eIF5A Promotes Translation Elongation, Polysome Disassembly and Stress Granule Assembly
Figure 4
Hypusination of eIF5A is required for SG assembly.
(A) U2OS cells transfected with control (siCONT) or DHS-specific (siDHS-1 and siDHS-2) siRNAs were cultured in the absence (Mock) or presence (Arsenite) of 120 µM arsenite for 30 min before processing for immunofluorescence microscopy using antibodies reactive with eIF3b (SG marker) and Rck (PB marker). Scale bar, 10 µm. (B) Hypusination inhibitor GC7 inhibits SG assembly. U2OS cells were treated with GC7 (10 µM) or vehicle control (Mock) for 48 hours prior to culture in the absence (Untreat) or presence (Arsenite) of 0.1 mM sodium arsenite for 1 hour, then processed for immunofluorescence microscopy using antibodies reactive with Hedls (PB marker) and eIF3b (SG marker). Merged views are shown in the right panels. Scale bar, 10 µm. (C) Western blot analysis quantifying the knockdown efficiency of DHS (upper panel). Actin was used as loading control (lower panel). (D) Graphical representation of SG assembly in U2OS cells transfected with control (siCONT) or non-overlapping DHS-specific (siDHS-1 and siDHS-2) siRNAs prior to treatment with 120 µM arsenite for 30 min. Cell counting data are shown as means ± S.D. from different experiments.*, p<0.05; **, p<0.01 (siDHS-1, n = 3; siDHS-2, n = 2). (E) U2OS cells cultured with vehicle (Mock) or GC7 (10 µM) as in (B). The percentage of cells with SGs was quantified microscopically and graphed as an average ± S.D. (n = 2).