Figure 1. Validation and characteristics of circ-BPTF in BCa cells. (A) Gel electrophoresis analysis of the PCR products of circ-BPTF and linear BPTF. Divergent primers amplified circ-BPTF only in cDNA, whereas, convergent primers produced linear transcript in both cDNA and gDNA. GAPDH was applied as a linear control. (B) Sanger sequencing of circ-BPTF products amplified by PCR. The back-splice junction site was marked by the red arrow. (C) Schematic illustration showed that circ-BPTF was cyclized from exon 21 and exon 27 of BPTF. (D) The circular and linear form of BPTF levels were examined by qPCR after exposure to Actinomycin D in T24 cells. (E) Circ-BPTF and linear BPTF levels were detected by qPCR in T24 cells treated with or without RNase R. (F) qPCR analysis of circ-BPTF and linear BPTF using random or oligo dT (18) primers in the reverse transcription process. (G and H) Circ-BPTF is mainly presented in the cytoplasm of T24 cells verified by nuclear mass separation assay and FISH. (I) Expression of circ-BPTF in BCa cell lines (5637, EJ, UM-UC-3, T24) and normal urothelial cells (SV-HUC-1) was detected by qPCR. Data indicate means ± SEM. **P<0.01, ***P<0.001.