Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: MicroRNA-139-5p inhibits bladder cancer proliferation and self-renewal by targeting the Bmi1 oncogene.

doi: 10.1177/1010428317718414

Figure Lengend Snippet: Figure 5. MiR-139-5p inhibits c-MYC and Wnt signaling pathway via downregulation of Bmi1. (a) Expression of Bmi1 and c-MYC levels in T24 and 5637 cells was determined by western blotting. (b) Expression of Wnt signaling pathway was measured by TOP/ FOP detection in T24 and 5637 cells (*p < 0.05 compared with NC treatment (scrambled miRNA; Student’s t test)).

Article Snippet: TOP Flash (Addgene plasmid # 12456) and FOP Flash (TOP Flash mutant; Addgene plasmid # 12457) were gifted by Randall Moon.

Techniques: Expressing, Western Blot

Journal:

Article Title: Oncogenic Mutants of RON and MET Receptor Tyrosine Kinases Cause Activation of the ?-Catenin Pathway

doi: 10.1128/MCB.21.17.5857-5868.2001

Figure Lengend Snippet: (A) RON M1254T receptor mutant causes constitutive transactivation of Tcf consensus sequence (TOPFLASH)-driven transcription in MDCK cells. MDCK cells expressing RON WT or M1254T mutant were infected with adenovirus encoding β-Gal or DN Tcf-4, and 24 h later these cells were transfected with luciferase reporter plasmids containing WT (TOPFLASH) or mutated (FOPFLASH) Tcf promoter. Tcf activity was determined by a luciferase assay as described in Materials and Methods. Data were normalized for total protein concentration. Data for a negative control FOPFLASH are not shown. Luciferase activity was calculated in fold increase, where luciferase activity in cells expressing RON WT and β-Gal was taken for 1. Bar graph data are means ± standard errors of three independent experiments. (B) The increased level of c-myc and D1 expression in cells with mutated RON is mediated by Tcf. MDCK cells expressing RON WT or M1254T mutant were infected with adenovirus encoding β-Gal or DN Tcf-4. After 48 h the amount of c-myc and cyclin D1 was determined in total lysates from cells by Western blotting (WB) with anti-c-myc and anti-cyclin D1 antibodies. The β-actin panel serves as a control showing an equal amount of protein in each sample. Positions of molecular-weight markers are indicated on the right.

Article Snippet: The reporter plasmids 3× WT Tcf-binding site (TOPFLASH) and 3× mutated Tcf-binding site (FOPFLASH) were from Promega.

Techniques: Mutagenesis, Sequencing, Expressing, Infection, Transfection, Luciferase, Activity Assay, Protein Concentration, Negative Control, Western Blot, Molecular Weight

Journal:

Article Title: Oncogenic Mutants of RON and MET Receptor Tyrosine Kinases Cause Activation of the ?-Catenin Pathway

doi: 10.1128/MCB.21.17.5857-5868.2001

Figure Lengend Snippet: (A) MET M1268T receptor mutant causes constitutive transactivation of Tcf consensus sequence (TOPFLASH)-driven transcription in NIH 3T3 cells. Activity of Tcf-4 in NIH 3T3 cells expressing MET WT or M1268T was determined by luciferase assay as described in the legend to Fig. ​Fig.6A.6A. Luciferase activity in cells expressing MET WT and β-Gal was set equal to 1. Bar graph data are means ± standard errors of three independent experiments. (B) The increased level of c-myc and D1 expression in cells with mutated MET is mediated by Tcf. NIH 3T3 cells expressing MET WT or M1268T were infected with an adenovirus encoding β-Gal or DN Tcf-4. After 48 h the amount of c-myc and cyclin D1 was determined in total lysates from cells by Western blotting with anti-c-myc and anti-cyclin D1 antibodies. MET tyrosine phosphorylation was determined by anti-PY antibodies in MET IPs. To estimate the amount of the MET in precipitates, the blot was probed with anti-MET antibodies (upper band, immature MET [170 kDa]; lower band, mature MET [140 kDa]). Tyrosine phosphorylation of β-catenin was detected by Western blotting (WB) with anti-PY antibodies. The amount of β-catenin in precipitates was determined with anti-β-catenin antibodies. The β-actin panel serves as a control showing an equal amount of protein in each sample. Positions of molecular-weight markers are indicated on the right.

Article Snippet: The reporter plasmids 3× WT Tcf-binding site (TOPFLASH) and 3× mutated Tcf-binding site (FOPFLASH) were from Promega.

Techniques: Mutagenesis, Sequencing, Activity Assay, Expressing, Luciferase, Infection, Western Blot, Molecular Weight

Journal: Oncology Reports

Article Title: FGD5-AS1 is an oncogenic lncRNA in pancreatic cancer and regulates the Wnt/β-catenin signaling pathway via miR-577

doi: 10.3892/or.2021.8232

Figure Lengend Snippet: miR-577 targets LRP6 and β-catenin. (A) A dual-luciferase reporter assay was utilized to validate the binding sites between miR-577 and LRP6 3′UTR. (B) A dual-luciferase reporter assay was utilized to validate the binding sites between miR-577 and β-catenin 3′UTR. (C) Reverse transcription-quantitative PCR was employed to detect LRP6 and β-catenin mRNA expression levels in SW1990 cells transfected with miR-577 mimics. (D) A TOPFlash luciferase reporter assay was performed to detect the effects of miR-577 on the activity of the Wnt/β-catenin signaling. (E and F) Pearson's correlation analysis was utilized for the correlations between (E) FGD5-AS1 and LRP6 and between (F) FGD5-AS1 and β-catenin in pancreatic cancer tissues. (G and H) Pearson's correlation analysis was utilized for the correlations between (G) miR-577 and LRP6 and between (H) miR-577 and β-catenin in pancreatic cancer tissues. All of the experiments were performed in triplicate. **P<0.01 and ***P<0.001. LRP6, low-density lipoprotein receptor-related protein 6; miR, microRNA; FGD5-AS1, FGD5 antisense RNA 1; UTR, untranslated region.

Article Snippet: To determine the activity of Wnt/β-catenin pathway, the TOPflash and FOPflash system (Upstate Biotechnology, Inc.) was used.

Techniques: Luciferase, Reporter Assay, Binding Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Transfection, Activity Assay