Fig. 4

Characterization of circLIFR in solid tumors. A Schematic illustration showing circLIFR is derived from exons 2–5 of LIFR mRNA. The F1/R1 primers were used to measure circLIFR by RT-qPCR. DP, divergent primers; CP, convergent primers. B circLIFR expression measured by RT-qPCR in ESCC, HCC and their matched adjacent normal tissues, normalized to U6 RNA levels. N, normal tissues; T, tumor tissues. Data represent the mean ± S.D., and the p-value was determined by two-tailed paired Student’s t-test. C The presence of circLIFR was validated in KYSE150 cells by RT-PCR. Divergent primers were used to amplify circLIFR from cDNA, but not from genomic DNA. LIFR mRNA linear transcripts were used as a negative control. cDNA, complementary DNA; gDNA, genomic DNA. D Total RNA from KYSE150 cells with or without RNase R treatment was subjected to RT-PCR. E Agarose gel electrophoresis and Sanger sequencing of RT-PCR products of circLIFR in KYSE30 and SK-Hep-1 cells. F RT-qPCR analyses of circLIFR after cytoplasmic/nuclear fractionation of KYSE30 and SK-Hep-1 cells. β-actin mRNA and U6 RNA represent cytoplasmic and nuclear RNAs, respectively. Western blotting confirmed the efficiency of nuclear/cytoplasmic isolation. Data are shown as the mean ± SD. G Fluorescence in situ hybridization of circLIFR in KYSE30 and SK-Hep-1 cells. 18S and U6 RNA represent cytoplasmic and nuclear RNAs, respectively. H circLIFR and LIFR mRNA levels in KYSE30 and SK-Hep-1cells without or with actinomycin D treatment