- Cell culture
Human bladder cancer cell lines HTB-9 and T24 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in RPMI 1640 (Gibco Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Gibco Life Technologies) in a humidified atmosphere with 5% CO2 at 37°C.
- BKPyV infection and inactivation
BKPyV stocks were initially propagated in Vero cells from viruses obtained from ATCC (VR-837, Dunlop). Viral lysates were made through three cycles of freezing the infected cells and supernatant at −80°C and thawing at 37°C. The inactivation of BKPyV was performed at 100℃ for 10 minutes. Both T24 and HTB-9 cells were grown in culture dishes to 70% confluence, and infected with BKPyV. After infection for two hours, cells were washed three times with phosphate-buffered saline (Kaiji Co. Ltd., Shanghai, China), then were cultured using medium containing 2% serum with and without 15 mM KYA1797K (Med Chem Express, Princeton, NJ, USA),a potent and selective β-catenin inhibitor.
- Animal model
Male BALB/c nude mice (age 5 weeks, 18–20 g; Shanghai LC Laboratory Animal Co. Ltd., Shanghai, China) were housed in sterile filter-capped cages. Cultures of T24 and HTB-9, including their respective BKPyV infected cells (106 cells in 100 mL PBS) were injected subcutaneously into nude mice with and without subsequent intraperitoneal injections of KYA1797K (25 mg/kg) (n=5). Thirty days after implantation, tumors were surgically dissected and stored in liquid nitrogen before processing for histopathological examination. All animal experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Shanghai Public Health Clinical Center, Fudan University.
- Cell Counting Kit-8 assay
The Cell Counting Kit-8 assay (CCK-8; Xiang SAM, Shanghai, China) was used to determine cell viability. Cells (2×103 cells/well) were seeded in 96-well plates according to the manufacturer's instructions. Absorbance of the medium at 450 nm was detected using a spectrophotometer by assessing cell viability. All observations were reproduced at least three times in independent experiments.
- Colony-formation assay
Cells were seeded in six-well plates at an initial density of 200 cells/well. Colonies were clearly visible after 10–14 days and selected cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature and stained with 4 mg/mL crystal violet (Sigma-Aldrich). Colonies containing >50 cells were counted using light microscopy (100×; Olympus, Tokyo, Japan). The average number of colonies was determined from three independent experiments.
- Cell migration and invasion assays
Transwell chambers (8-µm pore size; Corning, Corning, NY, USA) pre-coated with and without Matrigel (BD Biosciences) was used to determine cell invasion and migration. Cells (105) in 200 μL FBS-free medium were seeded in the upper chamber, and 600 mL medium containing 10% FBS was added to the lower chamber. After several hours of incubation, cells that had migrated or invaded through the membrane were stained with methanol and 0.1% crystal violet solution. A light microscope (200×; Olympus, Tokyo, Japan) was used to count the number of cells in five random fields of view. The mean cell number was calculated for each group.
- Scratch wound healing assay
Cells were inoculated in six‐well plates and cultured at 37°C in a 5% CO2 cell incubator. After the cells reached 90% confluence, wounds of approximately 1 mm width were created using a sterile pipette tip. Cells were washed, incubated and continuously cultured in serum‐free medium. Cultures at 0, 8, and 24 hours were observed under an inverted microscope (40×; Olympus, Tokyo, Japan).
- Histological assessment
Hematoxylin and eosin(H&E) staining was performed for histological assessments. Tumor tissues were cut into small pieces and soaked in 4% neutral formaldehyde solution. Tissue blocks were washed with distilled water and stored in 70% ethanol overnight. After being dehydrated, embedded in transparent paraffin and sectioned, slides were sealed with polylysine and stained using H&E.
- Immunofluorescence staining
Cell slides and frozen sections in 4% formaldehyde (10% PFA, Polysciences, Eppelheim, Germany) were diluted in PBS for 10 min and permeabilized with 0.2% Triton X-100 (10%, Sigma-Aldrich) for 10 min at room temperature (RT). Fixed cells were blocked with blocking buffer containing milk powder and PBS for 15 minutes (37°C). Primary and secondary antibodies were diluted in blocking buffer and incubated at RT for 50 minutes each. Primary antibodies included the polyclonal human rabbit anti-β-catenin (1:1000; Cell Signaling Technology, Beverly, MA, USA) and the monoclonal mouse anti-Large tumor antibody (1:50; PAb416, Abcam, Cambridge, MA,USA). Secondary antibodies used were the anti-mouse IgG1–Alexa Fluor 647 (1:800; Abcam), anti-rabbit IgG–Alexa Fluor 488 (1:1000; Abcam) and direct staining with Hoechst 33342 dye (1:1000; Abcam). Fluorescence was detected using inverted fluorescence microscope (EVOS XL Core, Thermo Fisher Scientific, USA). All images were processed using ImageJ software (NIH, Bethesda, MD, USA).
- Western blot
Western blotting was carried out as previously described. Membrane proteins were separated on SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were blocked using 5% milk and incubated overnight with anti-β-catenin, anti-cMYC, anti-Slug, anti-claudin-1 and anti-β-actin antibodies (1:1000; all from Cell Signaling Technology). Results were analyzed following previously described methods.
- Statistical analysis
All generated data is presented as mean ± standard deviation(SD). Statistical analyses were performed using the unpaired Student’s t-test or one-way Analysis of Variance(ANOVA) for more than two groups. Significant differences were considered when vales had P<0.05. All analyses were carried out using Graph pad prism 7 software.