Fig. 5. Ectopic c-Rel expression in individual clones ablates TGF-ß1-mediated growth inhibition. Individual clones (C1C5) were isolated from the Hs578TR mixed population by limiting dilution. A and B, immunoblot analysis. Expression of c-Rel in the individual clones was determined by immunoblot analysis. Nuclear extracts were isolated from exponentially growing clones and from parental Hs578T cells, and equal samples (80 µg) were subjected to immunoblot analysis using an anti-c-Rel antibody (SC-070, Santa Cruz Biotechnology) in the absence (A) or presence (B) of a 1:1 molar ratio of cognate peptide. *, position of a nonspecific band. C, MTS proliferation assay. Parental Hs578T and clonal Hs578TR-C1 and Hs578TR-C2 cells were plated at 20% confluence and treated with 5 ng/ml TGF-ß1 or BSA as control for 24 and 48 h. The effects of TGF-ß1 on growth were measured by MTS assay. Cell numbers for TGF-ß1-treated cells are given as percent values relative to BSA-treated control cells. D, DNA synthesis. Parental Hs578T and clonal Hs578TR-C1 and Hs578TR-C2 cells were treated in duplicate with 5 ng/ml TGF-ß1 for 48 h or with BSA as control. Cells were then incubated in media containing 2 µCi of [3H]thymidine per ml for 6 h, fixed, and exposed for autoradiography. Percent labeled nuclei was determined by visual counting. Mean and SD were determined in two different experiments. Black columns, parental Hs578T cells; grey columns, clone 1; white columns, clone 2.