Fig. 3: FUS regulates the generation of circ-GRB10 in NP cells.

a qRT-PCR showing FUS mRNA levels in normal and IDD NP tissues. Three independent repeats were performed in each experiment. ***p < 0.001. b Western blot showing FUS protein amounts were decreased in IDD NP tissues. c qRT-PCR analysis of circ-GRB10 expression level after FUS overexpression or knockdown in NP cells. FUS overexpression led to significantly increased circ-GRB10 amounts in NP cells, while its knockdown reduced circ-GRB10 levels. Moreover, FUS had no linear effects on GRB10 expression. Three independent repeats were performed in each experiment. ***p < 0.001. d, e qRT-PCR analysis of the expression of Collagen-II, aggrecan, MMP-13 and ADAMT-5 in NP cells. f Schematic illustrating the putative FUS-binding sites on the flanking introns in the circ-GRB10-s minigene. The 5′ terminus of the circular exons of circ-GRB10 was defined as position 0. Putative FUS-binding sites A and B are located in the intron at the 5′ terminus of the circ-GRB10 exon (position: −441 to −438), and on the intron at the 3′ terminus of the circ-GRB10 exon (position: 872–875). g RIP analysis of FUS-binding to circ-GRB10-s and circGRB10-s-Em minigenes in NP cells. Bound complexes were pulled-down using an antibody against FUS. qRT-PCR was then used to measure circ-GRB10-s binding to FUS. Values were normalized to the level of background RIP, as detected by an IgG isotype control. h qRT-PCR analysis of the expression of circ-GRB10 relative to GAPDH in NP cells. Cells were co-transfected with FUS or FUS siRNA and a circ-GRB10 minigene (circ-GRB10-s), or circ-GRB10 minigene containing deleted FUS-binding sites (circ-GRB10-Em). Quantitative data from three independent experiments is presented as mean ± SEM (error bars). **P < 0.01; ***P < 0.001.