Fig. 1

Characterization of Circ-HER2. a The volcano plot of differentially expressed circRNAs between TNBC and tumor adjacent normal tissues (n = 5). X axis, −log10 P value; Y aixs, log2 fold change. b The volcano plot of differentially expressed host coding gene between TNBC and tumor adjacent normal tissues (n = 5). X axis, −log10 P value; Y aixs, log2 fold change. c KEGG pathways enriched among differentially expressed host coding genes. d Comparison of differentially expressed host coding genes and differentially expressed circRNAs. Numbers in brackets in Venn diagram represent the number of differentially expressed circRNAs derived from corresponding host coding genes. e Illustration of the annotated genomic region of HER2 gene, the putative different mRNA splicing forms (linear splicing and ‘head-to-tail’ splicing) and the validation strategy for the circular exon 3–7 (circ-HER2). Sanger sequencing following PCR using the indicated divergent flanking primers showed the ‘head-to-tail’ splicing of circ-HER2 in MDA-MB-231 cells. f Northern blots using the junction-specific probe to detect circ-HER2 in MDA-MB-231 and MDA-MB-468 cells endogenously or with indicated modifications. g Left, relative RNA level of circ-HER2 and linear HER2 at different time point; Right, relative RNA level of circ-HER2 and linear HER2 treated with RNase R. h Left, total RNA from MDA-MB-231 cells were reverse-transcribed with Oligo dT primers or random primers; circ-HER2 or linear HER2 mRNA levels were detected by q-PCR. Right, cytoplasmic and nuclear fractions were isolated, circ-HER2 and linear HER2 expression were detected. β-actin and U6 RNA served as cytoplasmic and nuclear RNA markers. i Fluorescence in situ hybridization (FISH) with junction-specific probes (green) or linear specific probes (red) were used to detect the localization of circ-HER2 and HER2 in MDA-MB-231 TNBC cells. Circ-HER2 and linear HER2 plasmids were transfected as positive controls. Scale bars, 20 μm. Lines show the mean ± SD. ***p < 0.001. In (E), (F), (G), (H), (I), Data are representative from at least 2–3 experiments with similar results