Figure 1.

TRIM21 is localized to SGs under oxidative stress and its overexpression inhibits SG formation. (A) TRIM21 is recruited to SGs under oxidative stress. U-2 OS cells treated without or with arsenite were stained with TRIM21 (red) and G3BP1 (green). The arrows indicate colocalization of TRIM21 and G3BP1. Line graphs in the right panels show TRIM21 and G3BP1 signals along the indicated arrows in the insets. Scale bar: 10 μm. Inset scale bar: 2 μm. (B) Quantification of the percentage of SGs colocalized with TRIM21 from (A). Data are presented as means ± SEMs from three independent experiments with 100 cells counted. ***p < 0.001 (Student’s t-test). (C) Left, Z-stack projection of the representative image of TRIM21 and G3BP1 signals in U-2 OS cells under oxidative stress acquired by SIM. Cells were stained with G3BP1 (green), TRIM21 (red) and nuclei (DAPI, blue). Scale bar: 5 μm. Right, magnified orthogonal sectioning view of regions in the boxes; scale bar: 1 μm. (D) TRIM21 domain structures are shown and the TRIM21 truncations are made based on domain analysis: RING domain (16–55 aa), B-box domain (92–123 aa), coiled-coil (CC) domain (128–238 aa) and SPRY domain (268–465 aa). The level of structural disorder was predicted using the PONDR (predictor of natural disordered regions) VLXT algorithm (http://www.pondr.com/). (E) TRIM21ΔCC overexpression inhibits SG formation. U-2 OS cells were transfected with the empty vector or FLAG-TRIM21ΔCC and treated with arsenite before fixation. Cells were then stained with G3BP1 (green), FLAG (red) and nuclei (DAPI, blue). Scale bar: 20 μm. (F) Quantification of SG number per cell from (E). Data were pooled from three independent experiments with 120 cells counted. Error bars indicate SEM. ***p < 0.001 (Student’s t-test). (G) Knockdown of TRIM21 promotes the accumulation of SGs. A549 cells were infected with lentivirus-control shRNA or lentivirus-sh-TRIM21. Cells were then treated with arsenite before fixation. Cells were then stained with G3BP1 (green) and nuclei (DAPI, blue). Scale bar: 10 μm. (H) The knockdown efficiency of TRIM21. Quantifications of TRIM21 levels (normalized by ACTB) are shown. (I, J, K) Quantification of SG number per cell (I), SG number per cell area (J) and SG size (K) from (G). Data were pooled from three independent experiments with 120 cells counted. Error bars indicate SEM. ***p < 0.001 (Student’s t-test). (L) Immunoprecipitation analysis of the interaction between G3BP1 and TRIM21 in HEK293FT cells without or with arsenite treatment. Anti-G3BP1 antibody immunoprecipitated TRIM21 under normal and stress conditions. Relative fold change of immunoprecipitated TRIM21 is presented as mean of three replicate experiments ± SEM. ***p < 0.001 (Student’s t-test). (M) TRIM21 is enriched in SG fraction under oxidative stress. U-2 OS cells were treated without or with arsenite and SG fraction (S850) was extracted by serial centrifugations. The samples were analyzed by immunoblotting with the indicated antibodies. ACTB was used as the loading control. Protein levels of G3BP1, TRIM21 and TIA1 were quantified.