Fig. 4.

Ectopic DDX17 expression reduces insoluble cytoplasmic FUS formation and sequestration into stress granules. HEK cells were co-transfected with the indicated HA tagged FUS constructs (wild-type, R518K, or R521C) and either control EGFP or DDX17-EGFP over expression. a Representative confocal images showing FUS, DDX17 OE, and stress granule marker G3BP1 distributions in HEK cells. For IF, cells were co-transfected with HA tagged FUS (wild-type and mutant) and control EGFP, or EGFP tagged DDX17 OE at equal concentrations. White arrows indicate representative cytoplasmic FUS puncta in both FUS mutants that also co-localize with the stress granule marker, G3BP1. (b) Quantification of the average number of cytoplasmic FUS puncta per cell confirms that over expression of DDX17 significantly reduced the number of cytoplasmic FUS puncta in the R518K and R521C FUS mutants. (n = 55–70 cells per FUS group, One-way ANOVA w/ Turkey’s multiple comparisons). c Quantification of the percentage of G3BP1 positive cytoplasmic stress granules sequestering mutant FUS. Interestingly, overexpression of DDX17 significantly reduced mutant FUS integration into SGs (n = 55–70 cells per FUS group, One-way ANOVA w/ Turkey’s multiple comparisons). d Representative blots of soluble–insoluble fractionation of FUS protein for FUS-WT, FUS-R518K, and FUS-R521C plus EGFP control and FUS-WT, FUS-R518K, FUS-R521C plus DDX17 OE. Tubulin was used as a soluble loading control. e Quantification of soluble/insoluble (Sol/Insol) FUS formation from representative blots shown in panel (d) (n = 4 blots per condition, One-way ANOVA w/ Turkey’s multiple comparisons). Over expression of DDX17 significantly increases the sol/insol fraction of FUS expressing groups. Error bars indicate S.E.M. *p < 0.05, ****p < 0.0001