FIG. 1.

Rapamycin increases eIF4E phosphorylation under serum-free conditions (A), in the presence of IGF-1 (B) and an IGF-1R inhibitor (C), and in IRS-1-silenced (D) or -deficient (E) cells. (A) H157 cells were serum starved for 24 h and then treated with 10 nM rapamycin in the absence of FBS for the indicated times. In addition, H157 cells were cultured and treated with 10 nM rapamycin in medium containing 5% FBS for the given times. (B) H157 cells were serum starved for 20 h and then treated with 10 nM rapamycin alone, 1 ng/ml of IGF-1 alone, and their combination for 30, 60, 120, and 180 min. (C) A549 cells were pretreated with the given concentrations of IGF-1R inhibitor II (IGF-1Ri-II) for 30 min and then cotreated with 10 nM rapamycin (Rap) for the indicated times. (D) A549 cells were transfected with control (Ctrl) or IRS-1 siRNA. After 48 h, the cells were treated with 10 nM rapamycin (Rap) for 1 h before they were harvested for preparation of whole-cell protein lysates. In addition, untransfected cells (No) were included as a control. (E) WT and IRS^−/− murine 3T3 fibroblasts were treated with 10 nM rapamycin (R) or RAD001 (R1) for 8 h. After the aforementioned treatments, the cells were subjected to preparation of whole-cell protein lysates and subsequent Western blot analysis.