Hybond n nylon membrane

Northern blot analysis was performed with northern blot kit (Ambion, USA). Brie y, about total RNAs (30 µg) of osteosarcoma cells were denatured in formaldehyde and then electrophoresed in a 1% agaroseformaldehyde gel. The RNAs were then transferred onto a Hybond-N + nylonmembrane (Beyotime, China) and hybridized with biotin-labeled DNA probes. Biotin chromogenic detection kit (Thermo Scienti c, USA) was used to detect the bound RNAs. Finally, the membranes were exposed and analyzed using Image Lab software (Bio Rad, USA).

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Northern blot analysis was performed with a northern blot kit (Ambion, USA). Briefly, total RNA (~30 μg)
from NP cells was denatured in formaldehyde and then was electrophoresed in a 1% agarose-formaldehyde gel. The RNAs were then transferred onto a Hybond-N + nylon membrane (Beyotime, China) and were hybridized with biotin-labelled DNA probes. A biotin chromogenic detection kit (Thermo Scientific, USA)
was used to detect the bound RNAs. Finally, the membranes were exposed and analysed using Image Lab software (Bio Rad, USA).
2.12 RNA fluorescent in situ hybridization (FISH)
FISH assays were performed in NP cells or NP tissues. 29 Cy3-labelled CircGLCE probes and 488-labelled locked nucleic acid miR-587 probes were designed and synthesized by RiboBio (Guangzhou, China). The signals of the probes were detected by a Fluorescent in situ hybridization kit (RiboBio, Guangzhou, China)
according to the manufacturer's instructions. The images were acquired on a Nikon A1Si laser scanning confocal microscope (Nikon Instruments Inc., Japan). For in vivo FISH, tissue sections were deparaffinized, rehydrated, and permeabilized by 0.8% pepsin treatment at 37°C for 30 min before hybridization.