Smad4 is required for TGF-β signaling. (A) Activation of the 3TP reporter by TβRII plus TGF-β. Cells of the indicated genotypes were cotransfected with p3TP-lux together with a plasmid encoding TβRII (RII) or an identical plasmid devoid of TβRII (Con) as a control. The transfected cells were then cultured in the presence or absence of TGF-β1. Luciferase activity was measured 20 hr after transfection and ligand treatment, and was normalized to the control for each line. Bars and brackets represent the means and standard deviations, respectively, from triplicate transfections. (B) Activation of the 3TP reporter by constitutively activated receptors. Cells were transfected as in A, except that mutant forms of the RI receptors for TGF-β (TβRI*) or activin (ActR1B*) were used instead of RII. Results were normalized to the luciferase activity achieved with the control vector in each line. Heterozygote DPC4+/− cells behaved similarly to the parental HCT116 cells in these assays, and an additional clone of DPC4−/− cells gave results identical to those shown for clone 5–18.