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Figure 4 | Breast Cancer Research

Figure 4

From: Activator protein 1 (AP-1) contributes to EpCAM-dependent breast cancer invasion

Figure 4

EpCAM expression is associated with AP-1 transcription factor activity. Epithelial cell adhesion molecule (EpCAM) expression was specifically ablated in breast cancer cells using lentiviral constructs, and then rescued by cDNA or recombinant EpCAM. (a) A luciferase reporter pathway profiling system was used to measure the impact of EpCAM expression on the activity of specific transcription factors. Results from the pTA (control), activator protein 1 (AP-1) and SRE reporters are shown. AP-1 transcription factor activity was significantly decreased following specific ablation of EpCAM in MDA-231(sh2) cells. Other transcription factors were evaluated but specific ablation of EpCAM had minimal effect (data not shown). Stably transduced MDA-231 cells (scrambled control shRNA construct (SCR) and sh2) were plated for 12 hours, then transiently transfected with the reporters indicated. Luciferase activity was measured 48 hours later. (b) Following this same protocol, the AP-1 reporter was transfected into stably transduced MDA-231 cells (SCR and sh2). Twelve hours after transfection, AP-1 transcription factor activity was induced with phorbol-myristate acetate (PMA; 50 ng/mL) or epidermal growth factor (EGF; 10 ng/mL). 12 hours after induction, reporter activity was measured. (c) AP-1 transcription factor activity is rescued in a dose-dependent fashion by transfection with increasing concentrations of EpCAM cDNA. MDA-231(sh2) cells were transiently transfected with 200 ng of pcDNA3, or 50 to 200 ng of pCDNA-EpCAM as indicated. The total amount of DNA was constant at 200 ng, as indicated. (d) AP-1 transcription factor activity is rescued in a dose-dependent fashion by recombinant EpCAM. Twelve hours after transient transfection with the AP-1 reporter, the indicated doses of rEpCAM were added to MDA-231(sh2) cells. Fourteen hours after addition of rEpCAM, AP-1 reporter activity was measured. The results are representative of at least two independent experiments.

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