(A) N2A cells were transfected with FUS siRNA using
Lipofectamine™ RNAiMAX. 48h after transfection cells were harvested and
immunoblotting was performed using the indicated antibodies. (B) N2A and FUS KO
cells were cultured and immunoblotting was performed using the indicated
antibodies. (C) Confocal imaging of FUS and LC3 in N2A and FUS KO cells. Scale
bars are 10 μm. (D) Quantification of the percentage of cells containing
LC3-positive autophagosomes from multiple random viewfields (N > 150
cells). **, p ≤0.01. (E) FUS KO cells were transfected with FLAG-tagged
WT FUS, FUS-R495X, R521G, P525L. WT N2A cells and FUS KO cells with no
transfection were included as positive and negative controls. 48h after
transfections cells were collected and immunoblotting was performed with the
indicated antibodies. (F-G) Quantification of (E) from three independent
experiments. Results are shown in scatter plots with the bar representing the
median. Student’s t-test was performed for individual
comparisons against FLAG-FUS WT. **, p ≤0.01. N.S., no significance.