β catenin responsive firefly luciferase reporter plasmid top flash  (Addgene inc)

Journal: Iubmb Life

Article Title: Hypoxia inducible factor 3‐alpha promotes a malignant phenotype in colorectal cancer cells

doi: 10.1002/iub.70007

Figure Lengend Snippet: HIF‐3α modulates the canonical Wnt/β pathway in a cell context‐dependent manner. Stable scrambled‐sh control and HIF‐3α‐sh RKO (A) or SW480 cells (B) were transiently transfected with the pTOPFlash or pFOPFlash (control) reporter plasmids. Twenty‐four hours posttransfection, SW480 cells were washed and lysed, and the luciferase activity was assayed (B). For RKO cells, which display normal canonical Wnt that needs to be activated by Wnt3a ligand (A), cells were serum starved (2%) for 10 h, and then Wnt3a (100 ng/mL) or vehicle was added to the medium to stimulate cells for 12 h period; the cells were then washed and lysed, and the luciferase activity was assayed. The activity in both cell types was normalized with respect to the activity of Renilla luciferase or with respect to the protein content in each sample. The data represent the means ± SEM from at least five independent assays. *** p < .001.

Article Snippet: The β‐catenin transcriptional activity‐reporter plasmids pTOPFlash and pFOPFlash (control) were obtained from Upstate Biotechnology (Lake Placid, NY, USA).

Techniques: Control, Transfection, Luciferase, Activity Assay