Fig 3.

mTOR inactivation-induced hypophosphorylation of 4E-BP1 prevents eIF4E-mediated SG formation by disrupting its interaction with eIF4GI. (A and B) Depletion of 4E-BP1 rescues arsenite-induced SG formation in pp242-treated cells. HeLa cells were treated with nonspecific or 4E-BP1-selective siRNAs for 96 h. (A) Cells were lysed, and protein extracts were prepared and analyzed by Western blotting to detect 4E-BP1 using specific antibodies. Tubulin serves as a loading control. (B) Cells were then left untreated or were treated with pp242 (2. 5 μM) for 7 h or arsenite (150 μM) for 1 h or were incubated with pp242 (2. 5 μM) for 6 h before addition of arsenite (150 μM) together with pp242 (2.5 μΜ) for an additional 1 h. Cells were then processed for immunofluorescence to detect SG using anti-FMRP and anti-G3BP1 antibodies. Pictures were taken using a 63× objective with a 1.5 zoom. The percentage of cells harboring SG (>3 granules/cell) from 5 different fields and 5 different experiments containing a total of 2 × 10³ cells is indicated at the bottom of merged images. (C) pp242 disrupts eIF4F assembly, as monitored by cap-binding assays. HeLa cells were left untreated or were treated with pp242 (2. 5 μM) for 7 h or arsenite (150 μM) for 1 h or were incubated with pp242 (2.5 μM) for 6 h before addition of arsenite (150 μM) together with pp242 (2.5 μM) for an additional 1 h. Cells were lysed and then incubated with m⁷GTP-Sepharose beads. Eluted proteins were analyzed by Western blotting using specific antibodies. Total is 1% of the input used for cap pulldown. (D) HeLa cells were left untreated or were treated with pp242 (2. 5 μM) for 8 h or bortezomib (2 μM) for 4 h or were incubated with pp242 (2.5 μM) for 4 h before addition of bortezomib (2 μM) for an additional 4 h. Cells were lysed and then incubated with m⁷GTP-Sepharose beads. Eluted proteins were analyzed by Western blotting using specific antibodies. Total is 1% of the input used for cap pulldown.