Fig. 1

Identification of the circular structure. a Head-to-tail splicing of circSIPA1L1 and its genome size and sequences tested by Sanger sequencing. a. Schematic representation of SIPA1L1-expressing plasmid. b. Agarose gel electrophoresis of RT-PCR products from HEK293T cells transfected with SIPA1L1-expressing plasmid or empty vector. c. Sanger sequencing of RT-PCR products from 293T cells, the arrows indicate fusion sites. b We further confirmed that circSIPA1L1 was resistant to RNase R. rather than linear-SIPA1L1 could resist digestion by RNase R. c The existence of circSIPA1L1 was validated in 293T cell lines by RT-PCR. Divergent primers amplified circSIPA1L1 in cDNA but not genomic DNA (gDNA). GAPDH was used as a negative control. d FISH assay showed the localization of circSIPA1L1 in the cytoplasm. 18S and U6 were the internal control. e Three circSIPA1L1 small interfering RNAs (siRNAs) specifically targeting the backsplice junction sequences at different binding sites in circSIPA1L1 were designed. f. Small interfering RNA silencing efficiency was detected by RT-PCR. The results showed that si-circSIPA1L1-1 and si-circSIPA1L1-3 could effectively knock down the expression of circSIPA1L1(*P < 0.05, **P < 0.01). g The expression of circSIPA1L1 between NC and circSIPA1L1 group was detected by RT-PCR. h Dynamic expressions of ALP, RUNX2, and OSX during DPSC osteogenesis at days 0, 3, and 7. Dynamic expressions of circSIPA1L1 and miR-617 during DPSC osteogenesis at days 0, 3, and 7. RNA level was normalized to that at day 0. GAPDH and U6 were the internal controls, respectively. *P < 0.05, **P < 0.01