Journal: Cell

Article Title: Regulation of MicroRNA Machinery and Development by Interspecies S-Nitrosylation

doi: 10.1016/j.cell.2019.01.037

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Hmga2 3’UTR WT luciferase (Luc-wt) and Hmga2 3’UTR m7 luciferase (Luc-m7) were gifts from David Bartel (Addgene plasmid # 14785 and # 14788, respectively) ( Mayr et al., 2007 )).

Techniques: Western Blot, Immunoprecipitation, Recombinant, Purification, Lysis, Protease Inhibitor, Silver Staining, Staining, Reporter Assay, Clone Assay, esiRNA, Plasmid Preparation, Luciferase, Software

Journal: Cell

Article Title: Regulation of MicroRNA Machinery and Development by Interspecies S-Nitrosylation

doi: 10.1016/j.cell.2019.01.037

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Hmga2 3’UTR m7 luciferase , Mayr et al., 2007 , Addgene Plasmid #14788.

Techniques: Western Blot, Immunoprecipitation, Virus, Recombinant, Purification, Lysis, Protease Inhibitor, Silver Staining, Staining, Reporter Assay, Cloning, Control, esiRNA, Plasmid Preparation, Luciferase, Software

Journal: Disease Markers

Article Title: miR-1 Inhibits Cell Growth, Migration, and Invasion by Targeting VEGFA in Osteosarcoma Cells

doi: 10.1155/2016/7068986

Figure Lengend Snippet: The interaction between miR-1 and VEGFA. (a) Putative binding sites for miR-1 on VEGFA 3′-UTR were shown. The mutated region in VEGFA 3′-UTR was labeled by a horizontal line. (b) U2OS cells were cotransfected with a luciferase reporter containing the wild type (WT) or mutant (Mut) VEGFA 3′-UTR, along with miR-1 mimic or scramble mimic. The relative luciferase activities were measured 24 h after transfection. (c) The protein expression level of VEGFA was detected by western blot. A representative western blot and quantitative results of densitometry are shown. Data were expressed as the mean ± SD ( n = 3). ∗ P < 0.01; NS, no significant difference compared to control.

Article Snippet: The dual luciferase reporter plasmid fused with the wild type or mutant 3′-UTR segment of human VEGFA was purchased from GenePharma, Shanghai, China.

Techniques: Binding Assay, Labeling, Luciferase, Mutagenesis, Transfection, Expressing, Western Blot, Control

Journal: Oncotarget

Article Title: Inhibition of canonical WNT/β-catenin signaling is involved in leflunomide (LEF)-mediated cytotoxic effects on renal carcinoma cells

doi: 10.18632/oncotarget.10409

Figure Lengend Snippet: A. Heatmap of hierarchical clustering of gene expression from Caki-2 cells treated with 200 μM LEF or vehicle control. B. Real-time PCR for the expression of FZD1, FZD2 , and FZD10 in mRNA levels after LEF treatment. Data represent mean ± SD from three independent experiments C. Changes of FZD10 proteins after LEF treatment for 48 h. D. Caki-2 cells were transfected with FZD10 or scrambled siRNAs respectively. After 48 h, western blot detected the protein levels of FZD10 and β-catenin. Representative images from at least three independent experiments are shown. E. Cell growth was estimated by MST assay after siRNA transfection for 72 h. (* P <0.01, ** P <0.05, vs. the control).

Article Snippet: The FZD10 and scrambled siRNAs were purchased from RiboBio (RiboBio, China) and transfected at 100 nM.

Techniques: Gene Expression, Control, Real-time Polymerase Chain Reaction, Expressing, Transfection, Western Blot

Journal: Human Molecular Genetics

Article Title: Genome-wide association study with additional genetic and post-transcriptional analyses reveals novel regulators of plasma factor XI levels

doi: 10.1093/hmg/ddw401

Figure Lengend Snippet: Results from the luciferase assay of miRNA binding to F11 mRNA. The plot shows luciferase activity after 24h of cells co-transfected with either an empty luciferase reporter plasmid (pLS), or a plasmid containing the FXI 3’UTR (pLS-FXI-3’UTR), together with scramble control, miR-145 or miR-181.

Article Snippet: At 50–60% confluence, cells were transfected with luciferase reporter plasmid pLS, pLS-FXI-3’UTR (100 ng/well, Active Motif, Switchgear Genomics), together with scramble control, miR-145 or miR-181 (10 nM final concentration) using DharmaFECT Duo Transfection Reagent (Switchgear) according to the manufacturer’s protocol.

Techniques: Luciferase, Binding Assay, Activity Assay, Transfection, Plasmid Preparation, Control

Journal: Nature genetics

Article Title: 3′ UTR shortening represses tumor-suppressor genes in trans by disrupting ceRNA crosstalk

doi: 10.1038/s41588-018-0118-8

Figure Lengend Snippet: a, Western blot of 3′ US ceRNA tumor-suppressor genes (PHF6/LARP1) and 3′ US genes (LAMC1/YOD1) in NUDT21-knockdown cells. A representative image is shown from three independent experiments. b, Activity of the PHF6 3′ UTR and LARP1 3′ UTR luciferase reporter constructs in NUDT21-knockdown cells relative to control siRNA-transfected cells. The data are the average of luciferase activity ± standard deviation from three independent experiments (P = 0.037 and 0.05; P = 0.016 and 0.025, two-sided t-test). c, NUDT21 knockdown induces 3′ UTR shortening and upregulation of YOD1, allowing miR-3187–3p and miR-549 to repress PHF6. d, Western blot analysis using the indicated antibodies on lysates from HeLa cells transfected with siRNA for NUDT21 (si-NUDT21-4) and two antagomirs that block miR-549 and miR-3187–3p. The image is representative of two independent experiments. e, Activity of the PHF6 3′ UTR luciferase reporter construct in HeLa cells with the indicated siRNAs, miRNAs or antagomirs. The data are the average of luciferase activity ± standard deviation from three independent experiments (P = 0.004, P = 0.90, P = 0.018 and P = 0.015, two-sided t-test). f, Western blot analysis of cell lysates from cells transfected with either control siRNA or YOD1 siRNA. In the third lane, the cells were transfected with YOD1 siRNA and then transfected with YOD1 cDNA. The data are representative of three independent experiments. g, Activity of the PHF6 3′ UTR luciferase reporter in cells treated with the same experimental design as in f. The data are the average of luciferase activity ± standard deviation from three independent experiments (P = 0.016 and P = 0.01, two-sided t-test). h, Activity of the PHF6 luciferase reporter in cells transfected with the indicated siRNAs. The data are the average of luciferase activity ± standard deviation from three independent experiments (P = 0.025, P = 0.009 and P = 0.99, two-sided t-test).

Article Snippet: LightSwitch luciferase reporter constructs with PTEN, PHF6 and LARP1 3′ UTR were purchased from SWITCHGEAR genomics.

Techniques: Western Blot, Activity Assay, Luciferase, Construct, Transfection, Standard Deviation, Blocking Assay