JCS054437 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Ribosomal organization in HEK293 cells containing TIA1-induced stress granules. Ultrastructure of TIA1-GFP-transfected HEK293 cells was analysed by electron microscopy following Epon embedding. The plasma membranes limiting two adjacent cells are shown (white arrowheads). The upper cell contains a large stress granule (white star) and dispersed ribosomes, which indicate �polysomal run-off� and blockage of translation resulting from TIA1-GFP overexpression. By contrast, the neighbouring cell on the bottom left shows a typical rosette organization of the polysomes (black arrows). Scale bar: 1 µm.

• Supplemental Figure S2 -

  Fig. S2. Polysome disruption following arsenite treatment in HeLa cells. HeLa cells were treated or not with arsenite for 30 minutes. Cells were lysed in an NP40 lysis buffer containing cycloheximide, and protease and RNase inhibitors. The lysate was fractionated on a 15-50% sucrose density gradient, and analysed by spectrometry at 260 nm. The position of the ribosomal subunits and of the polysomes is indicated.

• Supplemental Figure S3 -

  Fig. S3. Specificity of in situ hybridization at the ultrastructural level. HeLa cells were processed for electron microscopy, and thin sections were reacted with a biotinylated rDNA probe to reveal the rRNA by immunodetection with an anti-biotin antibody coupled to gold particles. (A) Labelling of the 28S rRNA in untreated cells. As previously reported (Puvion-Dutilleul et al., 1991), in the nucleus (Nu), the gold particles which label the 28S are concentrated over the nucleolus (No), whereas the nucleoplasm is weakly labelled, reflecting the rapid transport of the 28S rRNA to the cytoplasm. In the cytoplasm, the ribosome-rich areas are highly labelled, whereas mitochondria (M) and cytoplasmic vacuoles (V) are not. (B) Labelling of the 18S rRNA in arsenite-treated cells. The picture is a less magnified view of the stress granule shown in Fig. 5B. The labelling is dense in the ribosome-rich areas of the cytoplasm and enriched in the stress granule, but absent from mitochondria and cytoplasmic vacuoles. Notice that the extracellular resin is unlabelled. Pm, plasma membrane. Scale bar: 500 nm.