Fig. 1

Validation and expression of circKEAP1 in OS tissues. (A). A circRNA microarray was used for 3 pairs of OS tissues and case-matched normal tissues. Circular heatmap showing the differentally-expressed circRNAs. (B). Expression of circKEAP1 was detected by qRT-PCR in OS (n = 26) and normal tissues (n = 11) from our own tissue bank. (C). The expression of circKEAP1 in normal (n = 3) and OS tissues (n = 3) was also found in GSE140256 from GEO database. (D). CircKEAP1 expression was detected by qRT-PCR in various human OS cell lines (HOS, Saos-2, MG63 and U2OS) and osteoblast cells (hFOB1.19). **, *** indicates significant differences compared with the hFOB1.19 at a p value < 0.01, < 0.001, respectively. (E). Schematic illustration showing the formation and head-to-tail splicing site of circKEAP1 via the circularization of exon 2 in KEAP1. The presence of circKEAP1 was validated by Sanger sequencing. (F). RT-PCR validated the existence of circKEAP1 in HOS and U2OS cell lines. CircKEAP1 was amplified by divergent and convergent primers in complementary DNA (cDNA) and genomic DNA (gDNA). (G). The expression of circKEAP1 and KEAP1 mRNA in both HOS and U2OS cell lines was detected by RT-PCR and qRT-PCR with/without RNase R (20 U/µl) treatment. (H). Expression of KEAP1 and circKEAP1 was detected in the cytoplasm and nuclei of OS cells. (I). FISH images demonstrating circKEAP1is mainly located in the cytoplasm of OS cells (scale bars, 20 μm). (J). Expression of circKEAP1 was detected by ISH in OS tissues (n = 72) and normal tissues (n = 6) (scale bars, 100 μm, 50 μm). (K). Overall survival analysis based on circKEAP1 expression in OS. (L). ROC curve was shown based on the expression of circKEAP1 in OS tissues. Error bars represent three independent experiments. *, **, *** indicates significant differences compared with the normal group at a p value < 0.05, < 0.01, < 0.001, respectively