nf-κb and β-catenin/tcf firefly luciferase reporter construct (topflash)  (Promega)

Journal: Arthritis Research & Therapy

Article Title: Small molecule inhibitors of WNT/β-catenin signaling block IL-1β- and TNFα-induced cartilage degradation

doi: 10.1186/ar4273

Figure Lengend Snippet: Small molecule inhibitors of WNT/β-catenin signaling effectively block TCF/Lef-mediated activity of β-catenin . (A) Small molecules dose-dependently inhibit transcription factor/lymphoid enhancer-binding factor (TCF/LEF) reporter activity in HEK-293t cells, induced by the glycogen synthase kinase 3β (GSK3β) inhibitor 6-bromoindirubin 3'-oxime (BIO) (1.0 µM). Data represent the means of three independent experiments with 95% confidence intervals (CIs). (B) Metabolic activity, measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in KS483-4C3 cells, was not affected by small molecules at lower concentrations; however, at 1.0 µM (except for CGP049090) and 3.0 µM, metabolic activity was significantly decreased. Data represent the means of three independent experiments with 95% CI. (C) Treatment with 50 mM LiCl induced nuclear translocation of β-catenin. Small molecules by themselves had no effect on cellular localization of β-catenin, whereas PKF118-310 and PKF115-584 blocked LiCl-induced translocation of β-catenin to the nucleus. CGP049090 did not affect nuclear accumulation of β-catenin after LiCl treatment. A representative example of three independent experiments is shown. Scale bar represents 10 µm * P < 0.05 placed in the order relative to the order of the data points below..

Article Snippet: HEK-293t cells were seeded at 7,500 cells/cm 2 into 96-well plates (Nalge Nunc International, Penfield, NY, USA) and cultured for 24 h in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) and 100 U of penicillin-streptomycin (Gibco/Life Technologies, Grand Island, NY, USA) prior to transfection with the TOPflash TCF/Lef luciferase reporter construct (Upstate Biotechnology/EMD Millipore, Lake Placid, NY, USA) and pRL-CMV control vector (Promega, Madison, WI, USA).

Techniques: Blocking Assay, Activity Assay, Binding Assay, Translocation Assay

Journal: American Journal of Cancer Research

Article Title: Long noncoding RNA lncARSR promotes epithelial ovarian cancer cell proliferation and invasion by association with HuR and miR-200 family

doi:

Figure Lengend Snippet: lncARSR activates Wnt/β-catenin signaling pathway in EOC cells. A. Luciferase activity of TOPflash reporter was evaluated in SKOV3 cells overexpressing lncARSR. B. Luciferase activity of TOPflash reporter was evaluated in CAOV3 cells with lncARSR knockdown. C. The mRNA and protein level of β-catenin was determined by qRT-PCR and western blot in SKOV3 cells overexpressing lncARSR, respectively. D. The mRNA and protein level of β-catenin was determined by qRT-PCR and western blot in CAOV3 cells with lncARSR knockdown, respectively. E. The correlation between lncARSR and β-catenin mRNA expression was determined in EOC tissue samples. F. The mRNA and protein level of c-myc and cyclin D1 was determined by qRT-PCR and western blot in SKOV3 cells overexpressing lncARSR, respectively. G. The mRNA and protein level of c-myc and cyclin D1 was determined by qRT-PCR and western blot in CAOV3 cells with lncARSR knockdown, respectively. Data are shown as means ± SEM, *P < 0.05.

Article Snippet: Luciferase reporter assays β-catenin/TCF firefly luciferase reporter construct (TOPflash) and pRL-TK reporter was purchased from Promega (Madison, WI, USA).

Techniques: Luciferase, Activity Assay, Knockdown, Quantitative RT-PCR, Western Blot, Expressing

Journal: American Journal of Cancer Research

Article Title: Long noncoding RNA lncARSR promotes epithelial ovarian cancer cell proliferation and invasion by association with HuR and miR-200 family

doi:

Figure Lengend Snippet: lncARSR interacts with miR-200 family. A. MS2-RIP followed by microRNA qRT-PCR to detect microRNAs endogenously associated with lncARSR. B. Luciferase activity in EOC cells co-transfected with miR-200s and luciferase reporters containing nothing, lncARSR or mutant transcript. Data are presented as the relative ratio of firefly luciferase activity to renilla luciferase activity. C. The relative expression of lncARSR in EOC cells transiently overexpressing miR-200s. D. Anti-AGO2 RIP was performed in EOC cells transiently overexpressing miR-200s, followed by qRT-PCR to detect lncARSR associated with AGO2. E. The relative expression of miR-200s in EOC cells transiently overexpressing lncARSR or lncARSR-mut. Data are shown as means ± SEM, *P < 0.05.

Article Snippet: Luciferase reporter assays β-catenin/TCF firefly luciferase reporter construct (TOPflash) and pRL-TK reporter was purchased from Promega (Madison, WI, USA).

Techniques: Quantitative RT-PCR, Luciferase, Activity Assay, Transfection, Mutagenesis, Expressing