Figure 8. In malignant mesothelioma cell line NCI-H28, mTOR inhibition blocks the formation of the cap complex, without affecting the ongoing translation.

(A) NCI-H28 cells were treated with 2 µM thapsigargin (Tg) and total proteins were extracted. Extracts were analyzed by WB to test eIF2α phosphorylation. (B) NCI-H28 cells were treated as indicated and for 45 minutes and pulsed with ³⁵S-methionine. Methionine incorporation in newly translated proteins was measured in triplicate. Thapsigargin blocks translation in NCI-H28 cells (C) Cells were treated for 45 minutes with 50 nM rapamycin (RAPA), 10 µM U0126, 5 µM 4-Amino-5-(4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine (Mnk1 inhib.) as indicated and total proteins were extracted. Total extracts were analyzed by WB to test activity of mTORc1, ERK1/2 and Mnk1 signaling. Rapamycin blocks mTORc1 activity. (D) NCI-H28 cells were treated as indicated and total proteins were incubated with 7-Methyl GTP-Sepharose beads. Input is 10% of the purification. Cap binding proteins were analyzed by WB with anti-4E-BP1 and with anti-eIF4G. eIF4E shows equal amount of purified proteins. (E) Cells were treated as indicated and pulsed with ³⁵S-methionine. Methionine incorporation in newly translated proteins was measured in triplicate. mTORc1 inactivation does not reduce translation. (F) Cells were treated with rapamycin or with PP242 and total proteins were extracted. Extracts were analyzed by WB to test activity of mTORc1 and mTORc2 signaling. PP242 blocks both rpS6 and 4E-BP1 phosphorylation by mTORc1 and Akt phosphorylation by mTORc2. (G) Cells were treated as indicated and pulsed with ³⁵S-methionine. Global translation was measured in triplicate. mTOR kinase inactivation reduces translation only at 1 µM concentration.