Fish kit

The RNA fluorescence in situ hybridization assay was performed by using a FISH kit (RiboBio, Guangzhou, China) according to the manufacturer’s guidelines. Cy3-labeled circRNA-RBCK1 probes and Dig-labeled locked nucleic acid miR-133a probes (Ribo-Bio, Guangzhou, China) were measured by the FISH kit, followed by visualized with a confocal microscopy.

We used Cy3-labeled circCASP9 and Cy5-labeled miR-589-5p probes (GenePharma) in GC cells using FISH kits (RiboBio). Probes specific for circCASP9 and/or miR-589-5p were mixed and incubated overnight, and nuclei were then stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired using an LSM800 laser scanning confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). The sequences of the Cy3-labeled circCASP9 and Cy5-labeled miR-589-5p probes are listed in Table 2.

According to the directions of FISH kits (Guangzhou RiboBio Co., Ltd., Guangdong, China), A549 and H157 cells were trypsinized, seeded into confocal dishes and cultured, and then the medium was discarded after cell adherence. Fixed with 1 mL formaldehyde for 15 min, the cells were permeabilized with prepared 1% Triton X-100 for 5 min and blocked with prepared prehybridization solution for 30 min, and the hybridization solution was preheated at 37 °C. The prehybridization solution was removed and cells were incubated with probe hybridization solution containing appropriate probes overnight, rinsed by sodium citrate buffer for 3 times (10 min/time), and stained with 4′,6-diamidino-2-phenylindole 2 hci (DAPI) for 10 min. Cells were photographed under a fluorescence microscope.