Figure 2.
NSC119893 inhibits translation in vivo. (A) NSC119893 inhibits cytoplasmic polysome levels. MEFs were incubated for 1 h with 50 μM NSC119893. Cell extracts were prepared and fractionated on 10–50% sucrose gradients, and the polysome profile was monitored by measuring the OD254. (B) Inhibition of protein synthesis by NSC119893 is reversible in vivo. MEFs were incubated for 1 h with 50 μM NSC119893, after which time cells were washed and fresh medium was added. Fifteen minutes before harvesting, cells were labeled with [35S]methionine and incorporation into protein detected by TCA precipitation. The rate of [35S]methionine incorporation obtained with the control reaction was 22245 cpm/μg protein/15-min incubation. Results are expressed relative to the incorporation in the presence of 0.5% DMSO and represent the average of two experiments (each performed in duplicate). (C) NSC119893 induces a transient phosphorylation of eIF2α. MEFs were treated with 25 μM NSC119893 for the indicated times and the level of phospho-eIF2α determined by Western blotting. Tunicamycin (2 μg/ml) treatment was for 12 h. Detection of the total eIF2α contained in the cell extracts is presented in the bottom panel. (D) Effect of NSC119893 on [35S]methionine incorporation into TCA precipitable counts in wt or eIF2αS51A/S51A MEFs. Cells were incubated with the indicated concentrations of compounds for 45 min, after which time [35S]methionine was added to the media and labeling proceeded for 15 min. Results are expressed relative to the incorporation of [35S]methionine in the presence of vehicle (0.5% DMSO) and represent the average of three experiments.