lenti x qrt pcr titration kit  (TaKaRa)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    Lenti X qRT PCR Titration Kit
    Description:
    The Lenti X qRT PCR Titration Kit provides a fast and simple method for measuring lentivirus titer The kit uses a quick RNA purification step before quantifying the number of lentiviral genome copies using real time PCR
    Catalog Number:
    631235
    Price:
    None
    Size:
    200 Rxns
    Category:
    qRT PCR Titration kits Lentivirus Viral transduction Gene function
    Buy from Supplier


    Structured Review

    TaKaRa lenti x qrt pcr titration kit
    Transduction and RNAi efficiency of two human Nav1.1 lenti-shRNA clones. ( A ) Evaluating the transduction efficiency of the lentiviral particles were tested in HEK293T cells based on the expression of the tRFP reporter. Scale bars, 20 microns. ( B ) The RNAi efficiency of lenti-shRNA1 and lenti-shRNA2 relative to a non-silencing clone was determined by first transducing HEK293T cells with the lentiviral clones and next expressing the human Na v 1.1 cDNA via transfection. Na v 1.1 mRNAs were quantified with <t>qRT-PCR.</t> By ANOVA and post hoc Sidak’s multiple comparisons, p = 0.003 for the comparison between control and shRNA1; p = 0.002 for the comparison between control and shRNA2. DOI: http://dx.doi.org/10.7554/eLife.13073.034
    The Lenti X qRT PCR Titration Kit provides a fast and simple method for measuring lentivirus titer The kit uses a quick RNA purification step before quantifying the number of lentiviral genome copies using real time PCR
    https://www.bioz.com/result/lenti x qrt pcr titration kit/product/TaKaRa
    Average 99 stars, based on 140 article reviews
    Price from $9.99 to $1999.99
    lenti x qrt pcr titration kit - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) Product Images from "A deleterious Nav1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients"

    Article Title: A deleterious Nav1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients

    Journal: eLife

    doi: 10.7554/eLife.13073

    Transduction and RNAi efficiency of two human Nav1.1 lenti-shRNA clones. ( A ) Evaluating the transduction efficiency of the lentiviral particles were tested in HEK293T cells based on the expression of the tRFP reporter. Scale bars, 20 microns. ( B ) The RNAi efficiency of lenti-shRNA1 and lenti-shRNA2 relative to a non-silencing clone was determined by first transducing HEK293T cells with the lentiviral clones and next expressing the human Na v 1.1 cDNA via transfection. Na v 1.1 mRNAs were quantified with qRT-PCR. By ANOVA and post hoc Sidak’s multiple comparisons, p = 0.003 for the comparison between control and shRNA1; p = 0.002 for the comparison between control and shRNA2. DOI: http://dx.doi.org/10.7554/eLife.13073.034
    Figure Legend Snippet: Transduction and RNAi efficiency of two human Nav1.1 lenti-shRNA clones. ( A ) Evaluating the transduction efficiency of the lentiviral particles were tested in HEK293T cells based on the expression of the tRFP reporter. Scale bars, 20 microns. ( B ) The RNAi efficiency of lenti-shRNA1 and lenti-shRNA2 relative to a non-silencing clone was determined by first transducing HEK293T cells with the lentiviral clones and next expressing the human Na v 1.1 cDNA via transfection. Na v 1.1 mRNAs were quantified with qRT-PCR. By ANOVA and post hoc Sidak’s multiple comparisons, p = 0.003 for the comparison between control and shRNA1; p = 0.002 for the comparison between control and shRNA2. DOI: http://dx.doi.org/10.7554/eLife.13073.034

    Techniques Used: Transduction, shRNA, Clone Assay, Expressing, Transfection, Quantitative RT-PCR

    2) Product Images from "Characterization of miR-122-independent propagation of HCV"

    Article Title: Characterization of miR-122-independent propagation of HCV

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006374

    Propagation of HCV 122KO in 751-122KO cells. (A) HCV was inoculated into Huh7-122KO (#2), Huh7-122KO-cured (#3 or #5), or 751-122KO (#1, #2 or #3) cells, and the levels of intracellular HCV-RNA replication (top) and infectious titers in the culture supernatants (bottom) were determined by qRT-PCR and focus formation assay, respectively, at 72 hpi. (B) Infectious titer in the culture medium on serial passage of each 751-122KO cell clone. (C) HCV and HCV 122KO were inoculated into 751-122KO and Huh7.5.1 cells and the levels of intracellular HCV-RNA replication (top) and infectious titers in the culture supernatants (bottom) were determined at 72 hpi. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P
    Figure Legend Snippet: Propagation of HCV 122KO in 751-122KO cells. (A) HCV was inoculated into Huh7-122KO (#2), Huh7-122KO-cured (#3 or #5), or 751-122KO (#1, #2 or #3) cells, and the levels of intracellular HCV-RNA replication (top) and infectious titers in the culture supernatants (bottom) were determined by qRT-PCR and focus formation assay, respectively, at 72 hpi. (B) Infectious titer in the culture medium on serial passage of each 751-122KO cell clone. (C) HCV and HCV 122KO were inoculated into 751-122KO and Huh7.5.1 cells and the levels of intracellular HCV-RNA replication (top) and infectious titers in the culture supernatants (bottom) were determined at 72 hpi. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P

    Techniques Used: Quantitative RT-PCR, Tube Formation Assay, Standard Deviation

    G28A mutants can replicate efficiently in an Ago2-independent manner. (A) Intracellular HCV-RNA levels (left panel) and infectious titers in the culture supernatants (right panel) of Huh7-122KO and Huh7-122KOR cells infected with either HCV or HCV 122KO in the presence of either control-LNA or LNA-miR-122 were determined at 72 hpi. (B) Ago2 complexes in 751-122KO and Huh7.5.1 cells infected with HCV were immunoprecipitated by either anti-IgG or anti-Ago2 mouse antibody at 12 dpi. Levels of Ago2 and HCV-RNA in the precipitates were determined by immunoblotting and qRT-PCR, respectively. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P
    Figure Legend Snippet: G28A mutants can replicate efficiently in an Ago2-independent manner. (A) Intracellular HCV-RNA levels (left panel) and infectious titers in the culture supernatants (right panel) of Huh7-122KO and Huh7-122KOR cells infected with either HCV or HCV 122KO in the presence of either control-LNA or LNA-miR-122 were determined at 72 hpi. (B) Ago2 complexes in 751-122KO and Huh7.5.1 cells infected with HCV were immunoprecipitated by either anti-IgG or anti-Ago2 mouse antibody at 12 dpi. Levels of Ago2 and HCV-RNA in the precipitates were determined by immunoblotting and qRT-PCR, respectively. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P

    Techniques Used: Infection, Immunoprecipitation, Quantitative RT-PCR, Standard Deviation

    Establishment of replicon cells derived from Huh7-122KO cells. (A) Subgenomic HCV replicon RNA was electroporated into Huh7-122KO and Huh7-122KOR cells, or into Huh7-122KO cells together with control- or miR-122-mimic, and G418-resistant colonies were stained with crystal violet at 21 days post-transduction. (B) Expression of miR-122 in Huh7-122KO cells electroporated with either control-mimic or miR-122-mimic at 72 h post-electroporation. Relative expression of miR-122 was determined by qRT-PCR by using U6 snRNA as an internal control. (C) Each of the three clones derived from each type of replicon cells was subjected to qRT-PCR after extraction of total RNA (top) and to immunoblotting by using anti-NS5A and β-actin (middle). The relative expression of miR-122 was determined by qRT-PCR by using U6 snRNA as an internal control (bottom). (D) Intracellular HCV-RNA replication in Huh7-122KO-SGR cells (#1, #3, #5) and Huh7-122KOR-SGR cells (#1, #5, #6) in the presence of 20 nM of either LNA-control or LNA-miR122 was determined by qRT-PCR. (E) The Ago2 complex was immunoprecipitated from Huh7-122KO-SGR#1 and Huh7-122KOR-SGR#1 cells by using either anti-IgG or anti-Ago2 mouse antibody. The HCV-RNA associated with Ago2 was determined by qRT-PCR and Ago2 was detected by immunoblotting. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P
    Figure Legend Snippet: Establishment of replicon cells derived from Huh7-122KO cells. (A) Subgenomic HCV replicon RNA was electroporated into Huh7-122KO and Huh7-122KOR cells, or into Huh7-122KO cells together with control- or miR-122-mimic, and G418-resistant colonies were stained with crystal violet at 21 days post-transduction. (B) Expression of miR-122 in Huh7-122KO cells electroporated with either control-mimic or miR-122-mimic at 72 h post-electroporation. Relative expression of miR-122 was determined by qRT-PCR by using U6 snRNA as an internal control. (C) Each of the three clones derived from each type of replicon cells was subjected to qRT-PCR after extraction of total RNA (top) and to immunoblotting by using anti-NS5A and β-actin (middle). The relative expression of miR-122 was determined by qRT-PCR by using U6 snRNA as an internal control (bottom). (D) Intracellular HCV-RNA replication in Huh7-122KO-SGR cells (#1, #3, #5) and Huh7-122KOR-SGR cells (#1, #5, #6) in the presence of 20 nM of either LNA-control or LNA-miR122 was determined by qRT-PCR. (E) The Ago2 complex was immunoprecipitated from Huh7-122KO-SGR#1 and Huh7-122KOR-SGR#1 cells by using either anti-IgG or anti-Ago2 mouse antibody. The HCV-RNA associated with Ago2 was determined by qRT-PCR and Ago2 was detected by immunoblotting. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P

    Techniques Used: Derivative Assay, Staining, Transduction, Expressing, Electroporation, Quantitative RT-PCR, Clone Assay, Immunoprecipitation, Standard Deviation

    miR-122-independent HCV replication in Huh7-122KO cells. (A) HCV was inoculated into Huh7-122KO and Huh7-122KOR cells at an MOI of 3, and intracellular HCV-RNA levels (left panel) and infectious titers in the culture supernatants (right panel) were determined by qRT-PCR and focus formation assay, respectively. (B) HCV-RNA replication was inhibited by the treatment with IFNα, BILN, BMS790052, PSI7977 and anti-CD81 antibody. (C) HCV replication in Huh7-122KO cells was resistant to treatment with an miR-122 inhibitor, LNA (HCV-RNA replication: left; infectious titer: right). Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P
    Figure Legend Snippet: miR-122-independent HCV replication in Huh7-122KO cells. (A) HCV was inoculated into Huh7-122KO and Huh7-122KOR cells at an MOI of 3, and intracellular HCV-RNA levels (left panel) and infectious titers in the culture supernatants (right panel) were determined by qRT-PCR and focus formation assay, respectively. (B) HCV-RNA replication was inhibited by the treatment with IFNα, BILN, BMS790052, PSI7977 and anti-CD81 antibody. (C) HCV replication in Huh7-122KO cells was resistant to treatment with an miR-122 inhibitor, LNA (HCV-RNA replication: left; infectious titer: right). Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P

    Techniques Used: Quantitative RT-PCR, Tube Formation Assay, Standard Deviation

    3) Product Images from "High-fidelity CRISPR/Cas9- based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis"

    Article Title: High-fidelity CRISPR/Cas9- based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05766-5

    Targeted Rasal1 promoter demethylation by dCas9/dHFCas9-TET3CD- Rasal1 -sgRNA fusion protein ameliorates kidney fibrosis. a Schematic showing the parenchymal injection of lentiviral particles containing dCas9/dHFCas9-TET3CD fusion protein into UUO-challenged kidneys. b Schedule of UUO mouse surgery, lentivirus injection, and analysis. c qRT-PCR results showing that Rasal1 mRNA expression was significantly induced in UUO-challenged kidneys transduced with dCas9/dHFCas9-TET3CD- Rasal1 -sgRNA, but not in UUO-challenged kidneys transduced with control dCas9/dHFCas9-TET3CD- LacZ- sgRNA. There is no significant difference between dCas9-TET3CD and dHFCas9-TET3CD constructs. Results were normalized to reference gene Gapdh (expression is presented as mean value; error bars represent S.D., n ≥ 5 in each group, # not significant, *** p
    Figure Legend Snippet: Targeted Rasal1 promoter demethylation by dCas9/dHFCas9-TET3CD- Rasal1 -sgRNA fusion protein ameliorates kidney fibrosis. a Schematic showing the parenchymal injection of lentiviral particles containing dCas9/dHFCas9-TET3CD fusion protein into UUO-challenged kidneys. b Schedule of UUO mouse surgery, lentivirus injection, and analysis. c qRT-PCR results showing that Rasal1 mRNA expression was significantly induced in UUO-challenged kidneys transduced with dCas9/dHFCas9-TET3CD- Rasal1 -sgRNA, but not in UUO-challenged kidneys transduced with control dCas9/dHFCas9-TET3CD- LacZ- sgRNA. There is no significant difference between dCas9-TET3CD and dHFCas9-TET3CD constructs. Results were normalized to reference gene Gapdh (expression is presented as mean value; error bars represent S.D., n ≥ 5 in each group, # not significant, *** p

    Techniques Used: Injection, Quantitative RT-PCR, Expressing, Transduction, Construct

    Induction of Klotho promoter hydroxymethylation by dHFCas9-TET3CD- Klotho -sgRNA in tubular epithelial cells ameliorates kidney fibrosis. a Schematic shows retrograde ureter injection of lentiviral particles containing dHFCas9-TET3CD- Klotho -sgRNA into UUO-operated kidneys. b Schedule of UUO mouse surgery, lentivirus injection, and analysis. c qRT-PCR results showing that Kl mRNA expression was significantly induced in UUO-operated kidneys transduced with dHFCas9-TET3CD- Kl -sgRNA but not in UUO-challenged kidneys transduced with LacZ- sgRNA control. Results were normalized to reference gene Gapdh (expression is presented as mean value, error bars represent S.E.M. , n = 6 in each group, *** p
    Figure Legend Snippet: Induction of Klotho promoter hydroxymethylation by dHFCas9-TET3CD- Klotho -sgRNA in tubular epithelial cells ameliorates kidney fibrosis. a Schematic shows retrograde ureter injection of lentiviral particles containing dHFCas9-TET3CD- Klotho -sgRNA into UUO-operated kidneys. b Schedule of UUO mouse surgery, lentivirus injection, and analysis. c qRT-PCR results showing that Kl mRNA expression was significantly induced in UUO-operated kidneys transduced with dHFCas9-TET3CD- Kl -sgRNA but not in UUO-challenged kidneys transduced with LacZ- sgRNA control. Results were normalized to reference gene Gapdh (expression is presented as mean value, error bars represent S.E.M. , n = 6 in each group, *** p

    Techniques Used: Injection, Quantitative RT-PCR, Expressing, Transduction

    Rasal1 gene disruption results in aggravated fibrosis level in the UUO model. a Schematic of knockout-first strategy for Rasal1 gene. The gene trapping LacZ cassette is alternatively spliced with exon 2 mediated by a splicing acceptor (SA). A promoter-driven Neomycin cassette is inserted after LacZ . The targeted exon 3 and 4 are flanked by loxP sites. Black arrows indicate the location of genotyping primers. b qRT-PCR analysis shows the Rasal1 mRNA expression in homozygous mice are significantly reduced as compared with wild-type mice, while there is no significant reduction in heterozygous mice. The data are presented as mean value, error bars represent S.D., n.s. not significant, *** p
    Figure Legend Snippet: Rasal1 gene disruption results in aggravated fibrosis level in the UUO model. a Schematic of knockout-first strategy for Rasal1 gene. The gene trapping LacZ cassette is alternatively spliced with exon 2 mediated by a splicing acceptor (SA). A promoter-driven Neomycin cassette is inserted after LacZ . The targeted exon 3 and 4 are flanked by loxP sites. Black arrows indicate the location of genotyping primers. b qRT-PCR analysis shows the Rasal1 mRNA expression in homozygous mice are significantly reduced as compared with wild-type mice, while there is no significant reduction in heterozygous mice. The data are presented as mean value, error bars represent S.D., n.s. not significant, *** p

    Techniques Used: Knock-Out, Quantitative RT-PCR, Expressing, Mouse Assay

    4) Product Images from "Partnership between epigenetic reader BRD4 and transcription factor CEBPD"

    Article Title: Partnership between epigenetic reader BRD4 and transcription factor CEBPD

    Journal: bioRxiv

    doi: 10.1101/2020.03.27.012674

    Silencing BRD4 reduces CEBPD mRNA and protein in SMCs MOVAS cells were transfected with scrambled or specific siRNA for 24h, cultured with fresh medium (no RNAi Max) for another 24-48h, and then harvested for qRT-PCR (A) or Western blot (B and C) analysis, respectively. Quantification: Readings from triplicate qRT-PCR reactions were normalized (to GAPDH) and averaged. The average values from 3 independent repeat experiments were then averaged again to calculate mean ± SEM (n =3). Densitometry of Western blots from independent repeat experiments was normalized (to β-actin, similar band intensity among blots) and then averaged to calculate mean ± SEM (n =3). Statistics: One-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test; *P
    Figure Legend Snippet: Silencing BRD4 reduces CEBPD mRNA and protein in SMCs MOVAS cells were transfected with scrambled or specific siRNA for 24h, cultured with fresh medium (no RNAi Max) for another 24-48h, and then harvested for qRT-PCR (A) or Western blot (B and C) analysis, respectively. Quantification: Readings from triplicate qRT-PCR reactions were normalized (to GAPDH) and averaged. The average values from 3 independent repeat experiments were then averaged again to calculate mean ± SEM (n =3). Densitometry of Western blots from independent repeat experiments was normalized (to β-actin, similar band intensity among blots) and then averaged to calculate mean ± SEM (n =3). Statistics: One-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test; *P

    Techniques Used: Transfection, Cell Culture, Quantitative RT-PCR, Western Blot

    Preferential regulation of PDGFRα over PDGFRβ in SMCs A - D . Effect of JQ1 pretreatment on TNFα-stimulated upregulation of CEBPD and PDGFRα. A shows concentration-dependent effect of JQ1 on reducing CEBPD mRNA levels. Representative Western blots are shown in C. E and F . CEBPD gain of function. Representative Western blots are shown in E. G and H . CEBPD loss of function. Representative Western blots are shown in G. MOVAS cells for stable expression of HA-tagged empty vector (EV) or HA-tagged CEBPD (OE-CEBPD) were starved in basal medium (0.5% FBS) for 24h, pretreated with vehicle or JQ1 (1 μM) for 2h, and then treated with TNFα (final 20 ng/ml) for 24h prior to harvest for qRT-PCR (mRNA) or Western blot (protein) analysis. For CEBPD silencing, MOVAS cells were transfected with siRNA for 24h and cultured in fresh starvation medium (0.5% FBS) for another 24h prior to TNFα treatment. Quantification: Readings from triplicate qRT-PCR reactions were normalized (to GAPDH) and averaged. The average values from at least 3 independent repeat experiments were then averaged again to calculate mean ± SEM (n =3-5). Densitometry of Western blots from independent repeat experiments was normalized (to β-actin, similar band intensity among blots) and then averaged to calculate mean ± SEM (n =4). Statistics: One-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test; *P
    Figure Legend Snippet: Preferential regulation of PDGFRα over PDGFRβ in SMCs A - D . Effect of JQ1 pretreatment on TNFα-stimulated upregulation of CEBPD and PDGFRα. A shows concentration-dependent effect of JQ1 on reducing CEBPD mRNA levels. Representative Western blots are shown in C. E and F . CEBPD gain of function. Representative Western blots are shown in E. G and H . CEBPD loss of function. Representative Western blots are shown in G. MOVAS cells for stable expression of HA-tagged empty vector (EV) or HA-tagged CEBPD (OE-CEBPD) were starved in basal medium (0.5% FBS) for 24h, pretreated with vehicle or JQ1 (1 μM) for 2h, and then treated with TNFα (final 20 ng/ml) for 24h prior to harvest for qRT-PCR (mRNA) or Western blot (protein) analysis. For CEBPD silencing, MOVAS cells were transfected with siRNA for 24h and cultured in fresh starvation medium (0.5% FBS) for another 24h prior to TNFα treatment. Quantification: Readings from triplicate qRT-PCR reactions were normalized (to GAPDH) and averaged. The average values from at least 3 independent repeat experiments were then averaged again to calculate mean ± SEM (n =3-5). Densitometry of Western blots from independent repeat experiments was normalized (to β-actin, similar band intensity among blots) and then averaged to calculate mean ± SEM (n =4). Statistics: One-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test; *P

    Techniques Used: Concentration Assay, Western Blot, Expressing, Plasmid Preparation, Quantitative RT-PCR, Transfection, Cell Culture

    Chromatin immunoprecipitation of Cebpd promoter DNA with CEBPD or endogenous BRD4 A-D . ChIP against HA-tagged CEBPD in SMCs. E-F . ChIP against endogenous BRD4 in SMCs (E) or HEK293 cells (F). MOVAS cells for stable expression of HA-tagged empty vector (EV) or HA-tagged CEBPD (OE-CEBPD) were starved in basal medium (0.5% FBS) for 24h, pretreated with vehicle or bromodomain blocker JQ1 (1 μM) for 2h, and then treated with TNFα (final 20 ng/ml) for 24h prior to harvest for ChIP-qPCR analysis. Quantification: Readings from triplicate qRT-PCR reactions were normalized (to GAPDH) and averaged. The average values from at least 3 independent repeat experiments were then averaged again to calculate mean ± SEM (n =3-5). Statistics: One-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test; *P
    Figure Legend Snippet: Chromatin immunoprecipitation of Cebpd promoter DNA with CEBPD or endogenous BRD4 A-D . ChIP against HA-tagged CEBPD in SMCs. E-F . ChIP against endogenous BRD4 in SMCs (E) or HEK293 cells (F). MOVAS cells for stable expression of HA-tagged empty vector (EV) or HA-tagged CEBPD (OE-CEBPD) were starved in basal medium (0.5% FBS) for 24h, pretreated with vehicle or bromodomain blocker JQ1 (1 μM) for 2h, and then treated with TNFα (final 20 ng/ml) for 24h prior to harvest for ChIP-qPCR analysis. Quantification: Readings from triplicate qRT-PCR reactions were normalized (to GAPDH) and averaged. The average values from at least 3 independent repeat experiments were then averaged again to calculate mean ± SEM (n =3-5). Statistics: One-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test; *P

    Techniques Used: Chromatin Immunoprecipitation, Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Role of CEBPD in the inflammatory SMC state transition A and B . CEBPD gain of function. A shows CEBPD overexpression. C and D . CEBPD loss of function. C indicates effective CEBPD silencing. E. Treatment of SMCs with TNFα upregulates BRD4 protein (Movas cell line). F. Effect of pretreatment with bromodomain blocker JQ1. MOVAS cells for stable expression of HA-tagged empty vector (EV) or HA-tagged CEBPD (OE-CEBPD) were starved in basal medium (0.5% FBS) for 24h, pretreated with vehicle or JQ1 (1 μM) for 2h, and then treated with TNFα (final 20 ng/ml) for 24h prior to harvest for qRT-PCR (mRNA) or Western blot (protein) analysis. For CEBPD silencing, MOVAS cells were transfected with siRNA for 24h and cultured in fresh starvation medium (0.5% FBS) for another 24h prior to TNFα treatment. Quantification: Readings from triplicate qRT-PCR reactions were normalized (to GAPDH) and averaged. The average values from at least 3 independent repeat experiments were then averaged again to calculate mean ± SEM (n =3-5). Statistics: One-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test; *P
    Figure Legend Snippet: Role of CEBPD in the inflammatory SMC state transition A and B . CEBPD gain of function. A shows CEBPD overexpression. C and D . CEBPD loss of function. C indicates effective CEBPD silencing. E. Treatment of SMCs with TNFα upregulates BRD4 protein (Movas cell line). F. Effect of pretreatment with bromodomain blocker JQ1. MOVAS cells for stable expression of HA-tagged empty vector (EV) or HA-tagged CEBPD (OE-CEBPD) were starved in basal medium (0.5% FBS) for 24h, pretreated with vehicle or JQ1 (1 μM) for 2h, and then treated with TNFα (final 20 ng/ml) for 24h prior to harvest for qRT-PCR (mRNA) or Western blot (protein) analysis. For CEBPD silencing, MOVAS cells were transfected with siRNA for 24h and cultured in fresh starvation medium (0.5% FBS) for another 24h prior to TNFα treatment. Quantification: Readings from triplicate qRT-PCR reactions were normalized (to GAPDH) and averaged. The average values from at least 3 independent repeat experiments were then averaged again to calculate mean ± SEM (n =3-5). Statistics: One-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test; *P

    Techniques Used: Over Expression, Expressing, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Transfection, Cell Culture

    5) Product Images from "Photoreceptor protection via blockade of BET epigenetic readers in a murine model of inherited retinal degeneration"

    Article Title: Photoreceptor protection via blockade of BET epigenetic readers in a murine model of inherited retinal degeneration

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-016-0775-4

    JQ1 pre-treatment inhibits N9 microglial cell inflammation, proliferation, and migration. N9 microglial cells were pre-incubated with vehicle (DMSO), JQ1 (0.5 μM), RVX208 (30 μM), or Olinone (30 μM) for 12 h followed by stimulation with LPS (1 μg/ml) for another 2 h, and then subjected to qRT-PCR ( a ) for determination of inflammatory cytokine expression. Proliferation assay ( b , BrdU ELISA) and migration assay ( c , Transwell) were performed after incubation for additional 24 h. Quantification: mean ± SEM; n = 3 experiments; * P
    Figure Legend Snippet: JQ1 pre-treatment inhibits N9 microglial cell inflammation, proliferation, and migration. N9 microglial cells were pre-incubated with vehicle (DMSO), JQ1 (0.5 μM), RVX208 (30 μM), or Olinone (30 μM) for 12 h followed by stimulation with LPS (1 μg/ml) for another 2 h, and then subjected to qRT-PCR ( a ) for determination of inflammatory cytokine expression. Proliferation assay ( b , BrdU ELISA) and migration assay ( c , Transwell) were performed after incubation for additional 24 h. Quantification: mean ± SEM; n = 3 experiments; * P

    Techniques Used: Migration, Incubation, Quantitative RT-PCR, Expressing, Proliferation Assay, Enzyme-linked Immunosorbent Assay

    BET2 knockdown in N9 microglial cells inhibits inflammatory cytokine expression. N9 cells were infected with lentivirus expressing BET2 or BET4 siRNAs for 3 days and then GFP-positive (infected) cells were purified by flow sorting and cultured for 2–3 days. The cells were either used for Western blotting or stimulated with LPS (1 μg/ml) for 2 h and then subjected to qRT-PCR. a , b Western blots showing BET protein knockdown. Quantification: mean ± SEM; n = 3 experiments; * P
    Figure Legend Snippet: BET2 knockdown in N9 microglial cells inhibits inflammatory cytokine expression. N9 cells were infected with lentivirus expressing BET2 or BET4 siRNAs for 3 days and then GFP-positive (infected) cells were purified by flow sorting and cultured for 2–3 days. The cells were either used for Western blotting or stimulated with LPS (1 μg/ml) for 2 h and then subjected to qRT-PCR. a , b Western blots showing BET protein knockdown. Quantification: mean ± SEM; n = 3 experiments; * P

    Techniques Used: Expressing, Infection, Purification, Flow Cytometry, Cell Culture, Western Blot, Quantitative RT-PCR

    JQ1 treatment inhibits microglial expression of inflammatory cytokines in the rd10 retina. Intravitreal injection of JQ1 (or vehicle) was performed at PN14, as described in Fig. 1 . Retinas were collected at PN24. a Homogenates were prepared from retinas (3 groups, total 12 mice) and used for qRT-PCR. Quantification: normalization to GAPDH mRNA; mean ± SEM; n = 3 independent homogenation/qRT-PCR experiments; * P
    Figure Legend Snippet: JQ1 treatment inhibits microglial expression of inflammatory cytokines in the rd10 retina. Intravitreal injection of JQ1 (or vehicle) was performed at PN14, as described in Fig. 1 . Retinas were collected at PN24. a Homogenates were prepared from retinas (3 groups, total 12 mice) and used for qRT-PCR. Quantification: normalization to GAPDH mRNA; mean ± SEM; n = 3 independent homogenation/qRT-PCR experiments; * P

    Techniques Used: Expressing, Injection, Mouse Assay, Quantitative RT-PCR

    6) Product Images from "Selective targeting of an oncogenic KRAS mutant allele by CRISPR/Cas9 induces efficient tumor regression"

    Article Title: Selective targeting of an oncogenic KRAS mutant allele by CRISPR/Cas9 induces efficient tumor regression

    Journal: bioRxiv

    doi: 10.1101/807578

    dCas9-KRAB mRNA-regulating system downregulated G12S transcription and inhibited tumor cell proliferation. a, b No off-target indels were detectably induced by CRISPR/Cas9 gene-cutting system at fourteen homologous sites that different from the on-target sites by up to 4 nt in the human genome. PAM sequences are shown in red and mismatched nucleotides are shown in green. On: on-target site. OT: off-target site. Cleavage position within the 20-bp target sequences is indicated by red arrow. Error bar indicates S.E.M. (n=3 to 4). c Diagram of knocking down KRAS G12S mutant allele specifically by dCas9-KRAB system. Blue strands: spacer; green strands: PAM sequence; red strands and star: single-nucleotide missense mutations. d qRT-PCR analysis of KRAS G12S mRNA expression. Error bars represent S.E.M. 0.01
    Figure Legend Snippet: dCas9-KRAB mRNA-regulating system downregulated G12S transcription and inhibited tumor cell proliferation. a, b No off-target indels were detectably induced by CRISPR/Cas9 gene-cutting system at fourteen homologous sites that different from the on-target sites by up to 4 nt in the human genome. PAM sequences are shown in red and mismatched nucleotides are shown in green. On: on-target site. OT: off-target site. Cleavage position within the 20-bp target sequences is indicated by red arrow. Error bar indicates S.E.M. (n=3 to 4). c Diagram of knocking down KRAS G12S mutant allele specifically by dCas9-KRAB system. Blue strands: spacer; green strands: PAM sequence; red strands and star: single-nucleotide missense mutations. d qRT-PCR analysis of KRAS G12S mRNA expression. Error bars represent S.E.M. 0.01

    Techniques Used: CRISPR, Mutagenesis, Sequencing, Quantitative RT-PCR, Expressing

    7) Product Images from "The role of Twist1 in mutant huntingtin–induced transcriptional alterations and neurotoxicity"

    Article Title: The role of Twist1 in mutant huntingtin–induced transcriptional alterations and neurotoxicity

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA117.001211

    Twist1 knockdown protects neurons from mutant Htt–induced toxicity in primary cortical neurons. A , DIV 5 cortical neurons were transduced with Htt- or control (vector)-expressing lentivirus. Five days later, RNA was prepared and subjected to qRT-PCR using β- actin and Hprt as reference genes. Twist1 was up-regulated by Htt-72Q expression (ANOVA; *, p = 0.0083 compared with Htt-25Q, n = 3). B , qRT-PCR was performed using RNA prepared as in A. Pou4f1 was up-regulated by Htt-72Q expression (ANOVA; *, p
    Figure Legend Snippet: Twist1 knockdown protects neurons from mutant Htt–induced toxicity in primary cortical neurons. A , DIV 5 cortical neurons were transduced with Htt- or control (vector)-expressing lentivirus. Five days later, RNA was prepared and subjected to qRT-PCR using β- actin and Hprt as reference genes. Twist1 was up-regulated by Htt-72Q expression (ANOVA; *, p = 0.0083 compared with Htt-25Q, n = 3). B , qRT-PCR was performed using RNA prepared as in A. Pou4f1 was up-regulated by Htt-72Q expression (ANOVA; *, p

    Techniques Used: Mutagenesis, Transduction, Plasmid Preparation, Expressing, Quantitative RT-PCR

    Increased Twist1 gene expression in mouse models of HD. A , qRT-PCR analysis was performed using RNA prepared from 6-week-old R6/2 HD and control WT mouse forebrains ( n = 6 (3 males, 3 females)/group). β- actin and Hprt were used as reference genes. Twist1 RNA was up-regulated in R6/2 compared with WT mice (unpaired t tests; *, p = 0.0043). Egr3 and Bdnf (exon IX, protein coding) RNAs were down-regulated in R6/2 compared with WT mice (unpaired t tests; *, p = 0.0013 for Egr3 ; *, p = 0.022 for Bdnf ). B and C , qRT-PCR analysis was performed using RNA prepared from 33-week-old zQ175 heterozygous ( Het ), homozygous ( Homo ), and control WT mouse cortex ( B ) and striatum ( C ) ( n = 6 (3 males and 3 females) for zQ175 heterozygous and homozygous; n = 7 (4 males and 3 females) for WT). Twist1 RNA was up-regulated in the cortex and striatum of zQ175 mice compared with those of WT (ANOVA; *, p = 0.0001 in B ; *, p = 0.0007 in C ). Egr3 and Bdnf (exon IX, protein coding) RNAs were down-regulated in the cortex of zQ175 compared with WT mice (ANOVA; *, p = 0.042; #, p = 0.011 for Egr3 ; *, p = 0.047 for Bdnf ) ( B ). Drd2 mRNA was down-regulated in zQ175 striatum compared with WT control (ANOVA; *, p = 0.014) ( C ). A trend toward decreased expression of Ppp1r1b in zQ175 mice (zQ175 homozygous compared with WT; p = 0.063) ( C ). D , immunoblot analysis ( IB ) was performed using forebrain lysates prepared from 9-week-old R6/2 HD and control WT mice ( n = 6 (3 males, 3 females)/group). Twist1 band intensity was quantified by densitometry (ImageJ) and normalized to that of GAPDH. Twist1 protein expression was increased in R6/2 compared with WT mice (unpaired t tests; *, p
    Figure Legend Snippet: Increased Twist1 gene expression in mouse models of HD. A , qRT-PCR analysis was performed using RNA prepared from 6-week-old R6/2 HD and control WT mouse forebrains ( n = 6 (3 males, 3 females)/group). β- actin and Hprt were used as reference genes. Twist1 RNA was up-regulated in R6/2 compared with WT mice (unpaired t tests; *, p = 0.0043). Egr3 and Bdnf (exon IX, protein coding) RNAs were down-regulated in R6/2 compared with WT mice (unpaired t tests; *, p = 0.0013 for Egr3 ; *, p = 0.022 for Bdnf ). B and C , qRT-PCR analysis was performed using RNA prepared from 33-week-old zQ175 heterozygous ( Het ), homozygous ( Homo ), and control WT mouse cortex ( B ) and striatum ( C ) ( n = 6 (3 males and 3 females) for zQ175 heterozygous and homozygous; n = 7 (4 males and 3 females) for WT). Twist1 RNA was up-regulated in the cortex and striatum of zQ175 mice compared with those of WT (ANOVA; *, p = 0.0001 in B ; *, p = 0.0007 in C ). Egr3 and Bdnf (exon IX, protein coding) RNAs were down-regulated in the cortex of zQ175 compared with WT mice (ANOVA; *, p = 0.042; #, p = 0.011 for Egr3 ; *, p = 0.047 for Bdnf ) ( B ). Drd2 mRNA was down-regulated in zQ175 striatum compared with WT control (ANOVA; *, p = 0.014) ( C ). A trend toward decreased expression of Ppp1r1b in zQ175 mice (zQ175 homozygous compared with WT; p = 0.063) ( C ). D , immunoblot analysis ( IB ) was performed using forebrain lysates prepared from 9-week-old R6/2 HD and control WT mice ( n = 6 (3 males, 3 females)/group). Twist1 band intensity was quantified by densitometry (ImageJ) and normalized to that of GAPDH. Twist1 protein expression was increased in R6/2 compared with WT mice (unpaired t tests; *, p

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

    8) Product Images from "Arabidopsis ERF4 and MYB52 Transcription Factors Play Antagonistic Roles in Regulating Homogalacturonan De-methylesterification in Seed Coat Mucilage"

    Article Title: Arabidopsis ERF4 and MYB52 Transcription Factors Play Antagonistic Roles in Regulating Homogalacturonan De-methylesterification in Seed Coat Mucilage

    Journal: bioRxiv

    doi: 10.1101/2020.01.21.914390

    Expression analysis of ERF4 and the erf4 mutants present a mucilage phenotype under vigorous shaking conditions. (A) Relative expression of ERF4 in developing siliques at 4, 7, 9, 11 and 13 DPA and major Arabidopsis organs with qRT-PCR analysis. Gene expressions were measured using the reference gene ACTIN2 . Values correspond to means ± SD of three replicates for each sample. The expression level at 4 DPA was set as 1. (B) Co-expression network of ERF4 with known mucilage genes based on GeneMANIA. GL2 , Glabra2; LUH/MUM1 , Mucilage-modified 1; MYB52 , MYB domain protein 52; STK , SEEDSTICK; FLY1 , Flying Saucer 1; CSLA2 , Cellulose synthase-like A2; CESA5 , Cellulose synthase 5; SBT1.7 , Subtilisin-like serine protease 1.7; MUCI10 , Mucilage-related 10; PER36 , Peroxidase 36; PMEI6, 13, 14, 15 , Pectin methylesterase inhibitor 6, 13, 14, 15; PME58 , Pectin methylesterase 58. Co-expression was indicated with purple lines, while proteins share similar domains with yellow green. (C) Gene model of ERF4 with T-DNA insertion sites. The black line shows coding region (CDS), while gray lines represent non-coding upstream and downstream regions. (D) Semi-quantitative RT-PCR was performed on cDNA from siliques of wild-type and erf4 mutants with primers flanking the full-length CDS. ACTIN2 was amplified as the loading control. (E) RR staining of adherent mucilage (AM) for wild-type and erf4 seeds. Mature dry seeds were shaking in distilled water with 0.01% RR at 200 rpm for 1 h before being photographed with an optical microscope. Bars = 500 μm and 100 μm, respectively. (F) Area of AM layers. Values are average area ± SD of at least 20 seeds which were measured with the optical microscope from the top to the bottom of the red halo (white lines in (E) ). (G) Comparison of total sugar contents in AM layers and whole mucilage of wild-type and erf4 seeds extracted by shaking at 200 rpm for 1 h in water or EDTA. *, P
    Figure Legend Snippet: Expression analysis of ERF4 and the erf4 mutants present a mucilage phenotype under vigorous shaking conditions. (A) Relative expression of ERF4 in developing siliques at 4, 7, 9, 11 and 13 DPA and major Arabidopsis organs with qRT-PCR analysis. Gene expressions were measured using the reference gene ACTIN2 . Values correspond to means ± SD of three replicates for each sample. The expression level at 4 DPA was set as 1. (B) Co-expression network of ERF4 with known mucilage genes based on GeneMANIA. GL2 , Glabra2; LUH/MUM1 , Mucilage-modified 1; MYB52 , MYB domain protein 52; STK , SEEDSTICK; FLY1 , Flying Saucer 1; CSLA2 , Cellulose synthase-like A2; CESA5 , Cellulose synthase 5; SBT1.7 , Subtilisin-like serine protease 1.7; MUCI10 , Mucilage-related 10; PER36 , Peroxidase 36; PMEI6, 13, 14, 15 , Pectin methylesterase inhibitor 6, 13, 14, 15; PME58 , Pectin methylesterase 58. Co-expression was indicated with purple lines, while proteins share similar domains with yellow green. (C) Gene model of ERF4 with T-DNA insertion sites. The black line shows coding region (CDS), while gray lines represent non-coding upstream and downstream regions. (D) Semi-quantitative RT-PCR was performed on cDNA from siliques of wild-type and erf4 mutants with primers flanking the full-length CDS. ACTIN2 was amplified as the loading control. (E) RR staining of adherent mucilage (AM) for wild-type and erf4 seeds. Mature dry seeds were shaking in distilled water with 0.01% RR at 200 rpm for 1 h before being photographed with an optical microscope. Bars = 500 μm and 100 μm, respectively. (F) Area of AM layers. Values are average area ± SD of at least 20 seeds which were measured with the optical microscope from the top to the bottom of the red halo (white lines in (E) ). (G) Comparison of total sugar contents in AM layers and whole mucilage of wild-type and erf4 seeds extracted by shaking at 200 rpm for 1 h in water or EDTA. *, P

    Techniques Used: Expressing, Quantitative RT-PCR, Modification, Amplification, Staining, Microscopy

    9) Product Images from "Various miRNAs are involved in efficient HCV replication"

    Article Title: Various miRNAs are involved in efficient HCV replication

    Journal: bioRxiv

    doi: 10.1101/2020.01.10.901488

    miR-25-5p and miR-4730 can enhance HCV-RNA replication via non-miR-122-type interaction. (A) Diagrams of possible interaction between HCV 5’UTR and synthetic miRNAs with 7nt-match and 8nt-match. Gray-shaded area and framed by dashed line in gray indicate possible interaction region and G-U wobble pair, respectively. Nucleotides in red are important region for the enhancement by miR-122 shown in Fig 1D . (B) Sequence alignment of miR-122 and synthetic miRNAs with 7nt-match and 8nt-match. Mismatching nucleotides were shown in red. (C) Intracellular HCV-RNA levels of 751-122KO cells infected with JFH1 in the presence of mimic control, miR-122, miR-122-mt6 and synthetic miRNAs with 7nt-match shown in Fig 3A . (D) Intracellular HCV-RNA levels of 751-122KO cells infected with JFH1 in the presence of mimic control, miR-122, miR-122-mt6, miR-25-5p, miR-652-5p, miR-1236-5p and miR-4730 were determined at 72 hpi by qRT-PCR. The data are representative of three independent experiments. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (**P
    Figure Legend Snippet: miR-25-5p and miR-4730 can enhance HCV-RNA replication via non-miR-122-type interaction. (A) Diagrams of possible interaction between HCV 5’UTR and synthetic miRNAs with 7nt-match and 8nt-match. Gray-shaded area and framed by dashed line in gray indicate possible interaction region and G-U wobble pair, respectively. Nucleotides in red are important region for the enhancement by miR-122 shown in Fig 1D . (B) Sequence alignment of miR-122 and synthetic miRNAs with 7nt-match and 8nt-match. Mismatching nucleotides were shown in red. (C) Intracellular HCV-RNA levels of 751-122KO cells infected with JFH1 in the presence of mimic control, miR-122, miR-122-mt6 and synthetic miRNAs with 7nt-match shown in Fig 3A . (D) Intracellular HCV-RNA levels of 751-122KO cells infected with JFH1 in the presence of mimic control, miR-122, miR-122-mt6, miR-25-5p, miR-652-5p, miR-1236-5p and miR-4730 were determined at 72 hpi by qRT-PCR. The data are representative of three independent experiments. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (**P

    Techniques Used: Sequencing, Infection, Quantitative RT-PCR, Standard Deviation

    miR-504-3p, miR-574-5p and miR-1236-5p can enhance HCV-RNA replication via miR-122-type interaction. (A) Diagrams of possible interaction between HCV 5’UTR and miR-504-3p, miR-574-5p and miR-1236-5p. (B) Sequence alignment of miR-122, miR-504-3p, miR-504-3p-mt6, miR-504-3p-mt8, miR-574-5p miR-574-5p-mt5, miR-574-5p-addG, miR-1236-5p, miR-1236-5p-mt5 and miR-1236-5p-addG. Mismatching nucleotides were shown in red. (C, D) Intracellular HCV-RNA levels of 751-122KO cells infected with JFH1 in the presence of mimic control, miR-122, miR-504-3p, miR-574-5p and miR-1236-5p or their mutant derivatives were determined at 72 hpi by qRT-PCR. The data are representative of three independent experiments. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (**P
    Figure Legend Snippet: miR-504-3p, miR-574-5p and miR-1236-5p can enhance HCV-RNA replication via miR-122-type interaction. (A) Diagrams of possible interaction between HCV 5’UTR and miR-504-3p, miR-574-5p and miR-1236-5p. (B) Sequence alignment of miR-122, miR-504-3p, miR-504-3p-mt6, miR-504-3p-mt8, miR-574-5p miR-574-5p-mt5, miR-574-5p-addG, miR-1236-5p, miR-1236-5p-mt5 and miR-1236-5p-addG. Mismatching nucleotides were shown in red. (C, D) Intracellular HCV-RNA levels of 751-122KO cells infected with JFH1 in the presence of mimic control, miR-122, miR-504-3p, miR-574-5p and miR-1236-5p or their mutant derivatives were determined at 72 hpi by qRT-PCR. The data are representative of three independent experiments. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (**P

    Techniques Used: Sequencing, Infection, Mutagenesis, Quantitative RT-PCR, Standard Deviation

    Identification of core sequence of miR-122 and 5’UTR of HCV-RNA required for the enhancement of viral RNA replication. (A) Diagrams of possible interaction between HCV 5’UTR and miR-122. (B) Sequence alignment of miR-122 and its mutant derivatives, miR-122-mt1, -mt2, -mt3, -mt4, -mt5, -mt6, -mt7, -mt8, -mt15 or -mt16. (C) Intracellular HCV-RNA levels of 751-122KO cells infected with JFH1 in the presence of mimic control, miR-122 or its mutant derivatives were determined at 72 hpi by qRT-PCR. (D) Various pHH-JFH1 plasmids encoding HCV mutants at miR-122 interaction sites were transfected into Huh7.5.1 cells and infectious titers in the culture supernatants at 3 dpi were determined by focus formation assay. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P
    Figure Legend Snippet: Identification of core sequence of miR-122 and 5’UTR of HCV-RNA required for the enhancement of viral RNA replication. (A) Diagrams of possible interaction between HCV 5’UTR and miR-122. (B) Sequence alignment of miR-122 and its mutant derivatives, miR-122-mt1, -mt2, -mt3, -mt4, -mt5, -mt6, -mt7, -mt8, -mt15 or -mt16. (C) Intracellular HCV-RNA levels of 751-122KO cells infected with JFH1 in the presence of mimic control, miR-122 or its mutant derivatives were determined at 72 hpi by qRT-PCR. (D) Various pHH-JFH1 plasmids encoding HCV mutants at miR-122 interaction sites were transfected into Huh7.5.1 cells and infectious titers in the culture supernatants at 3 dpi were determined by focus formation assay. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P

    Techniques Used: Sequencing, Mutagenesis, Infection, Quantitative RT-PCR, Transfection, Tube Formation Assay, Standard Deviation

    10) Product Images from "Inhibition of DNA Methyltransferases Blocks Mutant Huntingtin-Induced Neurotoxicity"

    Article Title: Inhibition of DNA Methyltransferases Blocks Mutant Huntingtin-Induced Neurotoxicity

    Journal: Scientific Reports

    doi: 10.1038/srep31022

    Inhibition of DNMTs restores the expression of Bdnf exon IV and VI transcripts in primary cortical neurons. ( A ) DIV 5 cortical neurons were infected with Htt lentivirus. RNA was harvested 5 days later and subjected to qRT-PCR for total Bdnf (coding exon IX) using β-actin and 18S rRNA as reference genes. Htt-72Q decreased the expression of total Bdnf transcripts (Mann-Whitney U test, * P = 0.008 vs. Htt-25Q, n = 5). ( B ) Cortical neurons transduced as in ( A ) were cultured with recombinant BDNF (50 ng/ml) and subjected to MTS assay at DIV 14. BDNF increased the viability of Htt-72Q-expressing neurons (ANOVA, *P
    Figure Legend Snippet: Inhibition of DNMTs restores the expression of Bdnf exon IV and VI transcripts in primary cortical neurons. ( A ) DIV 5 cortical neurons were infected with Htt lentivirus. RNA was harvested 5 days later and subjected to qRT-PCR for total Bdnf (coding exon IX) using β-actin and 18S rRNA as reference genes. Htt-72Q decreased the expression of total Bdnf transcripts (Mann-Whitney U test, * P = 0.008 vs. Htt-25Q, n = 5). ( B ) Cortical neurons transduced as in ( A ) were cultured with recombinant BDNF (50 ng/ml) and subjected to MTS assay at DIV 14. BDNF increased the viability of Htt-72Q-expressing neurons (ANOVA, *P

    Techniques Used: Inhibition, Expressing, Infection, Quantitative RT-PCR, MANN-WHITNEY, Cell Culture, Recombinant, MTS Assay

    DNMT inhibitors reactivate striatal gene expression in mutant Htt-expressing primary neurons and R6/2 HD mouse brain in vivo . ( A ) DIV 5 mouse primary striatal neurons were infected with lentivirus expressing Htt-25Q or Htt-72Q exon1 fragment; 5 days later, RNA was prepared and subjected to qRT-PCR analysis. β-actin and Hprt were used as reference genes. Decitabine restored the expression of downregulated genes in mutant Htt-expressing striatal neurons (ANOVA, *P
    Figure Legend Snippet: DNMT inhibitors reactivate striatal gene expression in mutant Htt-expressing primary neurons and R6/2 HD mouse brain in vivo . ( A ) DIV 5 mouse primary striatal neurons were infected with lentivirus expressing Htt-25Q or Htt-72Q exon1 fragment; 5 days later, RNA was prepared and subjected to qRT-PCR analysis. β-actin and Hprt were used as reference genes. Decitabine restored the expression of downregulated genes in mutant Htt-expressing striatal neurons (ANOVA, *P

    Techniques Used: Expressing, Mutagenesis, In Vivo, Infection, Quantitative RT-PCR

    11) Product Images from "High-fidelity CRISPR/Cas9- based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis"

    Article Title: High-fidelity CRISPR/Cas9- based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05766-5

    Targeted Rasal1 promoter demethylation by dCas9/dHFCas9-TET3CD- Rasal1 -sgRNA fusion protein ameliorates kidney fibrosis. a Schematic showing the parenchymal injection of lentiviral particles containing dCas9/dHFCas9-TET3CD fusion protein into UUO-challenged kidneys. b Schedule of UUO mouse surgery, lentivirus injection, and analysis. c qRT-PCR results showing that Rasal1 mRNA expression was significantly induced in UUO-challenged kidneys transduced with dCas9/dHFCas9-TET3CD- Rasal1 -sgRNA, but not in UUO-challenged kidneys transduced with control dCas9/dHFCas9-TET3CD- LacZ- sgRNA. There is no significant difference between dCas9-TET3CD and dHFCas9-TET3CD constructs. Results were normalized to reference gene Gapdh (expression is presented as mean value; error bars represent S.D., n ≥ 5 in each group, # not significant, *** p
    Figure Legend Snippet: Targeted Rasal1 promoter demethylation by dCas9/dHFCas9-TET3CD- Rasal1 -sgRNA fusion protein ameliorates kidney fibrosis. a Schematic showing the parenchymal injection of lentiviral particles containing dCas9/dHFCas9-TET3CD fusion protein into UUO-challenged kidneys. b Schedule of UUO mouse surgery, lentivirus injection, and analysis. c qRT-PCR results showing that Rasal1 mRNA expression was significantly induced in UUO-challenged kidneys transduced with dCas9/dHFCas9-TET3CD- Rasal1 -sgRNA, but not in UUO-challenged kidneys transduced with control dCas9/dHFCas9-TET3CD- LacZ- sgRNA. There is no significant difference between dCas9-TET3CD and dHFCas9-TET3CD constructs. Results were normalized to reference gene Gapdh (expression is presented as mean value; error bars represent S.D., n ≥ 5 in each group, # not significant, *** p

    Techniques Used: Injection, Quantitative RT-PCR, Expressing, Transduction, Construct

    Induction of Klotho promoter hydroxymethylation by dHFCas9-TET3CD- Klotho -sgRNA in tubular epithelial cells ameliorates kidney fibrosis. a Schematic shows retrograde ureter injection of lentiviral particles containing dHFCas9-TET3CD- Klotho -sgRNA into UUO-operated kidneys. b Schedule of UUO mouse surgery, lentivirus injection, and analysis. c qRT-PCR results showing that Kl mRNA expression was significantly induced in UUO-operated kidneys transduced with dHFCas9-TET3CD- Kl -sgRNA but not in UUO-challenged kidneys transduced with LacZ- sgRNA control. Results were normalized to reference gene Gapdh (expression is presented as mean value, error bars represent S.E.M. , n = 6 in each group, *** p
    Figure Legend Snippet: Induction of Klotho promoter hydroxymethylation by dHFCas9-TET3CD- Klotho -sgRNA in tubular epithelial cells ameliorates kidney fibrosis. a Schematic shows retrograde ureter injection of lentiviral particles containing dHFCas9-TET3CD- Klotho -sgRNA into UUO-operated kidneys. b Schedule of UUO mouse surgery, lentivirus injection, and analysis. c qRT-PCR results showing that Kl mRNA expression was significantly induced in UUO-operated kidneys transduced with dHFCas9-TET3CD- Kl -sgRNA but not in UUO-challenged kidneys transduced with LacZ- sgRNA control. Results were normalized to reference gene Gapdh (expression is presented as mean value, error bars represent S.E.M. , n = 6 in each group, *** p

    Techniques Used: Injection, Quantitative RT-PCR, Expressing, Transduction

    Rasal1 gene disruption results in aggravated fibrosis level in the UUO model. a Schematic of knockout-first strategy for Rasal1 gene. The gene trapping LacZ cassette is alternatively spliced with exon 2 mediated by a splicing acceptor (SA). A promoter-driven Neomycin cassette is inserted after LacZ . The targeted exon 3 and 4 are flanked by loxP sites. Black arrows indicate the location of genotyping primers. b qRT-PCR analysis shows the Rasal1 mRNA expression in homozygous mice are significantly reduced as compared with wild-type mice, while there is no significant reduction in heterozygous mice. The data are presented as mean value, error bars represent S.D., n.s. not significant, *** p
    Figure Legend Snippet: Rasal1 gene disruption results in aggravated fibrosis level in the UUO model. a Schematic of knockout-first strategy for Rasal1 gene. The gene trapping LacZ cassette is alternatively spliced with exon 2 mediated by a splicing acceptor (SA). A promoter-driven Neomycin cassette is inserted after LacZ . The targeted exon 3 and 4 are flanked by loxP sites. Black arrows indicate the location of genotyping primers. b qRT-PCR analysis shows the Rasal1 mRNA expression in homozygous mice are significantly reduced as compared with wild-type mice, while there is no significant reduction in heterozygous mice. The data are presented as mean value, error bars represent S.D., n.s. not significant, *** p

    Techniques Used: Knock-Out, Quantitative RT-PCR, Expressing, Mouse Assay

    12) Product Images from "A Sendai Virus-Based Cytoplasmic RNA Vector as a Novel Platform for Long-Term Expression of MicroRNAs"

    Article Title: A Sendai Virus-Based Cytoplasmic RNA Vector as a Novel Platform for Long-Term Expression of MicroRNAs

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2019.10.012

    SeVdp Vector-Mediated miRNA Expression (A) Genome structure of the SeVdp vectors. NP , P , and L genes are indispensable for the replication of the viral genome and mRNA synthesis. The P gene contains multiple open reading frames encoding P, C, and V proteins. The SeVdp genome encodes Bs r , EGFP, and murine miRNA as transgenes. (B) Expression of miR-124 in HCT116 cells infected with either SeVdp-Ctrl or SeVdp-124; miR-124 levels were determined by qRT-PCR; the level in SeVdp-Ctrl-infected cells was set to 1.0. **p
    Figure Legend Snippet: SeVdp Vector-Mediated miRNA Expression (A) Genome structure of the SeVdp vectors. NP , P , and L genes are indispensable for the replication of the viral genome and mRNA synthesis. The P gene contains multiple open reading frames encoding P, C, and V proteins. The SeVdp genome encodes Bs r , EGFP, and murine miRNA as transgenes. (B) Expression of miR-124 in HCT116 cells infected with either SeVdp-Ctrl or SeVdp-124; miR-124 levels were determined by qRT-PCR; the level in SeVdp-Ctrl-infected cells was set to 1.0. **p

    Techniques Used: Plasmid Preparation, Expressing, Infection, Quantitative RT-PCR

    The SeVdp Vector as a Platform for Expressing Functional miRNAs (A) miRNA expression in various human cells infected with SeVdp-Ctrl or SeVdp-124. miR-124 levels were determined by qRT-PCR; the level in SeVdp-Ctrl-infected cells was set to 1.0. **p
    Figure Legend Snippet: The SeVdp Vector as a Platform for Expressing Functional miRNAs (A) miRNA expression in various human cells infected with SeVdp-Ctrl or SeVdp-124. miR-124 levels were determined by qRT-PCR; the level in SeVdp-Ctrl-infected cells was set to 1.0. **p

    Techniques Used: Plasmid Preparation, Expressing, Functional Assay, Infection, Quantitative RT-PCR

    13) Product Images from "Selective targeting of the oncogenic KRAS G12S mutant allele by CRISPR/Cas9 induces efficient tumor regression"

    Article Title: Selective targeting of the oncogenic KRAS G12S mutant allele by CRISPR/Cas9 induces efficient tumor regression

    Journal: Theranostics

    doi: 10.7150/thno.42325

    dCas9-KRAB mRNA-regulating system downregulated G12S transcription and inhibited tumor cell proliferation. ( A, B ) No off-target indels were noticeably caused by the CRISPR/Cas9 gene-cutting system at fourteen homologous sites that differed from the on-target sites by up to 4 nt. PAM sequences are shown in red and mismatched nucleotides are shown in green. On: on-target site. OT: off-target site. Cleavage position within the 20 bp target sequences is indicated by a red arrow. Error bar indicates S.E.M. (n=3 to 4). ( C ) Diagram of knocking down KRAS G12S mutant allele specifically by the dCas9-KRAB system. Blue strands: spacer; green strands: PAM sequence; red strands and star: single-nucleotide missense mutations. ( D ) qRT-PCR analysis of KRAS G12S mRNA expression. Error bars represent S.E.M. (∗) 0.01
    Figure Legend Snippet: dCas9-KRAB mRNA-regulating system downregulated G12S transcription and inhibited tumor cell proliferation. ( A, B ) No off-target indels were noticeably caused by the CRISPR/Cas9 gene-cutting system at fourteen homologous sites that differed from the on-target sites by up to 4 nt. PAM sequences are shown in red and mismatched nucleotides are shown in green. On: on-target site. OT: off-target site. Cleavage position within the 20 bp target sequences is indicated by a red arrow. Error bar indicates S.E.M. (n=3 to 4). ( C ) Diagram of knocking down KRAS G12S mutant allele specifically by the dCas9-KRAB system. Blue strands: spacer; green strands: PAM sequence; red strands and star: single-nucleotide missense mutations. ( D ) qRT-PCR analysis of KRAS G12S mRNA expression. Error bars represent S.E.M. (∗) 0.01

    Techniques Used: CRISPR, Mutagenesis, Sequencing, Quantitative RT-PCR, Expressing

    14) Product Images from "Persistent elevation of intrathecal pro-inflammatory cytokines leads to multiple sclerosis-like cortical demyelination and neurodegeneration"

    Article Title: Persistent elevation of intrathecal pro-inflammatory cytokines leads to multiple sclerosis-like cortical demyelination and neurodegeneration

    Journal: Acta Neuropathologica Communications

    doi: 10.1186/s40478-020-00938-1

    Expression of cell death related proteins in cortical neurons. CNPase+/Cleaved Casp-3+ oligodendrocytes are illustrated at 28 dpi in the cortical layers of MOG immunised animals injected with cytokine viral vectors, indicating apoptosis ( a ), whereas no NeuN+/Cleaved Casp-3+ neurons were found ( b ). QRT-PCR analysis showed increased gene expression for the necroptosis genes RIPK3 and MLKL in cortical tissue from 28 dpi cytokine viral vector injected animals compared to GFP controls, with no significant difference between IFA or MOG immunised animals ( c ). Levels of endogenous rat IFNγ ( d ) and TNF ( e ) genes were upregulated at 28 and 56 dpi in both MOG and IFA immunised animals, with highest levels at 28dpi. Statistics C-E: t-tests on Ct values, * P
    Figure Legend Snippet: Expression of cell death related proteins in cortical neurons. CNPase+/Cleaved Casp-3+ oligodendrocytes are illustrated at 28 dpi in the cortical layers of MOG immunised animals injected with cytokine viral vectors, indicating apoptosis ( a ), whereas no NeuN+/Cleaved Casp-3+ neurons were found ( b ). QRT-PCR analysis showed increased gene expression for the necroptosis genes RIPK3 and MLKL in cortical tissue from 28 dpi cytokine viral vector injected animals compared to GFP controls, with no significant difference between IFA or MOG immunised animals ( c ). Levels of endogenous rat IFNγ ( d ) and TNF ( e ) genes were upregulated at 28 and 56 dpi in both MOG and IFA immunised animals, with highest levels at 28dpi. Statistics C-E: t-tests on Ct values, * P

    Techniques Used: Expressing, Injection, Quantitative RT-PCR, Plasmid Preparation, Immunofluorescence

    15) Product Images from "Transduction of Human Primitive Repopulating Hematopoietic Cells With Lentiviral Vectors Pseudotyped With Various Envelope Proteins"

    Article Title: Transduction of Human Primitive Repopulating Hematopoietic Cells With Lentiviral Vectors Pseudotyped With Various Envelope Proteins

    Journal: Molecular Therapy

    doi: 10.1038/mt.2010.48

    Genome concentrations and transducing titers of various pesudotypes on HeLa cells. ( a ) Comparison of the relative vector genome concentration, as determined by qRT-PCR of medium containing vector particles pseudotyped with various envelope proteins as
    Figure Legend Snippet: Genome concentrations and transducing titers of various pesudotypes on HeLa cells. ( a ) Comparison of the relative vector genome concentration, as determined by qRT-PCR of medium containing vector particles pseudotyped with various envelope proteins as

    Techniques Used: Plasmid Preparation, Concentration Assay, Quantitative RT-PCR

    16) Product Images from "MicroRNA-Mediated Transformation by the Kaposi's Sarcoma-Associated Herpesvirus Kaposin Locus"

    Article Title: MicroRNA-Mediated Transformation by the Kaposi's Sarcoma-Associated Herpesvirus Kaposin Locus

    Journal: Journal of Virology

    doi: 10.1128/JVI.03317-14

    Continued transgene expression is required to maintain transformed status. (A) Schematic outlining the experimental setup used for secondary focus formation assays. (B) TaqMan qRT-PCR confirms loss of miR-K10a/+1 expression after withdrawal (−)
    Figure Legend Snippet: Continued transgene expression is required to maintain transformed status. (A) Schematic outlining the experimental setup used for secondary focus formation assays. (B) TaqMan qRT-PCR confirms loss of miR-K10a/+1 expression after withdrawal (−)

    Techniques Used: Expressing, Transformation Assay, Quantitative RT-PCR

    Inhibition of KapA expression does not affect the transformed phenotype in WT-transformed NIH 3T3 cells. (A) Western blotting confirms reduction of KapA expression (Flag) in cells expressing a KapA-directed sh-miR (shKap). (B) TaqMan qRT-PCR shows that
    Figure Legend Snippet: Inhibition of KapA expression does not affect the transformed phenotype in WT-transformed NIH 3T3 cells. (A) Western blotting confirms reduction of KapA expression (Flag) in cells expressing a KapA-directed sh-miR (shKap). (B) TaqMan qRT-PCR shows that

    Techniques Used: Inhibition, Expressing, Transformation Assay, Western Blot, Quantitative RT-PCR

    17) Product Images from "Expression of CD24 in Human Bone Marrow-Derived Mesenchymal Stromal Cells Is Regulated by TGFβ3 and Induces a Myofibroblast-Like Genotype"

    Article Title: Expression of CD24 in Human Bone Marrow-Derived Mesenchymal Stromal Cells Is Regulated by TGFβ3 and Induces a Myofibroblast-Like Genotype

    Journal: Stem Cells International

    doi: 10.1155/2016/1319578

    Reciprocal regulation of CD24 and TGF β 3. (a) qRT-PCR analysis of CD24 mRNA expression after stimulation with 10 ng/mL TGF β 3 for 4 h, 24 h, and 96 h relative to unstimulated control cells (ΔΔCt). (b) Exemplary histogram showing induction of CD24 surface expression after treatment with 10 ng/mL TGF β 3 or with 2.5 ng/mL TGF β 1 for 7 days as analyzed by flow cytometry. (c) Flow cytometric analysis of CD24 surface protein expression on hBMSCs after 7 days in unstimulated control cells (Control), in cells treated with 10 ng/mL TGF β 3 (TGF β 3), in cells treated with 10 ng/mL TGF β 3 + 20 μ M SB431542 (SB431542), or in cells treated with 10 ng/mL TGF β 3 and 4 μ g/mL anti-TGF β 3 antibody (anti-TGF β 3). (d) and (e) qRT-PCR analysis of TGF β 3 (d) and CD24 (e) mRNA expression after knockdown (CD24 Down) or overexpression (CD24 Up) of CD24 in hBMSCs after 7 days relative to respective control hBMSCs ( n = 3 independent experiments with hBMSCs from different donors).
    Figure Legend Snippet: Reciprocal regulation of CD24 and TGF β 3. (a) qRT-PCR analysis of CD24 mRNA expression after stimulation with 10 ng/mL TGF β 3 for 4 h, 24 h, and 96 h relative to unstimulated control cells (ΔΔCt). (b) Exemplary histogram showing induction of CD24 surface expression after treatment with 10 ng/mL TGF β 3 or with 2.5 ng/mL TGF β 1 for 7 days as analyzed by flow cytometry. (c) Flow cytometric analysis of CD24 surface protein expression on hBMSCs after 7 days in unstimulated control cells (Control), in cells treated with 10 ng/mL TGF β 3 (TGF β 3), in cells treated with 10 ng/mL TGF β 3 + 20 μ M SB431542 (SB431542), or in cells treated with 10 ng/mL TGF β 3 and 4 μ g/mL anti-TGF β 3 antibody (anti-TGF β 3). (d) and (e) qRT-PCR analysis of TGF β 3 (d) and CD24 (e) mRNA expression after knockdown (CD24 Down) or overexpression (CD24 Up) of CD24 in hBMSCs after 7 days relative to respective control hBMSCs ( n = 3 independent experiments with hBMSCs from different donors).

    Techniques Used: Quantitative RT-PCR, Expressing, Flow Cytometry, Cytometry, Over Expression

    Analysis of CD24 expression in hBMSCs. (a) hBMSC mRNA from 18 different donors in passage 2 of in vitro culture were analyzed for the expression of CD24 mRNA by qRT-PCR and analyzed for gender-dependent differences in mRNA expression levels. The relative expression values are plotted as fold change in mRNA expression (ΔΔCt) relative to the sample with the lowest relative CD24 mRNA expression. (b) The mRNA expression of CD24 was analyzed from passage 1 to passage 4 of in vitro culture in 4 individual donors. The expression values are normalized to the expression levels of the respective donor in passage 1 (ΔΔCt). (c) The expression of CD24 protein was evaluated by western blot with a polyclonal rabbit anti-CD24 antibody. In order to determine which bands were specific for CD24 hBMSCs were transduced with either a shRNA directed against CD24 mRNA (CD24 Down) or a scrambled shRNA (Control). Cell extracts from these two groups were then compared for CD24 reactive bands: the western blot bands with reduced intensity in the CD24 Down Group are marked with black arrows and represent bands specific for CD24. β -actin was used as loading control.
    Figure Legend Snippet: Analysis of CD24 expression in hBMSCs. (a) hBMSC mRNA from 18 different donors in passage 2 of in vitro culture were analyzed for the expression of CD24 mRNA by qRT-PCR and analyzed for gender-dependent differences in mRNA expression levels. The relative expression values are plotted as fold change in mRNA expression (ΔΔCt) relative to the sample with the lowest relative CD24 mRNA expression. (b) The mRNA expression of CD24 was analyzed from passage 1 to passage 4 of in vitro culture in 4 individual donors. The expression values are normalized to the expression levels of the respective donor in passage 1 (ΔΔCt). (c) The expression of CD24 protein was evaluated by western blot with a polyclonal rabbit anti-CD24 antibody. In order to determine which bands were specific for CD24 hBMSCs were transduced with either a shRNA directed against CD24 mRNA (CD24 Down) or a scrambled shRNA (Control). Cell extracts from these two groups were then compared for CD24 reactive bands: the western blot bands with reduced intensity in the CD24 Down Group are marked with black arrows and represent bands specific for CD24. β -actin was used as loading control.

    Techniques Used: Expressing, In Vitro, Quantitative RT-PCR, Western Blot, Transduction, shRNA

    18) Product Images from "Various miRNAs compensate the role of miR-122 on HCV replication"

    Article Title: Various miRNAs compensate the role of miR-122 on HCV replication

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008308

    miR-504-3p, miR-574-5p and miR-1236-5p can enhance HCV-RNA replication via miR-122-type interaction. (A) Diagrams of possible interaction between HCV 5’UTR and miR-504-3p, miR-574-5p and miR-1236-5p. (B) Sequence alignment of miR-122, miR-504-3p, miR-504-3p-mt6, miR-504-3p-mt8, miR-574-5p miR-574-5p-mt5, miR-574-5p-addG, miR-1236-5p, miR-1236-5p-mt5 and miR-1236-5p-addG. Mismatching nucleotides were shown in red. (C, D) Intracellular HCV-RNA levels of 751-122KO cells infected with JFH1 in the presence of mimic control, miR-122, miR-504-3p, miR-574-5p and miR-1236-5p or their mutant derivatives were determined at 72 hpi by qRT-PCR. The data are representative of three independent experiments. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (**P
    Figure Legend Snippet: miR-504-3p, miR-574-5p and miR-1236-5p can enhance HCV-RNA replication via miR-122-type interaction. (A) Diagrams of possible interaction between HCV 5’UTR and miR-504-3p, miR-574-5p and miR-1236-5p. (B) Sequence alignment of miR-122, miR-504-3p, miR-504-3p-mt6, miR-504-3p-mt8, miR-574-5p miR-574-5p-mt5, miR-574-5p-addG, miR-1236-5p, miR-1236-5p-mt5 and miR-1236-5p-addG. Mismatching nucleotides were shown in red. (C, D) Intracellular HCV-RNA levels of 751-122KO cells infected with JFH1 in the presence of mimic control, miR-122, miR-504-3p, miR-574-5p and miR-1236-5p or their mutant derivatives were determined at 72 hpi by qRT-PCR. The data are representative of three independent experiments. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (**P

    Techniques Used: Sequencing, Infection, Mutagenesis, Quantitative RT-PCR, Standard Deviation

    miR-25-5p and miR-4730 can enhance HCV-RNA replication via non-miR-122-type interaction. (A) Diagrams of possible interaction between HCV 5’UTR and synthetic miRNAs with 7nt-match and 8nt-match. Gray-shaded area and framed by dashed line in gray indicate possible interaction region and G-U wobble pair, respectively. Nucleotides in red are important region for the enhancement by miR-122 shown in Fig 1D . (B) Sequence alignment of miR-122 and synthetic miRNAs with 7nt-match and 8nt-match. Mismatching nucleotides were shown in red. (C) Intracellular HCV-RNA levels of 751-122KO cells infected with JFH1 in the presence of mimic control, miR-122, miR-122-mt6 and synthetic miRNAs with 7nt-match shown in Fig 3A. (D) Intracellular HCV-RNA levels of 751-122KO cells infected with JFH1 in the presence of mimic control, miR-122, miR-122-mt6, miR-25-5p, miR-652-5p, miR-1236-5p and miR-4730 were determined at 72 hpi by qRT-PCR. The data are representative of three independent experiments. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (**P
    Figure Legend Snippet: miR-25-5p and miR-4730 can enhance HCV-RNA replication via non-miR-122-type interaction. (A) Diagrams of possible interaction between HCV 5’UTR and synthetic miRNAs with 7nt-match and 8nt-match. Gray-shaded area and framed by dashed line in gray indicate possible interaction region and G-U wobble pair, respectively. Nucleotides in red are important region for the enhancement by miR-122 shown in Fig 1D . (B) Sequence alignment of miR-122 and synthetic miRNAs with 7nt-match and 8nt-match. Mismatching nucleotides were shown in red. (C) Intracellular HCV-RNA levels of 751-122KO cells infected with JFH1 in the presence of mimic control, miR-122, miR-122-mt6 and synthetic miRNAs with 7nt-match shown in Fig 3A. (D) Intracellular HCV-RNA levels of 751-122KO cells infected with JFH1 in the presence of mimic control, miR-122, miR-122-mt6, miR-25-5p, miR-652-5p, miR-1236-5p and miR-4730 were determined at 72 hpi by qRT-PCR. The data are representative of three independent experiments. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (**P

    Techniques Used: Sequencing, Infection, Quantitative RT-PCR, Standard Deviation

    Identification of core sequence of miR-122 and 5’UTR of HCV-RNA required for the enhancement of viral RNA replication. (A) Diagrams of possible interaction between HCV 5’UTR and miR-122. (B) Sequence alignment of miR-122 and its mutant derivatives, miR-122-mt1, -mt2, -mt3, -mt4, -mt5, -mt6, -mt7, -mt8, -mt15 or -mt16. (C) Intracellular HCV-RNA levels of 751-122KO cells infected with JFH1 in the presence of mimic control, miR-122 or its mutant derivatives were determined at 72 hpi by qRT-PCR. (D) Various pHH-JFH1 plasmids encoding HCV mutants at miR-122 interaction sites were transfected into Huh7.5.1 cells and infectious titers in the culture supernatants at 3 dpi were determined by focus formation assay. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P
    Figure Legend Snippet: Identification of core sequence of miR-122 and 5’UTR of HCV-RNA required for the enhancement of viral RNA replication. (A) Diagrams of possible interaction between HCV 5’UTR and miR-122. (B) Sequence alignment of miR-122 and its mutant derivatives, miR-122-mt1, -mt2, -mt3, -mt4, -mt5, -mt6, -mt7, -mt8, -mt15 or -mt16. (C) Intracellular HCV-RNA levels of 751-122KO cells infected with JFH1 in the presence of mimic control, miR-122 or its mutant derivatives were determined at 72 hpi by qRT-PCR. (D) Various pHH-JFH1 plasmids encoding HCV mutants at miR-122 interaction sites were transfected into Huh7.5.1 cells and infectious titers in the culture supernatants at 3 dpi were determined by focus formation assay. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P

    Techniques Used: Sequencing, Mutagenesis, Infection, Quantitative RT-PCR, Transfection, Tube Formation Assay, Standard Deviation

    Related Articles

    Transduction:

    Article Title: Inhibition of DNA Methyltransferases Blocks Mutant Huntingtin-Induced Neurotoxicity
    Article Snippet: .. Viral copy number was adjusted for transduction of neurons on the basis of titer measured using the Lenti-X qRT-PCR titration kit (Clontech), and equal numbers of viral particles of Htt-25Q and Htt-72Q expressing lentiviruses were used for transduction. .. For the experiments to test effects of DNMT inhibitors, neurons were treated with inhibitors six hours after Htt lentiviral infection.

    Article Title: The role of Twist1 in mutant huntingtin–induced transcriptional alterations and neurotoxicity
    Article Snippet: .. Equal numbers of viral particles of Htt-25Q– and Htt-72Q–expressing lentiviruses were used for transduction of neurons on the basis of viral copy number measured using the Lenti-X qRT-PCR titration kit (Clontech). .. In lentivirus-mediated knockdown experiments in Htt-expressing neurons, DIV 5 primary neurons were cotransduced with shRNA- and Htt-expressing lentiviruses. pLKO.1-luciferase shRNA was used as control for pLKO.1- Twist1 shRNA and pLKO.1- Pou4f1 shRNA.

    Article Title: High-fidelity CRISPR/Cas9- based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis
    Article Snippet: .. Lentivirus titration was determined by the Lenti-X qRT-PCR Titration Kit (Clonetech, Heidelberg, Germany) and 8 μg/ml Polybrene (Sigma- Aldrich, Munich, Germany) was added to the viral solution for the in vivo and in vitro transduction experiments. .. Two different mouse embryonic stem cell clones (A03, H03) carrying targeted gene knockout alleles were received from EUCOMM international knockout mouse consortium.

    In Vivo:

    Article Title: High-fidelity CRISPR/Cas9- based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis
    Article Snippet: .. Lentivirus titration was determined by the Lenti-X qRT-PCR Titration Kit (Clonetech, Heidelberg, Germany) and 8 μg/ml Polybrene (Sigma- Aldrich, Munich, Germany) was added to the viral solution for the in vivo and in vitro transduction experiments. .. Two different mouse embryonic stem cell clones (A03, H03) carrying targeted gene knockout alleles were received from EUCOMM international knockout mouse consortium.

    In Vitro:

    Article Title: High-fidelity CRISPR/Cas9- based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis
    Article Snippet: .. Lentivirus titration was determined by the Lenti-X qRT-PCR Titration Kit (Clonetech, Heidelberg, Germany) and 8 μg/ml Polybrene (Sigma- Aldrich, Munich, Germany) was added to the viral solution for the in vivo and in vitro transduction experiments. .. Two different mouse embryonic stem cell clones (A03, H03) carrying targeted gene knockout alleles were received from EUCOMM international knockout mouse consortium.

    Isolation:

    Article Title: A Metabolic Basis for Endothelial-to-Mesenchymal Transition
    Article Snippet: .. For lentiviral titration, viral RNA was isolated using a NucleoSpin RNA Virus Kit (Clontech) and the copy number of the viral RNA genome determined using a Lenti-X qRT-PCR (quantitative RT-PCR) Titration Kit (Clontech). .. Cells were infected (approximately 1–2 × 109 copies per million cells) in the presence of Polybrene (Santa Cruz Biotechnology) at final concentration of 8 µg ml−1 and incubated with the virus mixture.

    Quantitative RT-PCR:

    Article Title: A Metabolic Basis for Endothelial-to-Mesenchymal Transition
    Article Snippet: .. For lentiviral titration, viral RNA was isolated using a NucleoSpin RNA Virus Kit (Clontech) and the copy number of the viral RNA genome determined using a Lenti-X qRT-PCR (quantitative RT-PCR) Titration Kit (Clontech). .. Cells were infected (approximately 1–2 × 109 copies per million cells) in the presence of Polybrene (Santa Cruz Biotechnology) at final concentration of 8 µg ml−1 and incubated with the virus mixture.

    Article Title: Inhibition of DNA Methyltransferases Blocks Mutant Huntingtin-Induced Neurotoxicity
    Article Snippet: .. Viral copy number was adjusted for transduction of neurons on the basis of titer measured using the Lenti-X qRT-PCR titration kit (Clontech), and equal numbers of viral particles of Htt-25Q and Htt-72Q expressing lentiviruses were used for transduction. .. For the experiments to test effects of DNMT inhibitors, neurons were treated with inhibitors six hours after Htt lentiviral infection.

    Article Title: The role of Twist1 in mutant huntingtin–induced transcriptional alterations and neurotoxicity
    Article Snippet: .. Equal numbers of viral particles of Htt-25Q– and Htt-72Q–expressing lentiviruses were used for transduction of neurons on the basis of viral copy number measured using the Lenti-X qRT-PCR titration kit (Clontech). .. In lentivirus-mediated knockdown experiments in Htt-expressing neurons, DIV 5 primary neurons were cotransduced with shRNA- and Htt-expressing lentiviruses. pLKO.1-luciferase shRNA was used as control for pLKO.1- Twist1 shRNA and pLKO.1- Pou4f1 shRNA.

    Article Title: Photoreceptor protection via blockade of BET epigenetic readers in a murine model of inherited retinal degeneration
    Article Snippet: .. The culture medium was collected after 24-h incubation and passed through a 0.45-μm filter (EMD Millipore, MA) and then concentrated and titrated using Lenti-X™ Concentrator and Lenti-X™ qRT-PCR Titration Kit (Clontech Laboratories, Inc., Mountain View, CA). .. The lentivirus preparation was then applied to the N9 microglial cell culture together with 8 μg/ml polybrene and incubated for 24 h. Infected (green fluorescent) cells were recovered in fresh DMEM medium containing 1% FBS for 3 days and subjected to flow sorting.

    Article Title: A Novel Bromodomain Inhibitor Reverses HIV-1 Latency through Specific Binding with BRD4 to Promote Tat and P-TEFb Association
    Article Snippet: .. On day 21, 5 days post treatment; the supernatant was collected for quantification of newly produced viruses using the Lent-X™ qRT-PCR Titration Kit (Clontech) following the manufacturer's instruction. .. Isolation of CD8-depleted PBMCs PBMCs were isolated from fresh whole blood of cART-treated HIV- infected aviremic patients using the Ficoll-Hypaque gradient method, as described previously (Reuse et al., ; Bouchat et al., ).

    Article Title: Characterization of miR-122-independent propagation of HCV
    Article Snippet: .. The infectious titer of lentivirus was determined by a Lenti-X qRT-PCR Titration Kit (Clontech, Mountain View, CA). .. The vesicular stomatitis virus (VSV) variant NCP12.1, derived from the Indiana strain, was provided by M. Whitt.

    Article Title: High-fidelity CRISPR/Cas9- based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis
    Article Snippet: .. Lentivirus titration was determined by the Lenti-X qRT-PCR Titration Kit (Clonetech, Heidelberg, Germany) and 8 μg/ml Polybrene (Sigma- Aldrich, Munich, Germany) was added to the viral solution for the in vivo and in vitro transduction experiments. .. Two different mouse embryonic stem cell clones (A03, H03) carrying targeted gene knockout alleles were received from EUCOMM international knockout mouse consortium.

    Article Title: A deleterious Nav1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients
    Article Snippet: .. Viral supernatants were concentrated using Lenti-X Concentrator (Clontech, Mountain View, California) and viral titers were determined with a Lenti-X qRT-PCR Titration Kit (Clontech). .. NgN2-mediated neural differentiation of hESC line H9 H9 hESCs with stably integrated doxycycline-inducible Ngn2 and the tet-transactivator were maintained in mTESR medium on matrigel.

    Titration:

    Article Title: A Metabolic Basis for Endothelial-to-Mesenchymal Transition
    Article Snippet: .. For lentiviral titration, viral RNA was isolated using a NucleoSpin RNA Virus Kit (Clontech) and the copy number of the viral RNA genome determined using a Lenti-X qRT-PCR (quantitative RT-PCR) Titration Kit (Clontech). .. Cells were infected (approximately 1–2 × 109 copies per million cells) in the presence of Polybrene (Santa Cruz Biotechnology) at final concentration of 8 µg ml−1 and incubated with the virus mixture.

    Article Title: Inhibition of DNA Methyltransferases Blocks Mutant Huntingtin-Induced Neurotoxicity
    Article Snippet: .. Viral copy number was adjusted for transduction of neurons on the basis of titer measured using the Lenti-X qRT-PCR titration kit (Clontech), and equal numbers of viral particles of Htt-25Q and Htt-72Q expressing lentiviruses were used for transduction. .. For the experiments to test effects of DNMT inhibitors, neurons were treated with inhibitors six hours after Htt lentiviral infection.

    Article Title: The role of Twist1 in mutant huntingtin–induced transcriptional alterations and neurotoxicity
    Article Snippet: .. Equal numbers of viral particles of Htt-25Q– and Htt-72Q–expressing lentiviruses were used for transduction of neurons on the basis of viral copy number measured using the Lenti-X qRT-PCR titration kit (Clontech). .. In lentivirus-mediated knockdown experiments in Htt-expressing neurons, DIV 5 primary neurons were cotransduced with shRNA- and Htt-expressing lentiviruses. pLKO.1-luciferase shRNA was used as control for pLKO.1- Twist1 shRNA and pLKO.1- Pou4f1 shRNA.

    Article Title: Photoreceptor protection via blockade of BET epigenetic readers in a murine model of inherited retinal degeneration
    Article Snippet: .. The culture medium was collected after 24-h incubation and passed through a 0.45-μm filter (EMD Millipore, MA) and then concentrated and titrated using Lenti-X™ Concentrator and Lenti-X™ qRT-PCR Titration Kit (Clontech Laboratories, Inc., Mountain View, CA). .. The lentivirus preparation was then applied to the N9 microglial cell culture together with 8 μg/ml polybrene and incubated for 24 h. Infected (green fluorescent) cells were recovered in fresh DMEM medium containing 1% FBS for 3 days and subjected to flow sorting.

    Article Title: A Novel Bromodomain Inhibitor Reverses HIV-1 Latency through Specific Binding with BRD4 to Promote Tat and P-TEFb Association
    Article Snippet: .. On day 21, 5 days post treatment; the supernatant was collected for quantification of newly produced viruses using the Lent-X™ qRT-PCR Titration Kit (Clontech) following the manufacturer's instruction. .. Isolation of CD8-depleted PBMCs PBMCs were isolated from fresh whole blood of cART-treated HIV- infected aviremic patients using the Ficoll-Hypaque gradient method, as described previously (Reuse et al., ; Bouchat et al., ).

    Article Title: Characterization of miR-122-independent propagation of HCV
    Article Snippet: .. The infectious titer of lentivirus was determined by a Lenti-X qRT-PCR Titration Kit (Clontech, Mountain View, CA). .. The vesicular stomatitis virus (VSV) variant NCP12.1, derived from the Indiana strain, was provided by M. Whitt.

    Article Title: High-fidelity CRISPR/Cas9- based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis
    Article Snippet: .. Lentivirus titration was determined by the Lenti-X qRT-PCR Titration Kit (Clonetech, Heidelberg, Germany) and 8 μg/ml Polybrene (Sigma- Aldrich, Munich, Germany) was added to the viral solution for the in vivo and in vitro transduction experiments. .. Two different mouse embryonic stem cell clones (A03, H03) carrying targeted gene knockout alleles were received from EUCOMM international knockout mouse consortium.

    Article Title: A deleterious Nav1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients
    Article Snippet: .. Viral supernatants were concentrated using Lenti-X Concentrator (Clontech, Mountain View, California) and viral titers were determined with a Lenti-X qRT-PCR Titration Kit (Clontech). .. NgN2-mediated neural differentiation of hESC line H9 H9 hESCs with stably integrated doxycycline-inducible Ngn2 and the tet-transactivator were maintained in mTESR medium on matrigel.

    Produced:

    Article Title: A Novel Bromodomain Inhibitor Reverses HIV-1 Latency through Specific Binding with BRD4 to Promote Tat and P-TEFb Association
    Article Snippet: .. On day 21, 5 days post treatment; the supernatant was collected for quantification of newly produced viruses using the Lent-X™ qRT-PCR Titration Kit (Clontech) following the manufacturer's instruction. .. Isolation of CD8-depleted PBMCs PBMCs were isolated from fresh whole blood of cART-treated HIV- infected aviremic patients using the Ficoll-Hypaque gradient method, as described previously (Reuse et al., ; Bouchat et al., ).

    Incubation:

    Article Title: Photoreceptor protection via blockade of BET epigenetic readers in a murine model of inherited retinal degeneration
    Article Snippet: .. The culture medium was collected after 24-h incubation and passed through a 0.45-μm filter (EMD Millipore, MA) and then concentrated and titrated using Lenti-X™ Concentrator and Lenti-X™ qRT-PCR Titration Kit (Clontech Laboratories, Inc., Mountain View, CA). .. The lentivirus preparation was then applied to the N9 microglial cell culture together with 8 μg/ml polybrene and incubated for 24 h. Infected (green fluorescent) cells were recovered in fresh DMEM medium containing 1% FBS for 3 days and subjected to flow sorting.

    Expressing:

    Article Title: Inhibition of DNA Methyltransferases Blocks Mutant Huntingtin-Induced Neurotoxicity
    Article Snippet: .. Viral copy number was adjusted for transduction of neurons on the basis of titer measured using the Lenti-X qRT-PCR titration kit (Clontech), and equal numbers of viral particles of Htt-25Q and Htt-72Q expressing lentiviruses were used for transduction. .. For the experiments to test effects of DNMT inhibitors, neurons were treated with inhibitors six hours after Htt lentiviral infection.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    TaKaRa lent x qrt pcr titration kit
    UMB-136 reverses HIV-1 latency from multiple cell models of HIV-1 latency. (A) Multiple J-Lat cell clones (6.3, 8.4, 9.2, and 10.4, CD4+ T cell background) were treated with UMB-136 (2.5 or 5 μM) or JQ1 (1 μM). GFP-positive cells were acquired and analyzed by flow cytometry. Percentage of GFP-positive cells was determined. One representative of three independent experiments was shown. (B) THP89GFP cells (monocyte/macrophage background) were treated with UMB-136 (5 μM) or JQ1 (1 μM). GFP-positive cells were acquired and analyzed by flow cytometry. The HIV-reactivated cell population was indicated. (C) HIV-1 latency was established in the CD4+ T cells isolated from the peripheral blood mononuclear cells (PBMCs) of 2 donors, following a previously reported primary cell model (Vicente Planelles). Cells latently infected with wild-type HIV-1 NL4-3 viruses were treated with UMB-136 (2.5 μM) or JQ1 (1 μM). The viral mRNAs from newly produced HIV-1 viruses in supernatant were measured by <t>qRT-PCR</t> assay. (D) The same assay in (D) was conducted using the CD4+ T cells isolated from the tonsillar mononuclear cells (TMCs) of 2 donors. Results were presented as mean ± s.e.m. ( n = 3 ); * p
    Lent X Qrt Pcr Titration Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 141 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lent x qrt pcr titration kit/product/TaKaRa
    Average 99 stars, based on 141 article reviews
    Price from $9.99 to $1999.99
    lent x qrt pcr titration kit - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    Image Search Results


    UMB-136 reverses HIV-1 latency from multiple cell models of HIV-1 latency. (A) Multiple J-Lat cell clones (6.3, 8.4, 9.2, and 10.4, CD4+ T cell background) were treated with UMB-136 (2.5 or 5 μM) or JQ1 (1 μM). GFP-positive cells were acquired and analyzed by flow cytometry. Percentage of GFP-positive cells was determined. One representative of three independent experiments was shown. (B) THP89GFP cells (monocyte/macrophage background) were treated with UMB-136 (5 μM) or JQ1 (1 μM). GFP-positive cells were acquired and analyzed by flow cytometry. The HIV-reactivated cell population was indicated. (C) HIV-1 latency was established in the CD4+ T cells isolated from the peripheral blood mononuclear cells (PBMCs) of 2 donors, following a previously reported primary cell model (Vicente Planelles). Cells latently infected with wild-type HIV-1 NL4-3 viruses were treated with UMB-136 (2.5 μM) or JQ1 (1 μM). The viral mRNAs from newly produced HIV-1 viruses in supernatant were measured by qRT-PCR assay. (D) The same assay in (D) was conducted using the CD4+ T cells isolated from the tonsillar mononuclear cells (TMCs) of 2 donors. Results were presented as mean ± s.e.m. ( n = 3 ); * p

    Journal: Frontiers in Microbiology

    Article Title: A Novel Bromodomain Inhibitor Reverses HIV-1 Latency through Specific Binding with BRD4 to Promote Tat and P-TEFb Association

    doi: 10.3389/fmicb.2017.01035

    Figure Lengend Snippet: UMB-136 reverses HIV-1 latency from multiple cell models of HIV-1 latency. (A) Multiple J-Lat cell clones (6.3, 8.4, 9.2, and 10.4, CD4+ T cell background) were treated with UMB-136 (2.5 or 5 μM) or JQ1 (1 μM). GFP-positive cells were acquired and analyzed by flow cytometry. Percentage of GFP-positive cells was determined. One representative of three independent experiments was shown. (B) THP89GFP cells (monocyte/macrophage background) were treated with UMB-136 (5 μM) or JQ1 (1 μM). GFP-positive cells were acquired and analyzed by flow cytometry. The HIV-reactivated cell population was indicated. (C) HIV-1 latency was established in the CD4+ T cells isolated from the peripheral blood mononuclear cells (PBMCs) of 2 donors, following a previously reported primary cell model (Vicente Planelles). Cells latently infected with wild-type HIV-1 NL4-3 viruses were treated with UMB-136 (2.5 μM) or JQ1 (1 μM). The viral mRNAs from newly produced HIV-1 viruses in supernatant were measured by qRT-PCR assay. (D) The same assay in (D) was conducted using the CD4+ T cells isolated from the tonsillar mononuclear cells (TMCs) of 2 donors. Results were presented as mean ± s.e.m. ( n = 3 ); * p

    Article Snippet: On day 21, 5 days post treatment; the supernatant was collected for quantification of newly produced viruses using the Lent-X™ qRT-PCR Titration Kit (Clontech) following the manufacturer's instruction.

    Techniques: Clone Assay, Flow Cytometry, Cytometry, Isolation, Infection, Produced, Quantitative RT-PCR

    Transduction and RNAi efficiency of two human Nav1.1 lenti-shRNA clones. ( A ) Evaluating the transduction efficiency of the lentiviral particles were tested in HEK293T cells based on the expression of the tRFP reporter. Scale bars, 20 microns. ( B ) The RNAi efficiency of lenti-shRNA1 and lenti-shRNA2 relative to a non-silencing clone was determined by first transducing HEK293T cells with the lentiviral clones and next expressing the human Na v 1.1 cDNA via transfection. Na v 1.1 mRNAs were quantified with qRT-PCR. By ANOVA and post hoc Sidak’s multiple comparisons, p = 0.003 for the comparison between control and shRNA1; p = 0.002 for the comparison between control and shRNA2. DOI: http://dx.doi.org/10.7554/eLife.13073.034

    Journal: eLife

    Article Title: A deleterious Nav1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients

    doi: 10.7554/eLife.13073

    Figure Lengend Snippet: Transduction and RNAi efficiency of two human Nav1.1 lenti-shRNA clones. ( A ) Evaluating the transduction efficiency of the lentiviral particles were tested in HEK293T cells based on the expression of the tRFP reporter. Scale bars, 20 microns. ( B ) The RNAi efficiency of lenti-shRNA1 and lenti-shRNA2 relative to a non-silencing clone was determined by first transducing HEK293T cells with the lentiviral clones and next expressing the human Na v 1.1 cDNA via transfection. Na v 1.1 mRNAs were quantified with qRT-PCR. By ANOVA and post hoc Sidak’s multiple comparisons, p = 0.003 for the comparison between control and shRNA1; p = 0.002 for the comparison between control and shRNA2. DOI: http://dx.doi.org/10.7554/eLife.13073.034

    Article Snippet: Viral supernatants were concentrated using Lenti-X Concentrator (Clontech, Mountain View, California) and viral titers were determined with a Lenti-X qRT-PCR Titration Kit (Clontech).

    Techniques: Transduction, shRNA, Clone Assay, Expressing, Transfection, Quantitative RT-PCR

    Propagation of HCV 122KO in 751-122KO cells. (A) HCV was inoculated into Huh7-122KO (#2), Huh7-122KO-cured (#3 or #5), or 751-122KO (#1, #2 or #3) cells, and the levels of intracellular HCV-RNA replication (top) and infectious titers in the culture supernatants (bottom) were determined by qRT-PCR and focus formation assay, respectively, at 72 hpi. (B) Infectious titer in the culture medium on serial passage of each 751-122KO cell clone. (C) HCV and HCV 122KO were inoculated into 751-122KO and Huh7.5.1 cells and the levels of intracellular HCV-RNA replication (top) and infectious titers in the culture supernatants (bottom) were determined at 72 hpi. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P

    Journal: PLoS Pathogens

    Article Title: Characterization of miR-122-independent propagation of HCV

    doi: 10.1371/journal.ppat.1006374

    Figure Lengend Snippet: Propagation of HCV 122KO in 751-122KO cells. (A) HCV was inoculated into Huh7-122KO (#2), Huh7-122KO-cured (#3 or #5), or 751-122KO (#1, #2 or #3) cells, and the levels of intracellular HCV-RNA replication (top) and infectious titers in the culture supernatants (bottom) were determined by qRT-PCR and focus formation assay, respectively, at 72 hpi. (B) Infectious titer in the culture medium on serial passage of each 751-122KO cell clone. (C) HCV and HCV 122KO were inoculated into 751-122KO and Huh7.5.1 cells and the levels of intracellular HCV-RNA replication (top) and infectious titers in the culture supernatants (bottom) were determined at 72 hpi. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P

    Article Snippet: The infectious titer of lentivirus was determined by a Lenti-X qRT-PCR Titration Kit (Clontech, Mountain View, CA).

    Techniques: Quantitative RT-PCR, Tube Formation Assay, Standard Deviation

    G28A mutants can replicate efficiently in an Ago2-independent manner. (A) Intracellular HCV-RNA levels (left panel) and infectious titers in the culture supernatants (right panel) of Huh7-122KO and Huh7-122KOR cells infected with either HCV or HCV 122KO in the presence of either control-LNA or LNA-miR-122 were determined at 72 hpi. (B) Ago2 complexes in 751-122KO and Huh7.5.1 cells infected with HCV were immunoprecipitated by either anti-IgG or anti-Ago2 mouse antibody at 12 dpi. Levels of Ago2 and HCV-RNA in the precipitates were determined by immunoblotting and qRT-PCR, respectively. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P

    Journal: PLoS Pathogens

    Article Title: Characterization of miR-122-independent propagation of HCV

    doi: 10.1371/journal.ppat.1006374

    Figure Lengend Snippet: G28A mutants can replicate efficiently in an Ago2-independent manner. (A) Intracellular HCV-RNA levels (left panel) and infectious titers in the culture supernatants (right panel) of Huh7-122KO and Huh7-122KOR cells infected with either HCV or HCV 122KO in the presence of either control-LNA or LNA-miR-122 were determined at 72 hpi. (B) Ago2 complexes in 751-122KO and Huh7.5.1 cells infected with HCV were immunoprecipitated by either anti-IgG or anti-Ago2 mouse antibody at 12 dpi. Levels of Ago2 and HCV-RNA in the precipitates were determined by immunoblotting and qRT-PCR, respectively. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P

    Article Snippet: The infectious titer of lentivirus was determined by a Lenti-X qRT-PCR Titration Kit (Clontech, Mountain View, CA).

    Techniques: Infection, Immunoprecipitation, Quantitative RT-PCR, Standard Deviation

    Establishment of replicon cells derived from Huh7-122KO cells. (A) Subgenomic HCV replicon RNA was electroporated into Huh7-122KO and Huh7-122KOR cells, or into Huh7-122KO cells together with control- or miR-122-mimic, and G418-resistant colonies were stained with crystal violet at 21 days post-transduction. (B) Expression of miR-122 in Huh7-122KO cells electroporated with either control-mimic or miR-122-mimic at 72 h post-electroporation. Relative expression of miR-122 was determined by qRT-PCR by using U6 snRNA as an internal control. (C) Each of the three clones derived from each type of replicon cells was subjected to qRT-PCR after extraction of total RNA (top) and to immunoblotting by using anti-NS5A and β-actin (middle). The relative expression of miR-122 was determined by qRT-PCR by using U6 snRNA as an internal control (bottom). (D) Intracellular HCV-RNA replication in Huh7-122KO-SGR cells (#1, #3, #5) and Huh7-122KOR-SGR cells (#1, #5, #6) in the presence of 20 nM of either LNA-control or LNA-miR122 was determined by qRT-PCR. (E) The Ago2 complex was immunoprecipitated from Huh7-122KO-SGR#1 and Huh7-122KOR-SGR#1 cells by using either anti-IgG or anti-Ago2 mouse antibody. The HCV-RNA associated with Ago2 was determined by qRT-PCR and Ago2 was detected by immunoblotting. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P

    Journal: PLoS Pathogens

    Article Title: Characterization of miR-122-independent propagation of HCV

    doi: 10.1371/journal.ppat.1006374

    Figure Lengend Snippet: Establishment of replicon cells derived from Huh7-122KO cells. (A) Subgenomic HCV replicon RNA was electroporated into Huh7-122KO and Huh7-122KOR cells, or into Huh7-122KO cells together with control- or miR-122-mimic, and G418-resistant colonies were stained with crystal violet at 21 days post-transduction. (B) Expression of miR-122 in Huh7-122KO cells electroporated with either control-mimic or miR-122-mimic at 72 h post-electroporation. Relative expression of miR-122 was determined by qRT-PCR by using U6 snRNA as an internal control. (C) Each of the three clones derived from each type of replicon cells was subjected to qRT-PCR after extraction of total RNA (top) and to immunoblotting by using anti-NS5A and β-actin (middle). The relative expression of miR-122 was determined by qRT-PCR by using U6 snRNA as an internal control (bottom). (D) Intracellular HCV-RNA replication in Huh7-122KO-SGR cells (#1, #3, #5) and Huh7-122KOR-SGR cells (#1, #5, #6) in the presence of 20 nM of either LNA-control or LNA-miR122 was determined by qRT-PCR. (E) The Ago2 complex was immunoprecipitated from Huh7-122KO-SGR#1 and Huh7-122KOR-SGR#1 cells by using either anti-IgG or anti-Ago2 mouse antibody. The HCV-RNA associated with Ago2 was determined by qRT-PCR and Ago2 was detected by immunoblotting. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P

    Article Snippet: The infectious titer of lentivirus was determined by a Lenti-X qRT-PCR Titration Kit (Clontech, Mountain View, CA).

    Techniques: Derivative Assay, Staining, Transduction, Expressing, Electroporation, Quantitative RT-PCR, Clone Assay, Immunoprecipitation, Standard Deviation

    miR-122-independent HCV replication in Huh7-122KO cells. (A) HCV was inoculated into Huh7-122KO and Huh7-122KOR cells at an MOI of 3, and intracellular HCV-RNA levels (left panel) and infectious titers in the culture supernatants (right panel) were determined by qRT-PCR and focus formation assay, respectively. (B) HCV-RNA replication was inhibited by the treatment with IFNα, BILN, BMS790052, PSI7977 and anti-CD81 antibody. (C) HCV replication in Huh7-122KO cells was resistant to treatment with an miR-122 inhibitor, LNA (HCV-RNA replication: left; infectious titer: right). Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P

    Journal: PLoS Pathogens

    Article Title: Characterization of miR-122-independent propagation of HCV

    doi: 10.1371/journal.ppat.1006374

    Figure Lengend Snippet: miR-122-independent HCV replication in Huh7-122KO cells. (A) HCV was inoculated into Huh7-122KO and Huh7-122KOR cells at an MOI of 3, and intracellular HCV-RNA levels (left panel) and infectious titers in the culture supernatants (right panel) were determined by qRT-PCR and focus formation assay, respectively. (B) HCV-RNA replication was inhibited by the treatment with IFNα, BILN, BMS790052, PSI7977 and anti-CD81 antibody. (C) HCV replication in Huh7-122KO cells was resistant to treatment with an miR-122 inhibitor, LNA (HCV-RNA replication: left; infectious titer: right). Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P

    Article Snippet: The infectious titer of lentivirus was determined by a Lenti-X qRT-PCR Titration Kit (Clontech, Mountain View, CA).

    Techniques: Quantitative RT-PCR, Tube Formation Assay, Standard Deviation

    Targeted Rasal1 promoter demethylation by dCas9/dHFCas9-TET3CD- Rasal1 -sgRNA fusion protein ameliorates kidney fibrosis. a Schematic showing the parenchymal injection of lentiviral particles containing dCas9/dHFCas9-TET3CD fusion protein into UUO-challenged kidneys. b Schedule of UUO mouse surgery, lentivirus injection, and analysis. c qRT-PCR results showing that Rasal1 mRNA expression was significantly induced in UUO-challenged kidneys transduced with dCas9/dHFCas9-TET3CD- Rasal1 -sgRNA, but not in UUO-challenged kidneys transduced with control dCas9/dHFCas9-TET3CD- LacZ- sgRNA. There is no significant difference between dCas9-TET3CD and dHFCas9-TET3CD constructs. Results were normalized to reference gene Gapdh (expression is presented as mean value; error bars represent S.D., n ≥ 5 in each group, # not significant, *** p

    Journal: Nature Communications

    Article Title: High-fidelity CRISPR/Cas9- based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis

    doi: 10.1038/s41467-018-05766-5

    Figure Lengend Snippet: Targeted Rasal1 promoter demethylation by dCas9/dHFCas9-TET3CD- Rasal1 -sgRNA fusion protein ameliorates kidney fibrosis. a Schematic showing the parenchymal injection of lentiviral particles containing dCas9/dHFCas9-TET3CD fusion protein into UUO-challenged kidneys. b Schedule of UUO mouse surgery, lentivirus injection, and analysis. c qRT-PCR results showing that Rasal1 mRNA expression was significantly induced in UUO-challenged kidneys transduced with dCas9/dHFCas9-TET3CD- Rasal1 -sgRNA, but not in UUO-challenged kidneys transduced with control dCas9/dHFCas9-TET3CD- LacZ- sgRNA. There is no significant difference between dCas9-TET3CD and dHFCas9-TET3CD constructs. Results were normalized to reference gene Gapdh (expression is presented as mean value; error bars represent S.D., n ≥ 5 in each group, # not significant, *** p

    Article Snippet: Lentivirus titration was determined by the Lenti-X qRT-PCR Titration Kit (Clonetech, Heidelberg, Germany) and 8 μg/ml Polybrene (Sigma- Aldrich, Munich, Germany) was added to the viral solution for the in vivo and in vitro transduction experiments.

    Techniques: Injection, Quantitative RT-PCR, Expressing, Transduction, Construct

    Induction of Klotho promoter hydroxymethylation by dHFCas9-TET3CD- Klotho -sgRNA in tubular epithelial cells ameliorates kidney fibrosis. a Schematic shows retrograde ureter injection of lentiviral particles containing dHFCas9-TET3CD- Klotho -sgRNA into UUO-operated kidneys. b Schedule of UUO mouse surgery, lentivirus injection, and analysis. c qRT-PCR results showing that Kl mRNA expression was significantly induced in UUO-operated kidneys transduced with dHFCas9-TET3CD- Kl -sgRNA but not in UUO-challenged kidneys transduced with LacZ- sgRNA control. Results were normalized to reference gene Gapdh (expression is presented as mean value, error bars represent S.E.M. , n = 6 in each group, *** p

    Journal: Nature Communications

    Article Title: High-fidelity CRISPR/Cas9- based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis

    doi: 10.1038/s41467-018-05766-5

    Figure Lengend Snippet: Induction of Klotho promoter hydroxymethylation by dHFCas9-TET3CD- Klotho -sgRNA in tubular epithelial cells ameliorates kidney fibrosis. a Schematic shows retrograde ureter injection of lentiviral particles containing dHFCas9-TET3CD- Klotho -sgRNA into UUO-operated kidneys. b Schedule of UUO mouse surgery, lentivirus injection, and analysis. c qRT-PCR results showing that Kl mRNA expression was significantly induced in UUO-operated kidneys transduced with dHFCas9-TET3CD- Kl -sgRNA but not in UUO-challenged kidneys transduced with LacZ- sgRNA control. Results were normalized to reference gene Gapdh (expression is presented as mean value, error bars represent S.E.M. , n = 6 in each group, *** p

    Article Snippet: Lentivirus titration was determined by the Lenti-X qRT-PCR Titration Kit (Clonetech, Heidelberg, Germany) and 8 μg/ml Polybrene (Sigma- Aldrich, Munich, Germany) was added to the viral solution for the in vivo and in vitro transduction experiments.

    Techniques: Injection, Quantitative RT-PCR, Expressing, Transduction

    Rasal1 gene disruption results in aggravated fibrosis level in the UUO model. a Schematic of knockout-first strategy for Rasal1 gene. The gene trapping LacZ cassette is alternatively spliced with exon 2 mediated by a splicing acceptor (SA). A promoter-driven Neomycin cassette is inserted after LacZ . The targeted exon 3 and 4 are flanked by loxP sites. Black arrows indicate the location of genotyping primers. b qRT-PCR analysis shows the Rasal1 mRNA expression in homozygous mice are significantly reduced as compared with wild-type mice, while there is no significant reduction in heterozygous mice. The data are presented as mean value, error bars represent S.D., n.s. not significant, *** p

    Journal: Nature Communications

    Article Title: High-fidelity CRISPR/Cas9- based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis

    doi: 10.1038/s41467-018-05766-5

    Figure Lengend Snippet: Rasal1 gene disruption results in aggravated fibrosis level in the UUO model. a Schematic of knockout-first strategy for Rasal1 gene. The gene trapping LacZ cassette is alternatively spliced with exon 2 mediated by a splicing acceptor (SA). A promoter-driven Neomycin cassette is inserted after LacZ . The targeted exon 3 and 4 are flanked by loxP sites. Black arrows indicate the location of genotyping primers. b qRT-PCR analysis shows the Rasal1 mRNA expression in homozygous mice are significantly reduced as compared with wild-type mice, while there is no significant reduction in heterozygous mice. The data are presented as mean value, error bars represent S.D., n.s. not significant, *** p

    Article Snippet: Lentivirus titration was determined by the Lenti-X qRT-PCR Titration Kit (Clonetech, Heidelberg, Germany) and 8 μg/ml Polybrene (Sigma- Aldrich, Munich, Germany) was added to the viral solution for the in vivo and in vitro transduction experiments.

    Techniques: Knock-Out, Quantitative RT-PCR, Expressing, Mouse Assay