FIG. 5.

Enhanced Wnt3a signaling shows growth retardation in untransformed cells expressing E8. U2OS cells (A and C), BALB/3T3 cells (B), FL cells (E), and MEF (G) in six-well plates were transfected with an E8 expression plasmid or an empty vector together with a neomycin resistance gene, as described in Materials and Methods. BALB/3T3 cells (D) and KB cells (F) were cotransfected with the expression plasmids for E8 and ICAT together with a neomycin resistance gene. Cells were seeded in 12-well plates (A, C, E-G) or 24-well plates (B, D) at appropriate densities and cultured with Wnt3a/L-CM or neo/L-CM containing 1.0 mg/ml (A, C, E), 0.8 mg/ml (F, G) or 0.5 mg/ml (B, D) of G418 for 14 days (A-E) or 21 days (F, G). Colonies were stained with Giemsa staining solution (A, B), or the number of cells/well was quantified using a hemacytometer (C-G). The bars represent means ± SD (n = 3 to 6). All the data are representative of more than three independent experiments that gave similar results.