Stress induces partial translocation of dFMRP from dissociating polysomes and its accumulation in SG.

<p>(A–C) Sucrose gradient analysis of polysomes and analysis of dFMRP distribution on polysomes. (A) Schneider cells were untreated (left panels) or treated either with arsenite (0.5 mM; center panel) or heat shock at 37°C (right panel) for 1.5 h. Cytoplasmic extracts were sedimented through sucrose density gradients and dFMRP distribution was analyzed by western blot using specific antibodies. (B) eIF2α phosphorylation. Schneider cells were treated with arsenite (0.5 mM) or incubated under heat shock conditions (37°C) for 1.5 h. Total cell lysates were then prepared and analyzed by western blot for the phosphorylation of eIF2α using specific antibodies. Total eIF2α was analyzed using the pan-eIF2α antibodies. The amount of phosphorylated eIF2α was determined by quantitation of the film signals by densitometry using the Adobe Photoshop and expressed as a percentage of total eIF2α. The results are representative of 5 different experiments. (C–D) Schneider cells were treated with 0.5 mM arsenite or heat shock (37°C) for 1.5 h, fixed, permeabilized, and processed for immunofluorescence using antibodies against different SG markers: dPABP and deIF4E; (red signal in merged pictures) and dFMRP (green signal in merged pictures). DAPI (blue signal in merged pictures) is used as a nuclear stain. Pictures were taken using a 63X objective at 1.5 zoom (C). The percentage of cells harboring SG (>3 granules/cell) from 5 different fields and 5 different experiments containing a total of 2,000 cells is indicated at the bottom of merged images. Scale bars are indicated. (D) Densitometry of dFMRP immunofluorescence signal in SG with Adobe Photoshop. The number of pixels and mean intensities were recorded for the selected regions (SG, cytoplasm and background) using Photoshop. The mean intensity was multiplied by the number of pixels for the region selected to obtain the absolute intensity. The absolute intensity of the background region was subtracted from each region of interest. To compare the intensity between two given regions of interest, relative intensities were next calculated. Relative intensities correspond to the absolute intensities normalized to the absolute intensity of the region of reference.