Fig 1: Distribution of Eif3e and intermediate filament labelling relative to ADF/cofilin-actin rods in primary cultured chick neurons. (A) EIF3e labelled puncta do not enrich in 12E8-labelled rods that are induced in primary cultured chick neurons by CCCP treatment. (B) α-internexin labelling shows some localisation to 12E8-labelled rods in CCCP-treated primary chick neurons. (C) Control and CCCP-treated primary chick neurons labelled with vimentin and 12E8 antibodies. Vimentin labelling localises in part to 12E8-labelled rods. Scale bars: 10 µm (inset C: 5 µm).
Fig 2: The 12E8 antibody immunoprecipitates a complex containing MAP2, tubulin and Eif3e proteins. (A) 12E8 antibody immunoprecipitated EIF3e and different tau isoforms from chick brain represented by at least five protein bands detected with rabbit polyclonal tau antibody (Dako) (bottom). (B) Monoclonal rabbit anti-tau antibody ab64193 weakly immunoprecipitated EIF3e (arrow) as well as several isoforms of tau from chick brain. Monoclonal rabbit anti-tau antibody ab76128 immunoprecipitated isoforms of tau but not EIF3e from chick brain. Asterisks denote the positions of rabbit Ig heavy chains. (C) Mouse monoclonal anti-α-tubulin antibodies strongly, and mouse monoclonal anti-Map2 antibodies weakly immunoprecipitated EIF3e from chick brain lysates (arrows). The asterisks denote the positions of rabbit Ig heavy chains. (D) Recombinant EIF3e protein is not directly detected by 12E8 on immunoblot. (E) 12E8 antibody weakly immunoprecipitated EIF3e from mouse brain.
Fig 3: Relative immunofluorescence localisation of 12E8, Eif3e and tubulin labelling in primary cultured chick neurons. Immunofluorescent labelling of primary cultured chick neurons. (A) EIF3e label is prominent in the nucleolus as shown by co-localisation with nucleolar protein fibrillarin labelling (arrow). EIF3e labelling also shows punctuate distribution throughout the nucleus and cytoplasm. (B) By contrast, FUS protein labelling does not co-localise with fibrillarin label in the nucleolus. (C) The 12E8 antibody label is not evident in the nucleolus, but like EIF3e labelling, it shows a punctuate appearance in the nucleus and cytoplasm. Co-localisation analysis Pearson's and Mander's coefficients of ∼0.8 (see text) suggest some degree of co-localisation between 12E8 and EIF3e labelling. (D) EIF3e labelling shows punctuate distribution associated with tubulin or microtubules in the cytoplasm. Arrows point to nucleolus. Scale bars: 10 µm.
Fig 4: Punctuate staining with SG marker eIF3e was commonly found adjacent to or surrounding FUS-GFP SGs.Scale bars = 10 µm; Insets = 1 µm.
Fig 5: eIF3e-associated RNA signature predicts poor prognosis(A and B) Overall survival rates of METABRIC patients (n = 1980) with breast tumor (A) eIF3e gains/amplifications or (B) eIF3d gains/amplifications compared to no copy-number alterations (CNAs) in the METABRIC dataset.(C and D) Overall survival of patients in the METABRIC datasets stratified by (C) eIF3e and (D) eIF3d RNA-seq signatures using the intersection of targets in both MCF7-SIX1 and MDA-MB-231 cells. For clarity, only the first and fourth quartiles are shown.(E and F) Overall survival rates of patients in the METABRIC dataset stratified by (E) the combination of hypoxia signature and eIF3e or (F) eIF3d enrichment/depletion. The p values and hazard ratios were calculated using a Cox proportional hazards regression where each group was compared to the control group (e.g., no CNAs for eIF3e, heterozygous (het) CNA loss for eIF3d, and bottom 25% for signatures).
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