these time points, D-α-Tocopherol polyethylene glycol 1000 succinate supporting the conclusion that mTOR inhibition decreases MCL-1 548472-68-0 structure protein in the OCI-LY1 and OCI-LY7 cell lines at the translational level. Next we assessed total protein synthesis using a non-radioactive assay that measures uptake of an azide-linked methionine by cells during nascent protein synthesis. The incorporated AHA was labeled with biotin using a Click- IT chemistry based reaction, followed by a western blot with antibiotin HRP. In control OCI-LY1 cells MLN0128 caused a profound inhibition of protein synthesis, with intermediate effects of rapamycin. The compound PP242 that is structurally related but, 5�C10-fold less potent than MLN0128 had similar effects when added at 500 nM. In comparison, asTORi or rapamycin caused much less suppression of total protein synthesis in VAL cells. Thus, mTOR inhibition in VAL cells is ineffective at decreasing cap dependent translation and has negligible effects on overall protein synthesis. The results above suggested that maintenance of cap dependent translation following mTOR inhibition might play a pivotal role in the resistance of VAL cells to asTORi. This led us to test whether modulating the stoichiometry of the cap translation complex would sensitize VAL cells to asTORi treatment. Our first approach was to achieve knockdown of eIF4E in VAL cells. Knocking down eIF4E did not affect basal survival or proliferation of VAL cells but increased sensitivity to cell death following MLN0128 treatment. The parental and scrambled-shRNA control VAL cells behaved as expected with no significant increase in death with MLN0128. In OCI-LY1 cells that are basally sensitive to inhibition of cap dependent translation by asTORi, knocking down eIF4E did not significantly augment the cell death response compared to the scrambled-shRNA control. The increase in asTORi sensitivity of VAL cells with eIF4E knockdown was corroborated when apoptosis was measured by sub-diploid DNA content. A cap binding assay suggested that
