Figure 1.
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.
Figure 2.
ATXN2L is a component of SGs under different stress conditions.
A) HeLa cells were subjected to treatment with 0.5 mM sodium arsenite or heat-shock at 44°C, or B) 0.5 M sorbitol, 2 mM dithiothreitol (DTT), 2 mM hydrogen peroxide, or 1 mM sodium selenite for 1 hour, respectively, or left untreated as control. Cells were fixed and proteins were stained with antibodies directed against ATXN2L (red) and ATXN2 (BD Biosciences, green). Nuclei were stained with Hoechst (blue) and scale bars represent 20 µm.
Figure 3.
ATXN2L behaves as a dynamic SG component like ATXN2. A)
HeLa cells were concurrently treated with cycloheximide and 0.5 mM sodium arsenite for 1 hour. As controls, cells were only treated with sodium arsenite, or cells were left untreated at 37°C. B) Cells were treated with 0.5 mM sodium arsenite for 1 hour and incubated at normal growth conditions for 90 or 180 min to allow recovery. Subsequently, cells were fixed and stained with antibodies directed against ATXN2L (red) and ATXN2 (BD Biosciences, green). Nuclei were stained with Hoechst (blue). Scale bars represent 20 µm.
Figure 4.
ATXN2L overexpression induces the formation of SGs. A)
For co-immunoprecipitation experiments cell lysates were prepared from HEK293T and HeLa cells as described in Materials and Methods, and experiments were carried out with either anti-ATXN2L or anti-G3BP antibody. Precipitated proteins were detected with anti-G3BP or anti-ATXN2L. B) Co-immunoprecipitation experiments were carried out with either anti-ATXN2 (Bethyl) or anti-G3BP antibody. Precipitated proteins were detected with anti-G3BP or anti-ATXN2 (Bethyl). C) HeLa cells were transfected with the expression plasmids RSV-ATXN2L-MYC, pCMV-MYC-ATXN2-Q22 or pCMV-MYC-G3BP1 and incubated for 48 hours to allow expression of the respective fusion proteins. Afterward, cells were fixed and stained for the MYC-tag (Millipore, green) and eIF4G (red) to monitor induction of SGs. Nuclei were stained using Hoechst (blue). Scale bars represent 20 µm.
Figure 5.
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.
Figure 6.
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.
Figure 7.
ATXN2L reduction affects P-body formation.
HeLa cells were left untreated (mock) or transfected with siRNAs against ATXN2L or ATXN2 transcripts, or non-targeting control siRNAs. The cells were fixed 72 hours post transfection, and analyzed A) by confocal microscopy or B–D) by quantitative high-content screening microscopy. In A) cells were stained with an antibody detecting DCP1. In B–D) cells were stained with antibodies detecting DCP1 and DDX6. B) Cell numbers and size of nuclei of analyzed cells. C, D) Quantification of DCP1- and DDX6-positive P-bodies 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.