FIG. 3.

Rapamycin cooperates with TGF-β to induce growth arrest of cells transformed by E2F1, c-Myc, and ^V12H-Ras. (A) Mv1Lu cells transformed with E2F1 (E2F1-13) were treated for 24 h with normal growth medium (C), 10 ng of TGF-β/ml (T), 100 nM rapamycin (R), or 10 ng of TGF-β/ml plus 100 nM rapamycin (T + R). The cells were then subjected to [³H]thymidine incorporation assays as for Fig. 1 (left panel), or cell extracts were subjected to immunoblot analyses using antibody to total Rb, Rb phosphorylated on Ser²⁴⁹/Thr²⁵² [P-Rb(S249/T252)], Rb phosphorylated on Thr⁸²¹[P-Rb(T821)], E2F1, or c-Myc or β-tubulin as a loading control (right panel). Results of [³H]thymidine incorporation assays are presented as the average percent control incorporation of six replicate determinations ± standard deviation. (B) Mv1Lu cells transformed with c-Myc (c-Myc-5) were subjected to [³H]thymidine incorporation analyses (left panel) or immunoblot analyses (right panel) as for panel A. (C) Mv1Lu cells transformed with c-Myc2(Ala⁵⁸) [c-Myc(58A)-7] were subjected to [³H]thymidine incorporation analyses (left panel) or immunoblot analyses (right panel) as for panel A. (D) Mv1Lu cells transformed with ^V12H-Ras (Ras-9) were subjected to [³H]thymidine incorporation analyses (left panel) or immunoblot analyses (right panel) as for panel A.