FIG. 5.

cFLIP-L enhances Wnt signaling. (A) Expression of cFLIP-L and cFLIP-S in stable transfectant clones. HT1080 cells were transfected with cFLIP-L, cFLIP-S, or empty vector, and stable transfectant clones were isolated as described in Materials and Methods. Cell lysates prepared from the clones were analyzed by Western blotting (WB). Endogenous cFLIP-L was hardly visible under these conditions, but clone L1 expresses ∼20-fold more cFLIP-L protein than do parental HT1080 cells. The blot with HSP90 shows the loading control. (B) Comparison of cFLIP-L expression levels. Whole-cell lysates were prepared from HT1080 cells (lane 1), cFLIP-L stable transfectant clone 1 (lane 2), and HT1080 cells that had been transiently transfected with 1 μg of cFLIP-L in six-well plates (lanes 3 and 4). The indicated amounts of protein were loaded onto the gel and analyzed by Western blotting with the indicated antibodies. (C) Resistance to Fas-mediated apoptosis in cFLIP transfectant clones. The cFLIP clones were treated with the indicated concentrations of anti-Fas antibody in the presence of 1 μg of cycloheximide/ml (left) and the indicated concentrations of etoposide (right) for 48 h. Viable cells were measured by MTS assay (Promega) and are expressed as percentages compared with untreated cells. (D) Enhanced Wnt signaling in cFLIP-L transfectant clones. The cFLIP clones and control cells were transfected with TOP-TK-Luc and pRL-TK and replated in 96-well plates after 12 h. After another 12 h, the cells were treated with conditioned medium containing the indicated concentrations of Wnt3a for 24 h. The luciferase activity was measured and is expressed as the increase (n-fold) compared with the level observed in cells treated without Wnt3a. The data represent means of quadruplicate determinations. The error bars indicate standard deviations.