Two of the three media formulations were complete media formulations

in 4EBP1 AVE-8062 binding to eIF4E in OCI-LY1 cells, but little displacement of eIF4G. We also observed that VAL cells expressed the isoform 4EBP2, and that asTORi MEDChem Express AZD5363 treatment did increase the amount of 4EBP2 bound to the cap complex. Nevertheless, the induced binding of 4EBP2 to eIF4E seemed to be ineffective at displacing eIF4G and was therefore unable to compensate for loss of 4EBP1. Blotting for total eIF4E was used as a control to confirm equal pulldown in the untreated and asTORi treated samples, and additional blotting of the total cell lysates confirmed inhibition of TORC1 and TORC2 substrate phosphorylation by asTORi. These results suggest that asTORi treatment in the VAL cells is ineffective at inhibiting the formation of the eIF4F translation initiation complex. We next tested whether maintenance of the eIF4F complex in VAL cells treated with MLN0128 preserved cap dependent translation and overall protein synthesis. First we used a dual luciferase reporter construct containing a cap independent firefly luciferase downstream of the 5 �� UTR of coxsackie virus B3 and an upstream cap dependent Renilla luciferase. Following transfection and a treatment with rapamycin or MLN0128, cell extracts were prepared after 16 hours to quantify peak renilla and firefly luciferase expression. Renilla luciferase activity was normalized to the cap independent firefly luciferase activity. Strikingly, mTOR inhibition did not decrease cap dependent translation in the VAL cells relative to the untreated controls. In contrast, the 4EBP1-expressing control cell lines OCI-LY1 and OCI-LY7 showed a significant, decrease in cap dependent translation with MLN0128, with an intermediate effect of rapamycin consistent with the lesser effect of rapamycin in the cap binding assay. Analysis of the raw luciferase values showed that MLN0128 caused a greater decrease in Renilla luciferase expression in the OCI-LY1 and OCI-LY7 cells compared to the VAL cells, while the decreases in firefly expression were smaller and comparable between the three cell lines. We also assessed whether maintenance of cap dependent translation upon mTOR inhibition in VAL cells correlates with protein levels of cap dependent transcripts like MCL-1 and Cyclin D3. Indeed, MLN0128 and rapamycin did not decrease MCL-1 protein amounts in VAL cells, in contrast to the 4EBP1

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