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, 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, 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, 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:

Journal: Scientific Reports

Article Title: miRNA-dependent regulation of STIM1 expression in breast cancer

doi: 10.1038/s41598-019-49629-5

Figure Lengend Snippet: The STIM1 3′UTR regulates STIM1 expression. ( A ) Cartoon of the different GFP-mCherry reporter constructs used to study post-transcriptional regulation mediated by the 3′UTRs. ( B ) Representative images of HEK-293 cells expressing the control vector (Control), or GFP followed by one (1x) or three copied (3x) of the STIM1 3′UTR. Also shown are images from cells expressing STIM1 3′UTR 1x where Ago2 has been knocked-down (Ago2 siRNA). ( C ) Normalized GFP/mCherry expression ratios in HEK293 cells expressing the control vector or the GAPDH 3′UTR vector. ( D ) Normalized GFP/mCherry expression ratios were assessed by single cell imaging and normalized to the average ratio for the control vector in each cell line studied. HEK-293 cells were transfected with the control vector, STIM1 3′UTR (1x or 3x). MCF7 and MDA-MB-231 cells were transfected with the control vector or the STIM1 3′UTR (1x). The data were normalized to the control vector for each cell line. Data are mean ± S.D.; ANOVA, p < 0.0002, n = 3–4). ( E ) Normalized GFP/mCherry intensity in MCF7 and MDA-MB321 cells overexpressing the control vector or the STIM1 3′UTR construct as analyzed by flow cytometry (mean ± S.D.; paired t-test; **p = 0.0046; *p = 0.0202; n = 3). ( F ) Ratio of the GFP/mCherry reporter expression from HEK-293, MDA-MB231 and MCF7 cells transfected with 3′UTR constructs of STIM1, STIM2, Orai1, Orai2 and Orai3.

Article Snippet: Ago2 siRNA was obtained from Santa Cruz and Ago2 Trilencer siRNA was obtained from Origene.

Techniques: Expressing, Construct, Plasmid Preparation, Imaging, Transfection, Flow Cytometry

Journal: Scientific Reports

Article Title: miRNA-dependent regulation of STIM1 expression in breast cancer

doi: 10.1038/s41598-019-49629-5

Figure Lengend Snippet: STIM1 3′UTR-mediated downregulation of expression requires Ago2. ( A ) RT-PCR quantification of Ago2 message levels after transfection with the control siRNA or siRNA targeting Ago2 in HEK-293, MCF7 and MDA-MB231 cells (mean ± S.D.; unpaired t-test; p ≤ 0.007; n = 3). ( B ) Expression of the GFP-mCherry reporter in cells transfected with the control vector (Control GFP vector) or the STIM1 3′UTR construct without siRNA treatment or after Ago2 siRNA transfection at 24 and 48 hours. Data are normalized to the control vector in each cell line (mean ± S.D.; ANOVA; *p ≤ 0.0406; ns p = 0.0697; n = 3). ( C ) Representative Western blots showing STIM1 expressions with (Ago2) or without (Con) Ago2 knockdown at 24 hours in HEK-293, MCF7 and MDA-MB231 cells (left) and quantification data of the ratio of STIM1/actin in the three cell lines (right) (mean ± S.D.; ratio paired t-test; **p = 0.0052; * 0.0371; n = 3–5).

Article Snippet: Ago2 siRNA was obtained from Santa Cruz and Ago2 Trilencer siRNA was obtained from Origene.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Construct, Western Blot

Journal: Scientific Reports

Article Title: miRNA-dependent regulation of STIM1 expression in breast cancer

doi: 10.1038/s41598-019-49629-5

Figure Lengend Snippet: Ago2 expression is downregulated in MDA-MB-231 cells. ( A ) Northern blotting with DIG-labeled STIM1 coding region probe to asses STIM1 transcripts in the three cell lines. ( B ) Western blot showing endogenous protein expression of Ago2, Dicer, and Drosha in HEK-293, MDA-MB231, and MCF7 cells. Actin is shown as a loading control. Representative of three or more similar gels. ( C ) STIM1 levels in HEK293 and MCF7 overexpressing GFP-Ago2. Actin is shown as a loading control. Representative of three similar experiments. ( D ) Western blots showing endogenous Ago2, untagged Ago2, tagged Ago2, actin and STIM1 levels in MDA-MB231 cells overexpressing untagged and GFP-tagged Ago2. Representative of 5 similar experiments. ( E ) Overexpression of Ago2 results in decreased STIM1 protein levels in MDA-MB-231 cells. Representative histograms (left) and summary data right) of flow cytometry analysis of endogenous STIM1 expression levels from GFP or GFP-Ago2 transfected MDA-231, MCF7 and HEK293 cells (Live GFP + cells) (Mean ± S.D.; ratio paired t-test; **p = 0.0017; ns: not significant; n = 3–5).

Article Snippet: Ago2 siRNA was obtained from Santa Cruz and Ago2 Trilencer siRNA was obtained from Origene.

Techniques: Expressing, Northern Blot, Labeling, Western Blot, Over Expression, Flow Cytometry, Transfection

Journal: Scientific Reports

Article Title: miRNA-dependent regulation of STIM1 expression in breast cancer

doi: 10.1038/s41598-019-49629-5

Figure Lengend Snippet: Breast cancer progression, STIM1 and Ago2. All analyses were performed on the Breast Cancer Gene-Expression Miner v4.2 platform (bc-GenExMiner v4.2) using publically available datasets. ( A ) Table outlining the correlation between the expression of STIM1 and Ago2 based on the breast cancer molecular subtype as listed. The correlation coefficient (r), statistical significant (p) and the number of patients used for each analysis are shown. Also shown at the hazard ratios associate with STIM1 and Ago2 expression based on Kaplan-Meier survival curves for each molecular subtype. ( B ) Scatter plot of Ago2 versus STIM1 expression for the basal-like breast cancer molecular subtype. ( C , D ) Box plots of mRNA levels for Ago2 ( C ) and STIM1 ( D ) classified based on the breast cancer molecular subtype. Below each subtype classification the number of patients (#) used for the analysis as well as the p-value compared to the luminal A subtype are shown. ( E ) Kaplan-Meier survival curve for patients with basal-like breast cancer subtype with the green curve showing event-free survival for patient with STIM1 levels below or equal to the median and the red-dash curve for patients with STIM1 levels above the median levels.

Article Snippet: Ago2 siRNA was obtained from Santa Cruz and Ago2 Trilencer siRNA was obtained from Origene.

Techniques: Expressing

Journal: Frontiers in Veterinary Science

Article Title: Analyzing the interactions of mRNAs, miRNAs and lncRNAs to predict ceRNA networks in bovine cystic follicular granulosa cells

doi: 10.3389/fvets.2022.1028867

Figure Lengend Snippet: Analysis of miR-664b sponge HSD17B7 3'-UTR and lncRNA NONBTAT027373.1. (A) : The networks of lncRNA-miRNA- HSD17B7 (B) : The expression of DE lncRNAs in lncRNA-miRNA- HSD17B7 networks. (C) : The expression of HSD17B7 mRNA. (D) : The expression of DE miRNAs in lncRNA-miRNA- HSD17B7 networks. (E) : Interaction between lncRNA NONBTAT027373.1 and miR-664b through Ago2 protein investigated using the RIP assay. lncRNA NONBTAT027373.1 and miR-664b expression quantified by RT-qPCR. (F) : Schematic view of the miR-664b seed region, the motif of HSD17B7 3'-UTR and lncRNA NONBTAT027373.1. (G,H) : Construction of WT and MUT plasmid vectors of HSD17B7 3'-UTR and lncRNA NONBTAT027373.1. (I) : Dual-luciferase reporter gene analysis of the regulatory relationship between HSD17B7 3'-UTR and miR-664b. (J) : Dual-luciferase reporter gene analysis of the regulatory relationship between lncRNA NONBTAT027373.1 and miR-664b. (K) : Transfection efficiency of miR-664b after overexpression and knockdown of miR-664b. (L) : miR-664b regulates the expression of HSD17B7 mRNA. (M) : RT-qPCR analysis of the effect on silence of lncRNA NONBTAT027373.1 expression by siRNA. (N) : Relative expression of HSD17B7 mRNA in groups. NS, Non-significant. Values represent means ± SEM for three individuals. * P < 0.05, ** P < 0.01. Yellow nodes represent HSD17B7 ; red nodes represent miRNAs and blue nodes represent lncRNAs in lncRNA-miRNA- HSD17B7 networks. WT, Wild Type; MUT, Mutant; Mimics, Overexpression of miR-664b; Inhibitor, Knockdown of miR-664b; NC, Negative control.

Article Snippet: Incubated A/G protein beads (MCE, USA) with Ago2 (dilution multiple 1:100) (BOSTER, China)/IgG (dilution multiple 1:100) antibody (ABclonal, China) overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Plasmid Preparation, Luciferase, Transfection, Over Expression, Mutagenesis, Negative Control

Journal: PLoS ONE

Article Title: Modulation of Gene Expression by Human Cytosolic tRNase Z L through 5′-Half-tRNA

doi: 10.1371/journal.pone.0005908

Figure Lengend Snippet: (A) Luciferase assays with the 293 cells. The ratio of the expression level from mlucT(tRNA) or mlucT(rRNA) to that from the unmodified luciferase mRNA mlucT 0 is shown in the 293 cells that were co-transfected with 5′-half-tRNA Glu (+tRNA), the 28S rRNA fragment (+rRNA), the tRNase Z L expression plasmid (+Z L ), the tRNase Z S expression plasmid (+Z S ), the tRNase Z L siRNA (−Z L ), or the Ago2 siRNAs (−Ago). Two independent sets of data are presented, and error bars indicate s.d. (n = 3). Asterisk, P <0.01. (B) Western blotting for tRNase Z L , Ago2, and tRNase Z S in the 293 cells that were transfected with the above plasmids or siRNAs. (C) The ratio of the mlucT(tRNA) amount to the mlucT 0 amount in the 293 cells quantitated by real-time PCR. Two independent sets of data are presented, and error bars indicate s.d. (n = 3). Asterisk, P <0.05. (D) Distribution of 3′ ends of 5′ cleavage products of mlucT(tRNA). Red line, expected cleavage site.

Article Snippet: The 5′-half-tRNA Glu ( 5′-UCCCUGGUGGUCUAGUGGUUAGGAUUCGGCGCUCU-3′ ), the 28S rRNA fragment ( 5′-UUGAAAGUCAGCCCUCGACACAAGGGUUUG-3′ ), and the four siRNAs targeting the human Ago2 mRNA were chemically synthesized by Nippon Bioservice.

Techniques: Luciferase, Expressing, Transfection, Plasmid Preparation, Western Blot, Real-time Polymerase Chain Reaction