Fluorescence secondary antibody

EC9706 cells were fixed with 4% paraformaldehyde for 10 minutes, 0.5% Triton perforated for 5 minutes, after that rinsed with PBS for three times and then sealed with 5% BSA at room temperature for 1.5 hours. Rabbit anti‐RACO‐1 polyclonal antibody (HAP030098, Sigma) and mouse anti‐YAP monoclonal antibodies (SC‐101199, Santa Cruz) were used to incubated and then combined with fluorescence secondary antibody (Invitrogen). As negative controls, the samples were incubated with the secondary antibodies without primary antibodies. After stained with DAPI, images were observed under laser scanning confocal microscopy (Nikon C2+/si+ Japan).

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.

4-week-old male Kunming mice were purchased from the Experimental Animal Center of Fourth Military Medical University and approved by the Animal Ethics Committee of Fourth Military Medical University (License number: IACUC-2020065). The animal experiment was complied with the ARRIVE guidelines and was performed in accordance with the National Institutes of Health guide for the care and use of Laboratory animals. 60% high-fat diet were purchased from Readydietech, China. Streptozocin (STZ) was purchased from Sigma, USA. Hematoxylin-Eosin (HE) was purchased from Solarbio, China. CD31 antibody was purchased from Abcam (28364, 1:100), USA. F4/80 antibody was purchased from Abcam (6640, 1:100), USA. CD16/32 antibody was purchased from BD Biosciences (553142, 1:50), USA. Fluorescence secondary antibody was purchased from Invitrogen, USA. IL-1β and TNF-α ELISA kits were purchased from Proteintech, USA.