Fish detection kit

To detect circ-0000437 RNA in EC cells, RNA FISH experiments were performed with FISH Detection Kit (Ribo) in EC cell lines according to the protocol. Cells cultured on coverslips were fixed with 4% paraformaldehyde at 4 °C for 15 min and washed three times with PBS. The samples were subsequently incubated with Pre-Hybridization Buffer at 37 °C for 30 min and Hybridization Buffer with FISH probes at 37 °C overnight in the dark using a RiboTM Fluorescent In Situ Hybridization Kit (RiboBio). On the next day, the coverslips were washed three times with wash buffer I (4 × SSC with 0.1% Tween-20), once each with wash buffer II (2 × SSC) and wash buffer III (1 × SSC) at 42 °C in the dark for 5 min and once with PBS at room temperature. Then, the cells were stained with DAPI in the dark for 10 min. The has-circ-000437-Cy3 FISH probes were designed and synthesized by RiboBio Co, Ltd. Human U6 FISH probes (RiboBio) and Human 18S FISH probes (RiboBio) were used as nuclear and cytoplasmic controls, respectively.

Cy3-CircCCT3 and fluorescein isothiocyanate (FITC)-miR-613 probes were synthesized and obtained from RiboBio (Guangzhou, PR China). Hybridization assays were performed using a FISH Detection Kit (RiboBio, PR China). All images were captured by a confocal microscope (FV1000; Olympus, Tokyo, Japan).