C 1 magnetic beads

The pull-down assay was performed with biotinylated RNA as previously described. Briefly, for circCELSR1 pulled-down miRNAs, the biotinylated-circCELSR1 probe was incubated with C-1 magnetic beads (Life Technologies, Carlsbad, CA, USA) at 25°C for 2 h to generate probe-coated beads. These were then incubated with sonicated cells at 4°C overnight, eluted with the wash buffer, and analyzed by quantitative real-time PCR. For miR-1252 pulled-down circCELSR1, cells with circCELSR1 overexpression were transfected with biotinylated miR-1252 mimics or mutant miR-1252 using Lipofectamine 3000. The cells were harvested 48 h after transfection, lysed, sonicated, and incubated with C-1 magnetic beads (Life Technologies, Carlsbad, CA, USA), eluted with the wash buffer, and analyzed by quantitative real-time PCR. The biotinylated circCELSR1 probe and biotinylated miR-1252 mimics or mutant were designed and synthesized by RiboBio (Guangzhou, China).

Biotinylated-miR-543 probe and biotinylated NC (Bio-NC) probe were generated by Guangzhou RiboBio Co., Ltd. The RNA pull-down was performed as previously described (28 (link)). To produce probe-coated beads, miR-543 (Guangzhou RiboBio Co., Ltd.) was incubated with C-1 magnetic beads (Thermo Fisher Scientific, Inc.) for 2 h at 25°C. The cell lysates were then harvested and treated with the 50 pmol miR-543 probe or Bio-NC probe overnight at 4°C. After washing with wash/binding buffer, the RNA complexes adsorbed in the beads were collected and used to conduct RT-qPCR assay and northern blot analysis. For northern blot analysis, 30 µg RNAs were separated in a 1% agarose-formaldehyde gel and transferred to Hybond-N+ membrane (Beyotime Institute of Biotechnology). Then, the membranes were hybridized with digoxin-labeled DNA oligonucleotides specific to circ-FOXO3 (Guangzhou RiboBio Co., Ltd.) at 37°C. The membranes were exposed to phosphorimager screens and analyzed using Image Lab V3.0 software (Bio-Rad Laboratories, Inc.).

The targeted binding effect between circTADA2A and miR-638 was verified by RNA pull-down assay as previously reported (25 (link)). Biotinylated miR-638 probe was purchased from Shanghai GenePharma Co., Ltd. A549 and H1299 cells (1×10⁶) were harvested, lysed by 200 µl Pierce^® IP lysis buffer (Thermo Fisher Scientific, Inc.) and sonicated. The pull-down assay was performed using a Pierce™ Magnetic RNA-Protein Pull-Down kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. In brief, 50 µl C-1 magnetic beads (Thermo Fisher Scientific, Inc.) were co-incubated with 50 pmol biotinylated miR-638 or NC probes at 25°C for 30 min to generate probe-coated beads. The beads were harvested by a magnetic stand (Thermo Fisher Scientific, Inc.), then incubated with cell lysate at 4°C for 60 min. The beads were harvested using a magnetic separation stand (Thermo Fisher Scientific, Inc.). The pulled down RNA complexes were eluted and extracted using a RNeasy Mini kit (Qiagen, Inc.), Pulled down circTADA2A and circular nucleolar protein 10 (NOL10) were determined by RT-qPCR, as aforementioned.