Journal: Experimental and Therapeutic Medicine

Article Title: Long non-coding RNA SNHG7 facilitates pancreatic cancer progression by regulating the miR-146b-5p/Robo1 axis

doi: 10.3892/etm.2021.9829

Figure Lengend Snippet: miR-146b-5p is a direct target of SNHG7. (A) Complementary sequences between miR-146b-5p and SNHG7, and the MUT sequences of SNHG7. Luciferase activity of SNHG7 WT or SNHG7 MUT reporter in (B) SW1990 and (C) AsPC-1 cells transfected with miR-146b-5p or miR-control was assessed via dual-luciferase reporter. (D) Enrichment of SNHG7 in anti-Ago2 or anti-IgG labeled SW1990 cells transfected with miR-146b-5p or miR-control was evaluated by RIP assay. (E) Enrichment of SNHG7 in SW1990 cells transfected with Bio-miR-146b-5p or Bio-NC was analyzed by RNA pull-down assay. (F) Enrichment of SNHG7 in anti-Ago2 or anti-IgG labeled AsPC-1 cells transfected with miR-146b-5p or miR-control was evaluated by RIP assay. (G) Enrichment of SNHG7 in AsPC-1 cells transfected with Bio-miR-146b-5p or Bio-NC was analyzed by RNA pull-down assay. Transfection efficiency of miR-146b-5p probe was examined in (H) SW1990 and (I) AsPC-1 cells transfected with miR-control or miR-146b-5p. miR-146b-5p expression was measured in SW1990 and AsPC-1 cells transfected with (J) miR-control and miR-146b-5p, or (K) inhibitor-control and miR-146b-5p inhibitor. (L) Expression of miR-146b-5p in cells transfected with sh-control, sh-SNHG7, pcDNA-control or pcDNA-SNHG7 were detected via reverse transcription-quantitative PCR. (M) Correlation between SNHG7 and miR-146b-5p was analyzed by Pearson test. * P<0.05 vs. their respective normal groups. WT, wild-type; MUT, mutant; SNHG7, small nucleolar RNA host gene 7; miR, microRNA; sh, short hairpin RNA; NC, negative control; Ago2, argonaute-2.

Article Snippet: Following lysis of the transfected SW1990 and AsPC-1 cells, the lysate samples were incubated with magnetic beads labelled with anti-argonaute-2 (Ago2; BIOSS) or negative control anti-IgG antibody (BIOSS) for 4 h at 4˚C, and a 10 µl aliquot of RIP mixture was saved as input.

Techniques: Luciferase, Activity Assay, Transfection, Labeling, Pull Down Assay, Expressing, Real-time Polymerase Chain Reaction, Mutagenesis, shRNA, Negative Control

Journal: Science Advances

Article Title: Enterovirus evolution reveals the mechanism of an RNA-targeted antiviral and determinants of viral replication

doi: 10.1126/sciadv.adg3060

Figure Lengend Snippet: ( A ) Single-point TROSY HSQC titration of A( 13 C)–selectively labeled SLII resist with DMA-135 at a fivefold excess. The black correlation peaks correspond to free SLII resist and the red to the (DMA-135)-SLII resist complex. The spectra were collected at 900 MHz in 10 mM K 2 HPO 4 (pH 6.5 before exchanging in D 2 O), 20 mM KCl, 0.5 mM EDTA, and 4 mM BME D 2 O buffer at 298 K. ( B ) Calorimetric titrations of A-RRM1,2 into SLII (left) and SLII resist (right). The A-RRM1,2-SLII data were fit to a 1:1 stoichiometric binding model in Affinimeter . ND, not determined. Reported values for K D and corresponding SD are from triplicate experiments. ( C ) RNA constructs used to assess determinants of specific and high-affinity AUF1-SLII interactions. Left, SLII CCC replaces the phylogenetically conserved UAG bulge motif with CCC. Right, a 7-nt oligonucleotide that mimics the SLII bulge loop sequence with adjacent nucleotides. ( D ) Calorimetric titrations of A-RRM1,2 into SLII CCC (left) and the 7-nt oligonucleotide (right). The A-RRM1,2-SLII CCC data were fit to a 1:1 stoichiometric binding model in Affinimeter . Reported values for K D and corresponding SD are from triplicate experiments. ( E ) Protein-biotinylated RNA pull-down experiments were performed to evaluate the influence of DMA-135 on the interaction between AUF1 and SLII resist . Biotinylated SLII and SLII resist RNAs were transfected into SF268 cells. Cells were cultured with increasing concentrations of DMA-135. Cell lysates were used for pull-down assays of SLII-associated proteins AUF1, hnRNP A1, Ago2, and HuR and detection by Western blotting.

Article Snippet: The primary antibodies used were as follows: anti-AUF1 rabbit polyclonal, 1:15,000 (Pocono Rabbit Farm & Lab, Canadensis, PA); anti-hnRNP A1 mouse monoclonal (Abcam), 1:200; anti-Ago2 rabbit polyclonal (Abcam), 1:200; and anti-HuR mouse monoclonal (Santa Cruz Biotechnology), 1:200.

Techniques: Titration, Labeling, Binding Assay, Construct, Sequencing, Transfection, Cell Culture, Western Blot

Journal: Cell

Article Title: Phase transitions in the assembly and function of human miRISC

doi: 10.1016/j.cell.2018.02.051

Figure Lengend Snippet: (A) Cartoon representation of Ago2 illustrates the position of the trp-binding region. (B) Close up view of the trp-binding region. Fo-Fc tryptophan omit map, contoured at 2.5 σ, (green mesh) shows well-ordered indole side chains of three bound tryptophan molecules. (C) Surface representation of the region illustrates the three trp-binding pockets. Curved lines indicate approximate distances between adjacent pockets. See also Figure S1 and Table S1.

Article Snippet: Rabbit polyclonal anti-human Ago2 , Cell Signaling , Cat# 2897S.

Techniques: Binding Assay

Journal: Cell

Article Title: Phase transitions in the assembly and function of human miRISC

doi: 10.1016/j.cell.2018.02.051

Figure Lengend Snippet: (A) Linear schematic of TNRC6B domain structure. MBP fusion of the ABD used in pull down assays, and sequence context of motif I (W623 and W634) are indicated. (B) Coomassie stained SDS gel showing pull down of ABD variants by wild type (WT) Ago2. AllA is an ABD variant in which all trp residues have been replaced with alanine. Ago2, wild type ABD variants, and AllA ABD variants are indicated by single, double, and triple asterix, respectively. (C) Schematic of trp-binding pockets in Ago2 with residues mutated to disable individual pockets indicated in blue (K660S, P590G, and R688S mutations in pockets 1, 2 and 3, respectively). (D) Pull down of ABD variants by Ago2 with individual trp-binding pockets inactivated. (E) Pull down of ABD by Ago2 with combinations of trp-binding pockets inactivated. (F) Schematic of ABD construct with sequence context motif II (W875, W896, and W910) indicated. (G) Pull down of W875, W896, and W910 AllA-ABD variants by WT Ago2, and (H) by trp-binding pocket Ago2 mutants. See also Figure S2.

Article Snippet: Rabbit polyclonal anti-human Ago2 , Cell Signaling , Cat# 2897S.

Techniques: Sequencing, Staining, SDS-Gel, Variant Assay, Binding Assay, Construct

Journal: Cell

Article Title: Phase transitions in the assembly and function of human miRISC

doi: 10.1016/j.cell.2018.02.051

Figure Lengend Snippet: (A) Initial observation of ABD-Ago2 phase separation. Solutions containing the TNRC6B ABD become turbid upon introduction of Ago2. Solutions of the AllA-ABD mutant remain clear. (B) Turbidity, measured by absorbance of 480 nm light, of solutions containing Ago2 (20 µM), ABD (40 µM), or ABD + Ago2 (40µM and 20µM, respectively) plotted as a function of temperature. (C) Turbidity versus temperature for ABD samples (10 µM) with various concentrations of Ago2. (D) Light microscopy images of mixtures of ABD (20 µM) and Ago2 taken at room temperature (~23 °C). Scale bar, 10 µm. (E) Time-lapse images showing fusion of three adjacent ABD-Ago2 droplets (left), and confocal microscopy images showing fusion of ABD-Ago2 droplets containing Alexa-488 labeled ABD (right). (F) Fluorescence microscopy images from a FRAP experiment in which an entire ABD-Ago2 droplet was bleached. 10% of Ago2 molecules were labeled with TMR, and ~10% ABD molecules were labeled with Alexa Fluor 488. (G) FRAP recovery curves for three ABD-Ago2 droplets with error bars indicating SEM. All droplets were formed in 100 mM KOAc and 20 nM NaCl. See also Figures S3, and Movies S1 and S2.

Article Snippet: Rabbit polyclonal anti-human Ago2 , Cell Signaling , Cat# 2897S.

Techniques: Mutagenesis, Light Microscopy, Confocal Microscopy, Labeling, Fluorescence, Microscopy

Journal: Cell

Article Title: Phase transitions in the assembly and function of human miRISC

doi: 10.1016/j.cell.2018.02.051

Figure Lengend Snippet: (A) Fluorescence microscopy images showing Ago2 (TMR labeled) promotes phase separation of full length TNRC6B (Alexa Fluor 488 labeled) in vitro. (B) Spherical miRISC droplets formed in vitro coalesced into larger clusters over time. (C) Representative fluorescence microscopy images from miRISC FRAP experiments. (D) FRAP recovery curves for three miRISC droplets with error bars indicating SEM. (E) Live cell images showing fusion of two GFP-TNRC6B cytoplasmic foci in HEK 293 cells over time. (F) Representative live cell images of GFP-TNRC6B cytoplasmic foci FRAP experiments. (G) FRAP recovery curves for three GFP-TNRC6B cytoplasmic foci with error bars indicating SEM. (H) Representative live cell images of GFP-TNRC6B/mCherry-Ago2 cytoplasmic foci FRAP experiments. (I) FRAP recovery curves for four GFP-TNRC6B/mCherry-Ago2 cytoplasmic foci with error bars indicating SEM. See also Figure S4, and Movies S3, S5 and S6.

Article Snippet: Rabbit polyclonal anti-human Ago2 , Cell Signaling , Cat# 2897S.

Techniques: Fluorescence, Microscopy, Labeling, In Vitro

Journal: Cell

Article Title: Phase transitions in the assembly and function of human miRISC

doi: 10.1016/j.cell.2018.02.051

Figure Lengend Snippet: (A) Ago2-TNRC6B droplets can be separated from the bulk solvent by centrifugation. Cartoon schematic of procedure (left), and images of droplets in input and supernatant fractions (right). (B) Droplets recruit full-length TNRC6B, Ago2, and miRNA target RNAs. TNRC6B (~1 mM, partially purified) was mixed with Ago2 (0.5 µM) loaded with either let-7 or miR122, and a 32P-labeled let-7 target RNA (8xlet7 target, ~3 nM). After centrifugation, supernatant and pellet fractions were analyzed by Coomassie stained SDS PAGE (right, top panel) and phosphorimaging of a denaturing gel (right, bottom panel). (C) Ago2 remains active in the separated phase. TNRC6B (~ 1 µM) was mixed with Ago2-miR122 (250 nM) and a 32P-labeled target RNA (~0.5 µM) with perfect complementarity to miR122 in the absence of divalent cations. After centrifugation MgCl2 (3 mM) was added to the separated phase. Target RNA was extracted and analyzed by denaturing PAGE and phosphorimaging (right panel). (D) TNRC6B-Ago2 droplets recruit other miRISC components. TNRC6B (40 nM) was mixed with Ago2 (40 nM) and soluble lysate from HEK 293 cells (OD260 ~3). Input, supernatant, and pellet fractions were analyzed by Western blot (right panel). See also Figure S5.

Article Snippet: Rabbit polyclonal anti-human Ago2 , Cell Signaling , Cat# 2897S.

Techniques: Solvent, Centrifugation, Purification, Labeling, Staining, SDS Page, Western Blot

Journal: Cell

Article Title: Phase transitions in the assembly and function of human miRISC

doi: 10.1016/j.cell.2018.02.051

Figure Lengend Snippet: (A) Schematic of experiment. (B) Deadenylation of a target RNA by miRISC. Ago2 (40 nM, final concentration), loaded with either let7 or miR122 (negative control), was mixed with a 32P-5'-cap-labeled target RNA harboring binding sites for let-7 and a 114 nt. poly(A) tail in the presence of soluble lysate from HEK 293 cells (OD260 ~3), with and without additional TNRC6B (~300 nM, partially purified). After a 15-minute incubation, supernatant and pellet fractions were isolated by centrifugation and target RNA was extracted and analyzed by denaturing PAGE and phosphorimaging. (C) Deadenylation timecourse. Reactions containing Ago2-let7 (40 nM) and soluble HEK 293 lysate (OD260 ~3), with and without exogenous TNRC6B (~300 nM, final concentration) were fractionated at various times, and analyzed by denaturing gel. (D) Estimation of deadenylation rates. Target RNA bands in (C) were quantified and fraction of total intact RNA (A114) for +/− TNRC6B conditions was plotted as a function of incubation time. Data were fit with a first order decay, yielding A114 half-lives of 40 and 4 minutes for plus and minus exogenous TNRC6B, respectively. Plotted data are the average of three independent experiments with SEM indicated as error bars. See also Figure S6.

Article Snippet: Rabbit polyclonal anti-human Ago2 , Cell Signaling , Cat# 2897S.

Techniques: Concentration Assay, Negative Control, Labeling, Binding Assay, Purification, Incubation, Isolation, Centrifugation

Journal: Cell

Article Title: Phase transitions in the assembly and function of human miRISC

doi: 10.1016/j.cell.2018.02.051

Figure Lengend Snippet: (A) PEG 8000 promotes TNRC6B-Ago2 phase separation. Images of droplets formed from Alexa-488 labeled TNRC6B (~20 nM) and Ago2 (200 nM) in the presence and absence of 5% (w/v) PEG 8000. (B) Effects of PEG 8000 on target deadenylation reactions. 8xlet7 deadenylation reactions containing combinations of Ago2 (20 nM), HEK 293 lysate (OD260 ~1.5), and exogenous TNRC6B (~30 nM) were treated with 5% (w/v) PEG 8000, separated into supernatant and pellet fractions, and analyzed by denaturing PAGE. (C) Effect of PEG 8000 on deadenylation rates. Reactions containing Ago2-let7 (20 nM), HEK 293 lysate (OD260 ~1.5), and exogenous TNRC6B (~30 nM) were treated with 5 % (w/v) PEG 8000, separated into supernatant and pellet fractions, and analyzed by denaturing PAGE at various times. (D) PEG 8000 accelerates deadenylation. Bands corresponding to target RNA species in (C) were quantified and fraction of intact target (A114) plotted as a function of time. Plotted data are the average of three independent experiments with SEM indicated as error bars. See also Figure S7.

Article Snippet: Rabbit polyclonal anti-human Ago2 , Cell Signaling , Cat# 2897S.

Techniques: Labeling

Journal: Cell

Article Title: Phase transitions in the assembly and function of human miRISC

doi: 10.1016/j.cell.2018.02.051

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-human Ago2 , Cell Signaling , Cat# 2897S.

Techniques: Recombinant, Protease Inhibitor, Protein Purification, Cloning, Expressing, Synthesized, Software

Journal: BioMed Research International

Article Title: Nrp-1 Mediated Plasmatic Ago2 Binding miR-21a-3p Internalization: A Novel Mechanism for miR-21a-3p Accumulation in Renal Tubular Epithelial Cells during Sepsis

doi: 10.1155/2020/2370253

Figure Lengend Snippet: miR-21a-3p and Ago2 levels of both TECs and plasma during sepsis. (a) Quantitative analysis of RT-PCR results of miR-21a-3p of either plasma or TECs at different time points. (b) Quantitative analysis of ELISA for Ago2 concentrations of plasma at different groups. (c) Representative RIP results of Ago2 binding miR-21a-3p in plasma of different groups. (d) Representative Western Blot results of Ago2 in TECs of the control group and CLP groups at different time points ( ∗ P < 0.05 vs. the control; Ctrl: control; 12 h: 12 hours after CLP; 18 h: 18 hours after CLP; 24 h: 24 hours after CLP).

Article Snippet: For the cytoplasm Ago2 measurement, Fixation/Permeabilization Solution Kit (BD) was used to permeabilized the cells accordingly; then, Ago2 mouse monoclonal antibody (Novus) and FITC-conjugated goat anti-mouse IgG antibody (Boster) were used to determine the cytoplasm Ago2 level with flow cytometry.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Binding Assay, Western Blot

Journal: BioMed Research International

Article Title: Nrp-1 Mediated Plasmatic Ago2 Binding miR-21a-3p Internalization: A Novel Mechanism for miR-21a-3p Accumulation in Renal Tubular Epithelial Cells during Sepsis

doi: 10.1155/2020/2370253

Figure Lengend Snippet: Ago2 and miR-21a-3p levels in TECs stimulated with differently treated plasma. (a) Quantitative analysis of the results from ELIA for Ago2 and RT-PCR for miR-21a-3p of plasma with or without treatments. (b) Quantitative analysis of RT-PCR results of miR-21a-3p of TECs treated with different plasma. (c) Representative results of flow cytometry for Ago2 in TECs treated with different plasma. (d) Quantitative analysis of the results of flow cytometry for Ago2 in TECs treated with different plasma. (e) Representative Western Blot results of Ago2 in TECs treated with different plasma ( ∗ P < 0.05 vs. the control; Ctrl: control; NP: normal plasma; 12 h: septic plasma from rats 12 hours after CLP; 12 h+Ab: septic plasma treated with Ago2 antibody; 12 h+IP: septic plasma treated with Ago2 immunoprecipitation).

Article Snippet: For the cytoplasm Ago2 measurement, Fixation/Permeabilization Solution Kit (BD) was used to permeabilized the cells accordingly; then, Ago2 mouse monoclonal antibody (Novus) and FITC-conjugated goat anti-mouse IgG antibody (Boster) were used to determine the cytoplasm Ago2 level with flow cytometry.

Techniques: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Flow Cytometry, Western Blot, Immunoprecipitation

Journal: BioMed Research International

Article Title: Nrp-1 Mediated Plasmatic Ago2 Binding miR-21a-3p Internalization: A Novel Mechanism for miR-21a-3p Accumulation in Renal Tubular Epithelial Cells during Sepsis

doi: 10.1155/2020/2370253

Figure Lengend Snippet: Ago2 and miR-21a-3p levels in TECs with or without Nrp-1 knockdown treated with septic plasma. (a) Representative Western Blot results of cytoplasm Ago2 of TECs with different treatments. (b) Quantitative analysis of RT-PCR results of miR-21a-3p of TECs with different treatments ( ∗ P < 0.05 vs. the control, ^ P < 0.05 between the two groups; Ctrl: control; 12 h: septic plasma from rats 12 hours after CLP; SiNrp-1: Nrp-1 siRNA transfection; SiRNA NC: siRNA negative control transfection).

Article Snippet: For the cytoplasm Ago2 measurement, Fixation/Permeabilization Solution Kit (BD) was used to permeabilized the cells accordingly; then, Ago2 mouse monoclonal antibody (Novus) and FITC-conjugated goat anti-mouse IgG antibody (Boster) were used to determine the cytoplasm Ago2 level with flow cytometry.

Techniques: Western Blot, Quantitative RT-PCR, Transfection, Negative Control

Journal: BioMed Research International

Article Title: Nrp-1 Mediated Plasmatic Ago2 Binding miR-21a-3p Internalization: A Novel Mechanism for miR-21a-3p Accumulation in Renal Tubular Epithelial Cells during Sepsis

doi: 10.1155/2020/2370253

Figure Lengend Snippet: Exogenous Ago2 in TECs with different treatments. (a) Representative fluorescent results of cytoplasm Ago2 of TECs with different treatments. (b) Representative flow cytometry results of Ago2 in TECs with different treatments. (c) Quantitative analysis of flow cytometry results of Ago2 in TECs with different treatments. (d) Representative Western Blot results of cytoplasm Ago2 in TECs with different treatments. (e) Representative Western Blot results of cytoplasm His residue at 99 Kd in TECs with different treatments. (f) Representative results of immunoprecipitation of Ago2 binding His residue of TECs in the control and exogenous Ago2-treated groups. (g) Quantitative analysis of RT-PCR results of intracellular miR-21a-3p of TECs with different treatments ( ∗ P < 0.05 vs. the control; Ctrl: control; SiNrp-1: Nrp-1 siRNA transfection).

Article Snippet: For the cytoplasm Ago2 measurement, Fixation/Permeabilization Solution Kit (BD) was used to permeabilized the cells accordingly; then, Ago2 mouse monoclonal antibody (Novus) and FITC-conjugated goat anti-mouse IgG antibody (Boster) were used to determine the cytoplasm Ago2 level with flow cytometry.

Techniques: Flow Cytometry, Western Blot, Immunoprecipitation, Binding Assay, Quantitative RT-PCR, Transfection

Journal: BioMed Research International

Article Title: Nrp-1 Mediated Plasmatic Ago2 Binding miR-21a-3p Internalization: A Novel Mechanism for miR-21a-3p Accumulation in Renal Tubular Epithelial Cells during Sepsis

doi: 10.1155/2020/2370253

Figure Lengend Snippet: Interaction of Nrp-1 with either Ago2 or miR-21a-3p. (a) Representative results of immunoprecipitation of the cell lysates of TECs with different treatments; the lysates were immunoprecipitated with Ago2 antibody and probed with Ago2 and Nrp-1 antibodies after electrophoresis. (b) Representative results of RNA pull-down assay for lysates of TECs treated with biotin-miR-21a-3p single strain mimics. Biotin-miR-21a-3p was precipitated; Ago2 and Nrp-1 were probed with immunoblotting. (c) Representative results of RNA pull-down assay for biotin-miR-21a-3p/Nrp-1 mixture. Biotin-miR-21a-3p was precipitated and Nrp-1 was probed with immunoblotting. (d) Representative results of RNA pull-down assay for biotin-miR-21a-3p/Ago2/Nrp-1 mixture. Biotin-miR-21a-3p was precipitated; Ago2 and Nrp-1 were probed with immunoblotting (Ctrl: control; SiNrp-1: Nrp-1 siRNA transfection; SiRNA NC: siRNA negative control transfection; NC: miR-21a-3p mimic negative control; Mimic: miR-21a-3p mimics).

Article Snippet: For the cytoplasm Ago2 measurement, Fixation/Permeabilization Solution Kit (BD) was used to permeabilized the cells accordingly; then, Ago2 mouse monoclonal antibody (Novus) and FITC-conjugated goat anti-mouse IgG antibody (Boster) were used to determine the cytoplasm Ago2 level with flow cytometry.

Techniques: Immunoprecipitation, Electrophoresis, Pull Down Assay, Western Blot, Transfection, Negative Control