Figure 2.

Mutant FUS Is Recruited to poly(I:C)-Induced SGs

(A) Endogenous mutant FUS is highly enriched in poly(I:C)-induced SGs in FUSΔNLS lines. Cells were analyzed 4 h after poly(I:C) transfection. Representative images for two heterozygous FUSΔNLS (ΔNLS_het) lines and two homozygous FUSΔNLS (ΔNLS_ho) lines are shown.

(B) Poly(I:C), but not sodium arsenite (SA), causes persistent SGs in mutant FUS-expressing cells. Quantification of cells containing SGs in ΔNLS_het lines (two lines combined) and WT cells over a 24 h follow-up period post-transfection or post-treatment is shown. Cells were treated with SA for 1 h, washed, and analyzed during recovery, with ≥500 cells analyzed per time point for each line. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 (Student’s t test).

(C–E) poly(I:C) induces a few large SGs per cell in FUSΔNLS lines. Representative images (C), quantification of the SG area (D), and the fraction of cells containing 1, 2, and >2 SGs (E) in FUSΔNLS cells are shown. In (D) and (E), data for two ΔNLS_het and two ΔNLS_ho lines were combined. Between 250 and 400 cells were analyzed in (D), and between 92 and 141 cells were analyzed in (E). ^∗∗∗∗p < 0.0001 (Student’s t test).

(F) Near-complete clearance of mutant FUS from the nucleus in poly(I:C)-stimulated, but not SA-treated, ΔNLS_ho cells. Nuclei are circled. Note the loss of nuclear FUS in two SG-containing cells in the poly(I:C)-stimulated culture (nuclei circled in blue).

(G) poly(I:C) induces FUS-positive SGs in human patient fibroblasts bearing P525L mutation. Fibroblasts were analyzed 24 h post-transfection. Fibroblasts treated with SA for 1 h are shown for comparison.

In (C)–(F), cells were analyzed 6 h after poly(I:C) transfection and 1.5 h after SA addition. In (B) and (D), data are represented as mean ± SEM. Scale bars, 10 μm.