lentiviral integrations  (Thermo Fisher)


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    Structured Review

    Thermo Fisher lentiviral integrations
    N-terminal NLS sequences increase the range and potency of target base editing. a. Schematic representation of RA enzyme (above) and two new variants carrying NLS sequences within the XTEN linker (2X) or at the N-terminus (FNLS) b. Immunofluorescent staining of Cas9 in NIH/3T3 cells expressing RA, 2X, or FNLS. Experiment was repeated twice with similar results. c. Sanger sequencing chromatogram showing increased editing of the cytosine at position 10 (blue arrow) within the protospacer of a CTNNB1.S45 sgRNA. d . Frequency (%) of C > T conversion in NIH/3T3 cells transduced with RA - or FNLS-P2A-Puro <t>lentiviral</t> vectors 6 days following introduction of different sgRNAs, as indicated. Editing in BE3-PGK-Puro cells (from Figure 1e ) is shown for comparison. e . Frequency (%) of C > T conversion in PC9 cells transduced with BE3-PGK-Puro , FNLS or BE4Gam RA -P2A-Puro lentiviral vectors 6 days following introduction of different sgRNAs, as indicated. For d and e , graphs show mean values. Error bars represent s.e.m., n = 3 biologically independent samples; asterisks (*) indicate a significant difference (p
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    Images

    1) Product Images from "Optimized base editors enable efficient editing in cells, organoids and mice"

    Article Title: Optimized base editors enable efficient editing in cells, organoids and mice

    Journal: Nature biotechnology

    doi: 10.1038/nbt.4194

    N-terminal NLS sequences increase the range and potency of target base editing. a. Schematic representation of RA enzyme (above) and two new variants carrying NLS sequences within the XTEN linker (2X) or at the N-terminus (FNLS) b. Immunofluorescent staining of Cas9 in NIH/3T3 cells expressing RA, 2X, or FNLS. Experiment was repeated twice with similar results. c. Sanger sequencing chromatogram showing increased editing of the cytosine at position 10 (blue arrow) within the protospacer of a CTNNB1.S45 sgRNA. d . Frequency (%) of C > T conversion in NIH/3T3 cells transduced with RA - or FNLS-P2A-Puro lentiviral vectors 6 days following introduction of different sgRNAs, as indicated. Editing in BE3-PGK-Puro cells (from Figure 1e ) is shown for comparison. e . Frequency (%) of C > T conversion in PC9 cells transduced with BE3-PGK-Puro , FNLS or BE4Gam RA -P2A-Puro lentiviral vectors 6 days following introduction of different sgRNAs, as indicated. For d and e , graphs show mean values. Error bars represent s.e.m., n = 3 biologically independent samples; asterisks (*) indicate a significant difference (p
    Figure Legend Snippet: N-terminal NLS sequences increase the range and potency of target base editing. a. Schematic representation of RA enzyme (above) and two new variants carrying NLS sequences within the XTEN linker (2X) or at the N-terminus (FNLS) b. Immunofluorescent staining of Cas9 in NIH/3T3 cells expressing RA, 2X, or FNLS. Experiment was repeated twice with similar results. c. Sanger sequencing chromatogram showing increased editing of the cytosine at position 10 (blue arrow) within the protospacer of a CTNNB1.S45 sgRNA. d . Frequency (%) of C > T conversion in NIH/3T3 cells transduced with RA - or FNLS-P2A-Puro lentiviral vectors 6 days following introduction of different sgRNAs, as indicated. Editing in BE3-PGK-Puro cells (from Figure 1e ) is shown for comparison. e . Frequency (%) of C > T conversion in PC9 cells transduced with BE3-PGK-Puro , FNLS or BE4Gam RA -P2A-Puro lentiviral vectors 6 days following introduction of different sgRNAs, as indicated. For d and e , graphs show mean values. Error bars represent s.e.m., n = 3 biologically independent samples; asterisks (*) indicate a significant difference (p

    Techniques Used: Staining, Expressing, Sequencing, Transduction

    2) Product Images from "Synchronized Orchestration of miR-99b and let-7g Positively Regulates Rotavirus Infection by Modulating Autophagy"

    Article Title: Synchronized Orchestration of miR-99b and let-7g Positively Regulates Rotavirus Infection by Modulating Autophagy

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-38473-8

    TSC1 is a direct target of let-7g. ( A ) 293 T cells were co-transfected with pMIR-REPORT luciferase construct containing the 3′UTR of TSC1 and different doses of the let-7g mimic (10, 20 or 40 nM). Relative luciferase activity was measured after 48 h of transfection. ( B ) Luciferase activity of the mutant 3′UTR of TSC1 was not altered in presence of mimic let-7g (40 nM) compared to the wild-type 3′UTR. ( C ) let-7g transfected HT29 cell lysates were immunoprecipitated with Ago2 specific monoclonal antibody or IgG2a isotype control. RNA was isolated from immunoprecipitates using RNeasy kit. let-7g bound TSC1 mRNA expression was analyzed by qRT-PCR (left panel). Expression of Ago2 and TSC1 were checked in each set of input sample lysates by immunoblotting (right panel). Relative expression was determined after normalization to control cells. ( D ) Relative expression of let-7g from Ago2 immunoprecipitates was analyzed by qRT-PCR. The results are shown as mean and standard deviation from representative of three technical replicates.
    Figure Legend Snippet: TSC1 is a direct target of let-7g. ( A ) 293 T cells were co-transfected with pMIR-REPORT luciferase construct containing the 3′UTR of TSC1 and different doses of the let-7g mimic (10, 20 or 40 nM). Relative luciferase activity was measured after 48 h of transfection. ( B ) Luciferase activity of the mutant 3′UTR of TSC1 was not altered in presence of mimic let-7g (40 nM) compared to the wild-type 3′UTR. ( C ) let-7g transfected HT29 cell lysates were immunoprecipitated with Ago2 specific monoclonal antibody or IgG2a isotype control. RNA was isolated from immunoprecipitates using RNeasy kit. let-7g bound TSC1 mRNA expression was analyzed by qRT-PCR (left panel). Expression of Ago2 and TSC1 were checked in each set of input sample lysates by immunoblotting (right panel). Relative expression was determined after normalization to control cells. ( D ) Relative expression of let-7g from Ago2 immunoprecipitates was analyzed by qRT-PCR. The results are shown as mean and standard deviation from representative of three technical replicates.

    Techniques Used: Transfection, Luciferase, Construct, Activity Assay, Mutagenesis, Immunoprecipitation, Isolation, Expressing, Quantitative RT-PCR, Standard Deviation

    Schematic diagram showing the crosstalk between miR-99b, let-7g, and RV. RV infection downregulates let-7g which in turn elevates TSC1 expression. Inhibition of let-7g also promotes TSC1 mediated suppression of Rheb-GTP expression. On the other hand, RV infection upregulates miR-99b that directly targets mTOR. RV mediated upregulation of miR-99b and suppression of Rheb-GTP leads towards containment of mTOR expression. These results lead towards activation of autophagy mediators during RV infection. Arrows denote upregulation, and blunt arrows denote inhibition.
    Figure Legend Snippet: Schematic diagram showing the crosstalk between miR-99b, let-7g, and RV. RV infection downregulates let-7g which in turn elevates TSC1 expression. Inhibition of let-7g also promotes TSC1 mediated suppression of Rheb-GTP expression. On the other hand, RV infection upregulates miR-99b that directly targets mTOR. RV mediated upregulation of miR-99b and suppression of Rheb-GTP leads towards containment of mTOR expression. These results lead towards activation of autophagy mediators during RV infection. Arrows denote upregulation, and blunt arrows denote inhibition.

    Techniques Used: Infection, Expressing, Inhibition, Activation Assay

    RV infection downregulates let-7g expression. ( A ) HT29 cells were mock treated or infected with RV for indicated time points. Whole cell lysates were prepared, followed by western blot analysis using specific antibodies against Rheb-GTP, TSC1, TSC2, and RV-VP6. The blot was reprobed with an antibody to GAPDH for normalization. ( B ) In silico analysis of the TSC1 3′UTR revealed a single putative let-7g binding site. The let-7g target region of TSC1 (GenBank accession number NM_000368) is indicated. ( C ) HT29 cells were transfected with mimic let-7g (40 nM) (left panel) or pmR-ZsGreen1-pre-let7g (right panel) followed by western blot analysis using specific antibody against TSC1. Relative fold differences of the TSC1 level was analyzed after normalization with GAPDH from at least three independent experiments. ( D ) HT29 cells were mock treated or infected with RV-SA11 for indicated time points. Total RNA from mock- or virus-infected cells was extracted and expression of let-7g was analyzed by qRT-PCR and normalized to the expression of U6 snRNA. ( E ) RV-VP6 RNA level was measured and plotted as relative RNA level in comparison to mock-infected cells. ( F ) Cells were infected with UV inactivated RV-SA11. At 8hpi the total RNA was extracted and the relative expression of let-7g was quantified by qRT-PCR analysis. ( G ) VP6 RNA level was measured as a marker of viral infection in UV-inactivated RV-SA11 infected cells. Results are presented as the means and standard deviations from at least two independent experiments.
    Figure Legend Snippet: RV infection downregulates let-7g expression. ( A ) HT29 cells were mock treated or infected with RV for indicated time points. Whole cell lysates were prepared, followed by western blot analysis using specific antibodies against Rheb-GTP, TSC1, TSC2, and RV-VP6. The blot was reprobed with an antibody to GAPDH for normalization. ( B ) In silico analysis of the TSC1 3′UTR revealed a single putative let-7g binding site. The let-7g target region of TSC1 (GenBank accession number NM_000368) is indicated. ( C ) HT29 cells were transfected with mimic let-7g (40 nM) (left panel) or pmR-ZsGreen1-pre-let7g (right panel) followed by western blot analysis using specific antibody against TSC1. Relative fold differences of the TSC1 level was analyzed after normalization with GAPDH from at least three independent experiments. ( D ) HT29 cells were mock treated or infected with RV-SA11 for indicated time points. Total RNA from mock- or virus-infected cells was extracted and expression of let-7g was analyzed by qRT-PCR and normalized to the expression of U6 snRNA. ( E ) RV-VP6 RNA level was measured and plotted as relative RNA level in comparison to mock-infected cells. ( F ) Cells were infected with UV inactivated RV-SA11. At 8hpi the total RNA was extracted and the relative expression of let-7g was quantified by qRT-PCR analysis. ( G ) VP6 RNA level was measured as a marker of viral infection in UV-inactivated RV-SA11 infected cells. Results are presented as the means and standard deviations from at least two independent experiments.

    Techniques Used: Infection, Expressing, Western Blot, In Silico, Binding Assay, Transfection, Quantitative RT-PCR, Marker

    miR-99b and let-7g regulate autophagy during RV infection. HT29 cells were transfected with scrambled-miR, mimic let-7g, anti-miR-99b (40 nM each) or in combination (antagonist miRNA cocktail −50 nM total) and infected with RV-SA11. ( A ) The expressions of TSC1, TSC2, mTOR, and p-mTOR were examined by immunoblot analysis. GAPDH was used as an internal control for obtaining the expression levels after normalization. VP6 expression was checked to ensure virus infection. ( B ) Expressions of LC3I/II, Beclin1 and ATG5 were checked in the same cell lysates by immunoblotting. Normalization was done by GAPDH. Virus infection was confirmed by analysing NSP3 expression in the same cell lysates. Results are presented as the means and standard deviations from three experimental replicates.
    Figure Legend Snippet: miR-99b and let-7g regulate autophagy during RV infection. HT29 cells were transfected with scrambled-miR, mimic let-7g, anti-miR-99b (40 nM each) or in combination (antagonist miRNA cocktail −50 nM total) and infected with RV-SA11. ( A ) The expressions of TSC1, TSC2, mTOR, and p-mTOR were examined by immunoblot analysis. GAPDH was used as an internal control for obtaining the expression levels after normalization. VP6 expression was checked to ensure virus infection. ( B ) Expressions of LC3I/II, Beclin1 and ATG5 were checked in the same cell lysates by immunoblotting. Normalization was done by GAPDH. Virus infection was confirmed by analysing NSP3 expression in the same cell lysates. Results are presented as the means and standard deviations from three experimental replicates.

    Techniques Used: Infection, Transfection, Expressing

    Ectopic expression of anti-miR-99b and let-7g blocks RV infection. ( A ) MA104 cells were transfected with anti-miR-99b and let-7g (50 nM) followed by RV-SA11 infection. Cells were then fixed, permeabilized at 4–8 hpi and stained with antibody against NSP5 to detect virus infection (green), endogenous LC3 (red). Scale bars: 20μm. ( B ) HT29 cells were transfected with control-miR, anti-miR-99b and/or mimic let-7g (50 nM) for 48 h followed by RV infection. Infectious virus particle was measured at indicated time points by virion quantification assay. ( C ) 3-MA treated HT29 cells were transfected with scrambled-miR, agonist or antagonist miRNA cocktail (50 nM) followed by infection with RV-SA11. The expression of mTOR, p-mTOR and LC3 lipidation were examined by immunoblot analysis. VP6 and NSP3 expressions were analysed in the same set of samples to ensure virus infection. ( D ) Infectious RV particle was measured by virion quantification assay in the same experimental conditions in presence of 3-MA at 6hpi. ( E ) HT29 cells were treated with STO-609 (10 µM and 25 µM) and/or antagonist miRNA cocktail (20 nM and 50 nM total concentration) in different combinations followed by immunoblotting using RV-VP6 specific antibody. ( F ) Virion quantification assay was done in the same experimental conditions for quantification of infectious RV particles. The results are presented as the means and standard deviations from at least three independent experiments.
    Figure Legend Snippet: Ectopic expression of anti-miR-99b and let-7g blocks RV infection. ( A ) MA104 cells were transfected with anti-miR-99b and let-7g (50 nM) followed by RV-SA11 infection. Cells were then fixed, permeabilized at 4–8 hpi and stained with antibody against NSP5 to detect virus infection (green), endogenous LC3 (red). Scale bars: 20μm. ( B ) HT29 cells were transfected with control-miR, anti-miR-99b and/or mimic let-7g (50 nM) for 48 h followed by RV infection. Infectious virus particle was measured at indicated time points by virion quantification assay. ( C ) 3-MA treated HT29 cells were transfected with scrambled-miR, agonist or antagonist miRNA cocktail (50 nM) followed by infection with RV-SA11. The expression of mTOR, p-mTOR and LC3 lipidation were examined by immunoblot analysis. VP6 and NSP3 expressions were analysed in the same set of samples to ensure virus infection. ( D ) Infectious RV particle was measured by virion quantification assay in the same experimental conditions in presence of 3-MA at 6hpi. ( E ) HT29 cells were treated with STO-609 (10 µM and 25 µM) and/or antagonist miRNA cocktail (20 nM and 50 nM total concentration) in different combinations followed by immunoblotting using RV-VP6 specific antibody. ( F ) Virion quantification assay was done in the same experimental conditions for quantification of infectious RV particles. The results are presented as the means and standard deviations from at least three independent experiments.

    Techniques Used: Expressing, Infection, Transfection, Staining, Concentration Assay

    3) Product Images from "Novel Less-Abundant Viral MicroRNAs Encoded by Herpes Simplex Virus 2 Latency-Associated Transcript and Their Roles in Regulating ICP34.5 and ICP0 mRNAs ▿"

    Article Title: Novel Less-Abundant Viral MicroRNAs Encoded by Herpes Simplex Virus 2 Latency-Associated Transcript and Their Roles in Regulating ICP34.5 and ICP0 mRNAs ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01723-08

    Time course of LAT-encoded miRNA expression in LAT mutant virus- and wild-type virus-infected cells. Vero cells were infected in triplicate with HSV-2 strain 333 wild-type virus or a mutant in which the LAT promoter (NotI-SalI sequences) was deleted. Total RNA was prepared at 0, 3, 6, 9, 14, and 18 hpi. Fifty nanograms of total RNA was used in specific real-time PCR systems to quantify miR-I, miR-II, and miR-III at each time point for each virus.
    Figure Legend Snippet: Time course of LAT-encoded miRNA expression in LAT mutant virus- and wild-type virus-infected cells. Vero cells were infected in triplicate with HSV-2 strain 333 wild-type virus or a mutant in which the LAT promoter (NotI-SalI sequences) was deleted. Total RNA was prepared at 0, 3, 6, 9, 14, and 18 hpi. Fifty nanograms of total RNA was used in specific real-time PCR systems to quantify miR-I, miR-II, and miR-III at each time point for each virus.

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

    ICP4 downregulates LAT-encoded miRNA expression. (A) HSV-2 ICP4 reduces LAT reporter activity. The schematic diagram illustrates the construction of HSV-1 and HSV-2 luciferase reporters. (Middle) pLAT2-Luc1, pRL-Ts ( Renilla luciferase control), and pGl3-Basic were cotransfected with the pLAT2-ICP4 or pcDNA3 vector in HEK 293 cells. pLAT2-Luc1R was also transfected as an additional negative control. (Bottom) pLAT1-Luc3, pLAT1-Luc4 (with deletion of ICP4-binding sites), pRL-Ts, and pGl3-Basic were cotransfected with HSV-2 ICP4 or pcDNA in HEK 293 cells. Firefly luciferase activity readings were normalized with Renilla luciferase activity. (B) Detection of HSV-2 LAT-encoded miRNAs in cells transfected with plasmids containing LAT promoter sequences, ORF-O, P promoter sequences, and the ICP4 gene. pSSK and pPst1-HincII were cotransfected with pICP4 or pcDNA 3 into HEK 293 cells. Total RNAs were hybridized with 32 P-labeled probes for miR-I, miR-II-5p, miR-III, and U6 snRNA after stripping. (C) An HSV-1 mutant virus with an ORF-O, P ICP4-binding site mutation expresses more miR-LAT-ICP34.5 in infected cells. Vero cells were infected with R7530 (ORF-O, P ICP4-binding site mutant) or R7531 (rescuant virus of R7530). miR-LAT-ICP34.5 (miR-H4-3p) expression was analyzed by real-time PCR. The asterisk indicates that all mock infection controls were below the assay detection limit of ∼1,000 copies.
    Figure Legend Snippet: ICP4 downregulates LAT-encoded miRNA expression. (A) HSV-2 ICP4 reduces LAT reporter activity. The schematic diagram illustrates the construction of HSV-1 and HSV-2 luciferase reporters. (Middle) pLAT2-Luc1, pRL-Ts ( Renilla luciferase control), and pGl3-Basic were cotransfected with the pLAT2-ICP4 or pcDNA3 vector in HEK 293 cells. pLAT2-Luc1R was also transfected as an additional negative control. (Bottom) pLAT1-Luc3, pLAT1-Luc4 (with deletion of ICP4-binding sites), pRL-Ts, and pGl3-Basic were cotransfected with HSV-2 ICP4 or pcDNA in HEK 293 cells. Firefly luciferase activity readings were normalized with Renilla luciferase activity. (B) Detection of HSV-2 LAT-encoded miRNAs in cells transfected with plasmids containing LAT promoter sequences, ORF-O, P promoter sequences, and the ICP4 gene. pSSK and pPst1-HincII were cotransfected with pICP4 or pcDNA 3 into HEK 293 cells. Total RNAs were hybridized with 32 P-labeled probes for miR-I, miR-II-5p, miR-III, and U6 snRNA after stripping. (C) An HSV-1 mutant virus with an ORF-O, P ICP4-binding site mutation expresses more miR-LAT-ICP34.5 in infected cells. Vero cells were infected with R7530 (ORF-O, P ICP4-binding site mutant) or R7531 (rescuant virus of R7530). miR-LAT-ICP34.5 (miR-H4-3p) expression was analyzed by real-time PCR. The asterisk indicates that all mock infection controls were below the assay detection limit of ∼1,000 copies.

    Techniques Used: Expressing, Activity Assay, Luciferase, Plasmid Preparation, Transfection, Negative Control, Binding Assay, Labeling, Stripping Membranes, Mutagenesis, Infection, Real-time Polymerase Chain Reaction

    miR-II and miR-III inhibit ICP34.5 and ICP0 expression, respectively. (A) Mapping of the transcription initiation site of the HSV-2 ICP34.5 gene. Ten micrograms of total RNA was obtained at 16 h after inoculation of Vero cells with HSV-2 strain 333-infected Vero cells and used to analyze the 5′ transcription initiation site of the ICP34.5 gene. Two partially overlapping primers, oST505 and oST506, were used as gene-specific primers in 5′ RACE. cDNA was amplified with a 5′ RACE outer primer and oST505 (lane 1) or oST506 (lane 2). The first PCR products were further amplified with a nested 5′ RACE inner primer and oST505 (lane 3) or oST506 (lane 4). The nested PCR product from primers oST506 and the 5′ RACE inner primer was cloned and sequenced. The transcription initiation sites of the ICP34.5 gene map to nt 126943 (16 of 22 clones sequenced) and 126941 (6 of 22 clones). Asterisks indicate the transcription start sites. The locations of the ICP34.5 gene open reading frame (ORF), miR-II, and the primers are also labeled. (B) miR-II specifically silences ICP34.5 expression. U2OS cells were transfected with 20 nM miR-II duplex with or without 40 nM miR-II-5p-specific inhibitor 16 h before infection with HSV-2 strain 333 at a MOI of 2. Twenty nanomolar nonspecific (NS) siRNA (Ambion) and the miR-I duplex were also transfected and used as negative and positive controls, respectively. Total proteins were extracted at 6 hpi and separated on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel before transfer to a membrane and incubation with an HSV-2 ICP34.5-specific antibody. The same membrane was stripped and incubated with an anti-β-tubulin antibody as a loading control. (C) miR-III specifically reduces ICP0 mRNA levels. U2OS cells were transfected with 20 nM miR-III duplex with or without 40 nM miR-III-specific inhibitor or NS miRNA inhibitor 16 h before infection with HSV-2 strain 333 at a MOI of 2. Twenty nanomolar NS siRNA (Dharmacon) was also transfected and used as a negative control. Total RNAs were prepared at 6 hpi and ICP0 and TK levels were analyzed by real-time PCR. Expression of ICP0 was normalized to TK and is presented as relative ICP0 expression. The panel summarizes five independent experiments.
    Figure Legend Snippet: miR-II and miR-III inhibit ICP34.5 and ICP0 expression, respectively. (A) Mapping of the transcription initiation site of the HSV-2 ICP34.5 gene. Ten micrograms of total RNA was obtained at 16 h after inoculation of Vero cells with HSV-2 strain 333-infected Vero cells and used to analyze the 5′ transcription initiation site of the ICP34.5 gene. Two partially overlapping primers, oST505 and oST506, were used as gene-specific primers in 5′ RACE. cDNA was amplified with a 5′ RACE outer primer and oST505 (lane 1) or oST506 (lane 2). The first PCR products were further amplified with a nested 5′ RACE inner primer and oST505 (lane 3) or oST506 (lane 4). The nested PCR product from primers oST506 and the 5′ RACE inner primer was cloned and sequenced. The transcription initiation sites of the ICP34.5 gene map to nt 126943 (16 of 22 clones sequenced) and 126941 (6 of 22 clones). Asterisks indicate the transcription start sites. The locations of the ICP34.5 gene open reading frame (ORF), miR-II, and the primers are also labeled. (B) miR-II specifically silences ICP34.5 expression. U2OS cells were transfected with 20 nM miR-II duplex with or without 40 nM miR-II-5p-specific inhibitor 16 h before infection with HSV-2 strain 333 at a MOI of 2. Twenty nanomolar nonspecific (NS) siRNA (Ambion) and the miR-I duplex were also transfected and used as negative and positive controls, respectively. Total proteins were extracted at 6 hpi and separated on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel before transfer to a membrane and incubation with an HSV-2 ICP34.5-specific antibody. The same membrane was stripped and incubated with an anti-β-tubulin antibody as a loading control. (C) miR-III specifically reduces ICP0 mRNA levels. U2OS cells were transfected with 20 nM miR-III duplex with or without 40 nM miR-III-specific inhibitor or NS miRNA inhibitor 16 h before infection with HSV-2 strain 333 at a MOI of 2. Twenty nanomolar NS siRNA (Dharmacon) was also transfected and used as a negative control. Total RNAs were prepared at 6 hpi and ICP0 and TK levels were analyzed by real-time PCR. Expression of ICP0 was normalized to TK and is presented as relative ICP0 expression. The panel summarizes five independent experiments.

    Techniques Used: Expressing, Infection, Amplification, Polymerase Chain Reaction, Nested PCR, Clone Assay, Labeling, Transfection, Polyacrylamide Gel Electrophoresis, Incubation, Negative Control, Real-time Polymerase Chain Reaction

    4) Product Images from "Inhibition of IL-1 Signaling by Antisense Oligonucleotide-mediated Exon Skipping of IL-1 Receptor Accessory Protein (IL-1RAcP)"

    Article Title: Inhibition of IL-1 Signaling by Antisense Oligonucleotide-mediated Exon Skipping of IL-1 Receptor Accessory Protein (IL-1RAcP)

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1038/mtna.2012.58

    Effect of AONs on mouse and human IL-1RAcP pre-mRNA splicing in vitro . ( a ) AONs targeted to mouse IL-1RAcP pre-mRNA exon 9 and exon-intron junctions. ( b ) Test of AONs on NIH-3T3 cells at 500 nmol/l concentration for 24 hours. RT-PCR analysis of samples shows the full-length upper band and the skipped product as a lower band that were amplified with primers specific for exon 8 and 10. Sequence analysis also confirmed exon 9 skipping. ( c ) RT-PCR results of RNA samples from NIH-3T3 cells transfected with AONs PS300, PS327, 25-mer counterparts of them and PS300L that was designed to increase efficiency of PS300. Triangles show increasing concentrations of 20, 50, 100, 200 and 500 nmol/l for full 2′- O -MePS oligos and additional 10 nmol/l for PS300L (+ = positive control PS300, 100 nmol/l; − = water control, NT = non-transfected cells, Mock tr = only Lipofectamine-2000). ( d ) Quantification of skipping levels of AON concentration from 20 to 500 nmol/l by qPCR analysis. β-Actin was used as the reference gene and each bar represents the mean value of three different experiments ± SEM. ( e ) AONs targeted to human IL-1RAcP pre-mRNA exon 9 and exon-intron junctions. ( f ) Test of AONs on HEPG2 cells at 500 nmol/l for 24 hours. RT-PCR analysis of samples shows the full-length upper band and the skipped product as a lower band that were amplified with exon 8- and 10-specific primers. The sequence analysis also confirmed exon 9 skipping. AON, antisense oligonucleotide; IL-1RAcP, interleukin-1 receptor accessory protein; qPCR, quantitative PCR; RT-PCR, reverse transcription-PCR.
    Figure Legend Snippet: Effect of AONs on mouse and human IL-1RAcP pre-mRNA splicing in vitro . ( a ) AONs targeted to mouse IL-1RAcP pre-mRNA exon 9 and exon-intron junctions. ( b ) Test of AONs on NIH-3T3 cells at 500 nmol/l concentration for 24 hours. RT-PCR analysis of samples shows the full-length upper band and the skipped product as a lower band that were amplified with primers specific for exon 8 and 10. Sequence analysis also confirmed exon 9 skipping. ( c ) RT-PCR results of RNA samples from NIH-3T3 cells transfected with AONs PS300, PS327, 25-mer counterparts of them and PS300L that was designed to increase efficiency of PS300. Triangles show increasing concentrations of 20, 50, 100, 200 and 500 nmol/l for full 2′- O -MePS oligos and additional 10 nmol/l for PS300L (+ = positive control PS300, 100 nmol/l; − = water control, NT = non-transfected cells, Mock tr = only Lipofectamine-2000). ( d ) Quantification of skipping levels of AON concentration from 20 to 500 nmol/l by qPCR analysis. β-Actin was used as the reference gene and each bar represents the mean value of three different experiments ± SEM. ( e ) AONs targeted to human IL-1RAcP pre-mRNA exon 9 and exon-intron junctions. ( f ) Test of AONs on HEPG2 cells at 500 nmol/l for 24 hours. RT-PCR analysis of samples shows the full-length upper band and the skipped product as a lower band that were amplified with exon 8- and 10-specific primers. The sequence analysis also confirmed exon 9 skipping. AON, antisense oligonucleotide; IL-1RAcP, interleukin-1 receptor accessory protein; qPCR, quantitative PCR; RT-PCR, reverse transcription-PCR.

    Techniques Used: In Vitro, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Transfection, Positive Control, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    5) Product Images from "Hepatocyte-targeted RNAi Therapeutics for the Treatment of Chronic Hepatitis B Virus Infection"

    Article Title: Hepatocyte-targeted RNAi Therapeutics for the Treatment of Chronic Hepatitis B Virus Infection

    Journal: Molecular Therapy

    doi: 10.1038/mt.2013.31

    Efficacy and the duration of knockdown after coinjection of chol-siHBVs and NAG-MLP in the pHBV mouse model of chronic hepatitis B virus (HBV) infection. NOD-SCID mice were given a hydrodynamic tail vein injection with ( a , e ) 13.5 µg pHBV1.3 or ( b – d ) 10 µg pHBV1.3. Three or more weeks thereafter, mice were given one 200 µl IV coinjection of 6 mg/kg NAG-MLP and 0.25 mg/kg, 1 mg/kg or 6 mg/kg chol-siHBV-74, -75, -76, or 77 ( n = 3–4). ( a , b ) HBsAg and ( c ) HBeAg in serum were measured by enzyme linked immunosorbent assay at the indicated times relative to injection on day 1; LOD, limit of detection. ( d ) DNA was isolated from serum and the concentration of HBV genomes was quantitated by qPCR. ( e ) RNA was isolated from the liver 14 days after chol-siRNA injection. The relative amount of HBV transcripts was determined by RT-qPCR using a probe that was within the S gene and normalizing to the endogenous β-actin mRNA. At each dose level the chol-siRNA injected mice were compared with isotonic glucose-injected mice with similar initial HBsAg levels (isotonic glucose groups A and B). Standard deviation bars are shown for HBsAg and HBV RNA quantitation. Serum HBV DNA and HBeAg levels for each group were determined by combining equal proportions of serum from each mouse within the group to obtain sufficient pooled sample at each time point ( n = 3–4).
    Figure Legend Snippet: Efficacy and the duration of knockdown after coinjection of chol-siHBVs and NAG-MLP in the pHBV mouse model of chronic hepatitis B virus (HBV) infection. NOD-SCID mice were given a hydrodynamic tail vein injection with ( a , e ) 13.5 µg pHBV1.3 or ( b – d ) 10 µg pHBV1.3. Three or more weeks thereafter, mice were given one 200 µl IV coinjection of 6 mg/kg NAG-MLP and 0.25 mg/kg, 1 mg/kg or 6 mg/kg chol-siHBV-74, -75, -76, or 77 ( n = 3–4). ( a , b ) HBsAg and ( c ) HBeAg in serum were measured by enzyme linked immunosorbent assay at the indicated times relative to injection on day 1; LOD, limit of detection. ( d ) DNA was isolated from serum and the concentration of HBV genomes was quantitated by qPCR. ( e ) RNA was isolated from the liver 14 days after chol-siRNA injection. The relative amount of HBV transcripts was determined by RT-qPCR using a probe that was within the S gene and normalizing to the endogenous β-actin mRNA. At each dose level the chol-siRNA injected mice were compared with isotonic glucose-injected mice with similar initial HBsAg levels (isotonic glucose groups A and B). Standard deviation bars are shown for HBsAg and HBV RNA quantitation. Serum HBV DNA and HBeAg levels for each group were determined by combining equal proportions of serum from each mouse within the group to obtain sufficient pooled sample at each time point ( n = 3–4).

    Techniques Used: Infection, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Isolation, Concentration Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation, Quantitation Assay

    6) Product Images from "Rational design of antisense oligonucleotides targeting single nucleotide polymorphisms for potent and allele selective suppression of mutant Huntingtin in the CNS"

    Article Title: Rational design of antisense oligonucleotides targeting single nucleotide polymorphisms for potent and allele selective suppression of mutant Huntingtin in the CNS

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt725

    Precise sequence of the PS DNA gap region is important for optimal allele selectivity. Dose-dependent reduction of mu HTT and wt HTT mRNA in human GM04022 fibroblasts for ( A ) ASOs A29 (8-base PS DNA gap) and A30 (seven-base PS DNA gap) (b) ASOs A31 , A32 and A33 (seven-base PS DNA gaps frame-shifted by one nucleotide). Summary of sequence, activity and selectivity for ( C ) ASOs A1 , A29 , A30 and ( D ) A31 , A32 and A33 . Underlined nucleotide indicates position across from the SNP site. Error bars are in ±SD.
    Figure Legend Snippet: Precise sequence of the PS DNA gap region is important for optimal allele selectivity. Dose-dependent reduction of mu HTT and wt HTT mRNA in human GM04022 fibroblasts for ( A ) ASOs A29 (8-base PS DNA gap) and A30 (seven-base PS DNA gap) (b) ASOs A31 , A32 and A33 (seven-base PS DNA gaps frame-shifted by one nucleotide). Summary of sequence, activity and selectivity for ( C ) ASOs A1 , A29 , A30 and ( D ) A31 , A32 and A33 . Underlined nucleotide indicates position across from the SNP site. Error bars are in ±SD.

    Techniques Used: Sequencing, Activity Assay

    Effect of R -5′-Me-DNA and S -5′-Me-DNA on allele selectivity is dependent on position of incorporation and on the absolute configuration of the 5′-methyl group. Dose-dependent reduction of mu HTT and wt HTT mRNA in human GM04022 fibroblasts for ( A ) ASOs A17 ( R -5′-Me-DNA at position 5) and A18 ( S -5′-Me-DNA at position 5) and ( B ) ASOs A19 ( R -5′-Me-DNA at position 6) and A20 ( S -5′-Me-DNA at position 6). Summary of sequence, activity and selectivity for ( C ) ASOs A1 , A17 and A18 ( D ) ASOs A1 , A19 and A20 . Underlined nucleotide indicates position across from the SNP site. Error bars are in ±SD.
    Figure Legend Snippet: Effect of R -5′-Me-DNA and S -5′-Me-DNA on allele selectivity is dependent on position of incorporation and on the absolute configuration of the 5′-methyl group. Dose-dependent reduction of mu HTT and wt HTT mRNA in human GM04022 fibroblasts for ( A ) ASOs A17 ( R -5′-Me-DNA at position 5) and A18 ( S -5′-Me-DNA at position 5) and ( B ) ASOs A19 ( R -5′-Me-DNA at position 6) and A20 ( S -5′-Me-DNA at position 6). Summary of sequence, activity and selectivity for ( C ) ASOs A1 , A17 and A18 ( D ) ASOs A1 , A19 and A20 . Underlined nucleotide indicates position across from the SNP site. Error bars are in ±SD.

    Techniques Used: Sequencing, Activity Assay

    Effect of FANA and FRNA on allele selectivity is dependent on position of incorporation and on the absolute configuration of the 2′-fluorine atom. Dose-dependent reduction of mu HTT and wt HTT mRNA in human GM04022 fibroblasts for ( A ) ASOs A21 (FANA at position 5) and A22 (FRNA at position 5) and ( B ) ASOs A23 (FANA at position 6) and A24 (FRNA at position 6). Summary of sequence, activity and selectivity for ( C ) ASOs A1 , A21 and A22 ( D ) ASOs A1 , A23 and A24 . Underlined nucleotide indicates position across from the SNP site. Error bars are in ±SD.
    Figure Legend Snippet: Effect of FANA and FRNA on allele selectivity is dependent on position of incorporation and on the absolute configuration of the 2′-fluorine atom. Dose-dependent reduction of mu HTT and wt HTT mRNA in human GM04022 fibroblasts for ( A ) ASOs A21 (FANA at position 5) and A22 (FRNA at position 5) and ( B ) ASOs A23 (FANA at position 6) and A24 (FRNA at position 6). Summary of sequence, activity and selectivity for ( C ) ASOs A1 , A21 and A22 ( D ) ASOs A1 , A23 and A24 . Underlined nucleotide indicates position across from the SNP site. Error bars are in ±SD.

    Techniques Used: Sequencing, Activity Assay

    2S-dT has a positional effect on allele selectivity. ( A ) Dose-dependent reduction of mu HTT and wt HTT mRNA in human GM04022 fibroblasts using ASOs A15 and A16 . ( B ) Human RNase H1 cleavage patterns for A16 with 2S-dT at position 6 and A15 with 2S-dT at position 5. ( C ) Pictorial representation of human RNase H cleavage patterns for A16 and A15 . 2S-dT at position 5 blocks cleavage sites (d and c), whereas 2S-dT at position 6 blocks cleavage sites (b and c) on the matched and mismatched duplexes. The GT base pair ablates cleavage sites (a and b) on the mismatched duplex for ASOs A15 and A16 . Underlined nucleotide indicates position across from the SNP site. ( D ) Summary of potency and selectivity profile of A15 and A16 . Error bars are in ±SD.
    Figure Legend Snippet: 2S-dT has a positional effect on allele selectivity. ( A ) Dose-dependent reduction of mu HTT and wt HTT mRNA in human GM04022 fibroblasts using ASOs A15 and A16 . ( B ) Human RNase H1 cleavage patterns for A16 with 2S-dT at position 6 and A15 with 2S-dT at position 5. ( C ) Pictorial representation of human RNase H cleavage patterns for A16 and A15 . 2S-dT at position 5 blocks cleavage sites (d and c), whereas 2S-dT at position 6 blocks cleavage sites (b and c) on the matched and mismatched duplexes. The GT base pair ablates cleavage sites (a and b) on the mismatched duplex for ASOs A15 and A16 . Underlined nucleotide indicates position across from the SNP site. ( D ) Summary of potency and selectivity profile of A15 and A16 . Error bars are in ±SD.

    Techniques Used:

    FHNA and S -cEt improve allele selectivity when incorporated at positions 5 and 6 of ASO A1 . Dose-dependent reduction of mu HTT and wt HTT mRNA in human GM04022 fibroblasts for ( A ) ASO A25 (FHNA at position 6) and A26 (S-cEt at position 5) ( B ) ASO A27 (FHNA at position 6) and A28 ( S -cEt at position 6). Summary of sequence, activity and selectivity for ASOs ( C ) A1 , A25 , A26 and ( D ) A27 and A28 . Underlined nucleotide indicates position across from the SNP site. Error bars are in ±SD.
    Figure Legend Snippet: FHNA and S -cEt improve allele selectivity when incorporated at positions 5 and 6 of ASO A1 . Dose-dependent reduction of mu HTT and wt HTT mRNA in human GM04022 fibroblasts for ( A ) ASO A25 (FHNA at position 6) and A26 (S-cEt at position 5) ( B ) ASO A27 (FHNA at position 6) and A28 ( S -cEt at position 6). Summary of sequence, activity and selectivity for ASOs ( C ) A1 , A25 , A26 and ( D ) A27 and A28 . Underlined nucleotide indicates position across from the SNP site. Error bars are in ±SD.

    Techniques Used: Allele-specific Oligonucleotide, Sequencing, Activity Assay

    Chemical design and analysis of human RNase H1 cleavage patterns for ASO A1. (A) Chemical design of starting ASO A1, black letters indicate DNA, orange letters indicate MOE and blue letters indicate S -cEt nucleotides; underlined letter is the nucleotide across from the SNP site; all internucleosidic linkages are PS. (B) T m of A1 with matched and mismatched RNA complements representing mu HTT and wt HTT mRNA and structures of the AT cognate and GT wobble base pairs. (C and D) Analysis of human RNase H1 cleavage patterns of the matched duplex (A1/RNA mu ) versus the mismatched duplex (A1/RNA wt ) containing a GT wobble base pair positioned in the center of the PS DNA gap. Peaks labeled a, b, c and d correspond to the RNA fragments arising from human RNase H1 cleavage and are depicted using colored arrows. The cleavage products were analysed using (C) LCMS or (D) gel electrophoresis using 5′-radiolabeled RNA.
    Figure Legend Snippet: Chemical design and analysis of human RNase H1 cleavage patterns for ASO A1. (A) Chemical design of starting ASO A1, black letters indicate DNA, orange letters indicate MOE and blue letters indicate S -cEt nucleotides; underlined letter is the nucleotide across from the SNP site; all internucleosidic linkages are PS. (B) T m of A1 with matched and mismatched RNA complements representing mu HTT and wt HTT mRNA and structures of the AT cognate and GT wobble base pairs. (C and D) Analysis of human RNase H1 cleavage patterns of the matched duplex (A1/RNA mu ) versus the mismatched duplex (A1/RNA wt ) containing a GT wobble base pair positioned in the center of the PS DNA gap. Peaks labeled a, b, c and d correspond to the RNA fragments arising from human RNase H1 cleavage and are depicted using colored arrows. The cleavage products were analysed using (C) LCMS or (D) gel electrophoresis using 5′-radiolabeled RNA.

    Techniques Used: Allele-specific Oligonucleotide, Labeling, Liquid Chromatography with Mass Spectroscopy, Nucleic Acid Electrophoresis

    7) Product Images from "A neonatal gnotobiotic pig model of human enterovirus 71 infection and associated immune responses"

    Article Title: A neonatal gnotobiotic pig model of human enterovirus 71 infection and associated immune responses

    Journal: Emerging Microbes & Infections

    doi: 10.1038/emi.2014.35

    Fever, limb paralysis, vesicles and lung lesions in EV71-infected neonatal gnotobiotic pigs. ( A ) Body temperature in the EV71-infected neonatal gnotobiotic pigs. Body temperature was measured using subcutaneously implanted microchips posterior to the ear. The body temperature at each time point represents an average of three measurements. The normal core body temperature of pigs ranges from 38 to 40 °C, with an average of 38.8 °C. A temperature higher than 40 °C is considered a fever. ( B ) Vesicles (indicated by black arrow) on the snouts of EV71-infected neonatal gnotobiotic pigs. ( C ) Forelimb weakness in neonatal gnotobiotic pigs infected with the EV71 BJ110 strain. ( D ) An age-matched mock-infected control neonatal gnotobiotic pig. B ( E ) Multifocal mottling with petechial hemorrhages (indicated by the circle) in the lung was observed in an oral-nasally inoculated gnotobiotic pig on PID 21. ( F ) Normal lung from a mock-infected gnotobiotic pig. * P
    Figure Legend Snippet: Fever, limb paralysis, vesicles and lung lesions in EV71-infected neonatal gnotobiotic pigs. ( A ) Body temperature in the EV71-infected neonatal gnotobiotic pigs. Body temperature was measured using subcutaneously implanted microchips posterior to the ear. The body temperature at each time point represents an average of three measurements. The normal core body temperature of pigs ranges from 38 to 40 °C, with an average of 38.8 °C. A temperature higher than 40 °C is considered a fever. ( B ) Vesicles (indicated by black arrow) on the snouts of EV71-infected neonatal gnotobiotic pigs. ( C ) Forelimb weakness in neonatal gnotobiotic pigs infected with the EV71 BJ110 strain. ( D ) An age-matched mock-infected control neonatal gnotobiotic pig. B ( E ) Multifocal mottling with petechial hemorrhages (indicated by the circle) in the lung was observed in an oral-nasally inoculated gnotobiotic pig on PID 21. ( F ) Normal lung from a mock-infected gnotobiotic pig. * P

    Techniques Used: Infection

    Microscopic lesions in neonatal gnotobiotic pigs infected with the EV71 BJ110 strain. ( A ) The upper panel shows a section of the lung of an orally infected gnotobiotic pig and a mock-infected control on PID 21. The infected pig has peribronchial and perivascular hemorrhage (indicated by the arrow). An adjacent alveolus contains scattered erythrocytes and macrophages (indicated by the black triangle). ( B ) The lower panel shows the small intestine of an oral-nasally infected gnotobiotic pig on PID 7, with a prominent presence of immune cells in the lamina propria and a significantly increased number of Peyer's patches (indicated by the asterisk). Lung tissues were stained with H E; small intestinal tissues were sections of resin-embedded tissue stained with toluidine blue. H E, hematoxylin and eosin.
    Figure Legend Snippet: Microscopic lesions in neonatal gnotobiotic pigs infected with the EV71 BJ110 strain. ( A ) The upper panel shows a section of the lung of an orally infected gnotobiotic pig and a mock-infected control on PID 21. The infected pig has peribronchial and perivascular hemorrhage (indicated by the arrow). An adjacent alveolus contains scattered erythrocytes and macrophages (indicated by the black triangle). ( B ) The lower panel shows the small intestine of an oral-nasally infected gnotobiotic pig on PID 7, with a prominent presence of immune cells in the lamina propria and a significantly increased number of Peyer's patches (indicated by the asterisk). Lung tissues were stained with H E; small intestinal tissues were sections of resin-embedded tissue stained with toluidine blue. H E, hematoxylin and eosin.

    Techniques Used: Infection, Staining

    Immune responses during EV71 infection in neonatal gnotobiotic pigs. ( A ) Representative dot plots showing the frequency of CD3 + CD4 + IFN-γ + and CD3 + CD8 + IFN-γ + T lymphocytes among the total CD3 + mononuclear cells in the blood. MNCs were stimulated with semi-purified whole EV71 antigen or control medium for 17 h prior to staining. ( B ) The frequency of IFN-γ-producing CD3 + CD4 + and CD3 + CD8 + T cells among the CD3 + mononuclear cells in the ileum, spleen, blood, brain and lung on PID 7 and PID 14 after EV71 BJ110 infection via the oral–nasal route in neonatal gnotobiotic pigs. The mean frequencies were calculated by subtracting the mean frequency value of medium/mock stimulated cells from the mean frequency value of virus-stimulated cells. A positive mean frequency value indicates that IFN-γ production was upregulated upon virus stimulation, whereas a negative mean frequency value indicates that IFN-γ production was downregulated upon virus stimulation. The mean value for the EV71-infected group is indicated by the solid line, and the mean value for the mock control group is indicated by the dashed line. ( C ) Serum neutralizing antibody response in EV71-infected neonatal gnotobiotic pigs. Different uppercase letters (i.e., A, B and C) indicate significant differences between different treatment groups and different time points within the same treatment group. Shared uppercase letters or no letters indicate that no significant differences were observed. (Kruskal–Wallis test, P
    Figure Legend Snippet: Immune responses during EV71 infection in neonatal gnotobiotic pigs. ( A ) Representative dot plots showing the frequency of CD3 + CD4 + IFN-γ + and CD3 + CD8 + IFN-γ + T lymphocytes among the total CD3 + mononuclear cells in the blood. MNCs were stimulated with semi-purified whole EV71 antigen or control medium for 17 h prior to staining. ( B ) The frequency of IFN-γ-producing CD3 + CD4 + and CD3 + CD8 + T cells among the CD3 + mononuclear cells in the ileum, spleen, blood, brain and lung on PID 7 and PID 14 after EV71 BJ110 infection via the oral–nasal route in neonatal gnotobiotic pigs. The mean frequencies were calculated by subtracting the mean frequency value of medium/mock stimulated cells from the mean frequency value of virus-stimulated cells. A positive mean frequency value indicates that IFN-γ production was upregulated upon virus stimulation, whereas a negative mean frequency value indicates that IFN-γ production was downregulated upon virus stimulation. The mean value for the EV71-infected group is indicated by the solid line, and the mean value for the mock control group is indicated by the dashed line. ( C ) Serum neutralizing antibody response in EV71-infected neonatal gnotobiotic pigs. Different uppercase letters (i.e., A, B and C) indicate significant differences between different treatment groups and different time points within the same treatment group. Shared uppercase letters or no letters indicate that no significant differences were observed. (Kruskal–Wallis test, P

    Techniques Used: Infection, Purification, Staining

    The human EV71 BJ110 strain infects and replicates in pig intestinal epithelial cells, PBMCs and neural cell culture in vitro . ( A ) Identification of virus inoculum. ( a ) RT-PCR detection of the EV71 BJ110 strain. The positive PCR products were purified and sequenced. The sequence shares 100% identity with the published EV71 BJ110 strain VP1 gene sequence. ( b ) Mock-infected Vero cell culture. ( c ) CPE in Vero cells 72 h post-inoculation with the EV71 BJ110 strain at a MOI of 10. ( d ) EV71-infected Vero cells were detected using CCIF. ( B ) Porcine cell cultures can be infected by the EV71 BJ110 strain. EV71 was detected in IPEC-J2 cells, PBMCs and neural cells using CCIF. ( C ) Growth curves for the EV71 BJ110 strain in Vero and IPEC-J2 cells suggest that EV71 infects and replicates efficiently in IPEC-J2 cells.
    Figure Legend Snippet: The human EV71 BJ110 strain infects and replicates in pig intestinal epithelial cells, PBMCs and neural cell culture in vitro . ( A ) Identification of virus inoculum. ( a ) RT-PCR detection of the EV71 BJ110 strain. The positive PCR products were purified and sequenced. The sequence shares 100% identity with the published EV71 BJ110 strain VP1 gene sequence. ( b ) Mock-infected Vero cell culture. ( c ) CPE in Vero cells 72 h post-inoculation with the EV71 BJ110 strain at a MOI of 10. ( d ) EV71-infected Vero cells were detected using CCIF. ( B ) Porcine cell cultures can be infected by the EV71 BJ110 strain. EV71 was detected in IPEC-J2 cells, PBMCs and neural cells using CCIF. ( C ) Growth curves for the EV71 BJ110 strain in Vero and IPEC-J2 cells suggest that EV71 infects and replicates efficiently in IPEC-J2 cells.

    Techniques Used: Cell Culture, In Vitro, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Purification, Sequencing, Infection

    EV71 BJ110 strain fecal shedding, tissue distribution and dynamics in infected neonatal gnotobiotic pigs. ( A ) RT-PCR detection of EV71 in rectal swab samples. Left, RT-PCR detection of EV71 viral RNA in one orally infected pig (M, marker; lane 1, positive control; lanes 2–10, PID 0–8). Middle, RT-PCR detection of EV71 viral RNA in one oral–nasally infected pig (M, marker; lanes 1–8, PID 0–7). Right, RT-PCR detection of EV71 viral RNA in a mock control pig (lanes 1–8, PID 0–7; M, marker). ( B ) Taqman real-time PCR detection of virus shedding in rectal swab samples from different treatment groups. The mean viral RNA titer for each group at a specific time point is presented. The error bar indicates the standard error of the mean. No virus shedding was detected for any mock control group pigs at any time point; therefore, no bars are visible. ( C ) Detection of viral antigen on PID 7 and PID 14 in the ileum of gnotobiotic pigs infected with the EV71 BJ110 strain through the oral–nasal route at a dose of 5×10 8 FFU by immunofluorescence staining. A mouse anti-EV71 capsid protein VP1 monoclonal antibody (Abcam) was used as the primary antibody, and a goat anti-mouse IgG1 antibody labeled with fluorescein isothiocyanate (Sigma-Aldrich) was used as the secondary antibody. Nuclei were stained red by propidium iodide (Invitrogen). ( D ) Taqman real-time PCR detection of EV71 viral RNA in tissues of infected gnotobiotic pigs at PID 7 or PID 14. The route of inoculation and the euthanasia time (in parentheses) of the pigs are marked in the legends. Viral titers are presented as the mean of two replicates for the same sample. The negative samples are shown as blank on the bar graph. All data are representative of at least two independent experiments. BG, basal ganglia; CCC, caudal cerebral cortex; Duo, duodenum; Jej, jejunum; MLN, mesenteric lymph nodes; OB, olfactory bulb; RCC, rostral cerebral cortex; SC-C, spinal cord-cervical; SC-L, spinal cord-lumbar; SC-T, spinal cord-thoracic; SC-S, spinal cord-sacral.
    Figure Legend Snippet: EV71 BJ110 strain fecal shedding, tissue distribution and dynamics in infected neonatal gnotobiotic pigs. ( A ) RT-PCR detection of EV71 in rectal swab samples. Left, RT-PCR detection of EV71 viral RNA in one orally infected pig (M, marker; lane 1, positive control; lanes 2–10, PID 0–8). Middle, RT-PCR detection of EV71 viral RNA in one oral–nasally infected pig (M, marker; lanes 1–8, PID 0–7). Right, RT-PCR detection of EV71 viral RNA in a mock control pig (lanes 1–8, PID 0–7; M, marker). ( B ) Taqman real-time PCR detection of virus shedding in rectal swab samples from different treatment groups. The mean viral RNA titer for each group at a specific time point is presented. The error bar indicates the standard error of the mean. No virus shedding was detected for any mock control group pigs at any time point; therefore, no bars are visible. ( C ) Detection of viral antigen on PID 7 and PID 14 in the ileum of gnotobiotic pigs infected with the EV71 BJ110 strain through the oral–nasal route at a dose of 5×10 8 FFU by immunofluorescence staining. A mouse anti-EV71 capsid protein VP1 monoclonal antibody (Abcam) was used as the primary antibody, and a goat anti-mouse IgG1 antibody labeled with fluorescein isothiocyanate (Sigma-Aldrich) was used as the secondary antibody. Nuclei were stained red by propidium iodide (Invitrogen). ( D ) Taqman real-time PCR detection of EV71 viral RNA in tissues of infected gnotobiotic pigs at PID 7 or PID 14. The route of inoculation and the euthanasia time (in parentheses) of the pigs are marked in the legends. Viral titers are presented as the mean of two replicates for the same sample. The negative samples are shown as blank on the bar graph. All data are representative of at least two independent experiments. BG, basal ganglia; CCC, caudal cerebral cortex; Duo, duodenum; Jej, jejunum; MLN, mesenteric lymph nodes; OB, olfactory bulb; RCC, rostral cerebral cortex; SC-C, spinal cord-cervical; SC-L, spinal cord-lumbar; SC-T, spinal cord-thoracic; SC-S, spinal cord-sacral.

    Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction, Marker, Positive Control, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Labeling, Countercurrent Chromatography

    8) Product Images from "A neonatal gnotobiotic pig model of human enterovirus 71 infection and associated immune responses"

    Article Title: A neonatal gnotobiotic pig model of human enterovirus 71 infection and associated immune responses

    Journal: Emerging Microbes & Infections

    doi: 10.1038/emi.2014.35

    Fever, limb paralysis, vesicles and lung lesions in EV71-infected neonatal gnotobiotic pigs. ( A ) Body temperature in the EV71-infected neonatal gnotobiotic pigs. Body temperature was measured using subcutaneously implanted microchips posterior to the ear. The body temperature at each time point represents an average of three measurements. The normal core body temperature of pigs ranges from 38 to 40 °C, with an average of 38.8 °C. A temperature higher than 40 °C is considered a fever. ( B ) Vesicles (indicated by black arrow) on the snouts of EV71-infected neonatal gnotobiotic pigs. ( C ) Forelimb weakness in neonatal gnotobiotic pigs infected with the EV71 BJ110 strain. ( D ) An age-matched mock-infected control neonatal gnotobiotic pig. B ( E ) Multifocal mottling with petechial hemorrhages (indicated by the circle) in the lung was observed in an oral-nasally inoculated gnotobiotic pig on PID 21. ( F ) Normal lung from a mock-infected gnotobiotic pig. * P
    Figure Legend Snippet: Fever, limb paralysis, vesicles and lung lesions in EV71-infected neonatal gnotobiotic pigs. ( A ) Body temperature in the EV71-infected neonatal gnotobiotic pigs. Body temperature was measured using subcutaneously implanted microchips posterior to the ear. The body temperature at each time point represents an average of three measurements. The normal core body temperature of pigs ranges from 38 to 40 °C, with an average of 38.8 °C. A temperature higher than 40 °C is considered a fever. ( B ) Vesicles (indicated by black arrow) on the snouts of EV71-infected neonatal gnotobiotic pigs. ( C ) Forelimb weakness in neonatal gnotobiotic pigs infected with the EV71 BJ110 strain. ( D ) An age-matched mock-infected control neonatal gnotobiotic pig. B ( E ) Multifocal mottling with petechial hemorrhages (indicated by the circle) in the lung was observed in an oral-nasally inoculated gnotobiotic pig on PID 21. ( F ) Normal lung from a mock-infected gnotobiotic pig. * P

    Techniques Used: Infection

    Microscopic lesions in neonatal gnotobiotic pigs infected with the EV71 BJ110 strain. ( A ) The upper panel shows a section of the lung of an orally infected gnotobiotic pig and a mock-infected control on PID 21. The infected pig has peribronchial and perivascular hemorrhage (indicated by the arrow). An adjacent alveolus contains scattered erythrocytes and macrophages (indicated by the black triangle). ( B ) The lower panel shows the small intestine of an oral-nasally infected gnotobiotic pig on PID 7, with a prominent presence of immune cells in the lamina propria and a significantly increased number of Peyer's patches (indicated by the asterisk). Lung tissues were stained with H E; small intestinal tissues were sections of resin-embedded tissue stained with toluidine blue. H E, hematoxylin and eosin.
    Figure Legend Snippet: Microscopic lesions in neonatal gnotobiotic pigs infected with the EV71 BJ110 strain. ( A ) The upper panel shows a section of the lung of an orally infected gnotobiotic pig and a mock-infected control on PID 21. The infected pig has peribronchial and perivascular hemorrhage (indicated by the arrow). An adjacent alveolus contains scattered erythrocytes and macrophages (indicated by the black triangle). ( B ) The lower panel shows the small intestine of an oral-nasally infected gnotobiotic pig on PID 7, with a prominent presence of immune cells in the lamina propria and a significantly increased number of Peyer's patches (indicated by the asterisk). Lung tissues were stained with H E; small intestinal tissues were sections of resin-embedded tissue stained with toluidine blue. H E, hematoxylin and eosin.

    Techniques Used: Infection, Staining

    Immune responses during EV71 infection in neonatal gnotobiotic pigs. ( A ) Representative dot plots showing the frequency of CD3 + CD4 + IFN-γ + and CD3 + CD8 + IFN-γ + T lymphocytes among the total CD3 + mononuclear cells in the blood. MNCs were stimulated with semi-purified whole EV71 antigen or control medium for 17 h prior to staining. ( B ) The frequency of IFN-γ-producing CD3 + CD4 + and CD3 + CD8 + T cells among the CD3 + mononuclear cells in the ileum, spleen, blood, brain and lung on PID 7 and PID 14 after EV71 BJ110 infection via the oral–nasal route in neonatal gnotobiotic pigs. The mean frequencies were calculated by subtracting the mean frequency value of medium/mock stimulated cells from the mean frequency value of virus-stimulated cells. A positive mean frequency value indicates that IFN-γ production was upregulated upon virus stimulation, whereas a negative mean frequency value indicates that IFN-γ production was downregulated upon virus stimulation. The mean value for the EV71-infected group is indicated by the solid line, and the mean value for the mock control group is indicated by the dashed line. ( C ) Serum neutralizing antibody response in EV71-infected neonatal gnotobiotic pigs. Different uppercase letters (i.e., A, B and C) indicate significant differences between different treatment groups and different time points within the same treatment group. Shared uppercase letters or no letters indicate that no significant differences were observed. (Kruskal–Wallis test, P
    Figure Legend Snippet: Immune responses during EV71 infection in neonatal gnotobiotic pigs. ( A ) Representative dot plots showing the frequency of CD3 + CD4 + IFN-γ + and CD3 + CD8 + IFN-γ + T lymphocytes among the total CD3 + mononuclear cells in the blood. MNCs were stimulated with semi-purified whole EV71 antigen or control medium for 17 h prior to staining. ( B ) The frequency of IFN-γ-producing CD3 + CD4 + and CD3 + CD8 + T cells among the CD3 + mononuclear cells in the ileum, spleen, blood, brain and lung on PID 7 and PID 14 after EV71 BJ110 infection via the oral–nasal route in neonatal gnotobiotic pigs. The mean frequencies were calculated by subtracting the mean frequency value of medium/mock stimulated cells from the mean frequency value of virus-stimulated cells. A positive mean frequency value indicates that IFN-γ production was upregulated upon virus stimulation, whereas a negative mean frequency value indicates that IFN-γ production was downregulated upon virus stimulation. The mean value for the EV71-infected group is indicated by the solid line, and the mean value for the mock control group is indicated by the dashed line. ( C ) Serum neutralizing antibody response in EV71-infected neonatal gnotobiotic pigs. Different uppercase letters (i.e., A, B and C) indicate significant differences between different treatment groups and different time points within the same treatment group. Shared uppercase letters or no letters indicate that no significant differences were observed. (Kruskal–Wallis test, P

    Techniques Used: Infection, Purification, Staining

    The human EV71 BJ110 strain infects and replicates in pig intestinal epithelial cells, PBMCs and neural cell culture in vitro . ( A ) Identification of virus inoculum. ( a ) RT-PCR detection of the EV71 BJ110 strain. The positive PCR products were purified and sequenced. The sequence shares 100% identity with the published EV71 BJ110 strain VP1 gene sequence. ( b ) Mock-infected Vero cell culture. ( c ) CPE in Vero cells 72 h post-inoculation with the EV71 BJ110 strain at a MOI of 10. ( d ) EV71-infected Vero cells were detected using CCIF. ( B ) Porcine cell cultures can be infected by the EV71 BJ110 strain. EV71 was detected in IPEC-J2 cells, PBMCs and neural cells using CCIF. ( C ) Growth curves for the EV71 BJ110 strain in Vero and IPEC-J2 cells suggest that EV71 infects and replicates efficiently in IPEC-J2 cells.
    Figure Legend Snippet: The human EV71 BJ110 strain infects and replicates in pig intestinal epithelial cells, PBMCs and neural cell culture in vitro . ( A ) Identification of virus inoculum. ( a ) RT-PCR detection of the EV71 BJ110 strain. The positive PCR products were purified and sequenced. The sequence shares 100% identity with the published EV71 BJ110 strain VP1 gene sequence. ( b ) Mock-infected Vero cell culture. ( c ) CPE in Vero cells 72 h post-inoculation with the EV71 BJ110 strain at a MOI of 10. ( d ) EV71-infected Vero cells were detected using CCIF. ( B ) Porcine cell cultures can be infected by the EV71 BJ110 strain. EV71 was detected in IPEC-J2 cells, PBMCs and neural cells using CCIF. ( C ) Growth curves for the EV71 BJ110 strain in Vero and IPEC-J2 cells suggest that EV71 infects and replicates efficiently in IPEC-J2 cells.

    Techniques Used: Cell Culture, In Vitro, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Purification, Sequencing, Infection

    EV71 BJ110 strain fecal shedding, tissue distribution and dynamics in infected neonatal gnotobiotic pigs. ( A ) RT-PCR detection of EV71 in rectal swab samples. Left, RT-PCR detection of EV71 viral RNA in one orally infected pig (M, marker; lane 1, positive control; lanes 2–10, PID 0–8). Middle, RT-PCR detection of EV71 viral RNA in one oral–nasally infected pig (M, marker; lanes 1–8, PID 0–7). Right, RT-PCR detection of EV71 viral RNA in a mock control pig (lanes 1–8, PID 0–7; M, marker). ( B ) Taqman real-time PCR detection of virus shedding in rectal swab samples from different treatment groups. The mean viral RNA titer for each group at a specific time point is presented. The error bar indicates the standard error of the mean. No virus shedding was detected for any mock control group pigs at any time point; therefore, no bars are visible. ( C ) Detection of viral antigen on PID 7 and PID 14 in the ileum of gnotobiotic pigs infected with the EV71 BJ110 strain through the oral–nasal route at a dose of 5×10 8 FFU by immunofluorescence staining. A mouse anti-EV71 capsid protein VP1 monoclonal antibody (Abcam) was used as the primary antibody, and a goat anti-mouse IgG1 antibody labeled with fluorescein isothiocyanate (Sigma-Aldrich) was used as the secondary antibody. Nuclei were stained red by propidium iodide (Invitrogen). ( D ) Taqman real-time PCR detection of EV71 viral RNA in tissues of infected gnotobiotic pigs at PID 7 or PID 14. The route of inoculation and the euthanasia time (in parentheses) of the pigs are marked in the legends. Viral titers are presented as the mean of two replicates for the same sample. The negative samples are shown as blank on the bar graph. All data are representative of at least two independent experiments. BG, basal ganglia; CCC, caudal cerebral cortex; Duo, duodenum; Jej, jejunum; MLN, mesenteric lymph nodes; OB, olfactory bulb; RCC, rostral cerebral cortex; SC-C, spinal cord-cervical; SC-L, spinal cord-lumbar; SC-T, spinal cord-thoracic; SC-S, spinal cord-sacral.
    Figure Legend Snippet: EV71 BJ110 strain fecal shedding, tissue distribution and dynamics in infected neonatal gnotobiotic pigs. ( A ) RT-PCR detection of EV71 in rectal swab samples. Left, RT-PCR detection of EV71 viral RNA in one orally infected pig (M, marker; lane 1, positive control; lanes 2–10, PID 0–8). Middle, RT-PCR detection of EV71 viral RNA in one oral–nasally infected pig (M, marker; lanes 1–8, PID 0–7). Right, RT-PCR detection of EV71 viral RNA in a mock control pig (lanes 1–8, PID 0–7; M, marker). ( B ) Taqman real-time PCR detection of virus shedding in rectal swab samples from different treatment groups. The mean viral RNA titer for each group at a specific time point is presented. The error bar indicates the standard error of the mean. No virus shedding was detected for any mock control group pigs at any time point; therefore, no bars are visible. ( C ) Detection of viral antigen on PID 7 and PID 14 in the ileum of gnotobiotic pigs infected with the EV71 BJ110 strain through the oral–nasal route at a dose of 5×10 8 FFU by immunofluorescence staining. A mouse anti-EV71 capsid protein VP1 monoclonal antibody (Abcam) was used as the primary antibody, and a goat anti-mouse IgG1 antibody labeled with fluorescein isothiocyanate (Sigma-Aldrich) was used as the secondary antibody. Nuclei were stained red by propidium iodide (Invitrogen). ( D ) Taqman real-time PCR detection of EV71 viral RNA in tissues of infected gnotobiotic pigs at PID 7 or PID 14. The route of inoculation and the euthanasia time (in parentheses) of the pigs are marked in the legends. Viral titers are presented as the mean of two replicates for the same sample. The negative samples are shown as blank on the bar graph. All data are representative of at least two independent experiments. BG, basal ganglia; CCC, caudal cerebral cortex; Duo, duodenum; Jej, jejunum; MLN, mesenteric lymph nodes; OB, olfactory bulb; RCC, rostral cerebral cortex; SC-C, spinal cord-cervical; SC-L, spinal cord-lumbar; SC-T, spinal cord-thoracic; SC-S, spinal cord-sacral.

    Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction, Marker, Positive Control, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Labeling, Countercurrent Chromatography

    9) Product Images from "Targeting Cell Cycle Proteins in Breast Cancer Cells with siRNA by Using Lipid-Substituted Polyethylenimines"

    Article Title: Targeting Cell Cycle Proteins in Breast Cancer Cells with siRNA by Using Lipid-Substituted Polyethylenimines

    Journal: Frontiers in Bioengineering and Biotechnology

    doi: 10.3389/fbioe.2015.00014

    Combinations of siRNA (20 + 20 nM) treatments among KSP, CDC20, RAD51, and CHEK1 siRNA in MDA-MB-435WT (A) with siRNA:PEI-LA ratio of 1:2 and MDA-MB-435R (B) with siRNA:PEI–LA ratio of 1:8 . Scrambled siRNA (CsiRNA) was used as a control on its own as well as in combination with other siRNAs. The SD (not shown) was
    Figure Legend Snippet: Combinations of siRNA (20 + 20 nM) treatments among KSP, CDC20, RAD51, and CHEK1 siRNA in MDA-MB-435WT (A) with siRNA:PEI-LA ratio of 1:2 and MDA-MB-435R (B) with siRNA:PEI–LA ratio of 1:8 . Scrambled siRNA (CsiRNA) was used as a control on its own as well as in combination with other siRNAs. The SD (not shown) was

    Techniques Used: Multiple Displacement Amplification

    Inhibition of cell growth using DsiRNAs against CDC20, RAD51, and CHEK1 at 20 and 40 nM DsiRNA concentrations with different DsiRNA:PEI–LA ratios in MDA-MB-435WT (A,B) and MDA-MB-435R (C,D) . For each target proteins, three different DsiRNA isoforms were used. The significance (*) at p
    Figure Legend Snippet: Inhibition of cell growth using DsiRNAs against CDC20, RAD51, and CHEK1 at 20 and 40 nM DsiRNA concentrations with different DsiRNA:PEI–LA ratios in MDA-MB-435WT (A,B) and MDA-MB-435R (C,D) . For each target proteins, three different DsiRNA isoforms were used. The significance (*) at p

    Techniques Used: Inhibition, Multiple Displacement Amplification

    Digital droplet PCR (ddPCR) analysis in MDA-MB-435WT cells after 24 h (A) and 48 h (B) of treatment with indicated siRNAs . The percentage of quantity of transcripts was calculated based on the transcripts level of untreated cells (100%). The significance (*) at p
    Figure Legend Snippet: Digital droplet PCR (ddPCR) analysis in MDA-MB-435WT cells after 24 h (A) and 48 h (B) of treatment with indicated siRNAs . The percentage of quantity of transcripts was calculated based on the transcripts level of untreated cells (100%). The significance (*) at p

    Techniques Used: Polymerase Chain Reaction, Multiple Displacement Amplification

    Effect of CDC20 DsiRNA treatment in vivo . Xenografts of MDA-MB-435WT were established in nude mice, and were treated with a scrambled DsiRNA (CsiRNA) and CDC20-1 DsiRNA. Relative tumor volume for weekly injection [ (A) ; n = 6 and n = 5 in CsiRNA and CDC20-1 groups, respectively] and bi-weekly injection [ (B) ; n = 3 and n = 4 in CsiRNA and CDC20-1, respectively] groups, are summarized (only positive SDs are shown for clarity). The time points that showed a significant decrease in the volume of CDC20-1 DsiRNA treated tumors compared to CsiRNA treated tumor are indicated with an asterisk ( p
    Figure Legend Snippet: Effect of CDC20 DsiRNA treatment in vivo . Xenografts of MDA-MB-435WT were established in nude mice, and were treated with a scrambled DsiRNA (CsiRNA) and CDC20-1 DsiRNA. Relative tumor volume for weekly injection [ (A) ; n = 6 and n = 5 in CsiRNA and CDC20-1 groups, respectively] and bi-weekly injection [ (B) ; n = 3 and n = 4 in CsiRNA and CDC20-1, respectively] groups, are summarized (only positive SDs are shown for clarity). The time points that showed a significant decrease in the volume of CDC20-1 DsiRNA treated tumors compared to CsiRNA treated tumor are indicated with an asterisk ( p

    Techniques Used: In Vivo, Multiple Displacement Amplification, Mouse Assay, Injection

    (A) Uptake of FAM-labeled siRNA complexes at 20 and 40 nM siRNA using 1:2, 1:4, and 1:8 siRNA:PEI–LA ratios. The MDA-MB-435WT cells were incubated with the complexes for 24 h and recovered for flow cytometry analysis. The results were summarized as mean FAM-siRNA per cell (top) and FAM-siRNA positive cell population (bottom). (B) Confocal microscopy to determine the uptake of FAM-labeled siRNA complexes at 40 nM siRNA with 1:2 (ii) and 1:8 (iii) siRNA:PEI–LA ratios after 24 h treatment. Purple, red, and green colors represent nuclei, cytoplasm, and siRNA complexes, respectively. Non-labeled scrambled siRNA was transfected as a control in MDA-MB-435WT (i). (C) The number of visible complexes per cell (as quantitated from confocal microscopy images) at 1:2 and 1:8 siRNA:PEI–LA ratios. The uptake was significantly different between 1:2 and 1:8 ratios (* p
    Figure Legend Snippet: (A) Uptake of FAM-labeled siRNA complexes at 20 and 40 nM siRNA using 1:2, 1:4, and 1:8 siRNA:PEI–LA ratios. The MDA-MB-435WT cells were incubated with the complexes for 24 h and recovered for flow cytometry analysis. The results were summarized as mean FAM-siRNA per cell (top) and FAM-siRNA positive cell population (bottom). (B) Confocal microscopy to determine the uptake of FAM-labeled siRNA complexes at 40 nM siRNA with 1:2 (ii) and 1:8 (iii) siRNA:PEI–LA ratios after 24 h treatment. Purple, red, and green colors represent nuclei, cytoplasm, and siRNA complexes, respectively. Non-labeled scrambled siRNA was transfected as a control in MDA-MB-435WT (i). (C) The number of visible complexes per cell (as quantitated from confocal microscopy images) at 1:2 and 1:8 siRNA:PEI–LA ratios. The uptake was significantly different between 1:2 and 1:8 ratios (* p

    Techniques Used: Labeling, Multiple Displacement Amplification, Incubation, Flow Cytometry, Cytometry, Confocal Microscopy, Transfection

    Validation of KSP, CDC20, RAD51, and CHEK1 in MDA-MB-435WT cells at 20 nM (A,C) and 40 nM (B,D) siRNA concentrations . The results from PEI–LA were summarized in (A,B) (siRNA:polymer ratios of 1:2, 1:4, and 1:8) while the results from PEI–CA were summarized in (C,D) (siRNA:polymer ratios of 1:2 and 1:4). Scrambled siRNA (CsiRNA) was used as a control. Significant (* p
    Figure Legend Snippet: Validation of KSP, CDC20, RAD51, and CHEK1 in MDA-MB-435WT cells at 20 nM (A,C) and 40 nM (B,D) siRNA concentrations . The results from PEI–LA were summarized in (A,B) (siRNA:polymer ratios of 1:2, 1:4, and 1:8) while the results from PEI–CA were summarized in (C,D) (siRNA:polymer ratios of 1:2 and 1:4). Scrambled siRNA (CsiRNA) was used as a control. Significant (* p

    Techniques Used: Multiple Displacement Amplification

    Screening of polymeric carriers in MDA-MB-435WT cells at 1:2 (A) and 1:4 (B) siRNA to carrier ratios with 20 nM of scrambled siRNA (control; CsiRNA) and KSP siRNA . Polyethylenimines (PEI) substituted with linoleic acid (LA), caprylic acid (CA), and α-linoleic acid (αLA) were used in the initial screen along with two commercially available carriers.
    Figure Legend Snippet: Screening of polymeric carriers in MDA-MB-435WT cells at 1:2 (A) and 1:4 (B) siRNA to carrier ratios with 20 nM of scrambled siRNA (control; CsiRNA) and KSP siRNA . Polyethylenimines (PEI) substituted with linoleic acid (LA), caprylic acid (CA), and α-linoleic acid (αLA) were used in the initial screen along with two commercially available carriers.

    Techniques Used: Multiple Displacement Amplification

    10) Product Images from "C(5) modified uracil derivatives showing antiproliferative and erythroid differentiation inducing activities on human chronic myelogenous leukemia K562 cells"

    Article Title: C(5) modified uracil derivatives showing antiproliferative and erythroid differentiation inducing activities on human chronic myelogenous leukemia K562 cells

    Journal: European Journal of Pharmacology

    doi: 10.1016/j.ejphar.2011.09.024

    Accumulation of α-globin mRNA (A, black columns) and γ-globin mRNA (A, gray columns) in K562 cells treated for 6 days with 30 μM compound 9 . qRT-PCR amplifications were performed on RNA from untreated or treated cells using primers amplifying 18S ribosomal RNA as reference gene. Results are presented as fold increase of α-globin and γ-globin mRNAs with respect to untreated cells. (B,C) Effect of compounds 9 , 11 and 10 on accumulation of α-globin mRNA (B) and γ-globin mRNA (C). The results of untreated cells were taken as 1. Results represent the average ± S.D. of three independent experiments.
    Figure Legend Snippet: Accumulation of α-globin mRNA (A, black columns) and γ-globin mRNA (A, gray columns) in K562 cells treated for 6 days with 30 μM compound 9 . qRT-PCR amplifications were performed on RNA from untreated or treated cells using primers amplifying 18S ribosomal RNA as reference gene. Results are presented as fold increase of α-globin and γ-globin mRNAs with respect to untreated cells. (B,C) Effect of compounds 9 , 11 and 10 on accumulation of α-globin mRNA (B) and γ-globin mRNA (C). The results of untreated cells were taken as 1. Results represent the average ± S.D. of three independent experiments.

    Techniques Used: Quantitative RT-PCR

    11) Product Images from "Early Indicators of Disease in Ferrets Infected with a High Dose of Avian Influenza H5N1"

    Article Title: Early Indicators of Disease in Ferrets Infected with a High Dose of Avian Influenza H5N1

    Journal: Scientific Reports

    doi: 10.1038/srep00972

    Change in body temperature and body weight following infection with A/Vietnam/1203/04 virus. Temperatures were taken twice daily prior to challenge and then at approximately eight-hour intervals from 8–96 hours by use of subcutaneous implantable temperature transponders. After 96 hours, temperatures were recorded twice daily. Body weights were taken every other day, beginning on day 0 until the end of the study (day 18) or death of the animal. Weights taken immediately prior to challenge on day 0 served as baseline. (A) Mean body temperature, (B) Mean temperature comparison between survivors and animals that succumbed to disease, (C) mean body weight, (D) mean change from baseline weight for influenza-infected animals by survival status.
    Figure Legend Snippet: Change in body temperature and body weight following infection with A/Vietnam/1203/04 virus. Temperatures were taken twice daily prior to challenge and then at approximately eight-hour intervals from 8–96 hours by use of subcutaneous implantable temperature transponders. After 96 hours, temperatures were recorded twice daily. Body weights were taken every other day, beginning on day 0 until the end of the study (day 18) or death of the animal. Weights taken immediately prior to challenge on day 0 served as baseline. (A) Mean body temperature, (B) Mean temperature comparison between survivors and animals that succumbed to disease, (C) mean body weight, (D) mean change from baseline weight for influenza-infected animals by survival status.

    Techniques Used: Infection

    Survival of ferrets following challenge with 7 × 10 5 TCID 50 of A/Vietnam/1203/04 virus. Kaplan-Meier Curves representing the survival distribution for ferrets over the 18-day period following challenge.
    Figure Legend Snippet: Survival of ferrets following challenge with 7 × 10 5 TCID 50 of A/Vietnam/1203/04 virus. Kaplan-Meier Curves representing the survival distribution for ferrets over the 18-day period following challenge.

    Techniques Used:

    Clinical chemistry changes associated with A/Vietnam/1203/04 virus infection: (A) AT, (B) ALT, (C) glucose.
    Figure Legend Snippet: Clinical chemistry changes associated with A/Vietnam/1203/04 virus infection: (A) AT, (B) ALT, (C) glucose.

    Techniques Used: Infection

    Hematology changes associated with A/Vietnam/1203/04 virus infection: (A) RDW, (B) platelets, (C) WBC count, (D) eosinophils, (E) monoocytes, (F) lymphocytes, (G) LUC, (H) neutrophils and (I) N/L ratio.
    Figure Legend Snippet: Hematology changes associated with A/Vietnam/1203/04 virus infection: (A) RDW, (B) platelets, (C) WBC count, (D) eosinophils, (E) monoocytes, (F) lymphocytes, (G) LUC, (H) neutrophils and (I) N/L ratio.

    Techniques Used: Infection

    Replication of A/Vietnam/1203/04 in the upper respiratory tract of ferrets. Following challenge, nasal washes were performed at 8, 16, 24, 48, 72 and 96 hours for determination of virus shedding. Results represent viral titers for nasal wash specimens by (A) TCID 50 analysis, and (B) qPCR.
    Figure Legend Snippet: Replication of A/Vietnam/1203/04 in the upper respiratory tract of ferrets. Following challenge, nasal washes were performed at 8, 16, 24, 48, 72 and 96 hours for determination of virus shedding. Results represent viral titers for nasal wash specimens by (A) TCID 50 analysis, and (B) qPCR.

    Techniques Used: Real-time Polymerase Chain Reaction

    Clinical signs of disease in ferrets following infection with A/Vietnam/1203/04 virus. Animals were monitored twice daily for clinical observations and at eight-hour intervals until 96 hours, using the following scoring system; 0 = alert and playful, 1 = alert but playful only when stimulated, 2 = alert but not playful when stimulated, and 3 = neither alert nor playful when stimulated. (A) Percentage of animals exhibiting clinical observations, (B) time of onset for clinical observations presenting during the first 96 hours following infection, and (C) activity score changes for influenza-infected animals.
    Figure Legend Snippet: Clinical signs of disease in ferrets following infection with A/Vietnam/1203/04 virus. Animals were monitored twice daily for clinical observations and at eight-hour intervals until 96 hours, using the following scoring system; 0 = alert and playful, 1 = alert but playful only when stimulated, 2 = alert but not playful when stimulated, and 3 = neither alert nor playful when stimulated. (A) Percentage of animals exhibiting clinical observations, (B) time of onset for clinical observations presenting during the first 96 hours following infection, and (C) activity score changes for influenza-infected animals.

    Techniques Used: Infection, Activity Assay

    12) Product Images from "Granulosa cell-derived induced pluripotent stem cells exhibit pro-trophoblastic differentiation potential"

    Article Title: Granulosa cell-derived induced pluripotent stem cells exhibit pro-trophoblastic differentiation potential

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-015-0005-5

    In vitro and in vivo differentiation of human granulosa cell-derived induced pluripotent stem cells. (A) Immunofluorescence staining against three germ layer markers in a representative induced pluripotent stem cell line (iGRA1) following in vitro differentiation. (B) Hematoxylin and eosin staining of teratoma derived from a representative iGRA line (iGRA1). (C) RT-PCR analysis of expression of genes associated with three germ layers in in vitro differentiated iGRAs. 1, 2 and 7, iGRA1, iGRA2, and iGRA7, respectively. N, RNA transcripts untreated with reverse transcriptase (no RT control) served as a negative control for genomic DNA contamination. Scale bars = 50 μm (A) and 30 μm (B) .
    Figure Legend Snippet: In vitro and in vivo differentiation of human granulosa cell-derived induced pluripotent stem cells. (A) Immunofluorescence staining against three germ layer markers in a representative induced pluripotent stem cell line (iGRA1) following in vitro differentiation. (B) Hematoxylin and eosin staining of teratoma derived from a representative iGRA line (iGRA1). (C) RT-PCR analysis of expression of genes associated with three germ layers in in vitro differentiated iGRAs. 1, 2 and 7, iGRA1, iGRA2, and iGRA7, respectively. N, RNA transcripts untreated with reverse transcriptase (no RT control) served as a negative control for genomic DNA contamination. Scale bars = 50 μm (A) and 30 μm (B) .

    Techniques Used: In Vitro, In Vivo, Derivative Assay, Immunofluorescence, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control

    13) Product Images from "The Gdap1 knockout mouse mechanistically links redox control to Charcot-Marie-Tooth disease"

    Article Title: The Gdap1 knockout mouse mechanistically links redox control to Charcot-Marie-Tooth disease

    Journal: Brain

    doi: 10.1093/brain/awt371

    The paralogue of Gdap1 , Gdap1l1 , is expressed in the CNS. ( A ) Relative Gdap1l1 messenger RNA (mRNA) expression levels in mouse tissues were determined by quantitative reverse transcriptase PCR in relation to 18S ribosomal RNA. ( B ) GDAP1L1 protein expression was determined by western blot using 12 µg protein lysate of indicated murine tissues and anti-GDAP1L1 antiserum ( Supplementary Fig. 4A ). β-actin served as loading control. Arrowhead = predicted molecular weight of GDAP1L1.
    Figure Legend Snippet: The paralogue of Gdap1 , Gdap1l1 , is expressed in the CNS. ( A ) Relative Gdap1l1 messenger RNA (mRNA) expression levels in mouse tissues were determined by quantitative reverse transcriptase PCR in relation to 18S ribosomal RNA. ( B ) GDAP1L1 protein expression was determined by western blot using 12 µg protein lysate of indicated murine tissues and anti-GDAP1L1 antiserum ( Supplementary Fig. 4A ). β-actin served as loading control. Arrowhead = predicted molecular weight of GDAP1L1.

    Techniques Used: Expressing, Polymerase Chain Reaction, Western Blot, Molecular Weight

    Translocation of GDAP1L1 to mitochondria. ( A ) N1E-115 cells were transiently transfected with GDAP1L1 expression constructs and treated for 2 h as indicated. Cells were fixed, stained for GDAP1L1 and cytochrome c and examined by immunofluorescence. Single plane confocal images are shown. GDAP1L1 has mainly a cytosolic localization, which is altered upon treatment with menadione ( right ). ( B ) Primary hippocampal neurons were infected with lentivirus encoding mitochondrially targeted DsRed (MtDsRed). Four days post-infection, cells were treated for 2 h with menadione or left untreated, fixed and stained for endogenously expressed GDAP1L1. Single plane confocal images are shown. ( C ) Twenty-four hours post-transfection with GDAP1L1 expression constructs and subsequent 2 h treatment with the indicated reagents, N1E-115 cells were analysed on single plane confocal pictures. 2,3-dimethoxy-1,4-naphthoquinoe (DNMQ, 20 µM) led to a redistribution of GDAP1L1 to mitochondria as visualized by the colocalization with the mitochondrial marker cytochrome c (arrows). In contrast, tert -butylhydroquinone (TBHQ, 100 µM) had no impact on the subcellular distribution of GDAP1L1. Broken line indicates area shown in higher magnification. ( D ) N1E-115 cells were transiently transfected with GDAP1L1 expression constructs and homogenized using a Dounce homogenizer on the next day. The post-nuclear supernatant of the lysate was divided in two and incubated with reduced glutathione (GSH) or oxidized glutathione (GSSG). In differential sedimentation steps, cytosolic proteins were removed as demonstrated by western blot of all purification steps against the cytosolic marker β-actin. In the oxidized glutathione (GSSG)-treated fraction of the post nuclear supernatant, more GDAP1L1 co-sediments with the mitochondrial marker Porin compared with the reduced glutathione (GSH)-treated fraction. ( E ) Pellet 2 of GDAP1L1 expressing cells treated with oxidized glutathione was resuspended in buffer plus 5 mM dithiothreitol (DTT), in 0.1 M carbonate (pH 11), or in buffer containing 0.1% Triton™ X-100 and sedimented to separate soluble protein supernatants from membranous pellets. GDAP1L1 and the known transmembrane protein Porin are only solubilized by detergent. ( A–C ) Scale bars = 10 µm.
    Figure Legend Snippet: Translocation of GDAP1L1 to mitochondria. ( A ) N1E-115 cells were transiently transfected with GDAP1L1 expression constructs and treated for 2 h as indicated. Cells were fixed, stained for GDAP1L1 and cytochrome c and examined by immunofluorescence. Single plane confocal images are shown. GDAP1L1 has mainly a cytosolic localization, which is altered upon treatment with menadione ( right ). ( B ) Primary hippocampal neurons were infected with lentivirus encoding mitochondrially targeted DsRed (MtDsRed). Four days post-infection, cells were treated for 2 h with menadione or left untreated, fixed and stained for endogenously expressed GDAP1L1. Single plane confocal images are shown. ( C ) Twenty-four hours post-transfection with GDAP1L1 expression constructs and subsequent 2 h treatment with the indicated reagents, N1E-115 cells were analysed on single plane confocal pictures. 2,3-dimethoxy-1,4-naphthoquinoe (DNMQ, 20 µM) led to a redistribution of GDAP1L1 to mitochondria as visualized by the colocalization with the mitochondrial marker cytochrome c (arrows). In contrast, tert -butylhydroquinone (TBHQ, 100 µM) had no impact on the subcellular distribution of GDAP1L1. Broken line indicates area shown in higher magnification. ( D ) N1E-115 cells were transiently transfected with GDAP1L1 expression constructs and homogenized using a Dounce homogenizer on the next day. The post-nuclear supernatant of the lysate was divided in two and incubated with reduced glutathione (GSH) or oxidized glutathione (GSSG). In differential sedimentation steps, cytosolic proteins were removed as demonstrated by western blot of all purification steps against the cytosolic marker β-actin. In the oxidized glutathione (GSSG)-treated fraction of the post nuclear supernatant, more GDAP1L1 co-sediments with the mitochondrial marker Porin compared with the reduced glutathione (GSH)-treated fraction. ( E ) Pellet 2 of GDAP1L1 expressing cells treated with oxidized glutathione was resuspended in buffer plus 5 mM dithiothreitol (DTT), in 0.1 M carbonate (pH 11), or in buffer containing 0.1% Triton™ X-100 and sedimented to separate soluble protein supernatants from membranous pellets. GDAP1L1 and the known transmembrane protein Porin are only solubilized by detergent. ( A–C ) Scale bars = 10 µm.

    Techniques Used: Translocation Assay, Transfection, Expressing, Construct, Staining, Immunofluorescence, Infection, Marker, Incubation, Sedimentation, Western Blot, Purification

    Translocation of GDAP1L1 is needed to compensate for the loss of GDAP1 expression. ( A ) Spinal cords of 19-month-old wild-type or Gdap1 −/− animals were fractionated by differential centrifugation. In homogenates from Gdap1 −/− animals, more GDAP1L1 sediments with the mitochondrial marker Porin compared with age-matched wild-type controls. β-actin served as maker for the cytosolic fraction. ( B ) The relative amounts of GDAP1L1 were quantified by densitometry in relation to β-actin in the post-nuclear supernatant and the relative enrichment from GDAP1L1 was determined for pellet 2. The values obtained from wild-type animals were set to 1 in each of four independent experiments. Means and standard error of the mean, n = 4. n.s. = not significant, ** P
    Figure Legend Snippet: Translocation of GDAP1L1 is needed to compensate for the loss of GDAP1 expression. ( A ) Spinal cords of 19-month-old wild-type or Gdap1 −/− animals were fractionated by differential centrifugation. In homogenates from Gdap1 −/− animals, more GDAP1L1 sediments with the mitochondrial marker Porin compared with age-matched wild-type controls. β-actin served as maker for the cytosolic fraction. ( B ) The relative amounts of GDAP1L1 were quantified by densitometry in relation to β-actin in the post-nuclear supernatant and the relative enrichment from GDAP1L1 was determined for pellet 2. The values obtained from wild-type animals were set to 1 in each of four independent experiments. Means and standard error of the mean, n = 4. n.s. = not significant, ** P

    Techniques Used: Translocation Assay, Expressing, Centrifugation, Marker

    14) Product Images from "Imbalance of Wnt/Dkk Negative Feedback Promotes Persistent Activation of Pancreatic Stellate Cells in Chronic Pancreatitis"

    Article Title: Imbalance of Wnt/Dkk Negative Feedback Promotes Persistent Activation of Pancreatic Stellate Cells in Chronic Pancreatitis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0095145

    β-catenin-targeting siRNA inhibits the activation of PSCs in vitro . PSCs were transfected with negative control and β-catenin siRNA. (A) Transfection efficiency of PSCs. (B) β-catenin and α-SMA levels in cells were measured by western blotting. Blots were re-probed for GAPDH to confirm equal protein loading. (C) Hoechst 33342 staining. (D) The levels of α-SMA, PDGFRβ, TGFβRII and collagen1α1 mRNA were measured by quantitative RT-PCR. Data are presented as mean±SD from three independent experiments. *p
    Figure Legend Snippet: β-catenin-targeting siRNA inhibits the activation of PSCs in vitro . PSCs were transfected with negative control and β-catenin siRNA. (A) Transfection efficiency of PSCs. (B) β-catenin and α-SMA levels in cells were measured by western blotting. Blots were re-probed for GAPDH to confirm equal protein loading. (C) Hoechst 33342 staining. (D) The levels of α-SMA, PDGFRβ, TGFβRII and collagen1α1 mRNA were measured by quantitative RT-PCR. Data are presented as mean±SD from three independent experiments. *p

    Techniques Used: Activation Assay, In Vitro, Transfection, Negative Control, Western Blot, Staining, Quantitative RT-PCR

    15) Product Images from "MicroRNA-181c targets Bcl-2 and regulates mitochondrial morphology in myocardial cells"

    Article Title: MicroRNA-181c targets Bcl-2 and regulates mitochondrial morphology in myocardial cells

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.12563

    Genes included in the TNF-α-induced apoptosis pathway. The levels of miR-181c in the whole cells significantly increased after transfected with mimic ( P
    Figure Legend Snippet: Genes included in the TNF-α-induced apoptosis pathway. The levels of miR-181c in the whole cells significantly increased after transfected with mimic ( P

    Techniques Used: Transfection

    The levels of miR-181c (A) and pre-miR-181c (B) expression were titrated in myocardial cells transfected with mimic or inhibitor. Protein levels of Bcl-2 (C) changed in myocardial cells transfected with mimic or inhibitor (* P
    Figure Legend Snippet: The levels of miR-181c (A) and pre-miR-181c (B) expression were titrated in myocardial cells transfected with mimic or inhibitor. Protein levels of Bcl-2 (C) changed in myocardial cells transfected with mimic or inhibitor (* P

    Techniques Used: Expressing, Transfection

    The mouse myocardial cells, transfected with miR-181c mimic/inhibitor, were intervened with TNF-α and underwent the TEM scan. There were no significant changes in the gross morphology of myocardial cells and the number of mitochondria between groups (A–F, magnification: 4000 × to 30000 × ). The mitochondria showed abnormal appearance such as disorganization, rupture of the double membrane and reduction or vanish of the crista after TNF-α intervention. The severity of mitochondrial damage were for mimic group > control group > inhibitor group (G, magnification: 50000 × ).
    Figure Legend Snippet: The mouse myocardial cells, transfected with miR-181c mimic/inhibitor, were intervened with TNF-α and underwent the TEM scan. There were no significant changes in the gross morphology of myocardial cells and the number of mitochondria between groups (A–F, magnification: 4000 × to 30000 × ). The mitochondria showed abnormal appearance such as disorganization, rupture of the double membrane and reduction or vanish of the crista after TNF-α intervention. The severity of mitochondrial damage were for mimic group > control group > inhibitor group (G, magnification: 50000 × ).

    Techniques Used: Transfection, Transmission Electron Microscopy

    The seed sequences of Bcl-2 3′UTRs targeted by miR-181c are highly conserved across species (from TargetScan). With the computational miRNA target prediction algorithms at TargetScan, Bcl-2 was identified as one of potential targets of miR-181c.
    Figure Legend Snippet: The seed sequences of Bcl-2 3′UTRs targeted by miR-181c are highly conserved across species (from TargetScan). With the computational miRNA target prediction algorithms at TargetScan, Bcl-2 was identified as one of potential targets of miR-181c.

    Techniques Used:

    Setting cell membrane of the transfected cardiomyocytes labelled by the membrane fluorescence probe Dil as the boundary, the fluorescence intensity of FAM in the transfected cells were measured by the Cellomics ArrayScan™ Vti. FAM fluorescence (lane 1), Dil staining of cell membrane (lane 2), In the overlays (lane 3), FAM signal are visualized in green, Dil staining of cell membrane are in red. The fluorescence intensity of FAM signal was measured within the scope of the red fluorescence of Dil staining. The rows are scramble miRNA (A; negative control), mimic 50 nM (B), mimic 100 nM (C), mimic 150 nM (D), inhibitor 50 nM (E), inhibitor 50 nM (F) and inhibitor 150 nM (G). The H row show that the intracellular fluorescence intensity improved with the increase in miR-181c mimic/inhibitor concentration. These images are not scaled to the same intensity range. Fluorescence intensity was normalized by negative control intensity. FI: fluorescence intensity.
    Figure Legend Snippet: Setting cell membrane of the transfected cardiomyocytes labelled by the membrane fluorescence probe Dil as the boundary, the fluorescence intensity of FAM in the transfected cells were measured by the Cellomics ArrayScan™ Vti. FAM fluorescence (lane 1), Dil staining of cell membrane (lane 2), In the overlays (lane 3), FAM signal are visualized in green, Dil staining of cell membrane are in red. The fluorescence intensity of FAM signal was measured within the scope of the red fluorescence of Dil staining. The rows are scramble miRNA (A; negative control), mimic 50 nM (B), mimic 100 nM (C), mimic 150 nM (D), inhibitor 50 nM (E), inhibitor 50 nM (F) and inhibitor 150 nM (G). The H row show that the intracellular fluorescence intensity improved with the increase in miR-181c mimic/inhibitor concentration. These images are not scaled to the same intensity range. Fluorescence intensity was normalized by negative control intensity. FI: fluorescence intensity.

    Techniques Used: Transfection, Fluorescence, Staining, Negative Control, Concentration Assay

    EGFP Reporter Assay. The intensity of EGFP fluorescence in NIH3T3 cells transfected with pcDNA3/EGFP-Bcl2 3′UTR and miR-181c mimic reduce the luciferase activity compared with the control group. Luciferase assays were performed three times in triplicate. (* P
    Figure Legend Snippet: EGFP Reporter Assay. The intensity of EGFP fluorescence in NIH3T3 cells transfected with pcDNA3/EGFP-Bcl2 3′UTR and miR-181c mimic reduce the luciferase activity compared with the control group. Luciferase assays were performed three times in triplicate. (* P

    Techniques Used: Reporter Assay, Fluorescence, Transfection, Luciferase, Activity Assay

    16) Product Images from "Universal Single-Probe RT-PCR Assay for Diagnosis of Dengue Virus Infections"

    Article Title: Universal Single-Probe RT-PCR Assay for Diagnosis of Dengue Virus Infections

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0003416

    Results of the DENV RT-PCR assay performed on serum samples obtained 1 to 9 days after onset of symptoms. (A) Archived serum samples that had been collected from 60 patients on days 1–3 (n = 5), 4 (n = 10), 5 (n = 7), 6 (n = 11), 7 (n = 12), 8 (n = 8), and 9 (n = 7) after disease onset were tested by the DENV RT-PCR assay, an NS1 antigen detection test, IgM capture ELISA, and IFA detecting DENV-specific IgG antibodies. The criteria for a positive result in each of these analyses are explained in Methods . The curves show the percent of samples testing positive in the individual assays for the specified days after disease onset. (B) Viral load in samples collected on days 1–9 after onset of symptoms. Each dot represents the mean of results for duplicate samples from a single patient. GCE = genome copy equivalents.
    Figure Legend Snippet: Results of the DENV RT-PCR assay performed on serum samples obtained 1 to 9 days after onset of symptoms. (A) Archived serum samples that had been collected from 60 patients on days 1–3 (n = 5), 4 (n = 10), 5 (n = 7), 6 (n = 11), 7 (n = 12), 8 (n = 8), and 9 (n = 7) after disease onset were tested by the DENV RT-PCR assay, an NS1 antigen detection test, IgM capture ELISA, and IFA detecting DENV-specific IgG antibodies. The criteria for a positive result in each of these analyses are explained in Methods . The curves show the percent of samples testing positive in the individual assays for the specified days after disease onset. (B) Viral load in samples collected on days 1–9 after onset of symptoms. Each dot represents the mean of results for duplicate samples from a single patient. GCE = genome copy equivalents.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunofluorescence

    Overview of the primers and probe in the DENV RT-PCR assay. The reverse primers DENV_R1–3 (A) and DENV_R4 (B) were specifically designed to target DENV serotypes 1–3 and serotype 4, respectively. Vertical bars and percentages show the fraction of sequences with nucleotides deviating from the consensus of DENV serotypes 1–3 (A) and serotype 4 (B). Percentages below 1 are not shown. Numbers indicate genomic positions.
    Figure Legend Snippet: Overview of the primers and probe in the DENV RT-PCR assay. The reverse primers DENV_R1–3 (A) and DENV_R4 (B) were specifically designed to target DENV serotypes 1–3 and serotype 4, respectively. Vertical bars and percentages show the fraction of sequences with nucleotides deviating from the consensus of DENV serotypes 1–3 (A) and serotype 4 (B). Percentages below 1 are not shown. Numbers indicate genomic positions.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    Dynamic range and limit of detection of the DENV RT-PCR assay. (A) The linear dynamic range of the DENV RT-PCR assay was determined by testing triplicates of 10-fold serially diluted in vitro transcribed RNA. The sequences of the transcript RNA (RNA[DENV_R1–3] and RNA[DENV_R4]) were matched with the two reverse primers DENV_R1–3 and DENV_R4, respectively. Each dot represents the mean Cq-value from three replicates, the error bars indicate the 95% confidence interval, and the lines illustrate the result of the lin-log regression analysis. (B and C) Limit of detection was determined by assaying eight replicates of twofold serially diluted RNA transcripts in three separate experiments, and the results of testing are shown for RNA[DENV_R1–3] (B) and RNA[DENV_R4] (C). Horizontal lines indicate mean values, boxes denote the 25th to 75th percentiles and whiskers the 5–95% percentiles, and dots represent outliers. The number of positives per total number of replicates tested is given above each box. Limit of detection was defined as the last dilution in which transcript RNA was detected in all 24 replicates. GCE = genome copy equivalents.
    Figure Legend Snippet: Dynamic range and limit of detection of the DENV RT-PCR assay. (A) The linear dynamic range of the DENV RT-PCR assay was determined by testing triplicates of 10-fold serially diluted in vitro transcribed RNA. The sequences of the transcript RNA (RNA[DENV_R1–3] and RNA[DENV_R4]) were matched with the two reverse primers DENV_R1–3 and DENV_R4, respectively. Each dot represents the mean Cq-value from three replicates, the error bars indicate the 95% confidence interval, and the lines illustrate the result of the lin-log regression analysis. (B and C) Limit of detection was determined by assaying eight replicates of twofold serially diluted RNA transcripts in three separate experiments, and the results of testing are shown for RNA[DENV_R1–3] (B) and RNA[DENV_R4] (C). Horizontal lines indicate mean values, boxes denote the 25th to 75th percentiles and whiskers the 5–95% percentiles, and dots represent outliers. The number of positives per total number of replicates tested is given above each box. Limit of detection was defined as the last dilution in which transcript RNA was detected in all 24 replicates. GCE = genome copy equivalents.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, In Vitro

    Flow-chart showing the laboratory test results for samples from returning travelers with dengue-compatible symptomatology. Serum samples collected during January and February 2014 were tested consecutively by the newly developed DENV RT-PCR method, an NS1 antigen detection test, IgM capture ELISA, and/or an in-house IFA detecting DENV-specific IgG antibodies. The criteria for a positive result in each individual assay are explained in Methods . The results of the laboratory analysis of the first sample arriving at the Public Health Agency of Sweden are shown. w/o = without.
    Figure Legend Snippet: Flow-chart showing the laboratory test results for samples from returning travelers with dengue-compatible symptomatology. Serum samples collected during January and February 2014 were tested consecutively by the newly developed DENV RT-PCR method, an NS1 antigen detection test, IgM capture ELISA, and/or an in-house IFA detecting DENV-specific IgG antibodies. The criteria for a positive result in each individual assay are explained in Methods . The results of the laboratory analysis of the first sample arriving at the Public Health Agency of Sweden are shown. w/o = without.

    Techniques Used: Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunofluorescence

    17) Product Images from "Binding of Cyclic Di-AMP to the Staphylococcus aureus Sensor Kinase KdpD Occurs via the Universal Stress Protein Domain and Downregulates the Expression of the Kdp Potassium Transporter"

    Article Title: Binding of Cyclic Di-AMP to the Staphylococcus aureus Sensor Kinase KdpD Occurs via the Universal Stress Protein Domain and Downregulates the Expression of the Kdp Potassium Transporter

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00480-15

    High levels of c-di-AMP inhibit expression of kdpA . Shown is the relative quantification (RQ) of transcript levels of kdpA measured by RT-qPCR for the wild-type S. aureus strain LAC* (WT) and an isogenic gdpP (Δ gdpP ) mutant strain (with constitutively high levels of c-di-AMP), normalized to gyrB transcript levels. WT and mutant S. aureus strains were grown to an OD 600 of 0.7 in LB medium without salt (0 M NaCl) or containing 1 M NaCl, and RNA was extracted and used for RT-qPCR experiments as described in Materials and Methods. Three independent experiments with triplicates were performed, and a representative result is shown. The data from one representative experiment are plotted and were analyzed by using a two-tailed Student t test. A statistically significant difference between the values was observed, with a P value of
    Figure Legend Snippet: High levels of c-di-AMP inhibit expression of kdpA . Shown is the relative quantification (RQ) of transcript levels of kdpA measured by RT-qPCR for the wild-type S. aureus strain LAC* (WT) and an isogenic gdpP (Δ gdpP ) mutant strain (with constitutively high levels of c-di-AMP), normalized to gyrB transcript levels. WT and mutant S. aureus strains were grown to an OD 600 of 0.7 in LB medium without salt (0 M NaCl) or containing 1 M NaCl, and RNA was extracted and used for RT-qPCR experiments as described in Materials and Methods. Three independent experiments with triplicates were performed, and a representative result is shown. The data from one representative experiment are plotted and were analyzed by using a two-tailed Student t test. A statistically significant difference between the values was observed, with a P value of

    Techniques Used: Expressing, Quantitative RT-PCR, Mutagenesis, Two Tailed Test

    18) Product Images from "Electrical Stimulation Counteracts Muscle Decline in Seniors"

    Article Title: Electrical Stimulation Counteracts Muscle Decline in Seniors

    Journal: Frontiers in Aging Neuroscience

    doi: 10.3389/fnagi.2014.00189

    Expression analyses of genes controlling muscle mass and metabolism . Real time PCR analysis for the expression of IGF-1 isoforms (total IGF-1pan, IGF-1Ea, IGF-1Eb, IGF-1Ec) (A) Atrogin-1, MurF-1, Beclin1, p62 (B) , Myostatin (C) , PGC1α (D) , and Nrf2 (E) . Data are represented as average ± SEM. n = 16. ** p
    Figure Legend Snippet: Expression analyses of genes controlling muscle mass and metabolism . Real time PCR analysis for the expression of IGF-1 isoforms (total IGF-1pan, IGF-1Ea, IGF-1Eb, IGF-1Ec) (A) Atrogin-1, MurF-1, Beclin1, p62 (B) , Myostatin (C) , PGC1α (D) , and Nrf2 (E) . Data are represented as average ± SEM. n = 16. ** p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    19) Product Images from "Developmental Validation of a novel 5 dye Y-STR System comprising the 27 YfilerPlus loci"

    Article Title: Developmental Validation of a novel 5 dye Y-STR System comprising the 27 YfilerPlus loci

    Journal: Scientific Reports

    doi: 10.1038/srep29557

    Electropherogram of allelic ladders and internal size standard in the STRtyper-27 system. The four dye panels for allelic ladders correspond to (from top to bottom) 6FAM (blue), HEX (green), TAMRA (yellow), ROX (red) dye-labeled peaks. The haplotype is shown with the allele number displayed underneath each peak. The fifth panel reserved for internal size standard labels an orange dye: SIZE500 (a total of eighteen fragments: 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 and 500 bp).
    Figure Legend Snippet: Electropherogram of allelic ladders and internal size standard in the STRtyper-27 system. The four dye panels for allelic ladders correspond to (from top to bottom) 6FAM (blue), HEX (green), TAMRA (yellow), ROX (red) dye-labeled peaks. The haplotype is shown with the allele number displayed underneath each peak. The fifth panel reserved for internal size standard labels an orange dye: SIZE500 (a total of eighteen fragments: 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 and 500 bp).

    Techniques Used: Labeling

    20) Product Images from "Human Chondrosarcoma Cells Acquire an Epithelial-Like Gene Expression Pattern via an Epigenetic Switch: Evidence for Mesenchymal-Epithelial Transition during Sarcomagenesis"

    Article Title: Human Chondrosarcoma Cells Acquire an Epithelial-Like Gene Expression Pattern via an Epigenetic Switch: Evidence for Mesenchymal-Epithelial Transition during Sarcomagenesis

    Journal: Sarcoma

    doi: 10.1155/2011/598218

    The methyltransferase inhibitor 5-Aza-dC reactivated both maspin and 14-3-3 σ mRNA expression in chondrocytes and chondrosarcomas cell lines. The CS8E chondrosarcoma cell line and SNM83 normal chondrocyte cell line were exposed to 10 uM 5-Aza-dC. RNA was harvested after 48, 72, and 120 hours and subjected to RT-PCR for maspin and 14-3-3 σ . GAPDH was used as loading control.
    Figure Legend Snippet: The methyltransferase inhibitor 5-Aza-dC reactivated both maspin and 14-3-3 σ mRNA expression in chondrocytes and chondrosarcomas cell lines. The CS8E chondrosarcoma cell line and SNM83 normal chondrocyte cell line were exposed to 10 uM 5-Aza-dC. RNA was harvested after 48, 72, and 120 hours and subjected to RT-PCR for maspin and 14-3-3 σ . GAPDH was used as loading control.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    21) Product Images from "Ex vivo screening for immunodominant viral epitopes by quantitative real time polymerase chain reaction (qRT-PCR)"

    Article Title: Ex vivo screening for immunodominant viral epitopes by quantitative real time polymerase chain reaction (qRT-PCR)

    Journal: Journal of Translational Medicine

    doi: 10.1186/1479-5876-1-12

    The kinetics of IFN-γ, IL-2, IL-4 and IL-10 transcription following FluM1 58–66 stimulation of three HLA-A*0201 donors. The standard concentration of 2 × 10 5 cells/200μl was ex vivo exposed to the peptide. At hour 3, IFN-γ transcript production precedes IL-2, IL-4 and IL-10. At hour 12 there is consistent production of T h 1 (IFN-γ, IL-2) cytokines. IL-4 and IL-10 transcript production increases above the baseline levels from 24 to 48 hours. For each donor and at each time point mRNA copy numbers were calculated relative to unstimulated cells after normalization by β-actin. The result represents the mean ± SEM of averages of combined values of three independent experiments carried out on each of the three donors.
    Figure Legend Snippet: The kinetics of IFN-γ, IL-2, IL-4 and IL-10 transcription following FluM1 58–66 stimulation of three HLA-A*0201 donors. The standard concentration of 2 × 10 5 cells/200μl was ex vivo exposed to the peptide. At hour 3, IFN-γ transcript production precedes IL-2, IL-4 and IL-10. At hour 12 there is consistent production of T h 1 (IFN-γ, IL-2) cytokines. IL-4 and IL-10 transcript production increases above the baseline levels from 24 to 48 hours. For each donor and at each time point mRNA copy numbers were calculated relative to unstimulated cells after normalization by β-actin. The result represents the mean ± SEM of averages of combined values of three independent experiments carried out on each of the three donors.

    Techniques Used: Concentration Assay, Ex Vivo

    22) Product Images from "TAp73 promotes cell survival upon genotoxic stress by inhibiting p53 activity"

    Article Title: TAp73 promotes cell survival upon genotoxic stress by inhibiting p53 activity

    Journal: Oncotarget

    doi:

    TAp73 suppresses apoptosis and the expression of p53 downstream target genes following extensive DNA damage (A) HCT116 cells with Flag-TAp73 KI were transfected with control or HA-TAp73α construct, and then treated with cisplatin at indicated concentrations for 24 hr. Left , analysis of apoptosis by nuclear staining; Right , analysis of caspase activity. (B) Following treatment as in (A), indicated proteins were analyzed by western blotting. TAp73 blots with short or long exposure (exp.) are presented to show transfected TAp73 and endogenous (En)/knock-in (KI) TAp73, respectively. (C) mRNA expression of PUMA , Bax , and p21 in cells treated as in (A) was analyzed by real-time RT-PCR and normalized to the control β-actin and untreated samples. (D) HCT116 cells with Flag-TAp73 KI were transfected with control or TAp73 siRNA, and treated with cisplatin at indicated concentrations for 24 hr. Left , analysis of apoptosis by nuclear staining; Right , analysis of caspase activity. (E) Following treatment as in (D), indicated proteins were analyzed by western blotting. (F) mRNA expression of PUMA , Bax and p21 in cells treated as in (D) was analyzed by real-time RT-PCR. (G) HCT116 cells with stable transfection of TAp73 or control shRNA were analyzed for the expression of TAp73 , ΔNp73 , TAp63 and ΔNp73 by RT-PCR. (H) HCT116 cells with stable transfection of TAp73 or control shRNA at inoculation (0 hr) or after culturing for 48 hr were analyzed for TAp73 and γH2Ax by western blotting. Lysates from cisplatin-treated HCT116 cells were used as a positive control (+) for γH2Ax. (I) HCT116 cells with stable transfection of TAp73 or control shRNA were treated with cisplatin at indicated concentrations for 24 hr. Apoptosis was analyzed by nuclear staining. (J) Following treatment as in (I), indicated proteins were analyzed by western blotting. Results in (A), (C), (D), (F) and (I) were expressed as means ± s.d. of three independent experiments. *** , P
    Figure Legend Snippet: TAp73 suppresses apoptosis and the expression of p53 downstream target genes following extensive DNA damage (A) HCT116 cells with Flag-TAp73 KI were transfected with control or HA-TAp73α construct, and then treated with cisplatin at indicated concentrations for 24 hr. Left , analysis of apoptosis by nuclear staining; Right , analysis of caspase activity. (B) Following treatment as in (A), indicated proteins were analyzed by western blotting. TAp73 blots with short or long exposure (exp.) are presented to show transfected TAp73 and endogenous (En)/knock-in (KI) TAp73, respectively. (C) mRNA expression of PUMA , Bax , and p21 in cells treated as in (A) was analyzed by real-time RT-PCR and normalized to the control β-actin and untreated samples. (D) HCT116 cells with Flag-TAp73 KI were transfected with control or TAp73 siRNA, and treated with cisplatin at indicated concentrations for 24 hr. Left , analysis of apoptosis by nuclear staining; Right , analysis of caspase activity. (E) Following treatment as in (D), indicated proteins were analyzed by western blotting. (F) mRNA expression of PUMA , Bax and p21 in cells treated as in (D) was analyzed by real-time RT-PCR. (G) HCT116 cells with stable transfection of TAp73 or control shRNA were analyzed for the expression of TAp73 , ΔNp73 , TAp63 and ΔNp73 by RT-PCR. (H) HCT116 cells with stable transfection of TAp73 or control shRNA at inoculation (0 hr) or after culturing for 48 hr were analyzed for TAp73 and γH2Ax by western blotting. Lysates from cisplatin-treated HCT116 cells were used as a positive control (+) for γH2Ax. (I) HCT116 cells with stable transfection of TAp73 or control shRNA were treated with cisplatin at indicated concentrations for 24 hr. Apoptosis was analyzed by nuclear staining. (J) Following treatment as in (I), indicated proteins were analyzed by western blotting. Results in (A), (C), (D), (F) and (I) were expressed as means ± s.d. of three independent experiments. *** , P

    Techniques Used: Expressing, Transfection, Construct, Staining, Activity Assay, Western Blot, Knock-In, Quantitative RT-PCR, Stable Transfection, shRNA, Reverse Transcription Polymerase Chain Reaction, Positive Control

    p53 is required for the effects of TAp73 on p53 target gene induction and apoptosis following extensive DNA damage (A) Flag-KI and p53 -knockout ( p53 -KO) HCT116 cells were treated with cisplatin at indicated concentrations for 24 hr. Indicated proteins were analyzed by western blotting. (B) Apoptosis in cells treated as in (A), (C) and (D) was analyzed by nuclear staining. ** , P
    Figure Legend Snippet: p53 is required for the effects of TAp73 on p53 target gene induction and apoptosis following extensive DNA damage (A) Flag-KI and p53 -knockout ( p53 -KO) HCT116 cells were treated with cisplatin at indicated concentrations for 24 hr. Indicated proteins were analyzed by western blotting. (B) Apoptosis in cells treated as in (A), (C) and (D) was analyzed by nuclear staining. ** , P

    Techniques Used: Knock-Out, Western Blot, Staining

    TAp73 inhibits p53 binding to its target gene promoters following excessive DNA damage (A) HCT116 cells with Flag-TAp73 KI were treated with cisplatin at indicated concentrations for 24 hr. Chromatin immunoprecipitation (ChIP) followed by quantitative real-time PCR was used to analyze the binding of p53 and TAp73 to the promoters of PUMA , Bax and p21 , using anti-p53 antibody and anti-Flag-conjugated beads, respectively. Results were normalized to those of IgG (for p53 ChIP) and parental HCT116 (for TAp73 ChIP), which were used as negative controls for ChIP, and plotted as fold of enrichment relative to the control. (B) Flag-KI HCT116 cells were transfected with control or HA-TAp73α construct, and then treated with cisplatin at indicated concentrations for 24 hr. Occupancy of p53 at the indicated promoters was analyzed by ChIP as in (A). (C) Flag-KI HCT116 cells were transfected with control or TAp73 siRNA, and then treated with cisplatin at indicated concentrations for 24 hr. Binding of p53 to the indicated promoters was analyzed as in (A). (D) HCT116 cells were transfected with p53 or TAp73α, and treated with cisplatin at indicated concentrations for 24 hr. Histone H4 acetylation, a marker of transcriptional activation, in PUMA and p21 promoters was analyzed by ChIP using an antibody specific for acetylated H4. The results were normalized to those of input. (E) p53 -KO HCT116 cells were co-transfected with a PUMA or p21 luciferase reporter, along with indicated amounts of p53 and TAp73α expression vectors, or control empty vectors. Activation of the PUMA and p21 reporters was measured 24 hr after transfection, as described in the Materials and Methods. Results in (A)-(E) were expressed as means ± s.d. of three independent experiments. *** , P
    Figure Legend Snippet: TAp73 inhibits p53 binding to its target gene promoters following excessive DNA damage (A) HCT116 cells with Flag-TAp73 KI were treated with cisplatin at indicated concentrations for 24 hr. Chromatin immunoprecipitation (ChIP) followed by quantitative real-time PCR was used to analyze the binding of p53 and TAp73 to the promoters of PUMA , Bax and p21 , using anti-p53 antibody and anti-Flag-conjugated beads, respectively. Results were normalized to those of IgG (for p53 ChIP) and parental HCT116 (for TAp73 ChIP), which were used as negative controls for ChIP, and plotted as fold of enrichment relative to the control. (B) Flag-KI HCT116 cells were transfected with control or HA-TAp73α construct, and then treated with cisplatin at indicated concentrations for 24 hr. Occupancy of p53 at the indicated promoters was analyzed by ChIP as in (A). (C) Flag-KI HCT116 cells were transfected with control or TAp73 siRNA, and then treated with cisplatin at indicated concentrations for 24 hr. Binding of p53 to the indicated promoters was analyzed as in (A). (D) HCT116 cells were transfected with p53 or TAp73α, and treated with cisplatin at indicated concentrations for 24 hr. Histone H4 acetylation, a marker of transcriptional activation, in PUMA and p21 promoters was analyzed by ChIP using an antibody specific for acetylated H4. The results were normalized to those of input. (E) p53 -KO HCT116 cells were co-transfected with a PUMA or p21 luciferase reporter, along with indicated amounts of p53 and TAp73α expression vectors, or control empty vectors. Activation of the PUMA and p21 reporters was measured 24 hr after transfection, as described in the Materials and Methods. Results in (A)-(E) were expressed as means ± s.d. of three independent experiments. *** , P

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Transfection, Construct, Marker, Activation Assay, Luciferase, Expressing

    23) Product Images from "A Helitron transposon reconstructed from bats reveals a novel mechanism of genome shuffling in eukaryotes"

    Article Title: A Helitron transposon reconstructed from bats reveals a novel mechanism of genome shuffling in eukaryotes

    Journal: Nature Communications

    doi: 10.1038/ncomms10716

    Functional analysis of the HUH nuclease and SF1B helicase domains. ( a ) Transposition by wild-type (WT) Helraiser transposase and its mutants in HeLa cells. Data are represented as mean±s.e.m., n =3 biological replicates. ( b ) Cleavage of single-stranded DNA oligonucleotides by the Helraiser transposase in vitro . ( c ) DNA-binding assay with the Helraiser transposase and its point mutant and truncated derivatives. 0.3–5 × values represent molar ratios of protein to DNA. ( d ) Colorimetric ATPase assay with the WT and K1068Q mutant transposase protein. PPi, pyrophosphate release. Data are represented as mean ± s.d., n =3 biological replicates.
    Figure Legend Snippet: Functional analysis of the HUH nuclease and SF1B helicase domains. ( a ) Transposition by wild-type (WT) Helraiser transposase and its mutants in HeLa cells. Data are represented as mean±s.e.m., n =3 biological replicates. ( b ) Cleavage of single-stranded DNA oligonucleotides by the Helraiser transposase in vitro . ( c ) DNA-binding assay with the Helraiser transposase and its point mutant and truncated derivatives. 0.3–5 × values represent molar ratios of protein to DNA. ( d ) Colorimetric ATPase assay with the WT and K1068Q mutant transposase protein. PPi, pyrophosphate release. Data are represented as mean ± s.d., n =3 biological replicates.

    Techniques Used: Functional Assay, In Vitro, DNA Binding Assay, Mutagenesis, ATPase Assay

    Proposed model of Helraiser transposition. ( a ) Helraiser transposase (light blue oval) binds the LTS (green) and nicks ssDNA donor site generating a 5′-phosphotyrosine intermediate between the tyrosine residue (orange line) in the HUH nuclease active site (dark blue oval) and the transposon end. ( b ) A free 3′-OH group at the donor site primes DNA synthesis, while the helicase domain unwinds the dsDNA helix in a 5′ to 3′ direction. ( c ) The hairpin structure in the RTS (purple) induces pausing of the helicase required for the recognition and nicking of the CTAG-3′ tetrad at the RTS by the second tyrosine (yellow arrow) of the HUH domain. This generates a free 3′-OH group at the transposon RTS that attacks the first 5′-phosphotyrosine linkage generating a free ssDNA circle. The ssDNA circle is possibly converted into dsDNA circle used for further rounds of transposition. Alternatively, the transposase reads through the RTS and mobilizes host flanking sequences, thereby generating an alternative, de novo 3′-end. Further steps in transposition of the canonical transposon and the transposon containing the captured host sequence are identical. ( d ) Two tyrosine residues in the nuclease active site catalyse cleavage of the single-stranded target DNA and the Helitron circle, mediating the strand-transfer reaction. ( e ) The single-stranded transposon DNA covalently bound to the target is passively replicated and converted into the double-stranded form during the DNA synthesis phase of the cell cycle, leading to the amplification of the transposon in the host genome and transduction of host genomic sequence.
    Figure Legend Snippet: Proposed model of Helraiser transposition. ( a ) Helraiser transposase (light blue oval) binds the LTS (green) and nicks ssDNA donor site generating a 5′-phosphotyrosine intermediate between the tyrosine residue (orange line) in the HUH nuclease active site (dark blue oval) and the transposon end. ( b ) A free 3′-OH group at the donor site primes DNA synthesis, while the helicase domain unwinds the dsDNA helix in a 5′ to 3′ direction. ( c ) The hairpin structure in the RTS (purple) induces pausing of the helicase required for the recognition and nicking of the CTAG-3′ tetrad at the RTS by the second tyrosine (yellow arrow) of the HUH domain. This generates a free 3′-OH group at the transposon RTS that attacks the first 5′-phosphotyrosine linkage generating a free ssDNA circle. The ssDNA circle is possibly converted into dsDNA circle used for further rounds of transposition. Alternatively, the transposase reads through the RTS and mobilizes host flanking sequences, thereby generating an alternative, de novo 3′-end. Further steps in transposition of the canonical transposon and the transposon containing the captured host sequence are identical. ( d ) Two tyrosine residues in the nuclease active site catalyse cleavage of the single-stranded target DNA and the Helitron circle, mediating the strand-transfer reaction. ( e ) The single-stranded transposon DNA covalently bound to the target is passively replicated and converted into the double-stranded form during the DNA synthesis phase of the cell cycle, leading to the amplification of the transposon in the host genome and transduction of host genomic sequence.

    Techniques Used: DNA Synthesis, Sequencing, Amplification, Transduction

    24) Product Images from "Human Choline Kinase-α Promotes Hepatitis C Virus RNA Replication through Modulation of Membranous Viral Replication Complex Formation"

    Article Title: Human Choline Kinase-α Promotes Hepatitis C Virus RNA Replication through Modulation of Membranous Viral Replication Complex Formation

    Journal: Journal of Virology

    doi: 10.1128/JVI.00960-16

    Requirement of hCKα for HCV RNA replication. (A) Huh7 cells transfected with control or hCKα- or CD81-specific siRNAs for 48 h were separately infected with HCVpp generated from the genotype 1a H77 strain and genotype 2a JFH1 strain or with VSVpp, and luciferase activities were measured. In parallel, a set of cells harvested at 48 h post-siRNA transfection was analyzed by Western blotting; a representative data set is shown. (B) Huh7 cells transfected with control or hCKα-specific siRNAs were infected with HCV, and the infectivities of the extracellular and intracellular viruses were assessed. This analysis was performed by inoculating culture medium that contained extracellular virions and cell extracts that contained intracellular virions, respectively, into naive Huh7.5-1 cells. The infected cells were harvested, and isolated total RNA was analyzed for the HCV RNA level by RT-PCR. (C) Huh7 cells were transfected with FL-Luc-JFH1 RNA or SGR-Luc-JFH1 RNA, as indicated. HCV RNA replication was measured by determining the fold change in firefly luciferase activity at different time points over that determined at 4 h after RNA transfection. (D) Huh7 cells stably replicating genotype 1b Con1 strain SGR remained untransfected or were transfected with the indicated siRNAs, and cell lysates were analyzed by Western blotting (top panel). The level of the indicated proteins in different settings relative to that detected in siRNA-untreated SGR-replicating cells, which was arbitrarily designated 100%, was expressed as indicated (bottom panel). (E and F) FL-Luc-JFH1 or SGR-Luc-JFH1 RNA-transfected cells were treated with different concentrations of CK37 or HDTAB, as indicated, and the luciferase activity was measured and expressed as the percentage of that detected in cells that received vehicle treatment. (G) Huh7 cells constitutively expressing hCKα shRNA were transfected with control vector (−) or wild-type (α-R) or D288A (D288A-R) hCKα resistant to hCKα shRNA, and cells were left uninfected or infected with HCV. Duplicate sets of cells were analyzed by Western blotting for protein expression (left panel) and by RT-PCR for the viral RNA level (right panel). *, P
    Figure Legend Snippet: Requirement of hCKα for HCV RNA replication. (A) Huh7 cells transfected with control or hCKα- or CD81-specific siRNAs for 48 h were separately infected with HCVpp generated from the genotype 1a H77 strain and genotype 2a JFH1 strain or with VSVpp, and luciferase activities were measured. In parallel, a set of cells harvested at 48 h post-siRNA transfection was analyzed by Western blotting; a representative data set is shown. (B) Huh7 cells transfected with control or hCKα-specific siRNAs were infected with HCV, and the infectivities of the extracellular and intracellular viruses were assessed. This analysis was performed by inoculating culture medium that contained extracellular virions and cell extracts that contained intracellular virions, respectively, into naive Huh7.5-1 cells. The infected cells were harvested, and isolated total RNA was analyzed for the HCV RNA level by RT-PCR. (C) Huh7 cells were transfected with FL-Luc-JFH1 RNA or SGR-Luc-JFH1 RNA, as indicated. HCV RNA replication was measured by determining the fold change in firefly luciferase activity at different time points over that determined at 4 h after RNA transfection. (D) Huh7 cells stably replicating genotype 1b Con1 strain SGR remained untransfected or were transfected with the indicated siRNAs, and cell lysates were analyzed by Western blotting (top panel). The level of the indicated proteins in different settings relative to that detected in siRNA-untreated SGR-replicating cells, which was arbitrarily designated 100%, was expressed as indicated (bottom panel). (E and F) FL-Luc-JFH1 or SGR-Luc-JFH1 RNA-transfected cells were treated with different concentrations of CK37 or HDTAB, as indicated, and the luciferase activity was measured and expressed as the percentage of that detected in cells that received vehicle treatment. (G) Huh7 cells constitutively expressing hCKα shRNA were transfected with control vector (−) or wild-type (α-R) or D288A (D288A-R) hCKα resistant to hCKα shRNA, and cells were left uninfected or infected with HCV. Duplicate sets of cells were analyzed by Western blotting for protein expression (left panel) and by RT-PCR for the viral RNA level (right panel). *, P

    Techniques Used: Transfection, Infection, Generated, Luciferase, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Stable Transfection, Expressing, shRNA, Plasmid Preparation

    25) Product Images from "The effects of first-line anti-tuberculosis drugs on the actions of vitamin D in human macrophages"

    Article Title: The effects of first-line anti-tuberculosis drugs on the actions of vitamin D in human macrophages

    Journal: Journal of Clinical & Translational Endocrinology

    doi: 10.1016/j.jcte.2016.08.005

    Expression of hCAP18 in PBMC and plasma LL-37 in subjects with pulmonary TB
    Figure Legend Snippet: Expression of hCAP18 in PBMC and plasma LL-37 in subjects with pulmonary TB

    Techniques Used: Expressing

    Plasma concentrations of LL-37 (the active human cathelicidin protein) in subjects recently diagnosed with pulmonary TB receiving conventional 4-drug initial anti-TB therapy (INH, rifampicin, pyrazinamide and ethambutol) and receiving at total of 1.2
    Figure Legend Snippet: Plasma concentrations of LL-37 (the active human cathelicidin protein) in subjects recently diagnosed with pulmonary TB receiving conventional 4-drug initial anti-TB therapy (INH, rifampicin, pyrazinamide and ethambutol) and receiving at total of 1.2

    Techniques Used:

    26) Product Images from "Tissue-specific expression of TRP channel genes in the mouse and its variation in three different mouse strains"

    Article Title: Tissue-specific expression of TRP channel genes in the mouse and its variation in three different mouse strains

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-7-159

    Summary of tissue specific TRP channel gene expression . Expression of TRPC1-7, TRPM1-8, TRPV1-6 and TRPA1 mRNAs in murine tissues was determined by real-time RT-PCR. Levels of mRNAs are given in relation to that of 18S rRNA; all data were multiplied by 1000. Means were calculated from n = 3–10 samples and classified into 5 categories. If the threshold level of fluorescence according to the TaqMan RT-PCR technique was not reached after 40 cycles of amplification, a resulting expression level of zero (0) was assigned to the corresponding channel transcript and tissue. For better illustration categories of gene expression levels are represented by different colors ranging from zero (white) to values higher than 10 (dark blue).
    Figure Legend Snippet: Summary of tissue specific TRP channel gene expression . Expression of TRPC1-7, TRPM1-8, TRPV1-6 and TRPA1 mRNAs in murine tissues was determined by real-time RT-PCR. Levels of mRNAs are given in relation to that of 18S rRNA; all data were multiplied by 1000. Means were calculated from n = 3–10 samples and classified into 5 categories. If the threshold level of fluorescence according to the TaqMan RT-PCR technique was not reached after 40 cycles of amplification, a resulting expression level of zero (0) was assigned to the corresponding channel transcript and tissue. For better illustration categories of gene expression levels are represented by different colors ranging from zero (white) to values higher than 10 (dark blue).

    Techniques Used: Expressing, Quantitative RT-PCR, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Amplification

    27) Product Images from "Acquired Resistance of Lung Adenocarcinomas to Gefitinib or Erlotinib Is Associated with a Second Mutation in the EGFR Kinase DomainHow Tumor Cells Acquire Resistance to Kinase InhibitorsEGFR Inhibition in Non-Small Cell Lung Cancer: Resistance, Once Again, Rears Its Ugly Head"

    Article Title: Acquired Resistance of Lung Adenocarcinomas to Gefitinib or Erlotinib Is Associated with a Second Mutation in the EGFR Kinase DomainHow Tumor Cells Acquire Resistance to Kinase InhibitorsEGFR Inhibition in Non-Small Cell Lung Cancer: Resistance, Once Again, Rears Its Ugly Head

    Journal: PLoS Medicine

    doi: 10.1371/journal.pmed.0020073

    A Novel PCR-RFLP Assay Independently Confirms Presence of the T790M Mutation in Exon 20 of the EGFR Kinase Domain (A) Design of the assay (see text for details). “F” designates the fluorescent label, FAM. At the bottom of this panel, the assay demonstrates with the 97-bp NlaIII cleavage product the presence of the T790M mutation in the H1975 cell line; this product is absent in H2030 DNA. The 106-bp NlaIII cleavage product is generated by digestion of wild-type EGFR . (B) The PCR-RFLP assay demonstrates that pre-drug tumor samples from the three patients lack detectable levels of the mutant 97-bp product, while specimens obtained after disease progression contain the T790M mutation. Pt, patient.
    Figure Legend Snippet: A Novel PCR-RFLP Assay Independently Confirms Presence of the T790M Mutation in Exon 20 of the EGFR Kinase Domain (A) Design of the assay (see text for details). “F” designates the fluorescent label, FAM. At the bottom of this panel, the assay demonstrates with the 97-bp NlaIII cleavage product the presence of the T790M mutation in the H1975 cell line; this product is absent in H2030 DNA. The 106-bp NlaIII cleavage product is generated by digestion of wild-type EGFR . (B) The PCR-RFLP assay demonstrates that pre-drug tumor samples from the three patients lack detectable levels of the mutant 97-bp product, while specimens obtained after disease progression contain the T790M mutation. Pt, patient.

    Techniques Used: Polymerase Chain Reaction, RFLP Assay, Mutagenesis, Generated

    28) Product Images from "Targeting Cell Cycle Proteins in Breast Cancer Cells with siRNA by Using Lipid-Substituted Polyethylenimines"

    Article Title: Targeting Cell Cycle Proteins in Breast Cancer Cells with siRNA by Using Lipid-Substituted Polyethylenimines

    Journal: Frontiers in Bioengineering and Biotechnology

    doi: 10.3389/fbioe.2015.00014

    (A) Uptake of FAM-labeled siRNA complexes at 20 and 40 nM siRNA using 1:2, 1:4, and 1:8 siRNA:PEI–LA ratios. The MDA-MB-435WT cells were incubated with the complexes for 24 h and recovered for flow cytometry analysis. The results were summarized as mean FAM-siRNA per cell (top) and FAM-siRNA positive cell population (bottom). (B) Confocal microscopy to determine the uptake of FAM-labeled siRNA complexes at 40 nM siRNA with 1:2 (ii) and 1:8 (iii) siRNA:PEI–LA ratios after 24 h treatment. Purple, red, and green colors represent nuclei, cytoplasm, and siRNA complexes, respectively. Non-labeled scrambled siRNA was transfected as a control in MDA-MB-435WT (i). (C) The number of visible complexes per cell (as quantitated from confocal microscopy images) at 1:2 and 1:8 siRNA:PEI–LA ratios. The uptake was significantly different between 1:2 and 1:8 ratios (* p
    Figure Legend Snippet: (A) Uptake of FAM-labeled siRNA complexes at 20 and 40 nM siRNA using 1:2, 1:4, and 1:8 siRNA:PEI–LA ratios. The MDA-MB-435WT cells were incubated with the complexes for 24 h and recovered for flow cytometry analysis. The results were summarized as mean FAM-siRNA per cell (top) and FAM-siRNA positive cell population (bottom). (B) Confocal microscopy to determine the uptake of FAM-labeled siRNA complexes at 40 nM siRNA with 1:2 (ii) and 1:8 (iii) siRNA:PEI–LA ratios after 24 h treatment. Purple, red, and green colors represent nuclei, cytoplasm, and siRNA complexes, respectively. Non-labeled scrambled siRNA was transfected as a control in MDA-MB-435WT (i). (C) The number of visible complexes per cell (as quantitated from confocal microscopy images) at 1:2 and 1:8 siRNA:PEI–LA ratios. The uptake was significantly different between 1:2 and 1:8 ratios (* p

    Techniques Used: Labeling, Multiple Displacement Amplification, Incubation, Flow Cytometry, Cytometry, Confocal Microscopy, Transfection

    Combinations of siRNA (20 + 20 nM) treatments among KSP, CDC20, RAD51, and CHEK1 siRNA in MDA-MB-435WT (A) with siRNA:PEI-LA ratio of 1:2 and MDA-MB-435R (B) with siRNA:PEI–LA ratio of 1:8 . Scrambled siRNA (CsiRNA) was used as a control on its own as well as in combination with other siRNAs. The SD (not shown) was
    Figure Legend Snippet: Combinations of siRNA (20 + 20 nM) treatments among KSP, CDC20, RAD51, and CHEK1 siRNA in MDA-MB-435WT (A) with siRNA:PEI-LA ratio of 1:2 and MDA-MB-435R (B) with siRNA:PEI–LA ratio of 1:8 . Scrambled siRNA (CsiRNA) was used as a control on its own as well as in combination with other siRNAs. The SD (not shown) was

    Techniques Used: Multiple Displacement Amplification

    (A) Screening of the library of siRNAs against cell cycle proteins using MDA-MB-231R and MDA-MB-435R cells. The identified cell cycle proteins, CDC20, RAD51, and CHEK1 are indicated with the positive control, PLK1. A specific siRNA against PLK1 (Polo-like kinase 1) was provided with the cell cycle proteins; it was meant to assure the functioning of siRNA delivery and was not pursued in this study. (B,C) The results of siRNA treatment alone and siRNA/doxorubicin combinational treatment in MDA-MB-231R (B) and MDA-MB-435R (C) . (D) The difference in cell growth between siRNA treatment alone and the combinational treatment. Inhibition of cell growth in all cases was expressed as a percentage of control (POC), which was calculated as a percentage of cell growth (from MTT Assay) for scrambled siRNA-treated cells.
    Figure Legend Snippet: (A) Screening of the library of siRNAs against cell cycle proteins using MDA-MB-231R and MDA-MB-435R cells. The identified cell cycle proteins, CDC20, RAD51, and CHEK1 are indicated with the positive control, PLK1. A specific siRNA against PLK1 (Polo-like kinase 1) was provided with the cell cycle proteins; it was meant to assure the functioning of siRNA delivery and was not pursued in this study. (B,C) The results of siRNA treatment alone and siRNA/doxorubicin combinational treatment in MDA-MB-231R (B) and MDA-MB-435R (C) . (D) The difference in cell growth between siRNA treatment alone and the combinational treatment. Inhibition of cell growth in all cases was expressed as a percentage of control (POC), which was calculated as a percentage of cell growth (from MTT Assay) for scrambled siRNA-treated cells.

    Techniques Used: Multiple Displacement Amplification, Positive Control, Inhibition, MTT Assay

    Validation of KSP, CDC20, RAD51, and CHEK1 in MDA-MB-435WT cells at 20 nM (A,C) and 40 nM (B,D) siRNA concentrations . The results from PEI–LA were summarized in (A,B) (siRNA:polymer ratios of 1:2, 1:4, and 1:8) while the results from PEI–CA were summarized in (C,D) (siRNA:polymer ratios of 1:2 and 1:4). Scrambled siRNA (CsiRNA) was used as a control. Significant (* p
    Figure Legend Snippet: Validation of KSP, CDC20, RAD51, and CHEK1 in MDA-MB-435WT cells at 20 nM (A,C) and 40 nM (B,D) siRNA concentrations . The results from PEI–LA were summarized in (A,B) (siRNA:polymer ratios of 1:2, 1:4, and 1:8) while the results from PEI–CA were summarized in (C,D) (siRNA:polymer ratios of 1:2 and 1:4). Scrambled siRNA (CsiRNA) was used as a control. Significant (* p

    Techniques Used: Multiple Displacement Amplification

    Effect of siRNA on doxorubicin cytotoxicity in MDA-MB-435R cells . The cells were first treated with siRNA at 20 nM (A,D) , 40 nM (B,E) , and 60 nM (C,F) with 1:2, 1:4, and 1:8 siRNA:PEI-LA ratios, followed by treatment with buffer [ (A–C) ; −DOX] or doxorubicin [ (D–F) ; +DOX]. Scrambled siRNA (CsiRNA) was used as a control.
    Figure Legend Snippet: Effect of siRNA on doxorubicin cytotoxicity in MDA-MB-435R cells . The cells were first treated with siRNA at 20 nM (A,D) , 40 nM (B,E) , and 60 nM (C,F) with 1:2, 1:4, and 1:8 siRNA:PEI-LA ratios, followed by treatment with buffer [ (A–C) ; −DOX] or doxorubicin [ (D–F) ; +DOX]. Scrambled siRNA (CsiRNA) was used as a control.

    Techniques Used: Multiple Displacement Amplification

    Screening of polymeric carriers in MDA-MB-435WT cells at 1:2 (A) and 1:4 (B) siRNA to carrier ratios with 20 nM of scrambled siRNA (control; CsiRNA) and KSP siRNA . Polyethylenimines (PEI) substituted with linoleic acid (LA), caprylic acid (CA), and α-linoleic acid (αLA) were used in the initial screen along with two commercially available carriers.
    Figure Legend Snippet: Screening of polymeric carriers in MDA-MB-435WT cells at 1:2 (A) and 1:4 (B) siRNA to carrier ratios with 20 nM of scrambled siRNA (control; CsiRNA) and KSP siRNA . Polyethylenimines (PEI) substituted with linoleic acid (LA), caprylic acid (CA), and α-linoleic acid (αLA) were used in the initial screen along with two commercially available carriers.

    Techniques Used: Multiple Displacement Amplification

    29) Product Images from "miR-29a Differentially Regulates Cell Survival in Astrocytes from Cornu Ammonis-1 and Dentate Gyrus by Targeting VDAC1"

    Article Title: miR-29a Differentially Regulates Cell Survival in Astrocytes from Cornu Ammonis-1 and Dentate Gyrus by Targeting VDAC1

    Journal: Mitochondrion

    doi: 10.1016/j.mito.2016.08.013

    Voltage-dependent anion channel 1 (VDAC1) expression in astrocytes from CA1 and DG following injury. (A) Micrographs of astrocytes cultured from CA1 (top) and DG (bottom) subregions following 8, 24 or 48 hrs of glucose deprivation (GD). Cells were triple stained with propidium iodide (red, dead cells) DAPI (blue, all cell nuclei) and anti-VDAC1 (green). (B) Areas that exhibited greater numbers of PI-positive cells tended to co-localize with areas of higher VDAC1 expression (arrow). Quantification of cell death (C) and VDAC1 (D) expression in CA1 and DG astrocytes following 8, 24 or 48 hrs of GD injury. (E) VDAC1 (red) co-localizes with the mitochondrial indicator ATP5a (green) in astrocytes following 48 hrs of GD injury. Bar = 15 µm. Graph represents pooled data from 3 independent experiments, n = 4 cultures/treatment. * = versus wash controls. Ψ = versus CA1, condition-matched. P
    Figure Legend Snippet: Voltage-dependent anion channel 1 (VDAC1) expression in astrocytes from CA1 and DG following injury. (A) Micrographs of astrocytes cultured from CA1 (top) and DG (bottom) subregions following 8, 24 or 48 hrs of glucose deprivation (GD). Cells were triple stained with propidium iodide (red, dead cells) DAPI (blue, all cell nuclei) and anti-VDAC1 (green). (B) Areas that exhibited greater numbers of PI-positive cells tended to co-localize with areas of higher VDAC1 expression (arrow). Quantification of cell death (C) and VDAC1 (D) expression in CA1 and DG astrocytes following 8, 24 or 48 hrs of GD injury. (E) VDAC1 (red) co-localizes with the mitochondrial indicator ATP5a (green) in astrocytes following 48 hrs of GD injury. Bar = 15 µm. Graph represents pooled data from 3 independent experiments, n = 4 cultures/treatment. * = versus wash controls. Ψ = versus CA1, condition-matched. P

    Techniques Used: Expressing, Cell Culture, Staining

    Effect of VDAC1 knockdown in astrocytes from CA1, DG, and cortex. (A) Immunofluorescence of astrocytes cultured from CA1 and DG hippocampal subregions pre-treated with 0, 30, or 50 pmol VDAC1 small interfering RNA (siRNA), and subjected to 48 hrs GD. Cells were stained for VDAC1 expression (green), cell death (red, PI) and normalized for total cell count by counter-staining with DAPI (blue). (B) Quantification of post-injury VDAC1 expression in CA1 and DG astrocytes with VDAC1 siRNA pre-treatment. (C) Quantification of cell death in CA1 and DG astrocytes pre-treated with VDAC1 siRNA. (D) VDAC1 protein expression following 48 hrs of GD in CA1 and DG astrocytes, with and without pre-treatment with VDAC1 siRNA. (E) Quantification of cell death from GD in CA1 and DG astrocytes with or without VDAC1 siRNA pre-treatment. Bar = 25 µm. Graphs represent n = 4–6 cultures/condition, all experiments repeated in triplicate. * = versus wash controls. # = versus post-injury controls. Ψ = versus CA1, condition-matched. P
    Figure Legend Snippet: Effect of VDAC1 knockdown in astrocytes from CA1, DG, and cortex. (A) Immunofluorescence of astrocytes cultured from CA1 and DG hippocampal subregions pre-treated with 0, 30, or 50 pmol VDAC1 small interfering RNA (siRNA), and subjected to 48 hrs GD. Cells were stained for VDAC1 expression (green), cell death (red, PI) and normalized for total cell count by counter-staining with DAPI (blue). (B) Quantification of post-injury VDAC1 expression in CA1 and DG astrocytes with VDAC1 siRNA pre-treatment. (C) Quantification of cell death in CA1 and DG astrocytes pre-treated with VDAC1 siRNA. (D) VDAC1 protein expression following 48 hrs of GD in CA1 and DG astrocytes, with and without pre-treatment with VDAC1 siRNA. (E) Quantification of cell death from GD in CA1 and DG astrocytes with or without VDAC1 siRNA pre-treatment. Bar = 25 µm. Graphs represent n = 4–6 cultures/condition, all experiments repeated in triplicate. * = versus wash controls. # = versus post-injury controls. Ψ = versus CA1, condition-matched. P

    Techniques Used: Immunofluorescence, Cell Culture, Small Interfering RNA, Staining, Expressing, Cell Counting

    (A) Voltage-dependent anion channel 1 (VDAC1) staining before and following 48 hrs of GD in astrocytes from CA1 and DG with either miR-29a mimic or transfection control pre-treatment. Cells are counterstained with DAPI for total cell count. (B) Immunoblot of VDAC1 protein expression following 48 hrs of GD in CA1 and DG astrocytes, with or without miR-29a mimic pre-treatment. (C) Quantification of VDAC1 protein expression following 48 hrs of GD in CA1 and DG astrocytes, with or without miR-29a mimic pre-treatment. Bar = 15 µm. Graph represents pooled data from 4 independent experiments, n = 4 cultures/treatment. * = versus wash controls, # = versus injury controls. Ψ = versus CA1, condition-matched. P
    Figure Legend Snippet: (A) Voltage-dependent anion channel 1 (VDAC1) staining before and following 48 hrs of GD in astrocytes from CA1 and DG with either miR-29a mimic or transfection control pre-treatment. Cells are counterstained with DAPI for total cell count. (B) Immunoblot of VDAC1 protein expression following 48 hrs of GD in CA1 and DG astrocytes, with or without miR-29a mimic pre-treatment. (C) Quantification of VDAC1 protein expression following 48 hrs of GD in CA1 and DG astrocytes, with or without miR-29a mimic pre-treatment. Bar = 15 µm. Graph represents pooled data from 4 independent experiments, n = 4 cultures/treatment. * = versus wash controls, # = versus injury controls. Ψ = versus CA1, condition-matched. P

    Techniques Used: Staining, Transfection, Cell Counting, Expressing

    30) Product Images from "Reduced expression levels of PTEN are associated with decreased sensitivity of HCC827 cells to icotinib"

    Article Title: Reduced expression levels of PTEN are associated with decreased sensitivity of HCC827 cells to icotinib

    Journal: Oncology Letters

    doi: 10.3892/ol.2017.5829

    The altered sensitivity of HCC827 cells to icotinib, following transfection. The concentration of icotinib required to inhibit 50% of the HCC827 cells was defined as the IC50 value. The IC50 for the HCC827 cells in the control group was determined to be 4.99 nmol, 5.63 nmol for the group transfected with PTEN-NC and 22.52 nmol following transfection with PTEN-siRNA. *P
    Figure Legend Snippet: The altered sensitivity of HCC827 cells to icotinib, following transfection. The concentration of icotinib required to inhibit 50% of the HCC827 cells was defined as the IC50 value. The IC50 for the HCC827 cells in the control group was determined to be 4.99 nmol, 5.63 nmol for the group transfected with PTEN-NC and 22.52 nmol following transfection with PTEN-siRNA. *P

    Techniques Used: Transfection, Concentration Assay

    31) Product Images from "Evaluation of factor VIII polymorphic short tandem repeat markers in linkage analysis for carrier diagnosis of hemophilia A"

    Article Title: Evaluation of factor VIII polymorphic short tandem repeat markers in linkage analysis for carrier diagnosis of hemophilia A

    Journal: Biomedical Reports

    doi: 10.3892/br.2016.712

    Electrophoretogram of seven short tandem repeat markers in the family with hemophilia A. I, DXS1227; II, DXS15; III, STR22; IV, DXS993; V, DXS1073; VI, DXS8091; VII, DXS991. Father, healthy; mother, carrier; daughter, suspected carrier; son, proband.
    Figure Legend Snippet: Electrophoretogram of seven short tandem repeat markers in the family with hemophilia A. I, DXS1227; II, DXS15; III, STR22; IV, DXS993; V, DXS1073; VI, DXS8091; VII, DXS991. Father, healthy; mother, carrier; daughter, suspected carrier; son, proband.

    Techniques Used:

    32) Product Images from "Aberrant histone acetylation, altered transcription, and retinal degeneration in a Drosophila model of polyglutamine disease are rescued by CREB-binding protein"

    Article Title: Aberrant histone acetylation, altered transcription, and retinal degeneration in a Drosophila model of polyglutamine disease are rescued by CREB-binding protein

    Journal: Genes & Development

    doi: 10.1101/gad.1087503

    Aggregation of polyglutamine is associated with neuronal dysfunction and death. It remains unclear whether the toxic species is an unfolded intermediate, oligomer, or large, organized aggregate. The data presented here suggest that CBP rescues toxicity at an upstream point by preventing aggregation. CBP may inhibit aggregation directly (like a blocking peptide) or indirectly (by influencing the expression of genes that regulate aggregation). Reduced aggregation results in a higher proportion of monomeric 127Q, an unstable peptide that is rapidly degraded. Alternatively, CBP may work indirectly by influencing the expression of genes involved in 127Q degradation.
    Figure Legend Snippet: Aggregation of polyglutamine is associated with neuronal dysfunction and death. It remains unclear whether the toxic species is an unfolded intermediate, oligomer, or large, organized aggregate. The data presented here suggest that CBP rescues toxicity at an upstream point by preventing aggregation. CBP may inhibit aggregation directly (like a blocking peptide) or indirectly (by influencing the expression of genes that regulate aggregation). Reduced aggregation results in a higher proportion of monomeric 127Q, an unstable peptide that is rapidly degraded. Alternatively, CBP may work indirectly by influencing the expression of genes involved in 127Q degradation.

    Techniques Used: Blocking Assay, Expressing

    Expression profiles of degenerating and rescued flies. ( A ) Each row shows patterns of gene expression in GMR-Gal4 (1,2,3), (EP 1149 )CBP;GMR-Gal4;127Q (4,5,6,7), (EP 1179 )CBP;GMR-Gal4;127Q (8,9,10), and GMR-Gal4;127Q (11,12,13) flies. The expression ratios of two rescued fly lines (1179 and 1149) are closely related to control flies, but are distant from degenerated flies. ( B ) Transcriptional dysregulation of degenerating flies is restored in the rescued flies.
    Figure Legend Snippet: Expression profiles of degenerating and rescued flies. ( A ) Each row shows patterns of gene expression in GMR-Gal4 (1,2,3), (EP 1149 )CBP;GMR-Gal4;127Q (4,5,6,7), (EP 1179 )CBP;GMR-Gal4;127Q (8,9,10), and GMR-Gal4;127Q (11,12,13) flies. The expression ratios of two rescued fly lines (1179 and 1149) are closely related to control flies, but are distant from degenerated flies. ( B ) Transcriptional dysregulation of degenerating flies is restored in the rescued flies.

    Techniques Used: Expressing

    Histone acetylation in 127Q and rescued flies. ( A ) The level of acetylated H3 and H4 is decreased in flies having 127Q, but restored in flies overexpressing CBP. ( B ) No changes in the acetylation level of other nuclear proteins (as indicated by *) were observed. C, GMR-Gal4; Q, GMR-Gal4;127Q; R, (EP 1179 )CBP;GMRGal4;127Q.
    Figure Legend Snippet: Histone acetylation in 127Q and rescued flies. ( A ) The level of acetylated H3 and H4 is decreased in flies having 127Q, but restored in flies overexpressing CBP. ( B ) No changes in the acetylation level of other nuclear proteins (as indicated by *) were observed. C, GMR-Gal4; Q, GMR-Gal4;127Q; R, (EP 1179 )CBP;GMRGal4;127Q.

    Techniques Used:

    Polyglutamine-induced eye degeneration is rescued by up-regulation of dCBP. ( A ) Control fly expressing the transcription factor GAL4 under control of the eye-specific promoter GMR. ( B ) Expression of 127Q driven by GAL4 leads to progressive eye degeneration. ( C ) Up-regulation of dCBP expression in concert with 127Q prevents polyglutamine toxicity. ( D ) Down-regulation of dCBP expression in concert with 127Q exacerbates polyglutamine toxicity. LM, light microscopy; SEM, scanning electron microscopy. ( E ) Rescue of phototactic behavior by augmentation of dCBP expression. In the multiple-trial countercurrent distribution experiment, the ordinate represents flies that responded positively, and the number of positive responses from 1 to 6 is indicated by the abscissa. GMR-GAL4;UAS-127Q flies showed poor performance. GMR-GAL4;UAS-127Q;EP 1179 dCBP flies perform as well as control GMR-GAL4 flies. n = 100 flies, 5d of age, for each graph. ( F ) 127Q mRNA expression is not significantly altered by up-regulation of dCBP as demonstrated by real-time PCR quantitation. ( G ) Polyglutamine-induced degeneration is accompanied by reduced dCBP expression. Rescue by up-regulation of endogenous dCBP is accomplished by normalization of dCBP expression.
    Figure Legend Snippet: Polyglutamine-induced eye degeneration is rescued by up-regulation of dCBP. ( A ) Control fly expressing the transcription factor GAL4 under control of the eye-specific promoter GMR. ( B ) Expression of 127Q driven by GAL4 leads to progressive eye degeneration. ( C ) Up-regulation of dCBP expression in concert with 127Q prevents polyglutamine toxicity. ( D ) Down-regulation of dCBP expression in concert with 127Q exacerbates polyglutamine toxicity. LM, light microscopy; SEM, scanning electron microscopy. ( E ) Rescue of phototactic behavior by augmentation of dCBP expression. In the multiple-trial countercurrent distribution experiment, the ordinate represents flies that responded positively, and the number of positive responses from 1 to 6 is indicated by the abscissa. GMR-GAL4;UAS-127Q flies showed poor performance. GMR-GAL4;UAS-127Q;EP 1179 dCBP flies perform as well as control GMR-GAL4 flies. n = 100 flies, 5d of age, for each graph. ( F ) 127Q mRNA expression is not significantly altered by up-regulation of dCBP as demonstrated by real-time PCR quantitation. ( G ) Polyglutamine-induced degeneration is accompanied by reduced dCBP expression. Rescue by up-regulation of endogenous dCBP is accomplished by normalization of dCBP expression.

    Techniques Used: Expressing, Light Microscopy, Electron Microscopy, Real-time Polymerase Chain Reaction, Quantitation Assay

    127Q aggregation and inclusion formation are prevented by increased dCBP. ( A ) Western blot of adult head tissue demonstrates that up-regulation of dCBP prevents the formation of high-molecular-weight, insoluble 127Q protein complexes. ( B ) Western blot of whole animal tissue demonstrates that in degenerating flies, polyglutamine aggregate accumulates gradually throughout development, while the steady-state level of polyglutamine monomer remains constant. Flies rescued by up-regulation of dCBP do not show this accumulation of aggregate. ( C ) Immunofluorescence demonstrates that up-regulation of dCBP prevents the formation of intranuclear polyglutamine inclusions. ( Insets ) Diffuse polyglutamine staining remains visible in the cytoplasm and nucleus. Bar, 20 μm.
    Figure Legend Snippet: 127Q aggregation and inclusion formation are prevented by increased dCBP. ( A ) Western blot of adult head tissue demonstrates that up-regulation of dCBP prevents the formation of high-molecular-weight, insoluble 127Q protein complexes. ( B ) Western blot of whole animal tissue demonstrates that in degenerating flies, polyglutamine aggregate accumulates gradually throughout development, while the steady-state level of polyglutamine monomer remains constant. Flies rescued by up-regulation of dCBP do not show this accumulation of aggregate. ( C ) Immunofluorescence demonstrates that up-regulation of dCBP prevents the formation of intranuclear polyglutamine inclusions. ( Insets ) Diffuse polyglutamine staining remains visible in the cytoplasm and nucleus. Bar, 20 μm.

    Techniques Used: Western Blot, Molecular Weight, Immunofluorescence, Staining

    33) Product Images from "A purified, fermented, extract of Triticum aestivum has lymphomacidal activity mediated via natural killer cell activation"

    Article Title: A purified, fermented, extract of Triticum aestivum has lymphomacidal activity mediated via natural killer cell activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190860

    FWGP efficacy depends on NK cell activity in vivo . nu/nu mice bearing established Raji tumors were treated with FWGP (140 mg/kg) with or without the NK-cell depleting antibody anti-ASGM1. Controls received either PBS or the antibody only. Data points represent average tumor volume ± SEM (n = 8 animals/group, * p
    Figure Legend Snippet: FWGP efficacy depends on NK cell activity in vivo . nu/nu mice bearing established Raji tumors were treated with FWGP (140 mg/kg) with or without the NK-cell depleting antibody anti-ASGM1. Controls received either PBS or the antibody only. Data points represent average tumor volume ± SEM (n = 8 animals/group, * p

    Techniques Used: Activity Assay, In Vivo, Mouse Assay

    FWGP activates NK cell in immunocompetent mice. BALB/c animals were treated with FWGP (140 mg/kg) or PBS for 3 days before spleens were dissected. Splenocytes were T-cell depleted and used as effector cells in functional assays. (A) For killing assays, target cells were CFSE-labeled YAC-1. Data points represent the mean±SD of the % CFSE + FVD + cells. (B) For degranulation assays YAC-1 cells were used as target. Data points represent the median±SD fluorescence intensity (as % of max) of CD107a staining. Maximum degranulation was defined as the CD107a signal intensity in cells stimulated with PMA+ionomycin (n = 3; * p
    Figure Legend Snippet: FWGP activates NK cell in immunocompetent mice. BALB/c animals were treated with FWGP (140 mg/kg) or PBS for 3 days before spleens were dissected. Splenocytes were T-cell depleted and used as effector cells in functional assays. (A) For killing assays, target cells were CFSE-labeled YAC-1. Data points represent the mean±SD of the % CFSE + FVD + cells. (B) For degranulation assays YAC-1 cells were used as target. Data points represent the median±SD fluorescence intensity (as % of max) of CD107a staining. Maximum degranulation was defined as the CD107a signal intensity in cells stimulated with PMA+ionomycin (n = 3; * p

    Techniques Used: Mouse Assay, Functional Assay, Labeling, Fluorescence, Staining

    In vivo activity of FWGE and FWGP. Raji cells were implanted in nu/nu mice and animals were treated with 250, 500 or 1000 mg/kg of FWGE by gastric gavage 5 days/week; treatment started after tumor reached > 100 mm 3 (A) or on the day the xenograft was implanted (B); control animals were treated with PBS. (C) Overall survival of animals in A and B. Curves are labeled with dose (mg/kg) and preemptive (Pre) or established (Est) tumor model. (D) Tumor progression was recorded as complete response (CR) or partial response (PR) to treatment. (E) Toxicity was assessed by monitoring animal weight; additional parameters are presented in S6 Fig (F) Animals with established Raji tumors were treated with FWGE, the FWGP subfraction or the subfraction
    Figure Legend Snippet: In vivo activity of FWGE and FWGP. Raji cells were implanted in nu/nu mice and animals were treated with 250, 500 or 1000 mg/kg of FWGE by gastric gavage 5 days/week; treatment started after tumor reached > 100 mm 3 (A) or on the day the xenograft was implanted (B); control animals were treated with PBS. (C) Overall survival of animals in A and B. Curves are labeled with dose (mg/kg) and preemptive (Pre) or established (Est) tumor model. (D) Tumor progression was recorded as complete response (CR) or partial response (PR) to treatment. (E) Toxicity was assessed by monitoring animal weight; additional parameters are presented in S6 Fig (F) Animals with established Raji tumors were treated with FWGE, the FWGP subfraction or the subfraction

    Techniques Used: In Vivo, Activity Assay, Mouse Assay, Labeling

    34) Product Images from "Increased Circulating Levels of Interleukin-6 Induce Perturbation in Redox-Regulated Signaling Cascades in Muscle of Dystrophic Mice"

    Article Title: Increased Circulating Levels of Interleukin-6 Induce Perturbation in Redox-Regulated Signaling Cascades in Muscle of Dystrophic Mice

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2017/1987218

    Nrf2 antioxidant genes are differently regulated in the prenecrotic dystrophic muscle. (a) Real-time PCR analysis of Nrf2-dependent genes (SOD1, SOD2, CAT-1, Gpx1, GCL, and NQO1) performed on the diaphragm muscle of 2-week-old WT, mdx, and mdx/IL6 mice. Values are reported as fold change in expression relative to the calibrator (WT, horizontal dot line) and represent mean ± SEM; n = 3 to 5 per group. p value by unpaired statistical tests. ^ p
    Figure Legend Snippet: Nrf2 antioxidant genes are differently regulated in the prenecrotic dystrophic muscle. (a) Real-time PCR analysis of Nrf2-dependent genes (SOD1, SOD2, CAT-1, Gpx1, GCL, and NQO1) performed on the diaphragm muscle of 2-week-old WT, mdx, and mdx/IL6 mice. Values are reported as fold change in expression relative to the calibrator (WT, horizontal dot line) and represent mean ± SEM; n = 3 to 5 per group. p value by unpaired statistical tests. ^ p

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

    35) Product Images from "Magnetic Enrichment of Dendritic Cell Vaccine in Lymph Node with Fluorescent-Magnetic Nanoparticles Enhanced Cancer Immunotherapy"

    Article Title: Magnetic Enrichment of Dendritic Cell Vaccine in Lymph Node with Fluorescent-Magnetic Nanoparticles Enhanced Cancer Immunotherapy

    Journal: Theranostics

    doi: 10.7150/thno.15102

    Analysis of α-AP-fmNPs uptake by BMDCs and DC2.4 cells and in vitro evaluation of MPF-promoted DC migration. a) Flow cytometric assessment of cellular α-AP-fmNPs uptake. The mean fluorescence intensity (MFI) was recorded and presented as the mean ± SEM (n = 3). b) Effect of α-AP-fmNPs labeling on the viability of BMDCs and DC2.4 cells. c) Prussian blue staining of α-AP-fmNP-internalized DC2.4 cells. d-e) Comparison of intracellular uptake of FAM-α-AP-fmNPs and FAM-AP in DC2.4 cells (d) and BMDCs (e). All experiments were carried out in triplicate. f) Determination of the subcellular localization of α-AP-fmNPs. Confocal imaging of DC2.4 cells incubated with FAM-α-AP-fmNPs for 6 h and stained with LysoTracker and Hoechst 33258. g) Representative images of migrated cells in the absence (-MPF) or presence (+MPF) of MPF. α-AP-fmNP-loaded DC2.4 cells were resuspended in a cell culture flask with a magnet attached to one side of the flask for 24 h, and the photograph of the migrated cells was acquired. h) Calculations of the migrated cells from bright images. Data were acquired from nine fields of view of three independent images. i) Analysis of the migration ability of α-AP-fmNP-loaded BMDCs. The number of the migrated BMDCs in the lower chamber was counted via flow cytometry. All experiments were carried out in triplicate. Data are presented as mean ± SEM (n = 3).
    Figure Legend Snippet: Analysis of α-AP-fmNPs uptake by BMDCs and DC2.4 cells and in vitro evaluation of MPF-promoted DC migration. a) Flow cytometric assessment of cellular α-AP-fmNPs uptake. The mean fluorescence intensity (MFI) was recorded and presented as the mean ± SEM (n = 3). b) Effect of α-AP-fmNPs labeling on the viability of BMDCs and DC2.4 cells. c) Prussian blue staining of α-AP-fmNP-internalized DC2.4 cells. d-e) Comparison of intracellular uptake of FAM-α-AP-fmNPs and FAM-AP in DC2.4 cells (d) and BMDCs (e). All experiments were carried out in triplicate. f) Determination of the subcellular localization of α-AP-fmNPs. Confocal imaging of DC2.4 cells incubated with FAM-α-AP-fmNPs for 6 h and stained with LysoTracker and Hoechst 33258. g) Representative images of migrated cells in the absence (-MPF) or presence (+MPF) of MPF. α-AP-fmNP-loaded DC2.4 cells were resuspended in a cell culture flask with a magnet attached to one side of the flask for 24 h, and the photograph of the migrated cells was acquired. h) Calculations of the migrated cells from bright images. Data were acquired from nine fields of view of three independent images. i) Analysis of the migration ability of α-AP-fmNP-loaded BMDCs. The number of the migrated BMDCs in the lower chamber was counted via flow cytometry. All experiments were carried out in triplicate. Data are presented as mean ± SEM (n = 3).

    Techniques Used: In Vitro, Migration, Flow Cytometry, Fluorescence, Labeling, Staining, Imaging, Incubation, Cell Culture, Cytometry

    36) Product Images from "Host Range Restriction of Insect-Specific Flaviviruses Occurs at Several Levels of the Viral Life Cycle"

    Article Title: Host Range Restriction of Insect-Specific Flaviviruses Occurs at Several Levels of the Viral Life Cycle

    Journal: mSphere

    doi: 10.1128/mSphere.00375-16

    Analyses of NIEV replication and translation. (A) Top: schematic drawing of the NIEV Renilla replicon. The narrow dark gray boxes represent the remaining structural proteins, namely, the first 30 amino acids of the capsid protein (C*) and the last 23 amino acids of the E protein (E*), which serve as signal sequence for the following NS1 protein. The pink box indicates the ubiquitin protein (Ub) responsible for generation of the N terminus of the Renilla luciferase protein (Rluc; yellow box). The narrow gray box represents the 17-amino-acid-residue autoproteolytic peptide from the foot-and-mouth disease virus (FMDV2A), which cleaves at its own C terminus. Lines indicate the 5′ and 3′ UTRs. Bottom: replication of the NIEV Renilla replicon (NIEVR) in insect cells. C6/36 cells were electroporated with NIEVR RNA transcribed in vitro . For comparison, electroporation of YFV Renilla replicon RNA (YFVR) was performed. Cells were incubated at 28°C, and replication kinetics were monitored by analysis of replicon-derived Renilla luciferase expression at the indicated times p.e. Data represent means and ranges of results of duplicate electroporation experiments presented in this panel and in panels B to D. (B) Replication of NIEVR and YFVR in vertebrate cells. The indicated different vertebrate cells were electroporated with in vitro -transcribed NIEVR and YFVR RNAs. Cells were incubated at 37°C, and replication kinetics were monitored as described for panel A. (C) Replication of NIEVR and YFVR in Dicer- and miRNA-deficient 293T cells. Wild-type 293T cells or Dicer- and miRNA-deficient cell clones 2–20 or 4–25 were electroporated with in vitro -transcribed NIEVR and YFVR RNAs. Cells were incubated at 37°C, and replication kinetics were monitored as described for panel A. (D) Analysis of NIEVR translation. BHK cells were electroporated with replication-incompetent Renilla replicon RNAs bearing the exchange of the polymerase GDD motif to GAA, resulting in NIEVR(GAA) or YFVR(GAA). Wild-type NIEVR and YFVR RNAs were electroporated for comparison. Cells were incubated at 37°C, and replication kinetics were monitored as described for panel A. (E) Analysis of NIEVR/YFV 3′ UTR translation. BHK or C6/36 cells were electroporated with NIEVR containing a YFV 3′ UTR insertion downstream of its ORF stop codon. Wild-type NIEVR was electroporated for comparison. Replication kinetics were monitored as described for panel A. Data represent means and ranges of results of duplicate electroporation experiments. Left axis, BHK; right axis, C6/36.
    Figure Legend Snippet: Analyses of NIEV replication and translation. (A) Top: schematic drawing of the NIEV Renilla replicon. The narrow dark gray boxes represent the remaining structural proteins, namely, the first 30 amino acids of the capsid protein (C*) and the last 23 amino acids of the E protein (E*), which serve as signal sequence for the following NS1 protein. The pink box indicates the ubiquitin protein (Ub) responsible for generation of the N terminus of the Renilla luciferase protein (Rluc; yellow box). The narrow gray box represents the 17-amino-acid-residue autoproteolytic peptide from the foot-and-mouth disease virus (FMDV2A), which cleaves at its own C terminus. Lines indicate the 5′ and 3′ UTRs. Bottom: replication of the NIEV Renilla replicon (NIEVR) in insect cells. C6/36 cells were electroporated with NIEVR RNA transcribed in vitro . For comparison, electroporation of YFV Renilla replicon RNA (YFVR) was performed. Cells were incubated at 28°C, and replication kinetics were monitored by analysis of replicon-derived Renilla luciferase expression at the indicated times p.e. Data represent means and ranges of results of duplicate electroporation experiments presented in this panel and in panels B to D. (B) Replication of NIEVR and YFVR in vertebrate cells. The indicated different vertebrate cells were electroporated with in vitro -transcribed NIEVR and YFVR RNAs. Cells were incubated at 37°C, and replication kinetics were monitored as described for panel A. (C) Replication of NIEVR and YFVR in Dicer- and miRNA-deficient 293T cells. Wild-type 293T cells or Dicer- and miRNA-deficient cell clones 2–20 or 4–25 were electroporated with in vitro -transcribed NIEVR and YFVR RNAs. Cells were incubated at 37°C, and replication kinetics were monitored as described for panel A. (D) Analysis of NIEVR translation. BHK cells were electroporated with replication-incompetent Renilla replicon RNAs bearing the exchange of the polymerase GDD motif to GAA, resulting in NIEVR(GAA) or YFVR(GAA). Wild-type NIEVR and YFVR RNAs were electroporated for comparison. Cells were incubated at 37°C, and replication kinetics were monitored as described for panel A. (E) Analysis of NIEVR/YFV 3′ UTR translation. BHK or C6/36 cells were electroporated with NIEVR containing a YFV 3′ UTR insertion downstream of its ORF stop codon. Wild-type NIEVR was electroporated for comparison. Replication kinetics were monitored as described for panel A. Data represent means and ranges of results of duplicate electroporation experiments. Left axis, BHK; right axis, C6/36.

    Techniques Used: Sequencing, Luciferase, In Vitro, Electroporation, Incubation, Derivative Assay, Expressing, Clone Assay

    Analyses of the YF/NIEV reporter virus construct in vertebrate cells. (A) RNA replication of YF/NIEV_Ren in BHK cells. BHK cells were electroporated with the indicated in vitro -transcribed reporter virus RNAs, and Renilla luciferase expression levels were measured at 24 h p.e. Data represent means and ranges of results of duplicate electroporation experiments. (B) Plaque formation of YFV_Ren and YF/NIEV_Ren in BHK cells. BHK cells were electroporated with in vitro RNA transcripts of the indicated constructs and processed for ICA analyses. Seeded cells were overlaid with agarose. At 3 days p.e., cells were fixed and subjected to crystal violet staining. (C) Infectivity assay. Supernatants from BHK cells electroporated with either YF_Ren or YF/NIEV_Ren in vitro -transcribed RNAs were used to infect C6/36 cells. To test for infectivity, Renilla luciferase levels were determined at the indicated time points p.i. Data represent means and ranges of results of duplicate infection experiments. (D) Intra- and extracellular infectivity titers. In vitro transcripts from the full-length clones specified at the bottom were electroporated in C6/36 or BHK cells as indicated. At 6 (C6/36) or 2 (BHK) days p.e., cell lysates and supernatants were subjected to three freeze and thaw cycles. Titers were determined by TCID 50 assay using C6/36 cells and Renilla luciferase as the readout. Dashed line, detection limit. Data represent means and ranges of results of duplicate electroporation experiments.
    Figure Legend Snippet: Analyses of the YF/NIEV reporter virus construct in vertebrate cells. (A) RNA replication of YF/NIEV_Ren in BHK cells. BHK cells were electroporated with the indicated in vitro -transcribed reporter virus RNAs, and Renilla luciferase expression levels were measured at 24 h p.e. Data represent means and ranges of results of duplicate electroporation experiments. (B) Plaque formation of YFV_Ren and YF/NIEV_Ren in BHK cells. BHK cells were electroporated with in vitro RNA transcripts of the indicated constructs and processed for ICA analyses. Seeded cells were overlaid with agarose. At 3 days p.e., cells were fixed and subjected to crystal violet staining. (C) Infectivity assay. Supernatants from BHK cells electroporated with either YF_Ren or YF/NIEV_Ren in vitro -transcribed RNAs were used to infect C6/36 cells. To test for infectivity, Renilla luciferase levels were determined at the indicated time points p.i. Data represent means and ranges of results of duplicate infection experiments. (D) Intra- and extracellular infectivity titers. In vitro transcripts from the full-length clones specified at the bottom were electroporated in C6/36 or BHK cells as indicated. At 6 (C6/36) or 2 (BHK) days p.e., cell lysates and supernatants were subjected to three freeze and thaw cycles. Titers were determined by TCID 50 assay using C6/36 cells and Renilla luciferase as the readout. Dashed line, detection limit. Data represent means and ranges of results of duplicate electroporation experiments.

    Techniques Used: Construct, In Vitro, Luciferase, Expressing, Electroporation, Staining, Infection, Clone Assay

    37) Product Images from "Regulation of ethylene-related gene expression by indole-3-acetic acid and 4-chloroindole-3-acetic acid in relation to pea fruit and seed development"

    Article Title: Regulation of ethylene-related gene expression by indole-3-acetic acid and 4-chloroindole-3-acetic acid in relation to pea fruit and seed development

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/erx217

    Effect of seed removal and hormone treatment on the relative transcript abundance of the ethylene biosynthesis genes PsACS1 (A), PsACS2 (B), PsACS4 (C), PsACO1 (D), PsACO2 (E), and PsACO3 (F) in the pericarps of pollinated pea ovaries. Pericarps at 2 DAA were either left intact, split (SP), or split and deseeded (SPNS), and treated 12 h after deseeding with 4-Cl-IAA, IAA, or ethephon alone or in combination. When fruits were treated with both auxin and ethephon, ethephon was applied 90 min after the auxin treatment and samples were collected based on the time after auxin treatment. Because of the delayed ethephon application, the auxin- plus ethephon-treated pericarps were not studied at the 2 h time point. Deseeded pericarps were also pre-treated with STS at pericarp splitting and deseeding (STS treatment), and IAA was applied to STS-pre-treated pericarps (IAA plus STS treatment). The SP and SPNS controls were treated with 0.1% aqueous Tween-80. All the samples were collected with respect to the time after hormone treatment. Data are means ±SE, n =3–8, with the exception of STS+IAA 2 h treatment, where n =2.
    Figure Legend Snippet: Effect of seed removal and hormone treatment on the relative transcript abundance of the ethylene biosynthesis genes PsACS1 (A), PsACS2 (B), PsACS4 (C), PsACO1 (D), PsACO2 (E), and PsACO3 (F) in the pericarps of pollinated pea ovaries. Pericarps at 2 DAA were either left intact, split (SP), or split and deseeded (SPNS), and treated 12 h after deseeding with 4-Cl-IAA, IAA, or ethephon alone or in combination. When fruits were treated with both auxin and ethephon, ethephon was applied 90 min after the auxin treatment and samples were collected based on the time after auxin treatment. Because of the delayed ethephon application, the auxin- plus ethephon-treated pericarps were not studied at the 2 h time point. Deseeded pericarps were also pre-treated with STS at pericarp splitting and deseeding (STS treatment), and IAA was applied to STS-pre-treated pericarps (IAA plus STS treatment). The SP and SPNS controls were treated with 0.1% aqueous Tween-80. All the samples were collected with respect to the time after hormone treatment. Data are means ±SE, n =3–8, with the exception of STS+IAA 2 h treatment, where n =2.

    Techniques Used:

    Relative transcript abundance of the ethylene biosynthesis genes PsACS1 (A and B), PsACS2 (C and D), and PsACS4 (E and F) in pollinated and non-pollinated pea fruits. Whole pericarps were assessed at –2 DAA, and pericarp tissues (ventral vascular suture, dorsal vascular suture, and wall) were assessed at 0–3 DAA. For non-pollinated fruits, flowers were emasculated at –2 DAA to prevent pollination. Data are means ±SE, n =3, with n =2 for a few samples where the tissues were limited due to small tissue size. Each sample is composed of a minimum of four pericarp tissues.
    Figure Legend Snippet: Relative transcript abundance of the ethylene biosynthesis genes PsACS1 (A and B), PsACS2 (C and D), and PsACS4 (E and F) in pollinated and non-pollinated pea fruits. Whole pericarps were assessed at –2 DAA, and pericarp tissues (ventral vascular suture, dorsal vascular suture, and wall) were assessed at 0–3 DAA. For non-pollinated fruits, flowers were emasculated at –2 DAA to prevent pollination. Data are means ±SE, n =3, with n =2 for a few samples where the tissues were limited due to small tissue size. Each sample is composed of a minimum of four pericarp tissues.

    Techniques Used:

    38) Product Images from "Knock-down of ELMO1 in Paediatric Rhabdomyosarcoma Cells by Nanoparticle Mediated siRNA Delivery"

    Article Title: Knock-down of ELMO1 in Paediatric Rhabdomyosarcoma Cells by Nanoparticle Mediated siRNA Delivery

    Journal: Nanobiomedicine

    doi: 10.5772/62690

    Assessment of invasion after transfection with siRNA. RMS cells were incubated with naked siRNA, non-targeted (NT) siRNA, or ELMO1 targeted siRNA. The lipid transfection agent, DharmaFECT, was compared with MSNPs of different morphologies for efficacy. The percentage closure of a wound inflicted in a confluent cell layer was measured after 24 hours. Data are presented as the mean ± SD of triplicate samples. Significance was tested using a two tailed t-test compared to the untreated cells for each cell line (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005).
    Figure Legend Snippet: Assessment of invasion after transfection with siRNA. RMS cells were incubated with naked siRNA, non-targeted (NT) siRNA, or ELMO1 targeted siRNA. The lipid transfection agent, DharmaFECT, was compared with MSNPs of different morphologies for efficacy. The percentage closure of a wound inflicted in a confluent cell layer was measured after 24 hours. Data are presented as the mean ± SD of triplicate samples. Significance was tested using a two tailed t-test compared to the untreated cells for each cell line (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005).

    Techniques Used: Transfection, Incubation, Two Tailed Test

    qRT-PCR assessment of ELMO1 knock-down. RMS cells were treated with nanoparticles coated with PEI (PEI- HMSNP/WMSNP), or non-targeting (NT) siRNA, or ELMO1 targeted siRNA. The lipid transfection agent, DharmaFECT, was compared with MSNPs of different morphologies for efficacy. The fold-difference, as determined by qRT-PCR, is shown. Data are presented as the mean ± SD of triplicate samples. Significance was tested using a two tailed t-test compared to the cells treated with ELMO1@DharmaFECT for each cell line (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005).
    Figure Legend Snippet: qRT-PCR assessment of ELMO1 knock-down. RMS cells were treated with nanoparticles coated with PEI (PEI- HMSNP/WMSNP), or non-targeting (NT) siRNA, or ELMO1 targeted siRNA. The lipid transfection agent, DharmaFECT, was compared with MSNPs of different morphologies for efficacy. The fold-difference, as determined by qRT-PCR, is shown. Data are presented as the mean ± SD of triplicate samples. Significance was tested using a two tailed t-test compared to the cells treated with ELMO1@DharmaFECT for each cell line (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005).

    Techniques Used: Quantitative RT-PCR, Transfection, Two Tailed Test

    Scratch test showing cell invasion under different test conditions. RH30 cells were transfected with ELMO1 or a non-targeted siRNA for 72 hours. Subsequently, the confluent cell layer was scratched with a pipette tip and the images were recorded at (i) t=0 and (ii) t= 24 hours. Representative images are shown for: (A) Untreated control cells, (B) Bare ELMO1-targeted siRNA, (C) non-targeted siRNA and DharmaFECT, (D) ELMO1 siRNA and DharmaFECT, (E) ELMO1 and SNP, (F) ELMO1 and HMSNP, (G) ELMO1 and BMSNP, (H) ELMO1 and CMSNP and (I) ELMO1 and WMNSP. The arrows indicate cell free gap. Scale bar = 200 μm.
    Figure Legend Snippet: Scratch test showing cell invasion under different test conditions. RH30 cells were transfected with ELMO1 or a non-targeted siRNA for 72 hours. Subsequently, the confluent cell layer was scratched with a pipette tip and the images were recorded at (i) t=0 and (ii) t= 24 hours. Representative images are shown for: (A) Untreated control cells, (B) Bare ELMO1-targeted siRNA, (C) non-targeted siRNA and DharmaFECT, (D) ELMO1 siRNA and DharmaFECT, (E) ELMO1 and SNP, (F) ELMO1 and HMSNP, (G) ELMO1 and BMSNP, (H) ELMO1 and CMSNP and (I) ELMO1 and WMNSP. The arrows indicate cell free gap. Scale bar = 200 μm.

    Techniques Used: Transfection, Transferring

    39) Product Images from "Biodiversity of cyanobacteria and other aquatic microorganisms across a freshwater to brackish water gradient determined by shotgun metagenomic sequencing analysis in the San Francisco Estuary, USA"

    Article Title: Biodiversity of cyanobacteria and other aquatic microorganisms across a freshwater to brackish water gradient determined by shotgun metagenomic sequencing analysis in the San Francisco Estuary, USA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0203953

    Phylogenetic tree for the six Microcystis genotypes generated based on the internal transcribed spacer (ITS) region. The number at each node represents the posterior probability value. The scale bar indicates inferred nucleotide substitution rate. A portion of ITS sequence from Gloeocapsa sp. (GenBank accession number: KJ746508.1) was used as an outgroup for generating the rooted phylogenetic tree. The numbers in the parentheses are the internal reference ID number.
    Figure Legend Snippet: Phylogenetic tree for the six Microcystis genotypes generated based on the internal transcribed spacer (ITS) region. The number at each node represents the posterior probability value. The scale bar indicates inferred nucleotide substitution rate. A portion of ITS sequence from Gloeocapsa sp. (GenBank accession number: KJ746508.1) was used as an outgroup for generating the rooted phylogenetic tree. The numbers in the parentheses are the internal reference ID number.

    Techniques Used: Generated, Sequencing

    40) Product Images from "Deficient humoral responses and disrupted B-cell immunity are associated with fatal SFTSV infection"

    Article Title: Deficient humoral responses and disrupted B-cell immunity are associated with fatal SFTSV infection

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05746-9

    Schematic presentation of the mechanisms in the defective humoral response to SFTSV infection. a In the pre-germinal center (pre-GC) phase, persistent viremia primes large quantity of naive B cells into PBs and causes massive monocytic apoptosis, resulting in impaired mDC differentiation, accompanied by the downregulation of TLR3, IL-4 and GM-CSF. Due to insufficient MHC-II antigen presentation by mDCs and B cells, the differentiation and function of Tfh are inhibited, characterized by the decreased numbers and downregulation of IL-21 secretion. Additionally, the expression of PD-1 on Tfh, which act as an important inhibitory molecule of T-cell function, was observed to be persistently upregulated in deceased patients. The expression of three key transcriptional factors, BLIMP-1, IRF-4 and XBP-1, was down-modulated due to the lack of Tfh stimulation. PBs failed to differentiate into non-GC plasma cells and were unable to secret IgM antibodies. b In the GC phase, Ag-experienced B cells interact with cognate Tfh and mDC. However, the B-cell maturation is impaired due to the dysfunction of cognate Tfh and mDC, whi ch, together with the downregulation of TLRs and IL-4, inhibited the induction of activation-induced cytidine deaminase (AID), resulting in the complete inhibition of immunoglobulin class-switch in the deceased patients. Red upward arrow and blue downward arrow denote up and downregulation, respectively
    Figure Legend Snippet: Schematic presentation of the mechanisms in the defective humoral response to SFTSV infection. a In the pre-germinal center (pre-GC) phase, persistent viremia primes large quantity of naive B cells into PBs and causes massive monocytic apoptosis, resulting in impaired mDC differentiation, accompanied by the downregulation of TLR3, IL-4 and GM-CSF. Due to insufficient MHC-II antigen presentation by mDCs and B cells, the differentiation and function of Tfh are inhibited, characterized by the decreased numbers and downregulation of IL-21 secretion. Additionally, the expression of PD-1 on Tfh, which act as an important inhibitory molecule of T-cell function, was observed to be persistently upregulated in deceased patients. The expression of three key transcriptional factors, BLIMP-1, IRF-4 and XBP-1, was down-modulated due to the lack of Tfh stimulation. PBs failed to differentiate into non-GC plasma cells and were unable to secret IgM antibodies. b In the GC phase, Ag-experienced B cells interact with cognate Tfh and mDC. However, the B-cell maturation is impaired due to the dysfunction of cognate Tfh and mDC, whi ch, together with the downregulation of TLRs and IL-4, inhibited the induction of activation-induced cytidine deaminase (AID), resulting in the complete inhibition of immunoglobulin class-switch in the deceased patients. Red upward arrow and blue downward arrow denote up and downregulation, respectively

    Techniques Used: Infection, Expressing, Activated Clotting Time Assay, Cell Function Assay, Activation Assay, Inhibition

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    Transfection:

    Article Title: Newcastle Disease Virus V Protein Inhibits Cell Apoptosis and Promotes Viral Replication by Targeting CacyBP/SIP
    Article Snippet: .. Transfection and viral infection Plasmid or siRNA was transfected to DF-1 and/or Vero cells using Lipofectamine 2000 (Thermo Scientific, NH, USA) according to the manufacturer's instruction. ..

    Article Title: 25-Hydroxycholesterol and 27-hydroxycholesterol inhibit human rotavirus infection by sequestering viral particles into late endosomes
    Article Snippet: .. Briefly, extraction of total RNA from transfected or untransfected Caco2 cells was performed 72 h after transfection using TRIzol Reagent (Applied Biosystems, Monza, Italy). .. Concentration and purity of the extracted RNA were assessed by spectrophotometry (A260/A280).

    Mutagenesis:

    Article Title: Tomato DCL2b is required for the biosynthesis of 22-nt small RNAs, the resulting secondary siRNAs, and the host defense against ToMV
    Article Snippet: .. High-throughput sequencing of RNAs and sRNAs The total RNA samples were prepared from WT and DCL2b mutant adult leaves using TRIzol reagent (Invitrogen, USA). .. Paired-end mRNA libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s recommendations and were sequenced on an Illumina HiSeq 4000 platform; 150 bp reads were generated.

    Isolation:

    Article Title: KDELR2 Competes with Measles Virus Envelope Proteins for Cellular Chaperones Reducing Their Chaperone-Mediated Cell Surface Transport
    Article Snippet: .. Isolated RNA was reverse transcribed in cDNA using the RevertAid first strand cDNA synthesis kit (Fermentas). ..

    Article Title: Complement Receptor C5aR1 Inhibition Reduces Pyroptosis in hDPP4-Transgenic Mice Infected with MERS-CoV
    Article Snippet: .. Isolation of RNA and Proteins THP-1 differentiated macrophages were lysed in TRIzol™ Reagent (Life Technologies, Carlsbad, CA, USA) at 24 h post-infection with MERS-CoV. .. Total RNA and proteins were isolated according to the reagent user guide.

    Multiplex Assay:

    Article Title: Decidual Interleukin-22-Producing CD4+ T Cells (Th17/Th0/IL-22+ and Th17/Th2/IL-22+, Th2/IL-22+, Th0/IL-22+), Which Also Produce IL-4, Are Involved in the Success of Pregnancy
    Article Snippet: .. Briefly, the mRNA expression of IL-4, IL-17A, IL-17F, IL-23R, IFN-γ, RORC, GATA3, AHR, IL-22, and Actb (high expression housekeeping gene) was measured using the QuantiGene multiplex assay (Thermo Fisher, Waltham, MA, USA). .. Samples (biopsies of decidua from those experiencing successful pregnancy and tubal biopsies of those experiencing ectopic pregnancies at the implantation site and away from the implantation site) were lysed after treatment in a lysis mixture; mRNA expression in lysates was detected and measured according to the manufacturer’s instructions.

    Infection:

    Article Title: Newcastle Disease Virus V Protein Inhibits Cell Apoptosis and Promotes Viral Replication by Targeting CacyBP/SIP
    Article Snippet: .. Transfection and viral infection Plasmid or siRNA was transfected to DF-1 and/or Vero cells using Lipofectamine 2000 (Thermo Scientific, NH, USA) according to the manufacturer's instruction. ..

    Sequencing:

    Article Title: Dual-modified cationic liposomes loaded with paclitaxel and survivin siRNA for targeted imaging and therapy of cancer stem cells in brain glioma
    Article Snippet: .. Survivin siRNA (sequence: 5′-GCAUUCGUCCGGUUGCGCUdTdT-3′) and a scrambled siRNA (sequence: 5′-AUGAACUUCAGGGUCAGCUdTdT-3′) were purchased from Thermo Scientific Dharmacon (Shanghai, China). .. The following primer probe sets (Integrated DNA Technologies, Coralville, IA, USA) were used: survivin, forward: 5′-CAACCGGACGAATGCTTTT-3′; reverse: 5′-AAGAACTGGCCCTTCTTGGA-3′; probe: 5′-/5HEX/CCAGATGAC/ZEN/GACCCCATAGAGGAA/3IABkFQ/-3′; GAPDH, forward: 5′-AATCCCATCACCATCTTCCAG-3′; reverse: 5′-AAATGAGCCCCAGCCTTC-3′; probe: 5′-/5Cy5/CCAGCATCGCCCCACTTG ATTTT/3IAbRQSp/-3′; β-actin primers, forward: 5′-CATCGTGGGCCGCCCTAGGC-3′, reverse: 5′-GGGCCTCGGTGAGCAGCACA-3′ (Sangon Biotech, Shanghai, China).

    Article Title: Tomato DCL2b is required for the biosynthesis of 22-nt small RNAs, the resulting secondary siRNAs, and the host defense against ToMV
    Article Snippet: .. High-throughput sequencing of RNAs and sRNAs The total RNA samples were prepared from WT and DCL2b mutant adult leaves using TRIzol reagent (Invitrogen, USA). .. Paired-end mRNA libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s recommendations and were sequenced on an Illumina HiSeq 4000 platform; 150 bp reads were generated.

    Expressing:

    Article Title: Decidual Interleukin-22-Producing CD4+ T Cells (Th17/Th0/IL-22+ and Th17/Th2/IL-22+, Th2/IL-22+, Th0/IL-22+), Which Also Produce IL-4, Are Involved in the Success of Pregnancy
    Article Snippet: .. Briefly, the mRNA expression of IL-4, IL-17A, IL-17F, IL-23R, IFN-γ, RORC, GATA3, AHR, IL-22, and Actb (high expression housekeeping gene) was measured using the QuantiGene multiplex assay (Thermo Fisher, Waltham, MA, USA). .. Samples (biopsies of decidua from those experiencing successful pregnancy and tubal biopsies of those experiencing ectopic pregnancies at the implantation site and away from the implantation site) were lysed after treatment in a lysis mixture; mRNA expression in lysates was detected and measured according to the manufacturer’s instructions.

    High Throughput Screening Assay:

    Article Title: Tomato DCL2b is required for the biosynthesis of 22-nt small RNAs, the resulting secondary siRNAs, and the host defense against ToMV
    Article Snippet: .. High-throughput sequencing of RNAs and sRNAs The total RNA samples were prepared from WT and DCL2b mutant adult leaves using TRIzol reagent (Invitrogen, USA). .. Paired-end mRNA libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s recommendations and were sequenced on an Illumina HiSeq 4000 platform; 150 bp reads were generated.

    Plasmid Preparation:

    Article Title: Newcastle Disease Virus V Protein Inhibits Cell Apoptosis and Promotes Viral Replication by Targeting CacyBP/SIP
    Article Snippet: .. Transfection and viral infection Plasmid or siRNA was transfected to DF-1 and/or Vero cells using Lipofectamine 2000 (Thermo Scientific, NH, USA) according to the manufacturer's instruction. ..

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    Thermo Fisher gene exp scn8a mm00488110 m1
    GS967/Prax330 extends the survival of <t>Scn8a</t> cond / + , EIIa-Cre mice and reduces activity of the mutant channel in transfected cells. ( A ) GS967/Prax330 was added to the mouse chow at 8 mg/kg beginning on P1. Untreated mice received normal chow. Mean survival was increased from 15 days for untreated mice ( n = 55) to 21 days for treated mice ( n = 14). Kaplan-Meier log-rank (Mantel-Cox) test, χ 2 (1) = 32, P
    Gene Exp Scn8a Mm00488110 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gene exp il6 mm00446190 m1
    Expression of known or suspected TTP targets in LPS-stimulated BMDMs. ( A ) Cells were either left unstimulated or were stimulated with LPS (1 µg/mL), RNA was extracted, and real-time RT-PCR was performed using transcript-specific primers for Tnf, Il1b, Cxcl2, Il10, <t>Il6,</t> and Il23a mRNAs. Values were normalized to Gapdh mRNA, and the fold changes were calculated relative to basal levels ( n = 4). Statistical analysis was performed by two-way ANOVA (** P
    Gene Exp Il6 Mm00446190 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 461 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GS967/Prax330 extends the survival of Scn8a cond / + , EIIa-Cre mice and reduces activity of the mutant channel in transfected cells. ( A ) GS967/Prax330 was added to the mouse chow at 8 mg/kg beginning on P1. Untreated mice received normal chow. Mean survival was increased from 15 days for untreated mice ( n = 55) to 21 days for treated mice ( n = 14). Kaplan-Meier log-rank (Mantel-Cox) test, χ 2 (1) = 32, P

    Journal: Brain

    Article Title: Prominent role of forebrain excitatory neurons in SCN8A encephalopathy

    doi: 10.1093/brain/awy324

    Figure Lengend Snippet: GS967/Prax330 extends the survival of Scn8a cond / + , EIIa-Cre mice and reduces activity of the mutant channel in transfected cells. ( A ) GS967/Prax330 was added to the mouse chow at 8 mg/kg beginning on P1. Untreated mice received normal chow. Mean survival was increased from 15 days for untreated mice ( n = 55) to 21 days for treated mice ( n = 14). Kaplan-Meier log-rank (Mantel-Cox) test, χ 2 (1) = 32, P

    Article Snippet: The adult-specific Scn8a transcript containing exon 18A ( ) was assayed using VIC labelled gene expression assay Mm00488119 and standardized to FAM-labelled total Scn8a transcript Mm00488110_m1.

    Techniques: Mouse Assay, Activity Assay, Mutagenesis, Transfection

    Ubiquitous activation of the conditional Scn8a allele by EIIA-Cre. ( A ) Genotyping of genomic DNA samples from brain and tail of heterozygous Scn8a cond/+ , EIIa-Cre ) and the PCR product was digested with KpnI. ( B ) RT-PCR of brain RNA using primer pair 6 in exons 25 and 26. There is expression of exon 26b from the conditional allele, due to the excision of exon 26a by Cre recombinase. In addition, exon 26 is expressed from the wild-type allele in the heterozygous mice. B = Bsrb1 . ( C ) Sanger sequencing of the RT-PCR products demonstrates the heterozygous expression of wild-type exon 26 and exon 26b. Heterozygous synonymous mutations are boxed and the heterozygous R1872W mutation is marked with an arrow. The location of the sequenced nucleotides within the amplified exon is indicated by dotted lines.

    Journal: Brain

    Article Title: Prominent role of forebrain excitatory neurons in SCN8A encephalopathy

    doi: 10.1093/brain/awy324

    Figure Lengend Snippet: Ubiquitous activation of the conditional Scn8a allele by EIIA-Cre. ( A ) Genotyping of genomic DNA samples from brain and tail of heterozygous Scn8a cond/+ , EIIa-Cre ) and the PCR product was digested with KpnI. ( B ) RT-PCR of brain RNA using primer pair 6 in exons 25 and 26. There is expression of exon 26b from the conditional allele, due to the excision of exon 26a by Cre recombinase. In addition, exon 26 is expressed from the wild-type allele in the heterozygous mice. B = Bsrb1 . ( C ) Sanger sequencing of the RT-PCR products demonstrates the heterozygous expression of wild-type exon 26 and exon 26b. Heterozygous synonymous mutations are boxed and the heterozygous R1872W mutation is marked with an arrow. The location of the sequenced nucleotides within the amplified exon is indicated by dotted lines.

    Article Snippet: The adult-specific Scn8a transcript containing exon 18A ( ) was assayed using VIC labelled gene expression assay Mm00488119 and standardized to FAM-labelled total Scn8a transcript Mm00488110_m1.

    Techniques: Activation Assay, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Mouse Assay, Sequencing, Mutagenesis, Amplification

    Activation of Scn8a R1872W in adult mice leads to seizures and lethality. Scn8a cond / + , CAG-Cre-ER mice ( n = 6) were treated at 8 weeks of age with five daily injections of tamoxifen. Seizures were monitored visually for 8 h per day. ( A ) The first seizures were observed between 2 and 4 weeks after tamoxifen injection. ( B ) Death occurred between 1 and 3 months after seizure onset. No seizures or deaths were observed in Scn8a cond / + littermates lacking Cre ( n = 3). ( C ) RT-PCR of brain RNA using primer pair 6 in exons 25 and 26. Brain transcripts encoding tryptophan 1872 (273 bp RT-PCR fragment) are present only in tamoxifen-treated Scn8a cond / + , CAG-Cre-ER mice and not in littermate controls lacking CAG-Cre-ER .

    Journal: Brain

    Article Title: Prominent role of forebrain excitatory neurons in SCN8A encephalopathy

    doi: 10.1093/brain/awy324

    Figure Lengend Snippet: Activation of Scn8a R1872W in adult mice leads to seizures and lethality. Scn8a cond / + , CAG-Cre-ER mice ( n = 6) were treated at 8 weeks of age with five daily injections of tamoxifen. Seizures were monitored visually for 8 h per day. ( A ) The first seizures were observed between 2 and 4 weeks after tamoxifen injection. ( B ) Death occurred between 1 and 3 months after seizure onset. No seizures or deaths were observed in Scn8a cond / + littermates lacking Cre ( n = 3). ( C ) RT-PCR of brain RNA using primer pair 6 in exons 25 and 26. Brain transcripts encoding tryptophan 1872 (273 bp RT-PCR fragment) are present only in tamoxifen-treated Scn8a cond / + , CAG-Cre-ER mice and not in littermate controls lacking CAG-Cre-ER .

    Article Snippet: The adult-specific Scn8a transcript containing exon 18A ( ) was assayed using VIC labelled gene expression assay Mm00488119 and standardized to FAM-labelled total Scn8a transcript Mm00488110_m1.

    Techniques: Activation Assay, Mouse Assay, Injection, Reverse Transcription Polymerase Chain Reaction

    Phenotypic effects of activation of the conditional allele by ubiquitously expressed EIIa-Cre . ( A ) Scn8a cond / + heterozygotes expressing EIIa-Cre exhibited sudden lethality at the end of the second week of life, with median survival of 15 days. Scn8a cond / + heterozygotes that did not inherit EIIa-Cre exhibited normal life span. ( B ) EEG recording of a fatal seizure in an Scn8a cond / + , EIIa-Cre mouse on P15. The recording was obtained from bipolar insulated stainless-steel electrodes in the left parietal cortex ( top trace ) and right parietal cortex ( second trace ). The EMG artefact in the third trace reflects the period of running evolving to tonic extension. Death occurred 3 min 20 s after the onset of running. The bottom trace was obtained from an electrode placed subcutaneously in the right thorax and demonstrates sudden slowing of the heart rate from 756 bpm prior to the running fit to 156 bpm immediately afterwards.

    Journal: Brain

    Article Title: Prominent role of forebrain excitatory neurons in SCN8A encephalopathy

    doi: 10.1093/brain/awy324

    Figure Lengend Snippet: Phenotypic effects of activation of the conditional allele by ubiquitously expressed EIIa-Cre . ( A ) Scn8a cond / + heterozygotes expressing EIIa-Cre exhibited sudden lethality at the end of the second week of life, with median survival of 15 days. Scn8a cond / + heterozygotes that did not inherit EIIa-Cre exhibited normal life span. ( B ) EEG recording of a fatal seizure in an Scn8a cond / + , EIIa-Cre mouse on P15. The recording was obtained from bipolar insulated stainless-steel electrodes in the left parietal cortex ( top trace ) and right parietal cortex ( second trace ). The EMG artefact in the third trace reflects the period of running evolving to tonic extension. Death occurred 3 min 20 s after the onset of running. The bottom trace was obtained from an electrode placed subcutaneously in the right thorax and demonstrates sudden slowing of the heart rate from 756 bpm prior to the running fit to 156 bpm immediately afterwards.

    Article Snippet: The adult-specific Scn8a transcript containing exon 18A ( ) was assayed using VIC labelled gene expression assay Mm00488119 and standardized to FAM-labelled total Scn8a transcript Mm00488110_m1.

    Techniques: Activation Assay, Expressing

    Effects of expression of Scn8a R1872W in forebrain neurons. ( A ) Survival of conditional mice expressing cell-specific Cre constructs. Nestin-Cre is broadly expressed in neural cells; median survival 21 days. Emx1-Cre is expressed in excitatory neurons of the forebrain; median survival 46 days. Gad2-Cre is expressed in inhibitory neurons. ( B ) Brain transcripts encoding arginine 1872 (173 bp RT-PCR fragment) and tryptophan 1872 (273 bp RT-PCR fragment) in mice expressing various Cre recombinases.

    Journal: Brain

    Article Title: Prominent role of forebrain excitatory neurons in SCN8A encephalopathy

    doi: 10.1093/brain/awy324

    Figure Lengend Snippet: Effects of expression of Scn8a R1872W in forebrain neurons. ( A ) Survival of conditional mice expressing cell-specific Cre constructs. Nestin-Cre is broadly expressed in neural cells; median survival 21 days. Emx1-Cre is expressed in excitatory neurons of the forebrain; median survival 46 days. Gad2-Cre is expressed in inhibitory neurons. ( B ) Brain transcripts encoding arginine 1872 (173 bp RT-PCR fragment) and tryptophan 1872 (273 bp RT-PCR fragment) in mice expressing various Cre recombinases.

    Article Snippet: The adult-specific Scn8a transcript containing exon 18A ( ) was assayed using VIC labelled gene expression assay Mm00488119 and standardized to FAM-labelled total Scn8a transcript Mm00488110_m1.

    Techniques: Expressing, Mouse Assay, Construct, Reverse Transcription Polymerase Chain Reaction

    Structure and basal expression of the conditional R1872W allele of Scn8a . ( A ) The conditional allele contains two copies of exon 26, the final exon of Scn8a. Both exons 26a and 26b were derived from a targeting vector with eight silent substitutions in exon 26 that were introduced to prevent digestion by the TALEN endonucleases used for targeting of the Scn8a for details). The upstream exon 26a contains wild-type arginine codon 1872 (R) and the 3′ UTR from the bGH gene, and is flanked by loxP sites. The downstream exon 26b contains the same eight silent substitutions as well as the mutant tryptophan codon 1872 (W) and terminates in the endogenous 3′ UTR. Exposure to Cre recombinase will delete exon 26a and lead to expression of exon 26b encoding the mutant sodium channel. ( B ) Southern blot of genomic DNA from eight potential founder mice. Correct targeting of the endogenous Scn8a locus in DNA from founder 2176 (asterisk) is demonstrated by the 5.4 kb KpnI restriction fragment hybridizing with the 5′ probe 1 as well as the 3.5 kb HincII hybridizi ng C show the sequenced regions of long-range junction PCR products that confirmed the structure of the conditional allele. ( C ) RT-PCR products were amplified with forward primer in exon 25 and reverse primer either in the bGH 3′ UTR or the endogenous 3′ UTR. The wild-type allele expresses the endogenous 3′ UTR and the conditional allele expresses only the bGH 3′ UTR in the absence of Cre recombinase. ( D ) Sequences of the RT-PCR products amplified using primer pair 6 (forward primer in exon 25, reverse primer in exon 26) demonstrate expression of exon 26 from the wild-type allele and exon 26a from the conditional allele. Boxed nucleotides indicate the location of markers of the conditional allele: three of the synonymous nucleotide substitutions and tryptophan codon 1872. ( E ) Quantitative RT-PCR of whole brain RNA with the TaqMan TM assay for Scn8a and comparison with the control transcript of the Tata-binding protein ( Tbp ) demonstrates comparable level of transcript from the wild-type allele and the conditional allele. Three animals of each genotype were assayed.

    Journal: Brain

    Article Title: Prominent role of forebrain excitatory neurons in SCN8A encephalopathy

    doi: 10.1093/brain/awy324

    Figure Lengend Snippet: Structure and basal expression of the conditional R1872W allele of Scn8a . ( A ) The conditional allele contains two copies of exon 26, the final exon of Scn8a. Both exons 26a and 26b were derived from a targeting vector with eight silent substitutions in exon 26 that were introduced to prevent digestion by the TALEN endonucleases used for targeting of the Scn8a for details). The upstream exon 26a contains wild-type arginine codon 1872 (R) and the 3′ UTR from the bGH gene, and is flanked by loxP sites. The downstream exon 26b contains the same eight silent substitutions as well as the mutant tryptophan codon 1872 (W) and terminates in the endogenous 3′ UTR. Exposure to Cre recombinase will delete exon 26a and lead to expression of exon 26b encoding the mutant sodium channel. ( B ) Southern blot of genomic DNA from eight potential founder mice. Correct targeting of the endogenous Scn8a locus in DNA from founder 2176 (asterisk) is demonstrated by the 5.4 kb KpnI restriction fragment hybridizing with the 5′ probe 1 as well as the 3.5 kb HincII hybridizi ng C show the sequenced regions of long-range junction PCR products that confirmed the structure of the conditional allele. ( C ) RT-PCR products were amplified with forward primer in exon 25 and reverse primer either in the bGH 3′ UTR or the endogenous 3′ UTR. The wild-type allele expresses the endogenous 3′ UTR and the conditional allele expresses only the bGH 3′ UTR in the absence of Cre recombinase. ( D ) Sequences of the RT-PCR products amplified using primer pair 6 (forward primer in exon 25, reverse primer in exon 26) demonstrate expression of exon 26 from the wild-type allele and exon 26a from the conditional allele. Boxed nucleotides indicate the location of markers of the conditional allele: three of the synonymous nucleotide substitutions and tryptophan codon 1872. ( E ) Quantitative RT-PCR of whole brain RNA with the TaqMan TM assay for Scn8a and comparison with the control transcript of the Tata-binding protein ( Tbp ) demonstrates comparable level of transcript from the wild-type allele and the conditional allele. Three animals of each genotype were assayed.

    Article Snippet: The adult-specific Scn8a transcript containing exon 18A ( ) was assayed using VIC labelled gene expression assay Mm00488119 and standardized to FAM-labelled total Scn8a transcript Mm00488110_m1.

    Techniques: Expressing, Derivative Assay, Plasmid Preparation, Mutagenesis, Southern Blot, Mouse Assay, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Amplification, Quantitative RT-PCR, Binding Assay

    Expression of known or suspected TTP targets in LPS-stimulated BMDMs. ( A ) Cells were either left unstimulated or were stimulated with LPS (1 µg/mL), RNA was extracted, and real-time RT-PCR was performed using transcript-specific primers for Tnf, Il1b, Cxcl2, Il10, Il6, and Il23a mRNAs. Values were normalized to Gapdh mRNA, and the fold changes were calculated relative to basal levels ( n = 4). Statistical analysis was performed by two-way ANOVA (** P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Enhanced stability of tristetraprolin mRNA protects mice against immune-mediated inflammatory pathologies

    doi: 10.1073/pnas.1519906113

    Figure Lengend Snippet: Expression of known or suspected TTP targets in LPS-stimulated BMDMs. ( A ) Cells were either left unstimulated or were stimulated with LPS (1 µg/mL), RNA was extracted, and real-time RT-PCR was performed using transcript-specific primers for Tnf, Il1b, Cxcl2, Il10, Il6, and Il23a mRNAs. Values were normalized to Gapdh mRNA, and the fold changes were calculated relative to basal levels ( n = 4). Statistical analysis was performed by two-way ANOVA (** P

    Article Snippet: To amplify cDNA of mouse Il1b , Cxcl2 , Il6 , and Il23a , we used TaqMan Universal PCR Master Mix (Applied Biosystems) and predesigned TaqMan assays for the Il1b transcript (FAM/MGB probe Mm01336189_m1), the Cxcl2 mRNA (FAM/MGB probe Mm00436450_m1), the Il6 mRNA (FAM/MGB probe Mm00446190_m1), the Il23a mRNA (FAM/MGB probe Mm00518984_m1), and a mouse Gapdh mRNA control (catalog no. 4352932E), according to the manufacturer’s instructions.

    Techniques: Expressing, Quantitative RT-PCR

    AIMp1 deficiency in dendritic cells impairs T H 1 polarization in vitro . (A) Wild-type (WT) or AIMp1 KO bone marrow-derived dendritic cell (BMDC) were matured for 48 h with cytokine cocktail, then cocultured with naive WT CD3 + T-cells at a 1:10 ratio. IFN-γ from 3-day coculture supernatants was measured by ELISA ( n = 7, biological repeats). (B) WT or AIMp1 KO BMDC were loaded with ovalbumin (OVA) antigen and matured for 48 h with cytokine cocktail, then cocultured with OT-II T-cells at a 1:20 ratio. IFN-γ from 3-day coculture supernatants was measured by ELISA ( n = 8, biological repeats). (C) WT T-cells were harvested and lysed for Western blotting analysis of pSTAT1(Y701)/STAT1, pSTAT4(Y693), T-bet, and β-actin after 6 h of coculture with BMDC as in (A) (Representative of three independent experiments). (D) WT or AIMp1 KO BMDC were treated with LPS, and levels of proinflammatory cytokines IL-12p70, IL-6, and IL-1β were measured by ELISA ( n = 7, technical repeats, pooled from two independent experiments). (E) WT or AIMp1 KO BMDC were treated with maturation cocktail or LPS and levels of CD86, CD40, and MHC II (I-A b ) were measured by flow cytometry (normalized and pooled from multiple independent experiments). (F) Conditioned supernatant from WT or AIMp1 KO BMDC loaded with SIINFEKL + OVA antigen and matured for 48 h were mixed 1:1 with fresh media and no additional cytokines and used to treat fresh BMDC; Levels of il12a, il12b , and il6 transcripts were measured by RT-PCR after 2 days ( n = 3, biological repeats). Data are displayed as mean ± SEM. * p

    Journal: Frontiers in Immunology

    Article Title: AIMp1 Potentiates TH1 Polarization and Is Critical for Effective Antitumor and Antiviral Immunity

    doi: 10.3389/fimmu.2017.01801

    Figure Lengend Snippet: AIMp1 deficiency in dendritic cells impairs T H 1 polarization in vitro . (A) Wild-type (WT) or AIMp1 KO bone marrow-derived dendritic cell (BMDC) were matured for 48 h with cytokine cocktail, then cocultured with naive WT CD3 + T-cells at a 1:10 ratio. IFN-γ from 3-day coculture supernatants was measured by ELISA ( n = 7, biological repeats). (B) WT or AIMp1 KO BMDC were loaded with ovalbumin (OVA) antigen and matured for 48 h with cytokine cocktail, then cocultured with OT-II T-cells at a 1:20 ratio. IFN-γ from 3-day coculture supernatants was measured by ELISA ( n = 8, biological repeats). (C) WT T-cells were harvested and lysed for Western blotting analysis of pSTAT1(Y701)/STAT1, pSTAT4(Y693), T-bet, and β-actin after 6 h of coculture with BMDC as in (A) (Representative of three independent experiments). (D) WT or AIMp1 KO BMDC were treated with LPS, and levels of proinflammatory cytokines IL-12p70, IL-6, and IL-1β were measured by ELISA ( n = 7, technical repeats, pooled from two independent experiments). (E) WT or AIMp1 KO BMDC were treated with maturation cocktail or LPS and levels of CD86, CD40, and MHC II (I-A b ) were measured by flow cytometry (normalized and pooled from multiple independent experiments). (F) Conditioned supernatant from WT or AIMp1 KO BMDC loaded with SIINFEKL + OVA antigen and matured for 48 h were mixed 1:1 with fresh media and no additional cytokines and used to treat fresh BMDC; Levels of il12a, il12b , and il6 transcripts were measured by RT-PCR after 2 days ( n = 3, biological repeats). Data are displayed as mean ± SEM. * p

    Article Snippet: Primers: il6 (Mm00446190_m1, FAM), il12a (Mm00434165_m1, FAM), il12b (Mm01288989_m1, FAM), ifng (Mm01168134_m1, FAM), ifit1 (Mm00515153_m1, FAM), mx2 (Mm00488995_m1, FAM), oasl1 (Mm00455081_m1, FAM), irf7 (Mm00516793_g1, FAM), ifna1 (Mm03030145_gH, FAM), and 18s rRNA (4319413E, VIC).

    Techniques: In Vitro, Derivative Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction