Fig. 1

CircIPO11 is highly expressed in HCC tumors and liver CSCs. A Heat map of 46 differentially expressed circRNAs (P < 0.05) as well as highly conserved in human and mouse. HCC #1, HCC #2, and HCC #3 denoted HCC sample numbers. Top 6 upregulated circRNAs in tumor were shown. B Top 6 upregulated circRNAs in HCC primary cells were depleted using shRNA. Their proportions of sphere formation were measured. Data are shown as means ± SD. C Schematic representation of human circIPO11. Convergent primers for linear IPO11 were denoted in black arrowheads, and divergent primers specifically for circIPO11 were denoted in red arrowheads. E1, exon #1. (D, E) Expression levels of circIPO11 in HCC primary tumors and peri-tumors (D), or in oncosphere and non-sphere cells (E). Data of qRT-PCR were normalized to endogenous 18S rRNA unless noted in this study. Results are shown as means ± SD. Isoforms and size of circIPO11 were detected by Northern blot, using 18S rRNA as a loading control (right panel). F Expression levels of circIPO11 in liver CSCs (CD13⁺CD133⁺) and non-CSCs (CD13⁺CD133⁺) sorted from liver cancer cell lines and HCC primary cells. G Copy numbers of circIPO11 were analyzed by qRT-PCR. Black dots represent known copies of circIPO11 from a plasmid pcDNA3 containing circIPO11 sequences (left), and red and blue dots represent circIPO11 copies in spheres and liver CSCs (right). Average copies of circIPO11 per cell were calculated (right). H In situ hybridization of circIPO11 in human HCC tumor and peri-tumor tissues. Representative images are shown in the left panel. Quantitation of circIPO11 positive cells in sections from 10 different HCC samples was shown in the right panel using Image-Pro Plus 6. Results are shown as means ± SEM. Scale bar, 50 μm. I Nuclear-cytoplasmic separation assays were performed using HCC oncosphere cell lysates, followed by qRT PCR (left panel) and Western blot analysis (right panel). U1 RNA served as a positive control for nuclear location. EEA1, endosome antigen 1; H3, histone 3. Data are shown as means ± SD. J Representative immunofluorescence staining of circIPO11 in HCC tumor tissues (n = 3). CircIPO11 was visualized by RNA FISH, followed by Alexa594-conjugated phalloidin (1:3000, 23,122, aatbio). Scale bar, 50 μm. **P < 0.01; *** P < 0.001 by two-tailed Student’s t-test. Data are representative of at least three independent experiments