Figure 3: Characterization of circHIPK3 RNA in human cells.

(a) The genomic loci of five circRNAs in HIPK3 gene. The supported unique reads were presented. The expression of circHIPK3 was validated by RT–PCR followed by sanger sequencing. Arrows represent divergent primers binding to the genome region of circHIPK3. (b) Absolute quantification for circHIPK3 and HIPK3 mRNA in six human normal tissues. (c) qRT–PCR for the abundance of circHIPK3 and HIPK3 mRNA in HeLa cells treated with Actinomycin D at the indicated time points. (d) qRT–PCR for the abundance of circHIPK3 and HIPK3 mRNA in HeLa cells treated with RNase R. The amount of circHIPK3 and HIPK3 mRNA were normalized to the value measured in the mock treatment. (e) qRT–PCR data indicating the abundance of circHIPK3 and HIPK3 mRNA in either the cytoplasm or nucleus of HeLa cells. The amounts of circHIPK3 and HIPK3 mRNA were normalized to the value measured in the cytoplasm. Data in (c–e) are the means±s.e.m. of three experiments. (f) RNA fluorescence in situ hybridization for circHIPK3. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar, 5 μm.