Fig 2: A reduced ATXN2L level affects the size and number of SGs.HeLa cells were left untreated (mock) or transfected with siRNAs against ATXN2L or ATXN2 transcripts, or non-targeting control siRNA (siNT). 72 hours post transfection cells were treated with 0.5 mM sodium arsenite for 1 hour and fixed. A) For the confocal microscopy proteins were stained with an antibody detecting eIF4G. (B-D) For the quantitative high-content screening microscopy staining occurred with antibodies detecting eIF4G and TIAR. B) Cell number and size of nuclei was analyzed by quantitative high-content screening microscopy. C, D) Quantification of TIAR- and eIF4G-positive SGs regarding number and size. Results are expressed as mean ± SD from one representative experiment, n = 5 replicate wells, *p<0.05; **p<0.01; ***p<0.001, One-way ANOVA with Tukey’s Multiple Comparison post test.

Fig 3: P-body formation is influenced by ATXN2L overexpression.HeLa cells were transfected with expression constructs RSV-ATXN2L-MYC or pCMV-MYC-ATXN2-Q22 to overexpress ATXN2L or ATXN2, respectively. 48 h post transfection, cells were fixed and stained with antibodies directed against the MYC-tag to visualize ATXN2L or ATXN2 overexpressing cells and an antibody against DDX6. Nuclei were stained with Hoechst. Images shown are taken from one representative experiment. Scale bars represent 20 µm.

Fig 4: ATXN2L is found in association with PABP, DDX6 and ATXN2. A)Scheme of the domain architecture of ATXN2L and ATXN2 highlighting conserved functional motifs. Lines below indicate antibody epitopes for anti-ATXN2L and anti-ATXN2 (BD Biosciences). B) Cell lysates were prepared from HEK293T and HeLa cells as described in Materials and Methods. Co-immunoprecipitation experiments were carried out with an anti-ATXN2L antibody, and precipitated proteins were detected using specific antibodies against PABP, DDX6 or ATXN2 (BD Biosciences). C) HeLa cells were fixed and ATXN2L or ATXN2 were stained with an anti-ATXN2L antibody or an anti-ATXN2 antibody (Sigma, red). SR-splicing proteins were stained using an anti-SR antibody (green). Nuclei were visualized by Hoechst staining (blue). Scale bars represent 20 µm.

Fig 5: 5-FU-induced SGs contain bona fide SG marker proteins. (A) HeLa cells were treated with 0.1 mM 5-FU for 72 h and processed for immunostaining of the SG marker proteins G3BP (green) and eIF4G (red) (left panel), or ATXN2 (green) and ATXN2L (red) (right panel). Nuclei were stained with Hoechst. Scale bars represent 20 µm. (B) Representative view fields of the quantitative HCS microscopy are shown. Outer cell borders (green lines) were calculated by extending the nuclear region (blue lines). G3BP- and ATXN2-positive SGs were quantified. (C–E) Cell number (C), size of nuclei (D) and number of G3BP- or ATXN2-positive SGs (E) were analyzed by HCS and quantified. Results are expressed as mean ± SD from one representative experiment; n = 5 replicate wells, *P < 0.05; **P < 0.01; ***P < 0.001, one-way ANOVA with Tukey's Multiple Comparison post test.