Fig. 1

The identification and characteristics of circFNDC3B in BC cells. a The general scheme of the establishment of invasive and noninvasive cell sublines from the human BC T24 cell line. b Number of differentially changed circular RNAs identified via the T24 cell invasion model. c Relative expression of circFNDC3B in poorly and highly invasive T24 cell sublines. d The PCR products of circFNDC3B and linear FNDC3B were tested by gel electrophoresis. Divergent primers amplified circFNDC3B in cDNA but not genomic DNA (gDNA). Convergent primers amplified linear FNDC3B in both cDNA and gDNA. GAPDH was used as a linear control. e The formation of circFNDC3B. circFNDC3B derived from back-spliced exons 5 and 6 of genomic FNDC3B. The existence of circFNDC3B was confirmed by Sanger sequencing. The green arrow represents the back-splice junction of circFNDC3B. f qRT-PCR analysis of circFNDC3B using random primers and oligo-dT primers,respectively in reverse transcription experiments. CircFNDC3B is absent in polyA-enriched samples. g The expression of circFNDC3B and FNDC3B mRNA was measured by qRT-PCR in T24 and UM-UC-3 cells treated with or without RNase R. h qRT-PCR analysis of circFNDC3B and FNCDC3B mRNA in T24 and UM-UC-3 cells treated with actinomycin D at the indicated time points. i and j CircFNDC3B was mainly located in the cytoplasm, as confirmed by the nuclear mass separation assay and FISH, in T24 cells. Nuclei were stained with Hoechst33342. Scale bar, 50 μm. Data are shown as the means±SEM of three experiments. **P < 0.01 (Student’s t-test)