Fig. 1: Characterization of circRNA-vgll3 in ADSCs.

a Venn analysis of abundant (RPM > 0.1) exonic circRNAs with potential miRNA binding sites in ADSCs. b Bioinformatics prediction of 11 circRNAs, their predicted miRNAs, and miRNA targets. c CircRNA-vgll3 generated from the third exon of vgll3 gene locus. d-f PCR validation of circRNA-vgll3 using outward-facing primer and Sanger sequencing of the PCR product. g The relative gene expression of circRNA-vgll3 and vgll3 after RNase R treatment (n = 3, *P < 0.05 versus Mock group, statistical analysis was performed by Student’s t test). h The relative gene expression changes of circRNA-vgll3 and vgll3 after actinomycin D treatment for 6 h, 12 h,18 h, and 24 h, compared to that of 0 h (n = 3). i qPCR analysis of circRNA-vgll3 expression in BMP2-induced ADSCs and naive ADSCs. j qPCR analysis of circRNA-vgll3 expression in bone tissue development time points of postnatal day 1, month 1 and month 3. k-l PCR and qPCR analysis of cytoplasmic and nuclear RNA with circRNA-vgll3 primer, vgll3 primer, GAPDH primer (canonical marker of cytoplasmic fraction) and RNU6B primer (canonical marker of nuclear fraction) showed that circRNA-vgll3 mainly located in the cytoplasm and vgll3 located in the cytoplasm and nucleus. m qPCR analysis of the efficiency of the si-vgll3 in knocking down the mRNA levels of vgll3 (n = 3, *P < 0.05 versus Control group, statistical analysis was performed by Student’s t test). n FISH results depicted the cytoplasm location of circRNA-vgll3. Si-vgll3 treatment did not alter the labeling efficiency. Scale bars: 60 µm.