Fig. 3

CircPDHK1 encodes a novel peptide, PDHK1-241aa. A The predicted 815 nt circPDHK1 sequence encodes 241 amino acids (PDHK1-241aa). Potential m6A sites and IRES (Internal Ribosome Entry Site) are also indicated. B IRES constructs in luciferase expression vectors. Full-length IRES, IRES del-1, IRES del-2 and mutated IRES types inserted in Rluc and hLuc reporter vectors containing independent start and stop codons. C Relative luciferase activity detected using dual-luciferase reporter assays to tested circPDHK1 translation ability in 293 T cells. D Illustration of the synthetic circRNA expression plasmid pLC5-ciR, a commercial circRNA blank expression vector; We also established circPDHK1-3 × flag plasmid for pLC5-circPDHK1 fused with 3 × flag tag and circPDHK1-3 × flag ATG mut, the start codon ATG of circPDHK1-3 × flag was mutated to CTG with a commercial kit to validate the coding potential of circPDHK1. E Sanger sequencing of the circPDHK1-3 × flag ATG mut vector to verify its construction. F-G RT‒qPCR and western blot were used to analyze the efficiency of overexpressing circPDHK1-3 × flag and circPDHK1-3 × flag ATG mut vectors to verified circPDHK1 translation ability in 293 T cells. H Immunofluorescence staining using a Flag antibody to detect the intracellular localization of circPDHK1-3 × flag in Caki-1 and 786-O cells. The red (anti-flag) indicated the circPDHK1-3 × flag, the blue (Hochest) indicated the nucleus. Bars = 20 μm. I PDHK1-241aa expression detected by western blot following SDS‒PAGE of IP pull-downs from cell lysates as indicated. Gel bands corresponding to 31 kDa were manually removed and submitted to LC‒MS for identification. J Identification of the amino acid sequence of PDHK1-241 by LC‒MS. K Western blot analysis of PDHK1-241aa expression in 16 paired ccRCC samples and normal adjacent tissues. **P < 0.01; ***P < 0.001