Protease k

A specific circ_RPPH1 FISH probe labeled with Cy3 (Geneseed, China) was designed and used in the experiment. Cells attached to slides were immobilized with 4% paraformaldehyde, washed with PBS, and then digested by protease K (Sangon, Shanghai, China) at 37°C for 5 min. After washing with PBS, the cells were immobilized with 1% paraformaldehyde followed by successive dehydration in 70, 85, and 100% alcohol. Hybridization solution dilute probe was dripped onto the cell slide followed by denaturation at 73°C for 3 min and hybridization overnight at 60°C in the dark. The slides were then washed with 50% formamide/2 × SSC preheated to 43°C, 0.1% NP-40/2 × SSC preheated to 37°C, and DAPI staining solution at room temperature. Images were acquired using a laser confocal microscope (Leica, Mannheim, Germany).

The FISH probe for circRNF13 labeled with 6-FAM was utilized in our study. After the cultured cells were attached to slides, they were treated with 4% paraformaldehyde for immobilizing and washed with PBS. Consequently, the protease K (Sangon, Shanghai, China) was used to digest processed cells. After PBS washing, the cells were treated by 1% paraformaldehyde followed by alcohol dehydration. Eventually, the hybridization solution dilute probe was added onto the cell slides and denatured at 73°C for 3 min, then hybridized overnight at 42°C, and washed with preheated 50% formamide/2 × SSC, 0.1% NP40/2 × SSC, and DAPI staining solution at room temperature. Images were obtained using a laser confocal microscope (Leica, Mannheim, Germany).

We established pGL3‐LUC‐circSWT1, pGL3‐LUC‐SNAIL, mutant pGL3‐LUC‐circSWT1, and mutant pGL3‐LUC‐SNAIL for the dual‐luciferase reporter gene experiment. The wild‐type or mutant vectors were then co‐transfected into HEK‐293 T cells with miR‐370‐3p mimics or NC mimics using transfection reagents (Thermo Fisher, Cat: 11668–019). After 48 hours of incubation, cells were lysed and centrifuged, and cell supernatants were collected for detection. A dual‐luciferase reporter assay system was used to measure luciferase activity in cell supernatants (Promega).
For the FISH, cells were cultured on coverslips, immobilized with 4% paraformaldehyde, washed with PBS, and digested by protease K (Sangon) for 5 minutes at 37°C and 5% CO₂. Next, cells were washed again and fixed with 1% paraformaldehyde; thus, we dehydrated these cells. The coverslips were hybridized with a specific probe at 37°C overnight. Finally, at room temperature, the coverslips were rinsed and the cell nuclei stained with DAPI. A confocal fluorescence microscope was used to take the pictures (LSM510; Zeiss). Table S5 in Additional file 6 shows all of the probes.