ghrelin receptor 1a (upper panel). Control tissue incubated with rabbit IgG showed no significant staining (lower panel). Scale bar represents 100 μm.
    Figure Legend Snippet: Expression of ghrelin receptor in DMNV A , RT-PCR products of RNA extracted from 3- to 5-day-old rat DMNV tissues corresponding to ghrelin receptor coding sequences (116 bp) are shown in lanes numbered 1 to 5 (each lane represents result from one individual rat, the numbers correspond to each rat). Lanes M show molecular size markers. Other lanes represent the positive control (+) from hypothalamus and the negative control (–) (RT-PCR performed without RNA reverse transcription). The upper panel represents ghrelin receptor mRNA, whereas the lower panel shows β-actin mRNA (181 bp). B , ghrelin receptor immunoreactivity was localized in DMNV neurones by a specific antibody against the 330–366 amino acid sequence of the human ghrelin receptor 1a (upper panel). Control tissue incubated with rabbit IgG showed no significant staining (lower panel). Scale bar represents 100 μm.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Sequencing, Incubation, Staining

    38) Product Images from "Programmed Cell Death in the Leaves of the Arabidopsis Spontaneous Necrotic Spots (sns-D) Mutant Correlates with Increased Expression of the Eukaryotic Translation Initiation Factor eIF4B2"

    Article Title: Programmed Cell Death in the Leaves of the Arabidopsis Spontaneous Necrotic Spots (sns-D) Mutant Correlates with Increased Expression of the Eukaryotic Translation Initiation Factor eIF4B2

    Journal: Frontiers in plant science

    doi: 10.3389/fpls.2011.00009

    Expression levels of genes flanking the T-DNA/vector insert . (A) Relative expression levels of At1g13020 (dark gray bars) and At1g21550 (light gray bars) in wild-type plants (WT) and in sns-D mutants with weak and strong phenotypes as determined via Q-RT-PCR. The expression level in the wild-type was set on 1 and the SD in the mutants was indicated by error bars. (B) Northern blot analysis of At1g13020 in the wild-type (W) and the sns-D mutant (S) at different growth stages. G, just germinated; 1, 1 week after germination; 2, 2 weeks after germination; 3, 3 weeks after germination; 4, 4 weeks after germination; 5, 5 weeks after germination; Sw, sns-D plants with weak phenotype; Ss, sns-D plants with strong phenotype. Loading of RNA was determined by quantification of the bands after hybridization with At4g38740 (cyclophilin). Relative expression of At1g13020 in the mutant plants was shown for each developmental stage. The expression in the wild-type was set on 1.
    Figure Legend Snippet: Expression levels of genes flanking the T-DNA/vector insert . (A) Relative expression levels of At1g13020 (dark gray bars) and At1g21550 (light gray bars) in wild-type plants (WT) and in sns-D mutants with weak and strong phenotypes as determined via Q-RT-PCR. The expression level in the wild-type was set on 1 and the SD in the mutants was indicated by error bars. (B) Northern blot analysis of At1g13020 in the wild-type (W) and the sns-D mutant (S) at different growth stages. G, just germinated; 1, 1 week after germination; 2, 2 weeks after germination; 3, 3 weeks after germination; 4, 4 weeks after germination; 5, 5 weeks after germination; Sw, sns-D plants with weak phenotype; Ss, sns-D plants with strong phenotype. Loading of RNA was determined by quantification of the bands after hybridization with At4g38740 (cyclophilin). Relative expression of At1g13020 in the mutant plants was shown for each developmental stage. The expression in the wild-type was set on 1.

    Techniques Used: Expressing, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Mutagenesis, Hybridization

    39) Product Images from "The bacterial antitoxin HipB establishes a ternary complex with operator DNA and phosphorylated toxin HipA to regulate bacterial persistence"

    Article Title: The bacterial antitoxin HipB establishes a ternary complex with operator DNA and phosphorylated toxin HipA to regulate bacterial persistence

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku665

    S. oneidensis MR-1 HipA so is regulated conformationally upon autophosphorylation. ( a ) Structural detail of the AMPPMP (atom colored) and Mg 2+ (green spheres) binding site in HipA so (blue). Residues involved are shown in stick representation. ( b ) Detailed view of the ejection of the pLoop of HipA so upon autophosphorylation. Ser147 and phosphate represented as spheres. ( c ) Identification of the phosphorylated peptide by nanoLCMS. The peptide LSVAGVQPK was observed at charge state 2+ in two forms differing by 80 Da in molecular weight. Sequence alignment of the pLoop regions in S. oneidensis MR-1 and E. coli.
    Figure Legend Snippet: S. oneidensis MR-1 HipA so is regulated conformationally upon autophosphorylation. ( a ) Structural detail of the AMPPMP (atom colored) and Mg 2+ (green spheres) binding site in HipA so (blue). Residues involved are shown in stick representation. ( b ) Detailed view of the ejection of the pLoop of HipA so upon autophosphorylation. Ser147 and phosphate represented as spheres. ( c ) Identification of the phosphorylated peptide by nanoLCMS. The peptide LSVAGVQPK was observed at charge state 2+ in two forms differing by 80 Da in molecular weight. Sequence alignment of the pLoop regions in S. oneidensis MR-1 and E. coli.

    Techniques Used: Binding Assay, Molecular Weight, Sequencing

    40) Product Images from "Cell differentiation defines acute and chronic infection cell types in Staphylococcus aureus"

    Article Title: Cell differentiation defines acute and chronic infection cell types in Staphylococcus aureus

    Journal: eLife

    doi: 10.7554/eLife.28023

    Experimental workflow to sort BRcells and DRcells using F luorescence A ctivated C ell S orting (FACS) to analyze and compare their transcriptomic profile. ( A ) Schematic flow of the experimental approach used for cell sorting and RNA-sequencing of specific cell types. Multicellular aggregates were resuspended and disaggregated in RNAlater for cell fixation. A homogenous suspension of single cells was obtained using mild sonication. FACS analysis separated the subpopulation of cells expressing the reporter in a test tube and the subpopulation of cells that did not express the reporter in a different test tube. We collected and concentrated the cells in a filter and isolated total RNA using hot phenol extraction protocol ( Blomberg et al., 1990 ). Total RNA was used to construct cDNA libraries that were sequenced using the Illumina HiSeq 2500. Results were analyzed using diverse bioinformatics tools. For further experimental procedures see Materials and Methods. ( B ) Control experiment of cell sorting. In the first panel, S. aureus wild type living cells from liquid cultures (non-fluorescent cells) were mixed with P ica -yfp labeled cells from liquid cultures (fluorescence cells) in relation 3:1 and analyzed using FACS. Second panel shows the flow cytometry analysis of sorted cells from the initial 3:1 mixture. The FACS-sorted bacterial population reached 97% enrichment of fluorescent cells. ( C ) Cell sorting of multicellular aggregates of S. aureus . A 4-days-old multicellular aggregate of P ica -yfp labeled strain was fixed using RNA later . Cells were dispersed and their fluorescence signal was analyzed using flow cytometry (left panel). Flow cytometry analysis showed higher expression of the reporter in a subpopulation of cells (subpopulation P1), as evidenced by the shoulder observed in the fluorescence expression profile of the culture. FACS of this sample led to an efficient separation of the subpopulation of fluorescence cells (P1) and the subpopulation of non-fluorescent cells (P2) in two different tubes. Flow cytometry analyses of P1 and P2 fluorescence signal showed that most of the cells from the P1 sample were fluorescent, whereas most of the cells from the P2 sample were non-fluorescent (center panel). In further experiments, we used a P ica -yfp labeled strain to separate a P1 subpopulation (BR+ sample) and a P2 subpopulation (BR- sample). Likewise, we used a P psmα -yfp labeled strain to separate a P1 subpopulation (DR+ sample) and a P2 subpopulation (DR- sample). We sorted approximately 25 million cells per sample prior RNA isolation. ( D ) Sorted fractions were diluted and plated on TSB and the resulting colonies were examined for viability and for emission of fluorescence using a fluorescence stereoscope. Consistent with our flow cytometry enrichment data, more than 95% of the colonies carried the transcriptional fusion and were fluorescent. In addition, the non-fluorescence cells (P2 fraction) revealed similar viability as the its fluorescent counterpart and. ( E ) PCR analysis of total RNA samples showed that samples are free of DNA contamination after DNaseI treatment. Amplification of rrna 16s control gene was detected only in the positive control (genomic DNA from S. aureus ). Molecular weight = 500 bp ladder. ( F ) Quantification of RNA concentration using spectrophotometry. The RNA concentration and absorption ratios for each sample were determined using the Nanodrop. Concentration is shown in the top right section of each panel. ( G ) Quality check of the RNA samples. RNA samples were examined using MultiNA microchip electrophoresis (Shimadzu) to determine quality and concentration of the RNA. After analysis, cDNA was synthesized following the protocol described in Materials and Methods. Molecular weight = 200 bp ladder. ( H ) Analysis of the PCR-amplified cDNA samples on a Shimadzu MultiNA microchip electrophoresis system. For Illumina sequencing, the cDNA was size-fractionated in the size range of 150–600 bp using a differential cleanup system. ( I ) An aliquot of each cDNA was analyzed by capillary electrophoresis. Each double-stranded cDNA sample was flanked with different adapter sequences to generate a cDNA with a combined length of 100 bases. Length range and concentration are shown at the top left section of each panel.
    Figure Legend Snippet: Experimental workflow to sort BRcells and DRcells using F luorescence A ctivated C ell S orting (FACS) to analyze and compare their transcriptomic profile. ( A ) Schematic flow of the experimental approach used for cell sorting and RNA-sequencing of specific cell types. Multicellular aggregates were resuspended and disaggregated in RNAlater for cell fixation. A homogenous suspension of single cells was obtained using mild sonication. FACS analysis separated the subpopulation of cells expressing the reporter in a test tube and the subpopulation of cells that did not express the reporter in a different test tube. We collected and concentrated the cells in a filter and isolated total RNA using hot phenol extraction protocol ( Blomberg et al., 1990 ). Total RNA was used to construct cDNA libraries that were sequenced using the Illumina HiSeq 2500. Results were analyzed using diverse bioinformatics tools. For further experimental procedures see Materials and Methods. ( B ) Control experiment of cell sorting. In the first panel, S. aureus wild type living cells from liquid cultures (non-fluorescent cells) were mixed with P ica -yfp labeled cells from liquid cultures (fluorescence cells) in relation 3:1 and analyzed using FACS. Second panel shows the flow cytometry analysis of sorted cells from the initial 3:1 mixture. The FACS-sorted bacterial population reached 97% enrichment of fluorescent cells. ( C ) Cell sorting of multicellular aggregates of S. aureus . A 4-days-old multicellular aggregate of P ica -yfp labeled strain was fixed using RNA later . Cells were dispersed and their fluorescence signal was analyzed using flow cytometry (left panel). Flow cytometry analysis showed higher expression of the reporter in a subpopulation of cells (subpopulation P1), as evidenced by the shoulder observed in the fluorescence expression profile of the culture. FACS of this sample led to an efficient separation of the subpopulation of fluorescence cells (P1) and the subpopulation of non-fluorescent cells (P2) in two different tubes. Flow cytometry analyses of P1 and P2 fluorescence signal showed that most of the cells from the P1 sample were fluorescent, whereas most of the cells from the P2 sample were non-fluorescent (center panel). In further experiments, we used a P ica -yfp labeled strain to separate a P1 subpopulation (BR+ sample) and a P2 subpopulation (BR- sample). Likewise, we used a P psmα -yfp labeled strain to separate a P1 subpopulation (DR+ sample) and a P2 subpopulation (DR- sample). We sorted approximately 25 million cells per sample prior RNA isolation. ( D ) Sorted fractions were diluted and plated on TSB and the resulting colonies were examined for viability and for emission of fluorescence using a fluorescence stereoscope. Consistent with our flow cytometry enrichment data, more than 95% of the colonies carried the transcriptional fusion and were fluorescent. In addition, the non-fluorescence cells (P2 fraction) revealed similar viability as the its fluorescent counterpart and. ( E ) PCR analysis of total RNA samples showed that samples are free of DNA contamination after DNaseI treatment. Amplification of rrna 16s control gene was detected only in the positive control (genomic DNA from S. aureus ). Molecular weight = 500 bp ladder. ( F ) Quantification of RNA concentration using spectrophotometry. The RNA concentration and absorption ratios for each sample were determined using the Nanodrop. Concentration is shown in the top right section of each panel. ( G ) Quality check of the RNA samples. RNA samples were examined using MultiNA microchip electrophoresis (Shimadzu) to determine quality and concentration of the RNA. After analysis, cDNA was synthesized following the protocol described in Materials and Methods. Molecular weight = 200 bp ladder. ( H ) Analysis of the PCR-amplified cDNA samples on a Shimadzu MultiNA microchip electrophoresis system. For Illumina sequencing, the cDNA was size-fractionated in the size range of 150–600 bp using a differential cleanup system. ( I ) An aliquot of each cDNA was analyzed by capillary electrophoresis. Each double-stranded cDNA sample was flanked with different adapter sequences to generate a cDNA with a combined length of 100 bases. Length range and concentration are shown at the top left section of each panel.

    Techniques Used: FACS, Flow Cytometry, RNA Sequencing Assay, Sonication, Expressing, Isolation, Construct, Labeling, Fluorescence, Cytometry, Polymerase Chain Reaction, Amplification, Positive Control, Molecular Weight, Concentration Assay, Spectrophotometry, MicroChIP Assay, Electrophoresis, Synthesized, Sequencing

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: Cadherin-23 Mediates Heterotypic Cell-Cell Adhesion between Breast Cancer Epithelial Cells and Fibroblasts
    Article Snippet: .. qPCR Confluent monocultures of MCF-7s and NBFs were trypsinized and ∼20×104 cells were used for mRNA isolation (RNEasy with RNAse-Free DNase set, Qiagen). qPCR was performed using the QuantiFast SYBR Green RT-PCR kit (Qiagen) on a LightCycler 480 (Roche, Indianapolis, IN). ..

    Isolation:

    Article Title: Cadherin-23 Mediates Heterotypic Cell-Cell Adhesion between Breast Cancer Epithelial Cells and Fibroblasts
    Article Snippet: .. qPCR Confluent monocultures of MCF-7s and NBFs were trypsinized and ∼20×104 cells were used for mRNA isolation (RNEasy with RNAse-Free DNase set, Qiagen). qPCR was performed using the QuantiFast SYBR Green RT-PCR kit (Qiagen) on a LightCycler 480 (Roche, Indianapolis, IN). ..

    Purification:

    Article Title: Dual-functional peptide with defective interfering genes effectively protects mice against avian and seasonal influenza
    Article Snippet: .. Extracted RNA were treated with DNase I (QIAGEN, Cat# 79254, USA) according to the manufacturer’s protocol and purified by RNeasy Mini Kit (QIAGEN, Cat# 74106, USA) to exclude plasmid DNA contamination. .. Real-time RT-qPCR was performed as we described previously .

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    SYBR Green Assay:

    Article Title: Cadherin-23 Mediates Heterotypic Cell-Cell Adhesion between Breast Cancer Epithelial Cells and Fibroblasts
    Article Snippet: .. qPCR Confluent monocultures of MCF-7s and NBFs were trypsinized and ∼20×104 cells were used for mRNA isolation (RNEasy with RNAse-Free DNase set, Qiagen). qPCR was performed using the QuantiFast SYBR Green RT-PCR kit (Qiagen) on a LightCycler 480 (Roche, Indianapolis, IN). ..

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Cadherin-23 Mediates Heterotypic Cell-Cell Adhesion between Breast Cancer Epithelial Cells and Fibroblasts
    Article Snippet: .. qPCR Confluent monocultures of MCF-7s and NBFs were trypsinized and ∼20×104 cells were used for mRNA isolation (RNEasy with RNAse-Free DNase set, Qiagen). qPCR was performed using the QuantiFast SYBR Green RT-PCR kit (Qiagen) on a LightCycler 480 (Roche, Indianapolis, IN). ..

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Transformation of accessible chromatin and 3D nucleome underlies lineage commitment of early T cells
    Article Snippet: .. Total RNA was extracted and on-column digestion with DNase (QIAGEN, Cat79254) was performed, followed by elution with 10μl of RNase-free water. .. Total RNA from 1K cells was reverse transcribed by SuperScript II (Invitrogen, Cat#18064-014) with oligo-dT and LNA-containing TSO primers in a final reaction volume of 10μl using the condition: 42°C for 90min, 10 cycles of 50°C 2min to 42°C 2min, 70°C for 15min and hold at 4°C. cDNA was pre-amplified by PCR using KAPA HiFi HotStart ReadyMix (KAPABIOSYSTEMS Cat#KK2602) with IS PCR for 12 cycles in 25μl.

    Plasmid Preparation:

    Article Title: Dual-functional peptide with defective interfering genes effectively protects mice against avian and seasonal influenza
    Article Snippet: .. Extracted RNA were treated with DNase I (QIAGEN, Cat# 79254, USA) according to the manufacturer’s protocol and purified by RNeasy Mini Kit (QIAGEN, Cat# 74106, USA) to exclude plasmid DNA contamination. .. Real-time RT-qPCR was performed as we described previously .

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    Inhibition of LPS-induced <t>TNF-α,</t> ICAM-1, and MCP-1 production by piceatannol. (A) U373 cells were treated as in Figure 3, and TNF-α mRNA levels were analyzed by RT-PCR. (Band C) RAW cells were treated with 100 μM piceatannol or solvent in the presence of 50 μg/mL cycloheximide for 1 h before stimulation with 1 μg/mL LPS for 6 h. Total RNA was prepared, and ICAM-1 (B) and MCP-1 (C) mRNA levels were determined by RNase protection assay. GAPDH was used as an internal control.
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    Combined disruptions of the CR and BR abolish HEXIM1 oligomerization. ( A ) Schematic diagram of Hex1 proteins used. The sign at their N-termini depicts the FLAG tag. The asterisks within the CR1 and CR2 indicate the mutations of leucines to alanines. The numbering indicates the positions of the mutated leucines in f.Hex1 proteins. ( B ) HEXIM1 with the disrupted CR does not oligomerize in the absence of 7SK snRNA. Proteins with the disrupted CR1 or CR2 (lanes 9–12) or CR (lanes 7 and 8) were co-expressed with x.Hex1 in HeLa cells. The lysates were treated with <t>RNase</t> A where indicated, immunoprecipitated with anti-FLAG agarose beads and immunoprecipitates were subjected to SDS–PAGE and WB (upper panel). Lower panels represent 10% input of proteins. ( C ) Combined disruptions of the CR and the 7SK snRNA binding site abolish completely HEXIM1 oligomerization in cells. FRET analysis was performed as in Figure 2 . Representative images of the nuclei in which Hex1.YFP and Hex1.CFP mutant proteins were co-expressed are presented. The amounts of nuclear fluorescence were quantified in the yellow, cyan and FRET channels.
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    Inhibition of LPS-induced TNF-α, ICAM-1, and MCP-1 production by piceatannol. (A) U373 cells were treated as in Figure 3, and TNF-α mRNA levels were analyzed by RT-PCR. (Band C) RAW cells were treated with 100 μM piceatannol or solvent in the presence of 50 μg/mL cycloheximide for 1 h before stimulation with 1 μg/mL LPS for 6 h. Total RNA was prepared, and ICAM-1 (B) and MCP-1 (C) mRNA levels were determined by RNase protection assay. GAPDH was used as an internal control.

    Journal: Shock (Augusta, Ga.)

    Article Title: INHIBITION OF LIPOPOLYSACCHARIDE-INDUCED INTERFERON REGULATORY FACTOR 3 ACTIVATION AND PROTECTION FROM SEPTIC SHOCK BY HYDROXYSTILBENES

    doi: 10.1097/01.shk.0000123513.13212.83

    Figure Lengend Snippet: Inhibition of LPS-induced TNF-α, ICAM-1, and MCP-1 production by piceatannol. (A) U373 cells were treated as in Figure 3, and TNF-α mRNA levels were analyzed by RT-PCR. (Band C) RAW cells were treated with 100 μM piceatannol or solvent in the presence of 50 μg/mL cycloheximide for 1 h before stimulation with 1 μg/mL LPS for 6 h. Total RNA was prepared, and ICAM-1 (B) and MCP-1 (C) mRNA levels were determined by RNase protection assay. GAPDH was used as an internal control.

    Article Snippet: cDNA was prepared from total RNA by reverse transcription using InVitrogen’s SuperScript First-Strand Synthesis System. cDNAs for RANTES, TNF-α, TF, and β-actin were amplified by using the Taq PCR Core Kit (Qiagen) with primers (5′-GCTGTCATCCTCATTGCTAC-3′) and (5′-TCTCCATCCTAGCTCATCTC-3′) for RANTES, (5′-AGCCTCTTCTCCTTCCTGATCG-3′) and (5′-TATCTCTCAGCTCCACGCC ATT-3′) for TNF-α, (5′-CGGGTGCAGGCATTCCAGAG-3′) and (5′-CAGGAGAGACAGGG TGCCTC-3′) for TF, and (5′-AAGAGAGGCATCCTCACCCT-3′) and (5′-TACATGGCTGG GGTGTTGAA-3′) for β-actin.

    Techniques: Inhibition, Reverse Transcription Polymerase Chain Reaction, Rnase Protection Assay

    Combined disruptions of the CR and BR abolish HEXIM1 oligomerization. ( A ) Schematic diagram of Hex1 proteins used. The sign at their N-termini depicts the FLAG tag. The asterisks within the CR1 and CR2 indicate the mutations of leucines to alanines. The numbering indicates the positions of the mutated leucines in f.Hex1 proteins. ( B ) HEXIM1 with the disrupted CR does not oligomerize in the absence of 7SK snRNA. Proteins with the disrupted CR1 or CR2 (lanes 9–12) or CR (lanes 7 and 8) were co-expressed with x.Hex1 in HeLa cells. The lysates were treated with RNase A where indicated, immunoprecipitated with anti-FLAG agarose beads and immunoprecipitates were subjected to SDS–PAGE and WB (upper panel). Lower panels represent 10% input of proteins. ( C ) Combined disruptions of the CR and the 7SK snRNA binding site abolish completely HEXIM1 oligomerization in cells. FRET analysis was performed as in Figure 2 . Representative images of the nuclei in which Hex1.YFP and Hex1.CFP mutant proteins were co-expressed are presented. The amounts of nuclear fluorescence were quantified in the yellow, cyan and FRET channels.

    Journal: Nucleic Acids Research

    Article Title: Oligomerization of HEXIM1 via 7SK snRNA and coiled-coil region directs the inhibition of P-TEFb

    doi: 10.1093/nar/gki997

    Figure Lengend Snippet: Combined disruptions of the CR and BR abolish HEXIM1 oligomerization. ( A ) Schematic diagram of Hex1 proteins used. The sign at their N-termini depicts the FLAG tag. The asterisks within the CR1 and CR2 indicate the mutations of leucines to alanines. The numbering indicates the positions of the mutated leucines in f.Hex1 proteins. ( B ) HEXIM1 with the disrupted CR does not oligomerize in the absence of 7SK snRNA. Proteins with the disrupted CR1 or CR2 (lanes 9–12) or CR (lanes 7 and 8) were co-expressed with x.Hex1 in HeLa cells. The lysates were treated with RNase A where indicated, immunoprecipitated with anti-FLAG agarose beads and immunoprecipitates were subjected to SDS–PAGE and WB (upper panel). Lower panels represent 10% input of proteins. ( C ) Combined disruptions of the CR and the 7SK snRNA binding site abolish completely HEXIM1 oligomerization in cells. FRET analysis was performed as in Figure 2 . Representative images of the nuclei in which Hex1.YFP and Hex1.CFP mutant proteins were co-expressed are presented. The amounts of nuclear fluorescence were quantified in the yellow, cyan and FRET channels.

    Article Snippet: After 40 h, HeLa cells were harvested and lysed in 0.75 ml of lysis buffer [1% NP-40, 10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% protease inhibitor and either 0.5% RNase inhibitor (Roche, Indianapolis, IN) or RNase A (100 µg/ml final concentration) (Qiagen, Hilden, Germany)] for 1 h at 4°C.

    Techniques: FLAG-tag, Immunoprecipitation, SDS Page, Western Blot, Binding Assay, Mutagenesis, Fluorescence

    The C-terminal domain and 7SK snRNA mediate the oligomerization of HEXIM1. ( A ) Schematic diagram of Hex1 proteins used. The signs at their N-termini depict the respective tags. ( B ) HEXIM1 forms oligomers. The x.Hex1 and f.Hex1 proteins were either expressed alone (lanes 4 and 1, 2, 3, 8, respectively) or f.Hex1 was co-expressed with x.Hex1 in HeLa cells (lanes 5–7 and 9) as indicated. Lysates were co-immunoprecipitated with anti-FLAG agarose beads and immunoprecipitates of x.Hex1 were identified as presented on the upper western blot (WB). The middle and lower WB contain 10% of input proteins for immunoprecipitations (IP). Wild-type and mutant HEXIM1 proteins are identified by arrows. ( C ) 7SK snRNA and the C-terminal domain of HEXIM1 mediate the oligomerization of HEXIM1. x.Hex1 was expressed alone (lanes 1 and 2) or with the indicated f.Hex1 proteins (lanes 3–8). IP were performed as in (B) and were treated with RNase A where indicated.

    Journal: Nucleic Acids Research

    Article Title: Oligomerization of HEXIM1 via 7SK snRNA and coiled-coil region directs the inhibition of P-TEFb

    doi: 10.1093/nar/gki997

    Figure Lengend Snippet: The C-terminal domain and 7SK snRNA mediate the oligomerization of HEXIM1. ( A ) Schematic diagram of Hex1 proteins used. The signs at their N-termini depict the respective tags. ( B ) HEXIM1 forms oligomers. The x.Hex1 and f.Hex1 proteins were either expressed alone (lanes 4 and 1, 2, 3, 8, respectively) or f.Hex1 was co-expressed with x.Hex1 in HeLa cells (lanes 5–7 and 9) as indicated. Lysates were co-immunoprecipitated with anti-FLAG agarose beads and immunoprecipitates of x.Hex1 were identified as presented on the upper western blot (WB). The middle and lower WB contain 10% of input proteins for immunoprecipitations (IP). Wild-type and mutant HEXIM1 proteins are identified by arrows. ( C ) 7SK snRNA and the C-terminal domain of HEXIM1 mediate the oligomerization of HEXIM1. x.Hex1 was expressed alone (lanes 1 and 2) or with the indicated f.Hex1 proteins (lanes 3–8). IP were performed as in (B) and were treated with RNase A where indicated.

    Article Snippet: After 40 h, HeLa cells were harvested and lysed in 0.75 ml of lysis buffer [1% NP-40, 10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% protease inhibitor and either 0.5% RNase inhibitor (Roche, Indianapolis, IN) or RNase A (100 µg/ml final concentration) (Qiagen, Hilden, Germany)] for 1 h at 4°C.

    Techniques: Immunoprecipitation, Western Blot, Mutagenesis

    Oligomerization of HEXIM1 via its BR or CR2 is required for the inhibition of transcription. ( A ) Schematic diagram of Hex1 proteins used. The BR, CR1 and CR2 regions participating in the oligomerization are depicted. The wild-type and the mutated residues of the BR are depicted above and below the diagram, respectively. The mutated BR is indicated by asterisk. The schematic picture represents the f.Hex1 and mutant f.Hex1(1–314), f.Hex1mBR and f.Hex1mBR(1–315) proteins used. ( B ) HEXIM1 without the BR and the CR2 does not oligomerize. The x.Hex1 and f.Hex1 proteins were co-expressed as depicted. Lysates were treated with RNase A where noted and IP was performed as described. Upper panel represents WB with the immunoprecipitated x.Hex1 proteins, whereas the middle and lower panels show 10% input of proteins used for IP. ( C ) HEXIM1 without the BR and the CR2 does not inhibit P-TEFb. Bars represent CAT data obtained by co-transfection of HeLa cells with pG6TAR (0.3 µg), Gal.CycT1 (1 µg) and indicated f.Hex1 plasmids (2.7 µg). The lower panel presents the expression of f.Hex1 proteins.

    Journal: Nucleic Acids Research

    Article Title: Oligomerization of HEXIM1 via 7SK snRNA and coiled-coil region directs the inhibition of P-TEFb

    doi: 10.1093/nar/gki997

    Figure Lengend Snippet: Oligomerization of HEXIM1 via its BR or CR2 is required for the inhibition of transcription. ( A ) Schematic diagram of Hex1 proteins used. The BR, CR1 and CR2 regions participating in the oligomerization are depicted. The wild-type and the mutated residues of the BR are depicted above and below the diagram, respectively. The mutated BR is indicated by asterisk. The schematic picture represents the f.Hex1 and mutant f.Hex1(1–314), f.Hex1mBR and f.Hex1mBR(1–315) proteins used. ( B ) HEXIM1 without the BR and the CR2 does not oligomerize. The x.Hex1 and f.Hex1 proteins were co-expressed as depicted. Lysates were treated with RNase A where noted and IP was performed as described. Upper panel represents WB with the immunoprecipitated x.Hex1 proteins, whereas the middle and lower panels show 10% input of proteins used for IP. ( C ) HEXIM1 without the BR and the CR2 does not inhibit P-TEFb. Bars represent CAT data obtained by co-transfection of HeLa cells with pG6TAR (0.3 µg), Gal.CycT1 (1 µg) and indicated f.Hex1 plasmids (2.7 µg). The lower panel presents the expression of f.Hex1 proteins.

    Article Snippet: After 40 h, HeLa cells were harvested and lysed in 0.75 ml of lysis buffer [1% NP-40, 10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% protease inhibitor and either 0.5% RNase inhibitor (Roche, Indianapolis, IN) or RNase A (100 µg/ml final concentration) (Qiagen, Hilden, Germany)] for 1 h at 4°C.

    Techniques: Inhibition, Mutagenesis, Western Blot, Immunoprecipitation, Cotransfection, Expressing

    Positively charged patches are important for subcellular localization and deaminase activity of A3H. ( A ) A3H structure showing the positively charged residues mutated in three patch mutants (patch 1–3). ( B ) Cell fractionation analysis of A3H and various mutants, showing the distribution between nucleus and cytosol in HEK293T cells. Transfected 293T cells expressing wild-type A3H hap I, hap II, and various hap II mutants were fractionated into whole cell (WC), cytoplasmic (Cyto) and nuclear (Nuc) fractions. A3B (mostly nucleus) and A3G (both cytoplasm and nucleus) were also used as controls. FLAG-A3H proteins in each fraction were analyzed by Western blot. ( C ) The deaminase assay of selected A3H mutants using the cell lysates of transfected HEK293T cells with or without RNase A treatment. The deaminase reaction was performed with cell lysate range of 0–6 μg (total protein amount, 2-fold dilutions from 6 μg) and 300 nM ssDNA.

    Journal: Scientific Reports

    Article Title: Understanding the Structure, Multimerization, Subcellular Localization and mC Selectivity of a Genomic Mutator and Anti-HIV Factor APOBEC3H

    doi: 10.1038/s41598-018-21955-0

    Figure Lengend Snippet: Positively charged patches are important for subcellular localization and deaminase activity of A3H. ( A ) A3H structure showing the positively charged residues mutated in three patch mutants (patch 1–3). ( B ) Cell fractionation analysis of A3H and various mutants, showing the distribution between nucleus and cytosol in HEK293T cells. Transfected 293T cells expressing wild-type A3H hap I, hap II, and various hap II mutants were fractionated into whole cell (WC), cytoplasmic (Cyto) and nuclear (Nuc) fractions. A3B (mostly nucleus) and A3G (both cytoplasm and nucleus) were also used as controls. FLAG-A3H proteins in each fraction were analyzed by Western blot. ( C ) The deaminase assay of selected A3H mutants using the cell lysates of transfected HEK293T cells with or without RNase A treatment. The deaminase reaction was performed with cell lysate range of 0–6 μg (total protein amount, 2-fold dilutions from 6 μg) and 300 nM ssDNA.

    Article Snippet: To purify dimer and monomer of MBP-fused wild-type A3H hap II, E. coli cells expressing MBP-fused A3H hap II were harvested and lysed in buffer A (25 mM HEPES pH 7.5, 500 mM NaCl, 20 mM MgCl2 and 1 mM DTT) supplemented with 1 mg RNase A (Qiagen) per liter cells.

    Techniques: Activity Assay, Cell Fractionation, Transfection, Expressing, Western Blot

    Multimerization of A3H in HEK293T cells and RNA-dependent inhibition of A3H deaminase activity. ( A ) A3H formed enzymatically inactive high molecular weight (HMW) ribonucleoprotein complex. Cell lysates of HEK293T cells expressing A3H, untreated or treated with RNase A, were fractionated by SEC on Superdex 200 column and then analyzed by Western blot and deaminase activity assay. HMW complexes were observed, and essentially no obvious deaminase activity was detected. ( B ) After RNase A treatment, the HMW complexes of A3H were converted to enzymatically active low molecular weight (LMW) species. α-tubulin is an endogenous control.

    Journal: Scientific Reports

    Article Title: Understanding the Structure, Multimerization, Subcellular Localization and mC Selectivity of a Genomic Mutator and Anti-HIV Factor APOBEC3H

    doi: 10.1038/s41598-018-21955-0

    Figure Lengend Snippet: Multimerization of A3H in HEK293T cells and RNA-dependent inhibition of A3H deaminase activity. ( A ) A3H formed enzymatically inactive high molecular weight (HMW) ribonucleoprotein complex. Cell lysates of HEK293T cells expressing A3H, untreated or treated with RNase A, were fractionated by SEC on Superdex 200 column and then analyzed by Western blot and deaminase activity assay. HMW complexes were observed, and essentially no obvious deaminase activity was detected. ( B ) After RNase A treatment, the HMW complexes of A3H were converted to enzymatically active low molecular weight (LMW) species. α-tubulin is an endogenous control.

    Article Snippet: To purify dimer and monomer of MBP-fused wild-type A3H hap II, E. coli cells expressing MBP-fused A3H hap II were harvested and lysed in buffer A (25 mM HEPES pH 7.5, 500 mM NaCl, 20 mM MgCl2 and 1 mM DTT) supplemented with 1 mg RNase A (Qiagen) per liter cells.

    Techniques: Inhibition, Activity Assay, Molecular Weight, Expressing, Size-exclusion Chromatography, Western Blot

    Protein purification and the overall structure of A3H. ( A ) SEC elution profiles of MBP-A3H dimeric and monomeric mutants on Superdex 200. A3H m1 forms a stable dimer after extensive RNase A treatment (blue). The purified m1 dimer can dissociate to monomer and free RNA after RNase A treatment followed by 1.5 M or higher salt buffer (black). The RNA-bound m1 dimer was disrupted by two sets of mutations on loop 7: H114A (m1+H114A) or W115A/C116S (m1+W115A/C116S), and clean monomers were purified from m1+H114A (light blue) m1+W115A/C116S (green). ( B ) MALS of MBP-fused m1+W115A/C116S mutant, showing the clean monomeric form. The expected molecular mass of a monomer is 63.2 kDa. ( C,D ) Crystal structure of A3H m1+W115A/C116S monomer mutant ( C ) and the superimposition of the A3H (green) with A3A (PDB: 4XXO, yellow), A3B-CD2 (PDB: 5CQI, salmon) and AID (PDB: 5W0R, purple) ( D ), with secondary structures indicated (Supplementary Figure S2A ). The long helix 6 (h6), break of β5, and the long loop 1 of A3H can be visualized in panels C and D (Supplementary Figure S3A , B ).

    Journal: Scientific Reports

    Article Title: Understanding the Structure, Multimerization, Subcellular Localization and mC Selectivity of a Genomic Mutator and Anti-HIV Factor APOBEC3H

    doi: 10.1038/s41598-018-21955-0

    Figure Lengend Snippet: Protein purification and the overall structure of A3H. ( A ) SEC elution profiles of MBP-A3H dimeric and monomeric mutants on Superdex 200. A3H m1 forms a stable dimer after extensive RNase A treatment (blue). The purified m1 dimer can dissociate to monomer and free RNA after RNase A treatment followed by 1.5 M or higher salt buffer (black). The RNA-bound m1 dimer was disrupted by two sets of mutations on loop 7: H114A (m1+H114A) or W115A/C116S (m1+W115A/C116S), and clean monomers were purified from m1+H114A (light blue) m1+W115A/C116S (green). ( B ) MALS of MBP-fused m1+W115A/C116S mutant, showing the clean monomeric form. The expected molecular mass of a monomer is 63.2 kDa. ( C,D ) Crystal structure of A3H m1+W115A/C116S monomer mutant ( C ) and the superimposition of the A3H (green) with A3A (PDB: 4XXO, yellow), A3B-CD2 (PDB: 5CQI, salmon) and AID (PDB: 5W0R, purple) ( D ), with secondary structures indicated (Supplementary Figure S2A ). The long helix 6 (h6), break of β5, and the long loop 1 of A3H can be visualized in panels C and D (Supplementary Figure S3A , B ).

    Article Snippet: To purify dimer and monomer of MBP-fused wild-type A3H hap II, E. coli cells expressing MBP-fused A3H hap II were harvested and lysed in buffer A (25 mM HEPES pH 7.5, 500 mM NaCl, 20 mM MgCl2 and 1 mM DTT) supplemented with 1 mg RNase A (Qiagen) per liter cells.

    Techniques: Protein Purification, Size-exclusion Chromatography, Purification, Mutagenesis