Fig. 2

Characterization of circNSUN2 in CRC. a The genomic locus of circNSUN2. Left, the expression of circNSUN2 was detected by qRT-PCR followed by Sanger sequencing. Arrows represent divergent primers binding to the genome region of circNSUN2. Right, qRT-PCR products with divergent primers showing circularization of circNSUN2. cDNA complementary DNA. gDNA genomic DNA. b qRT–PCR analysis for the expression of circNSUN2 and NSUN2 mRNA after treatment with RNase R in HCT116 cells. Data represent mean ± S.D. from five independent experiments; dot plot reflects data points from independent experiment. The P value was determined by a two-tailed unpaired Student’s t test. c Northern blotting of circNSUN2 and NSUN2 transcripts by hybridization with exon5 (top, left) and exon 5−exon 4 junction (top, right) probes in the presence or absence of RNase R treatment. Right, RNA marker. GAPDH mRNA (bottom) was also blotted as an internal control. The samples for blotting GAPDH were aliquoted before RNase R treatment and loaded separately in two wells for validation. d qRT–PCR analysis for the expression of circNSUN2 and NSUN2 mRNAs after treatment with Actinomycin D at the indicated time points in HCT116 cells. Data represent mean ± S.D. from five independent experiments; dot plot reflects data points from independent experiment. The P value was determined by a two-way ANOVA. e Cytoplasmic and Nuclear mRNA Fractionation experiment showing that circNSUN2 localized in the nucleus and the cytoplasm. β-actin and U3 were applied as positive controls in the cytoplasm and nucleus, respectively. Data represent mean ± S.D. from five independent experiments; dot plot reflects data points from independent experiment. f RNA fluorescence in situ hybridization for circNSUN2. Nuclei were stained with DAPI. Scale bar, 10 µm. For c and f, junction probe is complementary to the junction sequence of circNSUN2. Source data are provided as a Source Data file