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. 2019 Dec 24;29(13):4496–4508.e4. doi: 10.1016/j.celrep.2019.11.094

Figure 6.

Figure 6

Type I Interferon Promotes Accumulation of Normal and Mutant FUS Protein

(A) FUS mRNA level is increased in poly(I:C)-stimulated WT SH-SY5Y cells 24 h post-transfection as measured by qRT-PCR. n = 4, ∗p < 0.05 (Mann-Whitney U test).

(B) IFN-beta treatment alone upregulates FUS mRNA in WT cells. FUS mRNA levels in IFN-beta-treated cells were measured by qRT-PCR at the indicated time points. n = 4–5. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (Mann-Whitney U test).

(C) IFN-beta treatment causes FUS protein accumulation in WT cells in a time-dependent manner. Representative western blot and quantification of FUS protein levels after 24 h of IFN-beta treatment are shown. Western blot also demonstrates degradation of the IFNAR subunit in IFN-treated cells. n = 3, ∗p < 0.05 (Mann-Whitney U test).

(D) FUS pre-mRNA is not upregulated during IFN-beta treatment. Two pairs of primers mapping to the intron sequences of the FUS gene were used for qRT-PCR. n = 3.

(E) FUS mRNA species with longer PATs accumulate in cells treated with IFN-beta as revealed by the PAT assay. The diagram shows the principle of the PAT assay. P1, P2, and P3 are FUS-specific forward, FUS-specific reverse, and universal reverse primers, respectively. PA stands for poly(A) tail (amplified with P1 and P3), and int stands for the internal FUS fragment (amplified with P1 and P2). The electrophoresis image demonstrates a similar band intensity for the internal FUS fragment but increased intensity of the smear corresponding to the longer PA tails; the intensity profile of the PA tail lanes is also shown. Cells were treated with IFN-beta for 8 h.

(F) Mutant FUS protein accumulates in FUSΔNLS cells upon IFN-beta treatment. Cells were treated with IFN-beta for 24 h. The FUS knockout line was included as a negative control.

(G) IFN-beta treatment does not alter the subcellular localization of normal and mutant FUS. Cells were treated with IFN-beta for 24 h. Scale bar, 10 μm.

(H) Levels of FUS ex7− mRNA transcript significantly increase in WT lines, but not in FUSΔNLS lines, upon IFN-beta exposure. Cells were treated with IFN-beta for 24 h and analyzed by qRT-PCR. n = 4, ∗p < 0.05 (Mann-Whitney U test).

In all panels, data are represented as mean ± SEM.