Figure 2

Reducing HRI levels by siRNA prevents bortezomib-induced SGs formation. (A, C) HeLa cells were transfected for 48 h with anti-HRI (HRI-1) or anti-GCN2 siRNAs (GCN2-1), or with a control siRNA. (A, B) q(RT)-PCR of HRI (A) and GCN-2 mRNAs (B). Transfected cells were collected and their mRNA content was isolated. The amount of HRI and GCN-2 mRNAs relative to GAPDH mRNA was quantified by real-time q(RT)-PCR using the ΔΔCt method. The results are presented as the mean of triplicate measurements, with error bars corresponding to the SEM. (C) Transfected cells were processed for immunofluorescence using antibodies against different SG markers, as above. (D) HeLa cells were transfected for 48 h with HRI-1, GCN2-1, or with a control siRNA, and then treated with bortezomib for 4 h. Cells were collected and protein extracts were analyzed by Western blot analysis for the amount of phospho- and total eIF2α as described in Figure 1. (E) HeLa cells were transfected for 48 h with HRI-1 siRNA or with control siRNA, and then treated with bortezomib for 3 h before a 30-min incubation with [³⁵S] methionine (50 μCi/ml). Proteins were resolved on SDS-polyacrylamide gels, stained with Coomassie Blue (bottom panel), and detected by autoradiography (top panel).