4.1 Reagents

CEP (Cepharanthine, purity > 98%, MB2077-S) was purchased from Meilunbio (Dalian, China). CHX (Cycloheximide, S7418), MG132 (S2619) and ActD (Actinomycin D, S8964) were obtained from Selleck (Houston, TX, USA). Promega (Madison, WI, USA) provided the product of dual-luciferase reporter assay kits (E1960). Invitrogen (Camarillo, CA, USA) supplied the product of Lipofectamine® 3000 reagent (L3000015).

The antibodies used in the present investigation are as follows: β-catenin (Proteintech, 51067-2-AP), Axin2 (Abclonal, A17022), CyclinD1 (Proteintech, 60186-1-Ig), β-actin (Sigma-Aldrich, A1978), Lamin A/C (Proteintech, 10298-1-AP), Phospho-β-Catenin (Cell Signaling Technology, 9561), Active-β-Catenin (Cell Signaling Technology, 4270).

4.2 Cell culture

The HEK293 cell lines originated from human embryonic kidneys; the SW480, SW620, and LoVo cell lines originated from human colorectal cancer; and the NCM460 cell lines originated from human normal colon epithelial cells. They were purchased through the Chinese Academy of Sciences. All the cells were cultivated following the manufacturer’s instructions.

4.3 Plasmids

pGL3-β-catenin reporter plasmid was constructed by cloning the β-catenin promoter’s 2000 bp upstream region into the pGL3 plasmid (Promega). The Renilla luciferase plasmid was acquired from Promega. SuperTOPFlash plasmid was gift from Dr. Wei Wu (Tsinghua University, Beijing, China).

4.4 Cell viability assay

The impact of CEP on cell survival was evaluated using an MTS reagent (Promega, G3581). 96-well plates were used to seed 5000 cells per well, which were then incubated overnight. Next, the cells underwent exposure to different amounts of CEP for 24 and 48 h. After removal of the culture medium, MTS solutions were introduced and cultured for one to four hours at 37 °C. The microplate reader (PerkinElmer) was applied to measure the optical density. Based on the dose–response curves, the IC50 values were derived and computed.

4.5 Colony formation assay

Cell seeded were administrated with various doses of CEP for a period of 1–2 weeks. When clones were readily visible, they were washed in PBS and exposed to 4% paraformaldehyde for a duration of 15 min. The clones that were fixed were subsequently treated with 0.1% crystal violet for a duration of 30 min and then rinsed with distilled water. Finally, these stained clones were photographed and counted.

4.6 Cell cycle analysis

Cells were seeded then treated with CEP for 24 h. Following the sample collection, the cell cycle analysis was conducted as previously [50]. The flow cytometer (BD Biosciences) was employed to measure the amount of DNA in the stained cells, and FlowJo software was utilized to analyze the results.

4.7 Dual-luciferase reporter assay

96-well plates were employed to plant cells, which were then transfected with corresponding plasmids. After 6 h of transfection, cells were exposed to CEP for 24 h. Following this, the subsequent assay was conducted as previously [51]. The luciferase activity of fireflies was standardized by comparing it to the luciferase activity of Renilla.

4.8 RNA interference

siRNAs targeting CTNNB1 were produced by Qingke Biotechnology Company (Beijing, China). For CTNNB1, the siRNA sequences validated in the laboratory were used [51]. Lipofectamine 3000 transfected siRNAs for 48 h following manufacturer’s instructions.

4.9 Western blotting assay

The collected cells, which had been subjected to either chemical treatment or siRNA transfection, got lysed with a potent RIPA buffer (Beyotime, Shanghai, China), including PMSF, and a cocktail of phosphatase inhibitors (Roche). Next, the supernatant extracts were measured using a BCA kit (Beyotime, P0009). Each of the protein extracts was divided into equal quantities and then uploaded to SDS-PAGE. After that, they were transferred to PVDF membranes (Millipore, ISEQ00010). Specific antibodies were exposed to membranes at 4 °C for an overnight period after blocking. Afterward, the membranes were exposed to suitable secondary antibodies under ambient temperature for a duration of 1 h. Eventually, an ImageQuant LAS 4000 mini (GE Healthcare) was used to visualize target protein bands that had been exposed to ECL substrate (Thermo Fisher, 32106). The grayscale of the indicated protein was quantified by ImageJ software.

4.10 Immunofluorescence assay

96-well plates were used to seed 1.2 × 104 cells per well, which were then exposed to various amounts of CEP for 48 h. Following the treatment, the subsequent immunofluorescence experiment was conducted as previously [51].

4.11 Real-time PCR and RNA extraction

The Trizol reagent (Absin, abs60154), the reverse transcription kit (Takara, RR036A), and the SYBR reagents (Genestar, A301-10) were used for the extraction of RNA, subsequent reverse transcription, and finally real-time PCR, following the instructions provided by the respective manufacturers. Relevant primer sequences and data analysis methods were described previously [50].

4.12 Cytoplasmic and nuclear protein extraction

To summarize, the cells were gathered and immersed in lysis buffer A. The mixture was then kept on ice for a duration of 10 min. Following centrifugation, the swollen cells were incubated for 2 min on ice in buffer A containing 0.2% NP-40. The liquid portion that represents the cytoplasmic fraction was obtained by spinning at 6000 rpm for 15 min. Following a rinse with lysis buffer A, the solid parts were suspended in lysis buffer B. Following a 15-min spin at 13,000 rpm, the liquid fraction was gathered as nuclear fraction. The procedure for preparing buffer A and B has been described in previous publications [51].

4.13 Statistical analysis

To evaluate significance, two-tailed Student’s t-tests and two-way ANOVAs were used. This study used GraphPad Prism 8 to analyze the data. **p < 0.01; ***p < 0.001; *p < 0.05. P-values < 0.05 were significant.