Figure 2. Translation Elongation Activity Is Sustained in Transformed Cells under ND and Reduces Their Survival.

(A) Protein synthesis levels in NIH 3T3 MSCV, EN, and Ras^V12 cells deprived of nutrients for 5 min or 1, 3, or 6 hr determined by [³⁵S]-methionine/cysteine(Met/Cys) incorporation. Results are expressed as a percentage of [³⁵S]-Met/Cys incorporation/mg protein relative to MSCV cells at 5 min (n = 2).

(B) Polysome profiles for NIH 3T3 MSCV, EN, and Ras^V12 cells grown in complete media or under ND for 70 min as described in Extended Experimental Procedures. P/S indicates ratio of polysomal to subpolysomal (40S, 60S, and 80S) fractions.

(C) Ribosome half-transit times for NIH 3T3 MSCV, EN, and Ras^V12 cells grown in complete media or under ND for 70 min. [³⁵S]-Met/Cys incorporation into all polypeptides (postmitochondrial supernatant, PMS) and into polypeptides released from ribosomes (postribosomal supernatant, PRS) was obtained by linear regression analysis. Representative results from three different experiments are shown.

(D and E) siRNA-mediated knockdown of eEF2 in NIH 3T3 EN and Ras^V12 cells. Cells were transiently transfected with 12.5 nM of control (CTRL) or eEF2-directed siRNAs, grown in complete media for 48 hr, and placed under ND for 48 hr. Lysates were either analyzed by immunoblotting (D) or assayed for caspase-3 activity (n = 3) (E).

(F and G) siRNA-mediated knockdown of eEF2 in NIH 3T3 EN-S/MSCV, EN-S/DN-AMPK, Ras^V12-S/MSCV, and Ras^V12-S/DN-AMPK cells. Cells were transfected, treated, and analyzed as in (D) and (E).

Where shown, data are reported as means ± SD with indicated significance (**p < 0.005). See also Figure S3.