Fig. 3

CircKEAP1 encodes a novel 259-amino acid protein denoted KEAP1-259aa. (A). Putative ORF in circKEAP1 and the sequences of the putative ORF are shown. (B). Putative IRES activity in circKEAP1 was tested by dual luciferase activity assay. (C). U2OS cells were transfected with control vector, circKEAP1 ATG-mutated vector, circKEAP1 wild-type vector and linear KEAP1-259aa vector. CircKEAP1 expression was detected by qRT-PCR. (D). Total proteins from circKEAP1 or control plasmid-transfected U2OS cells were separated via SDS-PAGE. The differential gel bands between 25 kDa and 35 kDa were cut and subjected to LC-MS/MS. The identified KEAP1-259aa amino acids are shown. (E). KEAP1 and KEAP1-259aa expression were detected in eight paired normal and case-matched OS samples. (F). MG63 and U2OS cells were transfected with control or two circKEAP1 shRNAs. KEAP1 and KEAP1-259aa levels were characterized by western blotting. (G). KEAP1 and KEAP1-259aa expression were detected in control-, circKEAP1-, circKEAP1 IRSE mut-, and circKEAP1 ATG mut-transfected HOS and U2OS cells by western blotting. (H). Flag-tagged KEAP1-259aa was transfected into HOS and U2OS cells. Immunofluorescence staining using anti-Flag was performed to show KEAP1-259aa cellular localization (scale bars, 20 μm). Error bars represent three independent experiments. *, **, *** indicates significant differences compared with the empty vector group at a p value < 0.05, < 0.01, < 0.001, respectively