Lentiviruses that inhibit or activate the Wnt signaling pathway.

<p>To measure Wnt signaling activity in HEK293T and HEK293A, cells were transfected with the reporter construct pMegaTOPFlash prior to Wnt3A addition. A) HEK293T cells were infected with the hEF1α-dnTcf4//SV40-mCherry (“EdTC”) virus, and mCherry-expressing cells were selected by fluorescence-activated cell sorting. Induction of a Wnt reporter by purified Wnt3A protein (500 ng/ml final) was impaired in EdTC-infected cells compared to mock-infected cells (top). All selected cells were expressing mCherry (bottom). B) Mouse LS/L or HEK293A cells were infected with the hEF1α-dnTcf4//SV40-Puro^R (“EdTP”) virus and selected with 1 µg/ml puromycin. Puromycin selection led to enrichment of LS/L cells with an impaired response to Wnt3A-conditioned medium (left, compare “+” with “−” puromycin). Induction of a Wnt reporter by purified Wnt3A protein (500 ng/ml final) was also impaired in EdTP-infected HEK293A cells compared to mock-infected cells (right). C) HEK293T cells were infected with the hEF1α-β-catenin^4A//SV40-mCherry (“EβC”) virus at different multiplicities of infection (1, 0.3 and 0.1 relative concentration of virus per cell). Increasing the number of infected cells (as seen by the amount of mCherry-positive cells in a confluent field, right) led to increasing activation of the Wnt signaling pathway in absence of any exogenous Wnt source (left). D) Mouse LS/L and HEK293A cells were infected with the hEF1α-β-catenin^4A//SV40-Puro^R (“EβP”) virus and selected with 1 µg/ml puromycin. Selection with puromycin led to the enrichment of cells with constitutive activation of the Wnt signaling pathway (left, compare “−” with “Puro”). Infection of HEK293A with the EβP virus similarly led to ligand-independent activation of the Wnt signaling pathway (right). In panels A and B, activity is relative to vehicle-treated cells. In panels C and D, activity is relative to mock-infected cells.