Inhibition of Ribosome Recruitment Induces Stress Granule Formation Independently of Eukaryotic Initiation Factor 2 Phosphorylation

Supplemental Material

This article contains the following supporting material:

• Figure 1 - Reduction of eIF4AI levels by siRNA induces SG formation. A. Cells were transfected with an siRNA, named eIF4AI-2, and fixed two days later. The distribution of HuR and G3BP was visualized by immunofluorescence. The percentage of cells harboring SGs is indicated to the right bottom of panel 4. B. Western blot analysis of protein extracts prepared from cells treated with siRNAs against eIF4AI or control siRNA (Ctr). The blot was first probed with the monoclonal anti-eIF4A antibody (5D3), stripped, and reprobed with an anti-G3BP antibody to control for variation in sample loading.

• Figure 2 - The cellular distribution of HuR and DCP1α. Yellow arrows indicate the location of granules induced by perturbing eIF4A activity, whereas the white arrows indicate the position of PBs.

• Figure 3 - Inhibition of eIF4A activity does not induce Hsp70 expression. Cells were either left untreated (lanes 1 and 6), heat shocked (lanes 2 and 7), or exposed to 0.5 mM arsenite (lanes 3 and 8), 1 μM hippuristanol (lanes 4 and 9), or 0.1 μM pateamine (lanes 5 and 10). Following a 30 min treatment, cells were allowed to recover for 6 h before extracts were prepared and probed for the presence of Hsp70 and HuR by Western blotting.

• Table 1