Fig. 1

Generation and characterisation of SH-SY5Y cell lines with targeted modifications of the endogenous FUS gene. a Structures of the FUS gene and FUS protein together with the positions of CRISPR/Cas9 target sites chosen to delete the NLS-encoding fragment. PAM sequences are in green, stop codon is highlighted in yellow and exons are in bold. b Subcellular distribution of FUS protein in FUS ΔNLS and FUS knockout (KO) clones detected with N-terminal FUS antibody. c PCR genotyping of FUS ΔNLS clones. PCR with primers flanking the fragment to be deleted (underlined in A) yields 595 and 265 bp fragments for WT and edited FUS alleles, respectively. d RNA-Seq reads for exons 14 and 15 of the FUS gene in WT cells as well as heterozygous and homozygous FUS ΔNLS lines. Dashed lines indicate the deletion. e Analysis of FUS mRNA levels by qRT-PCR in FUS ΔNLS lines. Diagram shows positions of primers for measuring total and WT mRNA (not drawn to scale, del denotes the deleted region). Average values for three heterozygous (“het pooled”) and three homozygous (“ho pooled”) lines are also shown. N = 4–6, *p < 0.05, ****p < 0.0001 (one-way ANOVA). f Western blot analysis of FUS in FUS ΔNLS and FUS KO lines using antibodies recognising its N-terminal (aa.1–50) or C-terminal (aa.500–526) segments. Note that mutant FUS possesses a FUS-unrelated C-terminal amino acid stretch both in ΔNLS1_ho and ΔNLS2_het lines causing slower migration of the mutant protein (for protein sequences see Additional file 1: Figure S1A). g FUS distribution in representative heterozygous and homozygous FUS ΔNLS lines. Nuclei border in homozygous cells is indicated with a dashed line. h FUS levels in total lysates and cytoplasmic fraction from WT and FUS ΔNLS lines. Ratio C/T, ratio cytoplasmic to total FUS levels. Note absence of histones (arrows) in the cytoplasmic fraction. Scale bars, 10 μm