Complete medium

HCT-116 and PMF-K014 cells were initially stimulated in complete-medium supplemented with 0.2 mM sodium butyrate (Sigma-Aldrich, St. Louis, USA). Butyrate treatment induced cancer cell death. However, some cells survived and continued to proliferate; these were considered BR. BR cells were subcultured till 80% confluent. Subsequently, the concentration of butyrate was increased twofold every three generations. After the concentration of butyrate reached 3.2 mM, BR cells were used in further experiments.

To compare the proliferation of A375^SPANX-KD and A375^CTRL cell lines, 5 × 10³ cells were seeded into 96-well plates in complete medium (DMEM supplemented with l-glutamine and 10% FBS) (Sigma-Aldrich) and incubated overnight. After 18 h, the cells were considered to be in T = 0. After further incubation for 24 h, cell viability was measured using PrestoBlue Viability Reagent (Life Technologies, Thermo Scientific, MA, USA) following the manufacturer’s instructions. Briefly, the medium was replaced with PrestoBlue diluted 1/10 in complete medium and the viability was measured after 2 h of incubation. The results shown are the means of three different experiments, and the error bars indicate the SEM.

The vertical migration of mMSCs was detected using the Transwell migration assay. Transwell inserts (6.5-mm diameter and 8-mm pore size, Corning Inc., Corning, NY, USA) were loaded with 2 × 10⁴ BM-MSCs in 200 μl of complete medium (Cyagen, Suzhou, China), and 600 μl of serum-free DMEM/F12 (Corning Inc., Corning, NY, USA) with or without 1.5 × 10⁶ cells in the lower chambers. The complete medium containing different drugs (10 μg/ml aristolochic acid I sodium salt (AA-I), Sigma, USA; 10 ng/ml TNF-α, Peprotech, USA; 5 ng/ml transforming growth factor-β1 (Tgroup). The sham control (sham+salineGF-β1), Sino Biological China) was placed in the lower well to induce cell migration. The cells were allowed to migrate at 37 °C in a humidified CO₂ incubator for 36 h. The nonmigrated cells on the upper side of the membranes were removed using cotton swabs. Migrated cells on the lower side of the membranes were fixed with 4% paraformaldehyde for 30 min at 4 °C and stained with 0.1% crystal violet for 20 min. Five different visual fields (× 200 magnification) were randomly selected for observation and photographed under a fluorescence microscope (Olympus Optical Co., Ltd.). Quantification was performed by calculating the percentage of stained area to the total area using FIJI software.

Spleens were harvested, and single-cell suspensions were obtained after the tissue was mashed and passed through a 70-μm cell strainer (Becton Dickinson Biosciences Pharmingen, San Diego, CA, USA) in 15 mL of PBS. Pelleted cells from spleen were resuspended in Tris-buffered 0.83% NH₄Cl solution to lyse erythrocytes and washed in PBS. The total number of spleen leukocytes was analyzed in complete medium (Iscoves medium with L-glutamin, mercaptoethanol, gentamycin, and fetal calf serum) (Sigma-Aldrich) by using an automated cell counter (Sysmex, Hamburg, Germany). Synovial tissue was dissected and placed in medium (RPMI) (FisherScientific, Västra Frölunda, Sweden). Medium with DNaseI (Sigma-Aldrich) and collagenase type IV (Roche AB, Stockholm, Sweden) was added, and the suspension was incubated for 1 hour at 37°C. A single-cell suspension was obtained after the tissue was mashed and passed through a 40-μm cell strainer (Becton Dickinson) in 4 mL of PBS.