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

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: Molecular and Cellular Biology

Article Title: Ribosomal Protein L11 Recruits miR-24/miRISC To Repress c-Myc Expression in Response to Ribosomal Stress ^▿

doi: 10.1128/MCB.05810-11

Figure Lengend Snippet: Mutual dependency of L11 and ago2 in regulating c-myc mRNA. (A) Ago2 regulates c-myc mRNA levels. U2OS cells transfected with scrambled or Ago2 siRNA were assayed for expression of c-Myc protein (bottom panels) and mRNA (top panel). (B) Overexpression of L11 increases association of Ago2 with c-myc mRNA. U2OS cells were transfected with control or Flag-L11 plasmid. The cell lysates were immunoprecipitated with anti-Ago2 antibodies followed by an RT-qPCR assay to determine the levels of c-myc mRNA. The protein expression results are shown in the bottom panels. (C) Knockdown of L11 reduces the association of Ago2 with c-myc mRNA. Lysates from U2OS cells transfected with scrambled or L11 siRNA were immunoprecipitated with anti-Ago2 antibodies followed by an RT-qPCR assay to determine the levels of c-myc mRNA. The protein expression results are shown in the bottom panels. (D and E) L11 suppression of c-myc mRNA requires Ago2. U2OS cells transfected with control or Flag-L11 plasmid together with scrambled or Ago2 siRNA were assayed for c-Myc protein expression by immunoblotting (IB) (D) and for mRNA expression by RT-qPCR assays (E). The c-Myc bands were quantified and normalized to tubulin. The ratios of lane 2 to lane 1 and of lane 4 to lane 3 for the results from three independent experiments are indicated in panel D. (F and G) Ectopically expressed L11 interacts with Ago2. 293 (F) or U2OS (G) cells transfected with control or Flag-L11 plasmid were subjected to IP with anti-Flag antibody followed by IB using anti-Ago2 antibodies. (H) Endogenous L11 interacts with endogenous Ago2. 293 cell lysates were immunoprecipitated with control mouse IgG or anti-Ago2 (left panels) or rabbit IgG or anti-L11 (right panels) antibody followed by IB detection performed using anti-L11 or anti-Ago2 antibodies. (I) Interaction of L11 with Ago2 requires RNA. U2OS cell lysates were immunoprecipitated with control mouse IgG or anti-Ago2 antibodies in the absence (lanes 2 and 4) or presence (lanes 3 and 5) of RNase followed by IB detection performed using anti-L11 or anti-Ago2 antibodies.

Article Snippet: Anti-Flag (M2; Sigma), rabbit polyclonal anti-Ago2 (Millipore), mouse monoclonal anti-Ago2 (Abcam), mouse monoclonal anti-Myc (9E10; Zymed), and mouse polyclonal anti-Myc (Y69; Abcam) antibodies were purchased.

Techniques: Transfection, Expressing, Over Expression, Plasmid Preparation, Immunoprecipitation, Quantitative RT-PCR, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Ribosomal Protein L11 Recruits miR-24/miRISC To Repress c-Myc Expression in Response to Ribosomal Stress ^▿

doi: 10.1128/MCB.05810-11

Figure Lengend Snippet: L11 downregulates c-myc mRNA in response to ribosomal stress. (A and B) c-Myc is downregulated by treatment with Act D or 5-FU. U2OS cells were treated with DMSO, Act D (5 nM), or 5-FU (10 μg/ml) for the indicated hours. The cells were assayed for c-Myc protein expression by IB (A) and c-myc mRNA expression by RT-qPCR (B) assays. (C and D) Knockdown of L11 rescues the downregulation of c-Myc by treatment with Act D or 5-FU. U2OS cells transfected with scrambled or L11 siRNA were treated with DMSO, Act D (5 nM), or 5-FU (10 μg/ml) for 12 h. The cells were assayed for c-Myc protein expression by IB (C) and c-myc mRNA expression by RT-qPCR (D) assays. (E and F) Knockdown of Ago2 rescues the downregulation of c-Myc by treatment with Act D or 5-FU. U2OS cells transfected with scrambled or Ago2 siRNA were treated with DMSO, Act D (5 nM), or 5-FU (10 μg/ml) for 12 h. The cells were assayed for c-Myc protein expression by IB (E) and c-myc mRNA expression by RT-qPCR (F) assays.

Article Snippet: Anti-Flag (M2; Sigma), rabbit polyclonal anti-Ago2 (Millipore), mouse monoclonal anti-Ago2 (Abcam), mouse monoclonal anti-Myc (9E10; Zymed), and mouse polyclonal anti-Myc (Y69; Abcam) antibodies were purchased.

Techniques: Expressing, Quantitative RT-PCR, Transfection

Journal: Molecular and Cellular Biology

Article Title: Ribosomal Protein L11 Recruits miR-24/miRISC To Repress c-Myc Expression in Response to Ribosomal Stress ^▿

doi: 10.1128/MCB.05810-11

Figure Lengend Snippet: L11 recruits miRISC to c-myc mRNA in response to ribosomal stress. (A) Treatment with Act D or 5-FU increases the binding of L11 to c-myc mRNA. U2OS cells were treated with DMSO, Act D (5 nM), or 5-FU (10 μg/ml) for 12 h. The cell lysates were immunoprecipitated with control IgG or anti-L11 antibodies followed by RT-qPCR detection of c-myc mRNA. (B) Treatment with Act D or 5-FU increases the binding of Ago2 to c-myc mRNA. U2OS cells were treated with DMSO, Act D (5 nM), or 5-FU (10 μg/ml) for 12 h. The cell lysates were immunoprecipitated with anti-Ago2 antibodies or control IgG followed by RT-qPCR detection of c-myc mRNA. (C) Knockdown of L11 abolishes Ago2 binding to c-myc mRNA in response to ribosomal stress. U2OS cells transfected with scrambled or L11 siRNA were treated with DMSO, Act D (5 nM), or 5-FU (10 μg/ml) for 12 h. The cell lysates were immunoprecipitated with anti-Ago2 antibodies or control IgG followed by RT-qPCR detection of c-myc mRNA. The data were normalized to the c-myc mRNA in IP with control IgG. (D) Treatment with Act D or 5-FU increases the binding of L11 to Ago2. U2OS cells were treated with DMSO, Act D (5 nM), or 5-FU (10 μg/ml) for 12 h. The cell lysates were immunoprecipitated with anti-Ago2 antibodies followed by IB using anti-L11 antibodies. (E) Treatment with Act D or 5-FU increases the association of L11 with miR-24. U2OS cells were treated with DMSO, Act D (5 nM), or 5-FU (10 μg/ml) for 12 h and subjected to IP with anti-L11 or control IgG, followed by RT-qPCR detection of miR-24 and U6 snRNA. (F) Treatment with Act D or 5-FU does not change the total levels of miR-24 in cells. U2OS cells treated with DMSO, Act D (5 nM), or 5-FU (10 μg/ml) for 12 h were examined for expression of miR-24 normalized to that of U6 snRNA by the use of RT-qPCR assays.

Article Snippet: Anti-Flag (M2; Sigma), rabbit polyclonal anti-Ago2 (Millipore), mouse monoclonal anti-Ago2 (Abcam), mouse monoclonal anti-Myc (9E10; Zymed), and mouse polyclonal anti-Myc (Y69; Abcam) antibodies were purchased.

Techniques: Binding Assay, Immunoprecipitation, Quantitative RT-PCR, Transfection, Expressing