Fig. 1

The validation and expression of circTADA2A in osteosarcoma tissues and cells. a CircRNA microarray based on osteosarcoma cell lines and hFOB1.19 in GSE96964. b The expression of circTADA2A was detected by qRT-PCR in 10 osteosarcoma and chondroma tissues (n = 10) (*P < 0.01, Student’s t-test). c Representative FISH images demonstrating circTADA2A expression detected by a junction probe in chondroma and osteosarcoma tissues; scale bars, 200 μm and 50 μm (FISH, fluorescence in situ hybridization). d CircTADA2A expression was detected by qRT-PCR in various human osteosarcoma cell lines (HOS, 143B, U2OS, SJSA-1 and MG63) and normal cells (osteoblast hFOB1.19 and HEK-293); circTADA2A mRNA levels were higher in OS cells than in hFOB1.19 and HEK-293 cells. e Schematic illustration demonstrates the formation of circTADA2A via the circularization of exons 5 and 6 in TADA2A (black arrow). The presence of circTADA2A was validated by RT-PCR, followed by Sanger sequencing. The head-to-tail splicing site of circTADA2A is indicated by the red arrow. f RT-PCR validated the existence of circTADA2A in HOS and 143B cell lines. CircTADA2A was amplified by divergent primers in cDNA but not gDNA. GAPDH was used as a negative control. g & h The expression of circTADA2A and TADA2A mRNA in both HOS and 143B cell lines was detected by RT-PCR or qRT-PCR in the presence or absence of RNase R. i FISH showed that circTADA2A was predominantly localized in the cytoplasm. Nuclei were stained with DAPI, and circTADA2A probes were labeled with Alexa Fluor 555; scale bars, 50 μm. Data are from three independent experiments (mean ± SEM) (d and h) or are representative of three independent experiments with similar results (c, f, g and i) (*P < 0.01 vs control or as indicated by Student’s t-test)