Figure 1. Characterization of hsa_circ_0085576 in ccRCC cells. (A) Schematic diagram of the genomic location and splicing pattern of has-hsa_circ_0085576; the specific primers of 0085576 were validated by Sanger sequencing. (B) The existence of hsa_circ_0085576 was validated by RT-PCR in ccRCC tumor tissue samples. (C, D) RT-qPCR was used to determine the abundance of hsa_circ_0085576 and linear ASAP1 mRNA in A498 cells treated with RNase R. (E) The levels of hsa_circ_0085576 in 40 paired ccRCC and matched adjacent normal tissues were examined by RT-qPCR. (F) The levels of hsa_circ_0085576 in 45 non-metastasis and 31 metastasis ccRCC were examined by RT-qPCR. (G) The expression of hsa_circ_0085576 in cell lines HK-2, 786-O, A498, Caki1, and ACHN was detected by RT-qPCR. (H) The cellular distribution of hsa_circ_0085576 in A498 cells was analyzed by fluorescence in situ hybridization (FISH). Green indicates hsa_circ_0085576 and blue indicates nuclei. (I) The cellular distribution of hsa_circ_0085576 was analyzed by cellular RNA fractionation assays. U6 and GAPDH were used as nuclear and cytoplasmic positive controls, respectively. * P<0.05 vs. HK-2; ** P<0.05 vs. mock.