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Fig. 7. Ofs has an eIF4G-like function in S2R+ cells. (A) Growth curves representing the average counts of GFP-(untransfected) cells from 10-13 wells with error bars representing s.e.m. (B) Western blot against eIF4G and GFP-Ofs assessing the degree of knockdown at days 4 and 7. The major eIF4G band is indicated with an arrow. We variably saw a second band migrating slightly faster (asterisk); its identity is unclear. (C) Matched growth curves for both GFP-(untransfected) and GFP+ (transfected) cells from wells transfected with GFP alone, GFP-Ofs or GFP-eIF4G. Curves were normalized for transfection efficiency at day 3. Key in A applies. Expression of GFP-Ofs or GFP-eIF4G completely rescues the defects caused by eIF4G or eIF4G+Ofs knockdown, but not by eIF4A knockdown. Each curve represents average counts from 3-6 wells, with error bars indicating s.e.m. (D) Cell cycle profiles for the cells represented in A showing a shift towards G₁ with either eIF4G or eIF4G+Ofs knockdown. eIF4A knockdown caused an increase in the percentage of cells with sub-G₁ DNA content, normally indicative of apoptotic cells (Oancea et al., 2006). (E) Summary of cell cycle profiling showing rescue by GFP-Ofs and GFP-eIF4G, but not by GFP alone. Gray bars (GFP-) and green bars (GFP+) are paired for each experimental condition. Within each set, from left to right: sub-G₁, G₁, S and G₂. Notice the relative shifts in the G₁ and G₂ bars: depletion of eIF4G-like proteins caused an increase in the proportion of G₁ cells (compare G₁ with G₂ bars), and this trend was reversed by transfection of either GFP-Ofs or GFP-eIF4G.

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