Confocal laser scanning microscopy

Fluorescence in situ hybridization (FISH) and in situ hybridization (ISH) analyses were conducted using the in situ hybridization kit (BOSTER, Wuhan, China) according to the manufacturer’s instructions. Dig-labeled probes (Sangon Biotech, Shanghai, China) were used to detect circCDYL2 expression in nasopharyngeal carcinoma cells or clinical tissue samples. FISH employed fluorescence secondary antibody (Invitrogen, CA, USA), while ISH used DAB staining reagents (Meixin Biology, Fujian, China). Cell nuclei were counterstained with DAPI (Invitrogen, CA, USA, for FISH) or hematoxylin (for ISH). FISH slides were observed and photographed using confocal laser scanning microscopy (Perkin Elmer, MA, USA). The ISH slides were independently evaluated by two pathologists and analyzed using a semi-quantitative integral method. The sequences of circCDYL2 probes used in this study are provided in Supplemental Table 3.

UCMSCs were seeded on glass-bottomed cell culture dishes (NEST, Jiangsu, China) for immunofluorescence staining of the cytoskeleton. The adherent cells were fixed with 0.4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.1% Triton X-100 (T8200, Solarbio Science and Technology, Beijing, China) in PBS for 15 min at room temperature. After removing the Triton X-100, the permeabilized UCMSCs were washed three times with PBS and blocked with 5% bovine serum albumin (A8010, Solarbio Science and Technology, Beijing, China) for 1 h at room temperature. A total of 200 μL of anti-β-tublin (ab18207, Abcam, Cambridge, UK) was added, and then the UCMSCs were refrigerated overnight and washed with PBS three times. Rhodamine-conjugated phalloidin (350–555, Thermo Fisher, MA, USA) and 488-conjugated goat anti-rabbit IgG (ab150077, Abcam, Cambridge, UK) were added to the glass bottom for 60 min. The cells were washed again and incubated with a 4′-6-diamidino-2-phenylindole (DAPI) medium (C0065, Solarbio Science and Technology, Beijing, China) to stain the nuclei. The samples were imaged using confocal laser-scanning microscopy (PerkinElmer, MA, USA) and analyzed by the Volocity Demo software.