Yp0250

Tissue or cellular protein was extracted using the RIPA lysis buffer (Beyotime, Shanghai, China), and the cytoplasmic and nuclear fractions were isolated by the extraction kit (P0027, Beyotime, Shanghai, China). The protein quantification was done with the BCA protein assay kit (Thermo Fisher Scientific, USA). The protein extracts were isolated with SDS‐PAGE gel (12%), which were subsequently transferred to the nitrocellulose membranes. The membranes were blocked for 1 h and incubated with following primary STAT3 (1:2000, 60199‐1‐Ig, Proteintech, USA), phospho‐STAT3 (1:1000, YP0250, Immunoway, USA), P65 (1:1000, 66535‐1‐Ig, Proteintech, USA), phospho‐P65 (1:1000, YP0847, Immunoway, USA), IκBα (1:1000, YM3718, Immunoway, USA), β‐catenin (1:1000, #8814, CST, USA), phospho‐β‐Catenin (1:1000, #4176, CST, USA), GSK3β (1: 5000, ab32391, Abcam, USA), Cyclin D1 (1:10000, ab134175, Abcam, USA), LEF‐1 (1:1000, ab131872, Abcam, USA), TCF‐1 (1:1000, #2203, CST, USA) antibodies overnight at 4°C and HRP‐labelled secondary antibodies (1:4000, Sigma, USA) for 2 h at room temperature. The ECL kit (Thermo Fisher Scientific, USA) was utilized to determine the immunoreactive bands, and the band densities were normalized to β‐actin (1:3000, cw0096, CWbiotech, China).

According to the instructions, the fluorescence in situ hybridization (FISH) analysis was performed by the FISH Kit (F21201, Genepharma, Shanghai, China). Cy3‐labeled mmu_circ_0001109 (5′‐CACGCUGAACACAAUCUUGUUUCCUGGAUGCAGU‐3′) or mmu_circ_0001845 (5′AUUUGCUAGGAACCGGAACAAUCCCCAAAGGGAGC‐3′) probes were also designed and obtained from Shanghai GenePharma (China). After the slides were dewaxed, the probes were added into the hybridization solution and incubated with cells overnight in the dark at 37°C. Subsequently, slides were incubated with the following primary phospho‐STAT3 (1: 200, YP0250, Immunoway, USA), phospho‐P65 (1: 400, YP0847, Immunoway, USA) and β‐catenin (1: 400, #8814, CST, USA) for 2 h at 37°C. Then, slides were incubated with secondary antibody (BS50950, 1: 100, Bioworld, USA). Finally, the slides were counter stained with DAPI and imaged. The fluorescence intensity was calculated by the Image J software.