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: Cancer Cell International

Article Title: OIP5-AS1 contributes to tumorigenesis in hepatocellular carcinoma by miR-300/YY1-activated WNT pathway

doi: 10.1186/s12935-020-01467-6

Figure Lengend Snippet: MiR-300 targets to YY1 and inactivates WNT pathway in HCC. a The upstream miRNAs of YY1 were predicted by five programs in starBase (including miRmap, TargetScan, PicTar, miRanda and microT), and the prediction results were showed by Venn. b RIP assay plus qRT-PCR analyzed the enrichment of candidate miRNAs in RISCs in HepG2 and MHCC97H cells. c MiR-300 expression in five HCC cell lines and one human liver epithelium was evaluated via qRT-PCR. d qRT-PCR detected miR-300 expression in HepG2 and MHCC97H cells transfected miR-300 mimics or NC mimics. e YY1 mRNA and protein levels were measured by qRT-PCR and western blot in HepG2 and MHCC97H cells with the transfection of miR-300 mimics or NC mimics. f RNA pull down assay helped to determine the interaction of miR-300 with YY1. g StarBase predicted the binding sequence of miR-300 in YY1 3′UTR. h Luciferase reporter assay was performed to verify the interaction between miR-300 and YY1. i , j The effect of miR-300 mimics on the expression of factors related to WNT pathway was detected by qRT-PCR and western blot analyses. k TOP/FOP flash assay determined the activity of WNT pathway in cells transfected with NC mimics or mi-300 mimics. *P < 0.05, **P < 0.01

Article Snippet: Cells were placed in a 96-well plate, followed by co-transfection with indicated plasmids (GenePharma) and TOP Flash or FOP Flash vector (Promega).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Western Blot, Pull Down Assay, Binding Assay, Sequencing, Luciferase, Reporter Assay, Activity Assay

Journal: Cancer Cell International

Article Title: OIP5-AS1 contributes to tumorigenesis in hepatocellular carcinoma by miR-300/YY1-activated WNT pathway

doi: 10.1186/s12935-020-01467-6

Figure Lengend Snippet: YY1-mediated OIP5-AS1 promotes HCC cell growth by activating WNT pathway. a Expression of OIP5-AS1 in YY1-silenced HCC cells was detected by qRT-PCR. b qRT-PCR was carried out to measure OIP5-AS1 expression in HCC cells upon YY1 upregulation. c The binding motif of YY1 and the binding site between YY1 and OIP5-AS1 promoter were predicted by JASPAR. d ChIP assay was conducted to determine the interaction between YY1 and OIP5-AS1 promoter. e The interaction between YY1 and OIP5-AS1 promoter was further verified by luciferase reporter assay. f , g The effect of OIP5-AS1 silencing on the mRNA and protein levels of Axin2, CTNNB1 (non-phosphorylated β-catenin and phosphorylated β-catenin), Cyclin D1 and c-myc was detected by qRT-PCR and western blot analyses. h , i The nuclear translocation of β-catenin was assessed by nuclear-cytoplasmic fractionation followed by western blot analysis in two OIP5-AS1-downregulated HCC cells. j The effect of OIP5-AS1 depletion on the activity of WNT pathway in HepG2 and MHCC97H cells was determined with the employment of TOP/FOP-flash luciferase reporter assay. k The proliferation of HCC cells transfected with sh-NC or sh-OIP5-AS1 when exposed to CHIR99021 was estimated by colony formation assay. l The apoptosis of indicated cells was examined by caspase-3 activity analysis. **P < 0.01

Article Snippet: Cells were placed in a 96-well plate, followed by co-transfection with indicated plasmids (GenePharma) and TOP Flash or FOP Flash vector (Promega).

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Luciferase, Reporter Assay, Western Blot, Translocation Assay, Fractionation, Activity Assay, Transfection, Colony Assay

Journal: Oncotarget

Article Title: Synergistic inhibition effect of TNIK inhibitor KY-05009 and receptor tyrosine kinase inhibitor dovitinib on IL-6-induced proliferation and Wnt signaling pathway in human multiple myeloma cells

doi: 10.18632/oncotarget.17056

Figure Lengend Snippet: (A) Cell viability of RPMI8226 cells treated with IL-6 (50 ng/mL) and KY-05009 (3 μM) or dovitinib (3 μM) alone or in combination for 24 or 48 h. Data are presented as mean±SD. Experiments were performed in triplicate. (B) Relative TCF/LEF luciferase activity measured by FOPflash-normalized TOPflash luciferase activityin RPMI8226 cells treated with IL-6 (50 ng/mL) and KY-05009 (3 μM) or dovitinib (3 μM) alone or in combination for 9 h. (C) RPMI8226 cells treated with IL-6 (50 ng/mL) and KY-05009 (3 μM) or dovitinib (3 μM) alone or in combination for 1 h. The mRNA expression of indicated genes was detected by qRT-PCR analysis. (D and E) The expression of TCF4-interacting proteins and phosphorylation of TNIK detected by immunoprecipitation and Western blot of RPMI8226 cells treated with IL-6 (50 ng/mL) and KY-05009 (3 μM) or dovitinib (3 μM) alone or in combination for 9 h. * P < 0.01, * P < 0.001 versus control; # P < 0.01, ## P <0.001 versus cells treated with IL-6 alone.

Article Snippet: RPMI8226 cells were trasfected with TOPflash TCF reporter plasmid (wild-type TCF binding site) (Merck Millipore, Darmstadt, Germany), FOPflash plasmid (mutant TCF binding site) (Merck Millipore, Darmstadt, Germany), and Lipofectamine 2000 (Thermo Fisher Scientific, Boston, MA, USA) in an antibiotic-free medium.

Techniques: Luciferase, Activity Assay, Expressing, Quantitative RT-PCR, Phospho-proteomics, Immunoprecipitation, Western Blot, Control