In Situ Hybridization of circHIPK3 and miR-124 in HeLa Cells

In situ hybridization was performed using specific probes to circHIPK3 sequence. PCR fragments with T7 promoter were amplified with specific primers for the back-splice region of circHIPK3. Primers are listed in Supplementary Table 3. Digoxin or Biotin-labelled RNA probes were transcribed from PCR fragments using the DIG or Biotin RNA labelling mix and T7 RNA polymerase (Roche) according to the manufacturers' instructions. HeLa cells were grown to the exponential phase and were 80–95% confluent at the time of fixation. After pre-hybridization (1 × PBS/0.5% Triton X-100), cells were hybridized in hybridization buffer (40% formamide, 10% Dextran sulfate, 1 × Denhardt's solution, 4 × SSC, 10 mM DDT, 1 mg ml⁻¹ yeast transfer RNA, 1 mg ml⁻¹ sheared salmon sperm DNA) with DIG-labelled probes specific to circHIPK3 at 60 °C overnight. Signals were detected using tyramide-conjugated Alexa 488 fluorochrome TSA kit (Life Technologies). The double FISH assay was performed in HeLa cells after co-transfection with circHIPK3 and miR-124 expressing vectors. Biotin-labelled probes specific to circHIPK3 and Dig-labelled locked nucleic acid miR-124 probes (Exiqon, Vedbaek, Denmark) were used in the hybridization. The signals of biotin-labelled probes were detected using Cy5-Streptavidin (Life Technologies). The signals of Dig-labelled locked nucleic acid miR-124 probes were detected using tyramide-conjugated Alexa 488 fluorochrome TSA kit. Nuclei were counterstained with 4,6-diamidino-2-phenylindole. The images were acquired on a Leica SP5 confocal microscope (Leica Micosystems, Mannheim, Germany).