FIG. 1.

pRB-deficient cells are not transformed by H-Ras^V12. (A) Pools of puromycin-selected wild-type 3T3 (WT) and pRB^−/− 3T3 cells transfected with vector alone or with H-Ras^V12 were assayed for proliferation. The proliferation rate of vector-transfected cells or H-Ras^V12-transfected cells was analyzed over a period of 6 days by counting cells every 2 days following plating. The results reflect the average of two independent experiments performed in triplicate. The right panel shows a Western blot verifying the expression of H-Ras^V12 in wild-type and pRB^−/− 3T3 cells as indicated. The bottom panels show the proliferation in soft agar of wild-type and pRB^−/− 3T3 cells transfected with vector and H-Ras^V12. A representative example of soft agar colonies photographed 2 weeks postplating is shown. (B) pRB-deficient MEFs are not transformed by H-Ras^V12 but requires a cooperating event. Early-passage wild-type and pRB^−/− MEFs were transfected with vector, H-Ras^V12 or H-Ras^V12 plus adenovirus E1A as indicated in the figure. Infected cells were selected in puromycin containing medium and 4 days postselection an equal number of cells were plated on 10-cm dishes, medium was changed every 3 days, and the dishes were stained with Giemsa after 17 days. Representative images of two independent experiments in duplicate are shown.