Fig. 1

Identification and characterization of circNEIL3 in PDAC cells and tissues. a. Volcano plots showing 79 upregulated and 124 downregulated circRNAs in PDAC tissue relative to matched normal tissue. b. Basic information for the ten most dysregulated circRNAs. c. RT-qPCR analysis of the ten most dysregulated circRNAs in PANC-1, MiaPaca-2, BxPC-3 and CFPAC-1 cells compared to HPNE cells. d. Relative circNEIL3 expression in cell lines was determined by RT-qPCR. e-f. Relative circNEIL3 expression in PDAC tissues (tumour) and adjacent nontumour tissues (adjacent) was detected by RT-qPCR (n = 104). g. Schematic illustration of the genomic location and back splicing of circNEIL3, with the splicing site validated by Sanger sequencing. h. PCR and agarose gel electrophoresis analysis were performed to detect the presence of circNEIL3 and NEIL3 in cDNA and gDNA samples from PDAC cells using divergent and convergent primers. i. CircNEIL3 and linear NEIL3 expression in PDAC cells was detected after RNase treatment R compared to the mock treatment. j. Actinomycin D treatment was used to evaluate the stability of circNEIL3 and NEIL3 mRNA in PDAC cells. k. Nuclear-cytoplasmic fractionation assay results indicated that circNEIL3 was primarily localized in the cytoplasm of PDAC cells. The 18S rRNA and U6 genes were used as cytoplasmic and nuclear controls, respectively. l. FISH results showed the cellular localization of circNIEL3. The circNIEL3 probe was labeled with Cy3 (red), while nuclei were stained with DAPI (blue). The samples were imaged at 1000× magnification. Scale bar = 10 μm. All data are presented as the means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001