Fig. 1

Identification and characterization of circTOLLIP in HCC. a Volcano plot of circRNAs from GEO datasets of GSE97332 and GSE78520. Significantly upregulated and downregulated circRNAs are separately denoted in red and green. b Venn diagram showing the overlap between the two datasets. CircRNAs with P < 0.01 and |log2(foldchange)| ≥ 2 were chosen. c qRT–PCR analysis of the circTOLLIP expression in tissues. T, tumor tissues; N, adjacent nontumor tissues. d Representative ISH images of circTOLLIP in microarrays containing 138 paired HCC specimens. e Kaplan–Meier curve of the correlation between circTOLLIP expression and overall survival (OS). low circTOLLIP group: n = 63, high circTOLLIP group: n = 49. f Schematic display of circTOLLIP formation and the principle of divergent and convergent primer design. The back-splicing junction sites were confirmed by Sanger sequencing. g PCR amplification of circTOLLIP and its linear isoform using divergent and convergent primers from cDNA and genomic DNA (gDNA). GAPDH was used as control. h qRT–PCR analysis of circTOLLIP and TOLLIP mRNA levels with or without RNase R treatment. i Stability of circTOLLIP and TOLLIP RNA with or without actinomycin D treatment at the specific time point measured by qRT–PCR. j The RNA level of circTOLLIP in the nucleus and cytoplasm of HLF and 97H cells. k The subcellular location of circTOLLIP was validated mainly in the cytoplasm by using a FISH assay. Nuclei, blue; circTOLLIP, red. Scale bars = 10 μm