Fig. 3.

Chronic nutrient starvation induces static SGs that do not exchange material with translating mRNPs. (A) U2OS cells were either treated with 250 μM arsenite for 1 h or starved for 16 h prior to fixation and staining with antibodies against G3BP1 (green) and TiaR (red). During the last hour of stress, 20 μM emetine or 50 μg/ml cycloheximide were added to trap SG constituents within translating mRNPs by stalling elongating ribosomes on mRNAs to assess exchange of material with stSGs. (B) Quantification of results shown in A. The y-axis depicts the percentage of cells with stSGs for each condition. Statistical analysis was performed with a Chi-test (NS, not significant; ***P<0.001). (C) U2OS cells were starved for 16 h before addition of glucose, cycloheximide or glucose and cycloheximide, as indicated, for 1 h prior to fixation. Cells were stained as in A. (D) Quantification of the results shown in C. Results from all panels are representative of a minimum of three experimental replicates, and data are presented as mean±s.d.