Journal: BMC Research Notes

Article Title: A useful approach to total analysis of RISC-associated RNA

doi: 10.1186/1756-0500-2-169

Figure Lengend Snippet: Size distribution of synthesized cDNA and categories of cDNAs isolated from HeLa cells . A: Size distribution pattern of synthesized cDNA. The amount and size distribution of cDNA were determined with the Bioanalyzer DNA 1000 Kit. The red line indicates cDNA synthesized from anti-Ago2 immunoprecipitates, while the blue line indicates that from control mouse IgG. B: Composition of isolated cDNA clones. The sequences of 91 clones were examined by BLAST search and categorized as mRNA, Alu RNA embedded mRNA, free Alu RNA, or hits in the genome. The percentage recovery of each categorized cDNA clone is shown. Alu RNA embedded mRNA (14.3%, not indicated in the figure) is included in the mRNA area (total 80%) and separated by a broken line.

Article Snippet: Twenty μl of ProteinG coupling beads (Dynal) bound with 5 μg of anti-human Ago2 antibody 4G8 (Wako) was added to cell lysate and mixed by rotation for 2 hours at 4°C.

Techniques: Synthesized, Isolation, Clone Assay

Journal: BMC Research Notes

Article Title: A useful approach to total analysis of RISC-associated RNA

doi: 10.1186/1756-0500-2-169

Figure Lengend Snippet: Changes in mRNA level in total and Ago2-associated RNA after miR-122 transfection . Seventeen clones with high score percentile for the miR-122 target site (> = 40) were picked, and the mRNA levels in total and immunoprecipitated RNA were quantified by RT-PCR. In the figure, the ratios of the amount of mRNA in the miR-122-transfected cells to that in the GL3-transfected cells are shown. Blue bars indicate the ratio for Ago2-associated RNA, while purple bars show that for total RNA. The left panel shows the clones picked from the miR-122-transfected cells, and the right panel shows the clones picked from the GL3-transfected cells (See Figure 6A). The clones are aligned in order of height of context score percentile from the left, with the ratio for GAPDH inserted as a standard.

Article Snippet: Twenty μl of ProteinG coupling beads (Dynal) bound with 5 μg of anti-human Ago2 antibody 4G8 (Wako) was added to cell lysate and mixed by rotation for 2 hours at 4°C.

Techniques: Transfection, Clone Assay, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Oncology

Article Title: Anisomycin inhibits angiogenesis in ovarian cancer by attenuating the molecular sponge effect of the lncRNA-Meg3/miR-421/PDGFRA axis

doi: 10.3892/ijo.2019.4887

Figure Lengend Snippet: Anisomycin attenuates the molecular sponge effect in the lncRNA-Meg3/miR-421/PDGFRA axis. (A) Heatmap showing that the expression of lncRNA-Meg3 in the anisomycin-treated group was significantly lower than that of the control group. (B) Expression levels in clinical tumour samples and (C) in xenograft tumours suggest an inverse trend in expression for lncRNA-Meg3 and miR-421. (D) Expression levels of miR-421 in HuOCSCs following anisomycin treatment. ** P<0.01 vs. non-treated (n=4). (E) Schematic of the complementary sites of mature miR-421 and the 3'UTR of lncRNA-Meg3 and PDGFRA mRNA. (F) Schematic of the structure of luciferase reporter plasmids. (G) The molecular sponge effect of lncRNA-Meg3/miR-421/PDGFRA. (H-J) Results of the luciferase reporter assays. ** P<0.01 vs. blank plasmid (n=3). (K) RIP results revealed that the binding between Meg3 and Ago2 decreased significantly following treatment of HuOCSCs with anisomycin. lncRNA, long non-coding RNA; Meg3, maternally expressed 3; miR, microRNA; PDGFRA, platelet derived growth factor receptor α; HuOCSCs, human ovarian cancer stem cells; RIP, RNA immunoprecipitation; Ago2, argonaute 2; ORF, open reading frame; UTR, untranslated region; mut, mutant; IgG, immunoglobulin G.

Article Snippet: The mouse anti-human Ago2 monoclonal antibody (clone C34C6; cat no. 2897; Cell Signaling Technology, Inc.; 1:100) was added for 90 min, before the re-addition of 20 μ g of protein A agarose beads to capture the immune complexes.

Techniques: Expressing, Control, Luciferase, Plasmid Preparation, Binding Assay, Derivative Assay, RNA Immunoprecipitation, Mutagenesis

Journal: Molecular Biology of the Cell

Article Title: Actin-dependent recruitment of AGO2 to the zonula adherens

doi: 10.1091/mbc.e22-03-0099-t

Figure Lengend Snippet: FIGURE 7: Contractile actin tension at the ZA is required for AGO2 junctional recruitment. (A–F) Immunofluorescence of Caco2 cells during a calcium switch assay in which DMSO or Blebbistatin (Blebb) were included in the calcium- containing recovery medium. Cells fixed at 30 and 60 min post Ca2+ reintroduction were stained for AGO2 (A), PLEKHA7 (B), E-cadherin (C), LMO7 (D), LIMCH1 (E), and PDLIM1 (F). Insets are marked by white rectangles and are 3× magnification of the original image. Fluorescence intensity of 6-µm line scans drawn perpendicular to cell–cell junctions was measured for each marker at the 60-min time point upon recovery from n = 30 cell–cell junctions (10 junctions/field) representative of three independent experiments; statistical analyses were performed using two-way ANOVA tests. Error bars represent mean ± SD. ****P < 0.0001; *P < 0.05; ns, non-significant. Scale bars = 20 µm.

Article Snippet: Antibodies The primary antibodies used in this study were PLEKHA7 (HPA038610; SigmaAldrich), Ecad (610182; BD Transduction Labs), AGO2 (ab57113; Abcam - optimal for immunoblotting), AGO2 (AP5281; ECM Biosciences - optimal for immunofluorescence), AGO2 (ab156870; Abcam - optimal for immunofluorescence and immunoprecipitation/RNA- immunoprecipitation); LMO7 (ab224113; Abcam), LIMCH1 (ab96178; Abcam), PDLIM1 (ab129015; Abcam), β-actin (4967L; Cell Signaling Technology), Myosin IIB (909901; Biolegend), Myosin IIA (909801; Biolegend), pS19-MRLC (3671; Cell Signaling Technology), α-catenin (610193; BD Transduction Labs), anti-Flag (clone M2, F1804; Sigma-Aldrich).

Techniques: Immunofluorescence, Staining, Fluorescence, Marker

Journal: Molecular Biology of the Cell

Article Title: Actin-dependent recruitment of AGO2 to the zonula adherens

doi: 10.1091/mbc.e22-03-0099-t

Figure Lengend Snippet: FIGURE 8: PLEKHA7, LMO7, LIMCH1, or PDLIM1 depletion each disrupt AGO2-Myosin IIB interaction at the ZA. (A) Immunoprecipitation (IP) of AGO2 and Myosin IIB from Caco2 cells, immunoblotted (IB) for the same markers; IgG is the negative control. Molecular masses (kD) are indicated on the right. (B–E) Wild type (WT) or PLEKHA7 KO Caco2 cells and control (NT) or LMO7, LIMCH1, and PDLIM1 knockdown (shLMO7, shLIMCH1, and shPDLIM1) cells stably expressing an AGO2-Flag construct (see Supplemental Figure S1, A–D) were subjected to PLA for AGO2-Flag, using an anti-Flag antibody, and Myosin IIB, followed by immunofluorescence staining for Ecad and confocal microscopy; DAPI was used to stain nuclei. Caco2 cells stably transduced with an empty vector were used as negative PLA control. Insets are marked by white rectangles and are 3× magnification of the original image. The ratio of junctional vs cytoplasmic PLA signals was quantified for each condition and from n = 30 cells (10 cells/field) representative of three independent experiments; statistical analyses were performed using either unpaired two-way t test (B–C) or one-way ANOVA test (D–E). Error bars represent mean ± SD. **P < 0.01; *P < 0.05. Scale bars = 20 µm.

Article Snippet: Antibodies The primary antibodies used in this study were PLEKHA7 (HPA038610; SigmaAldrich), Ecad (610182; BD Transduction Labs), AGO2 (ab57113; Abcam - optimal for immunoblotting), AGO2 (AP5281; ECM Biosciences - optimal for immunofluorescence), AGO2 (ab156870; Abcam - optimal for immunofluorescence and immunoprecipitation/RNA- immunoprecipitation); LMO7 (ab224113; Abcam), LIMCH1 (ab96178; Abcam), PDLIM1 (ab129015; Abcam), β-actin (4967L; Cell Signaling Technology), Myosin IIB (909901; Biolegend), Myosin IIA (909801; Biolegend), pS19-MRLC (3671; Cell Signaling Technology), α-catenin (610193; BD Transduction Labs), anti-Flag (clone M2, F1804; Sigma-Aldrich).

Techniques: Immunoprecipitation, Negative Control, Control, Knockdown, Stable Transfection, Expressing, Construct, Immunofluorescence, Staining, Confocal Microscopy, Transduction, Plasmid Preparation