Increases in eIF4AII levels occur in response to small molecule perturbation of eIF4AI/II activity. (A) RNAi-mediated suppression of eIF4AI activates S6K1 phosphorylation and leads to reduction of PDCD4. HeLa cells were transfected with si4AI, and 48 h later, extracts were prepared from cells and blotted for the indicated proteins. The Western blot analysis was performed on extracts prepared from the same experiment and analyzed on the same blot, but the lanes are juxtapositioned for clarity. α-eIF4AI, ab31217. (B) Immunoblot of extracts prepared from HeLa cells exposed to 125 nM hippuristanol (Hipp) for 24 h. α-eIF4AI, sc-14211. (C) Inhibition of global protein synthesis and induction of eIF4AII as a function of Hipp concentration. HeLa cells were treated with the indicated concentrations of Hipp for 24 h, after which they were labeled with 100 μCi/mL 35S-methionine/cysteine for 30 min. Cells were harvested, and the amount of radiolabeled protein was quantitated by TCA precipitation. The values presented are relative to cells treated with vehicle (DMSO). Error bars, SEM. Immunoblots were prepared from35S-methionine/cysteine protein extracts and probed for the indicated proteins. eIF4AII protein levels were quantitated measuring the chemiluminescence signal from Western blots (inset) on an AlphaImager HP. Values are relative to cells treated with DMSO; n = 2. (D) Relative eIF4AI/II levels in Hipp-treated HeLa cells. HeLa cells were exposed to 125 nM Hipp for 24 h; total RNA was prepared and analyzed by RT-qPCR for eIF4AI/II levels. Values are standardized to GAPDH and set relative to eIF4AI levels obtained in vehicle (DMSO)-treated cells. n = 3. (E) Perturbation of eIF4A activity by silvestrol increases eIF4AII protein levels. Immunoblot of extracts prepared from HeLa cells exposed to 25 nM silvestrol (EC75) for 24 h. α-eIF4AI, ab31217. (F) Increased eIF4AII levels require ongoing protein synthesis. HeLa cells were pretreated with vehicle or 1 μM CHX for 2 h and then exposed to 125 nM Hipp for 24 h. Extracts were prepared and probed for the indicated proteins. α-eIF4AI, sc-14211. (G) The Hipp-mediated increase in eIF4AII levels is not mTOR dependent. HeLa cells were exposed to vehicle (lane 1), 125 nM Hipp (lane 2), 20 nM Rap (lane 3), or a combination of Hipp and Rap (lane 4) for 24 h. Extracts were prepared and probed for the indicated proteins. Note that in this experiment, lane 4 is slightly under-loaded, as gauged by the GAPDH control blot. α-eIF4AI, sc-14211.
