super8xfopflash  (Addgene inc)

Journal: Genes & Development

Article Title: Fatty acid uptake activates an AXL–CAV1–β-catenin axis to drive melanoma progression

doi: 10.1101/gad.351985.124

Figure Lengend Snippet: Reagents used in this study

Article Snippet: Super8XFOPflash , Obtained from R. Moon , , Addgene_12457.

Techniques: Software, Transduction, Virus, Subcloning, Bacteria, Recombinant, Bradford Assay, Protease Inhibitor, RNA Extraction, Reporter Assay, Bicinchoninic Acid Protein Assay, Quantitative RT-PCR, Negative Control

Journal: Cancers

Article Title: A Novel Role for the Tumor Suppressor Gene ITF2 in Tumorigenesis and Chemotherapy Response

doi: 10.3390/cancers12040786

Figure Lengend Snippet: The basal status of the Wnt signaling pathway in A2780 and H23 cell lines. ( A – B ) Pharmacological activation (A) and functional activation (B) of β-catenin transcriptional activity in A2780 cells. ( C ) The expression levels of downstream genes regulated by ITF2 involved in Wnt signaling pathway in A2780S and A2780R cells measured by RNAseq in terms of “fragments per kilobase of transcript per million mapped reads” or FPKM values. ( D ) Pharmacological activation of β-catenin transcriptional activity in H23 cells. β-catenin transcriptional activity was measure in A2780 and H23 cells after treatment with LiCl (10 mM) 24 h or transfection of bcat-S33Y, transfecting with Super8xTopFlash (Top) or Super8xFopFlash (Fop). The results show the fold induction of the Top/Fop ratio with respect to untreated cells (= 1). Values represent the mean of three independent experiments measured by triplicate ± SD. *** p < 0.001; ** p < 0.01, * p < 0.05 (Students t -test) ns, non significant.

Article Snippet: Cells were serum-starved overnight and cotransfected with 0.2 μg of either Super8xTopFlash (containing 7 copies of the TCF/LEF binding site) or Super8xFopFlash (containing 6 mutates copies of the TCF/LEF binding sites) expression plasmids, and 0.1 μg pRL-TK (Renilla-TK-luciferase vector, Promega, Madison, WI, USA) as a control, using lipofectamine 2000.

Techniques: Activation Assay, Functional Assay, Activity Assay, Expressing, Transfection

Journal: Cancers

Article Title: A Novel Role for the Tumor Suppressor Gene ITF2 in Tumorigenesis and Chemotherapy Response

doi: 10.3390/cancers12040786

Figure Lengend Snippet: The effect of ITF2 on cisplatin resistance, cell viability, and Wnt pathway. ( A ) Viability curves of A2780 cell lines transfected with pCMV6 (S-Ø and R-Ø) and with the overexpression vector (R-ITF2). Each experimental group was exposed to six different CDDP concentrations for 48 h. Data were normalized to each untreated control, set to 100%. The data represent the mean ± SD of at least three independent experiments performed in quadruplicate at each drug concentration for each cell line analyzed. ( B ) Viability of A2780 cell lines transfected with pCMV6 (R-Ø) and with the overexpression vectors (R-ITF2). ( C ) Relative expression levels of ITF2 measured by quantitative RT-PCR represented in Log10 scale; in each experimental group, the sensitive cell line transfected with pCMV6 plasmid was used as a calibrator. Each bar represents the combined relative expression of two independent experiments measured in triplicate. ( D ) β-catenin transcriptional activity was measured in A2780 cells after ITF2 overexpression and treatment with LiCl (10 mM) for 24 h, transfected with Super8xTopFlash (Top) or Super8xFopFlash (Fop). The results show the fold induction of the Top/Fop ratio with respect to untreated cells (=1). Values represent the mean of three independent experiments measured by triplicate ± SD. ( E ) Expression analysis of the downstream gene DKK1 regulated by ITF2 in A2780 cell line transfected with pCMV6 (S-Ø and R-Ø) and with the overexpression vector (R-ITF2) for 24 and 72 h. Representative images of DKK1 and GAPDH measured by RT-PCR. ( F ) Expression levels of DKK1 measured by qRT-PCR. Each assay was performed at least three times to confirm the results. *** p < 0.001; * p < 0.05 (Students t -test).

Article Snippet: Cells were serum-starved overnight and cotransfected with 0.2 μg of either Super8xTopFlash (containing 7 copies of the TCF/LEF binding site) or Super8xFopFlash (containing 6 mutates copies of the TCF/LEF binding sites) expression plasmids, and 0.1 μg pRL-TK (Renilla-TK-luciferase vector, Promega, Madison, WI, USA) as a control, using lipofectamine 2000.

Techniques: Transfection, Over Expression, Plasmid Preparation, Control, Concentration Assay, Expressing, Quantitative RT-PCR, Activity Assay, Reverse Transcription Polymerase Chain Reaction