Fig. 2

Characterization of circPPFIA1-L and -S. A, B The stability of circPPFIA1-L and -S was examined by RNase R resistance (A) and actinomycin D experiments (B). The level of remaining circPPFIA1-L and -S was determined by semi-qPCR and RT-qPCR. Linear PPFIA1 mRNA, GAPDH, and ACTB were included as controls. C, E Complementary (cDNA) and genomic DNA (gDNA) were prepared using RNA isolated from KM12C cells. The level of circPPFIA1-L (C) and -S (E) was determined by semi-qPCR. D, F To verify the divergent region of circPPFIA1-L (D) and -S (F), Sanger sequencing was performed. (G) To determine the cellular localization of circPPFIA1s, the fractionation experiment was performed using digitonin. The level of a-Tubulin and lamin B were checked for verification of cytoplasmic and nuclear extracts, respectively. The levels of circPPFIA1-L, -S, and linear PPFIA1 mRNA were determined by RT-qPCR. GAPDH mRNA and 7SK were used as a marker of cytoplasmic RNA and NEAT1, MALAT1, and 7SL were used for nuclear RNA. Statistical analyses were performed using the Student’s t-test using three independent experiments (*p < 0.05). All data represent mean ± standard variation (SD)