Structured Review

Addgene inc piresneo flag ha ago4
KEY RESOURCES TABLE
Piresneo Flag Ha Ago4, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/piresneo flag ha ago4/product/Addgene inc
Average 91 stars, based on 1 article reviews
Price from $9.99 to $1999.99
piresneo flag ha ago4 - by Bioz Stars, 2024-04
91/100 stars

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1) Product Images from "Structural Basis for Target-Directed MicroRNA Degradation"

Article Title: Structural Basis for Target-Directed MicroRNA Degradation

Journal: Molecular cell

doi: 10.1016/j.molcel.2019.06.019

KEY RESOURCES TABLE
Figure Legend Snippet: KEY RESOURCES TABLE

Techniques Used: Western Blot, Protease Inhibitor, Reporter Assay, Expressing, Recombinant, Software, Transduction, Plasmid Preparation, Construct


Structured Review

Addgene inc plasmid flag ha tagged ago4
Viral titers and viral RNA of (A) influenza-A-infected (PR/8/1934), (B) encephalomyocarditis (EMCV)-infected, or (C) vesicular stomatitis virus (VSV)-infected (indicated multiplicities of infection [MOIs] for 16 h) bone-marrow-derived macrophages (BMDMs) from Ago1 +/+ (black) and Ago1 −/− (yellow), (D–F) Ago3 +/+ (black) and Ago3 −/− (green), or (G–I) <t>Ago4</t> +/+ (black) and Ago4 −/− (red) littermate mice. Representative plaque assay images are shown. Influenza Ns1, EMCV 2A-2B , or VSV G RNA levels were quantified by qPCR relative to TATA box protein ( Tbp ). All data are from two or three biological replicates performed in triplicate and combined. Errors bars represent SEM. *p < 0.05, **p < 0.01, and ***p < 0.001, as measured by two-way ANOVA with Bonferroni multiple comparison test.
Plasmid Flag Ha Tagged Ago4, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/plasmid flag ha tagged ago4/product/Addgene inc
Average 91 stars, based on 1 article reviews
Price from $9.99 to $1999.99
plasmid flag ha tagged ago4 - by Bioz Stars, 2024-04
91/100 stars

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1) Product Images from "A Requirement for Argonaute 4 in Mammalian Antiviral Defense"

Article Title: A Requirement for Argonaute 4 in Mammalian Antiviral Defense

Journal: Cell reports

doi: 10.1016/j.celrep.2020.01.021

Viral titers and viral RNA of (A) influenza-A-infected (PR/8/1934), (B) encephalomyocarditis (EMCV)-infected, or (C) vesicular stomatitis virus (VSV)-infected (indicated multiplicities of infection [MOIs] for 16 h) bone-marrow-derived macrophages (BMDMs) from Ago1 +/+ (black) and Ago1 −/− (yellow), (D–F) Ago3 +/+ (black) and Ago3 −/− (green), or (G–I) Ago4 +/+ (black) and Ago4 −/− (red) littermate mice. Representative plaque assay images are shown. Influenza Ns1, EMCV 2A-2B , or VSV G RNA levels were quantified by qPCR relative to TATA box protein ( Tbp ). All data are from two or three biological replicates performed in triplicate and combined. Errors bars represent SEM. *p < 0.05, **p < 0.01, and ***p < 0.001, as measured by two-way ANOVA with Bonferroni multiple comparison test.
Figure Legend Snippet: Viral titers and viral RNA of (A) influenza-A-infected (PR/8/1934), (B) encephalomyocarditis (EMCV)-infected, or (C) vesicular stomatitis virus (VSV)-infected (indicated multiplicities of infection [MOIs] for 16 h) bone-marrow-derived macrophages (BMDMs) from Ago1 +/+ (black) and Ago1 −/− (yellow), (D–F) Ago3 +/+ (black) and Ago3 −/− (green), or (G–I) Ago4 +/+ (black) and Ago4 −/− (red) littermate mice. Representative plaque assay images are shown. Influenza Ns1, EMCV 2A-2B , or VSV G RNA levels were quantified by qPCR relative to TATA box protein ( Tbp ). All data are from two or three biological replicates performed in triplicate and combined. Errors bars represent SEM. *p < 0.05, **p < 0.01, and ***p < 0.001, as measured by two-way ANOVA with Bonferroni multiple comparison test.

Techniques Used: Infection, Derivative Assay, Plaque Assay

(A) Flow cytometry of HEK293T transfected with empty vector (EV) or indicated concentrations of Ago4-Myc for 24 h prior to infection with influenza-GFP at MOI 1 for 24 h. GFP percentages of 7-AAD − (non-apoptotic) cells are indicated. Right: mean and SEM of percentage of Flu-GFP relative to EV control. (B and C) Ago4 and influenza Ns1 RNA expression in HEK293T transfected with EV or 10 μg of Ago4-Myc and infected with Influenza A (MOI 1, 24 h p.i.). (D) Myc immunoblot of Ago1, Ago3, or Ago4 transfected HEK293T cells. (E) Percentage of Flu-GFP + cells in HEK293T cells transfected with 10 μg of the indicated plasmid and infected at MOI 1 for 24 h. 7-AAD − , relative to EV. (F) Influenza viral titers of mouse embryonic fibroblasts (MEFs) transfected with a negative control (NC) siRNA or vsiRNA against NS1. Data are from three biological replicates performed in triplicate and combined. Errors bars represent SEM. *p < 0.05, **p < 0.01, and ***p < 0.001, as measured by an unpaired t test or one-way ANOVA with Bonferroni multiple comparison test.
Figure Legend Snippet: (A) Flow cytometry of HEK293T transfected with empty vector (EV) or indicated concentrations of Ago4-Myc for 24 h prior to infection with influenza-GFP at MOI 1 for 24 h. GFP percentages of 7-AAD − (non-apoptotic) cells are indicated. Right: mean and SEM of percentage of Flu-GFP relative to EV control. (B and C) Ago4 and influenza Ns1 RNA expression in HEK293T transfected with EV or 10 μg of Ago4-Myc and infected with Influenza A (MOI 1, 24 h p.i.). (D) Myc immunoblot of Ago1, Ago3, or Ago4 transfected HEK293T cells. (E) Percentage of Flu-GFP + cells in HEK293T cells transfected with 10 μg of the indicated plasmid and infected at MOI 1 for 24 h. 7-AAD − , relative to EV. (F) Influenza viral titers of mouse embryonic fibroblasts (MEFs) transfected with a negative control (NC) siRNA or vsiRNA against NS1. Data are from three biological replicates performed in triplicate and combined. Errors bars represent SEM. *p < 0.05, **p < 0.01, and ***p < 0.001, as measured by an unpaired t test or one-way ANOVA with Bonferroni multiple comparison test.

Techniques Used: Flow Cytometry, Transfection, Plasmid Preparation, Infection, RNA Expression, Western Blot, Negative Control


Figure Legend Snippet:

Techniques Used: Western Blot, Recombinant, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Plasmid Preparation, Software


Structured Review

Addgene inc piresneo flag ha ago4 plasmid
Association of the <t>AGO4-binding</t> genes and methylation levels. (A) Scheme showing that tetracycline-treated cells switch off AGO4 protein expression (Tet+) from day 1 to 12 and that 5’-azacytidine treatment from day 7 to 9 inhibits DNA methyltransferase (5-AC). (B) Detection of AGO4 mRNA in Tet- (AGO4 expression) and Tet+ (No AGO4 expression) cells; AGO4 was significantly expressed in the absence of Tet. (C) Confirmation of AGO4-binding genes showed that AGO4 only bounds to the selected gene from bioinformatics data as exemplified by C16ORF89 . (D) However, AGO4 did not bind to a gene lacking the AGO4-binding site, MSN . (E) The AGO4 methylation level of the gene, which contains AGO4-binding sites, was increased due to the presence of the AGO4 protein (Tet-). This increased methylation level was not associated with the presence of DNA methyltransferase (5-AC+). (F) These results were not observed for a gene lacking AGO4-binding sites, MSN . (G to H) Recovery of DNA methylation levels is found in cells expressing the AGO4 protein (Tet-) with 5’-azacytidine withdrawal in both of AGO4-binding gene, C16ORF89 and non-AGO4-binding gene, MSN ; AGO4 is inferred to methylate previously demethylated loci. The above data are the representative dataset from three independent experiments presented as the mean ± SEM. Statistical analyses were performed using a paired-sample t -test, where p < 0.05, p < 0.01, and p < 0.005 are represented as *, **, and ***, respectively, where Tet+ means tetracycline treatment, 5-AC+ means azacytidine treatment, and Tet- and 5-AC- mean untreated groups (I). Schematic overview of the role of AGO4 in de novo methylation. After DNA replication, DNA methylation is maintained by DNMT1 by the recognition of hemimethylated CpG on the parental strand as a template and the addition of newly methylated CpG to the daughter strand. DNMT1 is inhibited by the action of 5-AC, and the methylation level should be decreased by half under 5-AC treatment. In our study, when tetracycline was added, AGO4 shRNA was manipulated, resulting in AGO4 expression repression. Thus, without tetracycline, AGO4 is upregulated and binds to DNA loci, and DNA methylation is maintained, although 5-AC is added, suggesting that AGO4 involved in de novo methylation
Piresneo Flag Ha Ago4 Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/piresneo flag ha ago4 plasmid/product/Addgene inc
Average 91 stars, based on 1 article reviews
Price from $9.99 to $1999.99
piresneo flag ha ago4 plasmid - by Bioz Stars, 2024-04
91/100 stars

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1) Product Images from "Argonaute 4 as an Effector Protein in RNA-Directed DNA Methylation in Human Cells"

Article Title: Argonaute 4 as an Effector Protein in RNA-Directed DNA Methylation in Human Cells

Journal: Frontiers in Genetics

doi: 10.3389/fgene.2019.00645

Association of the AGO4-binding genes and methylation levels. (A) Scheme showing that tetracycline-treated cells switch off AGO4 protein expression (Tet+) from day 1 to 12 and that 5’-azacytidine treatment from day 7 to 9 inhibits DNA methyltransferase (5-AC). (B) Detection of AGO4 mRNA in Tet- (AGO4 expression) and Tet+ (No AGO4 expression) cells; AGO4 was significantly expressed in the absence of Tet. (C) Confirmation of AGO4-binding genes showed that AGO4 only bounds to the selected gene from bioinformatics data as exemplified by C16ORF89 . (D) However, AGO4 did not bind to a gene lacking the AGO4-binding site, MSN . (E) The AGO4 methylation level of the gene, which contains AGO4-binding sites, was increased due to the presence of the AGO4 protein (Tet-). This increased methylation level was not associated with the presence of DNA methyltransferase (5-AC+). (F) These results were not observed for a gene lacking AGO4-binding sites, MSN . (G to H) Recovery of DNA methylation levels is found in cells expressing the AGO4 protein (Tet-) with 5’-azacytidine withdrawal in both of AGO4-binding gene, C16ORF89 and non-AGO4-binding gene, MSN ; AGO4 is inferred to methylate previously demethylated loci. The above data are the representative dataset from three independent experiments presented as the mean ± SEM. Statistical analyses were performed using a paired-sample t -test, where p < 0.05, p < 0.01, and p < 0.005 are represented as *, **, and ***, respectively, where Tet+ means tetracycline treatment, 5-AC+ means azacytidine treatment, and Tet- and 5-AC- mean untreated groups (I). Schematic overview of the role of AGO4 in de novo methylation. After DNA replication, DNA methylation is maintained by DNMT1 by the recognition of hemimethylated CpG on the parental strand as a template and the addition of newly methylated CpG to the daughter strand. DNMT1 is inhibited by the action of 5-AC, and the methylation level should be decreased by half under 5-AC treatment. In our study, when tetracycline was added, AGO4 shRNA was manipulated, resulting in AGO4 expression repression. Thus, without tetracycline, AGO4 is upregulated and binds to DNA loci, and DNA methylation is maintained, although 5-AC is added, suggesting that AGO4 involved in de novo methylation
Figure Legend Snippet: Association of the AGO4-binding genes and methylation levels. (A) Scheme showing that tetracycline-treated cells switch off AGO4 protein expression (Tet+) from day 1 to 12 and that 5’-azacytidine treatment from day 7 to 9 inhibits DNA methyltransferase (5-AC). (B) Detection of AGO4 mRNA in Tet- (AGO4 expression) and Tet+ (No AGO4 expression) cells; AGO4 was significantly expressed in the absence of Tet. (C) Confirmation of AGO4-binding genes showed that AGO4 only bounds to the selected gene from bioinformatics data as exemplified by C16ORF89 . (D) However, AGO4 did not bind to a gene lacking the AGO4-binding site, MSN . (E) The AGO4 methylation level of the gene, which contains AGO4-binding sites, was increased due to the presence of the AGO4 protein (Tet-). This increased methylation level was not associated with the presence of DNA methyltransferase (5-AC+). (F) These results were not observed for a gene lacking AGO4-binding sites, MSN . (G to H) Recovery of DNA methylation levels is found in cells expressing the AGO4 protein (Tet-) with 5’-azacytidine withdrawal in both of AGO4-binding gene, C16ORF89 and non-AGO4-binding gene, MSN ; AGO4 is inferred to methylate previously demethylated loci. The above data are the representative dataset from three independent experiments presented as the mean ± SEM. Statistical analyses were performed using a paired-sample t -test, where p < 0.05, p < 0.01, and p < 0.005 are represented as *, **, and ***, respectively, where Tet+ means tetracycline treatment, 5-AC+ means azacytidine treatment, and Tet- and 5-AC- mean untreated groups (I). Schematic overview of the role of AGO4 in de novo methylation. After DNA replication, DNA methylation is maintained by DNMT1 by the recognition of hemimethylated CpG on the parental strand as a template and the addition of newly methylated CpG to the daughter strand. DNMT1 is inhibited by the action of 5-AC, and the methylation level should be decreased by half under 5-AC treatment. In our study, when tetracycline was added, AGO4 shRNA was manipulated, resulting in AGO4 expression repression. Thus, without tetracycline, AGO4 is upregulated and binds to DNA loci, and DNA methylation is maintained, although 5-AC is added, suggesting that AGO4 involved in de novo methylation

Techniques Used: Binding Assay, Methylation, Expressing, DNA Methylation Assay, shRNA

Alu methylation upon Alu siRNA transfection in Tet-controlled AGO4-expressing cells. The Alu methylation level was significantly increased when AGO4 was upregulated under Alu siRNA transfection. The above data are the representative dataset from six independent experiments presented as the mean ± SEM. Statistical analysis was performed using an unpaired t -test where p < 0.01 is represented as **.
Figure Legend Snippet: Alu methylation upon Alu siRNA transfection in Tet-controlled AGO4-expressing cells. The Alu methylation level was significantly increased when AGO4 was upregulated under Alu siRNA transfection. The above data are the representative dataset from six independent experiments presented as the mean ± SEM. Statistical analysis was performed using an unpaired t -test where p < 0.01 is represented as **.

Techniques Used: Methylation, Transfection, Expressing

Overexpression of HA-AGO4 increases global genomic methylation. (A) Confirmation of AGO4 mRNA expression in AGO4- or PC-overexpressing cells after transfection for 72 h by using real-time PCR showed that AGO4 mRNA was significantly upregulated in AGO4-overexpressing cells, and (B) Western blot analysis showed the expression of the HA-AGO4 protein in a time-course experiment. β-Actin was used to confirm equal protein loading of each lane. AGO4 protein expression was at the highest level at 48 h after transfection. (C) Overexpression of HA-AGO4 resulted in a significant increase in interspersed repetitive sequence methylation in the input (cell lysate) HA-AGO4 compared with untaransfected HeLa cells, detected at 72 h post-transfection. The above data in A and C are the representative dataset from five independent experiments presented as the mean ± SEM. Statistical analyses were performed using a paired t -test where p < 0.05, p < 0.01, and p < 0.005 are represented as *, **, and ***, respectively.
Figure Legend Snippet: Overexpression of HA-AGO4 increases global genomic methylation. (A) Confirmation of AGO4 mRNA expression in AGO4- or PC-overexpressing cells after transfection for 72 h by using real-time PCR showed that AGO4 mRNA was significantly upregulated in AGO4-overexpressing cells, and (B) Western blot analysis showed the expression of the HA-AGO4 protein in a time-course experiment. β-Actin was used to confirm equal protein loading of each lane. AGO4 protein expression was at the highest level at 48 h after transfection. (C) Overexpression of HA-AGO4 resulted in a significant increase in interspersed repetitive sequence methylation in the input (cell lysate) HA-AGO4 compared with untaransfected HeLa cells, detected at 72 h post-transfection. The above data in A and C are the representative dataset from five independent experiments presented as the mean ± SEM. Statistical analyses were performed using a paired t -test where p < 0.05, p < 0.01, and p < 0.005 are represented as *, **, and ***, respectively.

Techniques Used: Over Expression, Methylation, Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Sequencing

AGO4 localization is involved in RdDM by introducing genomic DNA methylation in human cells. (A) ChIP experiments using a hemagglutinin (HA) antibody showed elevation of the levels of AGO4-binding LINE-1 or ALU found in AGO4-overexpressing cells compared with their control counterparts. (B) Moreover, the binding of the AGO4 protein to interspersed repetitive sequence increased DNA methylation.
Figure Legend Snippet: AGO4 localization is involved in RdDM by introducing genomic DNA methylation in human cells. (A) ChIP experiments using a hemagglutinin (HA) antibody showed elevation of the levels of AGO4-binding LINE-1 or ALU found in AGO4-overexpressing cells compared with their control counterparts. (B) Moreover, the binding of the AGO4 protein to interspersed repetitive sequence increased DNA methylation.

Techniques Used: DNA Methylation Assay, Binding Assay, Sequencing

AGO4 protein specifically induced DNA methylation of target loci. By using CPP-AGO4 conjugated with Alu sgRNA (CPP-AGO4-Alu) transfected to HeLa cells, we found that (A) Alu methylation level was significantly increased in CPP-AGO4-Alu transfected cells, while Alu methylation was not promoted in control counterpart groups. (B) While LINE-1 methylation level was not changed, this means the complex has no off targets to LINE-1. The representative dataset was obtained from five independent experiments. Statistical analyses were performed using a paired-sample T -test where p < 0.05 and p < 0.01 are represented as * and **, respectively, where buffer means non-inducible CPP-AGO4 transfection.
Figure Legend Snippet: AGO4 protein specifically induced DNA methylation of target loci. By using CPP-AGO4 conjugated with Alu sgRNA (CPP-AGO4-Alu) transfected to HeLa cells, we found that (A) Alu methylation level was significantly increased in CPP-AGO4-Alu transfected cells, while Alu methylation was not promoted in control counterpart groups. (B) While LINE-1 methylation level was not changed, this means the complex has no off targets to LINE-1. The representative dataset was obtained from five independent experiments. Statistical analyses were performed using a paired-sample T -test where p < 0.05 and p < 0.01 are represented as * and **, respectively, where buffer means non-inducible CPP-AGO4 transfection.

Techniques Used: DNA Methylation Assay, Transfection, Methylation


Structured Review

Addgene inc piresneo flag ha ago4
Proposed function of sli-RISCs versus si-RISCs. ( A ) Northern blotting to detect chemically synthesized sli-887 and si-887. Processed products of sli-887 are longer than the 21-mer si-887. ( B ) Northern blotting to detect sli-887 and the 25-nt form of sli-887 (nts 1 to 25). Processed products of sli-887 are shorter than the 25-mer form. ( C ) Northern blotting to detect processed sli-1148 from a stably expressed sli-1148 driven by a U6 promoter, which mainly exists in an approximately 30-mer isoform, in HCT116 cells. ( D ) Proposed model of sli-RISC versus si-RISC function: si-RISCs can involve three non-slicing Agos (Ago1, Ago3, and <t>Ago4)</t> and slicing Ago2 loaded with uniform 21-mer guide RNAs; sli-RISCs contain the slicing Ago2 loaded with guide RNAs with various 3′ end lengths, and silencing may couple with 3′ end trimming/tailing by PARN. After the passenger strand is sliced, the hairpin might open during target binding, allowing nts 19 to 22 to base pair (open hairpin hypothesis), or the loop from nts 19 to 22 might remain, allowing only nts 2 to 14 to be used for target binding (partial hairpin hypothesis).
Piresneo Flag Ha Ago4, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/piresneo flag ha ago4/product/Addgene inc
Average 91 stars, based on 1 article reviews
Price from $9.99 to $1999.99
piresneo flag ha ago4 - by Bioz Stars, 2024-04
91/100 stars

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1) Product Images from "Differences in silencing of mismatched targets by sliced versus diced siRNAs"

Article Title: Differences in silencing of mismatched targets by sliced versus diced siRNAs

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky287

Proposed function of sli-RISCs versus si-RISCs. ( A ) Northern blotting to detect chemically synthesized sli-887 and si-887. Processed products of sli-887 are longer than the 21-mer si-887. ( B ) Northern blotting to detect sli-887 and the 25-nt form of sli-887 (nts 1 to 25). Processed products of sli-887 are shorter than the 25-mer form. ( C ) Northern blotting to detect processed sli-1148 from a stably expressed sli-1148 driven by a U6 promoter, which mainly exists in an approximately 30-mer isoform, in HCT116 cells. ( D ) Proposed model of sli-RISC versus si-RISC function: si-RISCs can involve three non-slicing Agos (Ago1, Ago3, and Ago4) and slicing Ago2 loaded with uniform 21-mer guide RNAs; sli-RISCs contain the slicing Ago2 loaded with guide RNAs with various 3′ end lengths, and silencing may couple with 3′ end trimming/tailing by PARN. After the passenger strand is sliced, the hairpin might open during target binding, allowing nts 19 to 22 to base pair (open hairpin hypothesis), or the loop from nts 19 to 22 might remain, allowing only nts 2 to 14 to be used for target binding (partial hairpin hypothesis).
Figure Legend Snippet: Proposed function of sli-RISCs versus si-RISCs. ( A ) Northern blotting to detect chemically synthesized sli-887 and si-887. Processed products of sli-887 are longer than the 21-mer si-887. ( B ) Northern blotting to detect sli-887 and the 25-nt form of sli-887 (nts 1 to 25). Processed products of sli-887 are shorter than the 25-mer form. ( C ) Northern blotting to detect processed sli-1148 from a stably expressed sli-1148 driven by a U6 promoter, which mainly exists in an approximately 30-mer isoform, in HCT116 cells. ( D ) Proposed model of sli-RISC versus si-RISC function: si-RISCs can involve three non-slicing Agos (Ago1, Ago3, and Ago4) and slicing Ago2 loaded with uniform 21-mer guide RNAs; sli-RISCs contain the slicing Ago2 loaded with guide RNAs with various 3′ end lengths, and silencing may couple with 3′ end trimming/tailing by PARN. After the passenger strand is sliced, the hairpin might open during target binding, allowing nts 19 to 22 to base pair (open hairpin hypothesis), or the loop from nts 19 to 22 might remain, allowing only nts 2 to 14 to be used for target binding (partial hairpin hypothesis).

Techniques Used: Northern Blot, Synthesized, Stable Transfection, Binding Assay


Structured Review

Addgene inc piresneo flag ha ago1
Proposed function of sli-RISCs versus si-RISCs. ( A ) Northern blotting to detect chemically synthesized sli-887 and si-887. Processed products of sli-887 are longer than the 21-mer si-887. ( B ) Northern blotting to detect sli-887 and the 25-nt form of sli-887 (nts 1 to 25). Processed products of sli-887 are shorter than the 25-mer form. ( C ) Northern blotting to detect processed sli-1148 from a stably expressed sli-1148 driven by a U6 promoter, which mainly exists in an approximately 30-mer isoform, in HCT116 cells. ( D ) Proposed model of sli-RISC versus si-RISC function: si-RISCs can involve three non-slicing Agos <t>(Ago1,</t> Ago3, and Ago4) and slicing Ago2 loaded with uniform 21-mer guide RNAs; sli-RISCs contain the slicing Ago2 loaded with guide RNAs with various 3′ end lengths, and silencing may couple with 3′ end trimming/tailing by PARN. After the passenger strand is sliced, the hairpin might open during target binding, allowing nts 19 to 22 to base pair (open hairpin hypothesis), or the loop from nts 19 to 22 might remain, allowing only nts 2 to 14 to be used for target binding (partial hairpin hypothesis).
Piresneo Flag Ha Ago1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/piresneo flag ha ago1/product/Addgene inc
Average 91 stars, based on 1 article reviews
Price from $9.99 to $1999.99
piresneo flag ha ago1 - by Bioz Stars, 2024-04
91/100 stars

Images

1) Product Images from "Differences in silencing of mismatched targets by sliced versus diced siRNAs"

Article Title: Differences in silencing of mismatched targets by sliced versus diced siRNAs

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky287

Proposed function of sli-RISCs versus si-RISCs. ( A ) Northern blotting to detect chemically synthesized sli-887 and si-887. Processed products of sli-887 are longer than the 21-mer si-887. ( B ) Northern blotting to detect sli-887 and the 25-nt form of sli-887 (nts 1 to 25). Processed products of sli-887 are shorter than the 25-mer form. ( C ) Northern blotting to detect processed sli-1148 from a stably expressed sli-1148 driven by a U6 promoter, which mainly exists in an approximately 30-mer isoform, in HCT116 cells. ( D ) Proposed model of sli-RISC versus si-RISC function: si-RISCs can involve three non-slicing Agos (Ago1, Ago3, and Ago4) and slicing Ago2 loaded with uniform 21-mer guide RNAs; sli-RISCs contain the slicing Ago2 loaded with guide RNAs with various 3′ end lengths, and silencing may couple with 3′ end trimming/tailing by PARN. After the passenger strand is sliced, the hairpin might open during target binding, allowing nts 19 to 22 to base pair (open hairpin hypothesis), or the loop from nts 19 to 22 might remain, allowing only nts 2 to 14 to be used for target binding (partial hairpin hypothesis).
Figure Legend Snippet: Proposed function of sli-RISCs versus si-RISCs. ( A ) Northern blotting to detect chemically synthesized sli-887 and si-887. Processed products of sli-887 are longer than the 21-mer si-887. ( B ) Northern blotting to detect sli-887 and the 25-nt form of sli-887 (nts 1 to 25). Processed products of sli-887 are shorter than the 25-mer form. ( C ) Northern blotting to detect processed sli-1148 from a stably expressed sli-1148 driven by a U6 promoter, which mainly exists in an approximately 30-mer isoform, in HCT116 cells. ( D ) Proposed model of sli-RISC versus si-RISC function: si-RISCs can involve three non-slicing Agos (Ago1, Ago3, and Ago4) and slicing Ago2 loaded with uniform 21-mer guide RNAs; sli-RISCs contain the slicing Ago2 loaded with guide RNAs with various 3′ end lengths, and silencing may couple with 3′ end trimming/tailing by PARN. After the passenger strand is sliced, the hairpin might open during target binding, allowing nts 19 to 22 to base pair (open hairpin hypothesis), or the loop from nts 19 to 22 might remain, allowing only nts 2 to 14 to be used for target binding (partial hairpin hypothesis).

Techniques Used: Northern Blot, Synthesized, Stable Transfection, Binding Assay


Structured Review

Addgene inc piresneo flag ha ago4
Piresneo Flag Ha Ago4, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/piresneo flag ha ago4/product/Addgene inc
Average 91 stars, based on 1 article reviews
Price from $9.99 to $1999.99
piresneo flag ha ago4 - by Bioz Stars, 2024-04
91/100 stars

Images


Structured Review

Addgene inc piresneo flag ha ago4 plasmids
Knockout of DUSP11 decreases AGO association with 5′ triphosphorylated BLV 5p miRNAs. ( A ) Immunoblot analysis of input lysate (0.1%) and RIP (5%) of endogenous AGO proteins (∼95 kDa) using pan-AGO antibody (2A8) in parental (wild-type [WT]) and DUSP11 knockout (D11-KO) HEK293T cells transfected with either pBLV-B2 or pBLV-B5 expression vectors. Asterisks indicate IgG light and heavy chains. ( B ) Northern blot analysis of input RNA (2.5%) and RNA recovered from RIP samples (50%) in A . The membrane was first probed with 5p probes, stripped, and reprobed with the 3p probes. ( C ) RIP–Northern blot analysis of BLV B2 and B5 miRNAs associated with individual AGO proteins. Parental (wild-type) and DUSP11 knockout HEK293T cells were cotransfected with pBLV-B2 and pBLV-B5 and each of the indicated AGO-Flag expression vectors. The individual AGOs were precipitated using the anti-Flag M2 antibody. Total input RNA (5% for AGOs1–3 and 10% for <t>AGO4)</t> and RIP samples (95%) were subjected to Northern blot analysis. Blots were first probed for BLV-miR-B5-5p, sequentially stripped, and reprobed for BLV-miR-B5-3p, BLV-miR-B2-5p, and BLV-miR-B2-3p. ( D ) RIP–Northern blot analysis of parental and DUSP11 knockout HEK293T cells cotransfected with pAGO1-Flag and the BLV-B5 pre-miRNA mimic pretreated with (+; 5′-p-B5) or without (−; 5′-ppp-B5) RNA 5′ polyphosphatase. RIP was performed using the anti-Flag M2 antibody 48 h after transfection of mimics and pAGO1-Flag. RNA recovered from RIP-Flag was subjected to Northern blot analysis. The membrane was first probed for the 5p miRNA arm, stripped, and reprobed for the 3p arm. Immunoblot analysis of the RIP-Flag samples is shown below the Northern blot to show the immunoprecipitation efficiency of AGO1-Flag between the RNA 5′ polyphosphatase-treated (+) and untreated (−) samples. ( E ) Similar to D except using the BLV-B5 duplexed miRNA mimics in which the 5p arm was pretreated with (+) or without (−) RNA 5′ polyphosphatase. All of the duplexes contained a 3p miRNA mimic that was treated with RNA 5′ polyphosphatase to mimic the 5′ monophosphate generated by Dicer processing.
Piresneo Flag Ha Ago4 Plasmids, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs"

Article Title: DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs

Journal: Genes & Development

doi: 10.1101/gad.282616.116

Knockout of DUSP11 decreases AGO association with 5′ triphosphorylated BLV 5p miRNAs. ( A ) Immunoblot analysis of input lysate (0.1%) and RIP (5%) of endogenous AGO proteins (∼95 kDa) using pan-AGO antibody (2A8) in parental (wild-type [WT]) and DUSP11 knockout (D11-KO) HEK293T cells transfected with either pBLV-B2 or pBLV-B5 expression vectors. Asterisks indicate IgG light and heavy chains. ( B ) Northern blot analysis of input RNA (2.5%) and RNA recovered from RIP samples (50%) in A . The membrane was first probed with 5p probes, stripped, and reprobed with the 3p probes. ( C ) RIP–Northern blot analysis of BLV B2 and B5 miRNAs associated with individual AGO proteins. Parental (wild-type) and DUSP11 knockout HEK293T cells were cotransfected with pBLV-B2 and pBLV-B5 and each of the indicated AGO-Flag expression vectors. The individual AGOs were precipitated using the anti-Flag M2 antibody. Total input RNA (5% for AGOs1–3 and 10% for AGO4) and RIP samples (95%) were subjected to Northern blot analysis. Blots were first probed for BLV-miR-B5-5p, sequentially stripped, and reprobed for BLV-miR-B5-3p, BLV-miR-B2-5p, and BLV-miR-B2-3p. ( D ) RIP–Northern blot analysis of parental and DUSP11 knockout HEK293T cells cotransfected with pAGO1-Flag and the BLV-B5 pre-miRNA mimic pretreated with (+; 5′-p-B5) or without (−; 5′-ppp-B5) RNA 5′ polyphosphatase. RIP was performed using the anti-Flag M2 antibody 48 h after transfection of mimics and pAGO1-Flag. RNA recovered from RIP-Flag was subjected to Northern blot analysis. The membrane was first probed for the 5p miRNA arm, stripped, and reprobed for the 3p arm. Immunoblot analysis of the RIP-Flag samples is shown below the Northern blot to show the immunoprecipitation efficiency of AGO1-Flag between the RNA 5′ polyphosphatase-treated (+) and untreated (−) samples. ( E ) Similar to D except using the BLV-B5 duplexed miRNA mimics in which the 5p arm was pretreated with (+) or without (−) RNA 5′ polyphosphatase. All of the duplexes contained a 3p miRNA mimic that was treated with RNA 5′ polyphosphatase to mimic the 5′ monophosphate generated by Dicer processing.
Figure Legend Snippet: Knockout of DUSP11 decreases AGO association with 5′ triphosphorylated BLV 5p miRNAs. ( A ) Immunoblot analysis of input lysate (0.1%) and RIP (5%) of endogenous AGO proteins (∼95 kDa) using pan-AGO antibody (2A8) in parental (wild-type [WT]) and DUSP11 knockout (D11-KO) HEK293T cells transfected with either pBLV-B2 or pBLV-B5 expression vectors. Asterisks indicate IgG light and heavy chains. ( B ) Northern blot analysis of input RNA (2.5%) and RNA recovered from RIP samples (50%) in A . The membrane was first probed with 5p probes, stripped, and reprobed with the 3p probes. ( C ) RIP–Northern blot analysis of BLV B2 and B5 miRNAs associated with individual AGO proteins. Parental (wild-type) and DUSP11 knockout HEK293T cells were cotransfected with pBLV-B2 and pBLV-B5 and each of the indicated AGO-Flag expression vectors. The individual AGOs were precipitated using the anti-Flag M2 antibody. Total input RNA (5% for AGOs1–3 and 10% for AGO4) and RIP samples (95%) were subjected to Northern blot analysis. Blots were first probed for BLV-miR-B5-5p, sequentially stripped, and reprobed for BLV-miR-B5-3p, BLV-miR-B2-5p, and BLV-miR-B2-3p. ( D ) RIP–Northern blot analysis of parental and DUSP11 knockout HEK293T cells cotransfected with pAGO1-Flag and the BLV-B5 pre-miRNA mimic pretreated with (+; 5′-p-B5) or without (−; 5′-ppp-B5) RNA 5′ polyphosphatase. RIP was performed using the anti-Flag M2 antibody 48 h after transfection of mimics and pAGO1-Flag. RNA recovered from RIP-Flag was subjected to Northern blot analysis. The membrane was first probed for the 5p miRNA arm, stripped, and reprobed for the 3p arm. Immunoblot analysis of the RIP-Flag samples is shown below the Northern blot to show the immunoprecipitation efficiency of AGO1-Flag between the RNA 5′ polyphosphatase-treated (+) and untreated (−) samples. ( E ) Similar to D except using the BLV-B5 duplexed miRNA mimics in which the 5p arm was pretreated with (+) or without (−) RNA 5′ polyphosphatase. All of the duplexes contained a 3p miRNA mimic that was treated with RNA 5′ polyphosphatase to mimic the 5′ monophosphate generated by Dicer processing.

Techniques Used: Knock-Out, Western Blot, Transfection, Expressing, Northern Blot, Immunoprecipitation, Generated


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Addgene inc piresneo flag ha ago4
Piresneo Flag Ha Ago4, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Addgene inc piresneo flag ha ago4
Piresneo Flag Ha Ago4, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/piresneo flag ha ago4/product/Addgene inc
Average 91 stars, based on 1 article reviews
Price from $9.99 to $1999.99
piresneo flag ha ago4 - by Bioz Stars, 2024-04
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    Addgene inc piresneo flag ha ago4
    KEY RESOURCES TABLE
    Piresneo Flag Ha Ago4, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/piresneo flag ha ago4/product/Addgene inc
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    91
    Addgene inc plasmid flag ha tagged ago4
    Viral titers and viral RNA of (A) influenza-A-infected (PR/8/1934), (B) encephalomyocarditis (EMCV)-infected, or (C) vesicular stomatitis virus (VSV)-infected (indicated multiplicities of infection [MOIs] for 16 h) bone-marrow-derived macrophages (BMDMs) from Ago1 +/+ (black) and Ago1 −/− (yellow), (D–F) Ago3 +/+ (black) and Ago3 −/− (green), or (G–I) <t>Ago4</t> +/+ (black) and Ago4 −/− (red) littermate mice. Representative plaque assay images are shown. Influenza Ns1, EMCV 2A-2B , or VSV G RNA levels were quantified by qPCR relative to TATA box protein ( Tbp ). All data are from two or three biological replicates performed in triplicate and combined. Errors bars represent SEM. *p < 0.05, **p < 0.01, and ***p < 0.001, as measured by two-way ANOVA with Bonferroni multiple comparison test.
    Plasmid Flag Ha Tagged Ago4, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Addgene inc piresneo flag ha ago4 plasmid
    Association of the <t>AGO4-binding</t> genes and methylation levels. (A) Scheme showing that tetracycline-treated cells switch off AGO4 protein expression (Tet+) from day 1 to 12 and that 5’-azacytidine treatment from day 7 to 9 inhibits DNA methyltransferase (5-AC). (B) Detection of AGO4 mRNA in Tet- (AGO4 expression) and Tet+ (No AGO4 expression) cells; AGO4 was significantly expressed in the absence of Tet. (C) Confirmation of AGO4-binding genes showed that AGO4 only bounds to the selected gene from bioinformatics data as exemplified by C16ORF89 . (D) However, AGO4 did not bind to a gene lacking the AGO4-binding site, MSN . (E) The AGO4 methylation level of the gene, which contains AGO4-binding sites, was increased due to the presence of the AGO4 protein (Tet-). This increased methylation level was not associated with the presence of DNA methyltransferase (5-AC+). (F) These results were not observed for a gene lacking AGO4-binding sites, MSN . (G to H) Recovery of DNA methylation levels is found in cells expressing the AGO4 protein (Tet-) with 5’-azacytidine withdrawal in both of AGO4-binding gene, C16ORF89 and non-AGO4-binding gene, MSN ; AGO4 is inferred to methylate previously demethylated loci. The above data are the representative dataset from three independent experiments presented as the mean ± SEM. Statistical analyses were performed using a paired-sample t -test, where p < 0.05, p < 0.01, and p < 0.005 are represented as *, **, and ***, respectively, where Tet+ means tetracycline treatment, 5-AC+ means azacytidine treatment, and Tet- and 5-AC- mean untreated groups (I). Schematic overview of the role of AGO4 in de novo methylation. After DNA replication, DNA methylation is maintained by DNMT1 by the recognition of hemimethylated CpG on the parental strand as a template and the addition of newly methylated CpG to the daughter strand. DNMT1 is inhibited by the action of 5-AC, and the methylation level should be decreased by half under 5-AC treatment. In our study, when tetracycline was added, AGO4 shRNA was manipulated, resulting in AGO4 expression repression. Thus, without tetracycline, AGO4 is upregulated and binds to DNA loci, and DNA methylation is maintained, although 5-AC is added, suggesting that AGO4 involved in de novo methylation
    Piresneo Flag Ha Ago4 Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/piresneo flag ha ago4 plasmid/product/Addgene inc
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    91
    Addgene inc piresneo flag ha ago1
    Proposed function of sli-RISCs versus si-RISCs. ( A ) Northern blotting to detect chemically synthesized sli-887 and si-887. Processed products of sli-887 are longer than the 21-mer si-887. ( B ) Northern blotting to detect sli-887 and the 25-nt form of sli-887 (nts 1 to 25). Processed products of sli-887 are shorter than the 25-mer form. ( C ) Northern blotting to detect processed sli-1148 from a stably expressed sli-1148 driven by a U6 promoter, which mainly exists in an approximately 30-mer isoform, in HCT116 cells. ( D ) Proposed model of sli-RISC versus si-RISC function: si-RISCs can involve three non-slicing Agos <t>(Ago1,</t> Ago3, and Ago4) and slicing Ago2 loaded with uniform 21-mer guide RNAs; sli-RISCs contain the slicing Ago2 loaded with guide RNAs with various 3′ end lengths, and silencing may couple with 3′ end trimming/tailing by PARN. After the passenger strand is sliced, the hairpin might open during target binding, allowing nts 19 to 22 to base pair (open hairpin hypothesis), or the loop from nts 19 to 22 might remain, allowing only nts 2 to 14 to be used for target binding (partial hairpin hypothesis).
    Piresneo Flag Ha Ago1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc piresneo flag ha ago4 plasmids
    Knockout of DUSP11 decreases AGO association with 5′ triphosphorylated BLV 5p miRNAs. ( A ) Immunoblot analysis of input lysate (0.1%) and RIP (5%) of endogenous AGO proteins (∼95 kDa) using pan-AGO antibody (2A8) in parental (wild-type [WT]) and DUSP11 knockout (D11-KO) HEK293T cells transfected with either pBLV-B2 or pBLV-B5 expression vectors. Asterisks indicate IgG light and heavy chains. ( B ) Northern blot analysis of input RNA (2.5%) and RNA recovered from RIP samples (50%) in A . The membrane was first probed with 5p probes, stripped, and reprobed with the 3p probes. ( C ) RIP–Northern blot analysis of BLV B2 and B5 miRNAs associated with individual AGO proteins. Parental (wild-type) and DUSP11 knockout HEK293T cells were cotransfected with pBLV-B2 and pBLV-B5 and each of the indicated AGO-Flag expression vectors. The individual AGOs were precipitated using the anti-Flag M2 antibody. Total input RNA (5% for AGOs1–3 and 10% for <t>AGO4)</t> and RIP samples (95%) were subjected to Northern blot analysis. Blots were first probed for BLV-miR-B5-5p, sequentially stripped, and reprobed for BLV-miR-B5-3p, BLV-miR-B2-5p, and BLV-miR-B2-3p. ( D ) RIP–Northern blot analysis of parental and DUSP11 knockout HEK293T cells cotransfected with pAGO1-Flag and the BLV-B5 pre-miRNA mimic pretreated with (+; 5′-p-B5) or without (−; 5′-ppp-B5) RNA 5′ polyphosphatase. RIP was performed using the anti-Flag M2 antibody 48 h after transfection of mimics and pAGO1-Flag. RNA recovered from RIP-Flag was subjected to Northern blot analysis. The membrane was first probed for the 5p miRNA arm, stripped, and reprobed for the 3p arm. Immunoblot analysis of the RIP-Flag samples is shown below the Northern blot to show the immunoprecipitation efficiency of AGO1-Flag between the RNA 5′ polyphosphatase-treated (+) and untreated (−) samples. ( E ) Similar to D except using the BLV-B5 duplexed miRNA mimics in which the 5p arm was pretreated with (+) or without (−) RNA 5′ polyphosphatase. All of the duplexes contained a 3p miRNA mimic that was treated with RNA 5′ polyphosphatase to mimic the 5′ monophosphate generated by Dicer processing.
    Piresneo Flag Ha Ago4 Plasmids, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    KEY RESOURCES TABLE

    Journal: Molecular cell

    Article Title: Structural Basis for Target-Directed MicroRNA Degradation

    doi: 10.1016/j.molcel.2019.06.019

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: pIRESneo-FLAG/HA Ago4 , ( Meister et al., 2004 ) , Addgene Cat# 10824.

    Techniques: Western Blot, Protease Inhibitor, Reporter Assay, Expressing, Recombinant, Software, Transduction, Plasmid Preparation, Construct

    Viral titers and viral RNA of (A) influenza-A-infected (PR/8/1934), (B) encephalomyocarditis (EMCV)-infected, or (C) vesicular stomatitis virus (VSV)-infected (indicated multiplicities of infection [MOIs] for 16 h) bone-marrow-derived macrophages (BMDMs) from Ago1 +/+ (black) and Ago1 −/− (yellow), (D–F) Ago3 +/+ (black) and Ago3 −/− (green), or (G–I) Ago4 +/+ (black) and Ago4 −/− (red) littermate mice. Representative plaque assay images are shown. Influenza Ns1, EMCV 2A-2B , or VSV G RNA levels were quantified by qPCR relative to TATA box protein ( Tbp ). All data are from two or three biological replicates performed in triplicate and combined. Errors bars represent SEM. *p < 0.05, **p < 0.01, and ***p < 0.001, as measured by two-way ANOVA with Bonferroni multiple comparison test.

    Journal: Cell reports

    Article Title: A Requirement for Argonaute 4 in Mammalian Antiviral Defense

    doi: 10.1016/j.celrep.2020.01.021

    Figure Lengend Snippet: Viral titers and viral RNA of (A) influenza-A-infected (PR/8/1934), (B) encephalomyocarditis (EMCV)-infected, or (C) vesicular stomatitis virus (VSV)-infected (indicated multiplicities of infection [MOIs] for 16 h) bone-marrow-derived macrophages (BMDMs) from Ago1 +/+ (black) and Ago1 −/− (yellow), (D–F) Ago3 +/+ (black) and Ago3 −/− (green), or (G–I) Ago4 +/+ (black) and Ago4 −/− (red) littermate mice. Representative plaque assay images are shown. Influenza Ns1, EMCV 2A-2B , or VSV G RNA levels were quantified by qPCR relative to TATA box protein ( Tbp ). All data are from two or three biological replicates performed in triplicate and combined. Errors bars represent SEM. *p < 0.05, **p < 0.01, and ***p < 0.001, as measured by two-way ANOVA with Bonferroni multiple comparison test.

    Article Snippet: Plasmid FLAG/HA-tagged Ago4 , Thomas Tuschl , Addgene #10824; RRID:Addgene_10824.

    Techniques: Infection, Derivative Assay, Plaque Assay

    (A) Flow cytometry of HEK293T transfected with empty vector (EV) or indicated concentrations of Ago4-Myc for 24 h prior to infection with influenza-GFP at MOI 1 for 24 h. GFP percentages of 7-AAD − (non-apoptotic) cells are indicated. Right: mean and SEM of percentage of Flu-GFP relative to EV control. (B and C) Ago4 and influenza Ns1 RNA expression in HEK293T transfected with EV or 10 μg of Ago4-Myc and infected with Influenza A (MOI 1, 24 h p.i.). (D) Myc immunoblot of Ago1, Ago3, or Ago4 transfected HEK293T cells. (E) Percentage of Flu-GFP + cells in HEK293T cells transfected with 10 μg of the indicated plasmid and infected at MOI 1 for 24 h. 7-AAD − , relative to EV. (F) Influenza viral titers of mouse embryonic fibroblasts (MEFs) transfected with a negative control (NC) siRNA or vsiRNA against NS1. Data are from three biological replicates performed in triplicate and combined. Errors bars represent SEM. *p < 0.05, **p < 0.01, and ***p < 0.001, as measured by an unpaired t test or one-way ANOVA with Bonferroni multiple comparison test.

    Journal: Cell reports

    Article Title: A Requirement for Argonaute 4 in Mammalian Antiviral Defense

    doi: 10.1016/j.celrep.2020.01.021

    Figure Lengend Snippet: (A) Flow cytometry of HEK293T transfected with empty vector (EV) or indicated concentrations of Ago4-Myc for 24 h prior to infection with influenza-GFP at MOI 1 for 24 h. GFP percentages of 7-AAD − (non-apoptotic) cells are indicated. Right: mean and SEM of percentage of Flu-GFP relative to EV control. (B and C) Ago4 and influenza Ns1 RNA expression in HEK293T transfected with EV or 10 μg of Ago4-Myc and infected with Influenza A (MOI 1, 24 h p.i.). (D) Myc immunoblot of Ago1, Ago3, or Ago4 transfected HEK293T cells. (E) Percentage of Flu-GFP + cells in HEK293T cells transfected with 10 μg of the indicated plasmid and infected at MOI 1 for 24 h. 7-AAD − , relative to EV. (F) Influenza viral titers of mouse embryonic fibroblasts (MEFs) transfected with a negative control (NC) siRNA or vsiRNA against NS1. Data are from three biological replicates performed in triplicate and combined. Errors bars represent SEM. *p < 0.05, **p < 0.01, and ***p < 0.001, as measured by an unpaired t test or one-way ANOVA with Bonferroni multiple comparison test.

    Article Snippet: Plasmid FLAG/HA-tagged Ago4 , Thomas Tuschl , Addgene #10824; RRID:Addgene_10824.

    Techniques: Flow Cytometry, Transfection, Plasmid Preparation, Infection, RNA Expression, Western Blot, Negative Control

    Journal: Cell reports

    Article Title: A Requirement for Argonaute 4 in Mammalian Antiviral Defense

    doi: 10.1016/j.celrep.2020.01.021

    Figure Lengend Snippet:

    Article Snippet: Plasmid FLAG/HA-tagged Ago4 , Thomas Tuschl , Addgene #10824; RRID:Addgene_10824.

    Techniques: Western Blot, Recombinant, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Plasmid Preparation, Software

    Association of the AGO4-binding genes and methylation levels. (A) Scheme showing that tetracycline-treated cells switch off AGO4 protein expression (Tet+) from day 1 to 12 and that 5’-azacytidine treatment from day 7 to 9 inhibits DNA methyltransferase (5-AC). (B) Detection of AGO4 mRNA in Tet- (AGO4 expression) and Tet+ (No AGO4 expression) cells; AGO4 was significantly expressed in the absence of Tet. (C) Confirmation of AGO4-binding genes showed that AGO4 only bounds to the selected gene from bioinformatics data as exemplified by C16ORF89 . (D) However, AGO4 did not bind to a gene lacking the AGO4-binding site, MSN . (E) The AGO4 methylation level of the gene, which contains AGO4-binding sites, was increased due to the presence of the AGO4 protein (Tet-). This increased methylation level was not associated with the presence of DNA methyltransferase (5-AC+). (F) These results were not observed for a gene lacking AGO4-binding sites, MSN . (G to H) Recovery of DNA methylation levels is found in cells expressing the AGO4 protein (Tet-) with 5’-azacytidine withdrawal in both of AGO4-binding gene, C16ORF89 and non-AGO4-binding gene, MSN ; AGO4 is inferred to methylate previously demethylated loci. The above data are the representative dataset from three independent experiments presented as the mean ± SEM. Statistical analyses were performed using a paired-sample t -test, where p < 0.05, p < 0.01, and p < 0.005 are represented as *, **, and ***, respectively, where Tet+ means tetracycline treatment, 5-AC+ means azacytidine treatment, and Tet- and 5-AC- mean untreated groups (I). Schematic overview of the role of AGO4 in de novo methylation. After DNA replication, DNA methylation is maintained by DNMT1 by the recognition of hemimethylated CpG on the parental strand as a template and the addition of newly methylated CpG to the daughter strand. DNMT1 is inhibited by the action of 5-AC, and the methylation level should be decreased by half under 5-AC treatment. In our study, when tetracycline was added, AGO4 shRNA was manipulated, resulting in AGO4 expression repression. Thus, without tetracycline, AGO4 is upregulated and binds to DNA loci, and DNA methylation is maintained, although 5-AC is added, suggesting that AGO4 involved in de novo methylation

    Journal: Frontiers in Genetics

    Article Title: Argonaute 4 as an Effector Protein in RNA-Directed DNA Methylation in Human Cells

    doi: 10.3389/fgene.2019.00645

    Figure Lengend Snippet: Association of the AGO4-binding genes and methylation levels. (A) Scheme showing that tetracycline-treated cells switch off AGO4 protein expression (Tet+) from day 1 to 12 and that 5’-azacytidine treatment from day 7 to 9 inhibits DNA methyltransferase (5-AC). (B) Detection of AGO4 mRNA in Tet- (AGO4 expression) and Tet+ (No AGO4 expression) cells; AGO4 was significantly expressed in the absence of Tet. (C) Confirmation of AGO4-binding genes showed that AGO4 only bounds to the selected gene from bioinformatics data as exemplified by C16ORF89 . (D) However, AGO4 did not bind to a gene lacking the AGO4-binding site, MSN . (E) The AGO4 methylation level of the gene, which contains AGO4-binding sites, was increased due to the presence of the AGO4 protein (Tet-). This increased methylation level was not associated with the presence of DNA methyltransferase (5-AC+). (F) These results were not observed for a gene lacking AGO4-binding sites, MSN . (G to H) Recovery of DNA methylation levels is found in cells expressing the AGO4 protein (Tet-) with 5’-azacytidine withdrawal in both of AGO4-binding gene, C16ORF89 and non-AGO4-binding gene, MSN ; AGO4 is inferred to methylate previously demethylated loci. The above data are the representative dataset from three independent experiments presented as the mean ± SEM. Statistical analyses were performed using a paired-sample t -test, where p < 0.05, p < 0.01, and p < 0.005 are represented as *, **, and ***, respectively, where Tet+ means tetracycline treatment, 5-AC+ means azacytidine treatment, and Tet- and 5-AC- mean untreated groups (I). Schematic overview of the role of AGO4 in de novo methylation. After DNA replication, DNA methylation is maintained by DNMT1 by the recognition of hemimethylated CpG on the parental strand as a template and the addition of newly methylated CpG to the daughter strand. DNMT1 is inhibited by the action of 5-AC, and the methylation level should be decreased by half under 5-AC treatment. In our study, when tetracycline was added, AGO4 shRNA was manipulated, resulting in AGO4 expression repression. Thus, without tetracycline, AGO4 is upregulated and binds to DNA loci, and DNA methylation is maintained, although 5-AC is added, suggesting that AGO4 involved in de novo methylation

    Article Snippet: Overexpression of AGO4 was performed by the using pIRESneo-FLAG/HA AGO4 plasmid (Addgene, MA, USA).

    Techniques: Binding Assay, Methylation, Expressing, DNA Methylation Assay, shRNA

    Alu methylation upon Alu siRNA transfection in Tet-controlled AGO4-expressing cells. The Alu methylation level was significantly increased when AGO4 was upregulated under Alu siRNA transfection. The above data are the representative dataset from six independent experiments presented as the mean ± SEM. Statistical analysis was performed using an unpaired t -test where p < 0.01 is represented as **.

    Journal: Frontiers in Genetics

    Article Title: Argonaute 4 as an Effector Protein in RNA-Directed DNA Methylation in Human Cells

    doi: 10.3389/fgene.2019.00645

    Figure Lengend Snippet: Alu methylation upon Alu siRNA transfection in Tet-controlled AGO4-expressing cells. The Alu methylation level was significantly increased when AGO4 was upregulated under Alu siRNA transfection. The above data are the representative dataset from six independent experiments presented as the mean ± SEM. Statistical analysis was performed using an unpaired t -test where p < 0.01 is represented as **.

    Article Snippet: Overexpression of AGO4 was performed by the using pIRESneo-FLAG/HA AGO4 plasmid (Addgene, MA, USA).

    Techniques: Methylation, Transfection, Expressing

    Overexpression of HA-AGO4 increases global genomic methylation. (A) Confirmation of AGO4 mRNA expression in AGO4- or PC-overexpressing cells after transfection for 72 h by using real-time PCR showed that AGO4 mRNA was significantly upregulated in AGO4-overexpressing cells, and (B) Western blot analysis showed the expression of the HA-AGO4 protein in a time-course experiment. β-Actin was used to confirm equal protein loading of each lane. AGO4 protein expression was at the highest level at 48 h after transfection. (C) Overexpression of HA-AGO4 resulted in a significant increase in interspersed repetitive sequence methylation in the input (cell lysate) HA-AGO4 compared with untaransfected HeLa cells, detected at 72 h post-transfection. The above data in A and C are the representative dataset from five independent experiments presented as the mean ± SEM. Statistical analyses were performed using a paired t -test where p < 0.05, p < 0.01, and p < 0.005 are represented as *, **, and ***, respectively.

    Journal: Frontiers in Genetics

    Article Title: Argonaute 4 as an Effector Protein in RNA-Directed DNA Methylation in Human Cells

    doi: 10.3389/fgene.2019.00645

    Figure Lengend Snippet: Overexpression of HA-AGO4 increases global genomic methylation. (A) Confirmation of AGO4 mRNA expression in AGO4- or PC-overexpressing cells after transfection for 72 h by using real-time PCR showed that AGO4 mRNA was significantly upregulated in AGO4-overexpressing cells, and (B) Western blot analysis showed the expression of the HA-AGO4 protein in a time-course experiment. β-Actin was used to confirm equal protein loading of each lane. AGO4 protein expression was at the highest level at 48 h after transfection. (C) Overexpression of HA-AGO4 resulted in a significant increase in interspersed repetitive sequence methylation in the input (cell lysate) HA-AGO4 compared with untaransfected HeLa cells, detected at 72 h post-transfection. The above data in A and C are the representative dataset from five independent experiments presented as the mean ± SEM. Statistical analyses were performed using a paired t -test where p < 0.05, p < 0.01, and p < 0.005 are represented as *, **, and ***, respectively.

    Article Snippet: Overexpression of AGO4 was performed by the using pIRESneo-FLAG/HA AGO4 plasmid (Addgene, MA, USA).

    Techniques: Over Expression, Methylation, Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Sequencing

    AGO4 localization is involved in RdDM by introducing genomic DNA methylation in human cells. (A) ChIP experiments using a hemagglutinin (HA) antibody showed elevation of the levels of AGO4-binding LINE-1 or ALU found in AGO4-overexpressing cells compared with their control counterparts. (B) Moreover, the binding of the AGO4 protein to interspersed repetitive sequence increased DNA methylation.

    Journal: Frontiers in Genetics

    Article Title: Argonaute 4 as an Effector Protein in RNA-Directed DNA Methylation in Human Cells

    doi: 10.3389/fgene.2019.00645

    Figure Lengend Snippet: AGO4 localization is involved in RdDM by introducing genomic DNA methylation in human cells. (A) ChIP experiments using a hemagglutinin (HA) antibody showed elevation of the levels of AGO4-binding LINE-1 or ALU found in AGO4-overexpressing cells compared with their control counterparts. (B) Moreover, the binding of the AGO4 protein to interspersed repetitive sequence increased DNA methylation.

    Article Snippet: Overexpression of AGO4 was performed by the using pIRESneo-FLAG/HA AGO4 plasmid (Addgene, MA, USA).

    Techniques: DNA Methylation Assay, Binding Assay, Sequencing

    AGO4 protein specifically induced DNA methylation of target loci. By using CPP-AGO4 conjugated with Alu sgRNA (CPP-AGO4-Alu) transfected to HeLa cells, we found that (A) Alu methylation level was significantly increased in CPP-AGO4-Alu transfected cells, while Alu methylation was not promoted in control counterpart groups. (B) While LINE-1 methylation level was not changed, this means the complex has no off targets to LINE-1. The representative dataset was obtained from five independent experiments. Statistical analyses were performed using a paired-sample T -test where p < 0.05 and p < 0.01 are represented as * and **, respectively, where buffer means non-inducible CPP-AGO4 transfection.

    Journal: Frontiers in Genetics

    Article Title: Argonaute 4 as an Effector Protein in RNA-Directed DNA Methylation in Human Cells

    doi: 10.3389/fgene.2019.00645

    Figure Lengend Snippet: AGO4 protein specifically induced DNA methylation of target loci. By using CPP-AGO4 conjugated with Alu sgRNA (CPP-AGO4-Alu) transfected to HeLa cells, we found that (A) Alu methylation level was significantly increased in CPP-AGO4-Alu transfected cells, while Alu methylation was not promoted in control counterpart groups. (B) While LINE-1 methylation level was not changed, this means the complex has no off targets to LINE-1. The representative dataset was obtained from five independent experiments. Statistical analyses were performed using a paired-sample T -test where p < 0.05 and p < 0.01 are represented as * and **, respectively, where buffer means non-inducible CPP-AGO4 transfection.

    Article Snippet: Overexpression of AGO4 was performed by the using pIRESneo-FLAG/HA AGO4 plasmid (Addgene, MA, USA).

    Techniques: DNA Methylation Assay, Transfection, Methylation

    Proposed function of sli-RISCs versus si-RISCs. ( A ) Northern blotting to detect chemically synthesized sli-887 and si-887. Processed products of sli-887 are longer than the 21-mer si-887. ( B ) Northern blotting to detect sli-887 and the 25-nt form of sli-887 (nts 1 to 25). Processed products of sli-887 are shorter than the 25-mer form. ( C ) Northern blotting to detect processed sli-1148 from a stably expressed sli-1148 driven by a U6 promoter, which mainly exists in an approximately 30-mer isoform, in HCT116 cells. ( D ) Proposed model of sli-RISC versus si-RISC function: si-RISCs can involve three non-slicing Agos (Ago1, Ago3, and Ago4) and slicing Ago2 loaded with uniform 21-mer guide RNAs; sli-RISCs contain the slicing Ago2 loaded with guide RNAs with various 3′ end lengths, and silencing may couple with 3′ end trimming/tailing by PARN. After the passenger strand is sliced, the hairpin might open during target binding, allowing nts 19 to 22 to base pair (open hairpin hypothesis), or the loop from nts 19 to 22 might remain, allowing only nts 2 to 14 to be used for target binding (partial hairpin hypothesis).

    Journal: Nucleic Acids Research

    Article Title: Differences in silencing of mismatched targets by sliced versus diced siRNAs

    doi: 10.1093/nar/gky287

    Figure Lengend Snippet: Proposed function of sli-RISCs versus si-RISCs. ( A ) Northern blotting to detect chemically synthesized sli-887 and si-887. Processed products of sli-887 are longer than the 21-mer si-887. ( B ) Northern blotting to detect sli-887 and the 25-nt form of sli-887 (nts 1 to 25). Processed products of sli-887 are shorter than the 25-mer form. ( C ) Northern blotting to detect processed sli-1148 from a stably expressed sli-1148 driven by a U6 promoter, which mainly exists in an approximately 30-mer isoform, in HCT116 cells. ( D ) Proposed model of sli-RISC versus si-RISC function: si-RISCs can involve three non-slicing Agos (Ago1, Ago3, and Ago4) and slicing Ago2 loaded with uniform 21-mer guide RNAs; sli-RISCs contain the slicing Ago2 loaded with guide RNAs with various 3′ end lengths, and silencing may couple with 3′ end trimming/tailing by PARN. After the passenger strand is sliced, the hairpin might open during target binding, allowing nts 19 to 22 to base pair (open hairpin hypothesis), or the loop from nts 19 to 22 might remain, allowing only nts 2 to 14 to be used for target binding (partial hairpin hypothesis).

    Article Snippet: S5A and S5E were generated by replacing a Kpn I to EcoRI fragment containing the S824–S834 region with Kpn I to EcoRI gBlock sequences (IDT). hAgo1, hAgo3, and hAgo4 expression plasmids were pIRESneo-FLAG/HA-Ago1 (Addgene #10820), pIRESneo-FLAG/HA-Ago3 (Addgene #10823), and pIRESneo-FLAG/HA-Ago4 (Addgene #10824), respectively. pcDNA3.1-neo was used as the control vector in cotransfection with Agos.

    Techniques: Northern Blot, Synthesized, Stable Transfection, Binding Assay

    Knockout of DUSP11 decreases AGO association with 5′ triphosphorylated BLV 5p miRNAs. ( A ) Immunoblot analysis of input lysate (0.1%) and RIP (5%) of endogenous AGO proteins (∼95 kDa) using pan-AGO antibody (2A8) in parental (wild-type [WT]) and DUSP11 knockout (D11-KO) HEK293T cells transfected with either pBLV-B2 or pBLV-B5 expression vectors. Asterisks indicate IgG light and heavy chains. ( B ) Northern blot analysis of input RNA (2.5%) and RNA recovered from RIP samples (50%) in A . The membrane was first probed with 5p probes, stripped, and reprobed with the 3p probes. ( C ) RIP–Northern blot analysis of BLV B2 and B5 miRNAs associated with individual AGO proteins. Parental (wild-type) and DUSP11 knockout HEK293T cells were cotransfected with pBLV-B2 and pBLV-B5 and each of the indicated AGO-Flag expression vectors. The individual AGOs were precipitated using the anti-Flag M2 antibody. Total input RNA (5% for AGOs1–3 and 10% for AGO4) and RIP samples (95%) were subjected to Northern blot analysis. Blots were first probed for BLV-miR-B5-5p, sequentially stripped, and reprobed for BLV-miR-B5-3p, BLV-miR-B2-5p, and BLV-miR-B2-3p. ( D ) RIP–Northern blot analysis of parental and DUSP11 knockout HEK293T cells cotransfected with pAGO1-Flag and the BLV-B5 pre-miRNA mimic pretreated with (+; 5′-p-B5) or without (−; 5′-ppp-B5) RNA 5′ polyphosphatase. RIP was performed using the anti-Flag M2 antibody 48 h after transfection of mimics and pAGO1-Flag. RNA recovered from RIP-Flag was subjected to Northern blot analysis. The membrane was first probed for the 5p miRNA arm, stripped, and reprobed for the 3p arm. Immunoblot analysis of the RIP-Flag samples is shown below the Northern blot to show the immunoprecipitation efficiency of AGO1-Flag between the RNA 5′ polyphosphatase-treated (+) and untreated (−) samples. ( E ) Similar to D except using the BLV-B5 duplexed miRNA mimics in which the 5p arm was pretreated with (+) or without (−) RNA 5′ polyphosphatase. All of the duplexes contained a 3p miRNA mimic that was treated with RNA 5′ polyphosphatase to mimic the 5′ monophosphate generated by Dicer processing.

    Journal: Genes & Development

    Article Title: DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs

    doi: 10.1101/gad.282616.116

    Figure Lengend Snippet: Knockout of DUSP11 decreases AGO association with 5′ triphosphorylated BLV 5p miRNAs. ( A ) Immunoblot analysis of input lysate (0.1%) and RIP (5%) of endogenous AGO proteins (∼95 kDa) using pan-AGO antibody (2A8) in parental (wild-type [WT]) and DUSP11 knockout (D11-KO) HEK293T cells transfected with either pBLV-B2 or pBLV-B5 expression vectors. Asterisks indicate IgG light and heavy chains. ( B ) Northern blot analysis of input RNA (2.5%) and RNA recovered from RIP samples (50%) in A . The membrane was first probed with 5p probes, stripped, and reprobed with the 3p probes. ( C ) RIP–Northern blot analysis of BLV B2 and B5 miRNAs associated with individual AGO proteins. Parental (wild-type) and DUSP11 knockout HEK293T cells were cotransfected with pBLV-B2 and pBLV-B5 and each of the indicated AGO-Flag expression vectors. The individual AGOs were precipitated using the anti-Flag M2 antibody. Total input RNA (5% for AGOs1–3 and 10% for AGO4) and RIP samples (95%) were subjected to Northern blot analysis. Blots were first probed for BLV-miR-B5-5p, sequentially stripped, and reprobed for BLV-miR-B5-3p, BLV-miR-B2-5p, and BLV-miR-B2-3p. ( D ) RIP–Northern blot analysis of parental and DUSP11 knockout HEK293T cells cotransfected with pAGO1-Flag and the BLV-B5 pre-miRNA mimic pretreated with (+; 5′-p-B5) or without (−; 5′-ppp-B5) RNA 5′ polyphosphatase. RIP was performed using the anti-Flag M2 antibody 48 h after transfection of mimics and pAGO1-Flag. RNA recovered from RIP-Flag was subjected to Northern blot analysis. The membrane was first probed for the 5p miRNA arm, stripped, and reprobed for the 3p arm. Immunoblot analysis of the RIP-Flag samples is shown below the Northern blot to show the immunoprecipitation efficiency of AGO1-Flag between the RNA 5′ polyphosphatase-treated (+) and untreated (−) samples. ( E ) Similar to D except using the BLV-B5 duplexed miRNA mimics in which the 5p arm was pretreated with (+) or without (−) RNA 5′ polyphosphatase. All of the duplexes contained a 3p miRNA mimic that was treated with RNA 5′ polyphosphatase to mimic the 5′ monophosphate generated by Dicer processing.

    Article Snippet: The pIRESneo-Flag/HA Ago1, pIRESneo-Flag/HA Ago2, pIRESneo-Flag/HA Ago3, and pIRESneo-Flag/HA Ago4 plasmids ( ) were obtained from Addgene.

    Techniques: Knock-Out, Western Blot, Transfection, Expressing, Northern Blot, Immunoprecipitation, Generated