Journal: Nucleic acids research

Article Title: Novel functional small RNAs are selectively loaded onto mammalian Ago1.

doi: 10.1093/nar/gku137

Figure Lengend Snippet: Figure 1. Equivalent amounts of human and EBV-encoded miRNAs associated with Ago1 and Ago2. The short RNAs that were co-immunopre- cipitated with Ago1 and Ago2 derived from L591 cells were analyzed. (a) The ratio of sRNAs mapped to the human genome and the EBV genome. (b) Equivalent amounts of human miRNAs associated with Ago1 and Ago2. The top 50 highly associated human miRNAs were plotted. (c) Equivalent amounts of EBV-encoded miRNAs associated with Ago1 and Ago2. The EBV-encoded miRNAs, co-immunoprecipitated with Ago1 and Ago2, with reads of >10 000 counts (42 miRNAs) were plotted. (d) The ratio of sRNAs mapped to miRNA-registered loci, non- miRNA nonannotated loci, snRNA, snoRNA, mt-tRNA and the other noncording sRNA loci.

Article Snippet: D ow nloaded from https://academ ic.oup.com /nar/article/42/8/5289/2903106 by guest on 19 M arch 2024 siRNA and transfection siRNA targeting human Ago1 and DROSHA was purchased from OriGene Technology, Inc. (MD, USA) and Cosmo Bio Co., Ltd. (Tokyo, Japan), respectively. siRNA against human Ago2 was kindly provided by Dr N. Kosaka (National Cancer Center Research Institute, Tokyo, Japan).

Techniques: Derivative Assay, Immunoprecipitation

Journal: Nucleic acids research

Article Title: Novel functional small RNAs are selectively loaded onto mammalian Ago1.

doi: 10.1093/nar/gku137

Figure Lengend Snippet: Figure 2. The sRNAs selectively associated with Ago1 are derived from tandem loci. (a) Definition of tandem loci and sRNA counts, which were mapped to the loci. When one locus (black arrow) with a read count of >10 reads and both flanking loci (gray arrows) located within 100 bases from the original locus also have >10 reads. (b) Representative loci are shown. Some Ago1 associating sRNAs are aligned to 228 776 000–228 783 000 bp area of chromosome 1 and 148 679 000–148 686 000 bp area of chromosome 7. The areas contain ASR3, ASR4 and many tandem locus. Black reads are aligned to forward strand, and gray reads to reverse strand. (c) Some unique sRNAs are selectively associated with Ago1. The counts of sRNAs associated with Ago1 and Ago2 (>2000 counts), derived from tandem loci, are listed. (d) The sequences of representative ASRs are shown. (e) Associations of ASR1, 2, 3 and 4 with Ago1 or Ago2 were determined by real-time PCR to validate the next-generation sequencing data. The data were normalized by the amount of RNA. Black bar indicates Ago1-associated RNA expression; white bar, Ago2.

Article Snippet: D ow nloaded from https://academ ic.oup.com /nar/article/42/8/5289/2903106 by guest on 19 M arch 2024 siRNA and transfection siRNA targeting human Ago1 and DROSHA was purchased from OriGene Technology, Inc. (MD, USA) and Cosmo Bio Co., Ltd. (Tokyo, Japan), respectively. siRNA against human Ago2 was kindly provided by Dr N. Kosaka (National Cancer Center Research Institute, Tokyo, Japan).

Techniques: Derivative Assay, Real-time Polymerase Chain Reaction, Next-Generation Sequencing, RNA Expression

Journal: Nucleic acids research

Article Title: Novel functional small RNAs are selectively loaded onto mammalian Ago1.

doi: 10.1093/nar/gku137

Figure Lengend Snippet: Figure 4. Characterization of ASRs. (a) The 50 base of the top 500 abundantly expressed ASRs. (b) The composition of the top 500 abundantly expressed ASRs. (c) The consensus motif of top 500 abundantly expressed ASRs. (d) Expression of representative ASRs in HeLa, THP1, L591, L1236 and PBMCs were analyzed by real-time PCR, normalized by GAPDH (n = 3). (e) Associations of ASR1, 2, 3 and 4 with Ago1 or Ago2 in L1236 cells were determined by next-generation sequencing and real-time PCR normalized by the amount of RNAs. The read counts of the ASRs listed in Figure 2c in Ago1-IP and Ago2-IP L1236 cells are summarized. Error bars indicate SD. *P < 0.05. (f) mRNA expression of Ago1 and Ago2 in Ago1-knockdown (black bar) and control (white bar) L591 cells (left). Expression of representative ASRs under each condition. Both data were normalized by GAPDH.

Article Snippet: D ow nloaded from https://academ ic.oup.com /nar/article/42/8/5289/2903106 by guest on 19 M arch 2024 siRNA and transfection siRNA targeting human Ago1 and DROSHA was purchased from OriGene Technology, Inc. (MD, USA) and Cosmo Bio Co., Ltd. (Tokyo, Japan), respectively. siRNA against human Ago2 was kindly provided by Dr N. Kosaka (National Cancer Center Research Institute, Tokyo, Japan).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Next-Generation Sequencing, Knockdown, Control

Journal: American Journal of Physiology - Cell Physiology

Article Title: RNA interference may suppress stress granule formation by preventing argonaute 2 recruitment

doi: 10.1152/ajpcell.00251.2018

Figure Lengend Snippet: Accumulation of Ago2 to SGs is inhibited by shRNA transfection. RPTC cells were treated with or without arsenite for 45 min. Cells were fixed and stained for Ago2 and eIF3η. A: representative confocal microscopy images of cells (scale bar = 10 μm). B: co-IP assay. Hsf1 shRNA knockdown cells and negative control cells were treated with or without arsenite for 45 min. Cell lysate was then collected for co-IP with anti-Ago2 antibody followed by immunoblotting of eIF3η, HuR, PKCα, and Ago2. C: HEK293 cells were transfected with Flag-Ago2 plasmid and then treated with or without arsenite for 45 min. The cell lysates were immunoprecipitated with Flag antibody and immunoblotted for eIF3, TIA1, HuR, and Ago2. Ago2, argonaute 2; co-IP, coimmunoprecipitation; eIF3, eukaryotic initiation factor 3; Hsf1, heat shock transcription factor 1; HEK293, human embryonic kidney 293; HuR, human antigen R; KD, knockdown; NC, negative control; RPTC, renal proximal tubule cells; SG, stress granules; TIA1, T-cell intracellular antigen 1.

Article Snippet: Ago2 KO was performed by using the CRISPR/Cas9 system (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"KN218078","term_id":"694262471","term_text":"KN218078"}} KN218078 ; Origene, Rockville, MD).

Techniques: shRNA, Transfection, Staining, Confocal Microscopy, Co-Immunoprecipitation Assay, Knockdown, Negative Control, Western Blot, Plasmid Preparation, Immunoprecipitation

Journal: American Journal of Physiology - Cell Physiology

Article Title: RNA interference may suppress stress granule formation by preventing argonaute 2 recruitment

doi: 10.1152/ajpcell.00251.2018

Figure Lengend Snippet: Kinetic interaction of Ago2 with RISC marker TRBP2 during arsenite treatment. A: co-IP assay. Hsf1 shRNA knockdown and negative control RPTC cells were treated with arsenite for 0, 15, 30, 45, or 60 min. Cell lysate was collected for co-IP with anti-TRBP2 antibody followed by immunoblotting of Ago2 and TRBP2. B: kinetic modeling of Ago2 dissociation from TRBP2 in Hsf1 KD cells and NC cells. The changes of TRBP2-associated Ago2 during arsenite treatment were semiquantified by densitometry. Ago2, argonaute 2; co-IP, coimmunoprecipitation; Hsf1, heat shock transcription factor 1; KD, knockdown; NC, negative control; RISC, RNA-induced silencing complex; RPTC, renal proximal tubule cells; TRBP2, trans-activation-responsive RNA-binding protein.

Article Snippet: Ago2 KO was performed by using the CRISPR/Cas9 system (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"KN218078","term_id":"694262471","term_text":"KN218078"}} KN218078 ; Origene, Rockville, MD).

Techniques: Marker, Co-Immunoprecipitation Assay, shRNA, Knockdown, Negative Control, Western Blot, Activation Assay, RNA Binding Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: RNA interference may suppress stress granule formation by preventing argonaute 2 recruitment

doi: 10.1152/ajpcell.00251.2018

Figure Lengend Snippet: SG formation is reduced in Ago2-deficient cells. A: schematic diagram of CRISPR-mediated Ago2-GFP/Puro recombination. G1 and G2 represent two guide RNA sequences in the chromosome that guide caspase-9 enzyme to cut its adjacent downstream sites. Donor vector contained 600-bp-long 5′- and 599-bp-long 3′-homologous arms flanking the gene of interest (2,588 bp). LHA, left homologous arm; RHA, right homologous arm. B: Western blot analysis of Ago2 expression in two Ago2−/− clones (#1, #2). Cyclophilin B served as a loading control. C: Ago2 knockout cells (Ago2 KO) and wild-type cells (WT) were treated with arsenite for 45 min. Cells were immediately fixed for eIF3η immunofluorescence. D: percentage of cells with SGs as calculated by 100 × [(number of cells with SGs)/(total number of cells)]. Data are presented as means ± SE (n = 3). *P < 0.05 vs. NC in Student’s t-test. Ago2, argonaute 2; eIF3, eukaryotic initiation factor 3; GFP, green fluorescent protein; NC, negative control; SG, stress granule.

Article Snippet: Ago2 KO was performed by using the CRISPR/Cas9 system (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"KN218078","term_id":"694262471","term_text":"KN218078"}} KN218078 ; Origene, Rockville, MD).

Techniques: CRISPR, Plasmid Preparation, Western Blot, Expressing, Clone Assay, Control, Knock-Out, Immunofluorescence, Negative Control

Journal: American Journal of Physiology - Cell Physiology

Article Title: RNA interference may suppress stress granule formation by preventing argonaute 2 recruitment

doi: 10.1152/ajpcell.00251.2018

Figure Lengend Snippet: Schematic diagram of the competing relationship between stress granules (SG) and RNA-induced silencing complex (RISC) for key protein components such as (Ago2). HuR, human antigen R; eIF3, eukaryotic initiation factor 3.

Article Snippet: Ago2 KO was performed by using the CRISPR/Cas9 system (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"KN218078","term_id":"694262471","term_text":"KN218078"}} KN218078 ; Origene, Rockville, MD).

Techniques: