Fig. 1

Verification of the structure and location of circ_0002669 and its clinical significance. A Circ_0002669 was generated from the DOCK1 gene located on chromosome 10. Sanger sequencing was used to identify the back-splice junction. B The closed loop structure of circ_0002669 was verified by PCR using convergent and divergent primers. C, D Upon RNase R treatment, expression of circ_0002669 and DOCK1 mRNA were detected by PCR and qRT-PCR in U2OS cells. E Circ_0002669 and DOCK1 mRNA expression were detected by qRT-PCR in OS cells treated with actinomycin D (1 μm) at the indicated time point. F Expression level of circ_0002669 in the nucleus and cytoplasm of cells was measured by qRT-PCR. G FISH was performed to identify the cellular location of circ_0002669 in OS cells (scale bar, 20 μm). H Circ_0002669 expression in 12 OS tissues and normal tissues (n = 5) was analyzed by qRT-PCR. (I) Circ_0002669 expression was detected in OS tissues and normal tissues by ISH (scale bar, 50 μm). J Circ_0002669 expression was detected in metastatic lesions and primary OS tissues (scale bar, 50 μm). K Survival analysis of 72 OS patients based on circ_0002669 ISH scores. L ROC curve based on the expression of circ_0002669 in OS tissues. Data shown are from three independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001