Fig. 1
CircMYO10 validation and expression in osteosarcoma tissue and cells. a The heatmap for top 30 differentially up-and down-regulated circRNAs. b CircMYO10 expression in hFOB1.19 and osteosarcoma (OS) cell lines (143B, HOS, MG63, U2OS, SJSA-1) was evaluated by qRT-PCR. Data represents the mean ± standard deviation (SD) (n = 9). c CircMYO10 expression in ten paired human osteosarcoma tissues (n = 10) and chondroma tissues (n = 10) was measured by qRT-PCR. Data was normalized to the levels of GAPDH and presented as 2-△CT. Data represents the mean ± SD (n = 90). d RNA fluorescence in situ hybridization (FISH) showed that circMYO10 expression is higher in human OS tissues than in chondroma tissues. Representative images are shown. Scale bars = 100 μm or 50 μm. e Schematic illustration demonstrated the circularization of exons 12–30 of MYO10 forms circMYO10 by “head-to-tail” junction and the upper black arrow represents the splicing sites. The presence of circMYO10 was validated by RT-PCR followed by Sanger sequencing. f The expression of circMYO10 and MYO10 in MG63 cells treated with or without Rnase R was detected by qRT-PCR. The levels of circMYO10 and MYO10 mRNA in cells treated with Rnase R were normalized to the value measured in the mock treatment. Data represents the mean ± SD (n = 9). g Northern blots for detecting circMYO10 in U2OS and MG63 cells treated with or without RNase R digestion. The upper panels show the probed blots of circMYO10 with a red line indicating the band size of circMYO10 (2867 nt). The lower panels show the gel electrophoretic results of RNA with or without RNase R digestion. h Agarose gel electrophoresis found that divergent primers (←→) amplify circMYO10 in complementary DNA (cDNA) but not genomic DNA (gDNA). i FISH showed that circMYO10 localizes mainly in the cytoplasm. CirMYO10 probes were labeled with cy3 with nuclei stained with DAPI. Scale bars, 50 μm. Three independent assays were performed in the above assays. b, c, f * P < 0.05. ** P < 0.01. *** P < 0.001 (Student’s t-test)