Fig 1.

Inactivating eIF4E or reducing its levels in HeLa cells impairs SG formation without affecting phosphorylation of eIF2α. (A and B) Cells were preincubated with 4EGI-1 (250 μM) for 6 h and then treated with 150 μM arsenite in the presence of 4EGI-1 (250 μM) for one additional hour. (A) Cells were then fixed, permeabilized, and processed for immunofluorescence using antibodies against different SG markers (FMRP in green and G3BP1 in red). DAPI (blue) was used as a nuclear stain. Pictures were taken using a 63× objective with a 1.5 zoom. The percentage of cells harboring SG (>3 granules/cell) from at least 5 different fields and 5 different experiments containing a total of 2 × 10³ cells is indicated at the bottom of the merged images. Typical SG are shown in enlarged pictures. (B) Cells were then lysed, and total cell lysates were analyzed by Western blotting for eIF2α phosphorylation using anti-phospho-eIF2α antibodies. Total eIF2α was analyzed using pan-eIF2α antibodies. The amount of phosphorylated eIF2α was determined by densitometry quantitation of the film signal and is expressed as a percentage of total eIF2α. The results are representative of 3 different experiments. (C to F) Cells were treated with nonspecific or eIF4E-selective siRNAs for 96 h. Cells were then incubated with 150 μM arsenite for 1 h (C and D) or with 2 μM bortezomib for 4 h (E and F). (C and E) Cells were fixed, permeabilized, and then processed for immunofluorescence to detect SG using anti-FMRP (green), anti-G3BP1 (red), and anti-FXR1 (red) antibodies. Anti-eIF4E (green) antibodies were used in order to detect SG and to assess eIF4E depletion. DAPI (blue) was used as a nuclear stain. 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 for a total of 2 × 10³ cells is indicated in the merged images. Typical SG are shown. (D and F) Cells were lysed, and protein extracts were prepared and analyzed by Western blotting to detect eIF4E and phospho-eIF2α. Total eIF2α was analyzed using the pan-eIF2α antibodies and served as a loading control. The percentage of eIF4E knockdown was determined by densitometry quantification of the film signal using Photoshop and expressed as a percentage of total eIF2α. The amount of phosphorylated eIF2α was determined as described for panels A and B. The results are representative of 3 different experiments.